CN116847887A - Methods and compositions for treating autoimmune diseases and cancers - Google Patents

Methods and compositions for treating autoimmune diseases and cancers Download PDF

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CN116847887A
CN116847887A CN202180072889.2A CN202180072889A CN116847887A CN 116847887 A CN116847887 A CN 116847887A CN 202180072889 A CN202180072889 A CN 202180072889A CN 116847887 A CN116847887 A CN 116847887A
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H·M·谢泼德
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Innosi Treatment Co
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Innosi Treatment Co
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Abstract

Molecular constructs targeting tumor necrosis factor receptor 1 (TNFR 1) and/or tumor necrosis factor receptor 2 (TNFR 2) are provided. The constructs are useful for treating diseases, disorders and conditions in which these receptors and/or TNF are involved in the etiology or in which their inhibition or activation may ameliorate the disease, disorder and condition or symptoms thereof. Among the constructs provided herein are TNFR1 antagonist constructs that are engineered to inhibit TNFR1 function and to eliminate any TNFR1 agonist activity. Constructs provided herein include agonists and antagonists of TNFR1 and TNFR 2. The TNFR1 antagonist construct is engineered to inhibit TNFR1 function, and in some embodiments, is engineered to avoid agonist activity. Agonists and antagonists of TNFR2 are also included. Agonists of TNFR2 increase regulatory T cell function to control acute or chronic inflammation. Antagonists of TNFR2 decrease regulatory T cell function, thereby increasing immunity, and are useful in the treatment of cancer and certain immunodeficiency disorders. Methods of treating various diseases in which TNF and its receptors play a role are also provided.

Description

Methods and compositions for treating autoimmune diseases and cancers
RELATED APPLICATIONS
Priority is claimed to U.S. provisional application serial No. 63/071,313, entitled "METHODS AND COMPOSITIONS TO TREAT AUTOIMMUNE DISEASES AND CANCER", filed by inventor h.michael sheplate and applicant Enosi Life Sciences Corp at day 27, 8 of 2020.
This application is related to International PCT publication No. WO 2020/172218, published as International PCT publication No. WO 2020/172218 at month 2 and 19 in 2020, and International PCT application No. PCT/US2020/018739 entitled "ANTIBODIES AND ENONOMERS", by the inventors H.Michael sheplate and by the applicant Enosi Life Sciences Corp. This application is also related to U.S. application Ser. No. 17/432,720, filed 8/20 in 2021 (U.S. national phase application No. 2020/018739, filed 19 in 2020, which claims the benefit of provisional application Ser. No. 62/808,635, filed 21 in 2019).
The subject matter of each of these applications is incorporated by reference in its entirety where permitted.
Incorporated by reference to an electronically provided sequence listing
The electronic version of the sequence listing is hereby submitted, the content of which is incorporated by reference in its entirety. An electronic file was created at 2021, 8, 26, and a size of 1.629 megabytes, titled 5301seqpc1.Txt.
Technical Field
The present application relates to nucleic acid constructs and encoded products useful as anti-TNF therapeutic agents. The diseases treated are diseases in which a TNF receptor and/or TNF/TNF receptor pathway or TNF receptor and/or TNF/TNF receptor pathway is involved, play a role in its etiology.
Background
anti-TNF therapeutic/TNF blocker (a biologically improving anti-rheumatic drug; DMARDs) is typically prescribed after failure of a conventional DMARD. These therapeutic agents include monoclonal antibodies (mAbs), such as the chimeric mAb infliximab @) The method comprises the steps of carrying out a first treatment on the surface of the Contains a murine variable region and a human IgG1 constant region and a fully humanized mAb (IgG 1) adalimumab (e.g., under the trademark +.>Sold) and golimumab (>An antibody); mAb cetuzumab targeting TNFAntibodies) a pegylated humanized Fab' fragment; and TNFR2 fusion proteins, for example the TNFR2-Fc fusion protein etanercept (under the trademark +.>Sold) which contains an extracellular receptor region containing the binding site of human TNFR2 fused to the Fc of human IgG 1. To->And->The drug marketed under the trademark infliximab is a biomimetic of infliximab and has been approved in the European Union for the treatment of a variety of autoimmune and chronic inflammatory diseases and conditions. These TNF inhibitors that sequester TNF are useful in the treatment of a variety of diseases and conditions including, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile Idiopathic Arthritis (JIA), and/or inflammatory bowel disease (IBD; e.g., crohn's disease and ulcerative colitis).
However, such therapeutic agents are associated with serious side effects, including, for example, increased risk of sepsis and serious infections, such as listeriosis, tuberculosis reactivation, hepatitis b/c reactivation, herpes zoster reactivation, and reactivation of invasive fungal and other opportunistic infections, including mycobacterium tuberculosis (m. These therapeutic agents have been shown to induce macrophage apoptosis in rheumatoid synovium. Infliximab (Infliximab) is associated with increased apoptosis of inflammatory cell infiltration in the intestine of patients with crohn's disease. Other antirheumatic drugs, such as methotrexate and glucocorticoids, can also induce immune apoptosis (see, e.g., vigna-Prez et al 2005 Clin. Exp. Immunol.141 (2): 372-380). These therapeutic agents can also lead to severe congestive heart failure, drug-induced lupus and demyelinating Central Nervous System (CNS) diseases, and exacerbation of lymphomas and non-melanoma skin cancers (see, e.g., benjamin et al disease Modifying Anti-Rheumatic Drugs (DMARDs) [ Updated 2020Feb 27]. In: statPearls [ Internet ]. Trecure Island (FL): statPearls Publishing); 2020Jan available from: other adverse side effects include liver injury, demyelinating diseases/CNS disorders, lupus, psoriasis, sarcoidosis and increased susceptibility to the development of other autoimmune diseases and cancers including lymphomas and solid malignancies (see e.g. Dong et al (2016) proc.Natl. Acad. Sci. U.S. 113 (43): 12304-12309;Zalevsky et al (2007) J.Immunol.179:1872-1883; zora et al (2019) Sci.Rep.9:17231), and therefore the use of these therapeutic agents, in particular for RA patients who require chronic diseases/conditions such as Arthritis and Inflammatory Bowel Disease (IBD) for which there is a limited time, is unresponsive when using Anti-TNF therapeutic agents or the therapeutic benefit is not sustained (see e.g. McCann et al (2014) Artid. Sci.6. U.113 (43):) J.Immunol.179:1872-1883; zora et al (2019) Sci.Rep.9:17231) and hence the therapeutic benefit is not sustained (see e.g. McCanton et al (2014) and hence no more than 13% of the therapeutic benefit is stopped In patients who receive Anti-TNF therapy (27.37) for which is a limited time-use of Anti-TNF therapeutic agent (e.g. for which there is no longer needed for chronic diseases/conditions such as Arthritis and Inflammatory Bowel Disease (IBD) for which is limited time, about 30%, there is a need for therapies with improved efficacy and safety.
Summary of The Invention
Molecular constructs targeting tumor necrosis factor receptor 1 (TNFR 1) and/or tumor necrosis factor receptor 2 (TNFR 2) and nucleic acids encoding the same are provided. The constructs are useful in the treatment of diseases, disorders and conditions in which these receptors and/or TNF are involved in the etiology or in which inhibition or activation thereof may ameliorate a disease, disorder and/or condition or symptoms thereof. Constructs provided herein include agonists and antagonists of TNFR1 and TNFR2. The TNFR1 antagonist construct is engineered to inhibit TNFR1 function and to avoid TNFR1 agonist activity. Agonists and antagonists of TNFR2 are also included. Agonists of TNFR2 increase regulatory T cell function to control acute or chronic inflammation. Antagonists of TNFR2 reduce regulatory T cell function, thereby enhancing immunity, for the treatment of cancer and certain immunodeficiency diseases.
Cells have two TNF receptors: TNFR1 and TNFR2. These pathways are balanced with each other in normal physiology. TNF/TNFR1 drives inflammation, while TNF/TNFR2 has anti-inflammatory effects. TNFR2 is usually activated later than TNFR1 and therefore does not immediately affect useful TNF-induced inflammation, but is activated later to inhibit excessive activation of the inflammatory pathway. Simultaneous inhibition of both pathways removes the inflammatory inhibition of TNFR2. Existing TNF blockers limit their own efficacy because the anti-inflammatory Treg generator (TNFR 2) is turned off/off.
The constructs provided herein, in addition to properties other than existing therapeutic agents targeting TNF/TNFR, inhibit TNFR1 signaling or activity without compromising the ability of the subject to resist opportunistic infections. Among the constructs provided herein, there is a type of modified single chain antibody that specifically targets and inhibits TNFR1, but does not antagonize TNFR2, thereby preventing transient activation of TNFR1 by receptor clustering. The constructs provided herein silence TNF inflammatory pathways mediated by TNFR1, but retain and in some embodiments enhance the healing pathway of TNFR 2. These constructs can be administered to treat indications for TNF blocker failure. The constructs provided herein are those that specifically inhibit type 1 tumor necrosis factor receptor; methods and uses of the constructs are provided for treating diseases, disorders, and conditions in which TNF or its receptor plays a role in etiology or symptoms.
The existing anti-TNF drug blocks excessive inflammation occurring in autoimmune diseases including rheumatoid arthritis, polyarthritis idiopathic arthritis, central axial type spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, crohn's disease, pediatric crohn's disease and ulcerative colitis. The constructs herein can be used to treat the same disease, but avoid deleterious or adverse side effects. The constructs provided herein are superior to existing therapeutic agents such as the TNFR2-Fc fusion protein etanercept (under the trademark Sales) are more efficient and maintain regulatory T cell function. The construct may include an activity modulator or property modulator to increase serum half-life, which has been demonstrated to be active in blocking TNFR1 signaling, for example in TNF assays that compare activity with adalimumab and/or etanercept.
As established in the mouse model, the constructs retained macrophage function better than adalimumab, indicating that they did not lead to opportunistic infections; they also retain Treg function better than adalimumab or etanercept and are equally effective in treating diseases, disorders and conditions such as rheumatoid arthritis. In some embodiments, kd.ltoreq.1 nM, t in vivo 1/2 About 10-12 days. The construct may be administered by any route appropriate for the particular indication. Such routes include, but are not limited to, subcutaneous,Intravenous, intratumoral, intrahepatic, topical, mucosal, intradermal, and any other suitable route.
Constructs provided herein are shown below. The constructs provided are tumor necrosis factor receptor 1 (TNFR 1) antagonist constructs of formula 1: (TNFR 1 inhibitor) n -a joint p - (activity modulating agent) q Wherein: n and q are each integers and are each independently 1, 2 or 3; p is 0, 1, 2 or 3; TNFR1 inhibitors are molecules that bind TNFR1 to inhibit (antagonize) TNFR1 activity; an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and the linker increases the flexibility of the construct and/or moderates or reduces the steric effect of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media. The linker may comprise a plurality of components. Linkers include chemical linkers, polypeptide linkers, and combinations thereof. The constructs may be linked by chemical and/or physical bonds. The construct may be a fusion protein.
The TNFR1 inhibitor may comprise a domain antibody (dAb) or a single chain antibody. Such constructs include those in which the TNFR1 inhibitor is a domain antibody (dAb), the activity modulator is not an unmodified single Fc region or a human serum albumin antibody. For example, the activity modulator (or property modulator) is a modified Fc region or is human serum albumin. In the construct, the TNFR1 inhibitor may be an inhibitor that inhibits TNFR1 signaling, and/or the activity modulator increases the serum half-life of the construct. For example, the constructs include those in which the activity modulator is albumin or is modified to have Fc with reduced or no ADCC (antibody dependent cellular cytotoxicity) activity and/or reduced or no CDC (complement dependent cytotoxicity) activity. The TNFR1 inhibitor may be an inhibitor that inhibits TNFR1 activity but does not antagonize tumor necrosis factor receptor 2 (TNFR 2) activity. The TNFR1 inhibitor may be an inhibitor that inhibits TNFR1 signaling.
Multispecific constructs are also provided. For example, a multispecific construct is provided comprising a TNFR1 inhibitor and a Treg amplicon, wherein the bispecific construct interacts with two different target receptors or antigens or epitopes on the receptor. The multispecific constructs are those that are bispecific to TFNR1 and Treg amplicons. Treg amplicons may be TNFR2 agonists.
The construct may include linkers to provide flexibility, increase solubility, and/or reduce and/or steric hindrance and/or van der waals interactions. The construct optionally but typically comprises an activity modulator to alter or modulate the activity or property of the construct. Constructs having formula 2 are provided: (TNFR 1 inhibitor) n - (activity modulating agent) r1 - (joint (L)) p - (activity modulating agent) r2 - (TNFR 2 agonists) q Or (TNFR 1 inhibitor) n - (activity modulating agent) r1 - (joint (L)) p - (activity modulating agent) r2 - (Treg amplificates) q Wherein: n=1, 2 or 3, p=1, 2 or 3, q=0, 1 or 2, and r1 and r2 are each independently 0, 1 or 2; and the components may be in the order specified or any other order, so long as the construct interacts with TNFR1 and TNFR2 to antagonize TNFR1 and activate TNFR2, or has Treg amplicon activity. For example, among any of those constructs provided herein, constructs in which a TNFR1 inhibitor moiety inhibits tnfα binding to TNFR1 and/or inhibits signaling are included.
Constructs of formula 3a or 3b are also provided: (TNFR 2 agonist or Treg amplificates) n -a joint p - (activity modulating agent) q Formula 3a, or (Activity modifier) q -a joint p - (TNFR 2 agonist or Treg amplimer) n Formula 3b, wherein: n and q are integers and are each independently 1, 2 or 3; p is 0, 1, 2 or 3; an activity modulator is a moiety that alters the pharmacological properties or activity of a construct; the TNFR2 agonist interacts with TNFR2 resulting in TNFR2 activity; treg amplicons, including TNFR2 agonists, are molecules that lead to an increase in Treg cells; and the linker increases flexibility and/or mitigates or reduces the steric effects of the construct or its interaction with the receptor; and/or altering the solubility of the construct. In some embodiments, the activity modulator is an Fc region or modifiedAn Fc region or short FcRnBP; the linker comprises a hinge region, or is a linker comprising G and S residues. Examples of linkers are those that increase the serum half-life of the construct. For example, the linker may have the sequence shown in any one of SEQ ID NOS 812-834 or be a PEG moiety linker. In some embodiments, the construct comprises an activity modulator that is a modified Fc region or a peptide that increases the serum half-life of the construct. The Fc region may be an Fc dimer; the Fc region may be modified to have reduced ADCC and/or CDC activity, e.g., fc modified to have reduced or no ADCC activity.
Included among the constructs provided herein are any constructs wherein the TNFR1 inhibitor is defined in the sequence listing, listed below, or known in the art; the Treg amplicons are any amplicons known in the art, TNFR2 agonists or any Treg amplicons listed in the sequence listing or known in the art; the linker is a sequence listing or any linker listed below or known in the art; the activity modulator is any activity modulator listed in the sequence listing, known in the art, and/or listed below.
Constructs are provided that are TNFR1 antagonist constructs comprising TNFR1 inhibitors that are single chain antibodies or antigen-binding portions thereof that specifically target and inhibit TNFR1 but do not antagonize TNFR2, thereby preventing transient activation of TNFR1 by receptor clustering. In such constructs, the antibody or antigen-binding portion thereof comprises modifications that improve the pharmacological properties and/or structure of the construct.
In any of the constructs provided herein, the construct includes a component that agonizes TNFR2 signaling thereby increasing regulatory T cell (Treg) expression, thereby providing TNFR1 antagonism and concomitant (or substantially concomitant) increase in Treg expression. In the constructs provided herein, the TNFR1 inhibitor can be a single chain antibody that inhibits TNFR1 by inhibiting TNFR1 signaling, e.g., an antibody portion or antigen-binding portion of the construct inhibits binding of tnfα to TNFR 1. In these constructs, the TNFR1 inhibitor is an antibody or antigen-binding portion that does not inhibit tnfα binding to TNFR1 but inhibits TNFR1 signaling. The property or activity that can be modulated/altered can be serum half-life.
The construct may comprise an Fc modified to eliminate ADCC and/or CDC activity. The construct may comprise Fc dimers, for example, one of the Fc monomers comprises a recess (holes) and the other comprises a projection (knobs) to form a heterodimer. For example, the convex mutation is selected from the group consisting of S354C, T366Y, T366W and T394W of EU numbering; and the concave mutation is selected from the group consisting of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V, EU numbering, whereby the Fc monomer forms a heterodimer. In some embodiments where the construct comprises Fc, the Fc is from trastuzumab. The construct may dimerize by fusing the N-terminus and the C-terminus of trastuzumab.
In some embodiments where the construct comprises a linker, the linker is or comprises a hinge region from the Fc region. For example, the hinge region is derived from trastuzumab, which is linked to the Fc region. Constructs include those comprising a linker linked to an anti-TNFR 1 antagonist antibody or antigen-binding portion thereof. The linker may be attached to the anti-TNFR 1 antagonist antibody, or antigen-binding portion thereof, and to the Fc region, either directly or through a hinge region. The Fc region or modified Fc region comprises, for example, the amino acid sequences shown in any of SEQ ID NOs 10, 12, 14, 16, 27, 30, 1469 and 1470.
Constructs that bind to neonatal Fc receptor (FcRn) are also provided. For example, provided are TNFR1 constructs comprising a short FcRn binding peptide (FcRnBP) that provides for interaction of the construct with FcRn and comprises 6-25 or 10-20 amino acid residues. For example, fcRnBP contains 12-20 residues or 15 residues or 16 residues. Examples of these are TNFR1 antagonist constructs in which the FcRn binding peptide (FcRnBP) comprises or consists of a peptide as set forth in any one of SEQ ID NOs 48-51. The constructs include TNFR1 constructs comprising Fc heterodimers, wherein one Fc monomer comprises a recess and the other comprises a projection, whereby the Fc dimer produced is a heterodimer.
The constructs provided are TNFR1 antagonist constructs comprising: TNFR1 inhibitors; fc dimers; and Treg amplifications, wherein: the Fc dimer comprises two complementary Fc monomers; the TNFR1 inhibitor is linked to one Fc monomer and the Treg expansion is linked to the other Fc monomer. In such constructs, the Treg expansion may be a TNFR2 agonist. It may also comprise a second Treg expansion linked to the same Fc monomer as the TNFR1 inhibitor, wherein the first and second Treg expansion are the same or different. The second Treg expansion may be a TNFR2 agonist. In some embodiments, the Treg amplicons are identical. The TNFR1 inhibitor may be an inhibitor that inhibits or blocks TNFR1 signaling. In some embodiments, the TNFR1 inhibitor binds TNFR1 and blocks or inhibits tnfa binding and TNFR1 signaling. In some embodiments, the TNFR1 inhibitor binds TNFR1, does not bind tnfα and does not interfere with tnfα binding, and blocks or inhibits TNFR1 signaling. In some embodiments of these constructs, wherein the Treg expansion product is a TNFR2 agonist. The TNRF2 agonist may be an agonist that stimulates or induces TNFR2 signaling. Exemplary Treg amplifications are TNFR2 agonists, which are Fab of scFv, VHH single domain antibodies or TNFR2 agonist monoclonal antibodies. In these constructs, the Treg amplificates may be TNFR2 agonists, or nucleic acid aptamers, or peptide aptamers, as small molecules.
Any of these constructs that are or are also TNFR2 agonists are also provided. The TNFR2 agonist is a construct of formula 3a or 3b, wherein: formula 3a is (Treg amplificate) n -a joint p - (activity modulating agent) q Formula 3b is (Activity regulator) q -a joint p - (Treg amplificates) n . In these formulae, n and q are each integers and are each independently 1, 2 or 3; p is 0, 1, 2 or 3; an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and the linker increases the flexibility of the construct and/or moderates or reduces the steric effect of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media. In any of these constructs, the Treg expansion in the construct is a TNFR2 agonist. For example, a TNFR2 agonist stimulates or induces TNFR2 signaling. In other examples, the Treg expansion is a TNFR2 agonist, which is a Fab of scFv, VHH single domain antibodies, or TNFR2 agonist monoclonal antibodies. Treg amplicons may be TNFR2 agonistsAn agent which is a small molecule or a nucleic acid or peptide aptamer. In constructs comprising all or part of trastuzumab, e.g., comprising an Fc portion and/or an Fc and hinge region or modified form thereof, the construct can dimerize by fusing with the N-terminus of the C-terminus of trastuzumab.
The constructs provided comprise a TNFR1 inhibitor moiety linked to one or more Treg amplicons via a central PEG linker, or comprise at least two identical or different TNFR1 inhibitors, or comprise two identical or different Treg amplicons. Constructs comprising a PEG moiety, such as a central PEG linker, may comprise a branched PEG moiety linking the TNFR1 inhibitor and one or more Treg amplicons. Exemplified are those constructs having a structure selected from formulas 4A to 4D:
formula 4A:
n is 1-5;
R 1 is H or CH 3 Or CH 2 CH 3 Or other C 1 -C 5 An alkyl group, a hydroxyl group,
is a TNFR1 inhibitor (TNFR 1 antagonist);
is a Treg amplicon; or (b)
Formula 4B:
is TNFR1 inhibitor (TNFR 1 antagonist)
Is a Treg amplicon;
n is 1-5; or (b)
Formula 4C:
is a TNFR1 inhibitor (TNFR 1 antagonist), or a Treg amplicon; and
n is 1-5; or (b)
Formula 4D:
or
wherein the method comprises the steps of
Each of which isIdentical or different, and are each independently selected from a TNFR1 inhibitor (TNFR 1 antagonist) and a TNFR2 agonist;
the activity modulator is optional and may be attached to any suitable site in the molecule; and n is 1 to 5.
In the TNFR1 antagonist constructs and other constructs provided herein, the Treg expansion may be a TNFR2 agonist. These constructs may include an activity modulator, for example, where the activity modulator is an Fc region, or an Fc region that includes a hinge region or other linker; and the Fc region or Fc region having a hinge region is an Fc modified to reduce or eliminate ADCC and/or CDC activity. Examples are constructs in which the Fc or modified Fc is an IgG Fc or an IgG1 Fc or an IgG4 Fc, and/or constructs that bind neonatal Fc receptor (FcRn). Exemplary of these constructs are those wherein: the construct comprises a short FcRn binding peptide (FcRnBP), wherein the short FcRn binding peptide (FcRnBP) provides for interaction of the construct with FcRn and comprises 6-25, e.g., 10-20 amino acid residues; wherein FcRnBP comprises 12-20 residues or 15 residues or 16 residues, e.g. wherein FcRn binding peptide (FcRnBP) comprises or consists of any of the peptides shown in any of SEQ ID NOs 48-51.
Also provided are TNFR1 antagonist constructs of any of the formulas above and in the present application comprising: a) Is a TNFR1 inhibitor moiety that is TNFR1 selective; b) Optionally one or more linkers; c) Optionally a half-life extending moiety, wherein the antagonist construct comprises at least one of b) and c). In such constructs, the TNFR 1-selective antagonist selectively binds to and inhibits TNFR1 signaling, but does not bind to and does not inhibit TNFR2 signaling. As described for the constructs above, TNFR1 inhibitors, linkers, and other components can be as described above. These include constructs wherein a TNFR1 inhibitor that is a selective antagonist comprises an antigen-binding fragment that selectively binds to and inhibits TNFR1 signaling but does not bind to and does not inhibit TNFR2 signaling. For example, an antigen binding fragment that selectively binds to and inhibits TNFR1 signaling but does not bind to and does not inhibit TNFR2 signaling may comprise a domain antibody (dAb), scFv, or Fab fragment. In any of the constructs described herein, the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR 1 antagonist monoclonal antibody. For example, the human anti-TNFR 1 antagonist monoclonal antibody is H398 comprising SEQ ID NO:678, or ATROSAB, or an antigen binding portion thereof, or a sequence having at least 95% sequence identity to SEQ ID NO:31 or 32 or 673 or 678, or an antigen binding portion thereof that binds TNFR 1. Exemplary TNFR1 inhibitors are those of: comprising a domain antibody (dAb) or antigen binding portion thereof or comprising the amino acid sequence shown in any one of SEQ ID NOS: 52-672 or a sequence having at least 95% sequence identity thereto and retaining TNFR1 inhibitor activity; and/or variants comprising the scFv shown in any one of SEQ ID NOS: 673-678 or a polypeptide having at least 90% or 95% sequence identity thereto and retaining TNFR1 inhibitor activity; and/or a sequence comprising or having at least 90% or 95% sequence identity to a Fab as set forth in any one of SEQ ID NOs 679-682 and retaining TNFR1 inhibitor or binding activity; and/or nanobodies comprising the sequence shown in SEQ ID NO. 683 or 684 or sequences having at least 90% or 95% sequence identity thereto and retaining TNFR1 inhibitor or binding activity. The TNFR1 inhibitors are, for example, those comprising a dominant negative tumor necrosis factor (DN-TNF) or TNF mutein, e.g., DN-TNF or TNF mutein is a soluble TNF molecule comprising one or more amino acid substitutions that confer selective inhibition of TNFR1 and are selected from the group consisting of:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, A84S/V85T/S86T/Y87H/Q88N/T89Q, reference is made to the sequence of soluble TNF shown in SEQ ID NO 2. For example, a TNFR1 inhibitor is a TNFR mutein comprising the residue sequence set forth in any one of SEQ ID NOS: 701-703 or a sequence having at least or at least about 90% or 95% sequence identity to the residue sequence set forth in any one of SEQ ID NOS: 701-703 or a fragment thereof that retains TNFR1 inhibitor activity.
Any of the foregoing constructs provided herein can include a linker, wherein the linker comprises all or part of the hinge sequence of trastuzumab, SCDKTH corresponding to residues 222-227 of SEQ ID No. 26 or the complete sequence up to the hinge region of trastuzumab, which contains or has the sequence epkscdkthtcpcp (corresponding to residues 219-233 of SEQ ID No. 26), or at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues thereof, or ESKYGPPCPPCP residues 212-223 of SEQ ID No. 29, or a sequence of the linker having at least 98% or 99% sequence identity thereto. For example, the construct may comprise a linker, wherein the linker comprises the sequence SCDKTH corresponding to residues 222-227 of SEQ ID NO. 26. Alternatively or in addition to another linker, the construct may comprise a GS linker I.e., a linker comprising Glycine and Serine (GS) residues. Exemplary GS linkers of any construct provided herein include those selected from the group consisting of: (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; GSSSGS. Also included are linkers comprising all or part of the GS linker and the hinge sequence of trastuzumab corresponding to residues EPKSCDKTHTCPPCP (residues 219-233 of SEQ ID NO: 26), e.g., the linker may comprise the GS linker and only or comprise the sequence SCDKTH from the hinge sequence corresponding to residues 217-222 of SEQ ID NO: 31. Such linkers include, for example, those comprising all or part of the hinge sequence of GS linker and Nivolumab (Nivolumab) corresponding to residues 212-223 of SEQ ID NO. 29.
Constructs herein may contain an activity modulator. Such activity modulators include any of the modulators described herein, including those described above and below, as well as other modulators known to those of skill in the art; the activity modulator alters the activity or property of the construct. The activity modulator may be a half-life extending moiety that is an IgG Fc, a polyethylene glycol (PEG) molecule, or Human Serum Albumin (HSA). Exemplary IgG Fc is IgG1 Fc or IgG4 Fc. IgG1 Fc can be that of trastuzumab, as shown in SEQ ID NO 27, or an amino acid sequence having at least 95% sequence identity thereto; igG4 Fc can be that of nivolumab, as shown in SEQ ID NO. 30, or an amino acid sequence having at least 95% sequence identity thereto. For example, igG1 Fc is that of human IgG1, as shown in SEQ ID NO:10, and IgG4 Fc is that of human IgG4, as shown in SEQ ID NO: 16.
Constructs described herein include those that are TNFR1 inhibitors or that comprise TNFR1 inhibitors. These include constructs in which the TNFR1 inhibitor is monovalent. These may include linkers, for example wherein the linker comprises (Gly 4 Ser) 3 And/or comprises (Gly) 4 Ser) 3 And SCDKTH (residues 217-222 of SEQ ID NO: 31); and/or comprises (Gly) 4 Ser) 3 And a linker of a hinge sequence of trastuzumab, said hinge sequence corresponding to residues 219-233 of SEQ ID No. 26; and/or those comprising (Gly) 4 Ser) 3 And a linker corresponding to the nivolumab hinge sequence of residues 212-223 of SEQ ID NO. 29. Exemplary TNFR 1-inhibiting constructs provided herein are those comprising the residue sequence set forth in any one of SEQ ID NOS: 704-764, or constructs that inhibit TNFR1 and have a sequence that has at least or at least about 95% sequence identity to the residue sequence set forth in any one of SEQ ID NOS: 704-764.
Provided herein are TNFR1 antagonist constructs. These include those wherein the TNFR1 construct comprises a short FcRn binding peptide (FcRnBP); and the short FcRn-binding peptide (FcRnBP) provides for interaction of the construct with FcRn and comprises 6-25, e.g. 10-20 amino acid residues, e.g. wherein FcRnBP comprises 12-20 residues or 15 residues or 16 residues, e.g. wherein FcRn binding peptide (FcRnBP) comprises or has at least about 95% sequence identity to a peptide as shown in any one of SEQ ID NOs 48-51 or an FcRn-binding peptide (FcRnBP) consisting of a peptide as shown in any one of SEQ ID NOs 48-51.
Other example TNFR1 inhibitory constructs provided herein include constructs comprising: a) Domain antibodies that inhibit TNFR 1; b) A linker that increases flexibility, reduces steric effects, or increases solubility; and c) a half-life extending moiety. Including constructs in which the half-life extending moiety is not a human serum albumin antibody or unmodified Fc. These constructs include those that are TNFR1 antagonists comprising: a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703; b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5;(SerGly 4 ) n wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and c) a half-life extending moiety that is an IgG Fc. In these constructs, or any of the constructs provided herein, comprising one or more of these components, the GS linker may be (GGGGS) 3 The method comprises the steps of carrying out a first treatment on the surface of the And the IgG Fc may be that of trastuzumab or that of nivolumab.
Other constructs provided herein are TNFR1 antagonist constructs, including constructs comprising: a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703; b) A linker selected from the group consisting of all or part of a hinge sequence of trastuzumab and all or part of a hinge sequence of nivolumab; and c) a half-life extending moiety that is an IgG Fc. In such constructs, the linker may comprise all or part of the hinge sequence of trastuzumab, wherein the IgG Fc is that of trastuzumab. In other embodiments, the linker may comprise all or part of the hinge sequence of nivolumab, wherein IgG Fc is that of nivolumab.
Any construct as provided herein is provided that is a TNFR1 antagonist construct comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) A first linker which is a GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) A second linker selected from the group consisting of all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; and
d) Half-life extending moiety, which is an IgG Fc.
In some embodiments, these constructs may contain a first linker that is a GS linker (GGGGS) 3 The method comprises the steps of carrying out a first treatment on the surface of the And a second linker comprising the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and IgG Fc is that of trastuzumab. In other embodiments, the first linker is a GS linker (GGGGS) 3 The method comprises the steps of carrying out a first treatment on the surface of the The second linker comprises all or part of the hinge sequence of nivolumab; and IgG Fc is that of nivolumab.
The constructs provided are TNFR1 agonists comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) Half-life extending moiety, which is a PEG molecule. The GS linker may be any linker described herein or known to those skilled in the art, e.g., a (GGGGS) 3 . The PEG molecule may be a molecule having a molecular weight of at least 25kDa, typically at least 30kDa or greater, e.g. at least 40kDa or 50kDa, or 60kDa, or 80kDa or greater.
The construct provided is a TNFR1 agonist construct comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) A half-life extending moiety that is human serum albumin. Examples of linkers are any of the linkers described herein, e.g., wherein the GS linker is (GGGGS) 3 。
The primary amino acid sequence of any of the constructs provided herein (described above and below) can be optimized or modified to eliminate immunogenic sequences or immunogenic epitopes. For example, in an IgG Fc-containing construct, the IgG Fc may be modified to include one or more of the following modifications: a) Introducing one or more modifications of the male and female; b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling; and c) one or more modifications that reduce or eliminate immune effector function. In such constructs, as well as in any IgG Fc-containing construct, the convex mutation may be selected from S354C, T366Y, T366W and T394W according to EU numbering; and the concave mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A, Y V according to EU numbering. These TNFR1 antagonist constructs may be constructs wherein the one or more modifications that increase or enhance FcRn recycling are selected from one or more of the following: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering. The TNFRl antagonist construct may be modified to reduce or eliminate an immune effector function, such as one or more immune effector functions selected from the group consisting of Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cell-mediated phagocytosis (ADCP). For example, in these TNFRl antagonist constructs, the one or more modifications that reduce or eliminate immune effector function are selected from one or more of the following:
In IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG 4: L235E, F234A/L235A, S228P/L235E and S228P/F234A/L235A according to EU numbering.
The TNFR1 antagonist or multispecific construct may comprise a central PEG linker moiety; and the construct may comprise a modified Fc region, such as those described above, wherein the Fc region is a modified IgG Fc and the modified IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that increase or enhance one or more immune effector functions, wherein:
the immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain, and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain, and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
In some embodiments of any construct comprising an Fc region, the construct may comprise an IgG1 Fc comprising one or more modifications to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib. For example, the one or more modifications that increase binding to fcyriib are selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
Constructs that are Treg amplicon constructs are also provided. Included among such constructs are those comprising: a) Treg amplicons; b) A linker, wherein the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with a receptor and/or increases the solubility of the construct in an aqueous medium; and c) an activity modulator, wherein the activity modulator is a moiety that modulates or alters the activity or pharmacological properties of the construct compared to the construct in the absence of the activity modulator. Treg amplicons may be TNFR2 agonists. These constructs may also comprise TNFR1 inhibitors. In some embodiments, the TNFR2 agonist is a TNFR2 selective agonist.
Provided are constructs described herein that are TNFR2 agonist constructs comprising: a) TNFR2 agonists; b) A linker, wherein the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with a receptor and/or increases the solubility of the construct in an aqueous medium; and c) an activity modulator, wherein the activity modulator is a moiety that modulates or alters the activity or pharmacological properties of the construct compared to the construct in the absence of the activity modulator. In the TNFR2 agonist construct, the TNFR2 agonist may be a TNFR2 selective agonist. The construct may comprise an activity modulator, for example an activity modulator of a half-life extending moiety. The construct may be a TNFR2 agonist construct that selectively activates or antagonizes TNFR2 but not TNFR 1. Including TNFR2 agonist constructs wherein the TNFR2 agonist binds one or more epitopes within TNFR2. These include human TNFR2. Such epitopes include, for example, epitopes selected from one or more of the amino acid sequences shown as SEQ ID NOS: 839-865, 1202 and 1204 or epitopes consisting of the amino acid sequences shown as SEQ ID NOS: 839-865, 1202 and 1204.
There is provided a TNFR2 agonist construct wherein the TNFR2 agonist comprises an antigen-binding fragment of an agonist human anti-TNFR 2 antibody or a humanized anti-TNFR 2 antibody, or an antigen-binding portion thereof, or a single-chain form thereof. Examples of such antibodies are agonist anti-TNFR 2 antibodies selected from MR2-1 (also known as ab8161; U.S. Pat. No. 9,821,010) or MAB2261 (U.S. Pat. No. 9,821,010). The TNFR2 agonist may be an antigen-binding fragment selected from dAb, scFv or Fab fragments. In some embodiments, the TNFR2 agonist is a TNFR2 selective agonist. The selective agonist may comprise a TNFR2 agonist TNF mutein. Exemplary TNFR2 selective agonist muteins include, but are not limited to, soluble TNF variants comprising one or more TNFR2 selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, and combinations of any of the foregoing, refer to SEQ ID NO:2. For example, a TNFR2 agonist is a TNF mutein comprising the mutation D143N/A145R.
In the TNFR2 agonist constructs, the linker includes any linker described herein or known to those of skill in the art to be useful as a linker. Exemplary linkers comprise all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID No. 26, or all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID No. 29, or sequences having at least 95% sequence identity thereto. Other exemplary linkers comprise or consist of the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID NO. 31. The linker may be a glycine-serine (GS) linker, such as, but not limited to: GS linker selected from (GlySer) n Wherein n=1 to 10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n, wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS. The linker may comprise a combination of linkers, e.g. a linker comprising all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID No. 26, or the sequence SCDKTH of GS linker and residues 217-222 of SEQ ID No. 31, or the whole or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID No. 29.
All constructs provided herein can include an activity modulator that alters or modulates a property or activity of the construct. For example, the half-life extending moiety is an activity or property modulator. Examples as discussed above and below are IgG Fc, polyethylene glycol (PEG) molecules and Human Serum Albumin (HSA), or parts or derivatives of variants thereof. For example, in some embodiments, the IgG Fc is an IgG1 Fc or an IgG4 Fc. An example of an IgG1 Fc is that of trastuzumab, as shown in SEQ ID NO: 27; an example of IgG4 Fc is that of nivolumab, shown as SEQ ID NO. 30, a human version, where IgG1 Fc is that of human IgG1, shown as SEQ ID NO. 10, and IgG4 Fc is that of human IgG4, shown as SEQ ID NO. 16.
In some embodiments of the TNFR2 agonist construct, the TNFR2 agonist is monovalent; in other cases, it is multivalent, e.g., divalent or trivalent. The TNFR2 construct may contain a linker as described herein. For example, the linker may comprise Gly-Ser, e.g. (Gly 4 Ser) 3 Or (Gly) 4 Ser) 3 And SCDKTH (residues 217-222 of SEQ ID NO: 31), or (Gly) 4 Ser) 3 And a hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO:26, or (Gly) 4 Ser) 3 And a hinge sequence of nivolumab, corresponding to residues 212-223 of SEQ ID NO. 29, or a variant of any of the foregoing linkers having at least 95% sequence identity thereto. These constructs may also include an activity modulator, such as a modulator of a half-life extending moiety, such as PEG or HSA as described above. The PEG moiety is at least 20kDa in size, typically at least 30kDa or greater, as described above and below.
Also provided is a TNFR2 agonist construct comprising:
a) A TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Which is provided withN=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) The activity modulator, which is a half-life extending moiety, is an IgG Fc.
As described above, in an exemplary embodiment, the GS linker may be (GGGGS) 3 The method comprises the steps of carrying out a first treatment on the surface of the IgG Fc is the Fc of trastuzumab or the Fc of nivolumab.
Other TNFR2 agonist constructs comprise:
a) A TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204;
b) A linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; and
c) The activity modulator, which is a half-life extending moiety, is an IgG Fc. Examples of linkers and activity modulators are hinge sequences of trastuzumab; igG Fc is the Fc of trastuzumab, or all or part of the hinge sequence of nivolumab; igG Fc is that of nivolumab.
In other embodiments, the TNFR2 construct comprises:
a) A TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204;
b) A first linker which is a GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) A second linker selected from the group consisting of all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; and
d) The activity modulator, which is a half-life extending moiety, is an IgG Fc. Such constructionAn example of a body is where the first GS linker is (GGGGS) 3 The second linker comprises those constructs of the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31) and constructs in which the IgG Fc is that of trastuzumab. In other embodiments, the first linker is (GGGGS) 3 The second linker comprises all or part of the hinge sequence of nivolumab and IgG Fc is that of nivolumab.
In some embodiments, the construct is a TNFR2 agonist construct comprising:
a) A TNFR2 agonist comprising an antigen-binding fragment of an agonist human anti-TNFR 2 antibody selected from MR2-1 or MAB 2261;
b) A joint, comprising:
i) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and
c) An activity modulator that is a half-life extending moiety selected from the group consisting of IgG1 Fc or IgG4 Fc, a PEG molecule, and Human Serum Albumin (HSA), wherein:
IgG1 Fc is human IgG1 Fc as shown in SEQ ID NO. 10, or trastuzumab Fc as shown in SEQ ID NO. 27; and
the PEG molecules have a molecular weight of at least or at least about 30 kDa.
In some embodiments, the construct is a TNFR2 agonist construct comprising:
a) A TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S;
b) A joint, comprising:
i) A GS linker selected from: (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and
c) An activity modulator that is a half-life extending moiety selected from the group consisting of IgG1 Fc or IgG4 Fc, a PEG molecule, and Human Serum Albumin (HSA), wherein:
IgG1 Fc is human IgG1 Fc as shown in SEQ ID NO. 10, or trastuzumab Fc as shown in SEQ ID NO. 27; and
the PEG molecules have a molecular weight of at least or at least about 30 kDa.
In some embodiments, the construct is a TNFR2 agonist construct comprising:
a) TNFR2 TNF muteins comprising the mutation D143N/A145R;
b) (GGGGS) 3 linker; and
c) An activity modulator, which is a half-life extending moiety, is the Fc of trastuzumab or the Fc of nivolumab.
In some embodiments, the construct is a TNFR2 agonist construct comprising
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b)(GGGGS) 3 a linker and a second linker comprising the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
c) An activity modulator, which is a half-life extending moiety, is the Fc of trastuzumab.
In some embodiments, the construct is a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b)(GGGGS) 3 a linker and a second linker comprising all or part of the hinge sequence of nivolumab; and
c) An activity modulator, which is a half-life extending moiety, is the Fc of nivolumab.
In some embodiments, the construct is a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b) A linker comprising all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID No. 26; and
c) A half-life extending moiety that is an Fc of trastuzumab.
In some embodiments, the construct is a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b) A linker comprising all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID No. 29; and
c) An activity modulator, which is a half-life extending moiety, is the Fc of nivolumab.
TNFR1 antagonist constructs, TNFR2 agonist constructs, and both are provided, wherein the IgG Fc is monomeric or dimeric. Constructs provided herein can comprise a dAb (or Vhh). The construct may comprise a Vhh single strand or double strand comprising a dAb. These constructs may contain HSA linked to dAb either directly or through a linker. The HSA and dAb can be linked in any order, e.g., the C-terminus of the dAb is linked directly or via a linker such as any of the above to the N-terminus of the HSA. Examples of such constructs are those comprising:
a) Residues 20-732, which are dAb Dom1h-131-206 of SEQ ID NO. 59, linked to HSA by a linker as shown in SEQ ID NO. 1475, or a construct having at least 95%, 96%, 97%, 98%, 99% sequence identity to the construct of SEQ ID NO. 1475 or to residues 20-732 of SEQ ID NO. 1475 and having TNFR1 antagonist activity; or alternatively
b) 53-83 and 503-671, and variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto, whereby the construct has TNFR1 antagonist activity; or alternatively
c) dAbs having the sequence shown in any one of SEQ ID NOs 57-59 and variants thereof having at least 95% sequence identity thereto, whereby the construct has TNFR1 antagonist activity; or alternatively
d) A dAb designated DOM1h-131-206 as shown in SEQ ID NO 59, a variant having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto and TNFR1 antagonist activity; or alternatively
e) A combination of any of the above a) to d); or alternatively
f) The humanized sequence of any one of a) to f) above, or wherein a sufficient portion of a construct for administration to a human is humanized, wherein the sufficient portion is sufficient to eliminate or reduce any immune response to the construct when administered to a human.
The construct provided herein as a TNFR1 construct may further comprise a TNFR2 agonist or the construct may be a TNFR2 agonist construct. In constructs comprising a TNFR2 agonist, the TNFR2 agonist can be modified to eliminate an amino acid sequence or epitope that is immunogenic in the subject to be treated, e.g., for administration to a human subject. In a construct containing a TNFR2 agonist, it may be a TNFR2 selective agonist. These constructs may comprise modified IgG Fc. For example, an IgG Fc may comprise one or more of the following modifications:
a) Introducing one or more modifications of the male and female;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) One or more modifications that reduce or eliminate immune effector function selected from one or more of Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cell-mediated phagocytosis (ADCP). Exemplary such modifications are:
a) The one or more modifications introducing the bulge and the recess are selected from:
one or more convex mutations selected from S354C, T366Y, T366W and T394W according to EU numbering; and
one or more concave mutations selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering, whereby Fc forms a dimer;
b) The one or more modifications that increase or enhance FcRn recycling are selected from one or more of the following: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) The one or more modifications that reduce or eliminate immune effector function are selected from one or more of the following:
in IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG 4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A according to EU numbering.
Constructs provided herein include TNFR2 agonist constructs comprising a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that increase or enhance immune effector function, wherein:
immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
Constructs provided herein as TNFR2 agonist constructs may comprise a modified IgG1 Fc, for example, wherein the Fc is modified to increase binding to an inhibitory fcγriib receptor (fcγr) fcγriib, which may include modifications that increase binding to fcγriib. Examples of such modifications are those selected from one or more of S267E, N297A, L328F, L351S, T366R, L H, P395K, S E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
The constructs provided are or comprise TNFR2 agonist constructs that selectively activate or agonize TNFR2 but do not activate or antagonize TNFR1. These constructs include those comprising: a) TNFR2 agonists; b) One or more joints; and c) an activity modulator that is a half-life extending moiety, wherein:
the TNFR2 agonist construct is a fusion protein comprising a single-chain TNFR 2-selective TNF mutein trimer fused to a multimerization domain, and comprises the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or (b)
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III),
MD is a multimerization domain, and each domain is the same or different; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different. The TNF mutein may comprise one or more TNFR 2-selective mutations selected from the group consisting of: reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, e.g., TNFR 2-selective mutation D143N/A145R. In these constructs, the multimerization domain may be selected from EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residue of SEQ ID NO: 804) 110-139; SEQ ID NO: 805), or the trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806), or variants having at least 95%, 96%, 97%, 98%,99% sequence identity thereto. For example, the multimerization domain is an IgG1 Fc or an IgG4 Fc, and IgG1 Fc or IgG4 Fc is also a half-life extending moiety. These constructs contain linkers, including any of the linkers described herein and known to those of skill in the art. Examples of such constructs are those wherein the L1, L2 and/or L3 linker is independently selected from (GGGGS) n Wherein n=1-5, and all or part of the TNF stem region (SEQ ID NO: 812) or variants having at least 95%, 96%, 97%, 98%,99% sequence identity thereto. These constructs include those in which the linker between the TNFR2 agonist and the half-life extending moiety is selected from the group consisting of: GS joint (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or a linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; or a combination thereof. The half-life extending moiety may be selected from: igG1 Fc, i.e., human IgG1 Fc, as shown in SEQ ID NO. 10, or trastuzumab Fc, as shown in SEQ ID NO. 27; igG4 Fc is human IgG4 Fc, as shown in SEQ ID NO. 16, or Nawuzumab Fc, as shown in SEQ ID NO. 30; a PEG molecule having a size of at least or at least about 30kDa; human Serum Albumin (HSA), and variants of polypeptide portions having at least 95%, 96%, 97%, 98%,99% sequence identity thereto.
The constructs provided are or comprise TNFR2 agonist constructs. These constructs include those comprising the following formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III), wherein:
a) MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, wherein:
i) MD is selected from the trimerization domains of EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or a trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806);
ii) L1, L2 and L3 are each (GGGGS) n Wherein n=1-5, or all or part of the TNF stem region (SEQ ID NO: 812), or mixtures thereof; and
iii) TNF muteins comprise the TNFR 2-selective mutation D143N/A145R;
b) The half-life extending moiety is selected from the group consisting of:
IgG1 Fc, which is the Fc of human IgG1, as shown in SEQ ID NO. 10, or Fc of trastuzumab, as shown in SEQ ID NO. 27;
IgG4 Fc, which is the Fc of human IgG4, shown as SEQ ID NO. 16, or the Fc of Nawuzumab, shown as SEQ ID NO. 30;
a PEG molecule having a size of at least or at least about 30kDa; and
Human Serum Albumin (HSA); and
c) A linker between a TNFR 2-selective agonist and a half-life extending moiety, wherein the linker comprises:
GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n, wherein n=1-5; (SerGly) 4 ) n, wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or (b)
A linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; or alternatively
A combination thereof.
The constructs include TNFR2 agonist constructs comprising the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III),
wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, wherein:
i) MD is selected from IgG1 Fc or IgG4 Fc;
II) L2 and L3 in formula II, and L1 and L2 in formula III are each independently (GGGGS) n Wherein n=1-5, or the stem region of all or part of TNF (SEQ ID NO: 812), or a combination thereof;
iii) L1 in formula II and L3 in formula III are each independently selected from:
GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or (b)
A linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; or alternatively
A combination thereof; and
iv) the TNF mutein comprises the TNFR 2-selective mutation D143N/A145R. In these constructs, MD may be selected from:
IgG1 Fc, which is the Fc of human IgG1, as shown in SEQ ID NO. 10, or Fc of trastuzumab, as shown in SEQ ID NO. 27; or alternatively
IgG4 Fc, which is the Fc of human IgG4, shown as SEQ ID NO. 16, or the Fc of Nawuzumab, shown as SEQ ID NO. 30; or alternatively
A combination thereof or a variant having at least 95%, 96%, 97%, 98%, 99% sequence identity thereto.
Exemplary constructs are those comprising MD which is trastuzumab IgG1 Fc and the linker between MD and the adjacent TNF mutein is trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26 All or part of the hinge sequence, or MD of the IgG1 Fc of trastuzumab, and the linker between MD and the adjacent TNF mutein comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31). An exemplary construct is one comprising MD, which is an IgG1 Fc of trastuzumab, wherein the linker between MD and adjacent TNF muteins comprises (Gly 4 Ser) 3 And a hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26. In some embodiments, the MD is an IgG1 Fc of trastuzumab, and the linker between the MD and the adjacent TNF mutein comprises (Gly 4 Ser) 3 And SCDKTH (residues 222-227 of SEQ ID NO: 31), wherein MD is IgG4 Fc of nivolumab, and the linker between MD and the adjacent TNF mutein comprises all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO:29, or those wherein: MD is IgG4 Fc of nivolumab, and the linker between MD and adjacent TNF muteins comprises (Gly) 4 Ser) 3 And all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO. 29.
Constructs herein, including agonist constructs, may be modified to eliminate immunogenic sequences, such as those that are immunogenic to humans. Provided herein are TNFR2 agonist constructs wherein the TNFR2 agonist is modified to eliminate an immunogenic sequence or epitope that is immunogenic in a subject, such as a human subject.
In the constructs provided herein, which are TNFR2 agonist constructs and comprise a modified IgG Fc, the IgG Fc may comprise one or more of the following modifications:
a) One or more modifications introducing a convex depression (knobs-into-holes), wherein:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that reduce or eliminate immune effector function, wherein:
immune effector function is selected from one or more of CDC, ADCC and ADCP; and
The one or more modifications that reduce or eliminate immune effector function are selected from one or more of the following:
in IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG 4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A according to EU numbering.
Any of the foregoing constructs is provided that is a TNFR2 agonist construct comprising a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modification is selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that increase or enhance immune effector function, wherein:
immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
Any of the foregoing TNFR2 agonist constructs of any claim are provided comprising an IgG1 Fc modified to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib. For example, modifications wherein increased binding to fcyriib is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
Constructs provided herein can be multispecific in that they interact with two or more targets. Examples of such multispecific constructs are those of the multispecific TNFRl inhibitor/TNFR 2 agonist constructs and have any of the following formulas:
(TNFR 1 inhibitor) n Joint (L) p - (TNFR 2 agonists) q (formula I), or
(TNFR 1 inhibitor) n Joint (L) p - (TNFR 2 agonists) q Or (b)
(TNFR 1 inhibitor) n - (TNFR 2 agonists) q Joint (L) p Or (b)
(TNFR 2 agonist) q - (TNFR 1 inhibitors) n Joint (L) p Or (b)
Any of the above comprising an optional activity modulator, wherein: n=1 or 2, p=1, 2 or 3, and q=1 or 2; TNFR1 inhibitors interact with TNFR1 to inhibit its activity; an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and linkers, e.g., to increase the solubility of the construct, or to increase flexibility, or to alter the steric effects of the construct. These include those that are multispecific TNFR1 inhibitor/TNFR 2 agonist constructs wherein: TNFR1 inhibitors selectively inhibit or antagonize TNFR1 signaling but not TNFR2 signaling; TNFR1 inhibitors do not interfere with TNFR2 activation or agonism; a TNFR2 agonist selectively activates or agonizes TNFR2 signaling and does not activate and agonize TNFR1 signaling; and TNFR2 agonists do not interfere with the inhibition or antagonism of TNFR 1. Examples of such constructs are those of the following a) to c):
a) The TNFR1 inhibitor is selected from:
i) An antigen binding fragment of a human anti-TNFR 1 antagonist monoclonal antibody selected from H398 or ATROSAB or a polypeptide having a sequence with at least 95% sequence identity thereto; or alternatively
ii) a domain antibody (dAb) as shown in any one of SEQ ID NOS.52-672, or a scFv as shown in any one of SEQ ID NOS.673-678, or a Fab as shown in any one of SEQ ID NOS.679-682, or a nanobody as shown in SEQ ID NOS.683 or 684, or a TNF mutein as shown in any one of SEQ ID NOS.701-703, or a polypeptide having a sequence that is at least 95% sequence identity to any of the foregoing polypeptides and that is a TNFR1 inhibitor; or alternatively
iii) A dominant negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule having one or more amino acid substitutions that confer TNFR1 selective inhibition and selected from the group consisting of:
referring to V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q;
b) The linker is selected from:
i) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26 or all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO. 29; and
iii) IgG1Fc or IgG4 Fc, wherein:
the IgG1Fc is selected from human IgG1Fc shown as SEQ ID NO. 10, or trastuzumab IgG1Fc shown as SEQ ID NO. 27;
the IgG4 Fc is selected from human IgG4 Fc shown as SEQ ID NO. 16, or Nawuzumab IgG4 Fc shown as SEQ ID NO. 30; and
Optionally, the Fc comprises one or more modifications to introduce convexities and concavities, and/or to increase or enhance neonatal Fc receptor (FcRn) recycling, and/or to reduce or eliminate immune effector function; and
c) The TNFR2 agonist is selected from:
i) An antigen binding fragment that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes shown in SEQ ID NOs 839-865, 1202 and 1204; or alternatively
ii) an antigen binding fragment of an agonistic human anti-TNFR 2 antibody selected from MR2-1 or MAB 2261; or alternatively
iii) A TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more selective TNFR2 mutations selected from the group consisting of: reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S; or (b)
iv) a single-chain TNFR 2-selective TNF mutein trimer comprising the mutation D143N/A145R, wherein the TNF mutein consists of (GGGGS) n Or all or part of the TNF stem region (SEQ ID NO: 812) where n=1-5; or alternatively
v) a TNFR 2-selective agonist comprising the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III);
Wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, and wherein:
MD is selected from the trimerization domain of EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), chicken tenascin C (TNC) (residues 110-139;SEQ ID NO:805 of SEQ ID NO: 804), or the trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806);
l1, L2 and L3 are each (GGGGS) n Wherein n=1-5, or all or part of the TNF stem region (SEQ ID812), or a mixture thereof; and
TNF muteins comprise the TNFR 2-selective mutation D143N/A145R.
Other such constructs include those multispecific TNFR1 antagonist/TNFR 2 agonist constructs wherein:
a) TNFR1 inhibitors comprise a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) Joint comprising (GGGGS) 3 A polypeptide comprising the sequence SCDKTH (residues 222-227 of SEQ ID No. 26) and Fc of trastuzumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from the group consisting of: reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S.
Other such multispecific constructs are those in which:
a) TNFR1 inhibitors comprise a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) Joint comprising (GGGGS) 3 All or part of the hinge sequence of nivolumab and Fc of nivolumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: referring to K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146T/S147D, A145R/S147T, E146D/S147D, E146D, A145D/S147D, A145D/E146D/S147D, A145T/E146D/S145D, A145D/E145D, A145D/S145D and C145D/S145C/S145V/S145D and C/S145G.
Other such multispecific constructs are those in which:
a) TNFR1 inhibitors comprise a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) Joint comprising (GGGGS) 3 And Fc of trastuzumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S.
Other such multispecific constructs are those in which:
a) TNFR1 inhibitors comprise a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) Joint comprising (GGGGS) 3 And Fc of nivolumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, as well as any combination of the foregoing mutations.
These multispecific constructs may comprise a modified Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the male and female;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) One or more modifications that reduce or eliminate immune effector function. Examples of modified fcs comprising raised dimples are:
The convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering.
Other examples are multispecific constructs comprising Fc, for example wherein the Fc comprises a modification that increases or enhances FcRn recycling, the modification selected from one or more of: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering. The Fc may comprise modifications to one or more immune effector functions selected from the group consisting of Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cell-mediated phagocytosis (ADCP). Fc may comprise one or more modifications to reduce or eliminate immune effector function in IgG1 and/or IgG 4:
In IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and/or
In IgG 4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A according to EU numbering.
The IgG Fc may comprise one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that increase or enhance immune effector function, wherein:
immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
Other such multispecific constructs are wherein: a construct comprising an IgG1 Fc modified to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib. Exemplary are those constructs in which the modification to increase binding to fcyriib is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
Also provided are constructs that are multispecific TNFR1 antagonists/TNFR 2 agonists, the TNFR1 antagonists being monovalent; TNFR2 agonists are monovalent. Also provided are multispecific constructs that are multispecific TNFR1 antagonist/TNFR 2 agonist constructs, wherein the TNFR1 antagonist is monovalent; TNFR2 agonists are bivalent.
In some embodiments, the multispecific construct is a multispecific TNFR1 antagonist/TNFR 2 agonist construct, wherein:
a) The TNFR1 antagonist is selected from the group consisting of:
i) An antigen binding fragment of a human anti-TNFR 1 antagonist monoclonal antibody selected from H398 or ATROSAB; or alternatively
ii) a domain antibody (dAb) as set forth in any one of SEQ ID NOS.52-672, or an scFv as set forth in any one of SEQ ID NOS.673-678, or a Fab as set forth in any one of SEQ ID NOS.679-682, or a nanobody as set forth in SEQ ID NOS.683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS.701-703, or a sequence having at least or at least about 95% sequence identity thereto; or alternatively
iii) A dominant negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule having one or more amino acid substitutions that confer TNFR1 selective inhibition and selected from the group consisting of:
referring to V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q;
b) The linker is a branched PEG molecule having a size of at least or at least about 30kDa; and
c) The TNFR2 agonist is selected from:
i) An antigen binding fragment that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204; or alternatively
ii) an antigen binding fragment of an agonistic human anti-TNFR 2 antibody selected from MR2-1 or MAB 2261; or alternatively
iii) A TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of:
reference is made to SEQ ID NO:2, K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S; or (b)
iv) a single-chain TNFR 2-selective TNF mutein trimer comprising the mutation D143N/A145R, wherein the TNF mutein consists of (GGGGS) n Or by all or part of the TNF stem region (SEQ ID NO: 812), wherein n=1-5; or alternatively
v) a TNFR 2-selective agonist comprising the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III);
wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, wherein:
MD is selected from the trimerization domain of EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), chicken tenascin C (TNC) (residues 110-139;SEQ ID NO:805 of SEQ ID NO: 804), or the trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806);
l1, L2 and L3 are each (GGGGS) n Or all or part of the TNF stem region (SEQ ID NO: 812) or mixtures thereof, wherein n = 1-5; and
TNF muteins comprise the TNFR 2-selective mutation D143N/A145R.
Also provided are multispecific constructs wherein each TNFR1 antagonist and TNFR2 agonist is monovalent. Also provided are constructs wherein the TNFR1 antagonist is monovalent and the TNFR2 agonist is bivalent.
The constructs provided herein are useful for and for the treatment of various diseases, disorders, and conditions. The multispecific constructs provided are multispecific TNFR1 antagonists/TNFR 2 agonists for use in the treatment of chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory diseases or disorders, or diseases, conditions or disorders in their etiology characterized by TNF overexpression or deregulation of TNFR1 signaling. There is provided the use of a multispecific TNFR1 antagonist/TNFR 2 agonist construct in the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder or a disease, condition or disorder characterized by TNF overexpression or a deregulation of TNFR1 signaling in its etiology.
Also provided are compositions comprising a construct of any of the constructs provided herein in a pharmaceutically acceptable carrier or vehicle. These compositions are useful in the treatment or therapy of diseases, disorders and conditions and methods for treating or treating diseases, disorders and conditions such as, but not limited to, chronic inflammatory, autoimmune, neurodegenerative, demyelinating, or respiratory diseases or disorders, as well as diseases, conditions, or disorders characterized by overexpression of TNF or deregulation of TNFR1 signaling in their etiology. Exemplary chronic inflammatory, autoimmune, neurodegenerative, demyelinating, or respiratory diseases or disorders are diseases, disorders, and conditions characterized by overexpression of TNF or deregulation of TNFR1 signaling in their etiology. These include diseases, disorders and conditions selected from the group consisting of: rheumatoid Arthritis (RA), psoriasis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA), spondyloarthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, inflammatory Bowel Disease (IBD), uveitis, fibrotic disease, endometriosis, lupus, multiple Sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial Infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute Respiratory Distress Syndrome (ARDS), severe Acute Respiratory Syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, alzheimer's disease, parkinson's disease, white plug disease, metaverse's disease, tumor necrosis factor receptor-related periodic syndrome (trap), pancreatitis, type I diabetes, chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, emphysema, graft rejection, anti-host disease, vhd, inflammatory disease and pulmonary disease, inflammatory bowel disease, pulmonary inflammation, and inflammatory disease, infectious disease, TNF-fulminating disease, pulmonary disease, or inflammatory disease as a pathological medium, infectious disease, TNF-specific disease, pulmonary disease, or infectious disease, or infectious disease, TNF-specific or a pathological condition. In particular, constructs provided herein, such as, but not limited to, TNFR1 antagonist constructs, are useful in uses, methods of treatment, and compositions for treating rheumatoid arthritis.
Also provided herein are constructs that are TNFR2 antagonist constructs comprising a TNFR2 antagonist and optionally a linker and optionally an activity modulator. For example, such a construct has formula 5:
(TNFR 2 antagonists) n -a joint p - (activity modulating agent) q Or (b)
Joint p - (activity modulating agent) q - (TNFR 2 antagonists) n Wherein:
n and q are integers and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
a TNFR2 antagonist is a molecule that interacts with TNFR2 to inhibit (antagonize) TNFR2 activity, thereby inhibiting Treg proliferation and/or inducing death thereof, and may also inhibit proliferation and induce death of tumor cells expressing TNFR 2;
an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and
the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media.
In these constructs, each activity modulator and linker is as defined and described above and below with respect to the construct. They are useful in such methods of treatment and uses and in pharmaceutical compositions.
TNFR2 antagonists are useful in different diseases, disorders and conditions, such as reducing and/or inhibiting proliferation of myelogenous suppressor cells (MDSCs); and/or inducing apoptosis within the MDSC by binding to TNFR2 expressed on the surface of the MDSC present in the tumor microenvironment; and/or induce expansion of T effector cells (including cytotoxic cd8+ T cells) by inhibiting Treg expansion and activity. TNFR2 antagonists in constructs include antibodies, antigen-binding fragments thereof, or single chain antibodies that bind to an epitope within human TNFR2 that contains one or more of residues KCRPG (residues 142-146 corresponding to SEQ ID NO: 4) or a larger epitope that contains residues 130-149, 137-144, or 142-149 or at least 5 consecutive or non-consecutive residues of these epitopes and do not bind to an epitope that contains residue KCSPG (residues 56-60 corresponding to SEQ ID NO: 4); or binding to the TNFR2 epitope PECLSCGS (corresponding to residues 91-98 of SEQ ID NO: 4), RICTCRPG (corresponding to residues 116-123 of SEQ ID NO: 4), CAPLRCR (corresponding to residues 137-144 of SEQ ID NO: 4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or an epitope comprising at least 5 contiguous or non-contiguous residues within residues 75-128, 86-103, 111-128, or 150-190 of SEQ ID NO: 4. For example, the antibody, fragment thereof, or single chain form thereof binds to an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 840) with an affinity that is at least 10-fold greater than the affinity of the same antibody or antigen binding fragment for a peptide (SEQ ID NO: 839) containing the KCSPG sequence of human TNFR 2. In some embodiments of the TNFR2 antagonist construct, the TNFR2 antagonist is an antibody or fragment or single chain form of an antibody selected from the group consisting of:
TNFRAB1 (see SEQ ID NOs: 1212 and 1213, heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2, and TNFR2A3 (see, e.g., U.S. patent publication No. 2019/0144556 for descriptions of these antibodies);
antibodies and antibody fragments and single chain forms thereof comprising the CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; residues 99-112 corresponding to SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223) or a CDR-H3 sequence having at least about 85% sequence identity thereto. For example, TNFRAB1 specifically binds residues 130-149 containing the TNFR2 residue KCRPG with an affinity 40-fold higher than the affinity for residues 48-67 containing the TNFR2 residue KCSPG. In some embodiments, the TNFR2 antagonist binds to one or more epitopes selected from the group consisting of:
an epitope comprising residues 137-144 (CAPLRKCR; SEQ ID NO: 851);
epitopes comprising one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248) and/or 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR 2; and
an epitope of TNFR2A3 is selected from a first epitope comprising residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and containing the KCRPG motif and/or a second epitope comprising residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464).
In some embodiments, the TNFR2 antagonist in the construct is an antibody, fragment thereof, or single chain form thereof that contains a CDR-H1 amino acid sequence having any one of the sequences set forth in SEQ ID NO 1214, 1215, and 1231-1233, a CDR-H2 sequence having any one of the sequences set forth in SEQ ID NO 1216, 1224, and 1230, a CDR-H3 sequence having any one of the sequences set forth in SEQ ID NO 1217, 1223, and 1225-1229, and/or a CDR-H3 of TNFRAB1 corresponding to residues 99-112 of SEQ ID NO 1212; the CDR-L1 sequences shown in either of SEQ ID NOS 1218 and 1234-1236, and/or the CDR-L1 sequence of TNFRAB1 corresponding to residues 24-33 of SEQ ID NO 1213; the CDR-L2 sequences shown in any one of SEQ ID NOS 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of SEQ ID NO 1213; and/or CDR-L3 sequences as shown in any one of SEQ ID NOS 1221, 1222 and 1241 to 1244 or CDR-L3 sequences of TNFRAB1 corresponding to residues 88 to 96 of SEQ ID NO 1213; and/or the CDR-H1 and CDR-H2 sequences of the consensus sequence of the human antibody heavy chain variable domain of SEQ ID NO 1245 replaced with the corresponding CDR sequences of a phenotypically neutral, TNFR2 specific antibody and/or the CDR-L1, CDR-L2 and CDR-L3 sequences of the human antibody light chain variable domain sequence of SEQ ID NO 1246 replaced with the corresponding CDR sequences of a phenotypically neutral, TNFR2 specific antibody to produce a humanized antagonistic TNFR2 antibody. For example, the construct comprises a TNFR2 antagonist that specifically binds an epitope within TNFR2 that is shown in any one of SEQ ID NOs 1247-1464. In some embodiments, the TNFR2 antagonist specifically binds to one or more epitopes selected from the group consisting of:
(a) One or more epitopes within human TNFR2 that contain one or more of residues KCRPG corresponding to residues 142-146 of SEQ ID NO. 4, or larger epitopes containing residues 130-149, 137-144 or 142-149 or at least 5 consecutive or discontinuous residues within these epitopes, and that do not bind epitopes containing residues KCSPG corresponding to residues 56-60 of SEQ ID NO. 4; and/or
(b) One or more TNFR2 epitopes comprising an amino acid sequence having the following:
PECLSCGS corresponding to residues 91-98 of SEQ ID No. 4, and/or RICTCRPG corresponding to residues 116-123 of SEQ ID No. 4, and/or
CAPLRKCR corresponding to residues 137-144 of SEQ ID NO. 4, and/or
LRKCRPGFGVA corresponding to residues 140 to 150 of SEQ ID NO. 4, and/or
VVCKPCAPGTFSN corresponding to residues 159-171) of SEQ ID NO. 4, and/or
An epitope comprising at least 5 contiguous or non-contiguous residues within residues 75-128, 86-103, 111-128 or 150-190 of SEQ ID NO. 4.
In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that is a small molecule. For example, the TNFR2 antagonist is thalidomide (thalidomide) or an analog thereof, such as lenalidomide (lenalidomide) and pomalidomide (pomalidomide).
In some embodiments, the TNFR2 antagonist construct comprises a TNFR2 antagonist that reduces FoxP3 expression and inhibits Treg inhibitory activity. An example of such an antagonist is a histone deacetylase inhibitor, which reduces FoxP3 expression and inhibits the inhibitory activity of tregs. Examples of such inhibitors are panobinostat (panobinostat) or cyclophosphamide or Triptolide (Triptolide).
The TNFR2 constructs are useful in methods and uses for treating infectious diseases and in methods and uses for treating TNFR2 expressing cancers. Examples of such cancers are cancers selected from the group consisting of: t cell lymphomas such as hodgkin's lymphoma and cutaneous non-hodgkin's lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer.
The claims and their subject matter presented in the following and priority applications are incorporated into this abstract by reference.
Brief Description of Drawings
FIG. 1 shows a plasmid map of pCBL-1 expression plasmid containing the CMV promoter, wherein TE19080L is an inserted fragment.
Figure 2 shows an exemplary bispecific construct-with a linker (part of the hinge region) linking the two ligands and an activity modulator, such as a TNFR1 inhibitor (TNFR 1 antagonist) and a TNFR2 agonist.
Figures 3A-3D illustrate exemplary PEG-centric multi-specific constructs for presenting/providing two or more moieties that interact with one or more targets or with one target at multiple sites. FIG. 3A shows an exemplary bivalent construct. One of which is, for example, a polypeptide agonist, antagonist or binding protein, such as an antibody or antigen binding fragment thereof, or an aptamer (nucleic acid or peptide). The other circle represents a polysaccharide or receptor ligand or other moiety that interacts with the target of interest. The bivalent nature provides clustering of targets for receptor activation. In embodiments provided herein, the targets include TNFR1 and TNFR2; and as described throughout the disclosure herein, moieties include TNFR1 inhibitors, e.g., moieties that inhibit TNFR1 signaling, and TNFR2 agonists or other moieties that are Treg amplicons. Figure 3B shows a monovalent single ligand, such as cd3+, linked to an agonist, antagonist or binding protein through a PEG moiety to prevent cytokine release syndrome, which is bivalent for receptor clustering. Likewise, exemplary targets include TNFR1 and/or TNFR2. Figure 3C shows heterobifunctional PEG for cross-linking two different cell targeting agents or two agents such as trastuzumab and pertuzumab or portions thereof bound to different sites on the same receptor. For example, such constructs may be used to cluster checkpointed receptors to stimulate or inhibit an immune response, or to crosslink two different receptors to achieve inhibition of receptor activity (i.e., CD3 and CD 450), or to deliver two different ligands such as a stimulating and co-stimulating ligand to two different receptors on the same cell. Figure 3D shows a homobifunctional PEG for clustering the same receptor (depending on chain length) on the same or different cells, or capturing circulating disease targets, e.g. soluble receptors or ligands such as TNF. Furthermore, in all of these embodiments, other PEG side chains, optionally linked to another reactive group or functional group, e.g., a serum half-life extending moiety, such as HSA or FcRn polypeptide, may be included in these constructs. The PEG moiety may be modified or replaced with a moiety having similar properties to present the binding moiety.
Fig. 4 shows other example configurations and structures of PEG-centric constructs for displaying or providing binding moieties or reactive moieties, such as TNFR1 inhibitors and/or TNFR2 agonists described herein.
Fig. 5 shows other example configurations and structures of PEG-centric constructs for displaying or providing binding or reactive moieties, e.g., TNFR1 inhibitors and/or TNFR2 agonists. X and Y may be ligands and reactive moieties.
Detailed Description
Outline
A. Definition of the definition
B. Construction and method overview
C. Tumor Necrosis Factor (TNF) and chronic inflammatory and autoimmune diseases and disorders
1. Tumor Necrosis Factor (TNF)
2. Tumor Necrosis Factor Receptor (TNFR)
a.TNFR1
b.TNFR2
3. Regulatory T cells (tregs) and their role in autoimmune microenvironment
4. Autoimmune/inflammatory diseases mediated or participated by TNF
a. Arthritis treatment
i. Rheumatoid arthritis and other types of arthritis
b. Inflammatory Bowel Disease (IBD) and uveitis
c. Fibrotic diseases
d. Tumor necrosis factor receptor-related periodic syndrome (TRAPS)
e. Other diseases mediated or involving TNF
i. Neurodegenerative diseases
a) Alzheimer's disease
b) Parkinson's disease
c) Multiple Sclerosis (MS)
Endometriosis
Cardiovascular diseases
Acute Respiratory Distress Syndrome (ARDS)
Severe Acute Respiratory Syndrome (SARS) and COVID 19
D. Treatment of rheumatoid arthritis and other chronic inflammatory and autoimmune diseases and conditions
1. Traditional synthetic antirheumatic drugs (csDMARD) to alter disease states
2. anti-TNF therapeutic/TNF blocker
E. Therapeutic agents targeting TNFR1/TNFR2
Tnfr1 selective antagonists
TNFR1 antagonistic antibodies
b. Monovalent TNFR1 antagonistic antibodies/antibody fragments
i. Fab and scFv-based TNFR1 antagonists
TNFR1 antagonists based on domain antibodies (dAbs)
a) anti-TNFR 1 dAb-anti-albumin dAb fusion constructs
b) Domain antibody fragments designated GSK1995057 and GSK2862277
Nanobody (Nb)
anti-TNFR 1 nanobody-anti-albumin nanobody fusion constructs
Dominant negative inhibitors of TNF (DN-TNF)/TNF muteins
Tnfr 2-selective agonists
TNFR2 antagonistic antibodies
Tnfr 2-selective TNF muteins and fusions thereof
3. anti-TNFR 2 antagonistic antibodies and small molecule inhibitors
Selective targeting of the TNFR1 and/or TNFR2 axes
1. Selective blocking of TNFR1 with TNFR1 antagonists
2. Selective activation of TNFR2 with TNFR2 agonists
TNFR1 antagonist construct, TNFR2 agonist construct; multispecific (including bispecific) TNFR1 antagonists and TNFR2 agonist constructs
TNFR1 antagonist construct, TNFR2 agonist construct and components of multispecific (including bispecific) TNFR1 antagonist/TNFR 2 agonist constructs
TNFR1 inhibitor moiety (TNFR 1 antagonist)
TNFR2 agonist construct and TNFR2 antagonist construct
c. Joint
i. Peptide linker
a) Flexible joint
b) Rigid joint
Chemical linker
d. Activity modulators
i. Modification of Fc portion
a) Protruding concave (Knobs-in-Holes)
b) Modifications to enhance neonatal Fc receptor (FcRn) recycling
c) Enhancement or reduction/removal of Fc immune effector function
Other modifications of the Fc part
Human serum albumin
e. Multispecific TNFR1 antagonist/TNFR 2 agonist construct
PEGylation of components to join multispecific constructs, PEG-centered multispecific constructs, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs
f. Other Activity modulators-fusion proteins comprising a partial or complete polypeptide that increases serum half-life
5. Prediction and removal of immunogenicity in protein therapeutics
a.B cell and T cell epitope
b. Epitope prediction method through computer
i. Computer-based B cell epitope prediction
Computer T cell epitope prediction
peptide-MHC class II binding prediction
c. In vitro epitope prediction method
i. In vitro B cell epitope prediction method
in vitro T cell epitope prediction method
MHC/HLA binding assays
in vitro T cell assay
d. In vivo epitope prediction method
e. Predictive B-cell and T-cell epitope removal (deimmunization)
G. Pan-growth factor trap polypeptides
1. Receptor Tyrosine Kinases (RTKs)
a. Human epidermal growth factor receptor (HER) family
b. Human epidermal growth factor receptor (HER) family and ligand related diseases thereof
Pan-growth factor inhibition
RB242 ligand trap
b. RB200 and RB242 for the treatment of autoimmune diseases
RB242 ligand trap
3. Optimized multi-specific, e.g., bispecific growth factor trap constructs
a. Extracellular domain (ECD) polypeptides
b. Extracellular domain modification
c. Multimerization domains
Fc domain modification
i. Introduction of the protruding dent
Modifications to enhance neonatal Fc receptor (FcRn) recycling
Effector function
4. Compositions, therapeutic uses and methods of treatment
a. Pharmaceutical composition
b. Therapeutic uses and methods of treatment
5. Combination therapy
H. Assessment of TNFR1 antagonists and TNFR1 antagonist/TNFR 2 agonist construct Activity and efficacy
1. Disease Activity score (DAS 28)
2.Proteomic analysis and other proteomic tools for quantifying analytes
3. Transcriptome analysis to predict response to treatment and select subjects likely to benefit from treatment
L929 cytotoxicity assay
HeLa IL-8 assay
HUVEC assay
Quantification and assessment of Treg cell Activity
Assessment of binding Properties of TNFR1 antagonist/TNFR 2 agonist constructs
9. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays
10. Disease model
a. Collagen-induced arthritis (CIA)
b. Rheumatoid arthritis synovial mononuclear cell culture
Tg197 mouse arthritis model
d. Delta ARE mouse arthritis/IBD model
e. Humanized TNF/TNFR2 mice
I. Methods of producing nucleic acids encoding TNFR1 antagonist constructs and TNFR1 antagonist/TNFR 2 agonist constructs
1. Isolation or preparation of nucleic acids encoding TNFR1 antagonists and TNRF2 agonist polypeptides
2. Production of mutant or modified nucleic acids and encoded polypeptides
3. Vectors and cells
4. Expression of
a. Prokaryotic cells
b. Yeast cells
c. Insect and insect cell
d. Mammalian expression cells
e. Plants and methods of making the same
5. Purification
6. Other modifications
PEGylation
b. Albumination
c. Purification tag
7. Nucleic acid molecules and gene therapy
J. Compositions, formulations and dosages
1. Preparing
Administration of TFNR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific constructs, and nucleic acids
3. Administration of nucleic acids encoding polypeptides (Gene therapy)
K. Therapeutic uses and methods of treatment
1. Treatment of chronic inflammatory/autoimmune diseases and disorders
2. Treatment of neurodegenerative and demyelinating diseases and disorders
3. Treatment of cancer and other immunosuppressive diseases, disorders and conditions
4. Combination therapy
L. examples
A. Definition of the definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, genBank sequences, databases, websites, and other published materials mentioned throughout the disclosure herein are incorporated by reference in their entirety, unless otherwise stated. If there are multiple definitions of terms herein, the definitions in this section control. When referring to a URL or other such identifier or address, it should be appreciated that such identifier may change and that specific information on the internet may come and go, but equivalent information may be found by searching the internet. Reference thereto demonstrates the availability and public dissemination of such information.
As used herein, a construct is a product that contains one or more components, typically at least two components. The components may be polypeptides, small molecules, aptamers, nucleic acids, and/or other such components described herein or known to those of skill in the art. Various constructs are described and exemplified herein; the composition and nature of which are apparent from the description herein. In view of this description, one of skill in the art can envision other constructs within the disclosure and claims herein. The term construct is used because the product may include a variety of different types of components.
As used herein, a construct is a TNFR1 construct or a TNFR2 antagonist construct, which is a construct comprising a TNFR1 inhibitor moiety, which is a moiety that inhibits or reduces TNFR1 activity, such as signaling.
As used herein, a construct is a TNFR2 construct or a TNFR2 agonist construct, which is a construct comprising a TNFR2 agonist moiety, which is a moiety that activates or induces TNFR2 activity, such as signaling or activity that results in Treg cell proliferation.
As used herein, a construct is a TNFR2 antagonist construct, which is a construct comprising a TNFR2 antagonist.
As used herein, a construct is a multispecific construct that is a construct that comprises more than one antagonist or agonist, or both, e.g., a construct that comprises a TNFR1 inhibitor and a TNFR2 agonist, or a construct that comprises two TNFR1 antagonists, e.g., wherein each antagonist interacts with a different epitope on TNFR1 or each antagonist has a different TNFR1 antagonist activity, or two TNFR2 agonists, e.g., wherein each agonist interacts with a different TNFR2 epitope, or each agonist has a different TNFR2 agonist activity.
As used herein, "tumor necrosis factor," "tumor necrosis factor alpha," "TNF-alpha," and "tnfα" are used interchangeably to refer to pleiotropic pro-inflammatory cytokines, which are members of the TNF superfamily, that are associated with inflammatory and immunomodulatory activities, including modulation of tumorigenesis/cancer, host defense against pathogen infection, apoptosis, autoimmunity, and septic shock. When referring to other members of the TNF superfamily, they will be identified by name. TNF is involved in the coordination of innate and adaptive immune responses, as well as in organogenesis, particularly in the genesis of lymphoid organs. TNF is a homotrimeric membrane-bound protein containing 233 amino acids, cleavable by the protease TACE (TNFa converting enzyme; also known as ADAM 17), releasing soluble TNF (solTNF) containing 157 amino acids; membrane-bound and soluble forms of TNF have biological activity. Homotrimers of TNF bind and signal through two high affinity specific receptors TNFR1 and TNFR 2; membrane-bound TNF activates TNFR2 primarily, whereas soluble TNF activates TNFR1 primarily. Uncontrolled or deregulated production of TNF is associated with a variety of chronic inflammatory and autoimmune diseases and conditions including, but not limited to, for example, infectious shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and Inflammatory Bowel Disease (IBD), as well as neurodegenerative and demyelinating diseases and conditions including, but not limited to, for example, alzheimer's disease, parkinson's disease, stroke, and multiple sclerosis.
As used herein, "TNF mutein" or "TNF-alpha mutein" or "modified TNF polypeptide" refers to a polypeptide whose amino acid sequence differs from the amino acid sequence of the corresponding wild-type TNF (tnfa) by one or more amino acids for TNF from a particular species. Typically, such modified TNF polypeptides retain the ability to activate or inhibit TNFR1 and/or TNFR 2. Specific mutations in TNF can cause the resulting TNF mutein to selectively bind TNFR1 or TNFR2 and can result in a TNF mutein having antagonistic or agonistic properties. For example, as described herein, there are TNFR 1-selective antagonistic TNF muteins and TNFR 2-selective agonistic TNF muteins.
As used herein, a "dominant negative inhibitor of TNF" or "DN-TNF" is a TNF mutein having one or more mutations that eliminate binding to and signaling through TNFR1 and/or TNFR 2. DN-TNF selectively inhibits soluble TNF (sTNF or solTNF) by rapidly displacing subunits with natural TNF homotrimers, forming inactive mixed TNF heterotrimers with disrupted receptor binding surfaces, thereby preventing interaction with TNF receptors. DN-TNF leaves transmembrane TNF (tmTNF) unaffected, and maintains protection of TNF signaling through TNFR 2. An example of DN-TNF is a mutant of TNF containing one or more of the substitutions L133Y, S162Q, Y163H, I173T, Y191Q and A221R with reference to the amino acid sequence shown in SEQ ID NO. 1 (reference to the solTNF sequence shown in SEQ ID NO. 2, corresponding to residues L57Y, S86Q, Y87H, I97T, Y Q and A145R), which impairs binding to TNFR.
As used herein, "modification" refers to modification of an amino acid sequence in a polypeptide or a nucleotide sequence in a nucleic acid molecule, and includes deletion, insertion, transposition, substitution of amino acids and nucleotides, respectively, and combinations thereof. Methods for modifying polypeptides or nucleic acids are routine to those skilled in the art, for example, by using recombinant DNA methods.
As used herein, when referring to a nucleic acid or polypeptide sequence, a "deletion" refers to the deletion of one or more nucleotides or amino acids as compared to the sequence (e.g., target polynucleotide or polypeptide) or natural or wild-type sequence.
As used herein, when referring to a nucleic acid or amino acid sequence, "insertion" describes the inclusion of one or more additional nucleotides or amino acids within a target sequence, native sequence, wild-type sequence, or other related sequence. Thus, a nucleic acid molecule containing one or more insertions contains one or more additional nucleotides within the linear length of the sequence compared to the wild-type sequence.
As used herein, when referring to a nucleic acid or amino acid sequence, "adding" describes adding one or more nucleotides or amino acids at either end as compared to the other sequence.
As used herein, "substitution" or "replacement" refers to the replacement of one or more nucleotides or amino acids in a native sequence, target sequence, wild-type or other nucleic acid or polypeptide sequence with a replacement nucleotide or amino acid without changing the length of the molecule (as described by the number of residues). Thus, one or more substitutions in a molecule will not change the number of amino acid residues or nucleotides of the molecule. Amino acid substitutions compared to a particular polypeptide may be expressed in terms of the number of amino acid residues along the length of the polypeptide sequence. For example, a modified polypeptide having tyrosine (Tyr; Y) substituted/replaced with glutamic acid (Glu; E) at amino acid position 100 of the amino acid sequence may be represented as Y100E, tyr100Glu or 100E. Y100 may be used to indicate that the amino acid modified at position 100 is tyrosine. For the purposes herein, since the modification is in the Heavy Chain (HC) or Light Chain (LC) of an antibody, reference may also be made to HC-or LC-representing a polypeptide chain to represent the modification.
As used herein, recitation of a "position corresponding to …," or a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a published sequence, e.g., as set forth in the sequence listing, refers to a nucleotide or amino acid position identified when aligned to a reference sequence to maximize identity using standard alignment algorithms, e.g., the GAP algorithm. By aligning the sequences, the person skilled in the art can identify the corresponding residues, for example using conserved and identical amino acid residues as guidance. Typically, to identify the corresponding position, the amino acid sequences are aligned so as to obtain the highest order match (see, e.g., computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W, ed., academic Press, new York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M, and Griffin, h.g., eds., humana Press, new Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G, academic Press,1987;Sequence Analysis Primer,Gribskov,M.and Devereux,J, eds., M Stockton Press, new York,1991; and carrilo et al (1988) SIAM j.applied Math 48:1073).
As used herein, sequence alignment refers to the use of homology to align two or more nucleotide or amino acid sequences. Typically, two or more sequences having 50% or more identity are aligned. An alignment sequence set refers to 2 or more sequences aligned at corresponding positions and may include RNA-derived alignment sequences aligned with genomic DNA sequences, such as ESTs and other cdnas. The related or variant polypeptides or nucleic acid molecules may be aligned by any method known to those of skill in the art. Such methods typically maximize matching and include such methods as using manual alignment and by using many available alignment programs (e.g., BLASTP) and other methods known to those skilled in the art. By aligning the sequences of polypeptides or nucleic acids, one skilled in the art can use conserved and identical amino acid residues as a guide to identify similar parts or positions. Furthermore, one skilled in the art can also use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between human and non-human sequences. The corresponding positions may also be based on structural alignment, for example, by using computer-simulated protein structural alignment. In other cases, the corresponding region may be identified. The skilled artisan can also use conserved amino acid residues as a guide to find corresponding amino acid residues between human and non-human sequences.
As used herein, the expression protein "compared under identical conditions" refers to the same or substantially the same treatment of different proteins such that any one or more conditions that may affect the activity or properties of the protein or formulation have no or substantially no change between test substances. For example, when comparing the activity of one antibody to another, any one or more conditions, such as the amount or concentration of a polypeptide; the presence of excipients, carriers, or other components in the formulation, including amounts, other than the active agent (e.g., antibody); a temperature; pH value; storage time; a storage container; storage properties (e.g., agitation); and/or other conditions associated with exposure or use, are identical or substantially identical between the compared polypeptides/antibodies.
As used herein, "adverse effect" or "side effect" or "adverse event" or "adverse side effect" refers to a deleterious, adverse and/or undesired effect associated with the administration of a therapeutic agent. For example, in combination with administration of an anti-TNF antibody such as adalimumab (e.g., under the trademarkSales) are known to those skilled in the art, and some side effects are described herein. Such adverse side effects include, for example, severe infections such as tuberculosis, and other infections caused by viruses, fungi, and bacteria, including upper respiratory tract infections, as well as dermatological and dermatological toxicities such as rashes, headaches, and nausea. Thus, "adverse effects" or "side effects" Refers to the deleterious, adverse and/or undesirable effects of the administration of a therapeutic agent. Side effects or adverse reactions are graded according to toxicity, there are various toxicity grade scales, and definitions are provided for each grade. Examples of such scales are the national cancer institute general toxicity standard (National Cancer Institute Common Toxicity Criteria) version 2.0 toxicity scale, and the world health organization or adverse event general term standard (CTCAE) scale. The specified severity level is within the skill of an experienced physician or other healthcare professional. The severity of symptoms can be quantified using the NCI adverse event common terminology standard (CTCAE) grading system. CTCAE is a descriptive term for Adverse Event (AE) reporting. A rating (severity) scale is provided for each AE term. CTCAE showed 1 to 5 grades and clinically described the severity of each adverse event according to the following general guidelines: grade 1 (mild AE); grade 2 (medium AE); grade 3 (severe AE); grade 4 (life threatening or disabling AE); and grade 5 (AE related death/mortality).
As used herein, the "property" of a polypeptide, such as an antibody, refers to any characteristic exhibited by the polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, proteolytic resistance, conformational stability, thermostability, and tolerance to pH conditions. The change in property may alter the "activity" of the polypeptide. For example, changes in the binding specificity of an antibody polypeptide may alter the ability to bind an antigen, and/or various binding activities, such as affinity or avidity, or in vivo activity of the polypeptide.
As used herein, the "activity" or "functional activity" of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. These activities may be determined empirically. Exemplary activities include, but are not limited to, the ability to interact with biomolecules, such as by antigen binding, DNA binding, ligand binding, or dimerization; and enzymatic activity, such as kinase activity or proteolytic activity. For antibodies (including antibody fragments), activities include, but are not limited to, the ability to specifically bind to a particular antigen, the affinity of antigen binding (e.g., high or low affinity), binding rate, dissociation rate, effector functions, such as the ability to promote antigen neutralization or clearance, virus neutralization, and in vivo activity, such as the ability to prevent infection or invasion by a pathogen, or the ability to promote clearance, or penetration of a particular tissue or fluid or cell in the body. Activity may be assessed in vitro or in vivo using well-established assays, such as ELISA, flow cytometry, surface plasmon resonance, or equivalent assays to measure binding or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry, and binding assays (e.g., panning assays). For example, for an antibody polypeptide, activity can be assessed by measuring binding affinity, avidity and/or binding coefficient (e.g., binding/dissociation rate) and other in vitro activities, or by measuring various effects in vivo, e.g., measuring immune effects, such as antigen clearance; the antibody permeates or localizes into the tissue; preventing diseases, such as infection; serum or other liquid antibody titer; or other assays known in the art. The results of such assays that indicate that a polypeptide exhibits activity may be correlated with the in vivo activity of the polypeptide, where in vivo activity may be referred to as therapeutic activity or biological activity. The activity of a modified polypeptide may be any percentage level of activity of an unmodified polypeptide, including but not limited to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%,300%, 400%, 500% or more of its activity compared to the unmodified polypeptide. Assays for determining the functionality or activity of modified (or variant) antibodies are well known in the art.
As used herein, "binding," "bound," and grammatical variations thereof refer to the participation of one molecule in any attractive interaction with another molecule, resulting in a stable association of two molecules in close proximity to each other. Binding interactions include, but are not limited to, non-covalent bonds, covalent bonds (e.g., reversible and irreversible covalent bonds), and include interactions between molecules, such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as compounds, including drugs. Exemplary bonds are antibody-antigen interactions and receptor-ligand interactions. When an antibody "binds" to a particular antigen, "binding" refers to the antibody specifically recognizing the antigen at the antibody combining site by homologous antibody-antigen interactions. Binding may also include association of multiple polypeptide chains, such as antibody chains, that interact through disulfide bonds.
As used herein, "binding activity" refers to a characteristic of a molecule, such as a polypeptide, that relates to whether and how it binds to one or more binding partners. Binding activity includes the ability to bind a binding partner, its affinity for binding to a binding partner (e.g., high affinity), its affinity for binding to a binding partner, the strength of a bond with a binding partner, and/or the specificity of binding to a binding partner.
As used herein, "affinity" or "binding affinity" describes the strength of interaction between two or more molecules, e.g., binding partners, and is typically the strength of non-covalent interaction between two binding partners. The affinity of an antibody or antigen binding fragment thereof for an epitope is a measure of the total non-covalent interaction strength between a single antibody combining site and the epitope. Low affinity antibody-antigen interactions are weaker and molecules tend to dissociate rapidly, while high affinity antibody-antigen binding is strong and molecules remain bound for longer periods of time. Binding affinity can be determined from binding kinetics, e.g., by measuring the binding rate (k a Or k on ) And/or dissociation rate (k) d Or k off ) Half maximum effective concentration (EC 5 0) Values and/or thermodynamic data (e.g., gibbs free energy (ΔG), enthalpy (ΔH), entropy (-TΔS), and/or computational binding (K) a ) Or dissociation (K) d ) A constant. EC (EC) 50 Also known as apparent K d Is the concentration of antibody (e.g., ng/mL), where 50% of maximum binding to a fixed amount of antigen is observed. Generally, EC 50 The values are determined from an S-shaped dose-response curve, in which EC 50 Is the concentration at the inflection point. High antibody affinity to its substrate and low EC 50 Value correlation, low affinity corresponds to high EC 50 Values. Affinity constants can be determined by standard kinetic methods of antibody reaction, e.g., immunoassays, such as ELISA, then subjected to curve fitting analysis.
As used herein, "affinity constant" refers to the association constant (K a ). The higher the affinity constant, the greater the affinity of the antibody for the antigen. Affinity constants are expressed in units of the inverse mole (i.e., M -1 ) And may be calculated from the rate constants of the binding-dissociation reactions, as measured by standard kinetic methods of antibody reactions (e.g., immunoassays, surface plasmon resonance, or other kinetic interaction assays known in the art). The binding affinity of an antibody can also be expressed as a dissociation constant or K d . The dissociation constant is the inverse of the association constant, i.e. K d ＝1/K a . Thus, the affinity constant can also be K d And (3) representing. Affinity constants can be determined by standard kinetic methods of antibody reaction, such as immunoassays, surface Plasmon Resonance (SPR) (see, e.g., rich and Myszka (2000) curr. Opin. Biotechnol 11:54; englebiene (1998) analysis 123:1599), isothermal Titration Calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., paul, ed., fundamental Immunology,2nd ed., raven Press, new York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for descriptions of example SPR and ITC methods for calculating binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (e.g., BIAcore 2000,BIAcore AB,Upsala,Sweden and GE Healthcare Life Sciences; malmeqvist (2000) biochem. Soc. Trans. 27:335).
Methods for calculating affinity are well known, e.g. determining EC 50 A method of value, or a method of determining association/dissociation constant. For example, with EC 50 By high binding affinity is meant that the antibody specifically binds to the target protein, its EC 50 Less than about 10ng/mL, 9ng/mL, 8ng/mL, 7ng/mL, 6ng/mL, 5ng/mL, 3ng/mL, 2ng/mL, 1ng/mL or less. The high binding affinity may also be characterized by 10 -6 M or lower equilibrium dissociation constant (K d ) For example 10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M, or 10 -12 M, or moreLow. Equilibrium association constant (K) a ) In other words, high binding affinities generally correlate with greater than or equal to about 10 6 M -1 Greater than or equal to about 10 7 M -1 Greater than or equal to about 10 8 M -1 Or greater than or equal to about 10 9 M -1 、10 10 M -1 、10 11 M -1 Or 10 12 M -1 K of (2) a The values are correlated. Affinity may be estimated empirically, or may be determined comparatively, for example by comparing the affinities of two or more antibodies to a particular antigen, for example by calculating the pairwise ratio of affinities of the antibodies tested. For example, such affinity can be readily determined using conventional techniques, such as by ELISA, equilibrium dialysis, surface plasmon resonance, by radioimmunoassay using radiolabeled target antigen, or by another method known to those skilled in the art. Affinity data may be analyzed, for example, by the method of Scatchard et al, (1949) Ann n.y. acad.sci.,51:660, or by curve fitting analysis, for example using a 4 parameter Logistic nonlinear regression model, using the equation: y= ((a-D)/(1+ ((x/C)/(B))) +d, where a is the minimum asymptote, B is the slope factor, and C is the inflection point (EC) 50 ) And D is the maximum asymptote.
As used herein, "antibody avidity" refers to the strength of multiple interactions between a multivalent antibody and its cognate antigen, e.g., an antibody that contains multiple binding sites associated with an antigen having a repeating epitope or epitope array. High affinity antibodies have higher intensity of such interactions than low affinity antibodies.
As used herein, "specificity for a target," such as TNFR1, refers to preferential, higher binding affinity to bind the target than to a non-target. By selectively binding is meant at least about 10 in general 7 -10 8 M -1 Is bound to the target. It may also refer to relative activity in which the affinity of one moiety or molecule for one target molecule is compared to the affinity for another molecule, if the difference is of a certain magnitude, e.g. about 10 times, then the moiety or molecule is for the firstThe individual targets have greater specificity relative to the second target.
As used herein, "specific binding" or "immunospecific binding" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein to refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody's antibody combining site and the antigen. Typically, antibodies that immunospecifically bind (or specifically bind) e.g., TNFR1 bind to the affinity constant (K) of TNFR1 a ) Is about or 1X 10 7 M -1 Or 1X 10 8 M -1 Or greater (or dissociation constant (K) d ) Is 1X 10 -7 M or 1X 10 -8 M or lower). Antibodies or antigen binding fragments that immunospecifically bind to a particular antigen may be identified by, for example, an immunoassay, such as a Radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), surface Plasmon Resonance (SPR), or other techniques known to those skilled in the art.
As used herein, "steric effect" refers to the effect of the size of an atom or group on a molecule. Steric effects include, but are not limited to, steric hindrance and van der Waals repulsion. The spatial effect is an effect resulting from the fact that atoms occupy space; when atoms are close to each other, this consumes energy, as electrons in the vicinity of the atoms repel each other.
As used herein, "exhibiting at least one activity" or "retaining at least one activity" refers to an activity exhibited by an antibody polypeptide, such as a variant antibody or other therapeutic polypeptide, as compared to a polypeptide that does not contain the modified target or unmodified polypeptide. Modified or variant polypeptides that retain the activity of the target polypeptide may exhibit improved activity, reduced activity, or retain the activity of the unmodified polypeptide. In some cases, the modified or variant polypeptide may retain increased activity as compared to the target polypeptide or unmodified polypeptide. In some cases, the modified or variant polypeptide may retain reduced activity as compared to the unmodified or target polypeptide. The activity of the modified or variant polypeptide may be any percentage level of activity of the unmodified or target polypeptide compared to the unmodified or target polypeptide, including but not limited to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more of the activity. In other embodiments, the change in activity is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more greater than the unmodified polypeptide or target polypeptide. The activity retention assay depends on the activity to be retained. Such assays may be performed in vitro or in vivo. The activity may be measured, for example, using assays known in the art and described below for activity, such as, but not limited to, ELISA and panning assays. The activity of a modified polypeptide or variant polypeptide as compared to an unmodified polypeptide or target polypeptide may also be assessed based on the in vivo therapeutic or biological activity or result after administration of the polypeptide.
As used herein, "surface plasmon resonance" refers to an optical phenomenon that allows for analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix. Commercial forms are available. For example, the BIAcore system (GE Healthcare Life Sciences) is an exemplary business system.
As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetic, e.g., recombinantly produced, including any fragment thereof comprising at least a portion of the variable heavy and/or variable light chain regions of an immunoglobulin molecule, sufficient to form antigen binding sites and to specifically bind antigen upon assembly. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody combining site). For example, an antibody refers to an antibody that contains two heavy chains (which may be represented as H and H ') and two light chains (which may be represented as L and L'), wherein each heavy chain may be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen-binding site (e.g., heavyChains include, but are not limited to, V H Chain, V H -C H 1 chain and V H -C H 1-C H 2-C H 3) and each light chain may be a full length light chain or a portion thereof sufficient to form an antigen binding site (e.g., a light chain including, but not limited to, V L Chain and V L -C L Chains). Each heavy chain (H and H ') is paired with a light chain (L and L', respectively). Typically, antibodies minimally include variable weights (V H ) Chains and/or variable lights (V L ) All or at least a portion of the chain. Antibodies may also include other regions, such as all or part of a constant region, and/or all or part of a hinge region (sufficient to provide flexibility).
For purposes herein, unless otherwise indicated, the term "antibody" includes full length antibodies and portions thereof, including antibody fragments, such as anti-TNFR 1 antibody fragments. Antibody fragments, including but not limited to, for example, fab fragments, fab 'fragments, F (ab') 2 Fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd' fragments, single chain Fv (scFv), single chain Fab (scFab), hsFv (helix stabilized Fv), single domain antibody (dAb or sdAb), minibody, diabody, anti-idiotype (anti-Id) antibody, nanobody and camelidae antibody, free light chain, V HH An antibody (or nanobody), or an antigen-binding fragment of any of the antibodies described above. Antibody fragments may also include combinations of any of the above fragments, e.g., tandem scFv, fab-scFv (HC C-terminal or LC C-terminal), fab- (scFv) 2 (C-terminal), scFv-Fab-scFv, fab-C H 2-scFv, scFv fusion (C-terminal or N-terminal), fab fusion (HC C-terminal or LC C-terminal), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv and trisomes. The term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific and heteroconjugate antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies and intracellular antibodies. Antibodies provided herein include members of any immunoglobulin class (e.g., igG, igM, igD, igE, igA and IgY), any subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b).
As used herein, "antibody form" refers to a particular structure of an antibody. Antibodies herein include full length antibodies and portions thereof, such as Fab fragments or other antibody fragments. Thus, fab is a specific form of antibody.
As used herein, reference to "corresponding forms" of antibodies refers to the use of the same form of antibody to compare properties or activities of two antibodies when comparing the properties. For example, if an antibody is stated to have a lower activity than the activity of the corresponding form of the first antibody, that means that the particular form of the antibody, e.g., fab, has a lower activity than the Fab form of the first antibody.
As used herein, a full length antibody is a polypeptide having two full length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) Two full length light chains (V L -C L ) And antibodies of the hinge region, such as human antibodies produced by antibody-secreting B cells, and synthetically produced antibodies having the same domains.
As used herein, a "multispecific construct" refers to a construct, such as an antibody or construct comprising an antibody moiety, that exhibits affinity for more than one target antigen so that it can specifically interact with a target. The multispecific constructs herein may have a structure similar to an intact immunoglobulin molecule and include an Fc region, e.g., an IgG Fc region, and an antigen-binding region, e.g., a portion that specifically binds TNFR1 or TNFR 2.
As used herein, a "bispecific construct" refers to a multispecific construct that has binding specificity for two different antigens. Bispecific constructs include, for example, monoclonal antibodies or antigen-binding fragments thereof linked to a polypeptide region of modified construct activity, such as Fc or modified Fc. For human therapeutics, the construct is derived from or humanized by a human source and the construct has binding specificity for at least two different antigens. Bispecific constructs/molecules provided herein can have binding specificity for TNFR1 and TNFR 2. For example, bispecific constructs include TNFR1 antagonists and TNFR2 agonists. Bispecific antibodies or constructs include antibodies and antigen-binding fragments thereof, which comprise two separate antigen-binding domains (e.g., two scFv, or two dabs, or two Fab, linked by a linker). The antigen binding domains may bind the same antigen or different antigens.
As used herein, "antibody fragment" or "antibody portion" refers to any portion of a full-length antibody that is less than full length, but contains at least a portion of the antibody variable region (e.g., one or more Complementarity Determining Regions (CDRs)) sufficient to form an antigen binding site, thus preserving the binding specificity and/or activity of the full-length antibody; antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetic, e.g., recombinantly produced, derivatives. Examples of antibody fragments include, but are not limited to, fab', F (ab) 2 Single chain Fv (scFv), fv, dsFv, diabodies, triabodies, affinity antibodies, nanobodies, aptamers, dAbs, fd and Fd fragments (see, e.g., methods in Molecular Biology, vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); chapter 1; pp.3-25, kipriyanov). The fragments may comprise multiple chains linked together, for example by disulfide bridges and/or by peptide linkers. Antibody fragments typically contain at least about 50 amino acids, such as at least about or at least 100 amino acids, and typically at least about or at least 110, 120, 150, 170, 180, or 200 amino acids.
As used herein, an "Fv antibody fragment" is composed of one heavy chain variable domain (V H ) And a light chain variable domain (V L ) Composition is prepared.
As used herein, dsFv (disulfide-linked Fv) refers to Fv with engineered intermolecular disulfide bonds that stabilize V H -V L For each pair.
As used herein, "scFv fragment" refers to a fragment comprising a variable light chain (V L ) And a variable heavy chain (V H ) Covalently linked in any order by a polypeptide linker. The length of the linker is such that the two variable domains are bridged without substantial interference. Exemplary linkers are (Gly-Ser) n Residues, some Glu or Lys residues are scattered throughout to increase solubility.
As used herein, a "diabody" is a dimeric scFv; diabodies typically have a shorter peptide linker than scFv, and preferably dimerize.
As used herein, "trisomy" is a trimeric scFv; it contains three peptide chains, each containing a V H Domain and a V L Domains, linked by short linkers (e.g. linkers consisting of 1-2 amino acids) to allow for V within the same peptide chain H And V L Intramolecular binding of the domain; trisomy is usually trimerized.
As used herein, a "Fab fragment" is an antibody fragment produced by papain digestion of full length immunoglobulin, or a fragment having the same structure produced synthetically, e.g., by recombinant methods. The Fab fragment contains a light chain (containing V L And C L ) Comprising a heavy chain variable domain (V H ) And a constant region (C) H 1) Is a chain of the other chain.
As used herein, "F (ab') 2 Fragments "are antibody fragments produced by pepsin digestion of immunoglobulins at a pH of 4.0-4.5, or fragments having the same structure, e.g., synthetically produced by recombinant methods. F (ab') 2 The fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, e.g. all or part of a hinge region sufficient to provide flexibility, including cysteine residues forming disulfide bonds linking the two fragments.
As used herein, fab 'fragments are F-containing (ab') 2 Fragments that are half of the fragments (i.e., one heavy chain and one light chain).
As used herein, fd fragment is a variable domain containing the antibody heavy chain (V H ) And a constant region domain (C H 1) Is a fragment of an antibody of (a).
As used herein, fd 'fragments are F (ab') 2 An antibody fragment of one heavy chain portion of the fragment.
As used herein, fv' fragment is V containing only antibody molecules H And V L Structure of theFragments of the domain.
As used herein, hsFv (helix stabilized Fv) refers to antibody fragments in which the constant domains normally present in Fab fragments have been replaced by heterodimeric coiled-coil domains (see, e.g., arndt et al (2001) J.mol.biol.7:312:221-228).
As used herein, "domain antibody," "single domain antibody," "sdAb," or "dAb" interchangeably refer to a heavy chain comprising an antibody (V H ) Or light chain (V) L ) A monomeric small antibody fragment of the variable domain of (a). dabs are the smallest antigen-binding fragments of antibodies; it is about 11-15kDa (about 100-150 amino acids) in size and about one tenth of the size of an intact monoclonal antibody (mAb). Each V H And each V L There are three Complementarity Determining Regions (CDRs). Each dAb contains three of the six CDRs, which are from V in the antibody H -V L Highly diverse circular regions of the pair that bind to the target antigen.
As used herein, camelidae antibodies, also known as nanobodies or VHHs, lack a light chain and consist of two identical heavy chains. They naturally occur in camelids such as camels and alpacas.
As used herein, a polypeptide "domain" is a portion (sequence of 3 or more, typically 5, 10 or more amino acids) of a polypeptide that is structurally and/or functionally distinguishable or definable. Exemplary polypeptide domains are portions of polypeptides that can form independent folding structures within polypeptides composed of one or more structural motifs (e.g., combinations of alpha helices and/or beta strands joined by loop regions) and/or recognized by specific functional activities such as enzymatic activity, dimerization, or antigen binding. The polypeptides may have one or more, typically more than one, different domains. For example, a polypeptide may have one or more domains and one or more functional domains. Individual polypeptide domains can be distinguished by structure and function. The domains may encompass a continuous linear amino acid sequence. Alternatively, a domain may encompass multiple non-contiguous amino acid portions that are non-contiguous along the linear amino acid sequence of a polypeptide. Typically, a polypeptide contains multiple domains. For example, each heavy chain and each light chain of an antibody molecule contains multiple immunoglobulin (Ig) domains, each about 110 amino acids long. Those skilled in the art are familiar with polypeptide domains and can identify them by virtue of structural and/or functional homology to other such domains. For the purposes of the examples herein, definitions are provided, but it should be understood that identifying a particular domain by name is within the skill of the art. Appropriate software can be used to identify domains, if desired.
As used herein, a "functional region" of a polypeptide is a region of the polypeptide that contains at least one functional domain (which confers a particular function, e.g., the ability to interact with a biological molecule, e.g., by antigen binding, DNA binding, ligand binding or dimerization, or by enzymatic activity, e.g., kinase activity or proteolytic activity); exemplary functional regions of polypeptides are antibody domains, e.g., V H 、V L 、C H 、C L And portions thereof, such as CDRs, including CDR1, CDR2, and CDR3, or antigen binding portions, such as antibody combining sites.
As used herein, a "structural region" of a polypeptide is a region of the polypeptide that contains at least one domain.
As used herein, an "Ig domain" is a domain recognized by those skilled in the art as being distinguished by a structure called an immunoglobulin (Ig) fold, which contains two β -sheet sheets, each sheet containing antiparallel β chains of amino acids joined by loops. The two β -sheets in an Ig fold are clamped together by hydrophobic interactions and conserved intrachain disulfide bonds. The individual immunoglobulin domains in an antibody chain can be further distinguished by function. For example, the light chain contains a variable region domain (V L ) And a constant region domain (C L ) While the heavy chain contains a variable region domain (V H ) And three or four constant region domains (C H ). Each V L 、C L 、V H And C H The domains are examples of immunoglobulin domains.
As used herein, a "variable domain" with respect to an antibody is a specific immunoglobulin (Ig) domain of an antibody heavy or light chain that contains a variable between different antibodiesAmino acid sequence of the chemical. Each light chain and each heavy chain has a variable region domain (V respectively L And V H ). The variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains Complementarity Determining Regions (CDRs) that are part of the antigen binding site domain and Framework Regions (FR).
As used herein, "hypervariable region," "HV," "complementarity determining region," "CDR," and "antibody CDR" are used interchangeably to refer to one of the multiple portions within each variable region that together form the antigen binding site of an antibody. Each variable region domain contains three CDRs, designated CDR1, CDR2 and CDR3. These three CDRs are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within loops connecting the β -sheet parallel chains of the variable domains.
As used herein, "antigen binding domain," "antigen binding site," "antigen binding fragment," "antigen combining site," and "antibody combining site" are used synonymously, and refer to a domain within an antibody that recognizes and physically interacts with a cognate antigen. The natural conventional full length antibody molecule has two conventional antigen binding sites, each containing a portion of the heavy chain variable region and a portion of the light chain variable region. Conventional antigen binding sites contain loops that join antiparallel β chains within the variable region domain. The antigen binding site may contain other portions of the variable region domain. Each conventional antigen binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions are also known as Complementarity Determining Regions (CDRs).
As used herein, "a portion thereof" with respect to an antibody heavy or light chain or variable heavy or light chain refers to a contiguous portion thereof sufficient to form an antigen binding site, whereby when assembled into an antibody comprising heavy and light chains, it comprises a variable heavy chain (V H ) And variable light chain (V L ) Is sufficient to retain at least a portion of the binding specificity of a corresponding full length antibody containing all 6 CDRs. Typically, sufficient antigen binding sites require CDR3 (CDRH 3) of the heavy chain. It also typically requires CDR3 (CDRL 3) of the light chain. As described herein, in the artCDRs are known to those skilled in the art and can be identified based on Kabat or Chothia numbering (see, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. device of Health and Human Services, NIH Publication No.91-3242; and Chothia, C.et al (1987) J.mol. Biol.196:901-917).
As used herein, a "framework region" or "FR" is a domain located within the variable region domain of an antibody within the β -sheet; the FR region is relatively more conserved than the hypervariable region in terms of its amino acid sequence. Each variable region contains four framework regions, separating the three hypervariable regions.
As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that contains an amino acid sequence that is relatively more conserved in antibodies than a variable region domain. Each light chain has a single light chain constant region (C L ) Domains, each heavy chain containing one or more heavy chain constant regions (C H ) Domain, comprising C H 1、C H 2、C H 3 and C H 4. Full length IgA, igD and IgG isotypes contain C H 1、C H 2 and C H 3 domain and hinge region, while IgE and IgM contain C H 1、C H 2、C H 3 and C H 4 domain. C (C) H 1 and C L The domain extends the Fab arm of the antibody molecule, thereby facilitating the interaction with the antigen and rotation of the antibody arm. The antibody constant regions may perform effector functions such as, but not limited to, clearing antigens, pathogens, and toxins to which the antibodies specifically bind, such as through interactions with various cells, biomolecules, and tissues.
As used herein, "antibody hinge region" or "hinge region" refers to the polypeptide region in the heavy chain of gamma, delta, and alpha antibody isotypes that occurs at C H 1 and C H 2, links the Fab and Fc regions, and has no homology with other antibody domains. This region is rich in proline residues and provides flexibility for IgG, igD and IgA antibodies, allowing the two "arms" of the Fab portion (each arm containing one antibody combining site) to be movable, assuming different angles between each other as they bind antigen. This flexibility allows for Fab arms Moving to align the antibody combining sites to interact with epitopes on the cell surface or other antigens. The two interchain disulfide bonds within the hinge region stabilize the interaction between the two heavy chains. In some embodiments provided herein, synthetically produced antibody fragments contain one or more hinge regions, for example, to promote stability through interaction between two antibody chains. The hinge region is an example portion of a dimerization domain and is part of a linker for purposes herein.
As used herein, "fragment crystallizable region" or "Fc region" or "Fc domain" refers to a polypeptide that contains an antibody heavy chain constant region but does not include the first constant region immunoglobulin domain. Fc refers to the last two constant region immunoglobulin domains of IgA, igD and IgG (C H 2 and C H 3, also known as cγ2 and cγ3), or the last three constant region immunoglobulin domains of IgE and IgM (C H 2、C H 3 and C H 4). Optionally, the Fc domain may include all or part of a flexible hinge region located N-terminal to these domains. For IgA and IgM, the Fc may include the J chain. For the Fc domain of an exemplary IgG, fc contains immunoglobulin domain C H 2 and C H 3, optionally C H 1 and C H 2 (also referred to as cγ1 and cγ2). The boundaries of the Fc region may vary, but generally include at least a portion of the hinge region. For purposes herein, fc also includes any allelic or species variant, or any variant or modified form, such as any variant or modified form of Fc that alters binding to an Fc receptor (FcR) or alters Fc-mediated effector function. Mutations in the Fc region and their effects are well documented in the art.
As used herein, "Fc chimeric" refers to chimeric polypeptides in which one or more polypeptides are linked directly or indirectly to an Fc region or derivative thereof. Typically, an Fc chimera combines the Fc region of an immunoglobulin with another polypeptide. Derivatives of Fc polypeptides or modified Fc polypeptides are known to those skilled in the art.
As used herein, "Kabat numbering" refers to the index numbering of IgG1 Kabat antibodies (see, e.g., kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. section of Health and Human Services, NIH Publication No. 91-3242); it allows easy comparison between antibodies, similar to the way chymotrypsin numbering allows comparison between proteases. The Kabat numbering can be used by those skilled in the art to identify regions of constant regions.
As used herein, "EU numbering" or "EU index" refers to the numbering scheme of EU antibodies, described in Edelman et al, (1969) Proc.Natl.Acad.Sci.USA 63:78-85. The "EU index in Kabat" refers to the EU index numbering of human IgG1 Kabat antibodies, as described by Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, U.S. section of Health and Human Services, NIH Publication No. 91-3242. The amino acid residues of the Fc region of the light and heavy chains of antibodies are often numbered using EU numbering or EU numbering as in Kabat. For example, the EU numbering can be used by those skilled in the art to identify regions of constant regions. For example, C of Ig kappa light chain according to Kabat and EU numbering L The domain corresponds to residues R108-C214 (see, e.g., table 2 below). IgG 1C H The 1 domain corresponds to residues 118-215 (EU numbering) or 114-223 (Kabat numbering); c (C) H 2 corresponds to residues 231-340 (EU numbering) or 244-360 (Kabat numbering); c (C) H 3 corresponds to residues 341-447 (EU numbering) or 361-478 (Kabat numbering).
The following table defines the numbering of the IgG1 and IgG4 heavy chain constant domains and Ig kappa light chain constant domains according to EU, kabat, and sequential numbering. Table 1 shows IgG1 heavy chain constant domains numbered according to EU, kabat and sequence numbering relative to the amino acid sequence shown in SEQ ID NO.9 and identifying C H 1、C H 2 and C H 3 domain and hinge region. Table 2 shows immunoglobulin (Ig) kappa light chain constant domains numbered according to EU, kabat and sequence numbering, wherein sequence numbering is relative to the amino acid sequence shown in SEQ ID NO. 17. In Table 2, the amino acid residue numbers are listed in order number in the first line (bold) with reference to SEQ ID NO: 17; the second line (bold) provides the single letter code for the amino acid residue at the position indicated by the number in the first lineThe method comprises the steps of carrying out a first treatment on the surface of the The third row (italics) shows Kabat numbering corresponding to Kabat numbering; the fourth line shows EU index numbers corresponding to EU numbering. Table 3 shows IgG4 heavy chain constant domains numbered according to EU, kabat and sequence numbering relative to the amino acid sequence shown in SEQ ID NO. 15 and identifying C H 1、C H 2 and C H 3 domain and hinge region.
Table 1: igG1 heavy chain constant domains numbered according to EU, kabat and order
Table 3: igG4 heavy chain constant domains numbered according to EU, kabat and order
As used herein, the phrase "derived from" when referring to an antibody fragment derived from another antibody, e.g., a monoclonal antibody, refers to an antibody fragment (e.g., fab, F (ab') which retains the binding specificity of the original antibody 2 Engineering of single chain Fv (scFv), fv, dsFv, dAb, diabodies, fd and Fd' fragments). Such fragments may be derivatized by a variety of methods known in the art, including but not limited to enzymatic cleavage, chemical crosslinking, recombinant means, or combinations thereof. Typically, the derivatized antibody fragment shares the same or substantially the same heavy chain variable region (V H ) And a light chain variable region (V L ) Whereby the antibody fragment and the parent antibody bind to the same epitope.
As used herein, "parent antibody" or "source antibody" refers to an antibody fragment (e.g., fab, F (ab'), F (ab)) derived therefrom 2 Single chain Fv (scFv), fv, dsFv, dAb, diabodies, fd and Fd' fragments).
As used herein, the term "epitope" refers to any antigenic determinant on an antigen or protein to which the paratope of an antibody may bind. Epitope determinants generally contain chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three dimensional structural features, as well as specific charge features.
As used herein, "humanized antibody" and human therapeutic agent refer to antibodies and other protein therapeutic agents that are modified to include a "human" amino acid sequence, thereby not eliciting an immune response when administered to a human. For example, humanized antibodies typically contain complementarity determining regions (CDRs or hypervariable loops) derived from a non-human species immunoglobulin, the remainder of the antibody molecule being predominantly derived from a human immunoglobulin. Methods for humanizing proteins, including antibodies, and producing them are well known and readily available to those skilled in the art. For example, DNA encoding a monoclonal antibody can be altered by recombinant DNA techniques to encode an antibody in which the amino acid composition of the non-variable region is based on a human antibody. Methods for identifying these regions are known and include computer programs designed to identify the variable and non-variable regions of immunoglobulins. Thus, in general, a humanized antibody will substantially comprise at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (e.g., CDRs) correspond to those of a non-human immunoglobulin and all or substantially all of the Framework Regions (FRs) are of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
As used herein, a "multimerization domain" refers to an amino acid sequence that facilitates stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each polypeptide molecule containing a complementary multimerization domain, which may be the same or a different multimerization domain, forming a stable multimer with the first domain. Typically, the polypeptide is linked directly or indirectly to the multimerization domain. Exemplary multimerization domains include immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain may be, for example, an immunoglobulin constant region or domain, such as an Fc domain from IgG or a portion thereof, including IgG1, igG2, igG3, or IgG4 subtypes, igA, igE, igD, and IgM, and modified versions thereof.
As used herein, a "dimerization domain" is a multimerization domain that facilitates interactions between two polypeptide sequences (e.g., without limitation, antibody chains). Dimerization domains include, but are not limited to, amino acid sequences containing cysteine residues that facilitate disulfide bond formation between two polypeptide sequences, e.g., all or part of a full length antibody hinge region, or one or more dimerization sequences, which are amino acid sequences known to facilitate interactions between polypeptides (e.g., leucine zipper, GCN4 zipper).
As used herein, "chimeric polypeptide" refers to a polypeptide that contains portions from at least two different polypeptides or from two discrete portions of a single polypeptide. Thus, a chimeric polypeptide generally comprises all or part of an amino acid residue sequence from one polypeptide, as well as all or part of an amino acid sequence from a different polypeptide. The two moieties may be linked directly or indirectly, and may be linked by peptide bonds, other covalent bonds, or other non-covalent interactions, with sufficient strength to maintain most of the integrity of the chimeric polypeptide under equilibrium conditions and physiological conditions such as isotonic pH 7 buffered saline.
As used herein, a "fusion protein" is a polypeptide that is engineered to contain amino acid sequences corresponding to two different polypeptides that are linked together, for example, by expression of the fusion protein from a vector that contains two nucleic acids encoding the two polypeptides in close proximity to each other, e.g., adjacent, along the length of the vector. Thus, a fusion protein refers to a chimeric protein containing two proteins or peptides or portions thereof linked directly or indirectly through peptide bonds. The two molecules may be adjacent in the construct or may be separated by a linker or spacer polypeptide.
As used herein, "linker," "linker unit," or "linking" refers to a peptide or chemical moiety containing an atomic chain that covalently links an antibody or antigen binding fragment thereof to another therapeutic moiety or another antibody or fragment thereof. Joints are included to, for example, increase flexibility, alter steric effects (including steric hindrance), and increase solubility in aqueous media.
As used herein, "linker peptide" or "spacer peptide" refers to a short amino acid sequence that connects two polypeptide sequences (or nucleic acids encoding such amino acid sequences). "peptide linker" refers to a short amino acid sequence that connects two polypeptide sequences. Examples of polypeptide linkers are linkers that link the peptide transduction domain to an antibody, or linkers that link two antibody chains in a synthetic antibody fragment, such as an scFv fragment. Linkers are well known and any known linker may be used in the provided methods. Exemplary polypeptide graftingHead includes (Gly-Ser) n Amino acid sequences in which some Glu or Lys residues are scattered throughout to increase solubility. Other example joints are described herein; any of these and other known linkers can be used with the polypeptides, antibodies, and other products and methods provided herein.
As used herein, "tag" or "epitope tag" refers to an amino acid sequence that is typically added to the N-or C-terminus of a polypeptide, such as antibodies and antibody fragments/constructs provided herein. The inclusion of a tag fused to the polypeptide may facilitate purification and/or detection of the polypeptide. In general, a tag or tag polypeptide refers to a polypeptide that has enough residues to provide an epitope recognized by an antibody or that is available for detection or purification, but is short enough so that it does not interfere with the activity of the polypeptide to which it is attached. The tag polypeptide is typically sufficiently unique that antibodies that specifically bind thereto do not substantially cross-react with epitopes in the polypeptide to which it is attached. Suitable tag polypeptides typically have at least 5 or 6 amino acid residues, and typically between about 8-50 amino acid residues, typically between 9-30 residues. The tag may be linked to one or more chimeric polypeptides in the multimer and allow detection of the multimer from the sample or mixture or recovery thereof. Such labels are well known and can be easily synthesized and designed. Exemplary tag polypeptides include those for affinity purification, including, for example, FLAG tags; his tag; influenza Hemagglutinin (HA) tag polypeptide and antibody 12CA5 (see, e.g., field et al (1988) mol. Cell. Biol. 8:2159-2165); c-myc tags and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (see, e.g., evan et al (1985) Molecular and Cellular Biology 5:3610-3616); and herpes simplex virus glycoprotein D (gD) tags and antibodies thereto (see, e.g., paborsky et al (1990) Protein Engineering 3:547-553). Antibodies for detecting epitope-tagged antibodies are generally referred to herein as secondary antibodies.
As used herein, a "label" or "detectable moiety" is a detectable label (e.g., a fluorescent molecule, a chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be directly or indirectly attached or linked to a molecule (e.g., an antibody or antigen binding fragment thereof, such as an anti-TNFR 1 antibody or antigen binding fragment thereof provided herein), or bound thereto, and can be detected in vivo and/or in vitro. The detection method can be any method known in the art, including known in vivo and/or in vitro detection methods (e.g., imaging by visual inspection, magnetic Resonance (MR) spectroscopy, ultrasound signals, X-rays, gamma ray spectroscopy (e.g., positron Emission Tomography (PET) scanning, single Photon Emission Computed Tomography (SPECT), fluorescence spectroscopy, or absorbance)), indirect detection refers to measuring a physical phenomenon of an atom, molecule, or composition that directly or indirectly binds to the detectable moiety, such as energy or particle emission or absorption (e.g., detecting a labeled secondary antibody or antigen-binding fragment thereof that binds to a primary antibody (e.g., an anti-TNFR antibody or antigen-binding fragment thereof provided herein).
As used herein, "nucleic acid" refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by phosphodiester bonds. Also included within the term "nucleic acid" are analogs of nucleic acids, such as Peptide Nucleic Acids (PNAs), phosphorothioate DNA, and other such analogs and derivatives, or combinations thereof. Nucleic acids also include DNA and RNA derivatives containing "backbone" linkages other than, for example, nucleotide analogs or phosphodiester linkages, such as phosphotriester linkages, phosphoramidate linkages, phosphorothioate linkages, thioester linkages, or peptide linkages (i.e., peptide nucleic acids). The term also includes equivalents, derivatives, variants and analogues of RNA or DNA made from nucleotide analogs, single (sense or antisense) and double stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For RNA, the uracil base is uridine.
As used herein, an "isolated nucleic acid molecule" is a molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen binding fragments.
As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements or domains refers to nucleic acid regions that are functionally related to each other. For example, a nucleic acid encoding a leader peptide may be operably linked to a nucleic acid encoding a polypeptide whereby the nucleic acid may be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects the secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acid is transcribed into a single mRNA transcript, but translation of the mRNA transcript can result in expression of one of the two polypeptides. For example, an amber stop codon may be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, whereby when introduced into a portion of an amber suppressor cell, the resulting single mRNA transcript may be translated to produce a fusion protein containing the first and second polypeptides or may be translated to produce only the first polypeptide. In another example, a promoter may be operably linked to a nucleic acid encoding a polypeptide, whereby the promoter modulates or mediates transcription of the nucleic acid.
As used herein, "synthesis" with respect to, for example, a synthetic nucleic acid molecule or synthetic gene or synthetic peptide refers to a nucleic acid molecule or gene or polypeptide molecule produced by recombinant means and/or by chemical synthesis means.
As used herein, naturally occurring α -amino acid residues are those of 20 α -amino acids found in nature that are incorporated into proteins by specific recognition of charged tRNA molecules and their cognate mRNA codons in humans.
As used herein, "polypeptide" refers to two or more amino acids that are covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.
As used herein, "peptide" refers to a polypeptide that is 2 to about or 40 amino acids in length.
As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. The polypeptide contains two or more amino acids. For purposes herein, amino acids in a polypeptide such as an antibody are provided to include twenty naturally occurring amino acids (table 4), unnatural amino acids, and amino acid analogs (e.g., amino acids in which the α -carbon has a side chain). As used herein, amino acids in the various amino acid sequences of polypeptides herein are identified according to their well-known three-letter or one-letter abbreviations (see table 4). Nucleotides found in various nucleic acid molecules and fragments are named by standard single-letter designations conventionally used in the art.
As used herein, "amino acid residue" refers to an amino acid formed after chemical digestion (hydrolysis) at peptide bonds of a polypeptide. The amino acid residues described herein are typically in the "L" isomeric form. The "D" isomeric form of the residues can be substituted for any L-amino acid residue, so long as the polypeptide retains the desired functional properties. NH (NH) 2 Refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of the polypeptide. Consistent with standard polypeptide nomenclature described in j.biol.chem.,243:3557-59 (1968), and using 37c.f.r. ≡ ≡1.821-1.822, amino acid residue abbreviations are shown in table 4:
table 4: correspondence table
All amino acid residue sequences represented by the formulae herein are oriented in a conventional amino-terminal to carboxy-terminal direction from left to right. Furthermore, the phrase "amino acid residue" is defined to include amino acids, modified, unnatural, and unusual amino acids listed in the corresponding table (table 4). Furthermore, a dash at the beginning or end of a sequence of amino acid residues indicates a sequence other than one or more amino acid residues or with an amino terminal group (e.g., NH 2 ) Or a peptide bond to a carboxyl end group (e.g., COOH). In peptides or proteins, suitable amino acid conservative substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule Is performed under the condition of (1). Those skilled in the art recognize that typically single amino acid substitutions in non-essential regions of a polypeptide do not significantly alter biological activity (see, e.g., watson et al Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings pub. Co., p. 224).
Such substitutions may be made according to the example substitutions listed in table 5 below:
table 5: exemplary conservative amino acid substitutions
Original residue Conservative substitutions
Ala(A) Gly；Ser
Arg(R) Lys
Asn(N) Gln；His
Cys(C) Ser
Gln(Q) Asn
Glu(E) Asp
Gly(G) Ala；Pro
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；Gln；Glu
Met(M) Leu；Tyr；Ile
Phe(F) Met；Leu；Tyr
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
Other substitutions are also permissible and may be determined empirically or based on other known conservative or non-conservative substitutions.
As used herein, "naturally occurring amino acids" refers to the 20L-amino acids present in a polypeptide.
As used herein, the term "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid, but has been structurally modified to mimic the structure and reactivity of the natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids, and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary unnatural amino acids are known to those of skill in the art and include, but are not limited to, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β -alanine/β -aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid/pipecolic acid (4 Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2, 4-diaminobutyric acid (Dbu), desmin (Des), 2' -diaminopimelic acid (Dpm), 2, 3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (Etasn), hydroxylysine (Hyl), perhydroxy lysine (Ahyl), 3-hydroxyproline (3 Hyp), 4-hydroxyproline (4 Hyp), isodesmin (Aib), alloisoleucine (Me), N-methyl, meGly), N-methyl isoleucine (MeN-methyl-valine (Mevalin), norvaline (Meva), and norvaline (Meva).
As used herein, a "DNA construct" is a single-or double-stranded, linear or circular DNA molecule containing DNA segments that are combined and juxtaposed in a manner not found in nature. DNA constructs are the result of human manipulation, including cloning and other copies of the manipulated molecule.
As used herein, a "DNA segment" is a portion of a larger DNA molecule having particular properties. For example, a DNA segment encoding a particular polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that when read in the 5 'to 3' direction encodes the amino acid sequence of the particular polypeptide.
As used herein, the term "polynucleotide" refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in nucleotides (abbreviated as "nt") or base pairs (abbreviated as "bp"). The term nucleotide is used for single-and double-stranded molecules, where the context permits. When the term is applied to a double stranded molecule, it is used to denote the total length and is understood to be equivalent to the term base pair. One skilled in the art will recognize that the two strands of a double-stranded polynucleotide may be slightly different in length and that their ends may be staggered; thus, all nucleotides in a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends will generally not exceed 20 nucleotides in length.
As used herein, recombinant production by using recombinant DNA methods refers to the use of well-known molecular biological methods to express proteins encoded by cloned DNA.
As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, coomassie blue staining after gel electrophoresis, lowry protein assay, and Bradford protein assay.
As used herein, a "host cell" is a cell that is used to receive, maintain, regenerate and/or amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid in the vector is replicated, thereby amplifying the nucleic acid.
As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Reference to vectors includes those into which a nucleic acid encoding a polypeptide or fragment thereof may be introduced, typically by restriction digestion and ligation. Vectors mentioned also include those containing nucleic acids encoding polypeptides such as modified anti-TNFR 1 antibodies. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but may be designed to effect integration of a gene or portion thereof into the chromosome of the genome. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors is well known to those skilled in the art. Vectors also include "viral vectors" or "viral vectors". Viral vectors are viruses engineered to be operably linked to a foreign gene to transfer (as a carrier or shuttle) the foreign gene into a cell.
As used herein, an "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences (e.g., promoter regions) that enable expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which upon introduction into a suitable host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells, as well as those that remain episomal or that integrate into the host cell genome.
As used herein, "primary sequence" refers to the amino acid residue sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule.
As used herein, "sequence identity" refers to the number of identical or similar amino acid or nucleotide bases when compared between test and reference polypeptides or polynucleotides. Sequence identity may be used to identify regions of similarity or identity by sequence alignment of nucleic acid or protein sequences. For purposes herein, sequence identity is typically determined by alignment to identify identical residues. The alignment may be a local alignment or a global alignment. Matches, mismatches, and gaps can be identified between the compared sequences. Gaps are void amino acids or nucleotides inserted between residues of aligned sequences to align the same or similar characters. In general, internal and terminal vacancies may exist. When a gap penalty is used, sequence identity can be determined without penalty for the end gap (e.g., without penalty for the end gap). Alternatively, sequence identity may be determined without consideration of gaps, calculated as the number of identical positions/total aligned sequence length x 100.
As used herein, "global alignment" is an alignment method that aligns two sequences from beginning to end, with each letter in each sequence aligned only once. An alignment is generated whether or not there is similarity or identity between sequences. For example, 50% sequence identity based on "global alignment" refers to that in a full sequence alignment of two compared sequences, each 100 nucleotides in length, 50% of the residues are identical. It will be appreciated that even when the lengths of the aligned sequences are different, global alignment may be used to determine sequence identity. Differences in sequence ends are considered in determining sequence identity unless a "no end gap penalty" is selected. Typically, global alignment is used for sequences that have significant similarity over most of their length. Exemplary algorithms for performing global alignment include Needleman-Wunsch algorithm (Needleman et al (1970) j.mol. Biol. 48:443). Example programs for performing global alignment are publicly available, including global sequence alignment tools (Global Sequence Alignment Tool) available on the National Center for Biotechnology Information (NCBI) website (ncbi.nl.gov /), and programs available on deepc2.psi.
As used herein, "local alignment" is an alignment method that aligns two sequences, but aligns only those portions of the sequences that have similarity or identity. Thus, local alignment determines whether a sub-segment of one sequence is present in another sequence. If there is no similarity, no alignment is returned. Local alignment algorithms include BLAST or Smith-Waterman algorithm (adv.appl. Math.2:482 (1981)). For example, 50% sequence identity based on "local alignment" means that in a complete sequence alignment of two compared sequences of arbitrary length, 50% of the residues in a region of similarity or identity of 100 nucleotides in length are identical in the region of similarity or identity.
For purposes herein, sequence identity may be determined by standard alignment algorithm procedure using default gap penalties established for each vendor. Default parameters of the GAP program may include: (1) A weighted comparison matrix of the unitary comparison matrix (having an identity value of 1 and a non-identity value of 0) and Grisskov et al Nucl. Acids Res. 14:6755 (1986), as described in Schwartz and Dayhoff, eds., atlas of Protein Sequence and Structure, national Biomedical Research Foundation, pp.353-358 (1979); (2) A penalty of 3.0 per gap, 0.10 per symbol per gap; and (3) no penalty for terminal gaps. Whether any two nucleic acid molecules have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" nucleotide sequences, or whether any two polypeptides have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical" amino acid sequences, or other similar variations stating percent identity, can be determined using known computer algorithms based on local or global alignments (see, e.g., wikipedia. Org/wiki/sequence_alignment_software, providing links to tens of known and publicly available alignments databases and programs). Generally, for purposes herein, sequence identity is determined using a computer algorithm based on global alignment, such as Needleman-Wunsch global sequence alignment tools available from NCBI/BLAST (blast.ncbi.nm.nih.gov/blast.cgicmd=web & page_type=blasthome); LAlign (William Pearson (adv. Appl. Math. (1991) 12:337-357) performing the Huang and Miller algorithms); and the procedure of the Xiaoqui Huang, available at deepc2.Psi. Typically, the full length sequences of each of the compared polypeptides or nucleotides are aligned across the full length of each sequence in a global alignment. Local alignment may also be used when the length of sequences being compared is substantially the same.
As used herein, the term "identity" refers to a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, "at least 90% identical" refers to a percentage of identity from 90% to 100% relative to a reference polypeptide or polynucleotide. The 90% or higher level of identity indicates the following facts: it is assumed for purposes of illustration that when a test and reference polypeptide or polynucleotide of 100 amino acids or nucleotides in length are compared, no more than 10% (i.e., 10/100) of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from the amino acids or nucleotides in the reference polypeptide or polynucleotide. A similar comparison can be made between the test and reference polynucleotides. Such differences may appear as point mutations randomly distributed throughout the length of the amino acid sequence, or they may cluster at one or more positions of different lengths up to a maximum allowable value, e.g. 10/100 amino acid differences (about 90% identity). Differences may also be due to deletions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. Depending on the length of the comparison sequence, the results may be independent of program and gap parameter sets at homology or identity levels above about 85-90%; such high-level identities can be easily assessed, typically without reliance on software.
As used herein, a "disulfide bond" (also referred to as an S-S bond or disulfide bridge) is a single covalent bond derived from thiol group coupling. Disulfide bonds in proteins form between thiol groups of cysteine residues and stabilize interactions between polypeptide domains (e.g., antibody domains).
As used herein, "coupled" or "conjugated" refers to attachment by covalent or non-covalent interactions.
As used herein, the phrase "conjugated to an antibody" or "linked to an antibody" or grammatical variations thereof, when referring to the linking of a moiety to an antibody or antigen-binding fragment thereof, such as a diagnostic or therapeutic moiety, refers to the linking of the moiety to the antibody or antigen-binding fragment thereof by any known means for linking peptides, such as by recombinant means or by chemical means, post-translational production of a fusion protein. Conjugation can be accomplished using any of a variety of linkers, including but not limited to peptide or compound linkers, or chemical cross-linking agents.
As used herein, "antibody-dependent cell-mediated cytotoxicity," "antibody-dependent cellular cytotoxicity," and "ADCC" are used interchangeably to refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell, which subsequently results in lysis of the target cell. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in page 464 table 3 of Ravetch et al (1991) Annu.Rev.Immunol, 9:457-492. In order to assess ADCC activity of a target molecule, an in vitro ADCC assay may be performed (see, e.g., U.S. Pat. nos. 5,500,362 and 5,821,337). Exemplary effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule may be assessed in vivo, for example in an animal model, for example as disclosed in Clynes et al (1998) Proc.Natl. Acad.Sci.USA 95:652-656.
As used herein, complement Dependent Cytotoxicity (CDC) is the effector function of IgG and IgM antibodies. When such antibodies bind to surface antigens on target cells (e.g., bacterial cells or virus-infected cells), the classical complement pathway is triggered by binding of the protein C1q to these antibodies, resulting in the formation of a Membrane Attack Complex (MAC) and subsequent cell lysis.
As used herein, antibody-dependent cellular phagocytosis (ADCP) is the cellular process by which effector cells (e.g., monocytes and macrophages) with phagocytic potential internalize target cells. Once phagocytized, the target cells reside in phagosomes, which fuse with lysosomes to degrade the target cells via an oxygen-dependent or non-dependent mechanism.
As used herein, "therapeutic activity" refers to the in vivo activity of a therapeutic polypeptide. In general, a therapeutic activity is an activity associated with the treatment of a disease or disorder. The therapeutic activity of the modified polypeptide may be any percentage level of the therapeutic activity of the unmodified polypeptide, including but not limited to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more of its activity compared to the therapeutic activity of the unmodified polypeptide.
As used herein, the term "assessing" is intended to include obtaining quantitative and qualitative determinations in the sense of obtaining absolute values of the activity of a protein, such as an antibody or antigen binding fragment thereof, present in a sample, as well as obtaining an index, ratio, percentage, visual value, or other value indicative of the level of activity. The evaluation may be a direct or an indirect evaluation.
As used herein, "disease or disorder" refers to a pathological condition in an organism caused by a cause or disorder including, but not limited to, infection, acquired disorder, and genetic disorder, and is characterized by identifiable symptoms.
As used herein, "treating" a subject having a disease or disorder means that the symptoms of the subject are partially or completely reduced or remain static after treatment. Thus, treatment encompasses prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or disease progression. Treatment also encompasses any pharmaceutical use of any antibody or antigen-binding fragment or composition thereof provided herein.
As used herein, treatment refers to an improvement in symptoms or manifestations of a disease, disorder, or condition.
As used herein, "preventing" or "prophylaxis" refers to a method of reducing the risk of developing a disease or disorder. Preventing the disease means reducing the risk of illness.
As used herein, "pharmaceutically effective agent" includes any therapeutic or bioactive agent including, but not limited to, for example, anesthetics, vasoconstrictors, dispersants, and conventional therapeutic agents, including small molecule drugs and therapeutic proteins.
As used herein, "therapeutic effect" refers to the effect of a treatment performed on a subject to alter, generally improve or reduce the symptoms of, or cure a disease or disorder.
As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or condition.
As used herein, "therapeutic efficacy" refers to the ability of an agent, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to whom the agent, compound, material, or composition containing a compound has been administered.
As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of an agent, compound, material, or composition containing a compound that, when administered to a subject, has a desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom, or reducing the incidence of a viral infection. The complete prophylactic effect does not necessarily occur by administration of one dose, and only occurs after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.
As used herein, the amelioration of symptoms of a particular disease or disorder by treatment, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to any alleviation of symptoms attributable to or associated with the administration of the composition or therapeutic agent, whether permanent or temporary, continuous or transient.
As used herein, a "prodrug" is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being enzymatically activated or converted to a more active parent form (see, e.g., wilman,1986,Biochemical Society Transactions,615th Meeting Belfast,14:375-382; and stilla et al, "pro drugs: AChemical Approach to Targeted Drug Delivery," Directed Drug Delivery, borchardt et al, (ed.), pp.247-267,Humana Press,1985).
As used herein, "anti-cancer agent" refers to any agent that is destructive or toxic to malignant cells and tissues. For example, anticancer agents include agents that kill cancer cells or otherwise inhibit or impair the growth of tumors or cancer cells. Exemplary anticancer agents are chemotherapeutic agents.
As used herein, an "anti-angiogenic agent" or "angiogenesis inhibitor" is a compound that blocks or interferes with vascular development.
As used herein, a TNF-related or TNF-mediated disease refers to a disease, condition, or disorder in which TNFR1 or TNFR1 signaling plays a role in etiology; including diseases, conditions and disorders in which inhibition of TNFR1 signaling can ameliorate a symptom of the disease, condition or disorder.
As used herein, a "TNFR2 agonist" or an "anti-TNFR 2 agonist" refers to a compound, including small molecules and TNFR2 antibodies or antigen-binding fragments thereof, as well as other polypeptides that initiate, promote or increase TNFR2 activation and/or enhance one or more signal transduction pathways mediated by TNFR 2. For example, TNFR2 agonists can promote or increase proliferation of Treg cell populations. TNFR2 agonists can promote or increase TNFR2 activation by binding to TNFR2, for example, to induce conformational changes that render the receptor biologically active. For example, a TNFR2 agonist can nucleate trimerization of TNFR2 in a manner similar to or mimicking the interaction between TNFR2 and its cognate ligand TNF (tnfα), thereby inducing TNFR 2-mediated signaling. TNFR2 agonists may also induce CD4 + 、CD25 + 、FOXP3 + Proliferation of Treg cells. TNFR2 agonists may also inhibit cytotoxic T lymphocytes (e.g., CD8 + T cells), for example by activating immunoregulatory Treg cells or by binding TNFR2 directly to the surface of autoreactive cytotoxic T cells and inducing apoptosis. The TNFR2 agonist antibody or fragment thereof used in the methods herein can specifically bind TNFR2 and is generally of sufficient specificity so that it does not specifically bind to another receptor of a Tumor Necrosis Factor Receptor (TNFR) superfamily member, such as TNFR1.
As used herein, a TNFR 2-selective agonist is a TNFR2 agonist that does not or substantially does not result in TNFR1 signaling activity.
As used herein, a Treg expansion is a molecule, including small molecules and polypeptides, that increases regulatory T cells (Treg cells or tregs), which are immunosuppressive subpopulations of T cells that have immunosuppressive properties by producing cytokines.
As used herein, the terms "pan-growth factor trap construct," "pan-EGFR ligand trap construct," "growth factor trap," "multispecific growth factor trap construct," "bispecific growth factor trap construct," "EGFR ligand trap construct," "pan HER therapeutic," "EGFR ligand trap construct," "HER ligand trap construct," and "growth factor trap construct" are used interchangeably to refer to pan-cell surface receptor molecules, including peptide-based compounds, that modulate the activity of two or more human Epidermal Growth Factor Receptors (EGFR), also known as HER or ErbB receptors. Typically, a pan-growth factor trap targets at least two different HER receptors, for example, by ligand binding and/or interaction with the receptor.
As used herein, an "extracellular domain" or "ECD" is a cell surface receptor moiety that appears on the surface of a receptor, including a ligand binding site. For purposes herein, reference to an "ECD polypeptide" includes any ECD-containing molecule or portion thereof, so long as the ECD polypeptide does not contain any contiguous sequence associated with another domain of a cognate receptor (e.g., a transmembrane domain, a protein kinase domain, or other domain).
As used herein, "bulge recesses" (KIH) or "bulges in recesses" refer to multimerization domains, such as immunoglobulin Fc domains, that are engineered such that steric interactions between and/or among these domains promote stable interactions and promote the formation of heterodimers (or heteromultimers) as compared to homodimers (or homomultimers) from a monomer mixture. This can be achieved, for example, by constructing projections or protrusions and depressions or cavities in the complementary multimerization domains. "bulge" can be constructed by replacing small amino acid side chains at the interface of a first multimerization domain polypeptide (e.g., a first Fc monomer) with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "recess" of the same or similar size as the protrusion is optionally created at the interface of a second complementary multimerizing polypeptide (e.g., a second Fc monomer).
As used herein, "tethered" refers to an interaction between two domains of an acceptor monomer whereby the monomer appears in a conformation that makes it less available for interaction. For example, subdomain II of HER1, HER3, and HER4 can interact with subdomain IV to form a tethered, inactive structure. When in a tethered state, the receptor or isomer thereof is less or unavailable for dimerization and/or ligand binding. ECD in monomeric form of HER1, HER3 and HER4 occurs in tethered form with ligand affinity lower than in unbound form. HER2 lacks certain residues in subdomain IV, appears in unbound form, and is available for dimerization with HER1, HER3 and HER 4. After binding of the ligand to the tethered (monomeric) form, the tethered interaction is released and the ECD (or receptor) is in a conformation available for dimerization, which involves an interaction between domain II of the two ECDs.
As used herein, a HER (ErbB) -related disease, HER-related disease, or HER-mediated disease is any disease, condition, or disorder in which an epidermal growth factor receptor (HER) and/or ligand is involved in certain aspects of its etiology, pathological progression, or symptoms thereof. Participation includes, for example, expression, overexpression or activity of HER family members or ligands. Diseases include, but are not limited to, proliferative diseases including cancers, such as, but not limited to, glioma and pancreatic cancer, gastric cancer, head and neck cancer, cervical cancer, lung cancer, colorectal cancer, endometrial cancer, prostate cancer, esophageal cancer, ovarian cancer, uterine cancer, bladder cancer, or breast cancer. Other conditions include those involving cell proliferation and/or migration, including those involving pathological inflammation and/or autoimmune responses, such as Rheumatoid Arthritis (RA), non-malignant hyperproliferative diseases, ocular conditions, skin conditions (e.g., psoriasis), conditions resulting from smooth muscle cell proliferation and/or migration, such as stenosis, including restenosis, atherosclerosis, thickening of the muscles of the bladder, heart, or other muscles, or endometriosis.
As used herein, the term "subject" refers to animals, including mammals, e.g., humans.
As used herein, "patient" refers to a human subject.
As used herein, "animal" includes any animal, such as, but not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; birds, such as chickens; ruminants, such as goats, cattle, deer and sheep; pig; and other animals. Non-human animals are excluded from humans as the intended animal. The polypeptides provided herein are from any source, such as from animals, plants, prokaryotes, and fungi. Most polypeptides are of animal origin, including mammalian origin, and are typically human or humanized for therapeutic use.
As used herein, "composition" refers to any mixture. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.
As used herein, "stabilizer" refers to a compound that is added to a formulation to protect an antibody or conjugate under conditions (e.g., temperature) in which the formulation is stored or used herein. Thus, formulations are included that prevent the protein from degrading other components in the composition. Examples of such formulations are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors or substrates, and other formulations described herein.
As used herein, "combination" refers to any association between or among two or more items. The combination may be two or more separate items, such as two compositions or two collections, mixtures thereof, such as a single mixture of two or more items, or any variant thereof. The elements of a combination are typically functionally related or related, such as for example, elements used in a method.
As used herein, "combination therapy" refers to the administration of two or more different therapeutic agents, such as an anti-TNFR construct provided herein or, for example, an antibody or antigen-binding fragment thereof, and one or more therapeutic agents or other therapies, such as radiation and surgery. The multiple therapeutic agents may be provided and administered separately, sequentially, intermittently, simultaneously, or in a single composition.
As used herein, a "kit" is a packaged combination that optionally includes other elements, such as additional reagents and instructions for using the combination or elements thereof, for purposes including, but not limited to, activating, administering, diagnosing, and assessing biological activity or properties.
As used herein, "unit dosage form" refers to physically discrete units suitable as unitary packages for human and animal subjects, as is known in the art.
As used herein, "single dose formulation" refers to a formulation that is directly administered.
As used herein, a "multi-dose formulation" refers to a formulation that contains multiple doses of a therapeutic agent and can be directly administered to provide several single doses of the therapeutic agent. The dose may be administered over a period of minutes, hours, weeks, days or months. The multi-dose formulation may allow for dose adjustment, dose combining, and/or dose splitting. Because multi-dose formulations are used over time, they typically contain one or more preservatives to prevent microbial growth.
As used herein, an "article of manufacture" is a product that is manufactured and sold. As used throughout the present application, the term is intended to encompass any composition provided herein contained in or for packaging articles.
As used herein, "fluid" refers to any composition that can flow. Thus, fluids encompass compositions in the form of semisolids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.
As used herein, an isolated or purified polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof) or biologically active portion thereof (e.g., an isolated antigen-binding fragment) is substantially free of cellular material or other contaminating proteins from cells or tissues from which the protein is derived, or substantially free of chemical precursors or other chemicals upon chemical synthesis. If the skilled person determines that the preparation does not appear to contain readily detectable impurities using standard analytical methods for assessing such purity, such as Thin Layer Chromatography (TLC), gel electrophoresis and High Performance Liquid Chromatography (HPLC), it can be determined that the preparation is substantially free of such impurities, or sufficiently pure that further purification does not detectably alter the physical and chemical properties of the substance, such as enzymatic and biological activity. Methods for purifying compounds to produce chemically substantially pure compounds are known to those skilled in the art. However, the chemically substantially pure compound may be a mixture of stereoisomers. In this case, further purification may increase the specific activity of the compound.
As used herein, "cell extract" or "lysate" refers to a preparation or fraction made from lysed or disrupted cells.
As used herein, "control" refers to a sample that is substantially identical to the test sample except that it has not been treated with the test parameters, or, in the case of a plasma sample, it may be from a normal volunteer that is not affected by the intended conditions. The control may also be an internal control.
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide containing an "immunoglobulin domain" includes a polypeptide having one or more immunoglobulin domains.
As used herein, the term "or" is used to mean "and/or" unless explicitly indicated to refer to items only or to the items being mutually exclusive.
As used herein, ranges and amounts can be expressed as "about" a particular value or range. "about" also includes precise amounts. Thus, "about 5 amino acids" means "about 5 amino acids" is also "5 amino acids". About the range within experimental error or acceptable to one skilled in the art for a particular parameter.
As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, optionally a variant moiety means that the moiety is a variant or a non-variant.
As used herein, unless otherwise indicated, any protecting group, amino acid, and other compound abbreviations are named according to their common usage, accepted abbreviations, or IUPAC-IUB biochemical nomenclature committee (see biochem. (1972) 11 (9): 1726-1732).
For clarity of illustration, and not to limit the disclosure, the detailed description is divided into the following subsections.
B. Construction and method overview
Autoimmune diseases occur when the body's immune system attacks itself. The resulting inflammation and tissue destruction is triggered by an inflammatory hormone known as Tumor Necrosis Factor (TNF). Autoimmune diseases have more than 100; in general, about 75% of patients with autoimmune disease are females. Drugs previously used for autoimmune diseases have adverse side effects including infections, heart problems, and other diseases and conditions.
TNF interacts with immune cells through two receptors, TNFR1 is overactive in autoimmune diseases, TNFR2 inhibits autoimmune diseases, but is inhibited when TNFR1 is overactive. TNF blockers, e.g. infliximab (in order to Sales), adalimumab (in +.>Sales) and etanercept (in +.>Marketing) blocks TNFR1 and TNFR2, resulting in adverse side effects. The constructs provided herein solve this problem. The constructs provided herein shut down only TNFR1, which results in increased TNFR2 activity, thereby not only treating autoimmune disease symptoms, but also providing improved treatment with reduced or no adverse side effects, as TNFR2 activity is not blocked. Provided herein are various constructs that address the problems of prior art TNF blockersA body. The following table summarizes the types of constructs identified based on their activity and specified and provided herein:
constructs are provided for treating TNF-mediated diseases, disorders, and conditions, or diseases, disorders, and conditions in which TNF plays a role in etiology or interferes with TNFR1 signaling. For example, TNFR1 antagonists are useful in the treatment of a variety of conditions, including autoimmune conditions, as well as, for example, endometriosis, brain fog from, for example, chemotherapy and COVID, alzheimer's disease, acute inflammation from, for example, influenza virus and SARS-COV2 infection, which causes long-term or permanent damage to the lung, kidneys and other tissues. Existing TNF blockers cannot be used for most of these indications due to their side effects and the attendant safety issues. The TNFR1 antagonist constructs provided herein can be used. These constructs described herein are monovalent in that they inhibit only TNFR1 without causing receptor clustering, are specific, non-immunogenic, and have a half-life of at least about 3-4 weeks, allowing for administration about once a month.
Thus, TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific constructs, such as bispecific constructs comprising TNFR1 antagonists and TNFR2 agonist activity, are provided. The construct includes at least one moiety that specifically interacts with TNFR1 or TNFR2 and another moiety that generally modulates the interaction directly or indirectly or provides the construct with pharmacological (pharmacodynamic or pharmacokinetic or both) properties. Thus, the constructs provided herein comprise at least two portions: a binding moiety that interacts with TNFR1 or TNFR2, and a second moiety that modulates or alters the pharmacological properties or activity of the construct or binding moiety.
Among the constructs provided herein are those that are antagonists of TNFR1 activity. The TNFR1 antagonist construct comprises a moiety that binds to or interacts with TNFR1 and inhibits TNFR 1-mediated signaling, and a second moiety that imparts additional properties such as extended serum half-life, abrogates ADCC and/or CDC activity, and modulates interactions with a particular receptor. TNFR1 antagonists and constructs also include one or more modifications such that they are not immunogenic or have reduced immunogenicity, particularly in humans, and may also include modifications that eliminate or reduce binding to pre-existing antibodies.
The TNFR1 antagonist construct is selected to specifically bind TNFR1 with minimal or no binding to TNFR2 or no TNFR2 antagonist activity. Thus, the construct only regulates TNFR1. In some embodiments, the TNFR1 antagonist construct is selected to also have or be linked to a second domain or portion having TNFR2 agonist activity. The TNFR1 constructs, including those designed or selected to interact with TNFR1 affinities, e.g., K d <50nM or<10nM or<5nM, especially with higher affinity (e.g. K d <1nM or<0.1nM or higher affinity) and/or is effective in inhibiting TNFR1 signaling (e.g., IC) 50 50nM or<10nM or<5nM or<3nM or 1nM or<0.5nM)。
Also provided are multispecific, e.g., bispecific constructs containing a TNFR1 antagonist moiety linked directly or through a linker to a TNFR2 agonist moiety. The linker provides advantageous properties to the molecule, such as increased serum half-life, increased stability, proper three-dimensional structure and flexibility, and improved pharmacological properties. These constructs solve problems associated with administration of other therapies, such as anti-TNF therapies ("TNF blockers", e.g., including Etanercept, adalimumab @) ) Infliximab), because these constructs increase the specificity of TNFR1 inflammatory blockade and result in protection or amplification of TNFR2 function, a natural immunosuppressant, at least in part by upregulating immunosuppressive tregs, and inducing protective and anti-inflammatory signaling pathways. In addition, TNF blockade results in inhibition of TNFR2 function, also reducing T cell-induced monocyte activation, and thus increasingPossibility of opportunistic infections (see, e.g., rossel et al (2007) J.Immunol.179:4239-48).
There are many differences between the activity of the exemplary TNFR1 antagonist constructs provided herein and existing approved TNF blockers: TNF blockers, such as etanercept, adalimumab, infliximab, which are not specific for TNFR 1. Other blockers, such as IL6, IL17, IL23, block only a portion, but not all, of their own cytokine cascade. Existing TNF blockers have the same mechanism of action on TNFR1 and TNFR2, blocking both activities. JAK inhibitors have similar problems; they have inflammatory and anti-inflammatory activity. For example, a JAK inhibitor does not block the inflammatory cytokine Il1, a JAK inhibitor blocks the inflammatory cytokine Il6 (a second line drug for rheumatoid arthritis treatment), and anti-inflammatory Il10 is not blocked by a JAK inhibitor. In contrast, the constructs provided herein combine the effectiveness of TNFR1 and TNF inhibitor therapies with the benefits of TNFR2 agonists, eliminate or reduce the side effects of anti-TNFR 1/anti-TNF therapies, and also provide additional therapeutic modality advantages, including up-regulation of immunosuppressive tregs and induction of protective and anti-inflammatory signaling pathways.
The TNFR1 antagonist construct comprises one or more TNFR1 inhibitors, one or more linkers, and one or more activity modulators. For example, the structure of the TNFR1 antagonist construct provided herein can be represented by formula 1:
(TNFR 1 inhibitor) n -a joint p - (activity modulating agent) q Of formula 1a, or
(Activity-controlling Agents) q -a joint p - (TNFR 1 inhibitors) n Formula 1b, wherein:
n and q are integers and are each independently 1, 2 or 3; p is 0, 1, 2 or 3; an activity modulator is a moiety, e.g., a polypeptide, such as albumin or Fc, modified to have reduced or no ADCC activity, which increases the serum half-life of the TNFRl inhibitor; an inhibitor of TNFR1 is a molecule, such as a polypeptide or a small pharmaceutical molecule, that binds to TNFR1 and inhibits its activity, such as signaling activity. The activity modulator is not a human serum albumin antibody or an unmodified single Fc. Modulators of activity include modified Fc regions, such as Fc, fc dimers, and other antibody domains modified to eliminate ADCC and/or CDC activity. Linkers include chemical linkers and polypeptides, such as GS linkers, as well as hinge regions, such as from antibodies, so the constructs include chemical conjugates, fusion proteins, and combinations of both.
Multispecific constructs are also provided. The construct of the multispecific, e.g., bispecific construct provided herein is represented by the following formula (formula 2):
(TNFR 1 inhibitor) n - (activity modulating agent) r1 - (joint (L) p - (activity modulating agent) r2 - (TNFR 2 agonists) q ，
Where n=1, 2 or 3, p=1, 2 or 3, q=0, 1 or 2, and each of r1 and r2 is independently 0, 1 or 2. As with the construct of formula 1, the order of the components may be changed and additional linkers may be provided as desired. The construct may include additional linkers as needed to impart properties such as flexibility. Each joint may contain multiple components. Formula 2 may also include an activity modulator instead of or in addition to the linker. The activity modulators and linkers include Fc or Fc with hinge regions, or Fc with GS linkers, or a combination of other components. The Fc in these constructs includes an unmodified Fc region; the joints are as described above and in detail below.
Also provided are TNFR2 agonist constructs having formula 3:
(TNFR 2 agonist) n -a joint p - (activity modulating agent) q Of formula 3a, or
(Activity-controlling Agents) q -a joint p - (TNFR 2 agonists) n The composition of 3b,
wherein n, p and q are as shown in formula 1, and the linker and the activity modulator are as described in formula 1.
The components of formulas 1-3 may be polypeptides or other molecules, such as small molecule drugs that specifically bind or interact with a target receptor, as will be discussed in detail in the following sections. Each component of the constructs/molecules provided herein is described in turn in the following sections.
The nature of each component of the constructs provided herein is discussed in detail in the following sections. Thus, components of the construct include, but are not limited to, the following components, which will be discussed in detail in the following sections:
TNFR1 antagonists
Tnfr2 agonists
3. Joint
a. Glycine-serine linker
b. Hinge region
c. Chemical joint
4. Activity modulators
a. Modified Fc
b. Polypeptides and other moieties that confer improved or altered pharmacological properties such as increased serum half-life, resistance to degradation by endogenous proteases, and other such properties.
Other constructs are also provided, see subsequent sections for details.
The constructs are useful in methods of treating diseases, disorders, and conditions, wherein TNF is a pathological modulator of the disease, disorder, or condition, thereby reducing or inhibiting inhibition of TNFR1 signaling, and/or wherein inhibiting TNF or TNFR1 signaling can inhibit or cause regression of the disease, disorder, or condition, and/or wherein inhibition ameliorates symptoms of the disease, disorder, and/or condition. Such diseases, disorders, and conditions, including inflammatory diseases, including autoimmune diseases, are discussed in the following sections.
Pharmaceutical compositions for use in the methods and uses are also provided, as are nucleic acids and vectors for producing constructs, including polypeptides and fusion proteins. The following sections describe diseases, disorders and conditions, TNFR1/TNFR2 activity and its role in the diseases, disorders and conditions, and existing methods of treatment of the diseases, disorders and conditions, constructs and components thereof provided herein, methods of producing the constructs, pharmaceutical compositions comprising the constructs and/or encoding nucleic acids, and methods of treatment.
C. Tumor Necrosis Factor (TNF) and chronic inflammatory and autoimmune diseases and disorders
This section describes the role of Tumor Necrosis Factor (TNF) and/or its receptors in inflammatory and autoimmune diseases, particularly in the exemplified diseases, and the problems of existing therapies, and demonstrates how the constructs provided herein address these problems.
1. Tumor Necrosis Factor (TNF)
Tumor necrosis factor (TNF; see, e.g., SEQ ID NO:1; also known as TNF alpha, TNF-alpha or TNF alpha) is a pleiotropic pro-inflammatory cytokine that is associated with inflammatory and immunomodulatory activities, including modulation of tumorigenesis/cancer, host defense against pathogen infection, apoptosis, autoimmunity and septic shock, and plays an important role in the coordination of innate and adaptive immune responses and the generation of organogenesis, particularly lymphoid organogenesis. In humans, TNF is produced primarily by macrophages, but also by monocytes, dendritic Cells (DCs), B cells, T cells, fibroblasts, and other cell types. It is a homotrimeric membrane-bound protein containing 233 amino acids (26 kDa) that is cleavable by the protease TACE (TNFa converting enzyme; also known as ADA 17) to release soluble TNF containing 157 amino acids (17 kDa); membrane-bound and soluble forms of TNF have biological activity. Human TNF comprises 233 amino acids and comprises cytoplasmic domains, corresponding residues 1-35, and transmembrane domains, corresponding residues 36-56, and extracellular domains, corresponding residues 57-233, see SEQ ID NO:1. The soluble form of TNF corresponds to amino acid residues 77-233 as shown in SEQ ID NO:1 (see the amino acid residue sequence of soluble TNF shown in SEQ ID NO: 2).
Uncontrolled TNF production is associated with a variety of inflammatory and autoimmune diseases and disorders including, for example, septic shock, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and Inflammatory Bowel Disease (IBD). Overexpression of TNF is also associated with neurodegenerative diseases and disorders such as alzheimer's disease, parkinson's disease, stroke and multiple sclerosis. In addition, TNF promotes osteoclastogenesis, while excessive TNF production is associated with bone loss. In Rheumatoid Arthritis (RA), TNF is overexpressed in synovial fluid and synovium, while TNF receptor (TNFR) expression is upregulated in synovium. For example, overexpression of human TNF in mice can lead to spontaneous RA-like lesions in joints, forming proliferative synovial membranes and disrupting cartilage and bone (see, e.g., blu ml et al (2010) Arthritis & Rheumatism 62 (6): 1608-1619;Keffer et al (1991) EMBO J.10 (13): 4025-4031;Esperito Santo et al. (2015) biochem. Biophys. Res. Commun.464:1145-1150; blu ml et al (2012) International Immunology (5): 275-281; dong et al (2016) Proc. Natl. Acad. Sci. USA 113 (43): 12304-12309).
TNF signals through two high affinity specific receptors TNFR1 and TNFR2, as discussed further below; TNFR1 is associated with deleterious inflammatory processes, while TNFR2 is associated with beneficial immunomodulatory processes. Membrane-bound TNF has been shown to activate predominantly TNFR2, whereas soluble TNF activates predominantly TNFR1 (bliml et al (2010) architis & rheomatism 62 (6): 1608-1619). Soluble TNF (solTNF; residues 77-233 corresponding to SEQ ID NO: 1; see also the sequence shown in SEQ ID NO: 2)) is involved in paracrine signaling (primarily through TNFR 1)) in chronic inflammation, whereas transmembrane TNF (tmTNF) induces a near-secretory signal by cell-to-cell contact (primarily through TNFR 2) and is associated with resolution of inflammation and induction of immunity to pathogens such as Listeria monocytogenes (Listeria monocytogenes) and Mycobacterium tuberculosis (Mycobacterium tuberculosis) (Zalevsky et al (2007) J. Immunol. 179:1872-1883). Thus, TNF signaling through TNFR1 and TNFR2 results in different results, depending on the receptor type.
Because of the association between TNF overexpression and the development of inflammatory and autoimmune diseases and disorders, blocking TNF has been used to treat a variety of such diseases and disorders, including, but not limited to, rheumatoid Arthritis (RA), psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile Idiopathic Arthritis (JIA), and inflammatory bowel disease (IBD; e.g., crohn's disease, ulcerative colitis). The use of TNF blockers can block TNF and prevent signaling through TNFR1 and TNFR2, which is associated with an increased risk of severe infections (such as tuberculosis and listeriosis) caused by immunosuppression. TNF blockers block not only detrimental inflammatory signals through TNFR1, but also beneficial immunomodulatory signals through TNFR 2. Thus, the use of TNF blockers may be limited, particularly in the case of chronic diseases/conditions (e.g., arthritis or IBD) where long-term administration is required. About one third of RA patients either do not respond to treatment with anti-TNF or the therapeutic effect does not persist. Thus, there is a need for therapies with improved efficacy and safety, particularly therapies that block the inflammatory effects of TNFR1 signaling but maintain or enhance the beneficial anti-inflammatory effects of TNFR2 signaling. Such therapies are provided herein.
2. Tumor Necrosis Factor Receptor (TNFR)
TNF homotrimers bind to and signal through two specific high affinity homotrimer receptors, TNFR1 (TNF receptor type 1; also known as TNFRI, p55, p60, CD120a, TNF receptor superfamily member 1A and TNFRSF 1A) and TNFR2 (TNF type 2 receptor; also known as TNFRI, p75, p80, CD120B, TNF receptor superfamily member 1B and TNFRSF 1B). TNFR1 is expressed by all nucleated cell types; TNFR2 expression is limited to immune cells (e.g., monocytes, macrophages, activated T cells, regulatory T cells (tregs), B cells, and Natural Killer (NK) cells), endothelial cells, specific Central Nervous System (CNS) cells, and specific cardiomyocytes. TNFR2 expression on tregs is induced upon T cell receptor activation.
In vivo, TNFR1 and TNFR2 exist as membrane-bound receptors and as soluble "decoy" (i.e., non-signaling) receptors after shedding from the cell surface. Soluble TNF preferentially/selectively binds TNFR1; however, binding of the membrane-bound and soluble forms of TNF activates TNFR1. The primary ligand of TNFR2 is membrane-bound TNF. Soluble TNF does not fully activate TNFR2, but soluble forms of TNFR2 (after TNFR2 shedding) have a high binding affinity for TNF, allowing it to clear and inhibit TNF from binding to membrane-bound signaling receptors, thereby contributing to the anti-inflammatory effects of TNFR 2. Membrane-bound TNFR2 binds TNF with rapid binding-dissociation kinetics such that TNFR2 concentrates TNF on the cell surface and delivers ligands to TNFR1, mediating TNFR1 signaling. TNFR1 and TNFR2 each contain extracellular, transmembrane and cytoplasmic domains. The extracellular domains of TNFR1 and TNFR2 contain four cysteine-rich domains (CRDs) required for ligand binding. In response to TNF ligand binding, the intracellular domains of TNFR1 and TNFR2 initiate different signaling cascades and mediate different effector functions.
Abnormal TNFR signaling is associated with a variety of autoimmune diseases and administration of TNF may be used as a therapeutic strategy for such diseases. For example, low doses of TNF selectively destroy autoreactive T cells in animal models of Sjogren's syndrome in blood samples of type I diabetes and scleroderma patients. Administration of TNF can lead to systemic toxicity in cancer patients, for example, with high TNF levels. As described herein, the toxicity is caused by the ubiquitous cellular expression of TNFR 1; as described herein, agonizing TNFR2 is a safer therapeutic option than administering TNF because TNFR2 is expressed by more restricted cells. Promotion of TNF signaling by TNFR2 can be achieved by administration of a TNFR1 antagonist (see, e.g., faustman et al (2013) front. Immunol. 4:478).
a.TNFR1
Human TNFR1 (see SEQ ID NO: 3) is the primary inflammatory receptor and accounts for the majority of the pro-inflammatory, cytotoxic and apoptotic effects of TNF. Human TNFR1 is a homotrimeric receptor whose binding to TNF induces a pro-inflammatory response (see, e.g., morton et al (2019) Sci Signal.12 (592): eaaw2418, description of TNFR1 signaling). TNFR1 contains 455 amino acid residues; residues 1-29 correspond to the signal peptide, residues 30-211 correspond to the extracellular domain, residues 212-232 correspond to the transmembrane domain, and residues 233-455 correspond to the cytoplasmic domain. Within the extracellular domain, TNFR1 contains cysteine-rich domains (CRDs) 1-4 corresponding to amino acid residues 43-82, 83-125, 126-166 and 167-196 of SEQ ID NO. 3, respectively. CRD 2 and 3 contact the bound TNF, CRD1, and in particular amino acid residues 30-82 of SEQ ID NO:3, forms a pro-ligand binding assembly domain (PLAD), which is a hemophilia interaction motif essential for ligand binding and receptor function. The cytoplasmic domain contains a death domain (corresponding to residues 356-441 of SEQ ID NO: 3) that binds to TNFR 1-related death domain (TRADD) and Fas-related death domain (FADD) upon binding of TNF to TNFR1, resulting in a signaling pathway that activates caspases and induces apoptosis. TNF binding to TNFR1 also initiates a proinflammatory cascade through MAPK (mitogen activated protein kinase; e.g., p38, JNK, ERK) and NF-. Kappa.B (nuclear factor kappa-light chain enhancer of activated B cells) signaling pathways. TNFR1 plays a role in lymphoid organogenesis and immune responses to pathogens and is a major receptor associated with host antiviral defense mechanisms. It has been shown that mycobacterial containment is dependent on TNF-derived signaling, and that patients receiving TNF blocker treatment may suffer from endogenous reactivation of latent tuberculosis.
TNFR1 is primarily involved in pro-inflammatory signaling and is the driving force for the development of arthritis. For example, knocking out TNFR1 in mice, and silencing TNFR1 expression by RNA interference, results in a reduction in collagen-induced arthritis (CIA), an animal model of arthritis. TNFR 1-deficient mice overexpressing TNF are protected from the occurrence of arthritis, and reintroducing TNFR1 into the mesenchymal cells results in the occurrence of TNF-dependent arthritis. Furthermore, TNFR1 enhances the production of osteoclasts, resulting in localized bone destruction, and it has been shown that hematopoietic cell deficiency TNFR1 reduces bone destruction in aggressive arthritis models. TNFR1 has also been associated with cardiac toxic effects in TNF-induced heart failure and myocardial infarction models, and has been shown to promote neurodegeneration in retinal ischemia animal models (see, e.g., schmidt et al (2013) Arthris & Rheumatism 65 (9): 2262-2273;Goodall et al (2015) PLoS ONE 10 (9): e0137065; mcCann et al (2014) Arthris & Rheumatology 66 (10): 2728-2738; ruspe et al (2014) Cellular Signaling 26:683-690;Faustman and Davis (2013) front. Immunol.4:478; blu ml et al (2012) International Immunology (5): 275-281; dong et al (2016) Proc. Natl. Acad. Sci. USA 113 (43): 12304-12309).
b.TNFR2
Human TNFR2 (see SEQ ID NO: 4) contains 461 amino acid residues; residues 1-22 correspond to the signal peptide, residues 23-257 correspond to the extracellular domain, residues 258-287 correspond to the transmembrane domain, and residues 288-461 correspond to the cytoplasmic domain. Unlike TNFR1, TNFR2 lacks the death domain but has a TNF receptor-related factor 2 (TRAF 2) binding site. TNFR2 signaling through TRAF2 promotes cell survival and proliferation through NF-. Kappa.B and activin 1 (AP 1) activation, and is associated with PI3K-PKB/Akt mediated repair and migration. TNF signaling through TNFR2 also promotes expansion and activation of regulatory T cells (tregs), which play an important role in inhibiting inflammatory and autoimmune diseases and disorders, as discussed elsewhere herein. TNFR2 signaling is involved in wound healing and repair and regeneration of the myocardial infarction model, whereas knockout of TNFR2 in the aggressive arthritis mouse model results in joint inflammation and bone destruction.
TNFR2 is primarily involved in anti-inflammatory signaling and is involved in neuroprotection, cardiac, intestinal and bone. TNFR2 has anti-inflammatory and protective effects; these effects have been demonstrated in, for example, experimental Autoimmune Encephalomyelitis (EAE), experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, demyelination and neurodegenerative diseases, and infectious diseases. For example, TNF activation TNFR2 inhibits seizures, alleviates cognitive dysfunction following brain injury, promotes survival following myocardial infarction in mice, prevents myocardial ischemia/reperfusion injury, and reduces remodeling and hypertrophy following heart failure. TNFR2 agonism is also associated with pancreatic regeneration, remyelination, neuronal subtype survival and stem cell proliferation. TNFR2 agonists selectively destroy autoreactive T cells in blood samples of patients with type I diabetes, multiple sclerosis, graves 'disease, and Sjogren's syndrome, but do not destroy healthy cells. In animal models of type I diabetes, the use of low doses of TNF to eliminate autoreactive T cells results in regeneration of pancreatic tissue. TNF signaling through TNFR2 has been shown to induce regeneration of oligodendrocyte precursors in myelin, and thus can be used to treat demyelinating diseases, such as Multiple Sclerosis (MS). TNFR2 has also been shown to promote neuroprotection in animal models of retinal ischemia.
TNFR2 also regulates osteoclastogenesis. Osteoclasts are bone cells that break down bone tissue. Modulation of osteoclast production is important for maintaining bone mass, preventing joint inflammation and erosive destruction. Mice lacking TNFR2 exhibit enhanced osteoclastic production, TNF-driven exacerbation of arthritis and localized bone destruction. Lack of TNFR2 in an animal model of erosive arthritis leads to disease progression and overexpression of TNF as compared to control miceThe R2 deficient mice may develop increased arthritis and joint destruction. Expression of TNFR2 on hematopoietic cells reduces TNF-driven arthritis, while loss of TNFR2 on hematopoietic cells increases recruitment of inflammatory cells to the synovium. In experimental colitis, CD4 + Lack of TNFR2 expression on T cells accelerates the onset of disease and increases the severity of inflammation, whereas in Experimental Autoimmune Encephalitis (EAE), symptoms of TNFR2 deficient mice are exacerbated (see, e.g., schmidt et al (2013) architis)&Rheumatism 65(9):2262-2273；Goodall et al.(2015)PLoS ONE 10(9):e0137065；McCann et al.(2014)Arthritis&Rheumatology 66 (10): 2728-2738; ruspi et al (2014) Cellular Signaling 26:683-690; faustman and Davis (2013) front. Immunol.4:478; blu ml et al (2012) International Immunology (5): 275-281; dong et al (2016) Proc.Natl.Acad.Sci.USA 113 (43): 12304-12309). Polymorphisms in the TNFR2 gene are associated with a variety of autoimmune diseases including, for example, RA, crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, and scleroderma; the polymorphism blocks TNF binding to TNFR2, thereby limiting NF- κB activation and blocking the TNFR2 signaling pathway in Treg (see, e.g., yang et al (2018) front. Immunol. 9:784).
TNFR1 contains an intracellular death domain that activates the apoptotic and/or inflammatory pathways, while TNFR2 binds TRAF and activates both the classical and non-classical NF-. Kappa.B pathways to control cell survival and proliferation. In general, TNFR 2-expressing cells also express TNFR1 in varying proportions, depending on the cell type and function. Since TNFR1 signaling generally induces cell death, while TNFR2 signaling generally induces cell survival, their co-expression ratios on cells shift the balance towards apoptosis or cell survival. As discussed above and elsewhere herein, TNFR1 has been shown to be the primary TNF receptor involved in RA pathogenesis, while TNFR2 plays an immunomodulatory role. However, both of these receptors are involved in mediating the antiviral activity of TNF. For example, animal disease models indicate that TNFR1 is associated with inflammatory neurodegeneration, while TNFR2 is associated with neuroprotection.
Selective inhibition of TNFR1 or TNFR2Selective activation by administration of ATROSAB (antagonistic TNF receptor 1 specific antibody), a TNFR1 selective antagonistic IgG1 antibody, or EHD2-scTNF R2 An agonistic TNFR 2-selective TNF mutein (i.e., a mutated protein) has been demonstrated in an NMDA-induced acute neurodegenerative mouse model. EHD2-scTNF R2 A human TNFR 2-containing selective single-chain TNF trimer containing covalent stability, having mutations D143N/A145R (residue numbers for soluble TNF are shown in SEQ ID NO:2, corresponding to D219N and A221R of SEQ ID NO:1, respectively, which mutations eliminate affinity for TNFR 1), fused to dimerization domain EHD2, EHD2 derived from heavy chain C of IgE H 2, and produces disulfide dimers containing hexameric TNF domains. In an in vivo mouse model, NMDA and ATROSAB, or NMDA and EHD2-scTNF, were compared to the control group R2 Simultaneous injection into the basal nuclei of large cells produces significant but incomplete neuroprotection. The incomplete nature of these responses is due to the agonistic activity of ATROSAB, a bivalent antibody by-product that induces aberrant receptor clustering and activation (Richter et al (2013) PLoS One 8 (8): e72156. Similarly, EHD2-scTNF R2 Because of their multiple fusion partners being immunogenic in humans, and immune responses to IgE fragments in toxicology studies lead to autoimmune responses (see, e.g., weeratna et al (2016) immun. Inflam. Dis.4 (2): 135-147). There is therefore a need for improved TNFR1 antagonists and improved TNFR2 agonists to overcome these limitations.
3. Regulatory T cells (tregs) and their role in autoimmune microenvironment
Regulatory T cells (Treg cells or tregs) are immunosuppressive subpopulations of T cells that have immunosuppressive properties by producing cytokines. These include transforming growth factor beta, interleukin 35 and interleukin 10. Induction of Treg function can inhibit a variety of pathological conditions. Induction can improve the success rate of transplantation, inhibit allergy, and control responses to infectious diseases and autoimmunity, such as severe acute respiratory syndrome. Treg inhibits and/or down regulates induction and proliferation of effector T cells (Teff), regulates the immune system, maintains immune homeostasis and tolerance to autoantigens, and canPreventing development of autoimmune diseases and tissue destruction. Treg expresses markers such as CD4, CTLA-4, CD25 (also known as IL-2 receptor alpha chain or IL2 RA) and FOXP3 (transcription factor fork P3), and expresses TNFR2 at a ten-fold higher intensity than it expresses TNFR 1. TNFR2 is expressed by only a subset of tregs, which is the most inhibitory subset; the subset contains TNFR2 expressing CD4 + FoxP3 + Treg. TNF promotes Treg cell proliferation, upregulates FoxP3 expression and Treg cell inhibitory activity/function through TNFR2 signaling. Autoimmune microenvironment contains specific immunosuppressive CD4 + Treg more autoreactive CD8 + Effector T cells, resulting in tissue destruction. Thus, a retained or enhanced TNFR2 function will expand tregs and eliminate autoreactive T cells, thereby restoring immune balance (see Shalma et al (2018) Front immunol.9:883). For these reasons, as well as others described below, the outcome of many acute and chronic inflammatory diseases (severe acute respiratory syndrome, autoimmune diseases) will be improved by selectively inhibiting TNFR1 and possibly pharmacologically preserving Treg function along with TNFR2 stimulation (agonism).
In addition to upregulating TNFR2 expression on tregs, TNF also upregulates expression of other co-stimulatory members of the TNF receptor superfamily (TNFRSF) on the Treg surface, such as 4-1BB and OX40, thereby achieving optimal activation and proliferation of tregs, as well as attenuation of excessive inflammatory responses. Neutralization of TNF (blocking TNFR 2) blocks in vivo expansion of tregs (e.g., hamano et al (2011) eur. J. Immunol.41: 2010-2020).
With CD4 + FoxP3 - CD4 compared with conventional T cells + FoxP3 + Tregs constitutively express TNFR2, promoting Treg cell activation, expansion and survival. TNF signaling through TNFR2 (i.e., TNFR2 agonism) promotes activation and expansion of tregs, whereas TNFR2 antagonism results in Treg contraction. For example, in human and autoimmune animal models of autoimmune disease, TNFR2 agonists selectively kill autoreactive T cells and expand inhibitory tregs. TNFR2 signaling promotes Tr in experimental autoimmune encephalomyelitis (EAE; animal models of inflammatory CNS demyelinating diseases, such as multiple sclerosis) and diabetic mouse models The eg cells expand and inhibit activity and induce human antigen specific Treg cells by tolerizing dendritic cells. TNFR 2-deficient tregs have reduced capacity to prevent experimental colitis in vivo, and TNFR2 is required for sustained FoxP3 expression on tregs, thus, to maintain the phenotypic and functional stability of tregs, TNFR2 was shown to be required for the immunosuppressive function of tregs in vivo (see, e.g., mcCann et al (2014) archlitis)&Rheumatology 66(10):2728-2738；Faustman and Davis(2013)Front.Immunol.4:478；Schmidt et al.(2013)Arthritis&Rheumatism 65 (9): 2262-2273; vanamee et al (2017) Trends in Molecular Medicine (11): P1037-P1046; chen et al (2013) J.Immunol.190 (3): 1076-1084). In one study, in vitro generated antigen-specific tregs were shown to inhibit disease and reduce joint inflammation and bone destruction in a well established antigen-induced arthritis (AIA) model in which mice were immunized with methylated bovine serum albumin (msa) to induce T cell-mediated tissue damage (see, e.g., wright et al (2009) proc.Natl. Acad. Sci. USA 106 (45): 19078-19083). The use of tregs in cell therapy, while promising, requires a traditional biologic therapy to provide the benefits of tregs without complications due to manufacturing and other complications.
As described and provided herein, TNFR2 and its expression by tregs are essential for inhibiting inflammatory and autoimmune diseases and disorders. For example, mycobacterium bovis BCG induces transient amplification of tregs. In one clinical trial, BCG elicits Treg production in type I diabetics, thereby inhibiting disease and temporarily restoring islet cell function, indicating the use of Treg and/or modulators of enhanced Treg function in the treatment of type I diabetes (see, e.g., spenc et al (2016) Curr Diab Rep 16 (11): 110.Doi:10.1007/s 11892-016-0807-6).
Modulation of Treg function is described and established herein to provide a therapeutic approach for the prevention or treatment of inflammatory and autoimmune diseases and disorders. However, tregs only account for total CD4 in blood + 1-5% of T cells. The small number hinders its clinical application. The ex vivo generation of tregs and/or stimulation of their production in vivo are factors limiting their therapeutic use. For example, IL-2 is used,In vivo stimulation of anti-CD 3 or anti-CD 28 is too toxic, whereas in vitro stimulation with these formulations would produce heterogeneous CD4 + A population of cells that release a proinflammatory cytokine and have antagonistic properties. Other approaches use TL1A-Ig, a naturally occurring TNF receptor superfamily agonist, or a TNFR2 monoclonal antibody agonist, which amplifies tregs in vivo and ex vivo, respectively. The TNFR2 agonist constructs and multispecific constructs provided herein can preserve and/or expand Treg populations in vivo without interfering with the therapeutic activity of anti-TNFR 1 activity. As described and provided herein, selective inhibition of inflammatory TNFR1 activity, while maintaining or increasing TNFR 2-related Treg inhibitory activity, is beneficial in the treatment of inflammatory and autoimmune diseases and disorders. These diseases and conditions include, but are not limited to, RA, type I diabetes, heart failure, and multiple sclerosis (see, e.g., goodall et al (2015) PLoS ONE 10 (9): e 0137065).
TNFR2 in Tumor Microenvironment (TME) and autoimmune microenvironment + Expansion of tregs prevents tissue destruction contrast, tumors are heavily immunosuppressive TNFR2 + Treg infiltration, which prevents tumor-killing CD8 + Proliferation of Cytotoxic T Lymphocytes (CTLs), also known as effector T cells (teffs), allows tumor growth. Antagonism of TNFR2 on lymphocytes in TME restores the balance between the two types of T cells by inhibiting or eliminating tregs and allowing activation and expansion of effector T cells, a condition that can control or reverse tumor growth. For use as a therapeutic agent, TNFR2 inhibitors must not aggregate immune cells by ADCC for two reasons: 1) Aggregation can transiently lead to "super induction" of TNFR 2-mediated immunosuppression; 2) Eventually leading to systemic depletion of tregs, which is detrimental to the patient, as basal levels of Treg activity must be maintained to maintain immune homeostasis. Tumor cells and myeloid-derived suppressor cells (MDSCs) also express TNFR2, inhibiting TNFR2 in MDSCs controls metastasis as shown in the mouse liver cancer model. Thus, blocking of TNFR2, for example, through the use of non-aggregation antagonistic antibodies or other therapeutic agents as provided herein, provides a useful treatment for certain types of cancers by inhibiting immunosuppressive tregs. However, TNFR2 antagonism The agent should only be administered to patients whose tumors show TNFR2 overexpression compared to adjacent normal tissues as judged by immunohistochemistry. Thus, such treatment should be accompanied by diagnosis to confirm an exemplary assay of over-expression (see, e.g., zhang et al (2019) Thorac Cancer10 (3): 437-444.Doi:10.1111/1759-7714.12948;Yang et al. (2017) Oncol Lett.14 (2): 2393-2398.Doi:10.3892/ol.2017.6410; and Yang et al (2018) Oncol Lett.16 (3): 2971-2978.Doi: 10.3892/ol.2018.8998).
4 autoimmune/inflammatory diseases mediated by TNF or involving TNF
Elevated or uncontrolled expression of TNF levels and deregulation of TNF signaling can lead to chronic inflammation, which can lead to the occurrence of autoimmune diseases and tissue damage. TNF- α is involved in a variety of diseases, disorders and conditions. The constructs provided herein are useful in the treatment of such diseases, disorders, and conditions. The following discussion describes some example diseases, disorders, and conditions in which blocking TNF may have a therapeutic effect. TNF blockers, such as etanercept, infliximab, adalimumab, cetuximab, and golimumab, have side effects that may limit their use in the treatment of such diseases, disorders, and conditions. The constructs provided herein avoid some or all of these side effects and are useful in the treatment of these diseases, disorders and conditions (see, e.g., liset al (2014) Arch Med sci.10 (6): 1175-1185 review the effects of TNF in disease and the use of TNF blockers in therapy).
Inflammatory diseases include a range of disorders and conditions characterized by inflammation, and include autoimmune diseases. The immune system protects the body by producing antibodies and/or activating lymphocytes in response to invading microorganisms such as viruses and bacteria. In healthy individuals, the immune system does not trigger a response against the body's own (i.e. "self") cells; autoimmune diseases occur when the immune system attacks healthy, non-invasive, self cells and tissues. Autoimmune/inflammatory diseases and conditions associated with elevated TNF levels include, for example, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spondyloarthropathies), inflammatory bowel disease (e.g., crohn's disease and ulcerative colitis), uveitis, fibrotic disease, endometriosis, lupus, ankylosing spondylitis, psoriasis, multiple Sclerosis (MS), parkinson's disease, and alzheimer's disease, among others. Exemplary autoimmune and inflammatory diseases and disorders that can be treated with the constructs provided herein are discussed below.
a. Arthritis treatment
Rheumatoid arthritis and other types of arthritis
Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disease. Inflammation associated with rheumatoid arthritis affects the inner layers of the joint (i.e., synovial lining), as well as the inner membranes of blood vessels, heart, and may also lead to inflammation. RA is characterized by infiltration of immune cells (e.g., activated B cells) into the synovium and proliferation of synovial cells, which results in thickening of the inner layers of the synovium. The proliferative substances called pannus invade and destroy cartilage and bone, irreversibly destroying joint structure and function. This is mediated by the induction of pro-inflammatory cytokines such as TNF, IL-1 and IL-6. Tumor necrosis factor alpha (tnfα) is a key regulator of the induction and maintenance of pro-inflammatory activity associated with RA. TNF is overexpressed in synovial fluid and in the synovial membrane, where expression of TNFR is up-regulated (see, e.g., blu ml et al (2012) International Immunology (5): 275-281;Schmidt et al (2013) Arthritis & Rheumatism 65 (9): 2262-2273;Keffer et al (1991) EMBO J.10 (13): 4025-4031). Other types of arthritis that can be treated with the constructs herein include, for example, psoriatic arthritis, juvenile idiopathic arthritis, and spondyloarthritis.
b. Inflammatory Bowel Disease (IBD) and uveitis
Inflammatory Bowel Disease (IBD) includes crohn's disease and ulcerative colitis, which are inflammatory diseases of the intestine and colon. Mice overexpressing TNF may develop intestinal inflammation similar to Crohn's disease, whereas TNFR1 deficiency may prevent Crohn's disease (see, e.g., fischer et al (2015) Antibodies 4:48-70).
Uveitis is an ocular inflammation affecting the intermediate layers of the eye between the eye wall (uvea), retina and sclera (white of the eye) and may lead to vision loss. TNF- α is involved in its pathophysiology, and TNF blockers have been used in therapy.
c. Fibrotic diseases
The constructs herein are useful for treating fibrotic diseases. Dupuytren's disease is an example of such a disease. Dupuytren's Disease (DD) is a common hand fibrosis disease characterized by irreversible flexor contracture of the fingers; this condition is limited to the palm, resulting in irreversible curling of the fingers, severely compromising hand function. There is no approved treatment for early stage disease, which manifests as a node that stands still for a period of time, then becomes active and progresses to chordate and flexor deformities of the finger, resulting in loss of hand function. Treatments include surgical excision (fasciotomy) of diseased tissue or chordae, or destruction of chordae using collagenase or needle-punched fasciotomy. Surgical and non-surgical treatments have high recurrence rates and complications. It is advantageous to perform therapeutic interventions at an early stage of the disease to prevent progression to chordogenesis and subsequent finger flexion contractures (see, e.g., nanshal et al (2018) EBioMedicine 33:282-288).
Myofibroblasts expressing the contractile protein α -smooth muscle actin (α -SMA) and accumulated in nodules deposit excessive extracellular collagen matrix and cause its remodeling and contraction in all fibrotic disorders including DD. TNF converts palm fibroblasts in DD patients to myofibroblasts via Wnt signaling pathway, which exhibit a dose-dependent decrease in contractile force and a decrease in α -SMA and procollagen expression following anti-TNF therapy treatment. Treatment with fully humanized IgG mabs adalimumab and golimumab was most effective. However, the use of anti-TNF therapy (e.g., adalimumab) has been associated with an increased risk of infection, and in a phase 2a trial evaluating the efficacy of adalimumab therapy DD, 1 patient (of 21 patients receiving adalimumab therapy) had a wound infection requiring hospitalization (see, e.g., nanshahal et al (2018) EBioMedicine 33:282-288). Thus, other therapies are needed.
d. Tumor necrosis factor receptor-associated periodic syndrome (TRAPS)
Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is the second most common autosomal dominant inherited inflammatory disease caused by mutations in the TNFRSF1A gene encoding TNFR 1. The TRAPS is characterized by periodic continuous fever, systemic inflammation, abdominal pain, skin lesions, conjunctivitis, myalgia and pericarditis without cause, and the onset of inflammation can last for several weeks. A complication associated with the more severe clinical phenotype of trap is AA-type serum amyloidosis, which can lead to kidney injury and failure. Attacks usually occur in childhood, but TRAPS may also occur in adults. Most TRAPS-related mutations occur in the extracellular domain of TNFR1, which is involved in ligand binding. High-exon mutations associated with the most severe clinical phenotypes occur in the extracellular cysteine-rich domain (CRD). These mutations affect the folding and secondary structure of TNFR1, resulting in defective TNFR1 transport, altered ligand binding affinity, reduced activation-induced shedding, and impaired cell signaling. For example, ligand-independent function acquisition of TNFR1 induces TRAPS pathophysiology, and certain mutations result in constitutive activity of TNFR1, NF-. Kappa.B, and caspase 1. Traditional anti-TNF therapies, including etanercept, infliximab, etc., are only partially effective in the treatment of trap (see, e.g., greco et al (2015) Arthritis Research & Therapy 17:93), and therefore, require additional therapies.
e. Other diseases mediated or participated by TNF
i. Neurodegenerative diseases
Aging and several neurodegenerative diseases are associated with elevated levels of TNF in the Central Nervous System (CNS). TNF is involved in initiating and maintaining neuroinflammation, as well as modulating other neurological processes such as synaptic function and plasticity. TNFR1 levels in the hippocampus of aged rats were approximately 3-fold higher than TNFR2 levels. In animal models of disease, TNF is involved in chronic glial cell activation and impaired neuronal viability by its effect on TNFR 1. In older animals, nervous system changes include synaptic dysfunction and Ca 2+ Deregulation, these changes can be replicated in healthy young animals and in neuronal cultures artificially raised using TNF. TNF also enhances L-type voltage sensitive Ca 2+ Activity of channels (L-VSCC); similar effects were observed in hippocampal neurons of memory-impaired aged rats. In ratsStudies have shown that TNF blockade in the cerebellum accelerates learning in deferred blink tasks. XPro1595 using soluble dominant negative TNF (DN-TNF) that preferentially inhibits TNFR1 signaling selectively blocks TNFR1 signaling, resulting in improved performance of Morris swimming tasks, reduced microglial activation, prevention of hippocampal long-term depression (LTD), and reduced activity of L-VSCC in CA1 neurons. These results indicate that TNF signaling through TNFR1 is involved in altering the neurological phenotype of aged animals and can lead to pathological changes associated with neurological diseases (see, e.g., sama et al (2012) PLoS ONE 7 (5): e 38170).
a) Alzheimer's disease
TNF is a core participant in the inflammatory response; TNF protein levels are lower in healthy brains but elevated for long periods in many neuroinflammatory disorders, including Alzheimer's Disease (AD). In animal models of AD, TNF promotes microglial activation, synaptic dysfunction, neuronal cell death, accumulation of plaques and tangles, and cognitive decline. For example, in a triple transgenic AD mouse model (3 xTg-Ad) with mutations in presenilin 1, amyloid Precursor Protein (APP) and tau, TNF levels are elevated in the cortex of the entorhinal region, consistent with the earliest occurring pathology (see, e.g., mcCoy et al (2006) J.Neurosci.26 (37): 9365-9375). TNF-driven processes are involved in AD pathology and lead to cognitive dysfunction and accelerate AD progression. Bacterial endotoxin Lipopolysaccharide (LPS) that induces inflammation and TNF production accelerates the appearance and severity of AD pathology in several animal models of AD. Excessive production of pro-inflammatory mediators (including TNF) occurs in the brain when microglial cells (often closely physically associated with amyloid plaques in the AD brain) are activated for prolonged periods of time. Elevated TNF levels inhibit phagocytosis of amyloid (aβ) in the brain of AD patients, thereby preventing microglia from effectively removing plaque. Chronic inhibition of solTNF by administration of DN-TNF (e.g., XENP 345) or lentivirus encoding DN-TNF prevented acceleration of AD-like pathology induced by chronic systemic inflammation in AD animal models (3 xtglad mice) and reduced intra-neuronal accumulation of LPS-induced 6E10 immune response proteins, particularly the C-terminal Amyloid Precursor Protein (APP) fragment (β -CTF), in hippocampus, cortex and amygdala. Genetic deletion of TNFR1 in 3xTgAD mice also prevented LPS-induced accumulation of neurotoxic beta-CTF. Neuronal cells carrying Familial AD (FAD) mutations accumulate β -CTF in the cell, suggesting that it is involved in the pathogenesis of AD. These results indicate that soluble TNF is a mediator of the effects of neuroinflammation on early pre-plaque lesions in 3 xtglad mice, and that targeted inhibition of solTNF in the Central Nervous System (CNS) can slow down the appearance of amyloid-related lesions, cognitive impairment, and progressive loss of neurons in AD (see, e.g., mcAlpine et al (2009) neurobiol. Dis.34 (1): 163-177).
b) Parkinson's disease
Parkinson's Disease (PD) is the second most prevalent neurodegenerative disease in the united states, with a 5% incidence in people over 65 years old. The clinical manifestations of parkinson's disease are due to loss of selectivity of dopaminergic neurons in the ventral midbrain substantia nigra pars compacta (SNpc), leading to reduction of striatal dopamine. Cerebrospinal fluid (CSF) and postmortem brains of PD patients and PD animal models show elevated TNF levels. A group of early-onset PD patients in japan showed an increase in the frequency of polymorphic alleles (-1031C) in TNF gene promoters, resulting in higher transcriptional activity and higher TNF levels. TNFR1 is highly expressed in nigrostriatal dopaminergic neurons, which increases vulnerability to TNF-induced neuroinflammation and dopaminergic neuron toxicity. The dominant negative TNF mutein (XENP 345) had neuroprotective effects on in vivo neutralization of soluble TNF (solTNF) and reduced retrograde melanins by 50% in rats caused by striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA) and attenuated amphetamine-induced rotational behavior in rats, indicating that striatal dopamine levels were preserved. Delayed administration of XENP345 in embryonic rat midbrain neuron/glial cell cultures exposed to Lipopolysaccharide (LPS) can prevent degeneration of dopaminergic neurons, but has sustained microglial activation and solTNF secretion. XENP345 also attenuated 6-OHDA-induced dopaminergic neuronal toxicity in vitro. TNF is therefore associated with the development of parkinson's disease and it is possible to delay the progressive degeneration of the human nigrostriatal pathway, particularly in the early stages of parkinson's disease, by using TNF blocking therapy (see, for example McCoy et al (2006) j. Neurosci.26 (37): 9365-9375).
c) Multiple Sclerosis (MS)
CNS-specific overexpression of TNF in transgenic mice resulted in spontaneous demyelination, suggesting a role for TNF in Multiple Sclerosis (MS). The polymorphism of the gene encoding TNFR1 is associated with increased susceptibility to MS. TNFR1 is essential for the induction of Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS, and TNFR2 deficiency exacerbates the disease. Mice expressing non-cleavable membrane-bound TNF are protected from EAE, suggesting that soluble TNF interactions with TNFR1 are associated with disease pathology (see, e.g., fischer et al (2015) Antibodies 4:48-70).
Endometriosis
TNF- α is involved in the pathophysiology of endometriosis. TNF- α levels in the peritoneal fluid of women suffering from endometriosis are elevated and correlated with the severity of the disease (see, e.g., koninckx (2008) Hum reprod.23:2017-2023). The peritoneal fluid TNF-alpha is produced locally by activated peritoneal macrophages, and TNF-alpha induces the secretion of IL-8 by peritoneal mesothelial cells. The peritoneal fluid concentrations of TNF- α and IL-8 correlated with the size and number of active peritoneal lesions (Bullimore, (2003) Med hypotheses.60:84-88). Serum TNF- α levels were elevated and monocytes from endometriosis patients released more TNF- α in vitro than monocytes from the control group. The level of MCP-1 peritoneal fluid is elevated in endometriosis patients. TNF-alpha, IL-8 and MCP-1 drive inflammatory Th-1 type responses in peritoneal fluid of endometriosis patients. TNF- α mediated inflammation may be a causative factor for pain associated with endometriosis. Blocking TNF- α in animal models appears to inhibit disease progression and may be effective in humans. Because of the adverse side effects of existing TNF blockers, the use of such blockers to treat endometriosis is not recommended (see Koninckx (2008) Hum reprod. 23:2017-2023). However, the constructs provided herein are intended to avoid deleterious effects and are contemplated for use in the treatment of TNF- α mediated inflammation in endometriosis.
Cardiovascular diseases
Tnfα is the first cytokine identified in human atherosclerotic plaques; tnfα is involved in the activation of endothelial cells and in the upregulation of adhesion molecules, which occur early in the development of atherosclerotic disease. TNF is also involved in the pathogenesis of atherosclerosis by affecting lipid metabolism and inducing vascular inflammation. Blocking of TNFα by TNF binding proteins, or IL-1 by IL-1 receptor antagonists, partially protects apoE knockout mice from atherosclerosis. Atherosclerosis is primarily the result of tnfα production by bone marrow cells. apoE for high fat diets -/- And TNF (tumor necrosis factor) -/- Plaque area of mice was apoE -/- Half of the plaque area of the mice. Will come from apoE -/- And TNF (tumor necrosis factor) -/- Bone marrow transplantation of mice to apoE -/- In mice, the size of the atherosclerotic lesions was reduced by 83%. In the treatment of apoE with recombinant soluble p55 (TNFR 1) TNF blockers -/- After mice, the size of the atherosclerotic lesions was also reduced, suggesting a role for TNF in atherosclerosis. NF-. Kappa.B signaling is involved in the production of TNF-. Alpha.in human atherosclerotic plaques. Peripheral blood tnfα levels in patients with cardiovascular disease are also associated with the occurrence of myocardial infarction. Cardiotoxicity is mainly due to TNF-induced cardiomyocyte apoptosis. The use of anti-TNF therapies (such as infliximab and etanercept) in clinical trials for the treatment of heart failure failed and resulted in increased mortality; thus, anti-TNF therapies have not been tested for treatment of cardiovascular disease (see, e.g., udalova et al (2016) Microbial Spectrum 4 (4): MCHD-0022-2015;Kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol.12 (1): 49-62). Therefore, alternative therapies are needed.
Acute Respiratory Distress Syndrome (ARDS)
Acute Respiratory Distress Syndrome (ARDS) affects approximately 190,000 patients annually in the united states with mortality rates as high as 40%. There is currently no effective therapy for the underlying pathophysiological mechanisms of ARDS. ARDS is characterized by immune cell-mediated lung injury, which is associated with the release of inflammatory cytokines and proteases. Uncontrolled local inflammatory reactions in ARDS can lead to impaired alveolar capillary barrier and non-cardiac pulmonary edema. Pulmonary neutrophil recruitment is central to the pathogenesis of ARDS, mediated by the interactions of activated and activated neutrophils with the pulmonary microvascular endothelium, and increased by injury to the alveolar capillary barrier caused by pro-inflammatory mediators. TNF- α has been identified as a key effector in ARDS as well as sepsis, a common cause of ARDS. For example, TNF- α contributes to increasing endothelial cell permeability. Clinical trials involving the treatment of sepsis with non-selective anti-TNF antibodies failed to demonstrate any survival benefit, one trial indicated that higher doses were detrimental.
TNFR1 deficient mice are protected from lung injury, sepsis and other acute organ injury, while TNFR2 deficient mice are more susceptible to injury in these models, suggesting that selective antagonism of TNFR1 is therapeutically effective. GSK1995057 is a short-acting fully human domain antibody (dAb) fragment that selectively antagonizes TNFR1 but not TNFR2, lessens the severity of disease in a mouse model of acute respiratory distress syndrome, and can reduce signs of inflammation and lung injury in non-human primates. In a randomized, placebo controlled trial of nebulized GSK1995057 in 37 healthy subjects receiving low dose inhalation endotoxin challenge, GSK1995057 treatment reduced evidence of pulmonary neutrophilia, inflammatory cytokine release, and endothelial injury in bronchoalveolar lavage fluid and serum samples. These results demonstrate the potential of pulmonary delivery of selective TNFR1 antagonists for the prevention and treatment of ARDS (see, e.g., proudboot et al (2018) Thorax 73:723-730).
Severe Acute Respiratory Syndrome (SARS) and COVID-19
Severe acute respiratory syndrome coronavirus (SARS-CoV) infected subjects develop fever and respiratory disease, general malaise and lower respiratory symptoms, including cough and shortness of breath, with a total mortality of about 10%. TNF signaling promotes the pathogenesis of SARS by inducing excessive inflammation, resulting in severe tissue damage. Cytokine Release Syndrome (CRS) occurs in severe covd-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some subjects, sustained viral stimulation can result in elevated levels of circulating cytokines, such as IL-6 and tnfα, which can lead to decreased lymphocyte counts and the initiation of inflammatory organ damage, particularly lung and blood vessels. SARS-CoV-2 has several similarities to SARS-CoV, which is a strain of coronavirus that resulted in the pandemic of SARS in 2002. SARS-CoV and SARS-CoV-2 use spike (S) proteins to bind their cellular receptor ACE2 (angiotensin converting enzyme 2) for invading cells. ACE2 receptor expression is upregulated by SARS-CoV-2 infection and inflammatory cytokine stimulation. In SARS-CoV infection, the S protein induces TNF- α converting enzyme (TACE) dependent shedding of the ACE2 extracellular domain, a process strictly associated with TNF α production. Loss of ACE2 activity caused by shedding is associated with lung injury caused by increased activity of the renin-angiotensin system. ACE2 knockout mice are prone to severe respiratory failure following chemical challenge, and ACE2 has been shown to alleviate ACE-induced intracellular inflammation. ACE2 down regulation is associated with severe respiratory distress associated with SARS-CoV infection. Thus, increased tnfα production can promote viral infection and cause organ damage, such as lung injury.
As discussed, regulatory T cells (tregs) are an immunosuppressive cell that has shown a variety of clinical applications in transplantation, allergy, infectious diseases, GVHD, autoimmunity, and cancer. Treg co-expression of CD4 + And interleukin 2 receptor alpha chain CD25 hi And has inducible levels of intracellular transcription factor fork P3 (FOXP 3). Naturally occurring tregs express TNFR2 at a higher intensity than TNFR 1. TNF signaling through TNFR2 promotes Treg activity: TNF-mediated activation of TNFR2 and induction of Treg proliferation (100), and TNFR2 expression indicates maximum suppression of Treg. Thus, tregs can prevent excessive responses to inflammatory stimuli in case TNF is overproduced in response to infection (influenza, SARS-type virus, endotoxemia).
anti-TNF can treat SARS and COVID-19. Adalimumab is being used to treat covd-19 (chinese clinical trial, month 2 2020 (ChiCTR 2000030089)); see, e.g., lucchino et al (2020) Rheumatology (Oxford) 59 (6): 1200-1203; haga et al (2008) Proc.Natl.Acad.Sci.U.S. A.105:7809-7814). Knocking out TNFR2 in mice infected with SARS-CoV does not provide any protection; double knockout of TNFR1 and TNFR2 protects infected mice from weight loss associated with infection (see, e.g., mcDermott et al (2016) BMC Systems Biology 10:93). These results indicate that TNF signaling through TNFR1 promotes the pathogenesis of SARS-CoV infection primarily by increasing the pro-inflammatory process, and that selectively inhibiting TNFR1 rather than TNF is a better therapeutic approach. The constructs provided herein are useful for treating acute inflammation of SARS and COVID-19. The construct is used in combination with an anti-infective agent; the constructs are useful for inhibiting or ameliorating the acute effects of cytokine storms.
Tnfα inhibition reduces the severity of viral-caused pulmonary diseases, such as those caused by mouse Respiratory Syncytial Virus (RSV) or influenza virus. The use of anti-TNF antibodies to deplete TNF in these mouse models reduces the pulmonary recruitment of inflammatory cells, reduces the production of pro-inflammatory cytokines by T cells (e.g., ifnγ, IL-4, IL-5, TNF), and reduces the severity of the disease without interfering with viral clearance (see, e.g., humill et al (2001) eur. J. Immunol. 31:2566-2573). These results indicate that TNF inhibitors and TNF receptor antagonists can be beneficial in treating human viral lung diseases, such as those caused by SARS-CoV and SARS-CoV2, by preventing or reducing TNF-induced immune activation and lung injury.
Allogeneic hematopoietic stem cell transplantation is complicated by the occurrence of non-infectious Idiopathic Pneumonia Syndrome (IPS), an acute lung dysfunction similar to SARS pneumonia. Elevated levels of tnfα are found in the serum of patients with lung injury following allogeneic Stem Cell Transplantation (SCT), and donor-derived alloreactive T cells have been shown to be involved in this process. In humans, anti-TNF therapy with etanercept is beneficial for the treatment of IPS after allogeneic stem cell transplantation. Because of the immunosuppressive effects of SCT pretreatment regimens, the need for long-term immunosuppressive drugs to prevent or treat graft versus host disease (GvHD), recipients of allogeneic stem cell transplantation are at high risk of developing bacterial and fungal infections, as well as other SCT complications that may impair host defense, including acute GvHD (see, e.g., yanik et al (2002) biol. Blood Marrow Transmount.8:395-400). Other indications that can be treated by the constructs provided herein include chemotherapy of the brain, a condition experienced during and after chemotherapy, particularly in women receiving breast cancer treatment. Furthermore, the therapeutic methods and constructs herein may be used to treat long covd.
Thus, the use of selective TNFR1 antagonists that retain protective TNF signaling through TNFR2 and, unlike anti-TNF therapies, do not increase the risk of serious infection provides a safer, more effective treatment option for treating, preventing or alleviating viral and non-viral-induced lung injury. Thus, the constructs provided herein are ideal therapies for these indications.
D. Treatment of rheumatoid arthritis and other chronic inflammatory and autoimmune diseases and conditions
Rheumatoid Arthritis (RA) cannot be cured, but treatment can improve symptoms and slow disease progression, such as minimizing pain and swelling, preventing skeletal deformity, and maintaining daily function. The main treatment for RA is an improved anti-rheumatic drug (DMARD), which is also used to treat other chronic inflammatory and autoimmune diseases and disorders, such as psoriasis, plaque psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, behcet's disease, inflammatory bowel disease (IBD; e.g. crohn's disease and ulcerative colitis), multiple sclerosis and lupus, as well as the treatment of some cancers.
DMARDs are immunosuppressants and immunomodulators, and are classified as either conventionally synthesized DMARD (csDMARD) or biological DMARDs (bdmards; e.g., antibodies and fusion proteins). Conventional synthetic DMARDs include, for example, methotrexate (MTX), a chemotherapeutic agent, and immunosuppressants; hydroxychloroquine (HCQ; ) An antimalarial agent; sulfasalazine (>) An anti-inflammatory agent; and leflunomide (>) An immunosuppressant for inhibiting dihydroorotate dehydrogenase. Biological DMARDs include, for example, abacavir (+)>) A fusion protein that prevents T cell activation and comprises an Fc region of IgG1 fused to a CTLA-4 extracellular domain; anakinra (e.g. under the trademark +.>Sold), a recombinant human IL-1 receptor antagonist; rituximab (to include +.> Sold under the trademark CD 20), a chimeric monoclonal antibody against CD20, induces CD20 + Apoptosis of cells such as B cells; toxicillin (Toxicillin,/-for)>) A humanized monoclonal antibody directed against the IL-6 receptor (IL-6R); corticosteroids; tofacitinib (>) A small molecule inhibitor of Janus kinase (JAK), a protein tyrosine kinase involved in mediating cytokine signaling; and TNF inhibitors/anti-TNF agents, e.g. polyethylene glycol cetuximab (++>) Infliximab @) Adalimumab (/ ->) Golimumab (+)>) And etanercept ()>). Combination therapy, particularly of methotrexate with biological DMARDs, is more effective than either therapy alone. Combination therapies may also include multiple csdmards and combinations of multiple csdmards with one biological DMARD. Due to the risk of serious side effects (including serious infections), various biological DMARDs, particularly anti-TNF DMARDs, are not generally used in combination therapy methods.
The following section describes existing therapies and the problems associated with each therapy to emphasize how the therapies provided herein address these problems.
1. Conventionally synthesized antirheumatic drug (csDMARD) for improving illness state
Conventional synthetic disease-modifying antirheumatic drugs (csdmards) are often first-line therapeutic drugs for RA and other autoimmune and chronic inflammatory diseases and conditions. csDMARDS includes methotrexate, leflunomide, hydroxychloroquine, sulfasalazine, and the like, which are the most commonly used initial therapeutic agents, and whose mechanism of action includes stimulation of fibroblast release of adenosine, reduction of neutrophil adhesion, inhibition of neutrophil synthesis of leukotriene B4, inhibition of local IL-1 production, reduction of IL-6 and IL-8 levels, inhibition of cell-mediated immunity, and inhibition of synovial collagenase gene expression. Other conventionally synthesized DMARDs act by inhibiting lymphocyte proliferation or causing lymphocyte dysfunction. For example, leflunomide inhibits dihydroorotate dehydrogenase, thereby inhibiting pyrimidine synthesis and blocking lymphocyte proliferation. Sulfasalazine mediates its anti-inflammatory effects by preventing oxidative, nitrifying and nitrosylating damage, while hydroxychloroquine is a mild immunomodulator that inhibits intracellular Toll-like receptor 9 (TLR 9).
Hydroxychloroquine has optimal safety in conventional DMARDs, does not increase the risk of infection, and does not cause hepatotoxicity or renal dysfunction; common side effects of hydroxychloroquine include rashes and diarrhea. Retinopathy/maculopathy is a rare but serious side effect of hydroxychloroquine therapy, associated with doses exceeding 5 mg/kg/day, long-term use (treatment exceeding 5 years), older and chronic kidney disease. Other rare side effects of hydroxychloroquine include anemia, leukopenia, myopathy and cardiomyopathy. Treatment with methotrexate, leflunomide and sulfasalazine has been associated with nausea, abdominal pain, diarrhea, rash/allergic reactions, bone marrow depression, hepatotoxicity and a high incidence of common and sometimes serious infections. Methotrexate and leflunomide also cause alopecia. Other side effects associated with methotrexate therapy include interstitial lung disease, folate deficiency and cirrhosis. Leflunomide is also associated with hypertension, peripheral neuropathy, and weight loss. Sulfasalazine has a very high risk of gastrointestinal discomfort and may rarely cause DRESS syndrome (eosinophilia and a drug response to systemic symptoms) (see, e.g., benjamin et al, disease Modifying Anti-Rheumatic Drugs (DMARD) [ Updated 2020Feb 27]. In: statPearls [ Internet ]. Treasure Island (FL): statPearls Publishing;2020Jan. Available from: URL: ncbi.nlm.nih.gov/books/NBK507863 /). These drugs are effective because they have immunosuppressive effects. The constructs provided herein as selective anti-TNFR 1 antagonists advantageously retain TNFR2 immunosuppressive activity and may eliminate the need for such immunosuppressive drugs.
2. anti-TNF therapeutic/TNF blockers
anti-TNF therapy/TNF blocker (a biological DMARD) is typically prescribed after failure of conventional DMARDs, including monoclonal antibodies (mabs), such as the chimeric mAb infliximab @) Comprises a murine variable region and a human IgG1 constant region; and the fully humanized monoclonal antibody (IgG 1) adalimumab (, I)>) And golimumab (>) The method comprises the steps of carrying out a first treatment on the surface of the Pegylated humanized Fab' fragments of mAbs targeting TNF, polyethylene glycol cetuzumab (/ -)>) The method comprises the steps of carrying out a first treatment on the surface of the And TNFR2 fusion proteins, e.g. the TNFR2-Fc fusion protein etanercept (/ -)>) It contains an extracellular receptor region comprising the binding site of human TNFR2 fused to the Fc of human IgG 1.And->Is a biomimetic of infliximab and has been approved in the European Union for the treatment of a variety of autoimmune and chronic inflammatory diseases and conditions. These TNF inhibitors that sequester TNF are useful in the treatment of a variety of diseases and conditions including, for example, RA, psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile Idiopathic Arthritis (JIA), and/or inflammatory bowel disease (IBD; e.g., crohn's disease and ulcerative colitis).
Because therapies targeting TNF have immunosuppressive effects, such therapies are associated with serious side effects, including, for example, sepsis and increased risk of serious infections, such as listeriosis, tuberculosis reactivation, hepatitis b/c reactivation, herpes zoster reactivation, invasive fungi, and other opportunistic infections. TNF is a critical cytokine in the inflammation and immune response of infection and the use of drugs that remove TNF can impair the host's immunity to microorganisms, thereby increasing the risk of infection. For example, TNF blockers are associated with reactivation of mycobacterium tuberculosis infection. TNF plays an important role in resistance to mycobacterium tuberculosis and adalimumab treatment of RA patients significantly reduces responsiveness to mycobacterium tuberculosis. As described herein, reduced immunoreactivity may be associated with activation of tregs and induction of effector lymphocyte apoptosis. anti-TNF treatment has been shown to induce macrophage apoptosis in rheumatoid synovium. Infliximab is associated with increased apoptosis of inflammatory cell infiltration in the gut of patients with crohn's disease. Other antirheumatic agents such as methotrexate and glucocorticoids can also induce immune apoptosis (see, e.g., vigna-Prez et al 2005 Clin. Exp. Immunol.141 (2): 372-380). Adalimumab and infliximab, but not etanercept, are TNFR2-Fc fusion proteins that induce caspase-dependent apoptosis in cultured monocytes and down-regulate IL-10 and IL-12 produced by monocytes (see, e.g., shen et al (2005) Ailment pharmacol. Ther. 21:251-258). The most common fungal infections associated with TNF blockers are histoplasmosis, candidiasis and aspergillosis. Anti-TNF agents can also lead to severe congestive heart failure, drug-induced lupus and demyelinating Central Nervous System (CNS) diseases, and exacerbation of lymphomas and non-melanoma skin cancers (see, e.g., benjamin et al disease Modifying Anti-Rheumatic Drugs (DMARD) [ Updated 2020Feb 27]. In: statPearls [ Internet ]. Treasure Island (FL): statPearls Publishing;2020Jan. Available from ncbi.
Infliximab is also associated with the development of leukopenia, neutropenia, thrombocytopenia and whole blood cytopenia (some fatal). Etanercept is associated with an increased incidence of opportunistic bacterial and viral infections in RA patients. Etanercept is also used to treat severe refractory graft versus host disease (GvHD). The risk of developing Invasive Aspergillosis (IA) in severe GvHD subjects receiving etanercept treatment is very high (100% in one study, see zocan et al (2019) sci.rep.9:17231), a life threatening mold (i.e., fungal) infection caused by aspergillus fumigatus. Etanercept therapy results in down-regulation of genes involved in immune responses and TNF signaling, including genes involved in NF- κb signaling, antimicrobial humoral responses and apoptotic processes, and reduced secretion of chemokines (e.g., CXCL 10) from immune cells (see, e.g., zocan et al (2019) sci.rep.9:17231).
Other side effects associated with the use of TNF blocking therapies include congestive heart failure, liver injury, demyelinating diseases/central nervous system diseases, lupus, psoriasis, sarcoidosis, and increased susceptibility to other autoimmune diseases and cancers, including lymphomas and solid malignancies (see, e.g., dong et al (2016) proc. Natl. Acad. Sci. USA 113 (43): 12304-12309;Zalevsky et al (2007) J. Immunol.179:1872-1883; zora et al (2019) Sci. Rep. 9:17231). Thus, eliminating all TNF-mediated signaling by sequestering TNF is not an ideal therapeutic strategy because it can lead to severe immunosuppression, resulting in serious, sometimes fatal, infections and other dangerous side effects.
anti-TNF therapy improves RA but does not cure and requires continuous and costly treatment for many years. Inhibition/blocking of TNF in RA reduces inflammation and joint destruction, but, as noted above, is associated with increased risk of serious infections (e.g., tuberculosis and listeriosis) due to immunosuppression. Thus, the use of TNF blockers is limited, especially in cases of chronic diseases/conditions (e.g., arthritis and IBD) where long-term administration is required. About 30% of RA patients either do not respond when treated with anti-TNF, or the therapeutic effect is not sustained (see, e.g., mcCann et al (2014) Arthritis & Rheumatology 66 (10): 2728-2738). Anergy also occurs in non-RA patients receiving anti-TNF therapy. Depending on the anti-TNF agent, 13-33% of the patients receiving treatment do not respond to treatment, and up to 46% of the patients stop responding, resulting in withdrawal or dose increase (see, e.g., richter et al (2019) MABS 11 (4): 653-665). Thus, therapies with improved efficacy and safety are needed.
anti-TNF therapy blocks/sequesters TNF and inhibits soluble TNF (solTNF) and transmembrane TNF (tmTNF) signaling through TNFR1 and TNFR2, respectively; solTNF signaling is associated with chronic inflammation, while tmTNF signaling is associated with resolution of inflammation and induction of immunity against pathogens such as listeria monocytogenes and mycobacterium tuberculosis. The primary anti-inflammatory effect of anti-TNF therapy is achieved by blocking TNFR1, while blocking TNFR2 inhibits Treg cell activity. As discussed elsewhere herein, TNFR1 signaling is primarily inflammatory and is involved in the pathogenesis of inflammatory and autoimmune diseases and disorders, such as RA, psoriasis, IBD, and neurodegenerative diseases, such as MS; whereas TNFR2 signaling has anti-inflammatory and protective effects in a variety of cell and organ types, including neural, cardiac, intestinal and bone tissues, and also participates in host defense mechanisms against pathogen infection. Thus, as described herein, selective blocking of TNFR1 increases therapeutic efficacy compared to anti-TNF therapy by eliminating undesirable pro-inflammatory signaling associated with RA and other autoimmune and inflammatory diseases and conditions, while retaining the beneficial effects of TNFR2 signaling (see, e.g., mcCann et al (2014) Arthritis & Rheumatology 66 (10): 2728-2738;Schmidt et al (2013) Arthritis & Rheumatology 65 (9): 2262-2273; blu ml et al (2012) International Immunology (5): 275-281;Zalevsky et al. (2007) J. Immunol.179:1872-1883).
anti-TNF therapy fails to treat neurodegenerative diseases such as alzheimer's disease, parkinson's disease, stroke, and Multiple Sclerosis (MS), which are associated with overexpression of TNF. For example, in phase II trials for treatment of relapsing-remitting MS, the TNFR1 receptor-Fc IgG1 fusion protein anti-TNF therapeutic drug lenacil (Ro 45-2081) failed and symptoms worsened/worsened compared to placebo-treated patients, in whom neurological deficit was more severe. These results indicate that anti-TNF therapy can exacerbate demyelinating diseases. Although TNFR1 has been shown to mediate inflammatory neurodegeneration, TNFR2 induces neuroprotection, and thus blocking signaling through both receptors by anti-TNF therapy abrogates the neuroprotection of TNFR2 signaling. Blocking TNFR1 with ATROSAB (a humanized monoclonal antibody that blocks TNFR 1), or activating TNFR2 with EHD2-scTNFR2 (an agonistic TNFR 2-selective TNF mutein), protects cholinergic neurons from cell death in an NMDA-induced acute neurodegenerative mouse model, and reverses memory impairment associated with neurodegeneration. This may be immunogenic. ATROSAB is a partial TNFR1 agonist; one skilled in the art would not administer a TNFR1 agonist. However, blocking TNFR1 and TNFR2 abrogating therapeutic effects, indicating that TNFR2 plays an important role in neuroprotection, selective blocking TNFR1 may be useful in the treatment of neurodegenerative diseases where anti-TNF therapy failed (see, e.g., dong et al (2016) Proc. Natl. Acad. Sci. USA 113 (43): 12304-12309).
Other therapies are needed due to adverse effects associated with the use of anti-TNF drugs, anergy in some patients, lack of sustained response in patients with initial response, and failure and/or exacerbation of treatment of neurodegenerative diseases such as MS. Such therapies are provided herein.
E. Therapeutic agents targeting TNFR1/TNFR2
The following section discusses exemplary therapeutic agents targeting TNFR1/TNFR2 and describes some of the problems and limitations of these therapeutic agents. Existing therapeutic agents may be modified as described in section F and examples, or used in whole or in part, or modified to improve their use properties in the constructs provided herein.
TNFR 1-selective antagonists
As discussed and provided herein, treatment with TNF blockers, such as etanercept, infliximab, adalimumab, and the like, eliminates TNF signaling through TNFR1 and TNFR 2. Although TNFR1 signaling leads to inflammation, cytotoxicity and apoptosis, TNFR2 signaling is protective and anti-inflammatory, in part because of its expansion and immunosuppressive Treg activation, destroying effector T cells in the autoimmune environment, preventing tissue destruction and disease progression. TNF blockers are treated by inhibiting TNFR2 signaling with a concomitant depletion of immunosuppressive tregs (resulting in a pro-inflammatory microenvironment) that may not be able to treat and/or exacerbate autoimmune and inflammatory diseases and conditions. Double blocking of TNFR1 and TNFR2 also leads to opportunistic infections and cancers.
As provided herein, specific inhibition of TNFR1 signaling maintains normal TNFR2 function, which is necessary to maintain a balance between pro-inflammatory and anti-inflammatory activity through the production of regulatory and cytotoxic T cell subsets. Selective TNFR1 inhibition retains potent anti-inflammatory activity of TNFR2 signaling, reduces opportunistic infections and cancers, and retains TNF-induced Treg function.
TNFR1 antagonist antibodies
In the TNFR1 antagonist antibody, ATROSAB (antagonistic TNF receptor 1 specific antibody) is provided. ATROSAB is the first TNFR1 blocking antibody, a full length IgG1, a humanized version of the neutralizing mouse anti-human TNFR1 monoclonal antibody H398. It was abandoned as a therapeutic agent because it has partial agonist activity, activating TNFR1, thus mimicking TNF activity, a toxic pathway. ATROSAB maintains the conformation of TNFR1 in an inactive state and blocks TNF binding. The Fc region in ATROSAB was mutated to eliminate FcγR receptor binding and complement fixation, thereby avoiding unwanted immune system activation (see, e.g., kalliolias and Ivashkiv (2016) Nat. Rev. Rheumatol.12 (1): 49-62).
Full length antibodies have the advantage of increasing half-life in vivo, but as discussed elsewhere herein, the development of TNFR1 antagonists is not feasible due to the tendency to agonize rather than antagonize receptor cross-linking of TNFR 1. This is not caused by Fc cross-linking, as the Fc-interacting portion of the antibody has been removed by mutation. For example, igG ATROSAB exhibits some TNFR1 agonistic activity in the absence of TNF due to its divalent molecular structure, which is a limited extent observed in a narrow concentration range. Crosslinking of TNFR1 may also occur due to minor events, such as interactions with fcγr or an anti-drug antibody (ADA), which must be avoided to maintain the antagonistic properties of the TNFR1 inhibitor. The incidence of ADA was observed to be 50% and 31%, respectively, in patients receiving infliximab or adalimumab treatment (see, e.g., richter et al (2019) mAbs11 (4): 653-665;Richter et al, (2019) mAbs11 (1): 166-177).
b. Monovalent TNFR1 antagonist antibodies/antibody fragments
Small antibody fragments, such as domain antibodies and derivatives and modified forms thereof, have been developed and exemplary antibody fragments and modified forms are discussed in the following sections. However, small antibody fragments have not been successfully developed into drugs. They have limited therapeutic uses; they have a shorter serum half-life and a faster rate of external Zhou Qingchu due to their smaller size. For example, molecules of 50-60kDa or smaller are subjected to renal filtration; dabs and other antibody fragments of less than 50-60kDa in size will be cleared rapidly by the kidneys. For example, dabs and similar molecules called DMS5541 exhibit selectivity for TNFR1 and potentially can inhibit the deleterious effects of TNFR1 signaling. DMS5541 is formed from two dabs (anti-TNFR 1 and anti-human serum albumin), is only about 25kDa in size, and is too small to have the ideal pharmacokinetics for therapeutic purposes. Its binding to HSA is intended to stabilize its half-life, only 34nM, meaning that it is normally in a dissociated state from HSA. The single domain antibodies (sdabs) tested to date are expressed in e.coli and are prone to aggregation (unfolding) during manufacturing. Furthermore, sdabs prepared in a cytoplasmic manner (by direct expression in e.coli) often lack the conserved disulfide bonds found in variable heavy domains, which both lower their melting point and reduce their ability to refold. Rapid clearance and short elimination half-life (perhaps less than a few hours) of small antibody fragments can reduce in vivo efficacy and require frequent administration and/or continuous infusion, which can reduce patient compliance. Since these molecules (see, e.g., holland et al (2013) J Clin Immunol 33 (7): 1192-203) are produced in E.coli and are often not folded correctly, they result in poor solubility and immunogenicity, resulting in their clinical failure (see, e.g., adisinight. Spring. Com/drugs/800037882).
The constructs provided herein, such as TNFR1 antagonist constructs, address this problem as well as other problems, such as immunogenicity and reaction with pre-existing antibodies. Provided herein are constructs comprising small antibody fragments (e.g., dabs), specific for TNFR1 and/or TNFR2, which exhibit improved pharmacological, e.g., pharmacokinetic properties, including longer serum half-life, increased stability, reduced/slower outer Zhou Qingchu rates, and lower immunogenicity, as compared to prior art dabs.
Therapeutic antibodies of various structures may be effective and well-tolerated therapeutic agents. Antibodies are useful for treating a variety of diseases and conditions, including, for example, rheumatoid arthritis (e.g., adalimumab, under the trademarkSales); cancers, e.g. non-hodgkin lymphomas (e.g. rituximabAnd temozolomide, respectively under the trade mark +.>And->Sold) and breast and gastric cancers (e.g. trastuzumab under the trademark +.>Sales); and respiratory syncytial virus infection (e.g. palivizumab under the trademark +.>Sales). The production of intact antibodies has several limitations, such as reliance on expression in mammalian cells. Thus, antigen binding fragments of antibodies have been developed, such as Fab (-57 kDa) and single chain Fv fragments (scFv (-27 kDa) and other structures, can be selected in vitro, for example by phage display (circumventing animal immunity), and can be produced in large quantities using bacterial or yeast cell cultures. Fab fragment contains V H -C H 1 polypeptide linked to V by disulfide bonds L -C L A polypeptide; scFv is a fusion protein comprising V linked by a short polypeptide linker H Domain and V L A domain. Another class of therapeutic small antibody fragments are domain antibodies (dAbs; also known as single domain antibodies or sdAbs) which are monomeric and contain the heavy chain of the antibody (V H ) Or light chain (V) L ) Is a variable domain of (a). dabs are the smallest antigen-binding fragments of antibodies; the size is about 11-15kDa, which is about one tenth of the size of an intact monoclonal antibody (mAb) (see, e.g., holt et al (2003) Trends in Biotechnology 21 (11): 484-490). Similar to dabs, nanobodies (Nb) are small antigen-binding fragments derived from camelid heavy chain antibodies, free of light chains. Nanobodies are small (-15 kDa), have low immunogenicity and high affinity, are soluble and stable, are encoded by a single gene/exon (VHH), and are therefore modular, allowing high yield production in bacteria and yeast (see e.g. Steeland et al (2015) J. Bi)ol.Chem.290(7):4022-4037；Steeland et al.(2017)Sci.Reports 7:13646)。
i. Fab and scFv-based TNFR1 antagonists
As described above, the humanized semi-agonistic/antagonistic TNFR1 specific antibody ATROSAB inhibits TNFR 1-mediated cellular sub-populations. In the absence of TNF, ATROSAB exhibits some TNFR1 agonistic activity, possibly due to its bivalent molecular structure or to its binding to TNFR 1. Parent mouse antibody H398 has a greater inhibitory potential due to the faster rate of ATROSAB dissociation (i.e., k) compared to H398 off The value is higher). This was determined using Quartz Crystal Microbalance (QCM) measurements, where the antigen density on the chip was reduced to favor monovalent interactions; the slower dissociation of monovalent bound H398 from TNFR1, and the resulting longer receptor occupancy, helps to improve the blocking of TNFR 1. Accordingly, monovalent derivatives of ATROSAB have been developed in order to eliminate the TNFR1 agonistic activity of ATROSAB and to increase its TNFR1 antagonistic activity.
To increase affinity and antagonistic activity of ATROSAB, a single chain variable fragment (scFv) of ATROSAB was subjected to a first affinity maturation by site-directed mutagenesis of exposed residues in a single CDR or combination of CDRs, and selected by phage display against human TNFR 1-Fc. The scFv of ATROSAB contains V H The domain, corresponding to residues 1-115 of the ATROSAB heavy chain (see SEQ ID NO: 31), is linked to V by a short peptide linker L A domain corresponding to residues 1-113 of the ATROSAB light chain (see SEQ ID NO: 32). Clone scFv IG11 (see SEQ ID NO: 674), having 6 mutations within the CDR-H2 of the ATROSAB heavy chain, Y52V, Y54T, S55Q, H E, Y K and E62D, showed slower receptor dissociation and improved equilibrium binding to human TNFR1-Fc and improved inhibition of TNF-induced TNFR1 activation, with reference to SEQ ID NO: 31. The clone was further subjected to random mutagenesis to yield clone scFv T12B (see SEQ ID NO: 675), which was found to be at V H The domain (cf. SEQ ID NO: 31) contains the mutations Q1H, Y52V, Y54S, S Q, H57E, Y K and E62D and V L Mutation S96G in the domain (cf. SEQ ID NO: 32). Dissociation of scFv T12B from immobilized TNFR1-Fc compared to scFv of ATROSAB and scFv IG11Decreased, and TNFR1 inhibitory activity increased (see, e.g., richter et al (2019) mAbs 11 (1): 166-177; see also Richter, F.thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB") "Stuttgart,2015; obtainable from pdfs.semmantischolar.org/d 8e7/8b87d76d 36225c1d 497939ef375cfaa8a.pdf).
Humanization of H398 the CDR arrangement was optimized by re-engineering the VH and VL framework regions of H398 with another germline gene. scFv13.7 contains the VH domain of scFV T12B, a novel humanized VL domain linked to H398 via a short peptide linker, has similar binding to human TNFR1-Fc in ELISA and QCM, improved inhibition of TNF-induced TNFR1 activity, and improved thermostability, melting temperature 10℃higher than scFv T12B. Based on scFv13.7, igG and Fab (IgG 13.7 and Fab 13.7, respectively) were produced with the same constant region as ATROSAB, with increased binding to TNFR1 compared to ATROSAB (1.4 fold) and Fab of ATROSAB (Fab ATR;8.7 fold). Fab 13.7 also reduced dissociation from immobilized TNFR1-Fc compared to Fab ATR, increasing monovalent affinity 18.8 fold. Thus, affinity maturation and frame substitution resulted in improved binding of Fab 13.7 to TNFR1. Fab 13.7 and IgG13.7 showed selectivity for TNFR1-Fc and did not bind to TNFR2-Fc fusion proteins; fab 13.7 binds to human and rhesus TNFR1-Fc but not to mouse and rat TNFR1-Fc, showing a similar binding pattern as ATROSAB. In vitro, monovalent Fab ATR and Fab 13.7 did not activate TNFR1, whereas ATROSAB showed edge activation of TNFR1 activity, while IgG13.7 strongly activated TNFR1. The agonistic activity of igg13.7 may be due to increased affinity and slower TNFR1 dissociation, leading to the formation of stable signaling competent receptor-antibody complexes. Fab 13.7 showed improved inhibition of TNFR1 activity compared to Fab ATR and ATROSAB, and did not have any agonistic activity. Incubation of Fab 13.7 or ATROSAB with cross-linked anti-human Fab serum indicated that Fab 13.7 did not activate TNFR-1, whereas ATROSAB activated TNFR-1 (see, e.g., richter et al (2019) mAbs 11 (1): 166-177).
Fab13.7 (molecular weight approximately 47 kDa) was compared to ATROSAB with an initial half-life of 0.44 hours, a final half-life of 32.1 hours, and an area under the curve (AUC) of 181 μg/ml x h. The initial half-life of Fab13.7 was 0.08 hours, the final half-life was 1.4 hours, and the AUC was 4.2. Mu.g/ml.times.h, which was similar to that obtained for Fab ATR. To extend half-life, by the method described in C H Introduction of a free cysteine residue at the C-terminus of the 1 domain generates the Fab 'fragment Fab'13.7, which is associated with branched PEG 40kDa The moieties were chemically coupled to yield Fab13.7 PEG. Fab13.7 was also fused to the N-terminus of Mouse Serum Albumin (MSA) via its Fd and a short flexible linker, yielding Fab13.7-MSA. Monovalent Fab-Fc fusion proteins were generated by fusing Fab13.7 with a modified Fc that lacks cysteine residues in the hinge region and through C H Ability of 3 domains to dimerize, thereby generating single arm half-IgG molecules (IgG 1 half 13.7). Monovalent Fv-Fc molecules were also produced by fusing the VH and VL domains to heterodimeric raised-recessed (kih) Fc chains lacking cysteine residues in the hinge region (Fv 13.7-Fc) kih ). No derivatives showed any agonistic TNFR1 activity and Fab13.7PEG, fab13.7-MSA and IgG1 were observed compared to Fab13.7 half 13.7 binding to human TNFR1-Fc was slightly reduced; fv13.7-Fc kih The combination of (2) is not affected. TNF-mediated inhibition of TNFR1 activity was reduced by a factor of 1.5-3.3 compared to Fab 13.7; fab13.7PEG showed the strongest loss of function, while Fv13.7-Fc kih Exhibiting minimal changes in biological activity. IgG (immunoglobulin G) half 13.7 shows a half-life similar to that of Fab13.7, increasing the AUC value by a factor of 7.1. Fab13.7PEG, fab13.7-MSA and Fv13.7-Fc kih The terminal half-life was prolonged, 14.4 hours, 9.7 hours and 10.5 hours, respectively, and the AUC value was increased. Thus, fusion protein Fv13.7-Fc kih Is engineered by heterodimer assembly of two peptide chains using the bulge-recess technique, showing an optimal combination of improved pharmacokinetic properties and TNFR1 antagonistic activity (see, e.g., richter et al (2019) mAbs 11 (1): 166-177; see also Richter, F. Thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB") "Stuttgart,2015; obtainable from pdfs.semmantischolar.org/d 8e7/8b87d76d 36225c1d 497939ef375cfaa8a.pdf).
In another study, to improve the pharmacokinetic properties of Fab13.7, such as serum half-life, igG-like Fc was incorporated into the molecule while retaining Fab-like heterodimerization of the polypeptide chains. To achieve this, the variable domains of the heavy and light chains of Fab13.7 were fused to the N-terminus of the nascent heterodimerized Fc chain, designated Fc-one/kappa (Fc 1 kappa). The Fc heterodimerization approach is based on interspersed Ig domains, derived from heterodimerization IgG1 constant heavy chain domains CH1 and kappa light chain constant domain clk, and contains a portion of the IgG1 CH3 sequence to mediate FcRn binding and to recycle FcRn-mediated drugs within the body. Interspersed Ig domains include "CH31" which contains both CH1 and CH3 amino acid sequence fragments, and "CH3kappa" (CH 3 kappa) which contains both CL kappa and CH3 amino acid sequence fragments. The IgG1 CH2 domain is also fused to the N-terminus of the CH31 and CH3kappa domains to include the entire FcRn binding region of the IgG molecule. Addition of an IgG1 hinge region to the N-terminus of the CH2 domain results in a covalently linked heterodimerized Fc portion, referred to as Fc1 kappa. In contrast to other Fc heterodimerization techniques involving substitution of one or more amino acids at the CH3-CH3 interface (e.g., protruding dents), fc heterodimerization is achieved by substitution of a larger stretch of amino acid sequences derived from a human antibody sequence. Asymmetric scFv-Fc1 kappa fusion proteins were prepared and compared to scFv fusions with Fc in the bulge-recess, heterodimer formation was similar or improved compared to fusions with the bulge-recess technique (see, e.g., richter et al (2019) mAbs 11 (4): 653-665).
The variable domain of the TNFR 1-specific Fab 13.7 molecule was fused to the N-terminus of the CH2 domain of an Fc chain containing CH31 or CH3 kappa with a short peptide linker, and VL was fused to the CH2-CH31 chain (VL 13.7-CH2-CH31/VH13.7-CH2-CH3 kappa; VL1C/VH kappa C) by fusing VH to the CH2-CH3 kappa chain to give a monovalent TNFR 1-specific antagonistic antibody derived molecule (Fv-Fc 1 kappa fusion protein), designated Atrosimab (72 kDa). Atrosi due to mutations introduced into Fc1 kappamab lacks the ability to mediate Fc effector functions; lack of binding to immune system effector molecules prevents activation of TNFR1 due to secondary cross-linking of Atrosimab bound to fcγr expressing cells. Atrosimab has high affinity (K) D 2.7 nM) binds to TNFR1, inhibits TNF-induced activation of TNFR1, and IC in various in vitro assays and in the presence of anti-human IgG antibody (i.e., cross-linked antibody) antibodies 50 Values of 16-55nM and show improved pharmacokinetic properties. TNFR1 binding and inhibition was slightly reduced compared to the parental Fab 13.7 molecule, which can be attributed to the change in VH and VL pairing after fusion to the CH2 domain. The initial and final half-lives of Atrosimab were determined to be 2.2+/-1.2 hours and 41.7+/-18.1 hours, respectively, with an AUC of 5856+/-1369.9 μg/ml×h. The terminal half-life of Atrosimab was nearly 40-fold longer compared to Fab 13.7, 1.3-fold longer compared to ATROSAB; however, these values may be inaccurate because of the lower injection doses of Fab 13.7 and ATROSAB, which can affect pharmacokinetic properties (see, e.g., richter et al (2019) mAbs 11 (4): 653-665).
TNFR1 antagonists based on domain antibodies (dAbs)
Another class of therapeutic antibodies is small fragments of domain antibodies (dAbs; also known as single domain antibodies or sdAbs) which are monomeric and contain the heavy chain of the antibody (V H ) Or light chain (V) L ) Is a variable domain of (a). dabs are the smallest antigen-binding fragments of antibodies; the size of which is about 11-15kDa, which is about one tenth of the size of an intact monoclonal antibody (mAb). Similar to dabs, nanobodies found in camelids produce antibodies containing only heavy chains, where the antigen binding site is a single unpaired variable domain, termed V HH . In the dAb, each V H And each V L Three Complementarity Determining Regions (CDRs) thereon; thus, each dAb contains a polypeptide from antibody V H -V L Three of the six CDRs of a pair, which are highly diverse loop regions that bind to the target antigen.
Because of its smaller size, a dAb can be produced in higher yields from bacterial culture and is more suitable for phage display because only one polypeptide chain is produced. Specific dabs with high affinity and potency can be produced rapidly by protein engineering. The small size of dabs also allows for increased tissue penetration, stability and choice of delivery formulation. Because of its small size, molecules can be created that contain linked dabs specific for different antigens/targets, which is not possible with traditional antibodies, and is also difficult to achieve with other antibody fragments (e.g., fab and scFv). Because of the monomeric and monovalent binding modes of dabs, they are suitable for use where targets are not amenable to intervention with monoclonal antibodies. TNFR1 is one such target; TNFR1 is activated/stimulated by antibody-induced receptor cross-linking (see, e.g., holt et al (2003) Trends in Biotechnology (11): 484-490;Schmidt et al (2013) Arthritis & Rheumatism 65 (9): 2262-2273;Goodall et al (2015) PLoS ONE 10 (9): e 0137065).
Compared with macromolecules, small-sized antibody fragments such as dAb, scFvs, fvs, disulfide bond-bonded Fvs, fab and the like are easier to produce and process and can be rapidly distributed throughout the body; however, its short in vivo half-life limits its therapeutic effect. Like other antibody fragments, increasing the serum half-life of dabs can increase therapeutic efficacy and reduce dosing frequency, particularly in applications where binding to antigens in the bloodstream is desired, such as in the treatment of rheumatoid arthritis or cancer. This can be achieved by pegylation, conjugation to serum albumin, fusion to a second dAb that specifically binds serum albumin, or fusion to an Fc fragment or intact antibody constant region. Fusion to the Fc region also allows recruitment of Fc effector functions, including complement activation, antibody-dependent cytotoxicity, or Fc-mediated clearance of immune complexes (see, e.g., holt et al (2003) Trends in Biotechnology (11): 484-490;Goodall et al (2015) PLoS ONE 10 (9): e 0137065).
a) anti-TNFR 1 dAb-anti-albumin dAb fusion constructs
DMS5540 is a 25kDa mouse TNFR1 antagonist, which is a bispecific single variable domain antibody containing a non-competitive (non-interfering with TNF binding) anti-TNFR 1 dAb fused to an albumin binding dAb (AlbudAb; to extend serum half-life). DMS5540 does not bind human TNFR1, which was found to inhibit tnfα -mediated cytotoxicity in mouse fibroblast L929 (highly sensitive to tnfα -mediated cytotoxicity). DMS5540 was intravenously injected into mice, tnfα was intravenously injected four hours later, and serum IL-6 levels were assessed. DMS5540 exhibited a dose-dependent inhibition of tnfα -mediated signaling effects in vivo, as determined by IL-6 responses, as compared to mice administered with control dabs (DMS 5538) or no dabs that lack specific antigen binding but are fused to an AlbudAb (see, e.g., goodall et al (2015) PLoS ONE 10 (9): e 0137065).
In another study, mice with collagen-induced arthritis (CIA) were treated with DMS5540, isotype (negative) control dAb (DMS 5538) or murine TNFR2 genetically fused to a mouse IgG1 Fc domain (mTNFRII-Fc; mtnfr2. Fc) for 10 days starting from the day of arthritis onset, which blocked both receptors (TNFR 1 and TNFR 2) and inhibited mouse TNF, and monitored for disease progression. The concentration of systemic cytokines was measured, the number of T cell subsets in lymph nodes and spleen was assessed, and the intrinsic Treg cell function was assessed. Blocking TNFR1 with DMS5540 and TNFR1/2 with mTNFRII-Fc similarly inhibited disease progression compared to the negative control, indicating that blocking TNFR1 or TNF protects the joint from inflammatory mediators that cause joint damage in arthritis. Effector T cell activity, measured in terms of expression levels of pro-inflammatory cytokines (e.g., ifnγ, IL-10, and RANTES), increased following blocking of TNFR1/2 with mTNFRII-Fc, but not following selective blocking of TNFR1 with DMS5540, indicating immunomodulatory effects on TNFR2 signaling (e.g., T cell effector function inhibition). Furthermore, blocking TNFR1 instead of TNFR1/2 resulted in expansion and activation of Treg cells, while increased expression of FoxP3 and TNFR2 was observed in joints undergoing remission, both of which were expressed by Treg, suggesting a role in the resolution of inflammation. These results indicate that inhibition of TNFR1 (but not TNFR 2) signaling can inhibit inflammation and promote Treg cell inhibitory activity, enhancing therapeutic efficacy compared to traditional TNF inhibition methods (see, e.g., mcCann et al (2014) archlitis & rheometer 66 (10): 2728-2738).
DMS5540 is also more effective than mtnfr2.Fc (anti-TNF) in preventing inflammation-induced osteoclast formation and bone loss in an in vivo mouse model of Lipopolysaccharide (LPS) -induced osteolysis. TNFR2 deficient mice exhibited an LPS-induced increase in bone destruction. In vitro, the human equivalent DMS5541 of DMS5540 contains an anti-human TNFR1 dAb that is more effective in reducing human osteoclastogenesis than etanercept in the presence and absence of low doses of TNF. These results indicate that TNFR2 signaling has bone protective effects. Thus, selective inhibition of TNFR1 may also be useful in therapeutic interventions for inflammatory bone loss diseases such as osteomyelitis and peri-prosthetic osteolysis and aseptic loosening (see, e.g., esperito Santo et al., biochem. Biophys. Res. Commun. 464:1145-1150).
DMS5541 (also known as TNFRI-AlbudAb) contains a non-competing human TNFR1 specific dAb fused to an AlbudAb, selective blockade of TNF signaling by TNFR1 was assessed in ex vivo cultured human Rheumatoid Arthritis (RA) synovial Monocytes (MNCs) that express TNFR1 and TNFR2 and spontaneously produce inflammatory cytokines and chemokines in the absence of exogenous stimulation. DMS5541 inhibits the production of the pro-inflammatory cytokines GM-CSF, IL-10, IL-1 beta and IL-6 and chemokines IL-8, RANTES (CCL 5) and MCP-1 (CCL 2) at levels similar to etanercept blocking of TNF ligands. This inhibition is not due to cytotoxicity, as DMS5541 inhibits tnfα -induced cytotoxicity in human rhabdomyosarcoma KYM-1D4 in a dose-dependent manner, similar to TNF blocking effects of etanercept. In addition, DMS5541 inhibited the production of soluble TNFR1, but not soluble TNFR2, indicating selectivity for TNFR 1. These results indicate that the TNFR1 pathway is the primary inflammatory pathway leading to the TNF response observed in the in vitro cultured RA synovial MNC disease model (see, e.g., schmidt et al (2013) Arthritis & Rheumatism 65 (9): 2262-2273).
b) Domain antibody fragments designated GSK1995057 and GSK2862277
A domain antibody fragment called GSK1995057 (see SEQ ID NO: 55) is a short-acting fully human domain antibody (dAb) fragment (containing a VH chain) that selectively antagonizes TNF signaling through TNFR1 but not TNFR 2. Because of its small size, GSK1995057 can be nebulized directly into the lungs and has been studied in animal and human models for the treatment of Acute Respiratory Distress Syndrome (ARDS) by inhalation. GSK1995057 reduces pulmonary inflammation in non-human primate (cynomolgus monkey) and human ARDS models. Pulmonary neutrophil infiltration is central to the pathogenesis of ARDS and may be increased by impairment of alveolar capillary barrier caused by pro-inflammatory mediators. TNF- α contributes to an increase in endothelial permeability, while GSK1995057 prevents this increase, suggesting that TNFR1 signaling mediates TNF-induced endothelial permeability (see, e.g., proudboot et al (2018) Thorax 73:723-730). The test failed due to its inherently short half-life and neutralization by the autoantibody. The immunogenicity of GSK1995057 may be more due to improper folding of proteins produced in e.coli than failure of correct humanization of dabs; it is derived from a human antibody fragment, and only the hypervariable sequences are altered to accommodate the specificity of TNFR1 (see, e.g., international PCT publication No. WO 2008/1494148A 2).
In monkeys exposed to single inhalation Lipopolysaccharide (LPS) challenges, which is a mature model, clinical-related inflammatory responses triggered to mimic sub-clinical tissue injury, pretreatment with GSK1995057 reduced neutrophil infiltration, pro-inflammatory chemokine levels, endothelial injury markers, and alveolar-capillary leakage in a dose-dependent manner. The results indicate that inhalation of GSK1995057 can produce the same results as higher doses of parenterally administered antibodies. In one clinical trial, healthy human subjects received a single nebulized dose of GSK1995057 pretreatment followed by exposure to low dose of inhaled LPS, and the pretreated subjects experienced fewer signs of systemic, neutrophilic pulmonary and endothelial injury in response to challenges of LPS compared to placebo-received subjects. Despite these results, it is unlikely to translate into clinic. In experiments, GSK1995057 was administered prior to LPS challenge, but patients with ARDS generally required treatment after initial injury (see, e.g., proudboot et al (2018) Thorax 73:723-730), rather than prior treatment.
Another difficulty is the adverse effect of anti-drug antibodies (ADA) against TNFR1 formulations, which was observed in a clinical phase I study of GSK1995057, where cytokine release infusion reactions were observed at doses of 2-10 μg/kg due to the high levels of pre-existing, naturally occurring anti-immunoglobulin autoantibodies (i.e. ADA) present in approximately 50% of healthy subjects who were not drug tested. In particular, ADA is a human anti-VH (HAVH) autoantibody, the complex of which with the GSK1995057 framework sequence results in activation of TNFR1 signaling, and a mild to moderate transfusion response occurs in subjects with high HAVH autoantibody titers (see, e.g., cordy et al (2015) Clin. Exp. Immunol. 182:139-148).
Binding of HAVH autoantibodies to dAb GSK1995057 framework regions induces cytokine release in vitro. Autoantibodies were identified for GSK1995057 epitopes. A pre-existing drug-resistant antibody (ADA) binds to an epitope near the C-terminal region of a VH dAb, including dAb GSK1995057. To address this problem, a modified dAb, termed GSK2862277 (see SEQ ID NO: 56), was generated by adding a single alanine residue at the C-terminus of the modified dAb. Such modification reduces binding to HAVH autoantibodies. In serum samples of healthy subjects screened positive for HAVH autoantibodies that bind GSK1995057, the frequency of pre-existing autoantibodies was reduced from 51% for GSK 1995057-specific HAVH autoantibodies to 7% for GSK 2862277-specific autoantibodies. In vitro systems and in vivo experiments in animals showed that GSK2862277 did not induce TNFR1 activation even in the presence of GSK2862277 specific autoantibodies, and that pharmacological and biophysical properties of GSK2862277, including target affinity, in vitro potency and in vivo pharmacokinetics and pharmacodynamics, were comparable to that of the parent dAb (GSK 1995057).
One study of the safety, tolerability, pharmacokinetics and pharmacodynamics of single and repeated dose inhalation (i.h.) and intravenous injection (i.v.) phase I clinical trials of GSK2862277 found that GSK2862277 was generally well tolerated when administered by inhalation or intravenous injection. However, one subject developed a mild infusion response and cytokine release following repeated intravenous administration; the subject had high serum levels of pre-existing GSK2862277 antibodies, and the serum antibodies of the subject were shown to activate TNFR1 signaling in an in vitro assay. The interaction between GSK2862277 and autoantibodies results in antibody-mediated, GSK 2862277-dependent cellular TNFR1 cross-linking, agonizing the receptor and resulting in cytokine release. Thus, despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects are still associated with the presence of a new pre-existing antibody response specific for the modified dAb framework. These results highlight the challenges of developing biological antagonists against TNFR1 (see, e.g., cordy et al (2015) Clin. Exp. Immunol. 182:139-148). Thus, there remains a need for improved TNFR1 antagonists.
Nanobody (Nb)
Similar to dabs, nanobodies (Nb) are small antigen-binding fragments derived from camelid heavy chain antibodies, free of light chains. It is small in size (15 kDa), has low immunogenicity and high affinity, is soluble and stable, and is encoded by a single gene/exon (VHH), making it modular and allowing high-yield production in bacteria or yeast.
anti-TNFR 1 nanobody-anti-albumin nanobody fusion constructs
TROS (TNF receptor single silencing agent; also known as Nb Alb-70-96) is a trivalent high affinity nanobody-based human TNFR1 selective inhibitor that competes with TNF for binding to TNFR1. To generate TROS, two anti-human TNFR1 nanobodies (Nb 70 and Nb 96; see SEQ ID NOS: 683 and 684, respectively) were ligated, which have been generated from a VHH library constructed by immunizing alpaca with recombinant human soluble TNFR1 and by (Gly 4 Ser) 3 A linker is attached to the anti-albumin nanobody (Nb Alb) to increase serum half-life to produce trivalent TROS. The serum half-life of the produced TROS is about 24 hours; the serum half-life of monovalent Nb is only-1.5 hours. Treatment with TROS delays onset of experimental autoimmune encephalomyelitis (EAE; an MS model) in mice and prevents established disease; the therapeutic effect is due to the diversion of TNF signaling through TNFR2 and the effects of this signaling. TROS also inhibits inflammation in colon biopsies of patients with Crohn's disease in vitro culture and antagonizes inflammation in acute TNF-induced liver inflammation models of liver chimeric humanized mice (see, e.g., steeland et al (2015) J.biol. Chem.290 (7): 4022-4037;Steeland et al (2017) Sci. Reports 7:13646).
Dominant negative inhibitors of TNF (DN-TNF)/TNF muteins
Another class of TNF inhibitors are the signaling-ineffectively dominant negative inhibitors of TNF (DN-TNF), also known as TNF muteins. DN-TNF is an engineered variant of TNF that has mutations that abrogate binding to and signaling through TNFR1 and TNFR 2. DN-TNF selectively inhibits soluble TNF (sTNF or solTNF) by rapidly replacing subunits with natural TNF homotrimers, forming inactive mixed TNF heterotrimers with disrupted receptor binding surfaces, thereby preventing interaction with TNF receptors. DN-TNF leaves transmembrane TNF (tmTNF) unaffected, maintaining protection from TNF signaling through TNFR 2. DN-TNF inhibits TNF-induced NF- κB activity and caspase-mediated apoptosis, and reduces disease severity in animal models of arthritis and Parkinson's disease. These molecules may be immunogenic due to their structure.
As a selective inhibitor of soluble TNF, DN-TNF does not inhibit tmTNF signaling nor inhibit the resistance of mice to listeria monocytogenes infection, unlike anti-TNF therapies that bind solTNF and tmTNF. An example of DN-TNF is a TNF mutant containing one or more substitutions L133Y, S162Q, Y163H, I173T, Y191Q and A221R, referring to the amino acid sequence shown in SEQ ID NO. 1 (corresponding to residues L57Y, S86Q, Y87H, I97T, Y Q and A145R referring to the solTNF sequence shown in SEQ ID NO. 2), which impairs binding to TNFR. Additional modifications may also be included, for example to enhance expression, allowing site-specific PEGylation (see, e.g., zalevsky et al (2007) J.Immunol.179:1872-1883).
For example, referring to SEQ ID NO. 2, the TNF mutations R32W and S86T resulted in hundreds of fold loss of affinity for TNFR2, but did not affect binding to TNFR 1. The R32W/S86T double mutant eliminates all binding to TNFR2 without loss of binding to TNFR 1. The mutations L29S, L8238Y, R31E, R N, R32Y, R32W, S86T, L S/R32 8239S/S86T, R32W/S86T, L S/R32W/S86T, R N/R32T, R E/S86T, R N/R32T/S86T and E146R with reference to SEQ ID NO 2 also confer selectivity for TNFR 1. Mutations D143N, D143Y, A R and D143N/A145R, referenced to SEQ ID NO. 2, make TNF variants selective for TNFR2 (see, e.g., loetscher et al (1993) J.biol. Chem.268 (35): 26350-26357; U.S. Pat. No. 5,422,104).
One modified TNF (INmuneBio; see SEQ ID NO: 701), known as XPro1595, is a pegylated soluble DN-TNF mutein that preferentially inhibits TNFR1 signaling and contains the mutations V1M, R31C, C69V, Y87H, C101A and A1456R, see, for example, SEQ ID NO:2 (see, e.g., U.S. publication No. 2015/0239951). XPro1595 reduces neuroinflammation and is being investigated for treatment of Alzheimer's disease (see, e.g., clinical trial number NCT 03943264). XPro1595 blocks amyloid pathology in the Alzheimer's disease mouse model (3 xTgAD), prevents loss of neuronal communication and cognitive impairment in the different (tgCRND 8) Alzheimer's disease mouse model, alleviates dysfunction and cognitive deficit in normal neuronal communication in normal aged rats, and prevents amyloid pathology, cognitive impairment and neuronal communication dysfunction in young mice in the third Alzheimer's disease model (5 xFAD). In aged mice with alzheimer's disease-like pathology, XPro1595 reduced amyloid, improved cognition, saved neuronal communication, and also normalized the innate and adaptive immune response.
Compared to TNFR2, TNFR1 levels were higher in the hippocampus of aged (22 months) but not young adult (6 months) Fischer 344 rats. When treated with XPro1595, aged rats exhibited better Morris water maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased GluR1 glutamate receptor levels, and reduced L-type voltage-sensitive Ca 2+ The activity of channel (L-VSCC) hippocampal CA1 neurons suggests that changes in function associated with brain aging may occur due to selective changes in TNF signaling. XPro1595 inhibits neuroinflammation and microglial activation in animal models of Parkinson's disease and aging. In EAE (MS model), XPro1595 may improve disease, improve remyelination, and reduce CNS injury and neuroinflammation. XPro1595 may also improve inflammatory arthritis and reduce the susceptibility of treated animals to infection. Treatment with XPro1595 delays the onset of EAE and improves symptoms more effectively than etanercept without therapeutic effect. Administration of XPro1595 increased TNR2 expression levels in the diseased area of EAE, indicating that tmTNF signaling through TNFR2 is involved in nerve regeneration (see, e.g., yang et al (2018) front. Immunol.9:784; sama et al) al. (2012) PLoS ONE 7 (5): e 38170). XPro1595 does not block the inflammatory effects of TNFR1 because it does not inhibit the activity of transmembrane TNF (activates TNFR1 and TNFR 2). This also applies to other dominant negative TNF reagents, as described below.
XENP345 (see SEQ ID NO: 702) is a PEGylated DN-TNF mutein containing the mutation I97T/A145R, see SEQ ID NO:2.XENP345 has neuroprotective effects on in vivo neutralization of soluble TNF (solTNF) in animal models of Parkinson's disease and Alzheimer's disease, reduces neuronal degeneration and cognitive dysfunction, and slows down progression of neurodegenerative diseases (see, e.g., mcCoy et al (2006) J. Neurosci.26 (37): 9365-9375;McAlpine et al. (2009) neurobiol. Dis.34 (1): 163-177).
R1antTNF (see SEQ ID NO: 703) is a TNFR1 selective antagonistic mutant TNF identified from a phage library displaying structural human TNF variants in which each of the six amino acid residues at the receptor binding site corresponding to residues 84-89 of SEQ ID NO. 2 has been mutated. R1antTNF contains the mutations A84S, V85T, S86T, Y H, Q N and T89Q, has similar TNFR1 affinity to wild-type human TNF, and does not interfere with TNFR2 activity. R1antTNF ameliorates liver injury as evidenced by decreased serum levels of alanine aminotransferase and the pro-inflammatory cytokines IL-2 and IL-6 in two models of acute hepatitis. However, as with wild-type TNF, the plasma half-life of R1antTNF is very short (12 minutes). To increase the in vivo half-life of R1antTNF, PEG-R1antTNF was produced in pegylated form, wherein PEG was conjugated to the N-terminal site of R1 antTNF. PEG-R1antTNF reduces morbidity, improves disease symptoms, improves demyelination, and inhibits Th1 and Th17 cell activation and inflammatory T cell infiltration in spinal cord in the EAE mouse model. PEG-R1antTNF also inhibits NF- κB, inhibits smooth muscle cell proliferation, reduces chemokine and adhesion molecule expression, and thereby reduces intimal hyperplasia and arterial inflammation following induction of femoral artery injury in an external vascular cuff model in IL-1 receptor antagonist deficient mice. When comparing the effect of PEG-R1antTNF and etanercept on viral immunity using recombinant adenovirus vectors, PEG-R1antTMF does not reactivate viral infection nor affect clearance of injected adenovirus, whereas viral load increases following etanercept treatment. PEG-R1 anti-TNF treatment also delays and ameliorates CIA symptoms in a prophylactic and therapeutic setting and is more effective than etanercept when used to treat established CIA (see, e.g., yang et al (2018) front. Immunol.9:784;Shibata et al (2008) J. Biol. Chem.283 (2): 998-1007;Kitagaki et al (2012) J. Atherocler. Thromb.19 (1): 36-46; fischer et al (2015) Antibodies 4:48-70;Horiuchi et al (2010) 6249:1215-1228).
Soluble TNFR1 is also associated with an increased risk of developing MS; thus, neutralization of soluble TNFR1 by DN-TNF/TNF muteins may be beneficial. In contrast to solTNF inhibitors (e.g., DN-TNF), TNFR1 antagonists can block the binding of lymphotoxin-alpha (LT-alpha), another member of the TNF superfamily, to TNFR 1. LT- α has a pro-inflammatory effect in RA and animal disease models (e.g., CIA and EAE); thus, simultaneous blocking of TNF and LT- α binding to TNFR1 by TNFR1 antagonists has additional benefits in acute and chronic inflammatory diseases and conditions compared to solTNF inhibition (see, e.g., fischer et al (2015) Antibodies 4:48-70).
Tnfr 2-selective agonists
CD4 + FoxP3 + Regulatory T cells (tregs) maintain immune homeostasis and suppress autoimmune responses; tregs also modulate anti-tumor immune responses, allowing tumor immunity to escape. Tregs are thus therapeutic targets for the treatment of e.g. autoimmune and chronic inflammatory diseases and disorders, graft versus host disease (GvHD), graft rejection and cancer. TNF signaling through TNFR2 regulates Treg function and activity. TNFR2 agonists up-regulate Treg activity, while TNFR2 antagonists down-regulate Treg activity. Treg stimulation of the TNF-TNFR2 signaling pathway is useful for lever therapy in a variety of human diseases and conditions, including treatment of autoimmune and chronic inflammatory diseases by agonism, and treatment of cancer by antagonism (see, e.g., zou et al (2018) front. Immunol. 9:594).
TNFR2 agonists include antibodies, such as monoclonal TNFR2 agonist antibodies and antigen-binding fragments thereof, peptides and proteins, such as TNFR 2-selective TNF muteins, fusion proteins and small molecules. As described hereinProvides that specific agonism of TNFR2 induces expansion and activation of tregs, thereby modulating the immune system and reducing autoreactive CD8 of damaged tissue + T cell activity and induces signaling pathways with anti-inflammatory effects as well as cell survival, regeneration and protection, including neuroprotection, cardioprotection, gut protection and bone protection. Thus, enhancement of TNFR2 signaling with a TNFR 2-selective agonist is useful for enhancing the therapeutic effect of TNFR 1-specific antagonism, particularly in the treatment of autoimmune and chronic inflammatory diseases and conditions in which anti-TNF therapy/TNF-blocker therapy fails, including neurodegenerative diseases.
Tnfr2 agonist antibodies
Human TNFR2 selective agonist antibodies include commercially available MR2-1 (monoclonal mouse IgG1 that binds human, cynomolgus and rhesus TNFR 2; hycult Biotech), and clone MAB2261 (monoclonal mouse IgG2A that binds human TNFR 2; R&D Systems). TNFR2 agonists, such as antibodies, can be effective in stimulating expansion of FoxP3+ Treg homogeneous populations in CD4 cell culture and up-regulating expression of TNF, TRAF2, TRAF3, BIRC3 (cIAP 2) and FoxP3 mRNA. Magnetically Activated Cell Sorting (MACS) purified CD4 + CD25 + Cells, cultured using standard in vitro human Treg expansion protocols (i.e., using anti-CD 3 antibodies, anti-CD 28 antibodies, IL-2, and rapamycin), produced expanded tregs with higher levels of FoxP3 (and other characteristic Treg markers) and more potent inhibitory capacity when compared to the absence of TNFR2 agonist antibodies in the presence of TNFR2 agonist antibodies. Tregs isolated from type 1 diabetics exhibit a resting phenotype, which are activated and amplified after treatment with TNFR2 agonist antibodies in vitro; such tregs more effectively inhibit autologous CD8 + T cells (see, e.g., zou et al (2018) front. Immunol. 9:594).
Treatment of isolated tregs expanded using standard in vitro protocols with MR2-1, a commercially available agonistic human TNFR2 monoclonal antibody (mAb) containing mouse IgG1, resulted in homologous FoxP3 + Helios + CD127 low Treg populations; these tregs retain their phenotypical and highly inhibitory activity in a humanized mouse model. Because ofIn this regard, TNFR2 agonists may enhance ex vivo expansion of Treg cells from an impure cell population for Treg-based immunotherapy (see, e.g., zou et al (2018) front. Immunol. 9:594).
Tnfr 2-selective TNF muteins and fusions thereof
TNF can be engineered to selectively bind TNFR1 or TNFR2, as described herein; for example, a TNFR 2-selective TNF mutein is a variant of TNF that contains one or more mutations that increase binding to TNFR2 and/or reduce or eliminate binding to TNFR 1. TNFR2 selective mutations include non-conservative substitutions of the Asp residue at position 143 of soluble TNF (see SEQ ID NO: 2), such as D143Y, D F or D143N, or non-conservative substitutions of the Ala residue at position 145 of soluble TNF, such as A145R (see, e.g., U.S. Pat. No. 9,081,017). Other mutations in TNF that confer selectivity for TNFR2 include, but are not limited to, for example: K65W, D143E, D143W, D143V, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, and D143V/A145S, reference to SEQ ID NO:2 (see, e.g., U.S. patent publication 2020/0102362).
TNF ligand trimerization is critical for signaling through TNFR. At low concentrations, e.g. in serum, the trimer dissociates, leading to its degradation. In order to produce functionally active receptor-specific TNF muteins, stable trimers must be created. TNF07 is a soluble TNF (sTNF or solTNF) mutein containing the mutation S95C/G148C (relative to the residue sequence shown in SEQ ID NO: 2) that forms stable TNF trimers and acts as a TNFR2 agonist. The S95C/G148C mutation results in the formation of an intermolecular Cys-Cys covalent bond; due to covalent internal disulfide cross-linking of sTNF at key positions between TNF monomers, stable trimers are formed. Despite the lack of TNFR2 selective mutations, TNF07 acts as a TNFR2 agonist. TNF07 induced withEfficient TNFR2 signaling, amplification of FoxP3 + Treg cells and selectively induce autoreactive CD8 isolated from type 1 diabetics + T cell death (see, e.g., ban et al (2015) Molecular and Cellular Therapies 3:7; zou et al (2018) front. Immunol. 9:594).
Several TNFR2 agonists have been generated, comprising fusion of a single-chain TNFR 2-selective TNF mutein trimer with a multimerizing domain. As described herein, the primary ligand of TNFR2 is membrane-bound TNF (memtNF; also referred to herein as transmembrane TNF or tmTNF). Adding a multimerization domain, such as a dimerization or trimerization domain, to generate hexamer or nonamer molecules, respectively, for TNF subunits; these hexamers and nonamers of TNF mimic membrane-bound TNF trimers and are therefore able to effectively activate TNFR2 signaling. Commonly used dimerization domains include EHD2, which is derived from the heavy chain C of IgE H 2 domain, and MHD2, derived from heavy chain C of IgM H 2 fields. Dimerization domains may also include Fc domains, such as those derived from IgG1 and IgG4, optionally including modifications that alter immune effector function. Commonly used trimerization domains include chicken tenascin-C (TNC) and human TNC. Dimerization and trimerization enhance TNFR2 signaling and improve the half-life of the fusion protein, e.g., by increasing the molecular weight of the molecule and/or by introducing FcRn recycling, e.g., when the dimerization domain is Fc.
STAR2 (also known as TNC-sc-mTNNF (221N/223R)) is a nonamer agonistic TNFR 2-specific mouse TNF variant that does not bind TNFR1, is a single chain mouse TNF trimer, wherein each TNF subunit is residues 91-235 of SEQ ID NO:5 fused to the trimerization domain of chicken tenascin C (cTNC), corresponding to residues 110-139 of SEQ ID NO:804 (see also SEQ ID NO: 805). Three single chain mouse TNF subunits are composed of two (GGGS) 4 Peptide linkers are attached (see, e.g., residues 116-120 of SEQ ID NO: 707), and the TNC trimerization domain is attached to the N-terminus of the first TNF subunit in the single-chain trimer. The specificity of STAR2 for TNFR2 results from mutations D221N and A223R within a single TNF subunit (see the sequence of mouse TNF, shown in SEQ ID NO: 5), which creates a spatial conflict between STAR2 and mouse TNFR 1. Fusion to TNC Trimerization domains result in spontaneous oligomer formation, generate three covalently linked TNF trimers, and mimic membrane-bound TNF. STAR2 stimulates Treg proliferation in vitro and in vivo with a TNFR2 dependent, IL-2 independent mechanism. Pretreatment of allogeneic hematopoietic stem cells with STAR2 prior to transplantation in mice prolonged survival and reduced severity of GvHD in a TNFR-2 and Treg dependent manner. Human equivalent TNC-scTNF (143N/145R) of TNFR 2-specific STAR2 agonist consisting of residues 9-157 of soluble TNF (see SEQ ID NO: 2), contains a mutation D143N/A145R of reference SEQ ID NO:2 (solTNF) effective to stimulate CD4 isolated from healthy donors + In vitro expansion of CD4 in T cells + FoxP3 + Treg (see, e.g., chopra et al (2016) J.Exp. Med.213 (9): 1881-1900; zou et al (2018) front. Immunol. 9:594).
TNC-scTNF R2 Is a soluble human TNFR2 agonist that is a fusion of the trimerization domain of human tenascin C (hTNC) containing residues 110-139 of SEQ ID NO:806 (see also SEQ ID NO: 807) with the N-terminus of a TNFR 2-selective single-chain TNF variant (scTNFR 2; SEQ ID NO: 803) containing three TNF domains linked by two short peptide linkers (GGGGS). TNFR2 Selective TNF molecule scTNFR2 is similar to soluble trimeric TNF in that each TNF subunit comprises amino acids 80-233 of full-length TNF shown in SEQ ID NO:1 (corresponding to residues 4-157 of SEQ ID NO: 2) with a mutation D143N/A145R, referred to SEQ ID NO:2, that eliminates binding to TNFR 1. Since TNFR2 is fully activated only by membrane-bound TNF, not by soluble TNF trimers, the trimerization domain of TNC is fused to scTNF R2 Is used for generating TNC-scTNF at the N terminal of the sample R2 。TNC-scTNF R2 Exists in the trimeric assembly of single chain fusion proteins, resembling nonamer TNF molecules; such oligomeric TNF muteins mimic membrane-bound TNF (memnf) activity due to their increased avidity, induce TNFR2 clustering and formation of TNFR2 signaling complexes, effectively activating TNFR2.TNC-scTNF R2 Has neuroprotective properties; it protects neurons from superoxide-induced cell death and rescues neurons from catecholaminergic cell death. TNC-scTNF in vitro models of Parkinson's disease R2 Neurons were rescued after 6-OHDA induced cell death.These results indicate TNC-scTNF R2 The neurodegenerative process can be improved (see, for example, fischer et al (2011) PLoS ONE 6 (11): e 27621).
EHD2-scTNF R2 (see SEQ ID NO: 810) is an agonistic TNFR2 selective TNF mutein fusion protein comprising a covalently stabilized human TNFR2 selective single chain TNF trimer (scTNF) R2 The method comprises the steps of carrying out a first treatment on the surface of the 803) with a mutation D143N/A145R (residue number of soluble TNF, as shown in SEQ ID NO: 2) that eliminates binding to TNFR1, fused to dimerization domain EHD2 (SEQ ID NO: 808) derived from IgE heavy chain C H 2, and produces disulfide dimers containing hexameric TNF domains. scTNF (scTNF) R2 Each TNF subunit within contains residues 4-157 of SEQ ID NO. 2. EHD2 is fused to trivalent human single stranded scTNF via a peptide linker (GGGSGGGSGGGSGGGSGGGSGGSEFLA; SEQ ID NO: 809) R2 N-terminal of scTNF R2 Is linked by two GGGGS peptide linkers. EHD2-scTNF R2 Neuroprotective properties have been shown in NMDA-induced acute neurodegenerative mouse models (see, e.g., dong et al (2016) Proc. Natl. Acad. Sci. U.S. A.113 (43): 12304-12309; and U.S. patent publication No. 2020/0102362).
The TNFR2 agonist fusion proteins also include single chain TNFR2 agonists (scTNF) R2 ) Three TNF muteins (cf. SEQ ID NO: 2) containing the mutation D143N/A145R, which eliminate binding to TNFR1, fuse with the dimerization domain as Fc, resulting in a hexamer protein (scTNF) relative to the TNF domain R2 -Fc). The Fc may be an IgG4 or IgG1 Fc, optionally containing mutations that eliminate Fc effector functions, such as ADCC and CDC. Three TNF muteins comprising residues 12-157 of SEQ ID NO. 2 are linked together by two short peptide linkers and the dimerization domain is linked to a single chain trimeric TNF molecule (scTNF) by a third short peptide linker R2 ) N-terminal or C-terminal of (C-terminal). The three linkers may all be the same or may be different, and may include GS linkers, such as residues 116-121 of SEQ ID NO:707 (GGGGS) n And/or Gly and Ser, where n=1-5, or may include a TNF- α stalk region (GPQREEFPRDLLSLISPLAQAVRSSSRTPSDK (SEQ ID NO: 812), corresponding to SEQ ID NOResidues 57-87) of NO:1, at least 10, 15 or 20 consecutive residues. Dimerization enhances TNFR2 agonist signaling and also improves the half-life of the fusion protein. Alternative dimerization domains that may be used in the fusion protein include Fc fusion proteins derived from other dimerization molecules, such as IgE heavy chain domain 2 (EHD 2; see SEQ ID NO: 808) and IgM heavy chain domain 2 (MHD 2; see SEQ ID NO: 811) (see, e.g., international application publication No. WO 2019/226750).
3. anti-TNFR 2 antagonist antibodies and small molecule inhibitors
TNFR2 antagonists inhibit proliferation and induce death of tregs, and also inhibit proliferation and induce death of TNFR 2-expressing tumor cells. TNFR2 antagonists can reduce or inhibit proliferation of myelogenous suppressor cells (MDSCs), and/or induce apoptosis within MDSCs by binding to TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment. TNFR2 antagonists also induce T effector cells (including cytotoxic CD 8) by inhibiting Treg expansion and activity + T cells). Accordingly, TNFR2 antagonists are useful in the treatment of infectious diseases and certain cancers that express TNFR2, such as T-cell lymphomas (e.g., hodgkin's lymphoma and cutaneous non-Hodgkin's lymphoma), ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer (see, e.g., U.S. patent publication No. 2019/0144556;Torrey et al (2017) Sci.Signal.10: eaaf 8608).
As discussed herein, expression of TNFR2 is limited to specific immune cells, including tregs and MDSCs, endothelial cells, and specific neurons and cardiomyocytes. Limited expression of TNFR2 makes it an ideal drug target because systemic toxicity is less likely to occur with anti-TNFR 2 therapeutics.
TNFR2 antagonist antibodies and antigen-binding fragments thereof bind to an epitope within human TNFR2 that contains one or more residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4), or a larger epitope containing residues 130-149, 137-144, or 142-149 or at least 5 consecutive or non-consecutive residues within these epitopes, and do not bind to an epitope containing residue KCSPG (corresponding to residues 56-60 of SEQ ID NO: 4). TNFR2 antagonists may also bind to the TNFR2 epitopes PECLSCGS (corresponding to residues 91-98 of SEQ ID NO: 4), RICTCRPG (corresponding to residues 116-123 of SEQ ID NO: 4), CAPLRCR (corresponding to residues 137-144 of SEQ ID NO: 4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4) and VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or epitopes containing at least 5 consecutive or non-consecutive residues within residues 75-128, 86-103, 111-128, or 150-190 of SEQ ID NO:4 (see, e.g., U.S. patent publication No. 2019/0144556).
In general, an antagonistic TNFR2 antibody or antigen binding fragment thereof binds an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 840) with an affinity that is, for example, at least 10-fold higher than the affinity of the same antibody or antigen binding fragment for a peptide containing the KCSPG sequence (SEQ ID NO: 839) of human TNFR 2. An antibody or antibody fragment that binds an epitope containing one or more residues of a KCRPG sequence and an epitope containing a KCSPG motif with similar affinity (e.g., less than a 10-fold difference in affinity) is not an antagonistic TNFR2 antibody. Antagonistic TNFR2 antibodies include TNFRAB1 (heavy and light chain sequences of TNFRAB1 are shown in SEQ ID NOs:1213 and 1213, respectively), TNFRAB2, and TNFR2A3 (see, e.g., U.S. patent publication No. 2019/0144556 for a description of these antibodies). TNFR2 antagonists also include antibodies and antibody fragments comprising the CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; residues 99-112 corresponding to SEQ ID NO: 1212), TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217) or TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223) or a CDR-H3 sequence having at least about 85% sequence identity thereto. For example, TNFRAB1 specifically binds residues 130-149 containing the TNFR2 residue KCRPG with an affinity 40-fold higher than 48-67 binding residues containing the TNFR2 residue KCSPG (see, e.g., U.S. patent publication No. 2019/0144556).
TNFRAB1 (heavy and light chains see SEQ ID NOS: 1212 and 1213, respectively) is a murine antibody that antagonizes TNF-TNFR2 interactions and binds to an epitope within residues 161-169 (CKPCAPGTF; SEQ ID NO: 1258) of TNFR2 (SEQ ID NO: 4) in addition to the KCRPG sequence that binds to TNFR 2. TNFRAB2, another antagonistic TNFR2 antibody, binds an epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851), and an epitope comprising one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECNSGS; SEQ ID NO: 1248) and 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR 2. TNFR2A3 is a murine antagonistic human TNFR2 antibody found by immunization of mice with human TNFR2 and subsequent CDR mutagenesis, in which the CDR-H3 of the resulting precursor antibody is replaced by CDR-H3 sequence ARDDGSYSPFDYFG (SEQ ID NO: 1223). TNFR2A3 binds to two different epitopes in human TNFR 2; the first epitope comprises residues 140-150 of human TNFR2 (LRKCRPGFGVA; SEQ ID NO: 1463) and contains the KCRPG motif, and the second epitope is the downstream sequence, containing residues 159-171 of human TNFR2 (VVCKPCAPGTFSN; SEQ ID NO: 1464). These data indicate that the CDR-H3 sequence of antagonistic TNFR2 antibodies largely determines the antigen binding properties, and that the CDR-H3 motif is a modular domain that can be substituted in anti-TNFR 2 antibodies that do not exhibit antagonistic activity to confer TNFR2 dominant antagonistic characteristics to such antibodies or antigen binding fragments thereof. For example, replacement of the CDR-H3 sequence of a neutral anti-TNFR 2 antibody (i.e., an antibody that is neither antagonistic nor agonistic) with the CDR-H3 sequence of an antagonistic TNFR2 antibody, e.g., TNFRAB1, TNFRAB2, or TNFR2A3, converts a phenotypically neutral antibody into an antagonistic TNFR2 antibody, e.g., a dominant antagonistic TNFR2 antibody, which is an antagonist that inhibits TNFR2 activation even in the presence of a TNFR2 agonist, e.g., TNF or IL-2 (see, e.g., U.S. patent publication No. 2019/0144556).
The TNFR2 antagonist antibody or antigen-binding fragment thereof may comprise a CDR-H1 sequence as set forth in any one of SEQ ID NOS 1214, 1215 and 1231-1233; the CDR-H2 sequence shown in any one of SEQ ID NOS 1216, 1224 and 1230; the CDR-H3 sequences shown in any one of SEQ ID NOS 1217, 1223 and 1225-1229, or CDR-H3 of TNFRAB1, correspond to residues 99-112 of SEQ ID NO 1212; the CDR-L1 sequence shown in any one of SEQ ID NOS 1218 and 1234-1236, or the CDR-L1 sequence of TNFRAB1, corresponds to residues 24-33 of SEQ ID NO 1213; the CDR-L2 sequence of any one of SEQ ID NO 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1, corresponds to residues 49-55 of SEQ ID NO 1213; or any one of the CDR-L3 sequences shown in SEQ ID NOS 1221, 1222 and 1241-1244, or the CDR-L3 sequence of TNFRAB1, corresponding to residues 88-96 of SEQ ID NO 1213. Exemplary framework regions that may be used to develop humanized anti-TNFR 2 antibodies containing one or more of the above-described CDRs include, but are not limited to, those described in U.S. Pat. Nos. 7,732,578 and 8,093,068 and International application publication No. WO 2003/105782. Another approach to engineering an antagonistic humanized anti-TNFR 2 antibody is to align the heavy and light chain variable region sequences of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3, with the heavy and light chain variable regions of a consensus human antibody. Consensus human antibody heavy and light chain sequences are known in the art (see, e.g., the "VBASE" human germline sequence database; see also Kabat, et al Sequences of Proteins of Immunological Interest, fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242, (1991); tomlinson et al, (1992) J.mol. Biol.227:776-798; and Cox et al, (1994) Eur. J. Immunol. 24:827-836). In this way, variable domain framework residues and CDRs can be identified by sequence alignment. For example, the CDR-H3 of a consensus human antibody may be substituted for the CDR-H3 of an antagonistic TNFR2 antibody, such as the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, to produce a humanized TNFR2 antagonist antibody. Exemplary variable domains of consensus human antibodies include the heavy chain variable domain shown in SEQ ID NO:1245 and the light chain variable domain shown in SEQ ID NO:1246, identified in U.S. Pat. No. 6,054,297 (see, e.g., U.S. patent publication No. 2019/0144556). The CDR-H1 and CDR-H2 sequences of the exemplary consensus sequence of the human antibody heavy chain variable domain of SEQ ID No. 1245 may be replaced with, for example, the corresponding CDR sequences of a phenotypically neutral, TNFR 2-specific antibody, and the CDR-L1, CDR-L2 and CDR-L3 sequences of the exemplary consensus sequence of the human antibody light chain variable domain of SEQ ID No. 1246 may be replaced with the corresponding CDR sequences of a phenotypically neutral, TNFR 2-specific antibody to produce a humanized antagonistic TNFR2 antibody.
Other TNFR2 antagonists, such as those shown in any of SEQ ID NOS 1247-1464, may be identified by screening peptides that bind to an epitope within TNFR2 using techniques known in the art, such as phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display, or any other method known in the art, such as those described in U.S. patent publication No. 2019/0144556.
When the human TNFR2 antagonist mAb was added to standard Treg expansion culture conditions, expansion of tregs was inhibited and its inhibitory activity was reduced (see Zou et al (2018) front. Immunol. 9:594). Two potent dominant anti-human TNFR2 antagonistic antibodies outperform TNF (a natural agonist of TNFR 2), inhibit TNF-induced in vitro expansion of human Tregs, and induce in vitro death of the Tregs. The TNFR2 antagonist specifically binds TNFR2 through the F (ab) region, independently of the Fc region or crosslinking of the antibody, and blocks the binding of TNF to TNFR2 by binding to the anti-parallel dimer of TNFR 2. As a result, TNF-induced activation of NF- κb pathway in Treg was inhibited and conversion of transmembrane TNFR2 (tmTNFR 2) to soluble TNFR2 (sTNFR 2) was inhibited. Tregs isolated from ovarian cancer tissue were found to be more sensitive to TNFR2 antagonist mAb-induced cell death because of higher levels of TNFR2 expression on tumor-infiltrating tregs. TNFR2 antagonists also induce TNFR2 + OVCAR3 (ovarian cancer) tumor cells die, which also express TNFR2. These results demonstrate the therapeutic potential of TNFR2 antagonists to treat tumors by targeting tumor-infiltrating tregs and tumor cells (see, e.g., zou et al (2018) front. Immunol.9:594; torrey et al (2017) Sci. Signal.10:eaaf 8608).
In addition to anti-TNFR 2 antagonistic monoclonal antibodies, small molecules can also inhibit TNFR2. For example, thalidomide is a small molecule synthetic glutamic acid derivative with immunomodulatory and anti-inflammatory properties; thalidomide and its structural analogs lenalidomide and pomalidomide are classified as immunomodulating drugs. Thalidomide and its analogs inhibit TNF synthesis by down-regulating NF-k B, disrupting TNF mRNA, and targeting reactive oxygen species and alpha 1-acid glycoproteins, and also inhibit TNFR2 surface expression on T cells by inhibiting intracellular transport of TNFR2 to the cell surface. Thalidomide has been shown to reduce the number and function of tregs in chronic lymphocytic leukemia patients, whereas combination therapy of lenalidomide and azacitidine can down-regulate CD4 in acute myelogenous leukemia patients + TNFR2 expression on T cells and reduction of TNFR2 + The number of tregs enhances effector immune function. However, thalidomide and its analogues in patients with multiple myeloma increased Treg numbers, possibly due to elevated serum TNF levels following treatment, Indicating thalidomide pair TNFR2 + The effect of Treg is disease-specific (see, e.g., zou et al (2018) front. Immunol. 9:594).
Another small molecule inhibitor of TNFR2 is panobinostat, which is a histone deacetylase inhibitor that reduces FoxP3 expression and inhibits the inhibitory activity of tregs. Combination therapy of panobinostat and azacytidine reduces TNFR2 in blood and bone marrow of patients with acute myelogenous leukemia + The number of tregs, and thus ifnγ and IL-2 produced by effector T cells, is increased, resulting in a therapeutic effect on these patients (see, e.g., zou et al (2018) front. Immunol. 9:594). Cyclophosphamide is a DNA alkylating agent commonly used as a cytotoxic chemotherapeutic agent in cancer treatment that inhibits the immunosuppressive function of tregs at low doses and depletes the maximally inhibitory tregs in PROb colon cancer bearing mice after a single dose administration, resulting in an activated anti-tumor immune response. Cyclophosphamide treatment depletes TNFR2 in a mesothelioma mouse model hi Treg. The combination of cyclophosphamide with etanercept inhibits the growth of established CT26 tumors in mice by blocking TNF-TNFR2 interactions and abrogating Treg activity that expresses TNFR2 (see, e.g., zou et al (2018) front. Immunol. 9:594). Triptolide is an immunosuppressive molecule isolated from the Chinese herbal herb tripterygium wilfordii, inhibits TNF and TNFR2 expression in the colon of a mouse colitis model, also reduces Treg numbers and inhibits tumor growth in melanoma mice (see, e.g., zou et al (2018) front. Immunol. 9:594).
Selective targeting of the TNFR1 and/or TNFR2 axes
As described herein, existing anti-TNF therapeutic agents that block TNF and inhibit its signaling through TNFR1 and TNFR2 are limited in efficacy, tolerability, and safety. anti-TNF therapeutic agents improve RA and other autoimmune and inflammatory diseases and disorders by preventing TNF signaling through TNFR1 and eliminating apoptotic and inflammatory pathways. However, these anti-TNF therapeutic agents also block the beneficial effects of TNFR2 signaling, including protection, survival promotion, regeneration promotion, and anti-inflammatory signaling pathways, as well as expansion of immunosuppressive tregs associated with TNFR2, resulting in serious, sometimes fatal, side effects, including serious infections. Other side effects associated with the use of TNF blocking therapies include congestive heart failure, liver injury, demyelinating diseases/central nervous system diseases, lupus, psoriasis, sarcoidosis, and increased susceptibility to developing other autoimmune diseases and cancers, including lymphomas and solid malignancies. anti-TNF therapeutic agents fail in the treatment of demyelinating and neurodegenerative diseases and exacerbate the disease symptoms.
Provided herein are constructs, including TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and nucleic acids and methods for selectively inhibiting TNF signaling through TNFR1 (see, e.g., fig. 2 depicts an exemplary bispecific construct). Constructs and methods for selectively inhibiting TNF signaling through TNFR1 are also provided, including simultaneously maintaining or enhancing TNFR2 signaling. These constructs and methods provide improved therapeutic approaches for the treatment of diseases and conditions of the TNF/TNFR1 axis. These methods of treatment include, but are not limited to, the treatment of autoimmune, chronic inflammatory, neurodegenerative and demyelinating diseases, disorders and conditions, and cancers that also have an inflammatory component. As described herein, TNFR1 antagonism is accompanied or sequentially and selectively agonizing TNFR2 has a therapeutic effect and can enhance the therapeutic index of selective TNFR1 antagonists by activating desired signaling pathways (e.g., anti-inflammatory pathways that control cell survival and proliferation and NF- κb pathways) and by inducing expansion of immunosuppressive tregs, which remove excess autoreactive/effector T cells from the autoimmune microenvironment that lead to tissue destruction.
Sections 1 and 2 describe methods for TNRF1 and TNRF 2; section 3 outlines the constructs provided herein that solve the problems in previous approaches, particularly those directed to TNFR 1; section 4 describes the structure and components of the constructs provided herein.
1. Selective blocking of TNFR1 with TNFR1 antagonists
However, the use of multivalent agents such as antibodies to TNFR1 is not feasible. TNF trimers bind three TNFR1 chains as a pre-ligand assembly complex, mediated by the pre-ligand assembly domain (panels) of each monomeric TNFR. This is in contrast to most receptor systems where ligand binding is required before clustering can occur on the cell surface. TNF receptors are single transmembrane glycoproteins that have about 28% homology in their extracellular domain with two receptors containing four tandem repeats of a cysteine-rich motif. The intracellular sequences are largely uncorrelated, have little homology to each other, and previous studies have shown a description of their signaling function (Grell et al (1994) J.Immol.153 (5): 1963-72). They contain several motifs of known functional significance. Each of TNFR1 and TNFR2 contains an extracellular pre-ligand binding assembly domain (PLAD) of a pre-complex receptor (unlike the ligand binding domain). When trimeric TNF ligands bind to TNFR trimers in the cell membrane, conformational changes are induced, leading to signal activation (MacEwan (2002) Br J Pharmacol.135 (4): 855-875; and Lo et al (2019) Sci Signal.12 (592): eaav 5637).
Thus, antibodies and other multivalent agents that bind to TNFR1 may be unsuitable for use as antagonists because they may cause superclustering, thereby activating TNFR signaling. On the other hand, monovalent antagonists, such as single domain antibodies (dAb or sdAb), nanobodies (Nb; camelid single domain antibodies), scFv fragments and Fab fragments, bind to one TNFR1 molecule without inducing cross-linking or clustering of the receptor at the cell surface, eliminating any activation of TNFR1 signaling. Monovalent antagonists may bind to domains 1, 2, 3, or 4 of the extracellular domain of TNFR1, or to epitopes spanning multiple domains (see, e.g., U.S. patent nos. 9,028,817 and 9,028,822), but these existing antagonists are ineffective therapeutic agents. Various problems include short serum half-life, immunogenicity, and other issues. Selective blocking of TNFR1 can be achieved with TNFR1 antagonists having the properties described and provided herein.
2. Selective activation of NFR2 with TNFR2 agonists
As described herein, selective activation of TNFR2 can be achieved using TNFR 2-specific agonists, which can include, for example, TNFR2 agonistic antibodies and antigen binding fragments thereof, as well as TNFR 2-selective TNF muteins and fusion proteins thereof. Antigen binding fragments of antibodies that bind to the first and/or second epitopes of human TNFR2 may be used. The first epitope of TNFR2 comprises amino acid residues 48-67 of SEQ ID NO. 4 and the second epitope comprises position 135 of SEQ ID NO. 4, including, for example, residues 128-147, 130-149, 135-147 or 135-153 of SEQ ID NO. 4 (see, for example, international application publication No. WO 2014/124134; and U.S. Pat. No. 9,821,010). Other epitopes on TNFR2 have been identified and can be used to design antigen-binding fragments that are TNFR2 selective, as described below.
In contrast to antagonism of TNFR1, to agonize TNFR2, dimer and trimer molecules are used to mimic the effects of membrane-bound TNF, the primary ligand that activates TNFR 2. Thus, TNFR2 agonists include TNFR 2-selective TNF muteins and antibody fragments. Exemplary are TNF muteins and antibody fragments fused to multimerization domains, particularly dimerization or trimerization domains, as described below. To extend the half-life of these molecules, they can be conjugated or coupled to polyethylene glycols with or without cleavable linkers (see e.g., santi et al (2012) proc.Natl. Acad.Sci.U.S. A.109:6211-6216), or fused or conjugated to half-life extending proteins or peptides, such as human serum albumin (with or without FcRn optimization, and whether themselves are pegylated); and ADCC-inactivated/FcRn-optimized Fc domains of antibodies, with or without PEGylation (see, e.g., strohl (2015) BioDrugs 29 (4): reviews of 215-239). Half-life extenders include, for example, polyethylene glycol, glycosylation modifications, sialylation, PAS (polymers of PAS amino acids, about 100-200 residues in length), ELP (see, for example, floss et al (2010) J.trends Biotechnol.28 (1): 37-45), hapylation (glycine homopolymer), fusion with human serum albumin, fusion with GLK, fusion with CTP, GLP fusion with constant fragment (Fc) domains of human immunoglobulin (IgG), fusion with transferrin, fusion with unstructured polypeptides, such as XTEN (also known as rPEG, gene fusion of non-precisely repeated peptide sequences, containing A, E, G, P, S and T, see, for example Schellenberger et al. (2009) NatBiotechnol.27 (12): 1186-90), and other modifications and fusions that increase size, increase hydrodynamic radius, alter charge or target receptor for recycling instead of clearance, and combinations of such modifications. Specific examples of half-life extenders are discussed and illustrated in detail below.
TNFR1 antagonist construct, TNFR2 agonist construct; multispecific, including bispecific TNFR1 antagonists and TNFR2 agonist constructs
Thus, provided herein are constructs for inhibiting tnffr 1 signaling/activity and/or for agonizing TNFR 2. Included among the constructs provided herein are constructs discussed below that are multispecific, e.g., bispecific that inhibit TNFR1 signaling and agonize TNFR 2. Care was taken in designing these constructs because the bispecific antagonists TNFR1 or TNFR2 inhibit the ability of TNF to induce an activation change in resting trimeric TNFR conformation, thereby preventing its signaling. Other multimeric molecules risk receptor aggregation, forcing TNFR to signal cell inflammation and apoptosis. The multispecific constructs herein typically target a different receptor, such as each of TNFR1 and TNFR 2. By inhibiting TNFR1 signaling and advantageously agonizing TNFR2 activity, this provides improved treatment of diseases, disorders, and conditions involving TNF.
Among the constructs provided herein are TNFR1 antagonist constructs. These include fusion protein constructs, such as TNFR1 antagonist-Fc fusion constructs. As described herein and illustrated in the examples, TNFR1 antagonists that specifically target TNFR1, do not antagonize or substantially antagonize TNFR2, or comprise or exhibit TNFR2 agonist activity can be selected, generated, or designed. The TNFR1 antagonist constructs improve the therapeutic efficacy and safety of previous TNFR1 antagonists, including monovalent antagonists such as dAbs, scFvs, and Fab.
Also provided are selective TNFR2 agonist constructs, such as TNFR2-Fc fusion constructs that improve the efficacy of existing TNFR2 agonists. For example, as shown herein, the half-life of the Fc fusion construct increases the half-life of an existing TNFR1 antagonist or TNFR2 agonist, e.g., which reduces dosing frequency, increases patient compliance, and increases therapeutic index. Also provided are selective TNFR2 agonist constructs, such as TNFR2-Fc fusion constructs that improve the efficacy of existing TNFR2 agonists. For example, as shown herein, the half-life of the Fc fusion construct increases the half-life of an existing TNFR1 antagonist or TNFR2 agonist, e.g., which reduces dosing frequency, increases patient compliance, and increases therapeutic index. Alternative half-life extenders, including pegylation and peptide fusion candidates, are discussed above, exemplary extenders are detailed below (reviewed in Strohl (2015) BioDrugs 29 (4): 215-239, see also Tan et al (2018) Current Pharmaceutical Design: 4932-4946), but also include pegylation using linear or branched PEG (see, e.g., swierczewska et al (2015) Expert Opin Emerg Drugs (4): 531-536).
The TNFR1 agonist structure comprises an optional linker and an optional activity modulator. They may be assembled in any order. The structure of the TNFR1 antagonist construct can be represented by formula 1:
(TNFR 1 inhibitor) n -a joint p - (activity modulating agent) q Of formula 1a, or
(Activity regulator) q-linker p - (TNFR 1 inhibitors) n Formula 1b, wherein:
n and q are integers and are each independently 1, 2 or 3; p is 0, 1, 2 or 3; an activity modulator is a moiety, e.g., a polypeptide, such as albumin or Fc, modified to have reduced or no ADCC activity, which increases the serum half-life of the TNFRl inhibitor; an inhibitor of TNFR1 is a molecule, such as a polypeptide or a small pharmaceutical molecule, that binds to TNFR1 and inhibits its activity. The activity modulator is not a human serum albumin antibody or an unmodified Fc. Also provided are TNFR2 agonists of formula 3: (TNFR 2 agonist) n -a joint p - (activity modulating agent) q Wherein n, p and q, the linker and the activity modulator are as shown in formula 1.
Also provided are multispecific, including bispecific constructs containing a TNFR1 antagonist (TNFR 1 inhibitor) and a TNFR2 agonist linked directly or through a linker. Such constructs may include a TNFR1 antagonist of the formula above or may have a structure as shown in formula 2 below. The bispecific and multispecific constructs selectively inhibit inflammation and detrimental TNFR1 signaling, enhancing protective and anti-inflammatory TNFR2 signaling. They include moieties that provide favorable pharmacokinetic properties, including increased serum half-life and stability, and reduced external Zhou Qingchu rates, compared to previous TNFR1 antagonists and TNFR2 agonists.
The structure of the multispecific, e.g., bispecific molecule/construct provided herein is represented by the following formula (formula 2):
(TNFR 1 inhibitor) n - (activity modulating agent) r1 Joint (L) p - (activity modulating agent) r2 - (TNFR 2 agonists) q ，
Where n=1, 2 or 3, p=1, 2 or 3, r1 and r2 each independently=0, 1, 2, and q=0, 1 or 2.
The order of the components may be varied as in formula 1. The linker may contain multiple components, such as a GS linker, a polymeric moiety, such as PEG, or other such linker, or a hinge region, or other component combinations, and the activity modulator is a polypeptide that modifies the activity of the construct, such as an Fc region or modified Fc region, or increases half-life or resistance to endogenous inhibitors. The components of formulas 1 and 2 may be polypeptides or may contain other molecules, such as small molecule drugs that specifically bind to chemical linkers, or non-peptide activity modulators. Examples of each component are described below.
Also provided are constructs comprising (formula 5):
(TNFR 2 agonist) n -a joint p - (activity modulating agent) q Of formula 5a, or
(Activity-controlling Agents) q -a joint p - (TNFR 2 agonists) n The composition of 5b,
wherein each component is as defined in formula 1 above, and the TNFR2 agonist may be a small molecule or polypeptide, such as a TNFR2 single chain antibody agonist or portion thereof.
TNFR1 antagonist construct, TNFR2 agonist construct and multispecific, including bispecific TNFR1 antagonist/TNFR 2 agonist construct components
The descriptions and examples of the constructs provided herein and each component of the construct are described in the following sections. An example form of each construct is depicted and described by formulas 1 and 2 above and 3 and 4 below.
TNFR1 inhibitor moiety (TNFR 1 antagonist)
The TNFR1 inhibitor moiety in the multispecific molecules/constructs provided above in formula 1 and herein (formula 2 above) is any molecule, including a polypeptide or small molecule, that inhibits TNFR1 signaling. This includes TNFR1 inhibitors that selectively inhibit TNFR1 signaling but not TNFR2 signaling.
To avoid receptor clustering that agonizes TNFR1, TNFR1 antagonist constructs are typically monomeric/monovalent. The TNFR1 antagonist-inhibitor component of the construct may be a component known to have TNFR1 antagonist activity or may be identified, for example, by selection from a library, such as a phage library, antibody library, or aptamer library. In the TNFR1 inhibitor portion, those are modified or selected to have a higher specificity or affinity for TNFR1 and to have little or no (whereby such activity produces less than grade 2, typically grade 1 or less, adverse side effects according to the NCI adverse event common term Standard (CTCAE) classification system) agonist activity for TNFR1, and optionally also TNFR2 agonist activity. In those cases, the TNFR1 inhibitor moiety can be provided as a single chain antibody or any other form described herein, including, for example, linked to a half-life extender, such as any of the half-life extenders described above and below, e.g., a modified Fc region or Fc dimer, or to another moiety or moieties that increase serum half-life.
For example, as provided herein, the TNFR1 inhibitor component of the TNFR1 antagonist construct can be or can include a human domain antibody (dAb) that specifically binds TNFR 1. A dAb may contain a variable region heavy (VH) or light (VL) domain. dAbs for use herein include, for example, dAbs of the following names: DOM1h-574-208 (SEQ ID NO: 54) (from DMS5541; see SEQ ID NO: 38), GSK1995057 (see SEQ ID NO: 55) and GSK2862277 (see SEQ ID NO: 56), and dAbs shown in any one of SEQ ID NO: 57-672; see, for example: U.S. patent nos. 9,028,817 and 9,028,822; U.S. publication No.: 2006/0083747, 2010/0034831, and 2012/0107330; international application publication No.: applications and patents of WO 2004/058820, WO 2004/081026, WO 2005/035572, WO 2006/038027, WO 2007/049017, WO 2008/149444, WO 2008/14948, WO 2010/094720, WO 2011/051217, WO 2011/006914, WO 2012/172070, WO 2012/104322, and WO 2015/104322, as well as other related family members; see also Enever et al, (2015) Protein Engineering, design & Selection 28 (3): 59-66, which provides for the sequence and discussion of the various dAbs. Vh dabs comprising heavy chains are provided. These dabs can be linked directly or indirectly to a moiety that increases serum half-life, such as Fc or HSA, and can also confer other properties or activity on the construct.
The anti-TNFR 1 inhibitor component can be or include a nanobody. Examples thereof are (Nb) Nb 70 and/or Nb 96 (see SEQ ID NOS: 683 and 684, respectively). The immunogenicity of these dabs and Nb was investigated and modified, if necessary, using molecular modeling and mutagenesis to remove predicted immunogenic sequences. The immunogenic sequences may be eliminated by standard methods known in the art. For example, potentially antigenic peptides are identified and conservative substitutions are made for each amino acid to identify those that are not antigenic and retain activity. Other methods are known (see, e.g., schubert et al (2018) PLoS Comput biol.14 (3): e 1005983), which describes a protein deimmunization method).
Thus, for example, a TNFR1 antagonist dAb moiety can be a dAb as set forth in any one of SEQ ID NOS.54-672, or a dAb having about or at least about 85%, 90%, 95%, 98%, 99% or more sequence identity to a dAb as set forth in any one of SEQ ID NOS.54-672, or a TNFR1 antagonist dAb known to those of skill in the art.
Other TNFR1 antagonists include, for example, antigen-binding antibody fragments. For example, the TNFRl antagonist may be a Fab fragment, a Fab 'fragment, a single chain Fv (scFv), a disulfide linked Fv (dsFv), an Fd fragment, an Fd' fragment, a single chain Fab (scFab), an hsFv (helix-stabilized Fv), a free light chain, or an antigen-binding fragment of any of the foregoing. It may also include linkers, such as GS linkers in the construct, for example to increase flexibility.
For example, the TNFR1 inhibitor portion of an antagonist may contain an antigen-binding fragment from a TNFR1 antagonist antibody known as ATROSAB. The fragments include one or more (or all) of the heavy or light chain CDRs of ATROSAB, or CDRs that exhibit at least 85%, 90%, 95% or more sequence identity thereto (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity). TNFR1 antagonists may contain the VH (residues 1-115 of SEQ ID NO: 31) and/or the VL (residues 1-113 of SEQ ID NO: 32) of ATROSAB or a VH or VL having at least 85%, 90%, 95% or more sequence identity to the VH or VL of ATROSAB. For example, it may contain a dAb derived from ATROSAB. TNFR1 antagonists may contain other monovalent antibody fragments of ATROSAB, including, for example, fab or scFv fragments, such as ATROSAB Fab (FabATR) light and heavy chains, respectively, as shown in SEQ ID NO:679 and 680, or ATROSAB scFv (scFv IZI 06.1) as shown in SEQ ID NO: 673. For example, scFv contains a VH domain, corresponding to residues 1-115 of the ATROSAB heavy chain (see SEQ ID NO: 31), linked to a VL domain by a short peptide linker (e.g., GGGGSGGGGSGGSAQ, as shown in SEQ ID NO:673, or any of the linkers shown in SEQ ID NO: 813-834), corresponding to residues 1-113 of the ATROSAB light chain (see SEQ ID NO: 32). TNFR1 antagonists may comprise ATROSAB scFV variants having increased affinity or selectivity for TNFR1, or both, including scFv IG11, which comprises or has the sequence set forth in SEQ ID NO:674, scFv T12B, which comprises the sequence set forth in SEQ ID NO:675, or scFv 13.7, which comprises the sequence set forth in SEQ ID NO:676, or variants having at least 90% sequence identity to the sequences of scFv IG11, scFv T12B, and scFv 13.7. TNFR1 antagonists may also comprise amino acid residue sequences from the light and heavy chains of Fab 13.7 (derived from scFv 13.7) as shown in SEQ ID NOS 681 and 682, respectively.
TNFR1 inhibitors in TNFR1 antagonist constructs also include TNF variants (muteins) that bind to TNFR1 to reduce or inhibit signaling. These include, for example, TNF variants (muteins), such as, but not limited to, TNF variants containing one or more of the following mutations: L29S, L29G, L29Y, R31E, R31N, R32Y, R W, S86T, L S/R32W, L S/S86T, R W/S86T, L29S/R32W/S86T, R31N/R32T, R E/S86T, R31N/R32T/S86T and E146R, with reference to SEQ ID NO 2, which confer selectivity to TNFR 1. TNFR1 antagonists may comprise, for example, a TNFR 1-selective antagonistic TNF mutein derived from a mutein designated XPro1595 (see SEQ ID NO: 701). XPro1595 contains the mutations V1M, R31C, C69V, Y H, C A and A1456R, see SEQ ID NO:2. Other exemplary TNFR1 selective antagonistic TNF muteins are derived from XENP345 (see SEQ ID NO: 702) containing the mutation I97T/A145R, see SEQ ID NO:2; and TNFR 1-selective antagonistic TNF muteins designated R1antTNF (see SEQ ID NO: 703) containing the mutations A84S, V85T, S86T, Y H, Q N and T89Q, see SEQ ID NO:2. TNFR1 inhibitors for TNFR1 antagonists also include small molecule inhibitors that can be chemically conjugated to a linker.
As described herein, see, e.g., examples, the TNFR1 inhibitor (antagonist) moiety can be modified to increase its specificity/selectivity for TNFR1, and can also optionally be modified to have TNFR2 agonist activity. TNF has a low pM affinity (K d 19 pM) binds to TNFR 1; in general, antagonists herein have at least the same affinity as TNF, except that their activity is due to "locking" the receptor in an inactive conformation, which is not necessary because the receptor is already locked. TNFR1 antagonist constructs provided herein include those having a K of less than or less than about 100nM D TNFR1 antagonist constructs that specifically bind TNFR1 (e.g., less than or equal to 95nM,90nM,85nM,80nM,75nM,70nM,65nM,60nM,55nM,50nM,45nM,40nM,35nM,30nM,25nM,20nM,15nM,10nM,5nM,4nM,3nM,2nM, or 1 nM). In certain embodiments, the TNFR1 antagonist specifically binds TNFR1, K D A value of less than 1nM (e.g., less than or equal to: 990pM, 3000 pM,710pM, 650pM, 1500, 300, respectively. The process comprises, the process comprises, 160pM,150pM,140pM,130pM,120pM,110pM,100pM,90pM,80pM,70pM,60pM,50pM,40pM,30pM,20pM,10pM,5pM, or 1 pM.
The TNFR1 antagonist constructs provided herein are also selected or designed to lack or reduce binding to other TNFR superfamily members. For example, it is evaluated using any suitable in vitro binding assay to identify those TNFR1 antagonist constructs that do not specifically bind to another TNFR superfamily member, such as TNFR 2. Assays include, for example, ELISA-based methods. For example, a TNFR1 antagonist construct may specifically bind to human TNFR1 or a TNFR 1-derived peptide, with a TNFR1 affinity that is greater than the affinity for the other family member or its corresponding peptide. The increased affinity is, for example, at least or at least about 5-fold higher (e.g., at least or equal to 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold higher, or more) than the affinity of the TNFR1 antagonist for another TNFR superfamily member, such as TNFR 2.
Among the TNFR1 antagonist constructs provided herein are those that exhibit high k upon interaction with TNFR1 on Value sum low k off Constructs of the values were consistent with high affinity receptor binding. For example, the TNFR1 antagonist constructs provided herein can exhibit a TNFR1 activity of greater than or equal to or greater than about 10 in the presence of TNFR1 4 M -1 s -1 K of (2) on A value (e.g., greater than or equal to 1.0X10) 4 M - 1 s -1 ，1.5×10 4 M -1 s -1 ，2.0×10 4 M -1 s -1 ，2.5×10 4 M -1 s -1 ，3.0×10 4 M -1 s -1 ，3.5×10 4 M -1 s -1 ，4.0×10 4 M -1 s -1 ，4.5×10 4 M -1 s -1 ，5.0×10 4 M -1 s -1 ，5.5×10 4 M -1 s -1 ，6.0×10 4 M -1 s -1 ，6.5×10 4 M -1 s -1 ，7.0×10 4 M -1 s -1 ，7.5×10 4 M -1 s -1 ，8.0×10 4 M -1 s -1 ，8.5×10 4 M -1 s -1 ，9.0×10 4 M -1 s -1 ，9.5×10 4 M - 1 s -1 ，1.0×10 5 M -1 s -1 ，1.5×10 5 M -1 s -1 ，2.0×10 5 M -1 s -1 ，2.5×10 5 M -1 s -1 ，3.0×10 5 M -1 s -1 ，3.5×10 5 M -1 s -1 ，4.0×10 5 M -1 s -1 ，4.5×10 5 M -1 s -1 ，5.0×10 5 M -1 s -1 ，5.5×10 5 M -1 s -1 ，6.0×10 5 M -1 s -1 ，6.5×10 5 M -1 s -1 ，7.0×10 5 M -1 s -1 ，7.5×10 5 M -1 s -1 ，8.0×10 5 M -1 s -1 ，8.5×10 5 M -1 s -1 ，9.0×10 5 M - 1 s -1 ，9.5×10 5 M -1 s -1 ，1.0×10 6 M -1 s -1 ). For example, the TNFR1 antagonists provided herein can exhibit less than or equal to or less than about 10 when complexed with TNFR1 -3 s -1 K of (2) off A value (e.g., less than or less than about 1.0X10) -3 s -1 ，9.5×10 -4 s -1 ，9.0×10 -4 s -1 ，8.5×10 -4 s -1 ，8.0×10 -4 s -1 ，7.5×10 -4 s -1 ，7.0×10 -4 s -1 ，6.5×10 -4 s -1 ，6.0×10 -4 s -1 ，5.5×10 -4 s -1 ，5.0×10 -4 s -1 ，4.5×10 -4 s -1 ，4.0×10 -4 s -1 ，3.5×10 -4 s -1 ，3.0×10 -4 s -1 ，2.5×10 -4 s -1 ，2.0×10 -4 s -1 ，1.5×10 -4 s -1 ，1.0×10 -4 s -1 ，9.5×10 -5 s -1 ，9.0×10 -5 s -1 ，8.5×10 -5 s -1 ，8.0×10 -5 s -1 ，7.5×10 -5 s -1 ，7.0×10 -5 s -1 ，6.5×10 -5 s -1 ，6.0×10 -5 s -1 ，5.5×10 -5 s -1 ，5.0×10 -5 s -1 ，4.5×10 -5 s -1 ，4.0×10 -5 s -1 ，3.5×10 -5 s -1 ，3.0×10 -5 s -1 ，2.5×10 -5 s -1 ，2.0×10 -5 s -1 ，1.5×10 -5 s -1 Or 1.0X10 -5 s -1 )。
The TNFR1 antagonist (TNFR 1 inhibitor portion of the constructs of formulas 1 and 2), e.g., the C-terminus of any of the TNFR1 antagonist constructs described herein, can be directly linked or more typically linked to the activity modulator through a linker or combination linker element, or fused to the N-terminus of the TNFR2 agonist (or small molecule TNFR2 agonist) through one or more linkers, as discussed below and elsewhere herein. Alternatively, the N-terminus of the TNFR1 inhibitor moiety may be fused to the C-terminus of the TNFR2 agonist, or the C-terminus of the TNFR1 inhibitor moiety (or small molecule TNFR2 agonist) may be fused to an activity modulator or linker directly or through a linker.
The linker (L), discussed in more detail below, is any linker that improves pharmacological properties, including increased stability and flexibility and reduced steric hindrance, and optionally imparts additional properties to the construct. The linker may comprise more than one component, wherein each component imparts a specific property. For example, a TNFR1 antagonist can include any one or more of an Ig Fc region and/or an antibody hinge region and/or a short peptide linker, such as a glycine-serine linker. The Fc region is modified, e.g., to eliminate or reduce ADCC activity, and/or alter receptor binding, and/or other such activities and properties. The linker also includes chemical linkers, as described below. For example, in some embodiments, the linker is a poly (ethylene glycol) (PEG) molecule, or a branched PEG molecule, such as those molecules having a molecular weight of or about 30kDa or greater.
TNFR2 agonist construct and TNFR2 antagonist construct
The TNFR2 agonist (regulatory T cell generating agent) constructs are useful in the treatment of inflammatory and autoimmune diseases, and solid tumor-like diseases, disorders, and conditions. Regulatory T cells (tregs) suppress autoimmunity and have immunosuppressive effects, for example in the tumor microenvironment. Proliferation of tregs is upregulated by TNFR2, and loss of TNFR2 is associated with reduced Treg numbers and exacerbation of experimental arthritis. Thus, the TNFR2 agonist constructs are useful in the treatment of many autoimmune diseases, other chronic inflammatory diseases, and other acute inflammatory diseases (e.g., SARS, COVID-19).
The TNFR2 antagonist construct inhibits regulatory T cells for the treatment of cancer and other hyperproliferative diseases (TNFR 2 is a "checkpoint receptor"). Regulatory T cells accumulate in the tumor microenvironment and cause suppression of the anti-tumor immune response. The TNFR2 antagonist constructs are useful in the treatment of cancer and other hyperproliferative diseases, such as metacarpal tendinous Contracture (Dupuytren's Contracture) and idiopathic pulmonary fibrosis.
As described above, TNFR2 agonist constructs comprising TNFR2 agonists are also provided. These include TNFR2 agonists linked to an activity modulator either directly or through a linker, and also include multispecific constructs, e.g., bispecific constructs, that contain TNRF1 antagonists and TNFR2 agonists of various configurations and linkers, with appropriate structures and properties. In some embodiments, the TNFR2 agonist is in a bispecific construct. TNFR2 agonists, particularly in the multispecific, e.g., bispecific molecules/constructs provided herein, selectively activate or agonize TNFR2 without activating or substantially activating TNFR1 and/or without interfering with the inhibition of TNFR1 signaling through the TNFR1 antagonist portion of the multispecific, e.g., bispecific molecule.
The TNFR2 agonist may be any known to those skilled in the art, including agonist antibodies and antigen-binding portions thereof, as well as single chain and other conformational derivatives of antibodies, as well as small molecule agonists. TNFR2 agonists may also be produced, for example, by computer design and/or by preparing candidates and screening libraries. For example, phage libraries or antibody libraries or aptamer libraries can be screened to identify TNFR2 agonists. TNFR2 agonist antibodies or antigen-binding fragments thereof can be generated by screening libraries of antibodies and antigen-binding fragments thereof for functional molecules that bind to epitopes within TNFR2 and selectively promote receptor activation. Examples of such methods and molecules are those described in international application publication No. WO 2017/040312.
The development of TNFR2 selective agonists can include elucidation of epitopes within TNFR2 that promote agonistic receptor binding. Epitope mapping analysis using linear peptides derived from different regions of TNFR2, as well as constrained cyclic and bicyclic peptides, showed that agonistic TNFR2 antibodies bind in a conformation-dependent manner to epitopes from different regions of the TNFR2 polypeptide. For example, one identified TNFR2 epitope comprises residues 56-60 of SEQ ID NO. 4 (KCSPG). The agonistic TNFR2 antibody MR2-1 binds to this epitope; it does not bind to an epitope comprising residues 142-146 (KCRPG) of SEQ ID NO. 4. Human TNFR2 may be selected to bind an epitope (e.g., residues 56-60 comprising SEQ ID NO: 4). In general, human TNFR2 agonists may be selected or designed to bind to an epitope within human TNFR2 that contains at least five discrete or contiguous residues within residues 96-154 of SEQ ID NO:4 (CGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA RPGT; SEQ ID NO: 841) and/or may bind to an epitope within residues 111-150 of SEQ ID NO:4 (TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA; SEQ ID NO: 842) to which MR2-1 also binds. Human agonists may also bind to an epitope within residues 115-142 of SEQ ID NO:4 (NRICTCRPGWYCALSKQEGCRLCAPLRK; SEQ ID NO: 843), and/or an epitope within residues 122-136 of SEQ ID NO:4 (PGWYCALSKQEGCRL; SEQ ID NO: 844), and/or residues 96-122 of SEQ ID NO:4 (CGSRCSSDQVETQACTR; SEQ ID NO: 845), and/or an epitope within residues 101-107 of SEQ ID NO:4 (SSDQVET; SEQ ID NO:846; MR2-1 also binds thereto), and/or an epitope within residues 4 amino acids 48-67 (QTAQMCCSKCSPGQHAKVFC; SEQ ID NO: 847), and/or an epitope comprising residues 130-149 of SEQ ID NO:4 (KQEGCRLCAPLRKCRPGFGV; SEQ ID NO: 848), and/or residues 110-147 of SEQ ID NO:4 (CTREQNRICTCRPGWYCALSKQEGCRLCAPLRKC RPGF; SEQ ID NO: 849), and/or an epitope comprising at least 5 consecutive or non-consecutive residues from residues 106-155 of SEQ ID NO: 6 (ETQACTREQNRICT CRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTE; SEQ ID NO: 850), and/or an epitope within residues 130-149 of SEQ ID NO: 848), and/or an epitope within residues 1-141-149 (SEQ ID NO: 852).
On the other hand, TNFR2 agonist antibodies and antigen-binding fragments thereof specifically bind to an epitope within or containing an amino acid residue set forth in any one of SEQ ID NOS: 853-1211 whereby the antibody or antigen-binding fragment specifically binds to human TNFR2 but does not specifically bind to another TNFR superfamily member, particularly TNFR1. The human TNFR2 agonist antibody or antigen-binding fragment thereof does not bind or attenuate/reduce binding to other members of the TNFR superfamily, including TNFR1 (see, e.g., international application publication No. WO 2017/040312).
Epitopes within TNFR2 that can be used to screen for TNFR2 agonists include peptides having the sequences shown in any one of SEQ ID NOS: 853-1211. These peptides can be converted to cyclic and polycyclic forms (e.g., by incorporating cysteine residues at the N-and C-terminal positions, or at different internal positions within the peptide chain) in order to confine the peptide fragment to different three-dimensional conformations, mimicking the structural rigid framework of TNFR2 and the conformational constraints of the peptide fragment within TNFR 2. The cyclic and polycyclic peptide fragments can then be immobilized on a solid surface and screened for molecules that bind, for example, to the TNFR2 agonistic antibody MR2-1 using ELISA. Using this assay, peptides containing residues within the TNFR2 epitope that promote receptor activation can be pre-organized in structure with these amino acids to make them similar to the conformation of the corresponding peptide in the native protein. The cyclic and polycyclic peptides thus obtained (e.g., peptides having the sequence shown in any of SEQ ID NOS: 853-1194, particularly those containing KCSPG motifs such as SEQ ID NOS: 905, 921, 927, 970 and 1085) can be used to screen libraries of antibodies and antigen binding fragments thereof to identify TNFR2 agonists for use herein. The restricted peptide serves as a surrogate for an epitope within TNFR2 that promotes receptor activation, and thus antibodies or antigen-binding fragments generated using this screening technique bind to the corresponding epitope in TNFR2 and are agonists of receptor activity (see, e.g., international application publication No. 2017/040312). To produce TNFR2 agonists, phage display technology was used. Phage display libraries are contacted under conditions where specific binding occurs. TNFR 2-derived peptides (e.g., any of the peptides shown in SEQ ID NOS: 853-1194) were immobilized on a solid support or in phage. Phages containing the TNFR2 binding moiety form complexes with targets on the solid support and unbound phages are washed away. The bound phage is then released from the target by changing the buffer to an extreme pH (pH 2 or 10), changing the ionic strength, adding a denaturing agent, or by other known methods. To isolate the bound phage, protein elution may be performed (see, e.g., international application publication No. WO 2017/040312).
MR2-1 is an exemplary agonistic TNFR2 antibody that binds TNFR2 and enhances TNFR 2-mediated proliferation of Treg cells. However, MR2-1 binds to a osteoprotegerin and the heavy and/or light chain variable region of this antibody, or in particular the heavy and/or light chain CDRs of MR2-1, can be modified to eliminate the ability of the resulting antibody or fragment to bind to TNFR superfamily members other than TNFR2, resulting in an agonistic TNFR2 antibody or antigen-binding fragment thereof. This may be achieved using genetic engineering and/or antibody library screening techniques, for example as described in International application publication No. WO 2017/040312.
As provided herein, TNFR2 agonists may contain antigen-binding fragments of an agonistic human anti-TNFR 2 antibody, such as MR2-1 and MAB2261, such as commercially available MR2-1 from Hycult Biotech; and from R&MAB2261 of D Systems. For example, MR2-1 or MAB 2261V H And V L A domain, or one or more CDRs contained therein, for use in generating a TNFR2 agonist. Such agonists may comprise a human domain antibody (dAb) specific for TNFR 2; the dAb may contain the variable region heavy chain of MR2-1 or MAB2261 (V H ) Or light chain (V) L ) Domain, or V with MR2-1 or MAB2261 H Or V L V having at least or at least about 85%, 90%, 95% or more sequence identity H Or V L Provided that the resulting TNFR2 retains TNFR2 agonist activity. TNFR2 agonists may also contain other antigen binding derived from MR2-1 or MAB2261 antibodiesSynthetic fragments, or amino acid sequences having at least or at least about 85%, 90%, 95% or more sequence identity thereto, e.g., fab fragments, fab 'fragments, F (ab') 2 Fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd' fragments, single chain Fv (scFv), single chain Fab (scFab), hsFv (helix stabilized Fv), minibodies, diabodies, anti-idiotype (anti-Id) antibodies, free light chains, or antigen-binding fragments of any of the foregoing. Antibody fragments include combinations of any of the above fragments, e.g., tandem scFv, fab-scFv (HC C-terminal, or LC C-terminal), fab- (scFv) 2 (C-terminal), scFv-Fab-scFv, fab-C H 2-scFv, scFv fusion (C-terminal or N-terminal), fab fusion (HC C-terminal or LC C-terminal), scFv-scFv-dAb, scFv-dAb-scFv, dAb-scFv-scFv and trisomes. TNFR2 agonists include any dAb whose sequence is provided herein or known in the art, with about or at least about 85%, 90%, 95% or more sequence identity thereto, as well as TNFR2 agonist activity.
In some embodiments, the TNFR2 agonist may be an scFv of a TNFR2 agonist monoclonal antibody, including any scFv known in the art, or an scFv having about or at least about 85%, 90%, 95% or more than 95% sequence identity to such scFv, provided that the resulting construct retains TNFR2 agonist activity. In some embodiments, the TNFR2 agonist may be a Fab fragment of a TNFR2 agonist monoclonal antibody, or a Fab thereof, or a Fab having about or at least about 85%, 90%, 95% or more sequence identity and TNFR2 agonist activity.
A TNFR2 agonist may also be or include a TNF mutein modified to bind TNFR2 and have agonist activity (see, e.g., SEQ ID NO: 765-800). An example of such an embodiment is a TNFR2 agonist comprising a TNFR 2-selective TNF mutein, e.g., a TNF variant having one or more of the following TNFR 2-selective mutations: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, and D143V/A145S, and combinations thereof, such as combinations of D143V/A145S and S95C/G148C, refer to SEQ ID NO:2. For example, a TNF variant having the mutation D143N/A145R (SEQ ID NO: 781) binds to and activates TNFR2 and can be used in the constructs provided herein. Referring to SEQ ID NO. 2, TNF muteins having the mutation S95C/G148C in combination with any other mutations listed or known or identified are also TNFR2 selective agonists that may be included in the constructs provided herein.
The TNFR2 agonist may comprise a fusion of a single-chain TNFR 2-selective TNF mutein trimer with a multimerization domain. As described herein, the primary ligand of TNFR2 is membrane-bound TNF (memtNF; also referred to herein as transmembrane TNF or tmTNF). Adding a multimerization domain, such as a dimerization or trimerization domain, to generate hexamer or nonamer molecules associated with TNF subunits, respectively; these hexamers and nonamers of TNF mimic membrane-bound TNF trimers, activating TNFR2 signaling. The dimerization domain includes EHD2 (SEQ ID NO: 808) as discussed above. EHD2 is derived from heavy chain C of IgE H 2, and MHD2 (SEQ ID NO: 811), which is derived from heavy chain C of IgM H 2 domain. Dimerization domains also include Fc domains, such as those derived from IgG1 (see SEQ ID NO: 10) and IgG4 (see SEQ ID NO: 16), optionally including modifications, such as those that alter immune effector function and/or enhance FcRn recycling. Trimerization domains include, for example, the trimerization domain of chicken tenascin C (TNC) (SEQ ID NO: 805) and the trimerization domain of human TNC (SEQ ID NO: 807). Dimerization and trimerization enhance TNFR2 signaling and improve the pharmacological properties of the construct. For example, the half-life of the fusion protein is increased by increasing the molecular weight of the molecule and/or by introducing FcRn recycling, for example when the dimerization domain is Fc.
As provided herein, a TNFR2 agonist may contain a TNF mutein (tnfput) trimer chain, have any of the mutations described herein that confer selectivity for TNFR2 and/or reduce or eliminate binding to TNFR 1. An example of such a mutation is the substitution D143N/A145R (cf. SEQ ID NO: 2) fused to a Multimerization Domain (MD), e.g.a dimerization or trimerization domain. The multimerization domains may be fused to the N or C terminus of the TNF mutein trimer chain and include linkers between each TNF mutein and between the TNF mutein trimer chain and the multimerization domain.
Such TNFR2 agonists have formulas 4 and 5:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula 4), or
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula 5),
wherein MD is a multimerization domain (activity modulator); TNFput is a TNFR 2-selective TNF mutein, e.g., a mutein having the mutation D143N/A145R; l1, L2 and L3 are linkers, such as Gly-Ser linkers, which may be the same or different.
In specific embodiments, the multimerization domain is EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), a chicken TNC trimerization domain (SEQ ID NO: 805), a human TNC trimerization domain (SEQ ID NO: 807), igG1 Fc or IgG4 Fc. When the dimerization domain is an IgG1 Fc or an IgG4 Fc, it is the same Fc used to link the TNFR1 antagonist and TNFR2 agonist, but not additional Fc. IgG1 or IgG4 Fc may be modified to enhance or eliminate immune effector functions, such as ADCC, ADCP and/or CDC activity, and/or enhance FcRn binding. Multimerization domains, such as the Fc region, increase the in vivo stability and serum half-life of the construct. For purposes herein, the Fc region in the constructs of formulas 1-5, or variants thereof, is typically modified to alter or modulate the pharmacological properties or activity of the construct. Fc modification is discussed in more detail below. Any multimerization domain known in the art is also contemplated for use in the TNFR2 agonists herein.
The TNF mutein may be a TNF variant having any one or more mutations that confer TNFR2 selectivity. Mutations include, for example: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146D 147D, A145Q/E146D/S147D, A145T/E146D, A145D/S147D, A145K/E146D/S147D, A145T/E147D, A145D/E147D and A145D/E145D/S147D. TNF variants with the mutation D143N/a145R are contemplated for use herein. Any other mutation known in the art that confers TNFR2 selectivity is also contemplated herein. TNF muteins may comprise the complete sequence of soluble TNF (i.e., residues 1-157 of SEQ ID NO: 2), or may comprise the partial sequence of soluble TNF, such as residues 4-157, 9-157 or 12-157 of SEQ ID NO:2, of sufficient length to bind and/or agonize TNFR2.
The L1, L2 or L3 linkers may be the same or different. In particular, the linker may contain a short peptide linker, such as a GS linker. For example, the linker may comprise (GGGGS) n Wherein n=1-5 (SEQ ID NO: 1471). The linker may also contain all or a portion (at least 10, 15 or 20 consecutive residues) of the TNF-alpha stem region, containing amino acid sequence GPQREEFPRDLLSLISPLAQAVRSSSRTPSDK (SEQ ID NO: 812), residues 57-87 corresponding to the full length sequence of TNF (transmembrane TNF) shown in SEQ ID NO: 1. For example, a linker containing all or a portion of the stem region, containing at least 10, 15, or 20 consecutive amino acid residues, may be between the N-or C-terminus of the TNF mutein and the multimerization domain. All three linkers may be (GGGGS) n Where n is generally 1-10 (SEQ ID NO: 1472), or other combinations of Gly-Ser, or may contain mixtures of Gly-Ser residues, e.g., GGGGS n And all or a portion of the TNF stem region, containing at least 10, 15 or 20 contiguous amino acid residues. Exemplary linkers are shown in SEQ ID NOS 813-834, 1471 and 1472.
TNFR2 agonists provided herein include those having a K of less than or equal to or less than about 100nM D Agonists that specifically bind TNFR2 (e.g., 95nM, 90nM, 85nM, 80nM, 75nM, 70nM, 65nM, 60nM, 55nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM, 10nM, 5nM, 4nM, 3nM, 2nM, or 1 nM) are used. In certain instances, the TNFR2 agonist specifically binds TNFR2, K thereof D A value of less than 1nM (e.g990pM, 980pM, 970pM, 960pM, 950pM, 940pM, 930pM, 920pM, 910pM, 900pM, 890pM, 880pM, 870pM, 860pM, 850pM, 840pM, 830pM, 820pM, 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 700pM, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530, 520pM, 510pM, 500pM 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, 160pM, 150pM, 140pM, 130pM, 120pM, 110pM, 100pM, 90pM, 80pM, 70pM, 60pM, 50pM, 40pM, 30pM, 20pM, 10pM, 5pM, or 1 pM.
A TNFR2 agonist is a compound that induces Treg (e.g., CD4 + 、CD25 + FOXP3 + Treg) or contacting in vitro with a TNFR2 agonist in a sample containing Treg for testing purposes. The proliferation of tregs may be induced, for example, by about 0.00001% to 100.0% (e.g., 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.3%, 0.0.0.0.0.0.0.80%, 0.70%, 0.20%, 0.70%, 0.40%, 0.20%, or 0.70% by analysis relative to a subject or sample containing a cell population not treated with the TNFR2 agonist.
Thus, TNFR2 agonists can be used to promote Treg cell proliferation and can be administered to patients suffering from autoimmunityMammalian subjects, e.g., human patients, for epidemic or chronic inflammatory diseases or conditions to attenuate the intensity and duration of immune responses in the patient (e.g., CD8 produced in vivo in response to self or non-threatening foreign antigens) + Number of cytotoxic T lymphocytes). For example, administration of a TNFR2 agonist to a human patient, or by treatment of an ex vivo expanded population of Treg cells with a TNFR2 agonist, can result in a reduction or decrease in the amount of secreted immunoglobulin (e.g., igG) that cross-reacts with a self or non-threatening antigen relative to a subject not treated with a TNFR2 agonist of about 0.00001mg/mL to 10.0mg/mL (e.g., 0.00001mg/mL, 0.0001mg/mL, 0.001mg/mL, 0.01mg/mL, 0.1mg/mL, 1.0mg/mL, or 10.0 mg/mL), or 0.001-1.0mg/mL (e.g., 0.001mg/mL, 0.005mg/mL, 0.010mg/mL, 0.050mg/mL, 0.10mg/mL, 0.20mg/mL, 0.30mg/mL, 0.40mg/mL, 0.50mg/mL, 0.60mg/mL, 0.70mg/mL, 0.80mg/mL, 0.90mg/mL, or 1.90 mg/mL). Additionally or alternatively, the TNFR2 agonist can decrease the cytotoxic T cell count (e.g., CD 8) in the subject relative to a subject not treated with the TNFR2 agonist + T cell levels), for example by about 0.00001 to 100.0% (e.g., 0.00001%, 0.00002%, 0.00003%, 0.00004%, 0.00005%, 0.00006%, 0.00007%, 0.00008%, 0.00009%, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4%, 0.6%, 0.0009%, 0.0%, 0.70%, 0.40%, 0.30%, 0.80%, 0.20%, 0.70%, 0.20%, or 0.80% by FACS analysis. For example, TNFR2 agonists can be administered to a subject (e.g., a mammalian subject, such as a human) to treat an autoimmune or chronic inflammatory disease or disorder, such as those described herein. Treating a subject in this manner reduces autoreactive CD8 in the subject + Number of T cells.
The TNFR2 agonists provided herein can be evaluated to identify those agonists that lack specific binding to another TNFR superfamily member, particularly TNFR 1. This can be accomplished using any of a variety of in vitro binding assays known to those of skill in the art, such as ELISA-based methods. For example, TNFR2 agonists include those that specifically bind human TNFR2 or TNFR 2-derived peptides, such as peptide fragments (QTAQMCSKCSPGQHAKVFC, SEQ ID NO: 847) containing residues 48-67 of SEQ ID NO:4 within human TNFR2, with an affinity that is at least or at least about 2, 3, 4, or 5-fold higher (e.g., 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold or more) than the affinity of the agonist for another TNFR superfamily member such as TNFR 1.
TNFR2 agonists provided herein include those that exhibit high k upon interaction with TNFR2 on Value sum low k off Agonists of the values are consistent with high affinity receptor binding. For example, a TNFR2 agonist provided herein can exhibit k in the presence of TNFR2 on A value of greater than or equal to or greater than about 104M -1 s -1 (e.g., greater than or greater than about 1.0X10) 4 M -1 s -1 、1.5×10 4 M -1 s -1 、2.0×10 4 M -1 s -1 、2.5×10 4 M -1 s -1 、3.0×10 4 M -1 s -1 、3.5×10 4 M -1 s -1 、4.0×10 4 M -1 s -1 、4.5×10 4 M - 1 s -1 、5.0×10 4 M -1 s -1 、5.5×10 4 M -1 s -1 、6.0×10 4 M -1 s -1 、6.5×10 4 M -1 s -1 、7.0×10 4 M -1 s -1 、7.5×10 4 M -1 s -1 、8.0×10 4 M -1 s -1 、8.5×10 4 M -1 s -1 、9.0×10 4 M -1 s -1 、9.5×10 4 M -1 s -1 、1.0×10 5 M -1 s -1 、1.5×10 5 M -1 s -1 、2.0×10 5 M -1 s -1 、2.5×10 5 M -1 s -1 、3.0×10 5 M -1 s -1 、3.5×10 5 M -1 s -1 、4.0×10 5 M - 1 s -1 、4.5×10 5 M -1 s -1 、5.0×10 5 M -1 s -1 、5.5×10 5 M -1 s -1 、6.0×10 5 M -1 s -1 、6.5×10 5 M -1 s -1 、7.0×10 5 M -1 s -1 、7.5×10 5 M -1 s -1 、8.0×10 5 M -1 s -1 、8.5×10 5 M -1 s -1 、9.0×10 5 M -1 s -1 、9.5×10 5 M -1 s -1 Or 1.0X10 6 M -1 s -1 ). For example, a TNFR2 agonist provided herein can exhibit k when complexed with TNFR2 off A value of less than or less than about 10 -3 s -1 (e.g., less than or less than about 1.0X10) -3 s -1 、9.5×10 -4 s -1 、9.0×10 -4 s -1 、8.5×10 -4 s -1 、8.0×10 -4 s -1 、7.5×10 -4 s -1 、7.0×10 -4 s -1 、6.5×10 -4 s -1 、6.0×10 -4 s -1 、5.5×10 -4 s -1 、5.0×10 -4 s -1 、4.5×10 -4 s -1 、4.0×10 -4 s -1 、3.5×10 -4 s -1 、3.0×10 -4 s -1 、2.5×10 -4 s -1 、2.0×10 -4 s -1 、1.5×10 -4 s -1 、1.0×10 -4 s -1 、9.5×10 -5 s -1 、9.0×10 -5 s -1 、8.5×10 -5 s -1 、8.0×10 -5 s -1 、7.5×10 -5 s -1 、7.0×10 -5 s -1 、6.5×10 -5 s -1 、6.0×10 -5 s -1 、5.5×10 -5 s -1 、5.0×10 -5 s -1 、4.5×10 -5 s -1 、4.0×10 -5 s -1 、3.5×10 -5 s -1 、3.0×10 -5 s -1 、2.5×10 -5 s -1 、2.0×10 -5 s -1 、1.5×10 -5 s -1 Or 1.0X10 -5 s -1 )。
As provided herein, a TNFR2 agonist is linked directly or indirectly through a linker, in any order or suitable configuration, to, for example, any one of the TNFR1 antagonists described above. For example, the N-terminus of a TNFR2 agonist (e.g., any of the TNFR2 agonists described herein) is fused to the C-terminus of a TNFR1 antagonist by one or more linkers as discussed below and elsewhere herein. Alternatively, the C-terminus of the TNFR2 agonist may be fused to the N-terminus of the TNFR1 antagonist. When the TNFR2 agonist has the structure shown in formula 3, the N-terminus of the multimerization domain is linked to the C-terminus of the TNFR1 antagonist, and when the TNFR2 agonist has the structure shown in formula 4, the C-terminus of the multimerization domain is linked to the N-terminus of the anti-TNFR 1 antagonist. The linker (L) between the TNFR1 antagonist and the TNFR2 agonist may comprise any suitable linker, and combinations thereof, such as one or more of an Ig Fc region and/or an antibody hinge region and/or a short peptide linker, such as a glycine-serine linker. In some embodiments, the linker is a poly (ethylene glycol) (PEG) molecule or branched PEG molecule of 30kDa or greater. As described above, when the TNFR2 agonist has a structure represented by formula 3 or 4, if the multimerization domain is Fc, it is the same Fc used to link the TNFR1 antagonist to the TNFR2 agonist.
c. Joint
The above TNFR1 antagonist construct (e.g., formula 1), the multispecific TNFR1 antagonist-TNFR 2 agonist construct (e.g., formula 2), and the TNFR2 agonist construct (e.g., formulas 3-5), optionally including a linker and an activity modulator. The linker has a variety of functions including providing additional or improved biological and pharmacological properties, as well as for structural purposes for linking different molecules. Exemplary linkers are Gly-Ser polypeptides, hinge regions (see e.g., tables 1-4 above, which list the sequences of the various hinge regions, and combinations thereof).
Included are polypeptide linkers and chemical linkers for chemical conjugation. Including linker peptides as spacers between polypeptides may promote proper protein folding and stability of the polypeptides, improve protein expression, and enhance the biological activity of the construct components. Peptide linkers are primarily designed as unstructured flexible peptides. The linker may be included as shown in formulas 1-4 as exemplified above. For example, in the bispecific constructs provided, the components are fused N-terminally to the C-terminus or C-terminally to the N-terminus via a linker (L). The linker is typically a peptide linker, including a polypeptide alone, such as an Fc region, or in combination with one or more other linkers, including, for example, a short peptide linker, such as a glycine-serine (GS) linker, and/or a hinge region of an immunoglobulin (Ig). In embodiments herein, for example, the C-terminus of a TNFR1 antagonist is fused to the N-terminus of a peptide linker, and the C-terminus of a peptide linker is fused to the N-terminus of a TNFR2 agonist. In other embodiments, the C-terminus of the TNFR2 agonist is fused to the N-terminus of the peptide linker, and the C-terminus of the peptide linker is fused to the N-terminus of the TNFR1 antagonist. The linker provides increased molecular weight, increased stability and serum half-life, enhanced tissue retention, and reduced or decreased out Zhou Xiaochu rate, thereby improving the therapeutic index of the molecule. The linker also increases the flexibility of the molecule, allowing each portion of the molecule to interact with its target antigen/epitope, such as TNFR1 and TNFR2 provided herein. As discussed below and elsewhere herein, in embodiments where the linker contains an Fc region of an immunoglobulin (typically a modified Fc region), additional properties may be imparted, including, for example, neonatal Fc receptor (FcRn) recycling, which further increases serum stability and half-life, and/or enhances or eliminates immune effector function.
i. Peptide linker
The linkers of fusion proteins are well known to those skilled in the art. See, e.g., chen et al (2013) Adv. Drug. Deliv. Rev.65:1357-1369, entitled "Fusion Protein Linkers:Property, design and Functionality". The linker may be designed or may be derived from or based on a linker derived from a naturally occurring multidomain protein. Joints designed empirically by researchers are generally classified into 3 classes according to their structure: flexible linkers, rigid linkers, and in vivo cleavable linkers, e.g., for delivering a prodrug activated by in situ linker cleavage.
In addition to the effect of linking the functional domains together (e.g., flexible and rigid linkers) or releasing the free functional domains in vivo (e.g., in vivo cleavable linkers), the nature of the moiety to which the linker may be linked. These include, for example, improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Databases and methods for selecting linkers are known to those skilled in the art (see, e.g., george et al (2002) "An analysis of Protein domain linkers: their classification and role in Protein folding", protein Eng.15:871-879).
a) Flexible joint
Flexible linkers are typically used when the linked domains require a degree of movement or interaction. Flexible linkers are typically rich in small amino acids or polar amino acids, such as Gly and Ser, to provide good flexibility and solubility. Flexible linkers are a suitable choice when certain movements or interactions are required for the fusion protein domains (e.g., in scFv). Furthermore, while flexible linkers do not have a rigid structure, they can act as passive linkers to maintain the distance between the functional domains. The length of the flexible linker may be adjusted to allow for proper folding or to achieve optimal biological activity of the fusion protein.
As suggested by Argos (1990) J.mol.biol.211 (4): 943-958, flexible linkers typically consist of small non-polar (e.g., gly) or polar (e.g., ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the linked functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing adverse interactions between the linker and the protein moiety.
Exemplary flexible linkers are linkers containing predominantly or exclusively a sequence of Gly and Ser residues ("GS" linkers). One example is a polypeptide having (Gly-Gly-Gly-Gly-Ser) n Flexible linkers of the sequences. By adjusting the copy number "n", the length of this GS linker can be selected or chosen to achieve proper separation of functional domains, or to maintain the necessary inter-domain interactions. Flexible linkers are also rich in small amino acids or polar amino acids, such as Gly and Ser, and may also contain additional amino acids, such as Thr and Ala, to maintain flexibility, and polar amino acids, such as Lys and Glu, to improve solubility.
To confer protease resistance and increase the flexibility of the fusion protein, the SCDKTH hinge sequence and other hinge sequences may be replaced with or placed before a short polypeptide linker. Examples of polypeptide linkers are (Gly-Ser) n Amino acid sequence (GS linker) in which some Glu or Lys residues are dispersed to increase solubility. For example, polypeptide linkers include, but are not limited to (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS (see GS linker shown in SEQ ID NO: 816-827). The linker may be a poly Gly peptide of at least 2 to 18 residues in length or more, or a similar linker of the same length and flexibility. Exemplary polypeptide linkers in the molecules provided herein include, but are not limited to (Gly-Ser linkers are shown in SEQ ID NOS: 816-827): such as GSGS, GGGGS or GGGGSGGGGSGGGGS. Another linker that provides similar properties is a (GGGGS) 4 (SEQ ID NO: 819) linker. Another flexible linker rich in Gly and Ser is GSAGSAAGSGEF (SEQ ID NO: 828). This linker has been shown to maintain good solubility in aqueous solutions. A linker containing only glycine may be used. For example, it is known (Gly) 6 (SEQ ID NO: 1473) and (Gly) 8 (SEQ ID NO: 1474) linker and shows that it is stable to proteolytic enzyme digestion during purification of proteins from the expressing organism.
Some other types of flexible linkers include KESGSVSSEQLAQFRSLD (SEQ ID NO: 829) and EGKSSGSGSESKST (SEQ ID NO: 830). Gly and Ser residues in the linker provide flexibility, glu and Lys improve solubility.
b) Rigid joint
While flexible joints have the advantage of passively connecting functional domains and allowing some degree of movement, the lack of rigidity of these joints may be limited. When spatial separation of domains is required to maintain stability or biological activity of the fusion protein, then a rigid linker is selected. Rigid linkers exhibit relatively rigid structures by employing an alpha-helical structure or containing multiple Pro residues. The length of the linker can be easily adjusted by varying the copy number to achieve optimal distance between domains.
Has (EAAAK) n Formation of alpha-helical linkers of the (SEQ ID NO: 831) sequence has been employed in the construction of many recombinant fusion proteins. The α -helical structure is rigid and stable, with intra-segmental hydrogen bonding and a tightly packed backbone. The hard alpha-helical linker may act as a rigid spacer between protein domains. One example of a rigid joint is: a (EAAAK) n A (SEQ ID NO: 832) wherein n=2-5. This linker displays an alpha-helical conformation, consisting of Glu-Lys within the segment + The salt bridge is stable. Another type of rigid linker has a Pro-rich sequence (XP) n Wherein X represents any amino acid, typically Ala, lys or Glu. The presence of Pro in the non-helical linker increases stiffness and allows for efficient separation of protein domains. An example of such a linker is a 33 residue peptide containing repeats-Glu-Pro-and-Lys-Pro-.
One skilled in the art may choose from known linkers or designed linkers. The required properties and their requirements are known. The following discussion summarizes some example linkers (see Chen et al (2013) adv. Drug. Deliv. Rev. 65:1357-1369), where details of flexible and rigid linkers and cleavable linkers are provided and may be used). Flexible linkers are rich in small amino acids and/or hydrophilic amino acids, such as Gly or Ser, to provide structural flexibility and have been used to link functional domains that facilitate inter-domain interactions or movement. Where sufficient separation of protein domains is desired, rigid linkers may be used. Rigid linkers are designed or selected to be those that employ an alpha-helical structure or incorporate proline. The rigid linker may keep the protein portion at a distance. Flexible and rigid linkers are stable in vivo and do not allow separation of the attached proteins. Cleavable linkers allow release of the free functional domain in vivo by either reductive or proteolytic cleavage. They are typically used to deliver a prodrug to a target site.
In formula 2 above, additional linkers may be included, for example, between the TNFR1 antagonist and/or TNFR2 agonist moiety and an activity modulating moiety, such as an Fc moiety; such linkers may contain, for example, all or part of the hinge sequence of trastuzumab sufficient to provide flexibility, including at least residues SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26), or all or part of the hinge region of nivolumab sufficient to provide flexibility, sequences having sequence ESKYGPPCPPCP (residues 212-223 corresponding to SEQ ID NO: 29) or having at least 98% or 99% sequence identity thereto, or any other suitable antibody hinge region or sequence known in the art.
In certain embodiments, only GS linkers are included. Other short peptide linkers known in the art are also contemplated for use in the bispecific molecules provided herein. For example, an N-terminal or C-terminal extension of Fc may be used as a linker. A C-terminal extension ELQLEESSAEAQDGELDG (SEQ ID NO: 833) from human IgG or a sequence having at least 98% or 99% sequence identity thereto, or a variant containing sequence ELQLEESSAEAQGG (SEQ ID NO: 834) or a sequence having at least 98% or 99% sequence identity thereto may also be used as a linker.
A second Fc subunit may be included, which may or may not be a fusion protein (see, e.g., fig. 2, and may be modified to contain a bulge recess (see discussion below). That assembles in a mammalian cell expression system to form a bulge recess-mediated Fc dimer to produce an Fc dimer, further increasing the serum half-life and stability of the molecule.
Chemical linker
In some embodiments, the linker is a chemical linker. These include linkers that are non-cleavable moieties, chemical cross-linking agents, and polypeptide modifiers such as polymer molecules, including pegylated moieties, and the like. Chemical linkers are more suitable for creating branched constructs and other structures not achievable using peptide linkers.
Exemplary joints include non-cleavable joints. Non-cleavable linkers include, for example, amide linkers and amides and ester linkages with succinate spacers (see, e.g., dosio et al, (2010) Toxins 3:848-883). Exemplary chemical cross-linking linkers include, but are not limited to, SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) and SIAB (succinimidyl- (4-iodoacetyl) aminobenzoate). SMCC is an amine-sulfhydryl crosslinker containing NHS-ester and maleimide reactive groups at opposite ends of a mid-length cyclohexane stable spacer. SIAB is a short NHS ester and iodoacetyl crosslinker for conjugation of amine to sulfhydryl groups. Other exemplary cross-linking agents include, but are not limited to, thioether linkers, chemically labile hydrazone linkers, 4-mercaptopentanoic acid, BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC, and thio-SMPB, as well as SVSB (succinimidyl- (4-vinylsulfonyl) benzoate), and bismaleimide agents, such as DTME, BMB, BMDB, BMH, BMOE, BM (PEO) 3 And BM (PEO) 4 These are commercially available (Pierce Biotechnology, inc.). The bismaleimide reagent allows the free thiol groups of the cysteine residues of the antibody to be attached to thiol-containing targeting agents or linker intermediates in a sequential or simultaneous manner. In addition to maleimides, other thiol-reactive functional groups include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. Other exemplary linkers and methods of use are well known to those skilled in the art, such as the linkers described in U.S. patent publication 2005/0276812 and Ducry et al (2010) bioconjug. Chem.21:5-13And a method.
The linker may optionally be substituted with groups that modulate properties such as solubility and reactivity. For example, sulfonate substituents may increase the water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or drug moiety, and/or facilitate the coupling reaction. Linker reagents may also be obtained from commercial sources, such as Molecular Biosciences inc (Boulder, co.), or synthesized according to the following methods: toki et al (2002) J.org.chem.67:1866-1872; U.S. Pat. nos. 6,214,345; U.S. publication Nos. 2003/130189 and 2003/096743; and International application publication Nos. WO 02/088172, WO 03/026577, WO 03/043583 and WO 04/032828. For example, a linker reagent such as DOTA-maleimide (4-maleimidobutyramide benzyl-DOTA) can be prepared by reacting aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated with isopropyl chloroformate (Aldrich) according to the method described in Axworth et al (2000) Proc.Natl. Acad.Sci.U.S.A.97 (4): 1802-1807. The DOTA-maleimide reagent reacts with the free cysteine amino acids of the cysteine engineered antibody and provides a metal complexing ligand on the antibody (Lewis et al (1998) bioconj.chem.9:72-86). Chelating linker labeling reagents such as DOTA-NHS (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid mono (N-hydroxysuccinimide ester)) are commercially available (macrographics, dallas, TX).
The linker may be a dendritic linker for covalently linking more than one moiety to the antibody via a branched multifunctional linker moiety (see, e.g., sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; king et al (2002) Tetrahedron Letters 43:1987-1990). If the antibody carries only one reactive cysteine thiol group, many other moieties may be linked by a dendritic linker. Exemplary dendritic linker reagents are known (see, e.g., U.S. patent publication No. 2005/0276812).
Another example of chemical linkers (which may also be activity modulators for constructs herein) are PEG molecules and branched PEG molecules, particularly those having a molecular weight of 30kDa or greater. PEG linkers provide for the introduction of multispecific and bivalent properties (receptor clustering enhances signaling in the case of TNFR2 agonists) and increase the molecular weight of the molecule, thereby increasing serum half-life in vivo. PEG linkers also improve difficulties in antibody re-engineering, for example by avoiding the introduction of non-native structures that degrade and clear rapidly and/or cause immunogenicity.
d. Activity modulators
Among the components of the construct are portions or regions that modulate or alter the activity and/or pharmacological properties of the construct (see formulas 1 and 2 above). Examples of such are Fc regions, modified Fc regions, other multimerization domains, dimers of Fc and modified Fc, and other moieties, such as polymeric moieties, including polypeptides such as half-life extending polypeptides, albumin such as Human Serum Albumin (HSA) and transferrin, and polymers such as PEG discussed elsewhere herein, which can increase serum half-life. Modulators of activity may confer properties such as, but not limited to, increasing plasma half-life by reducing exposure to proteases, reducing renal filtration, and/or altering intracellular pathways through receptor-mediated recycling; by binding to a receptor undergoing transcytosis, providing transepithelial bilayer absorption; targeting in vivo sites that overexpress or uniquely express a particular receptor or antigen; and other properties, as exemplified in the discussion below, and are also known in the art.
As provided herein, a construct may include a human immunoglobulin, such as an Fc region of IgG, e.g., igG1 Fc (SEQ ID NO: 10), igG2 Fc (SEQ ID NO: 12), igG3 Fc (SEQ ID NO: 14), or IgG4 Fc (SEQ ID NO: 16), as a modulator of activity. In particular, the Fc is derived from an IgG1 or IgG4 antibody. For example, the linker may comprise an IgG1 kappa Fc region, such as an IgG1 Fc derived from trastuzumab, C containing trastuzumab heavy chain (see, e.g., residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27) H 2 and C H 3 domain. The Fc subunit in the bispecific molecules provided herein may also be an IgG4 Fc, e.g.derived from Nawuzumab @) Comprises an IgG4 Fc of Nawushu heavy antibodyChain (see, e.g., residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30) C H 2 and C H 3 domain.
As described below, the Fc region may be mutated or modified to eliminate, reduce, or enhance immune effector functions, including, for example, any one or more of antibody-dependent cellular cytotoxicity (ADCC; also known as antibody-dependent cell-mediated cytotoxicity), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In some embodiments herein, for example where the construct is a bispecific molecule for the treatment of inflammatory and autoimmune diseases and disorders, the immune effector function is eliminated or reduced. When the therapeutic agent is used for treating tumors or cancers, immune effector functions may be enhanced to improve anti-tumor immune responses and therapeutic effects. Additionally or alternatively, the Fc region is modified to enhance FcRn recycling to increase the in vivo serum stability and half-life of the molecules provided herein.
For the purposes herein, the Fc region or domain is modified, particularly to reduce or eliminate ADCC. Small molecule therapeutics, such as antibody fragments (e.g., fab, scFv, dAb) are advantageous. They can be produced in high yields and have other advantageous properties. They exhibit enhanced tissue penetration and target accessibility compared to monoclonal antibodies (mabs) and can prevent adverse effects of mabs such as receptor clustering, activation of immune effector functions, poor tissue penetration in poorly vascularized areas, and lack of access to targets. However, the pharmacokinetic properties of small antibody fragments are poor. For example, dAbs and other antibody fragments will be cleared rapidly by the kidney due to their small size, as molecules of 50-60kDa or smaller will be filtered by the kidney. Rapid clearance and short elimination half-life (possibly less than a few hours) of small antibody fragments can reduce in vivo efficacy and require frequent administration and/or continuous infusion.
Several methods can be used to increase the retention and in vivo half-life of small antibody fragments, such as dabs. For example, as provided herein, dabs in TNFR1 antagonists, TNFR2 agonists, and combination/multispecific constructs are fused to a linker that is or includes a half-life extender, e.g., an Fc region of an IgG such as IgG1 or IgG 4. Fc may be monomeric or dimeric. Fusion of small antibody fragments, such as dabs, to the Fc region of IgG molecules increases the size of the molecules, thereby preventing their clearance/excretion from the body, and mediates binding to neonatal Fc receptors (FcRn) expressed on endothelial cells, which protects the antibodies from lysosomal degradation and extends their in vivo half-life. However, the addition of Fc introduces undesirable properties such as induction of immune effector functions that can lead to complement activation, release of pro-inflammatory cytokines, and cytotoxicity. Since TNFR1 is almost universally expressed and TNFR2 is expressed by many tissues, it is generally not desirable to use an antibody whose ADCC is enhanced, but rather to rely on the antagonist activity of the antibody for efficacy.
As described herein, the Fc region in TNFR1 antagonists, TNFR2 agonists, and multispecific, e.g., bispecific constructs, is modified to improve pharmacokinetic and pharmacodynamic (i.e., pharmacological) properties and eliminate undesirable properties. For example, the Fc region is modified to utilize/enhance neonatal FcR recycling to increase in vivo half-life, and/or is mutated to eliminate Fc-related immune effector functions such as antibody dependent cellular cytotoxicity (ADCC; also known as antibody dependent cell-mediated cytotoxicity), antibody dependent cell-mediated phagocytosis (ADCP), and Complement Dependent Cytotoxicity (CDC). Furthermore, in embodiments where the construct is multispecific, e.g., bispecific, e.g., embodiments where it contains a TNFR1 antagonist and a TNFR2 agonist and contains an Fc dimer, the dimer is mutated to introduce a protruding recess to prevent homodimerization. Many modifications to the Fc portion (or region) are known to those skilled in the art (see, e.g., li et al, (2014) Expert Opin Ther Targets 18:335-350).
Modification of Fc portion
a) Convex and concave
Bispecific antibodies (bsAb) include two distinct antigen binding sites, allowing therapeutic approaches to be used in place of traditional therapeutic monoclonal antibodies (mabs), such that limitations associated with mabs, such as receptor co-clustering, can be avoided. Although small antibody fragments are easier and less costly to produce in high yields and can readily penetrate tissues, they have limitations such asStability, solubility and pharmacokinetic properties are poor. For example, its small size results in a shorter serum half-life, reduced tissue retention, and rapid clearance from the blood through the kidneys. Therefore, igG-like bispecific (bs) antibodies without the same limitations are advantageous. For example, bsAb may include an Fc region to increase serum half-life, and may also allow effector function if desired. However, high-yield production of purified bsAb can be challenging because homodimerization of the heavy chain must be prevented. The "dishing" (KiH; also known as "knobs-intos-holes") method provides a solution to this problem. C of heavy chain of antibody (IgG) H The 3 domain is engineered for heterodimerization to allow the construction of Fc-containing bifunctional therapeutic molecules that do not self-associate.
The bulge-recess method involves asymmetrically mutating the C of two parent heavy chains in a complementary manner H 3 in the 3 domain. By at C H The interface between the 3 domains creates a "bulge" by replacing an amino acid with a small side chain with an amino acid with a larger side chain, such as tyrosine or tryptophan, and a "recess" by replacing an amino acid with a large side chain with an amino acid with a smaller side chain, such as alanine or threonine. Variants of the bulge and the recess by inserting the bulge into partner C H 3 domain in a correspondingly designed recess. Due to steric repulsion, the bulge-bulge binding is prevented and the pit-pit homodimer is unstable. For example, the convex mutation may be S354C, T366Y, T366W or T394W, and the concave mutation may be Y349C, T366S, L368A, F405A, Y35407T, Y407A or Y407V (all numbering according to EU). It has been shown that a bulge created towards the center of the dimer interface, e.g. at residue T366, is more damaging for homodimer formation than a bulge located near the edge of the dimer interface. First C H Residue T366 on the 3 domain in the second or partner C H Within the hydrogen bond distance of residue Y407 on the 3 domain, therefore, T366Y and Y407T represent a common protrusion-depression pair; this pairing has been shown to produce heterodimers with yields exceeding 90% (see, e.g., ridgway et al (1996) Protein eng.9 (7): 617-621).
For example, the IgG Fc region in the bispecific TNFR1 antagonist/TNFR 2 agonist constructs provided herein can be modified using a bulge-recess approach to produce high yields of heterodimeric molecules. Table 6 below shows the corresponding convex and concave mutations according to Kabat numbering and sequence numbers, with reference to the sequence of the IgG1 heavy chain constant domain set forth in SEQ ID NO. 9. Any mutation known to those skilled in the art that introduces a bulge or recess may be used in the constructs herein.
TABLE 6
Ligand trap constructs
Fc modified as described above to have "in the bulge and recess" can also be used with other bispecific molecules to produce heterodimers. For example, U.S. patent publication nos. 2010/0055093 and Jin et al (2009) mol. Med.15:11-20 describe dual-specific "ligand" trap constructs targeting EGF receptor family ligands, including one referred to as RB200 and the other as RB242. One problem with these constructs is that they are heterogeneous and contain homodimers and heterodimers, the latter being the intended therapeutic agents. RB200 and RB242 are exemplary ligand traps that can be modified by replacing the Fc portion with a modified Fc region having complementary projections and recesses, such that the resulting dimers are heterodimers. RB242 targets HER1 (EGFR), HER2 and HER3 ligands, as well as some HER4 ligands. It is designed such that it does not capture HER4 specific ligands because HER4 has an effect in neuronal development that other members of the EGFR family do not. RB242 consisted of the extracellular domains (ECDs) of HER1/ErbB1 (amino acids 1 to 621 of SEQ ID NO: 41) and HER3/ErbB3 (amino acids 1 to 621 of SEQ ID NO: 45), fused to the Fc domain of human immunoglobulin G1 (IgG 1) (HER 1-HER 3/Fc), and served as chimeric bispecific ligand traps. The HER3/Fc component of RB242 comprises a 6 Xhistidine tag at the COOH terminus (see, e.g., jin et al (2009) mol. Med. 15:11-20). RB200 binds HER1/ErbB1 ligands (EGF, TGF-alpha, HB-EGF, AR, BTC, EPR and EPG) and HER3/ErbB3 ligands (NRG 1-alpha and NRG 1-beta 3) with high affinity. RB242 inhibits EGF-stimulated and NRG1- β1-stimulated tyrosine phosphorylation of HER family proteins (HER 1, HER2, and HER 3) and shows efficacy in a variety of cell proliferation assays. RB200 inhibits tumor growth in an in vivo animal model.
The Epidermal Growth Factor (EGF) ligand/receptor family plays a role in a variety of diseases, disorders and conditions, including Rheumatoid Arthritis (RA). The EGF family of cell surface receptors (ErbB and human epidermal growth factor receptor (HER)) belongs to the Receptor Tyrosine Kinase (RTK) superfamily, containing extracellular domains (ECD) and intracellular tyrosine kinase signaling domains. The EGF family has four members: EGF receptor (EGFR)/HER 1/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4, which are activated by a large family of ligands including EGF, transforming growth factor alpha (TGF-alpha), heparin binding epidermal EGF-like growth factor (HB-EGF), amphiregulin (AR), beta-animal cellulose (BTC), epithelial regulatory protein (EPR), epigenetic gene (EPG) and Neuregulin (NRG). There are four ECDs in EGFR; domains I and III are ligand binding domains, and domains II and IV mediate binding to each other and to other members of the receptor family. Ligand binding induces homodimer or heterodimer formation between receptors. For example, TGF- α and EGF bind EGFR/HER1/ErbB1, while NRG4 binds HER4/ErbB 4. Depending on the dimers formed, intracellular regions transphosphorylate, resulting in activation of many downstream signaling pathways, leading to cell proliferation, survival and differentiation (see, e.g., jin et al (2009) mol. Med. 15:11-20).
The epidermal growth factor receptor family consists of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 (Erb-B2), HER3 (ErbB-3) and HER 4 (ErbB-4). In many cancer types, mutation or expansion of one family member is associated with worsening survival of cancer patients. In autoimmune diseases, TNF signaling transactivates the EGFR signaling pathway by inducing synthesis of macrophage epiepidermal regulatory protein and heparin binding EGF (HB-EGF), both of which activate EGFR.
In a complementary manner, EGFR and HER2 are upregulated on synovial fibroblasts, driving their proliferation. EGFR, HER2 (ErbB 2) and EGF-like growth factors are overexpressed, for example, in RA synovial fibroblasts and macrophages. Thus, TNF and EGFR pathways act synergistically in the progression of lupus and rheumatoid arthritis, as well as other autoimmune diseases. The constructs provided herein are constructs referred to as "ligand traps". The ligand trap construct intercepts most inflammatory growth factors of the EGFR family, thereby inhibiting the growth of rapidly growing synovial fibroblasts in the affected RA joint. These ligand traps are useful for administration in combination therapy regimens with TNF blocking constructs provided herein that are TNFR1 and/or TNFR2 targeting constructs. Such combination therapies, for example, against rheumatoid arthritis, may be synergistically combined to achieve disease regression.
The EGFR family of growth factors is overexpressed in hyperproliferative/inflammatory diseases such as RA, as well as ovarian and other cancers. Elevated levels of the EGFR family and/or homologs thereof are a common component of a variety of cancers. When overexpressed (or sometimes mutated), these receptors are causally related to the shorter survival of a variety of malignancies. Examples of targeted therapies acting through the EGFR family are cetuximab @ (listing common names and exemplary trademark providing sources)) Panitumumab (>) Trastuzumab (++>) And pertuzumab (>). Small molecule inhibitors are also directed against intracellular tyrosine kinase activity of the EGFR family. Examples of small molecules include lapatinib (/ -herba Drift)>) Erlotinib (/ -)>) And lenatinib (>). These drugs are directed against only one member of the EGFR family, so other members of this family can up-regulate and compensate for tumor growth. Also, antibodies to a single growth factor (e.g., TGF-alpha, EGF, HB-EGF, etc.) inhibit only that growth factor, and tumor cells will be complemented by upregulating other growth factors. The ligand trap constructs provided herein address this problem by blocking HER1, HER2 and HER3 together. This results in the ubiquity of the EGFR family on cancer cells. Ovarian cancer is one of the cancers in need of treatment.
The ligand trap constructs provided herein are improved by optimizing heterodimer production and FcRn recycling using a modified Fc region as described below for the TNFR1/TNFR2 constructs. The ligand trap construct is administered in combination with a TNFR1 antagonist construct, and/or a TNFR2 agonist construct, and/or a multispecific TNFR1 antagonist/bispecific construct, and/or any other construct provided herein, for use in the treatment of diseases, disorders, and conditions in which TNF exerts an effect as described herein and/or known to those of skill in the art.
b) Modification to enhance neonatal receptor (FcRn) recycling
There are a number of ways in which the short serum half-life of a short polypeptide or protein therapeutic can be increased. PEGylation increases the serum half-life of small protein therapeutics, but has drawbacks. PEGylation may reduce the efficacy or activity of protein therapeutics, may result in heterogeneity, and may result in immunoreactivity of the protein. Other methods include fusion with albumin, which can improve protein circulation by increasing molecular weight and decreasing renal clearance.
Serum half-life can also be prolonged by fusion to the Fc portion of IgG. The long circulation half-life and slow clearance of IgG from about 2 to 3 weeks is at least in part due to its interaction with neonatal Fc receptor (FcRn), which binds IgG with high affinity at acidic pH and is neutral or higher IgG was released at pH. FcRn binds to the Fc portion (within the CH2-CH3 domain) of pinocytosis IgG in the acidic (-pH 6) endosome in the 2:1 FcRn-IgG configuration (bivalent interaction), which is transferred from the lysosomal degradation pathway to the cell surface and recycled into the circulation after exposure to extracellular physiological pH (about 7.4), during which the Fc-FcRn complex dissociates. Poor binding to FcRn under acidic pH conditions results in the antibody being transported to lysosomes where it is degraded. Recycling receptors, such as FcRn, also provide a pathway for IgG to pass through epithelial cells (transcytosis) and into the blood stream. The use of interactions with FcRn may improve transport of proteins across epithelial barriers, such as in the gut and lung, to enable non-invasive administration. Fc C H 2 and C H Residues in the 3 domain are involved in FcRn binding, and their mutations in mabs have been shown to affect serum half-life in vivo. By fusing the small protein therapeutics to the Fc domain of IgG, circulation and delivery of the small protein therapeutics can be improved, allowing the resulting fusion protein to bind FcRn and utilize the IgG serum stabilization pathway. Fusion to the Fc domain also increases the molecular weight of the therapeutic agent, reducing renal clearance, but may be undesirable due to the potentially reduced tissue penetration and specific activity of the fusion protein. Alternatively, studies have shown that short FcRn binding peptides (FcRnBP) allow small proteins to interact with FcRn, eliminating the need for fusion to the high molecular weight Fc domain. For example, fusion to FcRnBP increases the molecular weight by about 3kDa, and about 50-70kDa (see, e.g., datta-Mannan et al (2019) Biotechnol. J.14:1800007;Sockolosky et al. (2012) Proc. Natl. Acad. Sci. USA 109 (40): 16095-16100).
For example, short (16 residues) linear and cyclic FcRnBP (see, e.g., SEQ ID NO: 48-51) has been fused to the C-terminus, N-terminus, or both of the Fab heavy and light chains (FcRnBP-Fab constructs), with 1-4 FcRnBP per Fab. Pharmacokinetic studies in cynomolgus monkeys showed that FcRn binding of the FcRnBP-Fab construct increases with increasing number of peptides fused to the Fab. This is due to the increased avidity, where constructs containing four linear fcrnbps are fused to the N-and C-termini of the heavy and light chains of the Fab, showing the greatest improvement in the pharmacokinetics in cynomolgus monkeys over the parent Fab. For example, half-life increases from 3.7 hours for the parent Fab to 15-60 hours for the various FcRnBP-Fab constructs (see, e.g., datta-Mannan et al (2019) biotechnol.j.14: 1800007). Although these results indicate an improvement in serum half-life, it is still much lower than the half-life of IgG, which is about 2-3 weeks. The use of FcRnBP also does not reduce renal clearance because they do not significantly increase the molecular weight of the therapeutic agent.
As described above, fusion to IgGFc increases the half-life of small protein therapeutics by utilizing FcRn binding and by increasing the molecular weight of the therapeutic and allows it to be cleared less rapidly from the body, e.g., by the kidneys. To improve pharmacokinetic and overall pharmacological properties, residues within the Fc region may be mutated to increase affinity for FcRn, typically by more than 30-fold, further increasing the in vivo half-life. Crossing C H 2 and C H The Fc region of the 3 domain interface interacts with FcRn. Human Fc residues identified as playing a role in FcRn binding include, for example, L251, M252, I253, S254, L309, H310, Q311, L314, E380, N434, H435, and Y436 (according to EU numbering, see table 1). Residue mutations at the Fc-FcRn interface, including M252, S254, T256, H433, N434, and Y436 (numbering according to EU), improved the stability of the human FcRn-IgG1 complex. For example, the M252Y/S254T/T256E and H433K/N434F/Y436H substitutions bound to human FcRn at pH6.0 were 11-fold and 6.5-fold, respectively, improved relative to wild-type IgG1 and released efficiently at pH 7.4. The combination of these substitutions resulted in a 57-fold increase in binding affinity for FcRn. Other mutations in IgG1 Fc that show improved binding to FcRn include, for example, M252W, M252Y, M Y/T256Q, M F/T256D, E380A and N434F/Y436H (see, e.g., dall' Acqua et al (2002) J.Immunol.169:5171-5180).
Triple substitution M252Y/S254T/T256E, when introduced with MEDI-524 (a humanized anti-Respiratory Syncytial Virus (RSV) mAb) C H 2, the serum half-life of the mAb in cynomolgus monkeys is increased by about 4-fold when compared to unmodified MEDI-524. Substitution M252Y/S25 when introducing Fc portion of MEDI-522 (a humanized, affinity optimized mAb to human αvβ3 integrin complex) 4T/T256E (YTE) reduced its ADCC activity and its binding to the human FcgammaRIIIA (F158 isoform). By introducing the ADCC enhancing substitution S239D/A330L/I332E (according to EU numbering), ADCC activity of MEDI-522-YTE can be restored and increased compared to unmodified MEDI-522, indicating that the substitution YTE provides a reversible mechanism for modulating ADCC function of human IgG1 (see, e.g., dall' Acqua et al (2006) J.biol. Chem.281 (33): 23514-23524).
Residues at positions 250, 314 and 428 (numbering according to EU) of the human IgG heavy chain conserved in all four human IgG subtypes are also located near the Fc-FcRn interface. Introduction of mutations T250Q, M L and T250Q/M428L into the Fc of a human IgG2 mAb resulted in about 3, 7 and 28 fold increases in binding to FcRn at pH6.0, respectively, but no binding was observed at pH 7.5. When the pharmacokinetics of the mutants were evaluated in rhesus monkeys, it was found that the average clearance (i.e., serum antibody volume cleared per unit time) was about 1.8-fold lower at the M428L mutant, about 2.8-fold lower at the T250Q/M428L mutant, about 1.8-fold longer at the elimination half-life at the M428L mutant, and about 1.9-fold longer at the T250Q/M428L compared to the unmodified antibody. Since these residues are conserved among the IgG subtypes, mutations M428L and T250Q/M428L are expected to have similar effects in human IgG1, igG3, and IgG4 antibodies (see, e.g., hinton et al (2004) j.biol. Chem.279 (8): 6213-6216). Modification of T250R/M428L was shown to result in selective binding to FcRn at pH6.0 in rhesus monkeys, and 2.8-fold reduction in serum IgG2 and IgG1 degradation (see, e.g., saxena et al (2016) front. Immunol. 7:580).
When human anti-HER 2 IgG1 trastuzumab was introduced, mutation N434A (numbering according to EU) resulted in approximately 4-fold higher affinity for human FcRn than the unmodified antibody at pH6, but negligible binding at pH 7.4. N434A variants have increased exposure, decreased clearance (about 2-fold) and increased half-life (about 2-fold) compared to wild-type antibodies when tested in cynomolgus monkeys. In contrast, the mutation N434W resulted in an approximately 80-fold increase in binding to FcRn at pH6, exhibiting a clearance similar to wild type; the mutant also showed significant binding to FcRn at pH7.4, indicating that maintaining pH-dependent binding of the Fc mutant to FcRn is critical for improving pharmacokinetics in vivo (Yeung et al (2009) j.immunol.182:7663-7671). The N434A mutation also counteracts the poor FcRn affinity that may result from introducing mutations that increase binding to fcγr; N434A is typically added to the mutation S298A/E333A/K333A to create variants with enhanced FcγR binding and normal or improved FcRn binding. Fc mutations that improve FcRn binding also include N434Y, E del/T307P/N434Y and T256N/A378V/S383N/N434Y. Deletion of E294 resulted in a higher sialylation degree of N297 glycans on Fc, thereby extending the in vivo half-life of the antibody. Sialylation was shown to play a role in regulating serum half-life as well (see, e.g., saunders, K.O. (2019) front. Immunol. 10:1296).
When humanized anti-VEGF IgG1 antibody bevacizumab is introduced) When replaced M428L/N434S (numbering according to EU) resulted in an 11-fold increase in affinity for FcRn at ph6.0 and an increase in serum half-life in cynomolgus monkeys from 9.7 days to 31.1 days by a factor of 3.2. When the anti-EGFR antibody cetuximab was introduced, the M428L/N434S modification resulted in similar increased FcRn binding and half-life extension, with rapid clearance due to receptor-mediated internalization. The half-life extension of these anti-tumor antibodies is associated with an enhancement of in vivo tumor reduction in a mouse model, indicating that the in vivo therapeutic effect of the antibodies is increased when pharmacokinetics (e.g., clearance) is improved. Other mutations engineered in bevacizumab Fc include (according to EU numbering): N434S, which results in an increase of about 3-fold FcRn binding in mice and an increase of about 2.8-fold serum half-life; V259I/V308F, fcRn binding in mice and cyno increased by about 6-fold, serum half-life increased by about 3-fold and about 2-fold, respectively; M252Y/S254T/T256E, fcRn binding was increased about 7-fold in mice and cynomolgus monkeys, serum half-life was increased about 4-fold and 2.5-fold, respectively; and V259I/V308F/M428L, fcRn binding was increased approximately 20-fold in mice and cynomolgus monkeys, serum half-life was increased approximately 4-5-fold and 2.6-fold, respectively (Zalevsky et al (2010) Nat. Biotechnol.28 (2): 157-159).
The mutations identified above and other such mutations can be introduced into the IgG Fc region in the constructs provided herein. These include, for example, those of formulas 1 and 2, wherein the linker comprises an Fc or Fc dimer, depending on the structure of the construct.
In some embodiments, the IgG Fc region in the constructs herein, such bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and TNFR1 antagonist constructs provided herein are modified to enhance neonatal FcR recycling to increase in vivo half-life. This can be accomplished by mutating the C of IgG Fc H 2 and C H The 3 domain interface residues are implemented, these residues are responsible for binding to FcRn. These residues include, but are not limited to, residues T250, L251, M252, I253, S254, T256, V259, T307, V308, L309, H310, L314, Q311, a378, E380, S383, M428, H433, N434, H435, and Y436, numbered according to EU. Exemplary Fc modifications that increase binding to FcRn include, but are not limited to, one or more of the following: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V428L, E294del/T307P/N434Y, T256N/A378V/S383N/N Y, and combinations thereof, numbered according to EU. Table 7 below shows the sequence of the IgG1 heavy chain constant domain according to Kabat numbering and sequence numbering, with reference to SEQ ID NO. 9. Other modifications known in the art that confer enhanced or increased FcRn binding are also contemplated for use herein.
TABLE 7
c) Enhancement or reduction/elimination of Fc immune effector function
There are four human IgG subclasses that differ in effector function, circulatory half-life, and stability. IgG1 has Fc effector function, the most abundant subclass of IgG, the most common subclass of FDA-approved therapeutic proteins. IgG2 lacks Fc effector function, but dimerizes with other IgG2 molecules and is unstable due to confusion of disulfide bonds in the hinge region. IgG3 has Fc effector function and a very long rigid hinge region. IgG4 lacks Fc effector function, has a shorter circulation half-life than other subclasses, and IgG4 dimers are biochemically unstable due to the presence of a single disulfide bond in the hinge region, which results in H-chain substitution between different IgG4 molecules. Thus, the Fc regions from IgG2 and IgG4 do not have effector functions and can be used in situations where effector functions are not needed or detrimental, such as in the case of autoimmune and inflammatory diseases and disorders.
Most approved therapeutic mabs belong to the human IgG1 subclass and can interact with the humoral and cellular components of the immune system. For example, antibodies are involved in a humoral immune response by interacting with complement protein C1q, complement protein C1q initiates the complement cascade, leading to the formation of a membrane attack complex, thereby inducing cytolysis (i.e., complement Dependent Cytotoxicity (CDC)) in target cells, and in a cellular immune response by interacting with fcγ receptors (fcγr). Fcγr includes fcγri (CD 64), fcγrii (CD 32) and fcγriii (CD 16) classes, which differ in cell surface expression and Fc binding affinity. Five activating fcγrs include high affinity fcγri, which can bind monovalent antibodies, and low affinity fcγriia, fcγriic, fcγriiia, and fcγriiib, which require affinity-based interactions. Fcyriib is the only inhibitory receptor. Upon binding of Fc to an activating receptor, intracellular signaling pathways are regulated by phosphorylation of immune receptor tyrosine-based activating motifs (ITAMs), leading to effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC; also known as antibody-dependent cytotoxicity) and antibody-dependent cell-mediated phagocytosis (ADCP; also known as antibody-dependent cell phagocytosis), and by inflammation due to induction of cytokine secretion. Through the signaling of inhibitory fcγriib, phosphatase is recruited to counter the activated signaling pathway by modulation of the phosphorylation of the immunoreceptor tyrosine-based inhibition motif (ITIM) (see, e.g., wang et al (2018) Protein Cell 9 (1)): 63-73).
Hinge and proximal end C H 2 amino acid sequence (lower hinge-upper C H 2 domain region), and Fc region C H Glycosylation of the conserved N297 residue (numbering according to EU) of the 2 domain Asn-X-Ser/Thr glycosylation motif mediates the interaction of the antibody with FcγR and complement protein C1 q. antibody/Fc engineering has been used to alter immune effector functions of antibodies by altering their binding to C1q and various fcγ receptors. Thus, depending on the application, CDC, ADCC and ADCP activity of the therapeutic mAb may be increased or decreased. For example, the efficacy of anticancer mabs depends in part on their induction of fcγr effector function. Effector functions include activation of Natural Killer (NK) cells by fcγriiia and subsequent ADCC activity and release of inflammatory cytokines, induction of macrophage-mediated ADCP by interaction with multiple fcγrs, and recruitment and activation of other immune cells, such as neutrophils, which are the primary receptors for NK cell-mediated ADCC. There are two polymorphic variants of fcγriiia: one has V158, with higher affinity for IgG 1; one has F158, which has low affinity for IgG 1. Cancer patients with high affinity V158 polymorphism may obtain better results after treatment with cetuximab, trastuzumab, and rituximab than patients with low affinity F158 polymorphism. Results such as these highlight the role of fcγr mediated immune effector functions in therapy and suggest that engineering antibodies and related molecules to increase affinity for fcγr may enhance therapeutic effects (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73).
Lower hinge and proximal C of IgG have been determined H Residues in the 2 region are critical for binding fcγr. Residues within 5 angstroms (angstroms) of the Fc interface for Fcgamm, fcgamma RIIb and Fcgamma RIIIb include residues (according to EU numbering) P232, E233, L234, L235, G236, G237, P238, S239 (corresponding to residues P115-S122, reference SEQ ID NO: 9), D265, V266, S267, H268, E269, D270 (corresponding to residues D148-D153, reference SEQ ID NO: 9), Y296, N297, S298, T299 (corresponding to residues Y179-T182, reference SEQ ID NO: 9), and N325, K326, A327, L328, P329, A330, P331 and I332 (corresponding to residues N208-I215, reference SEQ ID NO: 9)SEQ ID NO: 9) (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73).
Fc modifications that enhance or reduce ADCC activity and/or enhance receptor affinity/binding are known to those skilled in the art. For example, fc modifications that increase the affinity of IgG1 for fcγriiia and bind and/or enhance ADCC function include the following substitutions (according to EU numbering): F243L/R292P/Y300L/V305I/P396L, L V/F243L/R292P/Y300L/P396L, F L/R292P/Y300L, S239D, I332E, S D/I332E, S239D/A330L/I332E, S298A/E333A/K334A, and L234Y/L235Q/G236W/S239M/H268D 270E/S298A of a heavy chain in combination with D270E/K326D/A330M/K334E of an opposing chain, L234Y/G236W/S298A of a heavy chain in combination with S239D/A330L/I332E of an opposing heavy chain. In addition, the mutations A327Q/P329A (interacting with FcgammaRI), D265A/S267A/H268A/D270A/K326A/S337A (interacting with FcgammaRIIa), G236A (interacting with FcgammaRIIa) and T256A/K290A/S298A/E333A/K334A (interacting with FcgammaRIIIa) lead to high affinity interactions with FcgammaR.
Fc modifications that increase binding to fcγriia and fcγriiia and enhance ADCC and ADCP include (according to EU numbering) G236A/I332E, G a/S239D/I332E (also increase binding to fcγri) and G236A/S239D/a330L/I332E (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73; saxena et al (2016) front. Immunol.7:580; and Saunders, k.o. (2019) front. Immunol.10:1296).
Engineering of the glycosyl groups of IgG at C H The inclusion of a conserved N-linked glycosylation site at residue N297 of the 2 domain may enhance Fc effector function. Glycosylation of N297 is critical for maintaining Fc conformation and mediating its interaction with fcγr (and C1 q). Glycans present at residue N297 typically have two N-acetylglucosamines (GlcNAc), three mannoses, and two other GlcNAc linked to mannose to form a double-antennary complex glycan. Additional fucose, galactose, sialic acid, and GlcNAc may be added to the core glycan structure. Circulating IgG found in human serum is typically fucosylated, but recombinant IgG production can alter glycan composition by expressing antibodies in plant cells, knocking in or knocking out specific glycosidases, or enzymatically digesting glycosylated IgG in vitro; because of two weights Chains are glycosylated, so a single IgG molecule may have glycan heterogeneity. Glycans directly affect fcγr binding. For example, N297 glycans on Fc can collide with glycans on fcγriii proteins, resulting in poor effector cell involvement in mediating ADCC. The Fc region containing different glycans at N297 adopts different hinge region conformations, which can affect the ability of Fc to interact with fcγr. When IgG is expressed, expression of β (1, 4) -N-acetylglucosaminyl transferase III results in an antibody that is glycosylated with a biantennary glycan at position N297; such antibodies increase binding to fcγriiia and enhance ADCC activity. Binding of fucose-deficient (defucosylated/nonfucosylated) IgG1 to fcγriiia has been demonstrated to increase 50-fold and enhance ADCC activity. Two glycoengineered (defucosylated) monoclonal antibodies, atozumab (anti-CD 20) and Mo Geli bead mab (anti-CCR 4), have been approved for clinical use, indicating that glycoengineering has the potential to enhance self-function and convert it to clinically approved therapeutic drugs (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73; saxena et al (2016) front. Immunol.7:580; and Saunders, k.o. (2019) front. Immunol.10:1296).
Fc may also be modified to bind to a wider range of Fc receptors. Some leukocytes have non-gamma isotypes of Fc receptors (i.e., igA, igM, and IgE) present and the effector cells are bound by modifying the Fc region to bind to multiple Fc receptors, producing antibodies with amplification capability. Neutrophils are the most abundant leukocytes in the body, and bind to the Fc of IgA antibodies via fcαri receptors. For example, to bind fcγr and fcαri, a single domain of IgA2 is added to the end of the IgG1 constant region, creating a four domain constant region, CH1g-CH2g-CH 3a. The CH1 domain of IgG1 is replaced with the alpha 1 constant region domain, resulting in a constant region (CH 1a-CH2g-CH3g-CH3 a) that is structurally closer to the alpha constant region. These four-domain, cross-isotype IgGA chimeric antibodies bind J-chains similarly to native IgA2, reducing transport through the polymeric Ig receptor, fcγri affinity is reduced 3-5 fold, and the short serum half-life of IgA2 replaces the long serum circulation of IgG 1. However, the four-domain, cross-isotype IgGA chimeric antibodies have the ability to mediate complement-dependent lysis of sheep erythrocytesAbility, and more pH resistant than IgG 1. Another cross-isotype Fc is produced by fusing the gamma 1 and alpha constant regions together to produce a tandem G1-AFc region in which the hinge, CH2 and CH3 domains of IgA2 are fused to the C-terminus of IgG 1. This tandem cross isotype IgG/IgA fusion shows similar expression levels, antigen binding and thermostability as IgG1 and binds fcαri and fcγri, fcγrii, fcγriiia and FcRn in vitro with similar affinities as wild-type IgA and IgG, respectively. Binding to various fcrs results in ADCC activity of polymorphonuclear and NK cells; however, C1q binding was reduced 3-fold compared to IgG 1. The in vivo half-life of tandem IgG/IgA was similar to that of IgG1 in BALB/c mice. By substitution of the IgG1 constant region C H 3 domain and C H 2 alpha 1 loop residues 245-258 (corresponding to sequence PKPKDTLMISRTPE according to EU numbering; residues 128-141 of SEQ ID NO: 9)), another cross-isotype antibody was produced with regions of similar structure to the IgA constant region. Such chimeric fcs are capable of binding fcγri, fcγriia and fcαri, as well as antibodies containing ADCC of chimeric Fc-mediated polymorphonuclear cells and ADCP of macrophages, as well as activated complement, but lack binding to FcRn that modulates antibody half-life; thus, further optimization is required to be effective for use in vivo (see, e.g., saunders, K.O. (2019) front. Immunol. 10:1296).
Another approach to enhance fcγr binding is multimerization of IgG, which has shown promise in the treatment of autoimmune diseases. For example, by adding a heteromultimerization domain such as an isoleucine zipper, or by adding another hinge region at the N-terminus of the natural hinge, or by adding a different chain region at C H The C-terminal end of the 3 domain adds another hinge region, producing an IgG multimer. IgG hexamers are produced by attaching IgM tails to the C-terminus of IgG1 Fc and forming a cysteine bond at position 309; the multimeric IgG binds strongly to fcyri, fcyriia and fcyriiia and binds weakly to fcyriib and fcyriiib. Binding of various multimeric IgG to fcyri, fcyriib, and fcyriii is increased compared to monomeric IgG and shows promise in preclinical models of arthritis, neuropathy, and autoimmune myasthenia gravis. This multimeric IgG design is being further optimized to fine tune which immunity is received The body (including FcRn) can bind to a multimer (see, e.g., sasonders, k.o. (2019) front. Immunol. 10:1296).
Residues in the IgG Fc region involved in interaction and binding with C1q (and hence CDC) include (according to EU numbering) S267, D270, K322, K326, P329, P331 and E333. Fc modifications that have been shown to enhance CDC by increasing C1q binding include, for example, K326A, E333A, K A/E333A, K326W, K W/E333S, K326M/E333S, C220D/D221C, H268F/S324T, S267E, H268F, S T, S E/H268F/S324T and G236A/I332E/S267E/H268F/S324T (both according to EU numbering). Substitution of Trp (i.e., K222W, T W and H224W, numbering according to EU) at positions 222, 223 and 224 in various combinations in the upper hinge region of IgG1 Fc increased C1q binding and CDC activity relative to wild-type IgG1 without affecting fcγriiia binding and ADCC activity. Specifically, the mutations included K222W/T223W, K W/T223W/H224W and D221W/K222W. Mutations C220D/D221C and C220D/D221C/K222W/T223W also increase C1q binding and CDC activity (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73; saxen et al (2016) front. Immunol.7:580; saunders, K.O. (2019) front. Immunol.10:1296; and Dall' Acqua et al (2006) J. Immunol.177:1129-1138).
In vitro binding of IgG3 to C1q was optimal; c of IgG1 H 1 and hinge region and IgG 3C H 2 and C H The 3 regions combine (to retain ADCC activity of IgG1 and CDC activity of IgG 3) to produce IgG1/IgG3 cross subtype antibodies while increasing C1q binding and enhancing CDC activity. Another IgG1/IgG3 cross-subtype antibody with increased C1q binding and enhanced CDC activity includes the C of IgG1 H 1. Hinge and C H 3, and C of IgG3 H 2; these modifications result in increased Cq1 binding, as C1q binds C H 2 domain, and is easy to purify because protein a binds C H 3 domain. Furthermore, modification of E345R/E430G/S440Y results in the formation of IgG hexamers, where K322 is oriented in a position to advantageously interact with the hexamer C1q head, enhancing CDC activity. Mutation E345R alone also resulted in IgG hexamer formation, with increased C1q binding and enhanced CDC activity (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73; saxena et al (2016) front. Immunol.7:580; saunders, K.O. (20)19)Front.Immunol.10:1296)。
Glycoengineering can also be used to improve complement fixation; c capable of modifying Fc H N297 glycans within the 2 domain to increase CDC activity. For example, excessive galactosylation in IgG1 Fc increases C1q binding and CDC activity and also increases thermostability compared to unmodified IgG1 glycoforms. Accordingly, galactosylation of Fc can be used to produce stable biologicals with enhanced CDC activity (see, e.g., saunders (2019) front. Immunol. 10:1296).
Table 8 below summarizes the Fc modifications that increase binding to FcγR or C1q and thus enhance immune effector functions including ADCC, ADCP and CDC, and provides corresponding modifications according to Kabat numbering and sequence numbers, reference to the IgG1 heavy chain constant domain sequence shown in SEQ ID NO: 9. Any one or more of these modifications may be incorporated into the IgG1 Fc portion of the constructs provided herein, alone or in various combinations. Other modifications known in the art that confer enhanced or increased immune effector function are also contemplated herein.
TABLE 8
Therapeutic antibodies can also be engineered to reduce or eliminate immune effector functions. For the purposes herein, in some embodiments, it is of interest to reduce or eliminate ADCC activity, for example. Constructs herein comprising Fc are typically modified to reduce or eliminate ADCC activity.
It is interesting to reduce or eliminate immune effector functions, for example, among others: therapeutic antibodies are antagonistic to prevent receptor-ligand interactions and signaling; antibodies are receptor agonists that crosslink receptors and induce signaling; antibodies are drug delivery vehicles that deliver a drug to a target cell expressing an antigen; also, the reduction or elimination of effector function may prevent target cell death or unwanted cytokine secretion. Reduced effector function may also prevent the antibody-drug conjugate from interacting with fcγr, thereby reducing off-target cytotoxicity. After the occurrence of adverse events associated with the administration of the first approved monoclonal antibody, moromonab (muromonab), the importance of reducing or eliminating effector function becomes apparent, which is designed to prevent T cell activation in transplanted patients receiving donor kidneys, lungs or hearts. Patients administered with moromilast underwent risk induction of pro-inflammatory cytokines (i.e., cytokine storm); this is due in part to the interaction of Moromolizumab with FcγR (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73; and Saunders, K.O. (2019) front.Immunol.10:1296).
There are many known mutations that reduce or eliminate receptor function. For example, L235E and F234A/L235A substitutions in human IgG4, and L235E and L234A/L235A substitutions in human IgG1 (both according to EU numbering) can reduce FcγR and C1q binding and reduce effector functions, such as inflammatory cytokine release. Inflammatory cytokines released by therapeutic antibodies can cause adverse reactions. The S228P/L235E substitution introduced into IgG4 also reduced binding to fcγr; the S228P mutation improved the stability of IgG 4. The mutation S228P/F234A/L235A in IgG4 Fc reduced binding to FcγRI, IIa and IIIa and reduced ADCC and CDC. Triple mutation L234E/L235F/P331S in IgG1Fc reduced binding to FcgammaRI, fcgammaRII, fcgammaRIII, and C1q and reduced CDC, and mutation L234A/L235A/P329G in IgG1Fc abrogated FcgammaRI, fcgammaRII, fcgammaRIII, and C1q binding and reduced ADCP. The mutation L234F/L235E/P331S also reduced binding to Fc gamma R and C1q and reduced effector function of IgG1 Fc. Mutations G237A and E318A in IgG1Fc both reduced binding to fcγrii and reduced ADCP; mutations D265A and E233P reduced binding to fcγri, fcγrii and fcγriii and reduced ADCC and ADCP, and mutation G236R/L328R reduced binding to all fcγr and reduced ADCC. The crystal structure data shows conformational changes of residue P329, which comprises a "proline sandwich" formed between two conserved tryptophan residues present in all FcgammaRs, may be detrimental to interactions with FcgammaRs, and modification of residue D270 may negatively affect interactions with C1q (see e.g., wang et al (2018) Protein Cell 9 (1): 63-73; saunders, K.O. (2019) front. Immunol.10:1296; international application publication No. WO 2019/226750).
Induction of the complement cascade is associated with adverse reactions at the antibody injection site and abrogates C1q binding to Fc, which is also initial in CDC activation. Modification of the Fc region eliminates C1q binding and may be used to eliminate CDC of constructs containing the Fc region. Many mutations that abrogate fcγr binding also abrogate C1q binding, as indicated above. For example, mutation a330L disrupts C1q binding and reduces CDC while also eliminating fcyriib binding. Mutations D270A, P329A, K A and P331A also lead to reduced C1q binding and reduced CDC activity (see, e.g., saunders, K.O. (2019) front. Immunol.10:1296).
Glycosyl engineering can be used to eliminate fcγr and C1q binding. As discussed elsewhere herein, the glycan at residue N297 is a complex biantennary glycan. Modification of the glycan to a high mannose glycan (i.e., high mannose glycosylation) reduces the affinity of IgG1Fc for C1q and reduces CDC activity. Mutations in Fc that reduce or eliminate C1q and fcyri binding can also lead to increased galactosylation and sialylation of N297 glycans; such mutations include, for example, F241A, V264A and D265A. According to EU numbering, mutations N297A, N297Q, N297D and N297G remove the glycosylation site at N297 and reduce effector functions such as CDC and ADCC by eliminating Fc interactions with C1q and fcγr, respectively. The combination of N297G/D265A almost completely eliminates binding to Fc gamma R and C1 q. The binding of the non-glycosylated IgG3Fc (aglycone Fc) to FcgammaRI and C1q is reduced (see, e.g., wang et al (2018) Protein Cell9 (1): 63-73; saunders, K.O. (2019) front. Immunol. 10:1296).
To reduce or eliminate Fc effector function, a large portion of the Fc region from different subclasses lacking the opposite function may be replaced to create a cross subclass Fc region. For example, igG2 binds poorly to fcγr but to C1q, whereas IgG4 lacks binding to C1q but reacts with fcγrs; thus, a lack of C can be constructed1q and fcγr binding IgG2 and IgG4 CH domains. Typically, in the IgG1/IgG4 chimera, the hinge and C H The 1 domain is from IgG2, C H 2 and C H The 3 domain is from IgG4. Cross subclass methods can reduce effector function because IgG1 and IgG3 recruit complement more efficiently than IgG2 and IgG4, and because IgG2 and IgG4 have limited ability to induce ADCC. For example, the anti-C5 mAb eculizumab contains IgG2 residues 118-260 (according to EU numbering; residues 114-273 corresponding to Kabat numbering and residues 1-139 referring to SEQ ID NO: 11), and IgG4 residues 261-447 (according to EU numbering; residues 274-478 corresponding to Kabat numbering and residues 141-327 referring to SEQ ID NO: 15), and has limited or undetectable effector function. Similarly, igG2 variants (IgG 2m 4) with the point mutation H268Q/V309L/A330S/P331S from IgG4 (according to EU numbering; H281Q/V328L/A349S/P350S corresponding to Kabat numbering, and H147Q/V188L/A209S/P210S referring to SEQ ID NO: 11) lack binding to all Fc gamma R and C1Q and exhibit reduced effector functions. Variants containing the IgG2 to IgG4 cross-subclass mutation V309L/A330S/P331S (referred to as IgG2 sigma) (according to EU numbering; V328L/A349S/P350S corresponding to Kabat numbering, and V188L/A209S/P210S reference to SEQ ID NO: 11) and the non-germline mutation V234A/G237A/P238S/H268A (according to EU numbering; V247A/G250A/P251S/H281A corresponding to Kabat numbering, and V114A/G116A/P117S/H147A reference to SEQ ID NO: 11) abrogate binding to FcgammaRs and C1q and exhibit undetectable CDC, ADCC and ADCP activity. IgG1/IgG4 cross subclass variant IgG1 sigma, including the mutation L234A/L235A/G237A/P238S/H268A/A330S/P331S, lacks binding to Fcgamm and IIIa, and binds very poorly to Fcgamma and IIb at high concentrations of antibody, resulting in reduced ADCC and CDC activity (see, e.g., wang et al (2018) Protein Cell 9 (1): 63-73; saunders, K.O. (2019) front. Immunol.10:1296).
Tables 9 and 10 below summarize some IgG1 and IgG4 Fc modifications that reduce or eliminate binding to fcγr and/or C1q, and thus reduce or eliminate immune effector functions, including ADCC, ADCP, and CDC, that can be introduced into the Fc region of the constructs herein. These tables provide corresponding modifications according to Kabat numbering and sequence numbers with reference to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO. 9 or the sequence of the IgG4 heavy chain constant domain shown in SEQ ID NO. 15. Any one or more of these modifications may be incorporated into the IgG1 Fc portion of the constructs provided herein, alone or in various combinations. Other modifications known in the art to reduce or eliminate immune effector function are also contemplated herein.
TABLE 9
Table 10
Other modifications of the Fc part
The Fc portion may also be modified to increase binding to an inhibitory fcγr, thereby suppressing an immune response. Therapeutic antibodies with immunosuppressive Fc modifications are useful in the treatment of inflammatory diseases. These mutations may be incorporated into the Fc portion of constructs herein that are intended to treat diseases and conditions having an inflammatory component or etiology or involvement. For example, an immunosuppressive version of the anti-CD 19 antibody (XmAb 5871; xencor) containing the mutation S267E/L328F (numbering according to EU) binds to inhibitory FcgammaRIIB, increases in affinity by approximately 430-fold, and depletes CD19+ B cells in patients with Systemic Lupus Erythematosus (SLE). When the same mutation is introduced into humanized anti-IgE antibodies (XmAb 7195; xencor), igE is prevented from binding to its high affinity receptor (fceri) present on basophils and mast cells, increasing the affinity to fcyriib by about 430-fold for the treatment of allergies, including allergic asthma. anti-CD 3 antibody TRX4 (tolex), containing the deglycosylated Fc mutation N297A (numbering according to EU) inhibits pathogenic T cells and restores normal Treg cell activity in type 1 diabetic (autoimmune) patients (see, e.g., saxena et al (2016) front. Immunol. 7:580).
Another example is monomeric IgG1 Fc (mFc), which contains the mutation L351S/T366R/L368H/P395K (numbering according to EU), binds FcRn and exhibits a similar in vivo half-life as dimer F, and selectively binds fcγri with high affinity, but does not bind fcγriiia, thereby eliminating Fc-mediated cytotoxicity, including ADCC and CDC. Fcyri is expressed on inflammation-associated cells such as inflammatory macrophages. Targeting the receptor may be useful in the treatment of chronic inflammatory diseases such as arthritis, multiple sclerosis and cancer. Variant mFc kills fcyri when fused to Pseudomonas exotoxin a fragment (PE 38) + Macrophage-like U937 cells. Neither the variant mFc nor the fusion protein showed any cytotoxicity (ADCC or CDC) in vitro (see e.g. Ying et al (2014) mAbs 6 (5): 1201-1210).
Modifications that increase binding to or confer selective binding to inhibitory fcyriib and/or fcyri but not fcyriiia may be engineered into the IgG Fc region in the TNFR1 antagonists and TNFR1 antagonist/TNFR 2 agonist constructs provided herein. Such modifications include, but are not limited to, one or more of S267E, N297A, L328F, L351S, T366R, L H, P395K, S E/L328F, L S/T366R/L368H/P395K and combinations thereof according to EU numbering. Table 11 below shows the corresponding substitutions by Kabat numbering and sequence reference to the IgG heavy chain constant domain sequence shown in SEQ ID NO. 9.
Table 11:
human Serum Albumin (HSA)
One problem with previously provided dabs (see, e.g., international PCT application No. 2008/149744) is that their serum half-life is insufficient for use as a therapeutic agent. Which is linked to an anti-HSA antibody to bind HSA; the half-life is insufficient. In this context, dAb or Vhh antibodies are linked to HSA. HSA has 33 cysteines; cys34 is the only cysteine with a free thiol group that does not participate in disulfide bonds. HSA can be linked to the dAb either directly through its N or C terminus or through a linker, such as a Gly-Ser linker, to extend the serum half-life of the dAb. It may also be linked by free cysteines. Example 6 illustrates a construct containing a dAb linked to the N-terminus of HSA via a Gly-Ser linker.
e. Multispecific TNFR1 antagonist/TNFR 2 agonist construct
To selectively inhibit TNFR1 signaling while enhancing the beneficial effects of TNFR2 signaling, multispecific, e.g., bispecific constructs comprising a TNFR1 antagonist and a TNFR2 agonist are provided (see, e.g., formula 2 above). As discussed above, these multispecific constructs may include linkers and activity modulators as desired to impart advantageous properties.
The TNFR1 inhibitor and TNFR2 agonist portions of the constructs provided herein can be polypeptides or small molecules or combinations thereof; they may be directly linked in any order, or indirectly linked through a linker, such as a Gly-Ser linker, including any of the linkers and/or hinge regions described herein, or they may be linked through a chemical linker. The construct may contain an activity modulator, such as an Fc region or modified Fc, and/or other activity modulator, such as a half-life extending polypeptide, such as HSA, and may be a polymer, such as PEG or a polymeric moiety.
A human TNFR1 antagonist, such as a TNFR1 antagonist shown in any one of SEQ ID NOS: 54-703, or a TNFR1 antagonist that has about or at least about 95% sequence identity to a TNFR1 antagonist shown in any one of SEQ ID NOS: 54-703, is fused at the C-terminus to the N-terminus of a first IgG1 Fc, such as an IgG1 Fc from trastuzumab. The order may be reversed.
Fc region C containing trastuzumab heavy chain H 2 and C H 3 (see, e.g., residues 234-450 of SEQ ID NO: 26). In some embodiments, the linker between the TNFR1 antagonist and the first Fc subunit contains all or part of the hinge sequence of an antibody, such as trastuzumab (SCDKTH; residues 222-227 corresponding to SEQ ID NO: 26). Proteases for imparting fusion proteinsResistance and increased flexibility, the SCDKTH hinge sequence or protease cleavage site or both may be replaced with Gly-Ser short peptide linkers, such as GSGS, GGGGS or GGGGSGGGGSGGGGS and other linkers described herein and/or known in the art. In other embodiments, only GS linkers are included. In another embodiment, the linker comprises PEG or branched PEG, having a molecular weight of 30kDa or greater.
In some embodiments, the Fc subunit (also referred to as a region or domain) may be multimerized. The first Fc subunit is attached to the second Fc subunit by disulfide bonds. For bispecific constructs, the C-terminus of the second Fc subunit is linked to the N-terminus of a TNFR2 agonist, e.g., a TNFR2 agonist as set forth in any one of SEQ ID NOS 765-801, 803 and 810, or a TNFR2 agonist having about or at least about 95% sequence identity to a TNFR2 agonist as set forth in any one of SEQ ID NOS 765-801, 803 and 810. The second Fc subunit and TNFR2 agonist are linked by a linker, such as the SCDKTH hinge sequence of trastuzumab, alone or in combination with a short GS linker, as described above. In other embodiments, only GS linkers are included. In alternative embodiments, single chain Fv fragments (scFv) or Fab regions or other antigen-binding fragments of TNFR2 agonistic monoclonal antibodies may be used; scFv or Fab dimerizes by fusion of the N-terminus to the C-terminus of Fc. As provided herein, the antigen binding fragment may be derived from TNFR2 agonist mAb MR2-1 and mAb226.
The Fc subunit may be modified to alter its activity. For example, dimers are modified to prevent homodimerization, and/or eliminate immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC), and/or enhance neonatal FcR (FcRn) recycling to increase the in vivo half-life and stability of recombinant constructs, as described below.
In embodiments of the constructs for treating inflammatory diseases, the Fc portion is modified to have reduced or eliminated effector function. For example, in embodiments where the construct is used to treat cancer, the Fc dimer is modified to enhance immune effector functions, such as ADCC, ADCP and/or CDC. The specific Fc modification depends on the intended disease target.
In some embodiments, the Fc subunit may contain an IgG4 Fc region, e.g., derived from Nawuzumab @) Comprises the IgG4 Fc of the heavy chain of the Nawustite H 2 and C H 3 (see, e.g., SEQ ID NO:29 residues 224-440). A short peptide linker containing all or a sufficient portion of the nivolumab hinge sequence ESKYGPPCPPCP (see, e.g., residues 212-223 of SEQ ID NO: 29) may be included between the nivolumab Fc region and the TNFR1 antagonist and/or TNFR2 agonist. Optionally or alternatively, a GS linker may also be included.
In exemplary embodiments, because TNFR2 may require receptor aggregation/clustering for signaling, bivalent antibody-like structures may be generated to achieve excellent agonism. In this embodiment, the C-terminus of the first and second Fc subunits are each fused to the N-terminus of a TNFR2 agonist, as described above. The Fc dimers are modified to prevent homodimerization, eliminate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and enhance neonatal FcR recycling to increase the in vivo half-life of the recombinant construct, as described elsewhere herein.
Pegylation of components for linking multispecific constructs, PEG-centered multispecific constructs such as bispecific TNFR1 antagonist/TNFR 2 agonist constructs
Pegylation refers to the covalent attachment of biocompatible and bioinert polymeric polyethylene glycol (PEG) to molecules such as proteins, peptides, drugs and other molecules, which is another modulator of the activity of the construct. It can increase the water solubility of molecules, increase molecular weight of molecules, extend circulation time in vivo, decrease peripheral clearance, minimize nonspecific uptake, and target tumors through Enhanced Permeability and Retention (EPR) effects. PEGylation of therapeutic agents, including protein therapeutic agents, can mask unwanted antigenic surface markers to protect the therapeutic agent from antibodies and antigen-treated cells and reduce degradation of proteolytic enzymes and other inactivation processes. PEGylation also increases the molecular weight of the protein therapeutic, increases the in vivo half-life and reduces the rate of Zhou Qingchu ex vivo, and allows for reduced frequency of administration.
Chemical conjugation of therapeutic molecules to polymers such as PEG can form stable ester or amide linkages, as well as disulfide linkages. Conjugation of PEG to molecules of interest, such as TNFR1 antagonists and TNFR2 agonists provided herein, may be achieved, for example, by using a coupling agent, such as Dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), HATU (1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-hexafluorophosphate), or other coupling agents known in the art, or by using an N-hydroxysuccinimide (NHS) ester, such as PEG NHS ester. Other methods include the use of PEG maleimide, which reacts with sulfhydryl groups on proteins or peptides; PEG pentafluorophenyl (PFP) ester, which reacts with primary and secondary amines; thiol PEG, which reacts with thiols on the side chain of cysteine residues; click chemistry techniques. PEG azides, propargyl PEG, aminoxyPEG, hydroxyPEG, aminopolyPEG, PEG acids, biotin PEG, PEG tosylate, and PEG with other functional groups are also commercially available and can be used for conjugation to peptides and other therapeutic molecules.
A common method for preparing PEG-protein conjugates is to apply-NH on the protein 2 Coupling the group to monomethoxy PEG (mPEG) having electrophilic functional groups; this approach results in the formation of polymer chains that are covalently linked to the core globular protein. This property is exploited herein to provide PEG-centric constructs in which PEG or chemically similar or suitable moieties display multiple binding or interacting moieties for one or more different targets. To increase drug (binding moiety) loading, multi-arm or branched PEG (or similar moiety or branched moiety) may be used. Alternatively, the drug may be conjugated to a small PEG dendrite (see, e.g.PEG conjugation protocol on websites, available at broadpharm.com/web/protocols.php; see also Banerjee et al (2012) Journal of Drug Delivery, arc ID 103973). Is thatWith multiple, e.g., two, different therapeutic moieties attached, a heteromultifunctional, e.g., heterobifunctional PEG moiety having different reactive groups at each terminus can be used, for example. The PEG moiety may have two, three or more different reactive groups. Such molecules can be used to deliver two or more different ligands, target two different receptors on the same cell or different cells, such as TNFR1 and TNFR2 described herein, or deliver two targeting agents that bind to different sites on the same receptor, or cluster receptors, such as to activate or inhibit a receptor, or crosslink two different receptors, such as to inhibit receptor activity. Homobifunctional PEG molecules have the same reactive groups at each end and can be used to cluster the same receptor on the same cell or on different cells where PEG chain length permits. Such constructs may be used to capture circulating soluble receptors or ligands, such as TNF. Fig. 3 herein provides an exemplary construct using a PEG moiety to display a drug (conjugated to a reactive moiety).
To increase reactivity and flexibility, enhance ligand-protein binding, and reduce steric hindrance, the construct may include a linker molecule or a plurality of linker molecules as described herein as spacer molecules. Such spacers include, for example, amino acid spacers such as alanine, glycine, and small peptides. Any of the linkers described herein, including GS linkers and other flexible linkers as well as rigid linkers, can be used to conjugate reactive moieties such as the TNFR1 inhibitor moieties described herein and/or TNFR2 agonists described herein to multifunctional PEG molecules. These constructs may also include an activity modulator, such as an Fc region.
The use of branched PEG moieties or multi-arm PEG moieties as described herein (see, e.g., fig. 5), with or without linkers, is not limited to use in constructs containing TNFR1 inhibitor moieties or TNFR2 agonists or a combination of both, but can be used to present other inhibitor and/or agonist moieties for any receptor of interest and/or can also be used to generate immunotoxins and other toxic conjugates. Methods for synthesizing a large number of PEG moieties and variants thereof are known (see, e.g., U.S. patent publication No. 2010/0221213;Han et al, (2014) Sci Rep 4:4387.).
For example, in some embodiments comprising a TNFR1 inhibitor and a TNFR2 agonist moiety, the construct comprises a bifunctional PEG moiety, and further comprises a linker between the PEG moiety and each of the TNFR1 antagonist and the TNFR2 agonist. The multispecific construct comprises a branched PEG polymer to which a linker is attached, one or both of a TNFR1 inhibitor moiety and a TNFR2 moiety being attached to the polymer. Suitable PEG moieties can have a molecular weight of 30kDa or greater, e.g., 30-40kDa or greater. Exemplary branched PEG molecules can be, for example, 3-arm, heterobifunctional PEG molecules containing one arm with one type of reactive group (RG 1; e.g., -NH) 2 ) Attached to the TNFR1 inhibitor moiety, and two arms, with different types of reactive groups (RG 2; e.g., -COOH), each linked to a TNFR2 agonist. Such 3-arm heterobifunctional branched PEG molecules are commercially available (e.g., from). The first PEG arm may be linked to the N-terminus or C-terminus of the TNFR1 inhibitor moiety and the other two arms may be linked to the N-terminus or C-terminus of the TNFR2 agonist or TNFR2 agonist (if a small molecule). In some embodiments, the construct may further comprise an optional linker, as described herein. Such linkers may be included between the PEG arm and the TNFR1 inhibitor moiety and/or TNFR2 agonist. This PEG-centered bispecific construct provides monovalent for TNFR1 antagonist activity, thereby preventing TNFR1 receptor clustering that leads to unwanted agonism, and divalent for TNFR2 receptor clustering, thereby enhancing TNFR2 signaling. An exemplary structure of the PEG-centered bispecific TNFR1 antagonist/TNFR 2 agonist construct described herein is depicted in fig. 3.
In another embodiment, the linker between the TNFR1 antagonist and the TNFR2 agonist comprises a branched PEG having a molecular weight of 30kDa or greater. Branched PEG molecules contain one branch attached to the N-terminus of the TNFR1 antagonist and two branches each attached to a TNFR2 agonist, providing bivalent properties to the clustering of TNFR2 receptors, thereby enhancing TNFR2 signaling.
Figures 3A-D depict various configurations of multispecific constructs in which PEG moieties are linked to functional moieties. PEGylation moieties and PEGylation procedures are discussed in more detail below in section H. Methods for preparing various PEG linkers and configurations are well known to those skilled in the art (see, e.g., createpengworks. Com/conjugation_nature. Php; and broadpharm. Com/web/protocols. Php). In FIG. 3, each n may independently be 1-10, such as 1-7, 1-5, and 1-3, 1, or 2. In fig. 3A, each n is typically 1-3, depending on the particular ligand displayed. In the remainder of fig. 3, n is typically 1 to 5. In all embodiments, N may be 1. Those skilled in the art will recognize other conventional variations of PEG moieties for use as a central linker; similar moieties may be used in place of PEG.
In fig. 3A:
R 1 is H or lower alkyl (C1 to C5, or C1 or C2), e.g. CH 3 N is typically 1 to 5, for example 1 or 2. Ligands or epitopes (i.e., epitopes on a receptor) that bind to multiple targets are depicted, e.g., targets (circles) 1a, b, c represent different epitopes on the same receptor. Target 2 may be an epitope on a different receptor. In fig. 3B and 3C, circles are ligands targeting an epitope or receptor, n is typically 1-5, typically n is 1 or 2, typically 1. FIG. 3D depicts homobifunctional constructs; n is typically 1-3, typically 1 or 2, e.g. 1. The activity modulator is optional, such as Fc or other activity modulator described herein or known to those of skill in the art. In all of these constructs, the PEG moiety is typically "inactive" except for providing activity modulating activity, such as half-life extension and/or steric ligation of an operative fragment that binds to the intended target (peptide, small molecule, aptamer, etc.).
Fig. 3A depicts an example bivalent construct, wherein PEG is the central moiety. For example, one circle is a polypeptide agonist, antagonist or binding protein, such as an antibody or antigen binding fragment thereof, or an aptamer (nucleic acid or peptide). The other circle represents a different moiety, such as a polysaccharide or a receptor ligand. The divalent structure provides target clustering for receptor activation. In some embodiments provided herein, the targets include TNFR1 and TNFR2, and the circles represent TNFR1 inhibitors and TNFR2 agonists described in the disclosure herein. Figure 3B depicts a monovalent single ligand, such as cd3+, which can prevent cytokine release syndrome, linked to an agonist, antagonist or binding protein through a PEG moiety, which is bivalent for receptor clustering. FIG. 3C depicts heterobifunctional PEG (or other such carrier) for crosslinking two different cell targeting agents, or two agents that bind to the same receptor or different sites on two receptors, such as trastuzumab and pertuzumab. For example, the constructs of fig. 3B and 3C can be used to cluster checkpoint control receptors to stimulate or inhibit an immune response, or to crosslink two different receptors to achieve inhibition of receptor activity (i.e., CD3 versus CD 450), or to deliver two different ligands, such as a stimulatory ligand and a co-stimulatory ligand, to two different receptors on the same cell. These constructs may also be used as prodrugs, directed to or accumulating in anoxic zones with lower pH values where the linked moieties may be chemically released by protonation. For example, for tumors, this may be a toxin, or a locally released TNF inactivating agent (i.e., an aptamer or peptide).
Fig. 3D depicts homobifunctional PEG for clustering the same receptors on the same or different cells, or capturing circulating disease targets, e.g., soluble receptors or ligands, e.g., TNF, depending on chain length. Furthermore, in all of these embodiments, additional PEG side chains, optionally linked to another reactive group or functional group, e.g. a serum half-life extending moiety such as HSA, or FcRn polypeptide, may be included in these constructs.
Other structures are also contemplated, wherein X and Y refer to reactive groups, such as binding moieties, i.e., molecules that interact with a target (see fig. 4):
other examples (see, e.g., fig. 5) are described below, and X and Y may be any targeting moiety or binding moiety or drug that interacts with a target as described above:
f. other Activity modulators-fusion proteins comprising a partial or complete polypeptide that increases serum half-life
The properties of the construct may be altered by the addition of a full-length polypeptide or portion thereof that increases serum half-life, but without substantially altering or altering the interaction of the construct with TNFR1 and/or TNFR 2. This includes albumin and other such modifications (see discussion above regarding half-life extenders; reviewed in Strohl (2015) BioDrugs 29 (4): 215-239; see also Tan et al (2018) Current Pharmaceutical Design 24:4932-4946).
5. Prediction and removal of immunogenicity in protein therapeutics
Many protein therapeutics, including those containing human germline sequences, such as recombinant human cytokines and human antibodies, are immunogenic and induce an immune response in the host to the therapeutic. As described herein, constructs provided herein, including TNFR1 antagonist molecules and TNFR2 agonists and multispecific constructs described and provided herein, can be modified, e.g., by amino acid substitutions, as needed, to remove or eliminate the immunogenicity or epitope that interacts with a pre-existing antibody: .
The constructs are predicted, identified and depleted of immunogenic B-cell and/or T-cell epitopes, thereby reducing or eliminating any potential immunogenicity and improving the safety, tolerability and efficacy of the therapeutic molecule. These molecules were tested in vitro assays and in vivo animal models to determine immunogenicity of the immunogenic sequences before and after removal.
As discussed in more detail below, the protein therapeutic may contain immunogenic B-cell and/or T-cell epitopes. When the immune system recognizes a protein therapeutic as a foreign substance, a coordinated, unwanted immune response is induced against the therapeutic. The response can lead to clinical complications including, for example, rapid clearance of the drug, reduced drug function and efficacy, delayed infusion-like allergic reactions, anaphylactic reactions, and in some cases life-threatening autoimmunity. Immune responses to protein therapeutics occur through two different mechanisms; traditional immune responses and breaks tolerance. The immunological distinction between self-proteins and non-self proteins determines the mechanism of the immune response, and proteins identified as foreign elicit a classical immune response, characterized by the formation of antibodies within days to weeks after administration, typically after a single injection of the protein therapeutic. This response is durable and is difficult to reverse once memory B cells are formed. Subsequent exposure to the protein elicits a secondary response characterized by a large amount of IgG that negatively affects treatment. Therapeutic proteins that induce traditional immune responses include alternative therapies, such as rhGAA (recombinant human acid alpha-glucosidase) and FVIII, as well as monoclonal antibody (mAb) therapies in which the Complementarity Determining Regions (CDRs) are highly immunogenic and result in the production of anti-idiotype alloantibodies due to the lack of central tolerance to the CDR regions. Therapeutic proteins homologous to endogenous proteins do not normally lead to an immune response due to established immune tolerance, but after repeated administration they can be rendered immunogenic by disruption of B cell tolerance, for example in the case of administration of IFN- γ, INF- β and Erythropoietin (EPO) (see e.g. Baker et al (2010) Self/noself 1 (4): 314-322; choi et al (2017) Methods mol. Biol.1529:375-398;Dingman et al (2019) j.pharm. Sci.108 (5): 1637-1654).
Factors that affect the immunogenicity of protein therapeutics include, for example, the duration of treatment, the route and frequency of administration; subcutaneous administration of protein therapeutics is more immunogenic than intravenous administration, and long-term, frequent administration of therapeutics is more immunogenic. Patient-related factors, such as the patient's immune status and polymorphism of MHC (or human HLA) molecules, can also affect protein immunogenicity. For example, MHC molecules are highly polymorphic, with MHC II present in a number of different alleles, including different subunits, such as DP, DM, DOA, DOB, DQ and DR; the binding affinity of these receptor subtypes for epitopes varies, and thus, differences in MHC subtypes among patients affect the binding to proteinsImmune response of the plasma therapeutic agent. The immune status of a patient can also affect immunogenicity because autoimmune patients respond more strongly to protein therapeutics than patients with low immune function. Other factors affecting immunogenicity include the nature of the protein product including, for example, the presence of immunogenic epitopes recognized by MHC II, the formation of aggregates in the final product, oxidation of the protein, aggregation in the formulation and post-translational modifications such as glycosylation. Recombinant proteins can be produced in several different cell types, including, for example, bacterial cells such as E.coli and mammalian cells such as CHO cells. Proteins expressed in bacteria do not undergo post-translational modifications such as glycosylation, but proteins produced in mammalian cells are affected, which can lead to different immunogenicity. For example under the trademark The interferon sold, interferon beta-1 b, is produced in E.coli cells, is not glycosylated and is later developed under the trademark ∈ ->The product sold (interferon beta-1 a) was produced in CHO cells using recombinant DNA technology. Immunogenicity ratio of BetaserunThe interferons were much higher, 35% and 5%, respectively. This difference may be due in part to differences in glycosylation patterns that may lead to aggregation (see, e.g., dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654).
The effect of the administration of protein therapy is the production of high affinity anti-therapeutic antibodies, also known as anti-drug antibodies or ADA. ADA production involves stimulation of adaptive and non-adaptive immune responses that are primarily polyclonal and can produce neutralizing and non-neutralizing effects on protein therapeutics. ADA can contain multiple isotypes (e.g., igM, igE, and IgG) as well as subclasses of heavy chain constant regions (e.g., igG 1-4) and contain variable regions that bind protein therapeutics with high affinity, thus undergoing somatic hypermutation of the variable region genes. The immune response is induced by recognition of immunogenic peptide fragments such as B-cell and T-cell epitopes in protein therapeutics. Thus, many protein therapeutics require deimmunization prior to application in the clinic, while retaining the desired therapeutic activity (see, e.g., baker et al (2010) Self/Nonself 1 (4): 314-322; choi et al (2017) Methods mol. Biol. 1529:375-398).
The formation of anti-drug antibodies (ADA) against protein therapeutics is mediated by Antigen Presenting Cells (APC), such as Dendritic Cells (DCs) and macrophages, and B and T lymphocytes. MHC class II restricted T cell epitopes present in the sequence of a protein therapeutic may lead to the occurrence of a humoral response against the protein therapeutic. For example, DCs stimulated by Pattern Recognition Receptors (PRRs) stimulate T cells and induce the generation of T cell-dependent high affinity ADA responses. In the first step of the T cell dependent antibody response, APCs phagocytose the protein therapeutics, process the antigen into peptide epitopes, and present the epitopes to naive T cells by coupling the epitopes to Major Histocompatibility Complex (MHC) class II molecules on the surface of the APC cells. In order to fully activate T cells required for B cell activation, the T Cell Receptor (TCR) must interact with the MHC II epitope complex and this must be accompanied by additional signals from costimulatory molecules provided by APCs, such as CD80 and CD86. Naive B cells are activated by interactions between IgM and IgD receptors on the B cell surface and their cognate antigens. Antigen-specific T cells then secrete cytokines that stimulate B cells to proliferate and mature into plasma cells, which results in the binding of CD40 and CD40 ligands, providing further signals that produce antibodies by B cell clonal expansion and differentiation into antibody-secreting plasma cells and memory B cells. Memory B cells remain dormant until subsequent exposure to the therapeutic protein, while plasma cells secrete antibodies recognizing specific epitopes on the therapeutic protein presented by APC MHC receptors. Many protein therapeutics, including recombinant human proteins, contain potent T cell epitopes. For example, after mapping and removal of a single immunodominant (but not sub-dominant) T cell epitope by amino acid mutation, the immunogenicity of ifnβ1b is improved.
Immunogenicity can also occur in T cell independent processes whereby antigens bind directly to B cells. The high molecular weight aggregates of protein therapeutics can induce T cell-dependent and independent anti-drug antibody responses by stimulating DCs or by crosslinking B cell receptors. For example, if the protein therapeutic forms a multimeric structure that can crosslink B Cell Receptors (BCR) sufficiently to effectively avoid the need for T cell co-stimulation, T cell-independent stimulation of B cells may occur, resulting in an ADA response. There is a correlation between enhanced immunogenicity and aggregated or multimerized proteins. For example, recombinant human Interferon (IFN) alpha, which is aggregated rather than monomeric, results in the production of IFN alpha-specific antibodies in human IFN alpha transgenic mice. Aggregate formation depends on drug solubility and manufacturing process (see, e.g., baker et al (2010) Self/Nonself 1 (4): 314-322;Dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654;De Groot,A.S.and Moise (2007) curr. Opin. Drug discovery.level.10 (3): 332-340).
The ADA response to the protein therapeutic may be in the form of a neutralizing or binding antibody. Neutralizing antibodies recognize regions necessary for biological activity of protein therapeutics and directly eliminate their activity. The humoral response is directed against B-cell epitopes within the protein therapeutic, thereby yielding the ability to neutralize the protein therapeutic. For example, human anti-mouse (HAMA) or human anti-human (HAHA) responses directed against antibody therapeutic idiotypes have a neutralizing effect and can be generated against humanized antibodies and fully human antibodies. For example, in 30% of hemophilia a patients, neutralizing ADA is generated against the administered recombinant FVIII, thereby eliminating its hemostatic efficacy, whereas in 89-100% of Pompe patients receiving rhGAA, the anti-rhGAA neutralizing antibodies undermine therapeutic efficacy. Binding antibodies alters the pharmacokinetic properties of the protein therapeutic and indirectly affects its efficacy by reducing systemic exposure, for example by promoting rapid clearance of the protein. For example, prolonged use of adalimumab results in about 28% of patients developing ADA, resulting in lower adalimumab concentrations and poorer clinical outcome (see, e.g., baker et al (2010) Self/noself 1 (4): 314-322;Dingman et al (2019) j.pharm.sci.108 (5): 1637-1654).
ADA levels can be assessed and monitored pre-, during-, and post-treatment. There are a variety of assays available. These assays include bridging immunoassays that involve the labeling of drugs, and the detection of ADA that forms a bridge between two labeled drug molecules. Bridging detection can be used for all antibody classes and any type of sample. Ligand Binding Assays (LBAs) for detecting binding to a target include Surface Plasmon Resonance (SPR), electrochemiluminescence, and bioluminescence interferometry, but also can be used to detect ADA. Protein specific assays, such as the Bethesda assay, which have been used to measure the concentration of neutralizing anti-FVIII antibodies, can be used. anti-PEG antibodies can also be measured in assays in which biotin-PEG is conjugated to magnetic beads, and the amount of anti-PEG antibody bound to the magnetic beads is measured using a sensor that detects changes in complex size. Drug tolerance assays overcome limitations imposed by the presence of drugs in the sample and improve the quantification of ADA. These assays include, for example, pH shift idiotype antigen binding assays, acidolysis assays, temperature shift assays, and electrochemiluminescence assays. Enzyme-linked immunosorbent assays (ELISA) can be used to detect ADA; protein therapeutics were coated on the plates and incubated with the samples to measure the bound ADA. ELISA for detecting ADA may be limited due to lack of standards for ADA. Other methods include immuno-PCR, an extension of the bridging assay, in which complexes are labeled with biotin and detected using anti-biotin antibodies conjugated to DNA. The DNA was then amplified using PCR and quantified to assess ADA levels. immune-LC/MS can be used to detect ADA in plasma samples; the sample must be enriched by labeling the drug with biotin or by incorporating an excess of the drug into the sample to saturate ADA binding (see, e.g., dingman et al (2019) j.pharm. Sci.108 (5): 1637-1654).
Predicting and removing immunogenic epitopes from protein therapeutics (i.e., deimmunization) can improve the effectiveness and safety of the therapeutics and prevent adverse reactions that can lead to failure of the clinical trial drug. For example, depletion of T cell epitopes from protein therapeutics by deimmunization has been successful in the entry of protein therapeutics, particularly antibodies, into clinical trials. These results indicate the importance of T cell epitopes in generating ADA responses, and that deimmunization provides safer, less immunogenic therapeutics (see, e.g., baker et al (2010) Self/nossell 1 (4): 314-322). These methods can be used to detect or identify or predict the immunogenicity of constructs provided herein, and can be used to identify amino acid mutations to eliminate or reduce the immunogenicity or immune response of a subject. Provided are constructs modified to reduce or eliminate immunogenicity. De-immunization of protein therapeutics involves the identification of highly immunogenic B-cell and/or T-cell epitopes, with deletion of the identified epitopes by mutagenic substitution of key amino acid residues. As described below, preclinical prediction and assessment of immunogenic regions within protein therapeutic sequences includes the use of computer tools focused on epitope mapping, in vitro methods such as epitope mapping, MHC/HLA affinity assays and T cell proliferation assays, and in vivo testing of animal models. To increase the efficiency of deimmunization of protein therapeutics, computational epitope prediction tools are used. The computer tools include databases and algorithms that can rapidly predict immunogenic sequences in peptide libraries. The results can then be confirmed and the specific immunogenic effect of the epitope on B cells or T cells can be further assessed using in vitro assays. In vivo assays can be used to assess the effect of immune responses to protein therapeutics in animal models such as transgenic mice and non-human primates.
Once an immunogenic epitope is identified, the amino acid sequence of the therapeutic agent may be modified to remove the epitope. The removal method involves random or site-directed mutagenesis to remove immunogenic sequences (i.e., to deimmunize epitopes). After mutagenesis, the immunogenic sequence was re-assessed to confirm that it was no longer immunogenic. There are some computer tools that can simplify this process; for example, a useful procedure is to replace each amino acid in an immunogenic sequence with one of the other 19 naturally occurring amino acids (especially alanine) in turn, and then re-evaluate the sequence for immunogenicity. In this way, non-immunogenic sequences can be effectively narrowed to the most promising candidates prior to peptide synthesis and re-evaluation of immunogenicity in vitro and/or in vivo. However, the predictive and mutagenic deletion of immunogenic epitopes is not necessarily sufficient to deimmunize the protein, as the protein must retain its folded, stable and active structure in order to retain its therapeutic efficacy. Therefore, it is necessary to select for epitope deletion mutations that are compatible with the structure and function of the protein.
The methods and means discussed below are used to predict, identify and eliminate epitopes of the constructs provided herein.
a.B cell and T cell epitope
The interaction between antigen and antibody is important for inducing a humoral immune response against the invading pathogen. Specific antibodies recognize a particular antigen in discrete regions called antigenic determinants or B cell epitopes. B cell epitopes comprise clusters of amino acids that are accessible on the surface and are recognized and bound by secreted antibodies or B Cell Receptors (BCR) that contain membrane-bound immunoglobulins and that induce cellular or humoral immune responses.
The identification of B cell epitopes is part of the development of antibodies and other protein-based therapeutics. B cell epitopes are divided according to spatial structure into continuous (also called linear) epitopes, which contain contiguous residues, and discontinuous (also called conformational) epitopes, which are nonlinear and conformational. Discontinuous B cell epitopes contain a set of solvent-exposed amino acid residues that are not completely continuous, but which are in close proximity when the protein/antigen is folded into its three-dimensional conformation. About 90% of the B cell epitopes are conformational. Linear B cell epitopes can be recognized by antibodies after antigen denaturation, but conformational epitopes will no longer be recognized if the antigen is denatured. The minimum amino acid sequence or contact residue span required to properly fold a discontinuous B cell epitope is in the range of about 20 to 400 residues in the native protein. Most of the linear B cell epitopes identified are considered to be part of conformational B cell epitopes and it has been shown that more than 70% of discontinuous B cell epitopes contain 1-5 linear segments, each segment being 1 to 6 amino acids in length (see, for example, potocnakova et al (2016) Journal of Immunology Research, particle ID 676830; sanchez-trincoado et al (2017) Journal of Immunology Research, particle ID 2680160).
T cell epitopes are linear and bind to Major Histocompatibility Complex (MHC) molecules by interaction of amino acid side chains with binding pockets in MHC epitope binding cells. The presence or absence of specific side chains determines whether an epitope binds to MHC and how tightly it binds (see, e.g., de Groot, A.S. and Moise, L. (2007) Curr.Opin. Drug discovery.level.10 (3): 332-340). T cells have a T Cell Receptor (TCR) that recognizes an antigen displayed on the surface of an Antigen Presenting Cell (APC) and bound to an MHC molecule. T cell epitopes are presented by MHC class I (MHC I) and class II (MHC II) molecules, which are each presented by CD8 + And CD4 + T cell recognition; thus, CD8 is present + And CD4 + T cell epitopes. CD8 + T cells in CD8 + Cytotoxic T Lymphocytes (CTL) are formed upon T cell epitope recognition, with simultaneous priming of CD4 + T cells form helper T (Th) cells that amplify the immune response, or form regulatory T (Treg) cells, which are immunosuppressive uses (see, e.g., sanchez-trincoado et al (2017) Journal of Immunology Research, arc ID 2680160).
b. Computer epitope prediction method
Experimental studies and computer analysis have shown that most epitopes span 15-25 residues and 600-Is cyclic in organization, and the epitope sequence is rich in tyrosine, tryptophan, charged and polar amino acids, has exposed side chains, and has specific amino acid pairs. However, the differences between epitope and non-epitope residues have been shown to be insignificant, with amino acid composition alone being insufficient to distinguish between epitope and non-epitope. The combination of epitope mapping techniques with bioinformatics has led to the development of epidemic informatics involving the use of computer methods in immunology to identify the structure of antibodies, B cells, T cells and allergens, to predict MHC binding, epitope modeling and immune network analysis (see, e.g., potocnakova et al (2016) Journal of Immunology Research, arc ID 6760830). / >
Computer prediction of i.B cell epitopes
B cell epitope prediction identifies immunogenic epitopes so that they can be replaced/deimmunized, for example for therapeutic protein production. Databases of known B cell epitopes have been developed, including manifold databases such as the Immune Epitope Database (IEDB) and IEDB-3D (available at IEDB. Org) and AntiJen (available at ddg-pharmfac. Net/anti jen/AntiJen/anti homepage. Htm); b cell-oriented databases such as BciPep (available at imtech. Res. In/raghava/BciPep/info. Html), epitome (available at rostlab. Org/services/Epitome) and allergen protein structure database (SDAP; available at fermi. Utmb. Edu.); and databases for single pathogenic organisms such as the HIV molecular immunology database (available at hiv.lanl.gov/content/immunology/index.html), FLAVIdB (available at cvc.dfci.harvard.edu/flavi), and the influenza sequence and epitope database (ISED; available at influzza.cdc.go.kr.). Other B cell epitope databases include conformational epitope databases (CED; available at immunenet. Cn/CED/available), protein databases (PDB; available at rcsb. Org) and structural epitope databases (SEDB; available at SEDB. Bicpu. Edu. In) (see, e.g., potocnakova et al (2016) Journal of Immunology Research, article ID 6760830).
Several algorithms are available for predicting B cell epitopes from their sequences or structures. The algorithms have been developed, initially relying on the identification of linear epitopes by means of a trend scale, but improved by the development of machine learning based methods such as Hidden Markov Models (HMMs), recurrent Neural Networks (RNNs) and Support Vector Machines (SVMs). Computer B cell epitope prediction tools include those that predict continuous/linear B cell epitopes, as well as those that predict discontinuous/conformational B cell epitopes. Prediction of discontinuous B cell epitopes requires information on amino acid statistics, spatial information and surface exposure. Web-usable tools for continuous/linear B-cell epitope prediction include, for example, abcpared (available at crdd. Osdd. Net/raghava/ABCPred/obtaining), APCPred (available at omicols. Com/agprred-tool), BCPREDs (BCPred and FBCPred, available at ailab. Ist. Psu/BCPred/obtaining), bepippred (available at cbs. Dtu/services/bepippred/obtaining), lbtouch (available at crdd. Osdd. Net/raghava/lbtouch/obtaining), bcePred (available at crdd. Osdd. Net/raghava/BcePred/obtaining), epr (available at bio-touch. Tsinghua. Edge/EPMLR/obtaining), bepiPred (available at a. Support vector, available at cbs. Dtu. Dseed/obtaining), bepiprad (available at svc. Support vector, available at svd. Osdd. Net/raw/obtaining at svcp. Window/obtaining), and bepiprad (available at svd. End). Networks useful for discontinuous/conformational B cell Epitope prediction include, for example, CEP (available at biological fluids in/portion.htm), discope (available at cbs. Dtu/service/discovery tool-2.0/available), BEpro (previously known as ito; available at pepo.proteins. Ics. Edu/available), ellimunope (available at biological fluids in/elpro/available), sepa (available at biological fluids in/htm), sepa (available at biological fluids in/cut. Htm/available), epoop top (available at functional fluids in/bea.tau/available at functional tissues/il/available), cbtop (available at functional tissues/available) in/suspended protocols in/available at the same as vascular protocols 2.0/available at the same time, and by eico-standard protocols (available at the same as vascular protocols/available at the same as vascular protocols 2.72), and by-standard protocols in/available at the same time, such as vascular protocols 2.mat. Support protocols/available at the same time, and available at the same time, may be used at the same time, such as vascular protocols as vascular standards in/available at the same time, and at the vascular standards, available at the same time, vascular standards, and at the vascular standards, such as vascular standards, and standard protocols.62.62. Standard protocols.available at the vascular standards.
Prediction methods based on sequence and binding sites can also be used to predict B cell epitopes. Sequence-based predictive tools rely on the primary sequence of an antigen and use a trend scale to measure the probability that each residue is part of an epitope. Sequence-based prediction tools include BEST, which can predict conformational B cell epitopes. Binding site prediction tools aimed at identifying binding sites for conformational B-cell epitopes on antibodies include, for example, proMate, conSurf, PINUP and PIER (see, for example, sun et al (2013) comp.math Method m., arc ID 943636).
Conformational B-cell epitopes in proteins or antigens having known 3D structures can be identified using mimotope-based epitope prediction methods. Mimotopes are peptides selected from a random peptide library based on their ability to bind antibodies raised against the native antigen. Mimotope-based methods require the import of a peptide selected for antibody affinity (i.e., mimotope) and the 3D structure of the selected antigen. There are epitope prediction methods based on phage display experiment derived mimotopes, which can indicate B cell epitope position by using the statistical features of mimotopes to map mimotopes to overlapping position patches on the antigen surface, or by aligning back to the antigen sequence using mimotope mapping. To identify the affinity-selected peptide or mimotope, random peptides are displayed on the surface of the filamentous phage and peptides that bind to monoclonal antibodies with a degree of affinity are screened, eluted and amplified. This selection process was repeated 3-5 times in total, narrowing the peptide range to those peptides with highest affinity. Mimotopes and epitopes can combine the same paratopes of monoclonal antibodies and elicit an immune response and thus have similar functions. The mimotopes selected have a higher sequence similarity, indicating that certain critical binding motifs and physicochemical preferences exist during interaction with the antibody. Thus, mapping mimotopes back to the source antigen can help more accurately find the true epitope. Mimotopes have similar physicochemical properties and spatial organization, but rarely show sequence similarity to the native antigen. Databases providing mimotope information include, for example, ASPD (available at mgs.bionet.nsc.ru/mgs/gnw/ASPD), RELIC Peptides (available on demand), pepBank (available at pepbank.mgh.harvard.edu), and MimosDB (available at immunt.cn/MimoDB). Computer mimotope-based predictive tools are critical for mapping mimotopes back to the surface of the source antigen in order to locate the best aligned sequences and predict the likely epitope regions. Computer simulated B cell Epitope prediction tools based on mimotope analysis include, for example, MIMOX (available at immune/MIMOX), mimoPro (available at information. Neu. Edu. Cn/MimoPro), pep-3D-Search (available at ky. Neu. Edu. Cn/Pep3 DSearch), MIMOP/MimCons (available on demand), MIMOP/Mimalign (available on demand), locap (available at atene. Modules. Upper. Es/#soft), epiSearch (available at temperature. Uted. Edu/ep), pepitope/PepSurif (available at Pepitope. Ac. Il/source. Html), pepMapp (available at information. Neu. Edu/map), pep-map (available at temperature, on-line), and map-3D-map (available at temperature-map/map on-line); on-line unavailable), mapitope (available at pepitope.tau.ac.il), siteLight (on-line unavailable) (see, e.g., potocnakova et al (2016) Journal of Immunology Research, arc ID 6768230; sanchez-trincoado et al (2017) Journal of Immunology Research, arc ID 2680160;Sun et al (2013) comp.math Method m., arc ID 943636).
in silico prediction of T cell epitopes
The linear T cell epitope binds to MHC by interaction of its amino acid side chains with binding pockets in the MHC epitope binding groove, the presence or absence of a particular side chain determining whether the T cell epitope binds to MHC and how tightly it binds. For computer prediction, there are databases that provide existing epitope libraries, such as IEDB, episome and SEDB, which provide information about two-dimensional T cell epitopes. Other computer T cell epitope databases include CED, antiJen, bcipep and HLA Epitope Registry. Several web pages and programs are available for analyzing the sequence of protein therapeutic candidates and predicting immunogenic epitopes. For example, many MHC binding motif-based tools are available that scan for potential T cell epitopes of protein sequences. These T cell epitope mapping tools include, for example, epiMatrix, IEDB, SYFPEITHI, MHC Thread, MHCPred, MHCPred 2.0.0, epiJen, netMHC, netCTL, nHLAPred, SVMHC and Bimas (see, for example, de Groot, A.S. and Moise, L. (2007) curr.Opin. Drug discovery.level.10 (3): 332-340). For example, the MHCPred algorithm provides information about the MHC binding potential of amino acid sequences to various alleles, epiMatrix/janus matrix predicts allele-specific binding of protein therapeutics to MHC class II receptors, and can assess T cell receptor interface binding. Other algorithms and programs for epitope prediction include, for example, proPred, MMBPred and protein 3D. Epitope prediction should be combined with in vitro methods and activity assessment to ensure that any modification to remove immunogenic sequences retains the activity of the therapeutic protein (see, e.g., dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654).
peptide-MHC class II binding prediction
For example, structure-based methods for identifying T cell epitopes rely on modeling peptide-MHC structures and assessing interactions using molecular dynamics modeling. The structure-based method is large in calculation amount and lower in prediction performance than the data driving method. Structural based T cell epitope prediction tools include EpiDOCK (available from EpiDOCK. Ddg-pharmfac. Net). Data-driven methods for peptide-MHC binding prediction are based on peptide sequences known to bind to MHC molecules; the peptide sequences may be obtained in epitope databases such as IEDB, EPIMHC, antiJen and other epitope databases described herein and known in the art. peptide-MHC binding predictions may be based on Sequence Motifs (SM) that include amino acids that frequently occur at specific positions (anchor residues) known to bind to MHC molecules. Motif Matrices (MMs) evaluate the contribution of each residue (including non-anchor residues) to MHC molecule binding, but without regard to binding affinity. Quantitative Affinity Matrix (QAM) predicts peptide-MHC binding and binding affinity. A quantitative structure-activity relationship (QSAR) additive model predicts the binding affinity of peptides to MHC as the sum of the contributions of amino acids at each position, plus the contribution of adjacent side chain interactions. Machine Learning (ML) is the most popular and powerful method using algorithms trained on datasets composed of peptides with or without MHC molecules, examples of ML-based discrimination models include those based on Artificial Neural Networks (ANN), support Vector Machines (SVM), decision Trees (DT) and Hidden Markov Models (HMM) (see, e.g., sanchez-trincoado et al (2017) Journal of Immunology Research, arc ID 2680160).
Models for predicting the immunogenicity of protein therapeutics include computer-simulated peptide-MHC class II algorithms that predict the ability of peptide sequences to bind to MHC class II with reasonable accuracy. Such an algorithm allows for a rapid screening of sequence libraries. However, in silico and in vitro MHC class II binding assays result in high levels of false positives, where the identified immunogenic peptides fail to stimulate T cell responses in vitro and in vivo. Such analysis does not take into account other factors that affect epitope formation, such as protein processing, T Cell Receptor (TCR) recognition, and T cell tolerance to peptides. To address this problem, an in vitro T cell assay was used. The combination of in silico analysis and in vitro assays is very useful for the identification of epitopes and the design of peptide variants with epitope-depleted protein sequences having reduced MHC binding capacity (see, e.g., baker et al (2010) Self/nossell 1 (4): 314-322).
Predicting T cell epitopes by peptide-MHC binding models is also complicated by MHC polymorphisms; in humans, MHC molecules are known as Human Leukocyte Antigens (HLA) and there are hundreds of allelic variants of HLA class I and II molecules that bind different peptides and require specific models to predict peptide-MHC binding (see, e.g., sanchez-trincoado et al (2017) Journal of Immunology Research, arc ID 2680160). T-cell epitope prediction tools based on peptide-MHC binding models include, for example, epiDOCK, motifScan, rankpep, SYFPEITHI, MAPPP, PREDIVAC, PEPVAC, EPISOPT, vaxign, MHCPred, epiTOP, BIMAS, TEPITOPE, propred, propred-1, epijen, IEDB-MHCI, IEDB-MHCII, IL4pred, MULTIPRED2, MHC2PRED, netMHC, netMHCII, netMHCpan, netMCHIIpan, nHLApred, SVMHC, SVRMHC, netCTL, and WAPP (see, e.g., sanchez-Trincado et al (2017) Journal of Immunology Research, article ID 2680160).
EpiSweep is a set of protein design algorithms that integrate computational predictions of immunogenic T cell epitopes with sequence-based or structure-based evaluation of the effects of epitope deletion mutations on protein stability, structure, and function, allowing selection of mutations that optimize protein therapeutics to achieve low immunogenicity and high activity and stability (see, e.g., choi et al (2017) Methods mol. Biol.1529:375-398, stepwise guidance in the application of the EpiSweep deimmunization algorithm set).
c. In vitro epitope prediction method
In vitro methods can be used to determine the cellular mechanisms of the immune response, identify immunogenic epitopes, and evaluate MHC affinity, T cell proliferation and immunogenic effects of whole protein therapeutics. Epitope mapping, for example, identifies immunogenic epitopes by analyzing peptide fragments alone. The peptide fragments are exposed to immune cells and immunogenicity is determined by measuring cytokines and surface markers that indicate an inflammatory immune response. Epitope mapping of intact proteins is laborious, and computer programs are used in conjunction with mapping to identify regions likely to be immunogenic and narrow down candidate epitopes. In vitro epitope prediction methods include, for example, structural epitope mapping methods such as X-ray crystallography, nuclear magnetic resonance and electron microscopy methods, and functional epitope mapping such as antigen fragmentation/antigen binding assays, competitive binding assays, modification testing/mutagenesis, display techniques, such as phage display and yeast display, and mimotope analysis.
i. In vitro B cell epitope prediction method
Experimental methods for identifying B cell epitopes include, for example, resolving the 3D structure of antigen-antibody complexes, screening peptide libraries for antibody binding, or performing functional assays in which an antigen is mutated and analyzed for effects on antigen-antibody interactions. Antibody-producing B cells recognize structural epitopes, which are about 16-22 residues in size, contain amino acids that contact the antibody, and functional epitopes, which are about 3-5 residues in size and affect the affinity between the protein and the antibody. The most accurate method of identifying structural epitopes is by X-ray crystallography of antigen-antibody complexes, which identifies sequences that bind to antibodies, can be used to locate the exact position of the epitope in the protein structure, can identify continuous and discontinuous epitopes, and provides information about the binding strength. Structural epitope mapping identifies residues that are in direct contact with an antibody, but does not always provide information about which residues contribute to the binding strength. The FTProd program provided for free can be used as a calculation alternative for X-ray crystallography. Nuclear Magnetic Resonance (NMR) can be used to identify structural epitopes without the formation of crystals, but its use is limited to small proteins and peptides of less than 25kDa in size. NMR provides data on the structure, kinetics and binding energy of the antigen-antibody complex and is performed in solution without the formation of crystals. Two other methods for epitope mapping with medium resolution include saturation transfer difference NMR and antibody inhibition of hydrogen-deuterium exchange in the antigen. Electron microscopy can also be used for epitope mapping, but it is a low resolution structural approach, typically for larger antigens, such as whole virus particles or virus capsids. Electron microscopy cannot detect the contact residues but can be used to confirm the surface accessibility of the epitope. Frozen electron microscopy is an alternative method in which the flash frozen antigen-antibody complex is observed in physiological buffer without staining and fixative (see, e.g., dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654;Potocnakova et al (2016) Journal of Immunology Research, artcle ID 6760830).
Functional epitopes can be identified by a variety of methods including antigen fragmentation, competitive binding and modification assays. For example, functional B cell epitope mapping methods typically involve screening antigen-derived proteolytic fragments or peptides for antibody binding, and testing antigen-antibody reactivity of mutant proteins that have been subjected to site-directed or random mutagenesis. Thus, functional epitope mapping tools are used to identify and identify residues within epitopes important for antibody binding. Most functional methods detect binding of antibodies to antigen fragments, synthetic peptides or recombinant antigens such as mutant variants, antigens arranged by in situ cell-free translation and/or antigens expressed using alternative systems such as phage display. For example, antigen fragmentation and binding assays involve immobilizing peptides on a solid support and determining whether an epitope fragment binds to an antibody using Western blotting, dot blotting, and/or ELISA; binding indicates that the peptide fragment may be immunogenic. Competitive binding assays provide low resolution mapping by assessing whether multiple antibodies can bind to an epitope on a protein simultaneously, or whether they compete with each other for binding to the same epitope, providing information about the number of potentially immunogenic epitopes on a protein (see, e.g., dingman et al (2019) J.pharm.Sci.108 (5): 1637-1654;Potocnakova et al (2016) Journal of Immunology Research, artcle ID 6760830).
Modification testing or mutagenesis is an epitope mapping method in which individual residues in a functional epitope (called hot spots) are substituted and the effect of the modification on the binding of the antibody to the immunogenic sequence is assessed. The hot spots most often include Tyr, arg, and Trp residues, which are energetically important residues comprising a portion of the intact protein-protein interface region. Mutation of individual residues allows identification of deleterious residues that can be replaced, provided that the protein retains its structure and activity. For epitope mapping by mutagenesis, peptide libraries are generated by random or site-directed mutagenesis; the combination of mutagenesis and display techniques allows the screening of a large number of mutated proteins. Saturation mutagenesis is another epitope mapping method in which amino acid residues at specific positions within an epitope are replaced with all 20 naturally occurring amino acids and the loss of antibody binding is monitored. The disadvantage of this approach is that the loss of immunoreactivity may be due to disruption of the antigenic structure, resulting in difficulty in interpreting the results. Most of the contact between epitope and antibody occurs through amino acid side chains, alanine scanning mutagenesis can be used to define the contribution of each residue side chain to antibody binding. This is accomplished by sequentially alanine substitution of each non-alanine residue at a time, thus truncating the side chain to the beta-carbon without increasing the flexibility of the protein backbone. This approach identified the key residues whose side chains caused the paratope-epitope interactions to make the highest energy contribution. A computational alanine scan can also be used to rapidly determine the effect of alanine mutations on the free energy of binding in protein-protein complexes by using a simple free energy function. Combinatorial mutagenesis is based on randomized combinations of discrete antigen regions, as well as grouping of mutated residues (proximity of primary sequences) to maximize the opportunity to identify combinatorial effects mediated by neighboring residues; this allows identification of residues that are not important for binding but contribute to epitope formation, or residues that form multiple interactions with the weaker paratope alone. Bird gun mutagenesis (Shotgun mutagenesis) is a high throughput method based on large scale mutagenesis, where each clone has a defined amino acid mutation (e.g., alanine substitution) and involves direct cellular testing of mAb reactivity against naturally folded proteins. The bird gun method mutation allows the identification of linear and conformational epitopes with a mapping rate exceeding 20 epitopes per month (see, e.g., potocnakova et al (2016) Journal of Immunology Research, arc ID 6760830).
Other techniques for epitope mapping include display techniques and mimotope analysis, which are inexpensive, flexible and rapid. Display techniques, such as phage display and yeast display, are based on testing the binding capacity of various peptides displayed on a display platform to a mAb of interest (e.g., tethering the peptide to a ribosome-mRNA complex, or to phage, bacteria, mammal, insect or yeast cells) by biopanning affinity selection methods (see, e.g., potocnakova et al (2016) Journal of Immunology Research, arc ID 6760830).
in vitro T cell epitope prediction method
In vitro methods, such as MHC or HLA binding assays and T cell assays, can be used to predict T cell epitopes and assess T cell responses to protein therapeutic antigens. The synthesis of hundreds or thousands of overlapping peptides for in vitro analysis is a limiting factor that can be overcome by using a computer epitope prediction tool that can accurately mimic the MHC: epitope interface and predict immunogenic peptide sequences (see, e.g., de Groot, a.s.and movie, l. (2007) curr.opin. Drug discovery.level.10 (3): 332-340).
MHC/HLA binding assays
T cell epitope prediction identified stimulation of CD8 + Or CD4 + The shortest peptide sequence in the antigen of T cells, which is therefore immunogenic. The immunogenicity of T cell epitopes depends on antigen processing, binding of peptides to MHC molecules, and recognition of cognate TCRs; MHC peptide binding is the most selective method and is also the primary basis for predicting T cell epitopes. MHC binding assays can be used to detect high affinity peptides and are typically used in epitope mapping binding applications to identify regions of a protein that are likely to be immunogenic. In vitro MHC class II binding assays include cell-based binding assays and soluble HLA binding assays. The high throughput MHC binding assay involves incubating different doses of a peptide of interest with a control peptide and a soluble MHC protein to assess binding affinity; the binding of high affinity peptides to MHC is stronger and epitopes are more easily accessible to T cellsAnd (5) identification. For example, MHC class II epitope binding can be assessed by measuring the ability of exogenously added peptides to bind to the surface of lymphoblastic-like B cells expressing MHC class II alleles, and high throughput screening can be performed using a competition-based HLA assay. MHC binding assays for identifying potentially immunogenic epitopes are commercially available, for example from ProImmune (see, e.g., dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654;De Groot,A.S.and Moise,L (2007) Curr. Opin. Drug discovery.level.10 (3): 332-340, sanchez-Trincado et al (2017) Journal of Immunology Research, artcle ID 2680160).
in vitro T cell assay
The presence of T cell epitopes in protein therapeutics can be detected by assessing in vitro T cell responses in T cell assays. T cells proliferate and release cytokines upon stimulation by the immunogenic proteins. T cell epitopes induce secretion of cytokines, such as IL-2, IL-4, IL-5 and ifnγ, by effector T cells, and secretion of cytokines tgfβ and tnfα, and chemokines, such as MIP1 α/1 β, by regulatory T cells (tregs). Proliferation of T cells responding to immunogenic peptides/epitopes can be measured by radiolabeling with thymidine or labelling with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). ELISA or ELISPot methods, as well as flow cytometry, can be used to measure the levels of cytokines such as IL-2 and IFN-gamma secreted by T cells to determine immunogenicity. The ELISpot method is highly sensitive and allows the detection of individual T cells directly from spleen cells or peripheral blood, as well as the measurement of the number of antigen-specific T cells secreting specific cytokines. For example, ELISPot assays for measuring IL-2 and IL-4 are commercially available. Flow cytometry can also be used to measure T cell responses so that tetramers (MHC class II: epitope complexes) can be used to directly label T cells that respond to a particular epitope. T cell proliferation and cytokine release assays can be used in combination with T cell phenotyping to classify the type of T cell response that occurs. The number and phenotype of antigen-responsive T cells can be determined using, for example, flow cytometry, by identifying cell surface markers such as CD25 for effector T cells and FoxP3 for Treg, and/or by identifying fines Intracellular cytokine expression. Thus, the identification of T cell epitopes can be combined with phenotypic studies to assess whether the immune response is inflammatory or inhibitory. Peripheral Blood Mononuclear Cell (PBMC) assays using PBMC formulations include several types of immune cells (e.g., CD4 + And CD8 + T cells) better mimics the immune system in vivo and can be used to assess the immunogenicity of proteins, as well as potential immune responses, without testing in humans. PBMCs cultured in vitro were stimulated with either intact therapeutic protein or peptides derived from therapeutic protein. Use of the innate cell system, e.g. for the deficiency of CD8 + PBMC preparations of reactive T cells or innate immune screening of Innate Lymphoid Cells (ILC) may be used to distinguish between innate and adaptive immune responses to immunogenic proteins. To be useful, in vitro T cell assays should test peptides against PBMCs from large groups of donors with broad spectrum of MHC class II isoforms. In vitro T cell assays can provide information about the number and efficacy of T cell epitopes that can be used to determine the risk of developing immunogenicity during preclinical periods and to direct removal of such epitopes by targeted amino acid substitutions (see, e.g., baker et al (2010) Self/noself 1 (4): 314-322;Dingman et al (2019) j.pharm. Sci.108 (5): 1637-1654;De Groot,A.S.and Moise,L (2007) curr. Opin. Drug discovery.development.10 (3): 332-340).
d. In vivo epitope prediction method
Animal models, such as mice, are used to assess the immunogenicity of protein therapeutics in humans. In general, any human or humanized protein therapeutic may be immunogenic when administered to a non-human animal. However, animal models can be used to predict immunogenicity, compare relative immunogenicity between products, pharmaceutical formulations or routes of administration, determine immunogenicity of aggregates and elucidate immune mechanisms. Adoptive transfer and T cell proliferation studies in animal models can be used to determine the role of T cells and B cells in protein immunogenicity. Immunogenicity of human protein therapeutics is difficult to assess in animals because animal MHC receptors do not directly mimic human HLA receptors, and because HLA and MHC genes are highly polymorphic, with a high degree of inter-subject variability in HLA/MHC expression. To overcome these limitations, HLA transgenic mice have been produced that mimic human subjects and can be made tolerant to specific proteins; the mice will be resistant to the protein therapeutic to be evaluated and any immunogenicity that occurs is due to disruption of self-tolerance, not due to classical immune responses to foreign antigens. In vivo methods of determining the immunogenicity of protein therapeutics include exposing HLA-transgenic mice to the entire protein or epitope peptide. Several transgenic mouse lines expressing common HLA gene products, such as HLA-A, HLA-B and HLA-DR molecules, have been generated and can be used to measure T cell responses and antibodies induced by exposure to protein therapeutics, as measured by ELISA and neutralizing antibody assays. B cell epitopes in protein therapeutics can also be identified by immunizing HLA transgenic mice with the protein (see, e.g., dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654;De Groot,A.S.and Moise,L (2007) Curr. Opin. Drug discovery.level.10 (3): 332-340).
NOD Scid Gamma (NSG) mice, which are hyperimmune compromised and lack most immune cells and complement and cytokine signaling, can be transfected to study the human immune system in an in vivo model. For example, CD34 + Humanized NSG mouse models were implanted with cord blood-derived hematopoietic stem cells to develop a functional immune system with normal T cell and inflammatory functions. Animal models also include non-human primates, such as rhesus monkeys and chimpanzees, which are more useful in predicting protein immunogenicity because their proteins exhibit higher homology to human proteins and their immune mechanisms are similar to those of humans (see, e.g., dingman et al (2019) J.Pharm. Sci.108 (5): 1637-1654).
e. Removal of predicted B-and T-cell epitopes (deimmunization)
As described herein, the prediction and removal (i.e., deimmunization) of immunogenic epitopes from protein therapeutics can improve the efficacy and safety of the constructs provided herein and reduce the likelihood of or prevent side effects. For example, removal of an identified epitope, such as a B cell epitope, may prevent the formation of an ADA that reduces the efficacy of the administered protein therapeutic by neutralizing the therapeutic and/or inducing its rapid elimination from the body.
Deimmunization of protein therapeutics involves identifying highly immunogenic B-cell and/or T-cell epitopes, and deleting the identified epitopes by mutagenesis to replace critical amino acid residues. As described above, the prediction and assessment of immunogenic regions in protein therapeutic sequences includes administration of various in silico, in vitro, and in vivo methods. After the immunogenic epitope is identified, the amino acid sequence of the epitope is modified by random or directed mutagenesis to remove the immunogenic sequence and deimmunize the epitope. For example, alanine scanning mutagenesis can be used to define the contribution of each residue side chain to antibody binding by most of the contacts made between the epitope and the antibody occur through the amino acid side chain. This is done by substituting alanine for each non-alanine residue, one at a time in turn, to identify the key residue whose side chain provides the highest energy contribution to the paratope interaction. However, because proteins must retain their folded, stable and active structure to retain their therapeutic efficacy, the predictive and mutagenic deletion of immunogenic epitopes is insufficient to deimmunize the protein; epitope deletion mutations must be selected that are compatible with the structure and function of the protein.
Existing computer tools may improve the efficiency of this process. For example, a procedure is available that can successively replace each amino acid in an immunogenic sequence with one of the other 19 naturally occurring amino acids, and then re-evaluate the immunogenicity of the new sequence. For example, optiMatrix is a tool to iteratively replace all 20 amino acids at any given position in a peptide sequence, and then re-analyze the modified sequence for predicted immunogenicity (see, e.g., de Groot, A.S. and Moise, L. (2007) Curr.Opin. Drug discovery.10 (3): 332-340). EpiSweep is a set of protein design algorithms that integrate computational predictions of immunogenic T cell epitopes with sequence-based or structure-based assessment of the impact of epitope deletion mutations on protein stability, structure, and function, whereby combinations of mutations that optimize the protein therapeutic for low immunogenicity and high activity and stability can be selected (see, e.g., choi et al (2017) Methods mol. Biol.1529:375-398, step-by-step guidance on the application of the set of EpiSweep deimmunization algorithms). Computing alanine scans can also be used to rapidly determine the effect of alanine mutations on binding free energy in protein-protein complexes by using simple free energy functions (see, e.g., potocnakova et al (2016) Journal of Immunology Research, arc ID 6760830).
G. Pan growth factor trap constructs
1. Receptor Tyrosine Kinases (RTKs)
Receptor Tyrosine Kinases (RTKs) are high affinity cell surface receptors for many polypeptides growth factors, cytokines and hormones. RTKs are involved in many signaling pathways and play a role in a variety of cellular processes, including cell division, proliferation, differentiation, migration, and metabolism. RTKs can be activated by ligands that specifically bind to their cognate receptors. This activation in turn activates events in signaling pathways, such as by triggering autocrine or paracrine cellular signaling pathways, such as activating second messengers, leading to specific biological effects. About 20 different classes of RTKs have been identified, including the Epidermal Growth Factor Receptor (EGFR) family (class I, also known as ErbB family); insulin receptor family (class II); platelet-derived growth factor receptor (PDGFR) family (class III); vascular Endothelial Growth Factor Receptor (VEGFR) family (class IV); fibroblast Growth Factor Receptor (FGFR) family (class V); a Hepatocyte Growth Factor Receptor (HGFR) family (class VIII); and Eph receptor family (Ephs, erythropoietin-producing human hepatocyte receptor; class IX), and the like.
RTKs are involved in regulating pathways involved in angiogenesis, including physiological and tumor angiogenesis, and in regulating cell proliferation, migration and survival. RTKs are associated with a number of diseases including autoimmune diseases and cancers, such as breast and colorectal cancers, gastric cancers, gliomas and mesoderm-derived tumors. Dysregulation of RTKs is associated with a variety of cancers. For example, breast cancer is associated with amplified expression of P185-HER 2. RTKs are also associated with ocular diseases including diabetic retinopathy and macular degeneration. Furthermore, it has been shown that members of the Epidermal Growth Factor Receptor (EGFR) family as well as EGF-like growth factors (ligands) are overexpressed in synovial fibroblasts and macrophages in Rheumatoid Arthritis (RA) patients.
a. Human epidermal growth factor receptor (HER) family
RTKs associated with disease are a family of human EGFR (HER, also known as ErbB) receptors of class I including HER1/EGFR (ErbB 1), HER2 (ErbB 2/Neu), HER3 (ErbB 3) and HER4 (ErbB 4). HER1, HER3 and HER4 bind together 11 canonical ligands, including Epidermal Growth Factor (EGF), transforming Growth Factor (TGF) - α, heparin Binding (HB) -EGF, amphiregulin, β -animal cellulose (BTC), epithelial regulatory protein, epigen and Neuregulin (NRG) 1-4.HER2 does not bind any of these ligands, but rather acts as a signal amplifier by heterodimerization with other HER family members such as HER3 and HER4 (see, e.g., jin et al (2009) mol. Med.15 (1-2): 11-20). HER1, HER2 and HER4 are active as tyrosine kinases, whereas HER3 is inactive as a kinase (albeit with a kinase domain), and signals via the phosphatidylinositol 3-kinase pathway.
All members of the HER family have an extracellular ligand binding domain, a transmembrane domain and a domain containing cytoplasmic tyrosine kinase. The extracellular region of each HER family member contains four subdomains, namely L1, CR1, L2 and CR2, where "L" refers to a leucine rich repeat domain and "CR" refers to a cysteine rich region/domain (also known as a furin-like repeat domain); these four subfields are also referred to as domains I-IV, respectively. Domains I and III are ligand binding domains, and domains II and IV mediate binding to each other and to other members of the receptor family. Domain II contains the sequence required for dimerization, termed the dimerization arm, and domain IV contains a sequence that allows domain II/IV tethering, except HER2, which does not undergo tethered conformation. In the absence of ligand, EGFR, HER3 and HER4 subdomains II and IV form intramolecular self-inhibiting tethers. Upon ligand binding, the subdomains undergo a conformational change such that subdomains I and III form a high affinity ligand binding pocket. It has been shown that mutagenic disruption of the tether formed by subdomains II and IV, or deletion of the C-terminus of subdomain IV, can increase ligand binding affinity up to 15-fold (see, e.g., jin et al (2009) mol. Med.15 (1-2): 11-20).
HER family members are expressed in tissues of various epithelial, mesenchymal and neuronal origin. Under normal physiological conditions, HER activation is controlled by the spatial and temporal expression of its ligand, an EGF family member of the growth factor. Ligand binding induces the formation of multiple combinations of receptor homodimers and heterodimers, leading to activation of intrinsic kinase domains, autophosphorylation of cytoplasmic tail specific tyrosine residues, recruitment and phosphorylation of some intracellular proteins, and coupling to multiple downstream signaling cascades. Activated signaling pathways include the Ras-Raf-mitogen-activated protein kinase mitogenic pathway, the phosphatidylinositol 3-kinase-AKT cell survival pathway, and the stress-activated protein kinase C and Jak/Stat pathways. The induced signaling pathways lead to a variety of cellular responses including, for example, cell migration, invasion, proliferation, survival and differentiation (see, for example, sarup et al (2008) mol. Cancer Ther.7 (10): 3223-3236).
b. Diseases associated with the human epidermal growth factor receptor (HER) family and ligands therefor
HER family members and their ligands are deregulated by overexpression or mutation and have been shown to play a role in cancer and other diseases. For example, HER1 and HER2 are associated with the occurrence and pathology of many human cancers, and alterations in these receptors are associated with more aggressive diseases and poor clinical outcomes. TGF- α overexpression is associated with prostate, pancreatic, lung, ovarian and colon cancers, while NRG1 overexpression is associated with breast cancer. HER1 overexpression is associated with glioma, head and neck cancer, breast cancer, bladder cancer, prostate cancer, kidney cancer, and non-small cell lung cancer, HER1 mutation is associated with glioma as well as lung cancer, breast cancer, and ovarian cancer. HER2 overexpression is associated with breast, lung, pancreatic, colon, esophageal, endometrial and cervical cancers; HER3 is associated with breast, colon, stomach, prostate and oral squamous cell carcinoma; HER4 is associated with breast and prostate cancer and childhood medulloblastoma (see, e.g., yarden et al (2001) nat.rev.mol. Cell biol.2:127-137).
EGF ligands and receptor families have been shown to play a role in the development of inflammatory arthritis. For example, expression of HER2 and the presence of the EGFR ligands EGF, amphiregulin and TGF- α have been detected in RA synovium. The human EGFR family inhibitor herstatin is an alternative splice variant of HER2, whose adenovirus delivery has been shown to eliminate all clinical symptoms of collagen-induced arthritis (CIA) in mice. Herstatin disrupts dimerization and acts as a natural inhibitor of natural HER1, HER2 and HER 3. One long-term RA patient had previously been treated with rituximab and adalimumab, and joint pain was significantly reduced after treatment of head and neck cancer with the anti-EGFR/HER 1 antibody cetuximab. These results indicate that HER targeted Therapy is therapeutically useful for the treatment of autoimmune and inflammatory diseases, such as Rheumatoid Arthritis (RA) (see, e.g., golpes et al (2011) Arthritis Research & Therapy 13: r 161).
Macrophages are a source of TNF in chronically inflamed RA joint tissue. Phenotypic analysis of synovial tissue macrophages in RA patients showed a large amount of HBEGF + (heparin-binding EGF-like growth factor + ) Inflammatory macrophages overexpressing the pro-inflammatory genes NR43A (nuclear receptor subfamily 4 group A member 3), PLAUR (plasminogen activator, urokinase receptor) and CXCL2, as well as the growth factors HB-EGF and the epithelial regulatory protein (EGFR family ligand). HBEGF + Inflammatory macrophages also produce the pro-inflammatory cytokine IL-1 and promote synovial fibroblast invasion in an epidermal growth factor receptor-dependent manner. It has been shown that most drugs for treating RA target HBEGF in an ex vivo synovial tissue assay + Macrophages, whereas EGFR inhibitors effectively block macrophage-induced fibroblast responses in RA tissue in an ex vivo assay, suggesting that blocking of EGFR responses may provide non-immunosuppressive therapeutic approaches for RA (see, e.g., kuo et al (2019) sci. Transl. Med.11 (491)). This approach is superior to the use of traditional anti-TNF therapies, which have immunosuppressive effects and are often associated with the occurrence of severe infections such as tuberculosis.
HER family signaling is also associated with coronary atherosclerosis, which involves migration of vascular smooth muscle cells in the intima of the artery. Smooth muscle cell migration and proliferation requires activation of thrombin receptor, and activation of such G protein-coupled receptor is dependent on transactivation of HER1/EGFR in response to HB-EGF. EGFR expression is also associated with psoriasis; in normal skin, EGFR expression is limited to basal lamina only, whereas in psoriatic patients EGFR and its ligand amphiregulin are highly expressed throughout the epidermis (see, e.g., yarden et al (2001) Nat. Rev. Mol. Cell biol. 2:127-137).
Other HER mediated diseases and conditions include neurodegenerative diseases and conditions such as multiple sclerosis, parkinson's disease, schizophrenia, and alzheimer's disease. For example, several diseases and conditions are associated with exposure to one or more Neuregulin (NRG) ligands such as NRG1 (including I, II and type III), NRG2, NRG3, and/or NRG 4. Examples of NRG-related diseases include neurological or neuromuscular diseases including schizophrenia and alzheimer's disease (see, e.g., U.S. patent publication No. 2010/0055093).
HER is a target for therapeutic intervention due to its role in cancer and other proliferative, rheumatoid arthritis, neurodegenerative and autoimmune diseases. anti-HER therapeutic agents include antibodies targeting the extracellular domain (or ectodomain), referred to herein as ECD, and small molecule tyrosine kinase inhibitors. Therapeutic agents approved for the treatment of cancers driven by the HER protein family include monoclonal antibodies such as trastuzumab (for HER 2), pertuzumab (for HER 2), panitumumab (for HER 1/EGFR) and cetuximab (for HER 1/EGFR), and small molecule tyrosine kinase inhibitors such as the HER1 kinase inhibitors gefitinib (gefitinib) and erlotinib (erlotinib), and dual HER2 kinase and HER1 kinase inhibitors lapatinib (lapatinib). For example, trastuzumab is used to treat HER2 over-expressed lymph node positive or lymph node negative breast cancer; cetuximab is used for the treatment of metastatic colorectal cancer and head and neck cancer; panitumumab for use in the treatment of metastatic colorectal cancer; lapatinib is used as a first line treatment for triple positive breast cancer, as an adjunct treatment for patients who progress after trastuzumab treatment; erlotinib is used for the treatment of non-small cell lung and pancreatic cancer.
anti-HER therapies exhibit limited efficacy and limited duration of response. TrastuzumAnti (e.g. asSold) is a humanized version of a murine monoclonal antibody that targets the extracellular domain of HER 2. However, the effectiveness of trastuzumab requires high expression of HER2 (at least 3 to 5-fold over-expression), so only less than 25% of breast cancer patients are eligible to receive treatment. In this population, a significant proportion of people do not respond to treatment. In addition, small molecule tyrosine kinase inhibitors often lack specificity. The efficacy observed with single target anti-HER antibodies or small molecule tyrosine kinase inhibitors was in the range of 10-15% except for patients that highly expressed HER2 and received trastuzumab combination chemotherapy. Treatments, particularly those directed against only one HER family member, are also affected by intrinsic or acquired resistance, which is associated with co-expression and ligand activation of other RTKs, particularly other HER family members. For example, resistance is typically associated with upregulation or compensation of other HER family members, such as HER3 and HER4, or with increased expression of HER1 or HER3 ligands by tumor cells. Homodimerization and heterodimerization between HER family members also have an impact on treatment against individual HER family receptors. Because of the limited effectiveness of available therapies, alternative anti-HER therapies are needed. Provided herein are alternative, more effective therapies for targeting the HER family of RTKs and their ligands.
2. Pan-growth factor inhibition
As described herein, resistance to single targeted anti-HER therapies (e.g., trastuzumab, cetuximab, gefitinib, and erlotinib) is typically associated with co-expression and/or up-regulation of other HER family members and/or over-expression of their ligands. One strategy to reduce or overcome this resistance and improve the efficacy of HER targeted therapies is to inhibit multiple ligand-induced HER family members simultaneously. This can be accomplished, for example, by chimeric HER ligand binding molecules that behave like receptor decoys and sequesters multiple HER family ligands, thereby preventing ligand-dependent receptor activation and down-regulating aberrant HER family activity.
RB242 ligand trap
Antagonists called RB242 are chimeric bispecific ligand traps, fc-mediated heterodimers of EGFR (HER 1) and HER3 ligand binding domains, targeting all four members of the EGFR/HER family. EGFR and HER3 ligand binding domains dimerize by fusing each ligand binding domain to the Fc domain of human IgG 1. In RB200, the extracellular domain (ECD) of EGFR (corresponding to residues 1-621 of the mature EGFR protein, shown as SEQ ID NO: 41) and the extracellular domain of HER3 (corresponding to the mature HER3 protein, shown as SEQ ID NO: 45) were fused to the N-terminus of the Fc fragment of human IgG1 (corresponding to residues P100-K330 of SEQ ID NO: 9), to which a Gly-Arg-Met-Asp (GRMD) linker was added. The HER3/Fc component of RB200 contains a 6xHis tag at the COOH terminus for purification.
RB200 has been shown to bind EGFR and HER3 ligands (including EGF, TGF-alpha, HB-EGF, amphiregulin, beta-cell proteins, epithelial regulatory proteins, and epigen proteins, and NRG 1-alpha, NRG 1-beta 1, and NRG 1-beta 3, respectively) with high affinity, inhibit ligand-induced tyrosine phosphorylation of HER family members (HER 1, HER2, and HER 3), inhibit proliferation of a variety of tumor cells in vitro in a nude mouse model, inhibit growth of tumor xenografts (epidermoid carcinoma and non-small cell lung carcinoma). RB200 also showed a synergistic effect with in vitro inhibition of tumor cell proliferation by tyrosine kinase inhibitors such as AG-825, erlotinib, gefitinib or lapatinib against EGFR/HER1 and HER2 kinases. RB200 is more effective in inhibiting ligand-stimulated phosphorylation of HER1, HER2 and HER3 than monoclonal antibodies that target HER1 (C225) or HER2 (trastuzumab and 2C 4) (see, e.g., sarup et al (2008) mol. Cancer Ther.7 (10): 3223-3236;Gompels et al (2011) Arthritis Research & Therapy 13: r 161).
To express the RB200 and RB242 heterodimeric chimeric fusion proteins, vectors encoding the HER1/Fc and HER3/Fc constructs were co-transfected into HEK293T cells at a ratio of 1:3 (HER 1/Fc: HER 3/Fc). This results in the expression of HER1/Fc and HER3/Fc homodimers in addition to the HER1/Fc of interest, HER3/Fc heterodimers. The expressed protein was purified by a combination of protein-A, ni-Sepharose and EGFR-affibody column chromatography methods. Analytical reverse phase High Performance Liquid Chromatography (HPLC) shows that RB242 heterodimers contain approximately 10% of the combined contamination of both homodimers (see, e.g., sarup et al (2008) mol. Cancer Ther.7 (10): 3223-3236). Thus, there is a need for improved methods to increase the yield and purity of heterodimers.
b. RB200 and RB242 for the treatment of autoimmune diseases
As discussed elsewhere herein, a significant portion of RA patients do not respond or cease responding to treatment with anti-TNF therapies such as anti-TNF antibodies, which is associated with an increased risk of serious infections (including tuberculosis). Therefore, alternative therapies are needed. Increased expression of EGF ligand and receptor (HER) in synovial membranes and synovial fluid of Rheumatoid Arthritis (RA) patients suggests that EGFR-targeting therapies may be useful in the treatment of RA and other autoimmune and inflammatory diseases and disorders.
Bispecific EGFR ligand trap RB200 (and its derivative RB 242) exhibits a dose-dependent decrease in disease severity in collagen-induced arthritis (CIA). Mice with CIA were treated with RB200 (or RB 242) by intraperitoneal injection at doses of 0.1mg/kg, 1mg/kg or 10mg/kg on the day of disease onset (day 1) and on days 4 and 7. Treatment with 1mg/kg or 10mg/kg RB200 inhibited the increase in clinical scores and paw swelling in a dose-dependent manner. EGF has been shown to promote angiogenesis, RB200 treated mice showed a decrease in CD31 immunopositive staining, reflecting a decrease in synovial blood vessels and inhibition of synovial angiogenesis. Joint sections of mice treated with PBS control showed a large number of infiltrating cells in the inflamed synovium, and invasion and erosion of bone by the synovium, which is associated with significant CD31 expression. The joints of mice treated with 1mg/kg or 10mg/kg RB200 were protected, the appearance was normal, the joint structure was well preserved, and there were few CD31 positive blood vessels. These results indicate that inhibition of EGFR-mediated responses can be used to treat RA (see, e.g., gompels et al (2011) Arthritis Research & Therapy 13: R161).
TNF inhibition in combination with inhibitors of EGFR-mediated signaling may enhance the efficacy of anti-TNF therapies and may be useful in the treatment of RA. It has been shown that the combined administration of low dose RB200 (0.5 mg/kg) with suboptimal dose of etanercept (1 mg/kg) inhibited the increase in clinical scores and paw swelling and completely eliminated CIA, similar to the effect observed with the optimal dose of etanercept (5 mg/kg) alone. In contrast, administration of low dose RB200 alone or low dose etanercept alone was ineffective. A fluorescent-labeled monoclonal antibody directed against E-selectin can be used to localize endothelial activation in inflamed tissue in vivo, a sensitive, specific and quantifiable molecular imaging technique for assessing CIA. The combination of low dose RB200 and low dose etanercept reduced the amount of E-selectin detected in the paw to the level of healthy animals, whereas E-selectin was detected in the paw of CIA mice receiving either low dose RB200 or low dose etanercept alone. While RB200 alone and etanercept alone had a dose-dependent effect on joint structure, severely damaged joints were progressively reduced, and mildly or moderately damaged joints were more and more, the most significant effect was observed in combination treatment, with 64% of joints exhibiting normal, and 0% in mice with either low dose RB200 alone or low dose etanercept alone. Combination Therapy is also more effective than large doses of etanercept alone, suggesting the effectiveness of combination pan-EGFR and TNF targeted Therapy in promoting joint protection (see, e.g., gompels et al (2011) Arthritis Research & Therapy 13: R161).
RB242 ligand trap
The ligand trap called RB242, derived from RB200, is an affinity-optimized Fc-mediated triple mutant EGFR: HER3 heterodimer, with reference to the mature EGFR sequence (SEQ ID NO: 41), contains mutations T15S and G564S in EGFR ECD subdomains I and IV, respectively, and with reference to the mature HER3 protein sequence (SEQ ID NO: 45), contains mutation Y246A in HER3 ECD subdomain II. The average 22-fold increase in RB242 affinity for various ligands (including EGF, TGF- α, HB-EGF and NRG1- β) compared to the parent molecule RB200, and demonstrated an increase in antiproliferative activity in cultured single layer BxPC3 pancreatic cancer cells and in human non-small cell lung cancer mouse models. RB242 also exhibits a 10 to 60-fold improvement in inhibiting ligand-induced HER phosphorylation compared to RB200 (see, e.g., jin et al (2009) mol. Med.15 (1-2): 11-20).
3. Optimized multi-specific, e.g., bispecific growth factor trap constructs
Provided herein are multispecific, e.g., bispecific, growth factor trap constructs designed to specifically target pan-cell surface receptor therapeutics for more than one cell surface receptor, e.g., by binding to and/or interacting with a ligand for one or more receptors, so long as the activity of more than one cell surface receptor is modulated. These constructs include those that target more than one HER family member, as well as those that target one or more HER and other receptors, e.g., to HER that contributes to or is involved in the development of resistance to anti-HER therapy. The growth factor trap constructs provided herein comprise a plurality, particularly two, chimeric fusion polypeptides, each comprising all or part of an extracellular domain (ECD) of a receptor, particularly a member of the HER family, such as EGFR/HER1, HER2, HER3 or HER4, fused to a multimeric domain, such as the Fc of a human immunoglobulin (Ig), such as the Fc of a human IgG. The ECD or portion thereof in the chimeric fusion polypeptide may be directly linked to the Fc, or indirectly linked through a linker, such as a peptide linker. Typically, the C-terminus of the ECD polypeptide is linked to the N-terminus of the multimerization domain, e.g., igG Fc.
Growth factor trap constructs herein are expressed and purified as described in Sarup et al (2008) mol. Cancer Ther.7 (10): 3223-3236, gompels et al (2011) Arthritis Research & Therapy 13: R161, jin et al (2009) mol. Med.15 (1-2): 11-20 and U.S. patent publication No. 2010/0055093. The following section describes each section of the multi-specific growth factor trap constructs provided herein.
a. Extracellular domain (ECD) polypeptides
Provided herein are multispecific, e.g., bispecific, growth factor trap constructs comprising two or more extracellular domains (ECDs) of Cell Surface Receptors (CSRs), or portions thereof. In particular embodiments, the construct is a bispecific heterodimer construct comprising two different cell surface receptors. The construct includes a first ECD polypeptide and a second ECD polypeptide each linked directly or indirectly through a linker to a multimerization domain. In some embodiments, the first ECD polypeptide comprises the ECD of HER1/EGFR (residues 1-621 corresponding to SEQ ID NO: 41) or a portion thereof, and the second ECD polypeptide comprises the ECD of HER2 (residues 1-628 corresponding to SEQ ID NO: 43), HER3 (residues 1-621 corresponding to SEQ ID NO: 45) or HER4 (residues 1-625 corresponding to SEQ ID NO: 47) or a portion thereof, particularly the ECD of HER3 or HER4 or a portion thereof. In embodiments where the ECD polypeptide comprises less than the full length ECD of the HER protein, it contains at least a sufficient portion of the subdomains I, II and III for ligand binding and receptor dimerization. For example, the ECD may contain a sufficient portion of the subdomains I and III for ligand binding, and/or may contain a sufficient portion of the ECD to dimerize with a cell surface receptor, including a sufficient portion of subdomain II. In some embodiments, the ECD contains at least module 1 of subdomains I, II and III and domain IV.
In some examples, the multispecific, e.g., bispecific growth factor trap construct contains a first ECD polypeptide containing all or part of the ECD of HER1/EGFR, HER2, HER3, or HER4, particularly EGFR/HER1, and a second chimeric polypeptide containing ECD from a different CSR, e.g., from HER2, HER3, HER4, insulin growth factor-1 receptor (IGF 1-R), vascular endothelial growth factor receptor (VEGFR, e.g., VEGFR 1), fibroblast growth factor receptor (FGFR, e.g., FGFR2 or FGFR 4), TNFR, platelet-derived growth factor receptor (PDGFR), hepatocyte Growth Factor Receptor (HGFR), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE, e.g., TIE-1 or TEK (TIE-2)), late glycosylated end product Receptor (RAGE), eph receptor, or T cell receptor.
In a particular embodiment, the first ECD polypeptide comprises the full length ECD of HER1/EGFR (corresponding to residues 1-621 of SEQ ID NO: 41) or a portion thereof (e.g., residues 1-501 of SEQ ID NO:41, which corresponds to residues 1-621 of subdomains I-III and domain IV), and the second ECD polypeptide comprises the full length ECD of HER3 (corresponding to residues 1-621 of SEQ ID NO: 45), or a portion thereof (e.g., residues 1-500 of SEQ ID NO:45, which corresponds to residues 1-III of subdomains I-III and domain IV), wherein the ECD portion comprises at least a sufficient portion of subdomains I and III to bind to a ligand of a HER receptor, and a sufficient portion of the ECD to dimerize with a cell surface receptor, including a sufficient portion of subdomain II. The first and second ECD polypeptides form multimers, e.g., dimers, by interaction of their multimerization domains. The resulting multimeric constructs provided herein bind additional ligand compared to the first or second chimeric polypeptide or homodimer thereof alone and/or dimerize with more cell surface receptors than the first or second chimeric polypeptide or homodimer thereof alone. For example, the first and second ECD polypeptides form a heterodimer that binds HER1 ligand and HER3 ligand.
b. Extracellular domain modification
In some embodiments, at least one ECD domain or portion thereof comprises a modification that alters ligand binding, specificity, or other activity or property as compared to the unmodified ECD polypeptide. In such multimeric constructs, the second ECD moiety may be the same ECD domain, wild-type or mutated form, or may be an ECD from any other cell surface receptor. The ECD or portion thereof of each monomer is linked to the multimerization domain directly or through a linker, or to the second ECD or portion thereof directly or through a linker. For example, the modification alters ligand binding, specificity or other activity or property of the ECD or full length receptor containing such ECD as compared to the unmodified ECD or full length receptor, whereby the heteromultimer exhibits altered activity or property, e.g., ligand binding or specificity alteration. Such modifications include any modification that eliminates or increases or enhances activity, such as binding to an additional ligand. An example of such multimeric construct is a construct comprising at least one HER1 ECD comprising a mutation in subdomain III that increases its affinity for a ligand other than EGF. This increase in affinity is at least 2 to 10 fold, typically 100, 1000, 10 4 、10 5 、10 6 Multiple or more.
In a particular embodiment, the growth factor trap construct is a heterodimer comprising a HER1 (EGFR) chimeric fusion polypeptide and a HER3 chimeric fusion polypeptide, wherein each chimeric fusion polypeptide comprises an ECD of a receptor linked to an Fc of human IgG1, optionally linked by a peptide linker. Such chimeric fusion polypeptides are referred to herein as HER1/Fc and HER3/Fc. Typically, the C-terminus of the ECD polypeptide is linked to the N-terminus of the multimerization domain, e.g., igG1 Fc.
In some examples, the HER1 moiety has been enhanced for ligand binding and/or biological activity. In other examples, ligand binding and/or biological activity of the HER3 moiety has been enhanced. In another example, both HER1 and HER3 moieties have enhanced ligand binding and/or biological activity.
Exemplary modifications include, for example, S418F (reference to the mature protein sequence shown in SEQ ID NO: 41) in HER1, which allows HER3 ligand NRG 2-beta to stimulate HER1. The resulting ECD binds to or interacts with at least two ligands, one for HER1, e.g., EGF, and the other for HER3, e.g., NRG2- β. Other modifications include, for example, mutations T15S and G564S in EGFR/HER1 ECD subdomains I and IV, respectively, reference to the sequence of the mature EGFR protein (SEQ ID NO: 41), and Y246A in HER3 ECD subdomain II, reference to the sequence of the mature HER3 protein (SEQ ID NO: 45), which when combined, results in an average 22-fold increase in affinity for various ligands, including EGF, TGF-alpha, HB-EGF and NRG 1-beta. Other mutations in HER1 ECD include E330D/G588S, S N/E330D/G588S and T43K/S193N/E330D/G588S, the precursor HER1 sequence (including signal peptide) shown in SEQ ID NO:40, and the sequences corresponding to E306D/G564S, S N/E306D/G564S and T19K/S169N/E306D/G564S, the mature HER1 polypeptide sequence shown in SEQ ID NO: 41. These mutations increase the binding affinity of HER1 for the ligands EGF, HB-EGF and TGF- α (see, e.g., U.S. patent publication No. 2010/0055093).
c. Multimerization domains
In certain embodiments, the multimerization domain is an Fc domain or variant thereof that effects multimerization. The Fc domain may be from any immunoglobulin (Ig) molecule, including from IgG, igM, or IgE. For example, the Fc domain may be from IgG1, igG2, igG3, or IgG4, and comprises C H 2 and C H 3 domain, and optionally all or part of the hinge region. In certain examples, the Fc portion is that of human IgG1, optionally including all or part of the hinge region, and corresponds to residues 99-330, 100-330, 104-330, 109-330, 111-330, 113-330, or 114-330 of, for example, SEQ ID NO 9. Also included is a modified Fc domain as described above, which is modifiedThe decorations are provided with convex and concave and changed properties.
Each ECD polypeptide in the multi-specific growth factor trap construct is linked to Fc directly or indirectly through a linker, such as a chemical or polypeptide linker, to form a chimeric fusion polypeptide (i.e., ECD/Fc fusion polypeptide). The multimerization domains of each chimeric fusion polypeptide, e.g., fc domains, interact (via disulfide bonds in the case of Fc domains) to form a heteromultimer, e.g., a heterodimer.
The linker between the ECD and Fc portion of each chimeric fusion polypeptide can be a flexible peptide linker, such as the hinge region of IgG, or other polypeptide linker composed of small amino acids in various lengths and combinations, such as glycine, serine, threonine, and/or alanine. For example, the joint may be (Gly) n 、(GGGGS) n 、(SSSSG) n Or (AlaAlaProAla) n Wherein n is 1-6, or may be GKSSGSGSESKS, GGSTSGSGKSSEGKG, GSTSGSGKSSSEGSGSTKG, GSTSGSGKPGSGEGSTKG, EGKSSGSGSESSKEF, gly-Arg-Met-Asp (GRMD), ser-Cys-Asp-Lys-Thr (SCDKT) or Glu-Lys-Thr-Ile-Ser (EKTIS) (see SEQ ID NO: 816-834) or any other linker described elsewhere herein or known in the art suitable for such purpose.
Fc domain modification
The Fc domains in the growth factor trap constructs provided herein are modified to improve or enhance protein expression and purity, as well as to improve pharmacodynamic and pharmacokinetic properties, including, for example, by extending in vivo half-life and/or altering immune effector function, as described below, and resulting in the production of heterodimers as the primary or sole product.
i. Introduction of protrusion-depression
The Fc domains in the growth factor trap constructs provided herein can be engineered such that the steric interactions promote stable interactions and promote the formation of heterodimers rather than homodimers from a mixture of chimeric ECD polypeptide monomers. As discussed elsewhere herein, "in the bulge" and "in the recess" (KiH; also known as "knobs-intos-holes") are introduced into the C of the heavy chain of an antibody (e.g., igG) H 3 domain optimizing heterodimer productionRaw materials. The method in the bulge recess involves asymmetrically mutating C of two Fc monomers in a complementary manner H 3 in the 3 domain. Typically, the "bulge" or protrusion is formed by the process described in C H Substitution of amino acids with small side chains with amino acids with larger side chains (e.g., tyrosine or tryptophan) at the interface between the 3 domains, and compensatory "recesses" or cavities of the same or similar size as the protrusions are created by substitution of amino acids with large side chains with amino acids with smaller side chains (e.g., alanine or threonine). Raised and recessed variants of Fc monomers by insertion of a raised into partner C H 3 domain in a correspondingly designed recess. The bulge-pit binding is prevented due to steric repulsion, and the pit-pit homodimer is unstable.
In some embodiments, the Fc portion of the heterodimeric growth factor trap constructs provided herein are designed to be contained in a protruding recess. The protruding mutation may be, for example, S354C, T366Y, T366W or T394W according to EU numbering, corresponding to S237C, T249Y, T249W or T277W, respectively, referring to the human IgG1 heavy chain constant region sequence as set forth in SEQ ID NO: 9. The concave mutation may be Y349C, T366S, L368A, F405A, Y407T, Y407A or Y407V according to EU numbering corresponding to Y232C, T249S, L A, F288A, Y290T, Y A or Y290V, respectively, referring to the human IgG1 heavy chain constant domain sequence shown in SEQ ID NO 9. For example, the introduction of the bulge recess increases the yield of heterodimers of interest, reduces the number of homodimeric impurities, and facilitates the protein purification process of the dual-specific heterodimeric growth factor trap constructs provided herein, as compared to RB200 and RB 242.
Modifications to enhance neonatal Fc receptor (FcRn) recycling
Fusion to IgG Fc, as described elsewhere herein, allows for slower clearance from the body, e.g., the kidneys, by utilizing neonatal Fc receptor (FcRn) binding and by increasing the molecular weight of the therapeutic agent to extend the half-life of the small protein therapeutic. To improve pharmacokinetics and overall pharmacology, residues within the Fc region of the growth factor trap constructs provided herein may be mutated to increase affinity for FcRn, typically by more than 30-fold, further increasing the in vivo half-life.
In some embodiments, the Fc portion of the growth factor trap constructs herein is modified to enhance neonatal FcRn recycling, thereby increasing in vivo half-life. This can be accomplished by mutating the C of IgG Fc H 2 and C H The 3 domain interface residues are implemented, these residues are responsible for binding to FcRn. Exemplary Fc modifications that increase binding to FcRn and that may incorporate the Fc portion of the growth factor trap constructs herein include, but are not limited to, one or more of the following: T250Q, T250R, M F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V380I, V428I, V433I, V434I, V434I, V434I, V436I, V Y/T256I, V F/T256I, V Y/S254T/T256I, V433K/N434F/Y436I, V250Q/M428I, V R/M428I, V L/N434I, V259I/V308I, V259I/V308F/M428I, V del/T307P/N434Y, and T256N/A378V/S383N/N434Y, and combinations thereof (according to EU numbering). The corresponding modifications are listed in Table 7 (FcRn binding enhancing IgG1 Fc modification) in the section describing Fc by corresponding mutations of Kabat numbering and sequence number, with reference to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO. 9. Other modifications known in the art that confer enhanced or increased FcRn binding are also contemplated for use herein.
Modification of the Fc portion of the growth factor trap constructs provided herein to enhance FcRn binding and recycling increases the in vivo half-life of the therapeutic agent compared to RB200 and RB242, requires lower doses and/or lower frequency of administration, and increases therapeutic efficacy.
Effector function
IgG Fcs-mediated immune effector functions, as described herein, include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC; also known as antibody-dependent cytotoxicity), and antibody-dependent cell-mediated phagocytosis (ADCP; also known as antibody-dependent cell phagocytosis). As discussed below and elsewhere herein, the Fc region of the growth factor trap constructs herein may be mutated or modified to eliminate, reduce or enhance immune effector functions, including, for example, any one or more of CDC, ADCC and ADCP.
Since the growth factor targeted by the growth factor trap construct exists as a membrane protein and as a free (i.e., soluble) ligand, in certain embodiments, immune effector function, particularly ADCC, of the Fc portion of the ECD/Fc fusion polypeptide is preserved. In alternative embodiments, other Fc regions besides human IgG1 Fc may be included in the ECD/Fc chimeric fusion polypeptides provided herein. For example, where effector function mediated by Fc/fcγr interactions is minimized, fusion with IgG isotypes that poorly recruit complement or effector cells is contemplated and effector functions, such as Fc of IgG2 or IgG4, are not exhibited. Such methods can be used in situations where effector function is not required or is detrimental, such as in the case of autoimmune and inflammatory diseases and conditions.
In certain examples, the Fc portion may be modified to enhance or increase immune effector function. This may be accomplished, for example, by adding modifications that bind C1q (for CDC) and/or that activate certain fcγrs (e.g., fcγri, fcγriia, fcγriic, fcγriiia, and fcγriiib). The Fc region modified to have increased binding to Fc receptors may be more effective in promoting destruction of cancer cells in a patient, even when linked to an ECD polypeptide. Antibodies destroy tumor cells by a number of possible mechanisms, including, for example, anti-proliferation by blocking growth pathways, intracellular signaling leading to apoptosis, enhanced receptor down-regulation and/or turnover, ADCC, ADCP, CDC, and promotion of an adaptive immune response. Thus, in embodiments of the growth factor trap constructs herein for use in treating cancer, the Fc portion of the construct may be modified to enhance or increase immune effector function. Table 8 (IgG 1 Fc modification to enhance immune effector function) in section (enhancing or reducing/eliminating F immune effector function) summarizes Fc modifications, including ADCC, ADCP and CDC, that increase binding to FcgammaR or C1q and thus enhance immune effector function, and provides corresponding modifications according to Kabat numbering and sequence number with reference to the sequence of the IgG1 heavy chain constant domain shown in SEQ ID NO: 9. Any one or more of these modifications may be incorporated into the IgG1 Fc portion of the growth factor trap constructs provided herein, alone or in various combinations. Other modifications known in the art that confer enhanced or increased immune effector function are also contemplated herein. These are listed in the previous section, describing IgG1 Fc modifications that enhance immune effector function.
In alternative embodiments, the Fc portion of the growth factor trap constructs provided herein is modified to reduce or eliminate immune effector function. This may be achieved, for example, by reducing or eliminating modifications that bind to C1q (for CDC) and/or that activate certain fcγrs (e.g., fcγri, fcγriia, fcγriic, fcγriiia, and fcγriiib). This is desirable, for example, where antagonism is desired rather than killing cells carrying the target antigen, or where reduction of unwanted or deleterious immune effector functions (e.g., unwanted pro-inflammatory cytokine release and off-target cytotoxicity) is desired. Thus, in embodiments of the growth factor trap constructs provided herein for use in the treatment of chronic inflammatory and autoimmune diseases and disorders, such as RA, the Fc portion of the construct may be modified to reduce or eliminate immune effector function.
Table 9 (IgG 1 Fc modification that reduces or eliminates immune effector function) in part (enhancement or reduction/elimination of Fc immune effector function) summarizes exemplary IgG1 Fc modifications that reduce or eliminate binding to and activation of fcγr and/or C1q, and thus reduce or eliminate immune effector function, including ADCC, ADCP and CDC, and can be incorporated into the Fc region of the growth factor trap constructs herein. The table provides the corresponding modifications according to the Kabat numbering and sequence number of the sequence of the IgG1 heavy chain constant domain shown in reference SEQ ID NO. 9. Any one or more of these modifications may be incorporated into the IgG1 Fc portion of the growth factor trap constructs provided herein, alone or in various combinations. Other modifications known in the art to reduce or eliminate immune effector function are also contemplated herein.
The Fc portion of the growth factor trap constructs provided herein may also be modified to increase binding to inhibitory fcγrs, resulting in suppression of immune responses. Therapeutic antibodies with immunosuppressive Fc modifications are useful in the treatment of inflammatory diseases. These mutations can be incorporated into the Fc portion of the growth factor trap constructs herein, with the aim of treating diseases and conditions having an inflammatory component or etiology or involvement, such as RA and other inflammatory and autoimmune diseases.
Modifications that increase binding to or confer selective binding to inhibitory fcyriib and/or fcyri but not fcyriiia may be engineered into the IgG1 Fc region in the growth factor trap constructs provided herein. Such modifications include, but are not limited to, one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F, L S/T366R/L368H/P395K and combinations thereof (according to EU numbering). Table 11 of section F.4. D.i.i. (IgG 1 Fc modification to increase binding to inhibitory FcgammaRIIB) shows the corresponding substitutions of the IgG1 heavy chain constant domain shown with reference to SEQ ID NO. 9 according to Kabat numbering and sequence number. These modifications are summarized in the section above describing IgG1 Fc modifications that increase binding to the inhibitory fcyriib.
4. Compositions, therapeutic uses and methods of treatment
Nucleic acid molecules encoding chimeric fusion polypeptides (i.e., ECDs/Fc) and growth factor trap constructs, vectors containing the nucleic acid molecules are provided. Also provided are cells comprising the vectors described herein, as well as pharmaceutical compositions comprising any of the growth factor trap constructs, encoding nucleic acid molecules, vectors, or cells described herein. Growth factor trap constructs herein are generated and purified as previously described, for example, in Sarup et al (2008) mol. Cancer Ther.7 (10): 3223-3236, gompels et al (2011) Arthritis Research & Therapy 13: R161, jin et al (2009) mol. Med.15 (1-2): 11-20 and U.S. patent publication No. 2010/0055093.
The multi-specific (including bispecific) growth factor trap constructs herein contain two or more, especially two, chimeric proteins produced by direct or indirect attachment of two or more, especially two, identical or different, ECD polypeptides to a multimerization domain. In some examples, where the multimerization domain is a polypeptide, such as an immunoglobulin Fc, the gene fusion encoding the ECD-multimerization domain chimeric polypeptide is inserted into a suitable expression vector. The resulting ECD-multimerization domain chimeric proteins may be expressed in host cells, particularly mammalian cells (e.g., HEK293T or CHO cells, or any other suitable mammalian cells described herein or known in the art), transformed with a recombinant expression vector, and assembled into multimers, e.g., dimers, wherein the multimerization domains interact to form multivalent polypeptides. The resulting chimeric polypeptides and multimers formed therefrom may be purified by any suitable method known in the art, for example by affinity chromatography on a protein a or protein G column. Additionally or alternatively, other techniques for protein purification may be used, including, for example, gel electrophoresis, dialysis, ion exchange chromatography, ethanol precipitation, HPLC such as reverse phase HPLC, silica chromatography, heparin sepharose chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation. When two nucleic acid molecules encoding different ECD chimeric polypeptides are transformed into a cell (e.g., HER1/Fc and HER 3/Fc), homodimers and heterodimers will form. Expression conditions can be adjusted so that heterodimer formation is better than homodimer formation. For example, the proportion of nucleic acid molecules encoding different ECD chimeric polypeptides can be adjusted such that an excess of one nucleic acid molecule results in the formation of fewer homodimers. Furthermore, as described above, the introduction of the protruding depressions into the Fc monomer favors the formation of heterodimers rather than homodimers.
The ECD chimeric polypeptides containing the Fc region can also be engineered to include tags with metal chelates or other epitopes, such as 6XHis tags, c-myc tags, FLAG tags, maltose Binding Protein (MBP), glutathione-S-transferase (GST), or Thioredoxin (TRX). The tagged domains can be used for rapid purification by metal chelate chromatography and/or antibodies and allow detection in Western blots, immunoprecipitation or active depletion/blocking in bioassays.
a. Pharmaceutical composition
Provided herein are pharmaceutical compositions containing a multispecific, e.g., bispecific growth factor trap construct or encoding nucleic acid molecule provided herein. Also provided are pharmaceutical compositions comprising isolated cells comprising a nucleic acid molecule or vector provided herein. Such compositions contain a therapeutically effective amount of a growth factor trap construct. The pharmaceutical composition may be formulated in any conventional manner by mixing a selected amount of the growth factor trap construct or nucleic acid molecule with one or more physiologically acceptable carriers or excipients. The pharmaceutical compositions are useful for therapeutic, prophylactic and/or diagnostic applications. The concentration of the active compound in the composition will depend on the absorption, inactivation, and excretion rates of the active compound, the dosing regimen and dosing amount, and other factors known to those of skill in the art.
Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art as suitable for the particular mode of administration. The choice of carrier or excipient is within the skill of the practitioner and may depend on a number of parameters. These include, for example, modes of administration (i.e., systemic, oral, nasal, pulmonary, focal, topical, or any other) and conditions of treatment. Pharmaceutical compositions comprising a therapeutically effective amount of a multispecific, e.g., bispecific growth factor trap construct or nucleic acid molecule described herein may also be provided as a lyophilized powder, which is reconstituted, e.g., with sterile water, immediately prior to administration.
The pharmaceutical compositions provided herein can be in a variety of forms, such as solid, semi-solid, liquid, powder, aqueous, or lyophilized forms. The pharmaceutical compositions provided herein may be formulated for single dose (direct) administration, or diluted or otherwise modified administration. The concentration of the compound in the formulation is an amount effective to deliver, upon administration, an effective amount for the intended treatment. Typically, the compositions are formulated for single dose administration. The compounds may be suspended in micronized or other suitable form, or may be derivatized to produce more soluble active products. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the carrier or vehicle chosen. The effective concentration is sufficient to ameliorate the targeted condition and can be determined empirically. To formulate the composition, the weight fractions of the compound are dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the targeted disorder is alleviated or ameliorated.
Methods for producing nucleic acids encoding the growth factor trap constructs provided herein include the methods described in section H. Section H also describes vectors and cells that can be used, as well as methods for protein expression and purification. The compositions, formulations, dosages and methods of administration described in section I may be adapted for use in the production of compositions and formulations comprising the growth factor trap constructs and encoding nucleic acid molecules described herein. Dosages and methods of administration may be determined by the practitioner and are known in the art and described elsewhere herein.
b. Therapeutic uses and methods of treatment
The multi-specific (including bispecific) growth factor trap constructs provided herein may be used for any purpose known to those of skill in the art using such molecules. For example, the growth factor trap constructs provided herein may be used for one or more of therapeutic, diagnostic, industrial, and/or research purposes. In particular, the multi-specific growth factor trap constructs provided herein are useful for treating a variety of diseases and conditions involving CSR, including RTKs and particularly the HER protein family, including those described herein. HER signaling is involved in the etiology of a variety of diseases and disorders, and any such disease or disorder thereof is contemplated to be treated by the growth factor trap constructs provided herein.
The growth factor trap constructs and encoding nucleic acid molecules and pharmaceutical compositions provided herein are useful for treating any condition for which anti-HER therapy (e.g., trastuzumab, cetuximab, gefitinib, erlotinib, and lapatinib, and other formulations described herein and/or known in the art) is useful, including, but not limited to, cancer and other proliferative diseases and conditions, angiogenesis-related diseases and conditions, rheumatoid arthritis, and other chronic inflammatory and autoimmune diseases and conditions, as well as neurodegenerative diseases and Central Nervous System (CNS) conditions. For example, treatments using the growth factor trap constructs provided herein include, but are not limited to, treatments of angiogenesis-related diseases and disorders, inflammatory diseases and disorders, autoimmune diseases and disorders, neurodegenerative diseases, and disorders associated with cell proliferation. Such diseases and conditions include, for example, ocular diseases, atherosclerosis, vascular injury, alzheimer's disease, cancer, smooth muscle cell related disorders, rheumatoid Arthritis (RA), and various autoimmune diseases.
Dosage levels and regimens may be determined based on known dosages and regimens, and if desired may be extrapolated from variations in the properties of the polypeptides and constructs provided herein, and/or may be empirically determined based on a variety of factors. These factors include, for example, the weight of the individual and its general health, age, sex and diet, as well as the activity of the particular compound used, the time of administration, the rate of excretion, drug combination, the severity and course of the disease, as well as the predisposition of the patient and the discretion of the attendant physician. The active ingredient is typically combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form or multiple dosage forms can vary depending upon the host treated and the particular mode of administration.
The dosage will depend on the particular disorder, disease or condition being treated, as well as the particular subject. Typical doses are similar to known anti-HER therapies, e.g., antibodies, including trastuzumab, cetuximab, pertuzumab, and panitumumab, as well as small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib, and lapatinib. For subjects, including humans and other animals, exemplary dosages range from about or 0.1mg/kg to 100mg/kg, such as 1mg/kg to about or 30mg/kg, such as 5mg/kg to 25mg/kg. The dosage may be determined on the assumption that the average human weight is about 75 kg. The dosage may be adjusted for children, infants and smaller adults.
After the patient's condition has been improved, a maintenance dose of the compound or composition may be administered, if necessary; and the dosage, dosage form, or frequency of administration, or combinations thereof, may be modified. In some cases, the subject may need to undergo long-term intermittent treatment according to any recurrence of disease symptoms or according to a predetermined dosage regimen.
Treatment of diseases and conditions with the multi-specific growth factor trap constructs provided herein may be accomplished by any suitable route of administration using the suitable formulations described herein, including but not limited to infusion, subcutaneous injection and inhalation, or intramuscular, intradermal, oral, topical and transdermal routes of administration.
Provided herein is a method of treating a HER-mediated or HER-related disease or disorder comprising testing a subject suffering from the disease to determine which HER receptors are expressed or overexpressed, and selecting a multispecific growth factor trap construct to target at least one, and typically two HER receptors based on the results. In one embodiment, the disease is cancer. Exemplary cancers for treatment herein include glioma, as well as pancreatic, gastric, head and neck, cervical, lung, colorectal, endometrial, prostate, esophageal, ovarian, uterine, bladder, or breast cancer. Cancers that can be treated with the growth factor (HER ligand) trap constructs herein are typically cancers that express at least one HER receptor, typically more than one HER receptor. Such cancers may be identified by any means known in the art for detecting HER expression. For example, HER2 expression can be assessed using commercially available diagnostic/predictive assays, such as herceptist TM (Dako). Immunohistochemical (IHC) assays were performed on paraffin-embedded tissue sections from tumor biopsies and were consistent with HER2 protein staining intensity standards. Tumors meeting below the threshold score were identified as overexpressing HER2, while those greater than or equal to the threshold score were identified as overexpressing HER2. In one treatment example, HER2 overexpressed tumors are evaluated as candidates for treatment with a multispecific growth factor trap construct, such as any of the constructs provided herein.
In another embodiment, the HER-mediated or HER-related disease or disorder is an inflammatory or autoimmune disorder, particularly rheumatoid arthritis. Animal models of arthritis, such as collagen-induced arthritis (CIA) mouse models, can be used to test the growth factor trap constructs provided herein. For example, a reduction in arthritic symptoms, including paw swelling, erythema, and rigidity, can be observed in mice treated with the growth factor trap constructs herein, e.g., by local injection of protein. A reduction in synovial angiogenesis and synovial inflammation can also be observed.
The multispecific, including bispecific growth factor trap constructs, encoding nucleic acid molecules, and pharmaceutical compositions provided herein are useful for treating HER (ErbB) related diseases or HER receptor mediated diseases, which are any disease, condition, or disorder in which a HER receptor and/or ligand is involved in certain aspects of its etiology, pathology, or occurrence of the disease. The HER-related disease treated includes cancer, such as glioma or pancreatic cancer, gastric cancer, head and neck cancer, cervical cancer, lung cancer, colorectal cancer, endometrial cancer, prostate cancer, esophageal cancer, ovarian cancer, uterine cancer, bladder cancer, renal cancer, or breast cancer. Other diseases that may be treated include non-cancerous proliferative diseases, such as those involving smooth muscle cell proliferation and/or migration, inflammatory or autoimmune diseases, skin disorders, and ocular disorders. Diseases and conditions treated include, for example, rheumatoid arthritis, diabetic retinopathy, anterior ocular disease, psoriasis, restenosis, stenosis, atherosclerosis, vascular thickening induced hypertension, bladder, cardiac or other muscle thickening, bladder diseases, endometriosis and obstructive airways diseases, as well as diseases or conditions associated with (e.g., causing or exacerbating) exposure to one or more Neuregulin (NRG) ligands such as NRG1 (including types I, II and III), NRG2, NRG3 and/or NRG4 or other HER family ligands. Examples of NRG-related diseases and diseases associated with other HER family ligands include neurological or neuromuscular diseases including schizophrenia, parkinson's disease and alzheimer's disease, cardiomyopathy, preeclampsia, neurological diseases and heart failure.
Examples of cancers that may be treated include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, such as squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small-cell lung cancer, non-small-cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, renal cell carcinoma, esophageal cancer, glioma, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, and head and neck cancer.
The multispecific growth factor trap constructs provided herein may generally result in increased therapeutic efficacy and reduced resistance when administered as compared to single targeted anti-HER therapies such as trastuzumab, cetuximab and other antibodies described or known herein, as well as small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and lapatinib. As described herein, resistance to single targeted anti-HER therapies is associated with co-expression and/or up-regulation of other HER family members and overexpression of their ligands. The HER-ligand binding constructs provided herein act as receptor decoys and sequesters multiple HER family ligands, preventing ligand-dependent receptor activation and down-regulating aberrant HER family activity, resulting in simultaneous inhibition of multiple ligand-induced HER family members. This improves the therapeutic effect and reduces the chances of developing resistance.
5. Combination therapy
Combination therapy may be used. Combination therapy includes administration of the multi-specific (including bispecific) growth factor trap constructs, nucleic acid molecules, and pharmaceutical compositions provided herein, in combination with another agent or therapy (including radiation and surgery). The additional agent or therapy may be administered simultaneously, prior to, subsequent to, or intermittently with the treatment provided herein. They may be in separate compositions or in a complex formulation.
The multispecific, e.g., bispecific heteromultimeric growth factor trap constructs, nucleic acid molecules, and pharmaceutical compositions provided herein may be administered before, after, intermittently, or simultaneously with one or more other therapeutic regimens or agents, including, but not limited to TNF antagonists/blockers, chemotherapeutic agents, single target anti-HER therapies (including antibodies and tyrosine kinase inhibitors), anti-angiogenic agents, antibodies, cytotoxic agents, anti-inflammatory agents, cytokines, growth inhibitors, anti-hormonal agents, cardioprotective agents, steroids, immunostimulatory agents, immunosuppressants, biological or non-biological disease modifying antirheumatic drugs (DMARDs), infectious disease treatment drugs (including antibodies), or other therapeutic drugs. In particular, the growth factor trap constructs are administered with the TNFR1/TNFR2 axis constructs provided herein. They may also be administered with other anti-TNF therapies, including any of the therapies described in the section above or known to those of skill in the art.
The TNFR1 antagonist construct, TNFR2 agonist construct, multispecific construct, nucleic acid, and other constructs provided herein may be administered in a regimen with other anti-TNF therapies. Exemplary anti-TNF therapeutic agents useful in combination therapy herein include, for example, conventionally synthesized DMARDs, such as Methotrexate (MTX), hydroxychloroquine (HCQ;) Sulfasalazine) And leflunomide (>) The method comprises the steps of carrying out a first treatment on the surface of the Biological DMARDs, e.g. Abacalcet (+)>) Anakinra extract) Rituximab (+)> ) Toxicillin (Toxicillin),) Corticosteroids (e.g., dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib (>) And TNF inhibitors/anti-TNF agents, e.g. polyethylene glycol cetuximab) Infliximab (++>) Adalimumab (/ ->) Golimumab (+)>) And etanercept ()>). Combination therapies may also include immunotherapeutic agents such as cyclosporin, methotrexate, doxorubicin or cisplatin, as well as immunotoxins.
In particular examples, the growth factor trap constructs provided herein are administered with any of the TNFR1 antagonist constructs, TNFR2 agonist constructs, or multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs provided herein for use in treating any of the chronic inflammatory, autoimmune, and/or neurodegenerative/demyelinating diseases and disorders described herein, particularly Rheumatoid Arthritis (RA).
In some examples, a growth factor trap construct provided herein is administered with one or more anti-angiogenic agents. For example, an anti-angiogenic factor may be a small molecule or protein (e.g., an antibody, fc fusion, or cytokine) that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. Examples of anti-angiogenic agents include, but are not limited to, antibodies that bind Vascular Endothelial Growth Factor (VEGF) or bind VEGF-R, RNA-based therapeutics that reduce the level of VEGF or VEGF-R expression, VEGF-toxin fusions, VEGF-TRAP from Regeneron, angiostatin (a plasminogen fragment), antithrombin III, angiozyme, ABT-627, bay 12-9566, benefin, bevacizumab, bisphosphonates, BMS-2791, cartilage Derived Inhibitors (CDI), CAI, CD59 complement fragment, CEP-7055, col 3, combretastatin A-4, endostatin (a collagen XVIII fragment), farnesyl transferase inhibitors, fibronectin fragments, GRO-beta, halofuginone (halofuginone), heparinase, heparin hexasaccharide fragments, HMV833, human chorionic gonadotrophin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon inducible protein 10 (IP-10), interleukin 12, kringle 5 (plasminogen fragment), marimastat (marimastat), metalloproteinase inhibitors (e.g., TIMP), 2-methoxyestradiol, MMI 270 (CGS 27023A), plasminogen Activator Inhibitor (PAI), platelet factor 4 (PF 4), pramipestat (prinomostat), prolactin 16kDa fragment, prolidin Related Protein (PRP), PTK 787/ZK 222594, retinoid, solimastat (solimastat), squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, SU5416, thalidomide (thalidomide), thrombospondin-1 (TSP-1), TNP470, transforming growth factor beta (TGF-beta), angiostatin, vascular inhibitor (calreticulin fragment), ZS6126, and ZD6474.
In some examples, a growth factor trap construct provided herein is administered with one or more tyrosine kinase inhibitors and optionally a TNFR1/TNFR2 axis construct provided herein. Examples of tyrosine kinase inhibitors include, but are not limited to, quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidine; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidines; 4- (phenylamino) -7H-pyrrolo (2, 3-d) pyrimidine; curcumin (bis-feruloylmethane, 4, 5-bis (4-fluoroanilino) phthalimide); tyrosinase containing nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB encoding nucleic acids); quinoxalines (see, for example, U.S. patent No. 5,804,396); tyrosine phosphorylation inhibitors (tyrphostin) (see, e.g., U.S. patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G); pan-ErbB inhibitors such as C1-1033 (Pfizer); affinitac (ISIS 3521; isis/Lilly); imatinib mesylate (STI 571,；Novartis)；PKI 166(Novartis); GW2016 (Glaxo SmithKline); c1-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering A G); INC-1C11 (ImClone); gefitinib @ ZD1839, astraZeneca); and OSI-774 (under the trademark +.>Sell, OSI Pharmaceuticals/Genentech), or any of the following patent publications: U.S. Pat. Nos. WO 99/09016, WO 98/43960, WO 97/38983, WO 99/06378, WO 99/06396, WO 96/30347, WO 96/33978, WO 96/33979, and WO 96/33980.
Other compounds useful in combination therapy include steroids such as angiogenesis inhibiting 4,9 (11) -steroids and C21-oxidized steroids, angiostatin, endostatin, angiostatin (canstatin) and mastoplatin (Maspin), angiogenin, bacterial polysaccharide CM101 and antibody LM609 (see, e.g., U.S. patent No. 5,753,230), thrombospondin (TSP-1), platelet factor 4 (PF 4), interferons, metalloproteinase inhibitors, drugs including AGM-1470/TNP-470, thalidomide and Carboxyamidotriazole (CAI), cortisone, e.g., in the presence of heparin or heparin fragments, anti-invasive factors, retinoic acid and paclitaxel, shark cartilage extracts, anionic polyamides or polyurea oligomers, oxindole derivatives, estradiol derivatives and thiazolopyrimidine derivatives.
Examples of anti-cancer antibodies that may be co-administered with the growth factor trap constructs provided herein include, but are not limited to, anti-17-IA cell surface antigen antibodies, such as ibrutinab (under the trademark @ ) Sales); an anti-4-1 BB antibody; an anti-4 Dc antibody; anti-a 33 antibodies, such as a33 and CDP-833; anti- α1 integrin antibodies, such as natalizumab; anti- α4β7 integrin antibodies such as LDP-02; anti- αvβ1 integrin antibodies such as F-200, M-200, SJ-749; anti-alpha V beta 3 integrinWhite antibodies, such as Acximab, CNTO-95, mab-17E6, and Medi-523 (sold under the trade name Vitaxin); anti-complement factor 5 (C5) antibodies, e.g., 5G1.1; anti-CA 125 antibodies, e.g. ago Fu Shan antibodies (under the trademark +.>Sales); anti-CD 3 antibodies, e.g. Wiceizumab (>) And Rexomab; anti-CD 4 antibodies, such as IDEC-151, MDX-CD4, and OKT4A; anti-CD 6 antibodies, such as oncolytic B and oncolytic CD6; anti-CD 7 antibodies, such as HB2; anti-CD 19 antibodies, such as B43, MT-103 and oncolytic B; anti-CD 20 antibodies, e.g. 2H7, 2H7.v16, 2H7.v114, 2H7.v115, tositumomab (>) Rituximab @) And ibritumomab (/ -)>) The method comprises the steps of carrying out a first treatment on the surface of the anti-CD 22 antibodies, e.g. epratuzumab (>) The method comprises the steps of carrying out a first treatment on the surface of the anti-CD 23 antibodies, such as IDEC-152; anti-CD 25 antibodies, e.g. basiliximab and +.>(daclizumab); anti-CD 30 antibodies, such as AC10, MDX-060 and SGN-30; anti-CD 33 antibodies, e.g. gemtuzumab ozogamicin (++>) Oncolytic M and Smart MI 95; an anti-CD 38 antibody; anti-CD 40 antibodies, such as SGN-40 and tolaguemab; anti-CD 40L antibodies, such as 5c8, lu Lizhu mab (Antova) and IDEC-131; anti-CD 44 antibodies, e.g. bivalirudin Resistance; an anti-CD 46 antibody; anti-CD 52 antibodies, e.g.>(alemtuzumab); anti-CD 55 antibodies, such as SC-1; anti-CD 56 antibodies, such as huN901-DM1; anti-CD 64 antibodies, such as MDX-33; anti-CD 66e antibodies, such as XR-303; anti-CD 74 antibodies, such as IMMU-110; anti-CD 80 antibodies, such as ganciclibab and IDEC-114; anti-CD 89 antibodies, such as MDX-214; an anti-CD 123 antibody; anti-CD 138 antibodies, such as B-B4-DM1; anti-CD 146 antibodies, such as AA-98; an anti-CD 148 antibody; anti-CEA antibodies, e.g. cT84.66, la Bei Zhushan and +.>The method comprises the steps of carrying out a first treatment on the surface of the anti-CTLA-4 antibodies, such as MDX-101; anti-CXCR 4 antibodies; anti-EGFR antibodies, e.g., ABX-EGF, < + >>(cetuximab), panitumumab, IMC-C225 and Merck Mab 425; anti-EpCAM antibodies, such as anti-EpCAM, ING-1, and IS-IL-2 of cruell; an anti-ephrin B2/EphB4 antibody; anti-HER 2 antibodies, e.g.)>(trastuzumab), pertuzumab and MDX-210; anti-FAP (fibroblast activation protein) antibodies, such as cetrimab; anti-ferritin antibodies, such as NXT-211; an anti-FGF-1 antibody; an anti-FGF-3 antibody; an anti-FGF-8 antibody; an anti-FGFR antibody; an anti-fibrin antibody; anti-G250 antibodies, e.g. WX-G250 and gemtuximab (>) The method comprises the steps of carrying out a first treatment on the surface of the anti-GD 2 ganglioside antibodies, such as EMD-273063 and TriGem; anti-GD 3 ganglioside antibodies, such as BEC2, KW-2871 and Mi Tuomo monoclonal antibodies; anti-gpIIb/IIIa antibodies, such as ReoPro; an anti-heparanase antibody; anti-HLA antibodies, such as Oncolym and Smart 1D10; anti-HM 1.24 antibody; anti-ICAM antibodies, such as ICM3; an anti-IgA receptor antibody; anti-IGF-1 antibodies, such as CP-751871 and EM-164; anti-IGF-1R antibodies, such as IMC-A12; anti-IL -6 antibodies, such as CNTO-328 and Ai Ximo monoclonal antibodies; anti-IL-15 antibodies, e.g.>-IL15 antibodies; an anti-KDR antibody; an anti-laminin 5 antibody; anti-Lewis Y antigen antibodies, such as Hu3S193 and IGN-311; an anti-MCAM antibody; anti-Muc 1 antibodies, such as BravaRex and TriAb; anti-NCAM antibodies, such as ERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn and Therex; an anti-PSA antibody; anti-PSCA antibodies, such as IG8; an anti-Ptk antibody; an anti-PTN antibody; anti-RANKL antibodies, such as AMG-162; an anti-RLIP 76 antibody; anti-SK-1 antigen antibodies, such as Monopharm C; an anti-STEAP antibody; anti-TAG 72 antibodies, such as CC49-SCA and MDX-220; anti-TGF-beta antibodies, such as CAT-152; anti-TNF-alpha antibodies, e.g. CDP571, CDP870, D2E7, adalimumab (++>) And infliximab (++>) The method comprises the steps of carrying out a first treatment on the surface of the anti-TRAIL-R1 and TRAIL-R2 antibodies; an anti-VE-cadherin-2 antibody; and anti-VLA-4 antibodies, e.g. +.>An antibody. Anti-idiotype antibodies may be used, including but not limited to GD3 epitope antibody BEC2 and gp72 epitope antibody 105AD7. Bispecific antibodies may also be used, including but not limited to the anti-CD 3/CD20 antibody Bi20.
Examples of antibodies that can be co-administered with the growth factor trap constructs provided herein that can treat autoimmune or inflammatory diseases, transplant rejection, and/or GvHD include, but are not limited to, anti- α4β7 integrin antibodies, such as LDP-02; anti- β2 integrin antibodies, such as LDP-01; anti-complement (C5) antibodies, such as 5G1.1; anti-CD 2 antibodies, such as BTI-322 and MEDI-507; anti-CD 3 antibodies, such as OKT3 and SMART anti-CD 3; anti-CD 4 antibodies, such as IDEC-151, MDX-CD4, and OKT4A; an anti-CD 11a antibody; anti-CD 14 antibodies, such as IC14; an anti-CD 18 antibody; anti-CD 23 antibodies, such as IDEC-152; anti-CD 25 antibodies, e.g. Zen apox; anti-CD 40L antibodies, such as 5c8, antova, and IDEC-131; anti-CD 64 antibodies, such as MDX-33; anti-CD 80 antibodies, such as IDEC-114; anti-CD 147 antibodies, such as ABX-CBL; anti-E-selectin antibodies, such as CDP850; anti-gpIIb/IIIa antibodies, e.g.Abcixima; anti-ICAM-3 antibodies, such as ICM3; anti-ICE antibodies, such as VX-740; anti-fcγr1 antibodies, such as MDX-33; anti-IgE antibodies, such as rhuMAb-E25; anti-IL-4 antibodies, such as SB-240683; anti-IL-5 antibodies, such as SB-240563 and SCH55700; anti-IL-8 antibodies, such as ABX-IL8; an anti-interferon gamma antibody; anti-tnfa antibodies, such as CDP571, CDP870, D2E7, adalimumab, infliximab, and MAK-195F; and anti-VLA-4 antibodies, such as Antegren. Examples of other Fc-containing molecules that may be co-administered to treat autoimmune or inflammatory diseases, transplant rejection, and GvHD include, but are not limited to, TNFRII receptor/Fc fusion->IL-1TRAP of (etanercept) and Regeneron.
Examples of antibodies that may be co-administered to treat infectious diseases include, but are not limited to, anti-anthrax antibodies, such as ABthrax; anti-CMV antibodies, such as CytoGam and span Wei Shankang; anti-Cryptosporidium antibodies, such as CryptoGAM and Sporidin-G; anti-helicobacter pylori antibodies, such as Pyloran; anti-hepatitis B antibodies, such as HepeX-B and Nabi-HB; anti-HIV antibodies, such as HRG-214; anti-RSV antibodies, such as ubiquitin, HNK-20, palivizumab and Respicam; and anti-staphylococcal antibodies such as Aurexis, aurograb, BSYX-A110 and SE-Mabs.
In some examples, a growth factor trap construct described herein is administered with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide @) The method comprises the steps of carrying out a first treatment on the surface of the Alkyl sulfonates such as busulfan (busulfan), imperoshu (imposulfan) and piposhu (pipos)ulfan); androgens, such as carbosterone (calibretone), drotasone propionate (dromostanolone propionate), thioandrosterol (epiostanol), melandrane (mepistane), and testosterone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane) and trilostane (trilostane); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; antibiotics such as aclacinomycin (aclacinomycin), actinomycin (actinin), anthracycline (anthracycline), diazoserine (azaserine), bleomycin (bleomycin), actinomycin (calicheamicin), calicheamicin (carubicin), carminomycin (carminomycin), eosinophil (carzinophilin), chromomycins (chromomycins), dactinomycin (dactinomycin), daunorubicin (daunorubicin), dithizocin (detorubicin), 6-diazo-5-oxo-L-norleucine, doxorubicin (doxorubicin), epirubicin (epirubicin), epothilone (escin), idarubicin (carubicin), dactinomycin (maromycin), streptomycin (62), streptomycin (streptomycin), and streptomycin (streptomycin); antiestrogens including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), 4 (5) -imidazole inhibiting aromatase, 4-hydroxy tamoxifen, tamoxifen (trioxifene), ketoxifene (keoxifene), LY 117018, onapristone (onapristone), and toremifene (toremifene, farston); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as methotrexate (denopterin), methotrexate (methotrexate), pterin (pteroprerin), and trimetrexate (trimerexate); aziridines such as benzodopa (benzodepa), carboquone (carboquone), mettuyepa (meturedepa) and uratepa (uredepa); ethyleneimine Methyl melamines including altretamine, trivinyl melamine, trivinyl phosphoramide, trivinyl thiophosphamide and trimethylol melamine; folic acid supplements, such as folinic acid (folinic acid); nitrogen mustards such as chlorambucil (chlorrambucil), chlornapazine (chloronaphazine), chlorphosphamide (chlorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine (mechlorethamine oxide hydrochloride), melphalan (melphalan), novobiocin (novemblic), phenyllactone (phenoterine), prednisone (prednimustine), trofosfamide (trofosfamide) and uracil mustard (uracilmustard); nitrosoureas, such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ranimustine (ranimustine); platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiominoprine (thiamiprine), and thioguanine (thioguanine); pyrimidine analogs such as, for example, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, execitabine, fluorouridine and 5-FU; taxanes (taxanes), e.g. paclitaxel () >Bristol-Myers Squibb Oncology, prencton, N.J.) and docetaxel (/ -)>Rhone-Poulenc Rorer, antonny, france); topoisomerase inhibitors such as RFS 2000; thymidylate synthase inhibitors, such as Tomudex; additional chemotherapeutic agents, including acetylacetone (aceglatone); aldehyde phosphoramidate glycoside (aldophosphamide glycoside); an aminopentanonic acid (aminolevulinic acid); amsacrine (amacrine); armustine (bestabucil); biobian (bisantre)ne); edatraxate (edatrexate); ifosfamide (defosfamide); desmethylcolchicine (demecolcine); deaquinone (diaziquone); difluoromethyl ornithine (DMFO); efluoornithine (eflornithine); ammonium elide (elliptinium acetate); etoposide (etoglucid); gallium nitrate; hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mo Pi dipyridamole (mopidamol); nitrocline (nitrocrine); penstatin (penstatin); egg ammonia nitrogen mustard (phenol); pirarubicin (pirarubicin); podophylloic acid (podophyllinic acid); 2-ethyl hydrazide (ethyl hydrazide); procarbazine (procarbazine);The method comprises the steps of carrying out a first treatment on the surface of the Raschig (razoxane); cilzopran (silzofiran); spirogermanium (spirogermanium); tenuazonic acid; trinquinone (triaziquone); 2,2',2 "trichlorotriethylamine; a urethane; vindesine (vindeline); dacarbazine (dacarbazine); mannomustine (mannomustine); mi Tuobu rotol (mitobronitol); mitolactol (mitolactol); pipobromine (pipobroman); a gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil (chloramucil); gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine (mercaptopurine); methotrexate (methotrexate); etoposide (VP-16); ifosfamide (ifosfamide); mitomycin C; mitoxantrone (mitoxantrone); vincristine (vincristine); vinorelbine (vinorelbine); novibine (Navelbine); novanone (Novantrone); teniposide (teniposide); daunomycin (daunomycin); aminopterin (aminopterin); hilded (Xeloda); ibandronate (ibandronate); CPT-11; retinoic acid; epothilones (esperamicins); capecitabine (capecitabine); topoisomerase inhibitors such as irinotecan (irinotecan). Any of the above pharmaceutically acceptable salts, acids or derivatives may also be used.
The chemotherapeutic agent may be administered as a prodrug. Examples of prodrugs that may be administered with the growth factor trap constructs described herein include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, and 5-fluorocytosine and other 5-fluorouridine prodrugs, which may be converted to more active non-cytotoxic drugs.
In some examples, the multispecific growth factor trap constructs described herein are administered with one or more immunomodulatory agents. Such agents may increase or decrease the production of one or more cytokines, up-regulate or down-regulate autoantigen presentation, mask MHC antigens, or promote proliferation, differentiation, migration, or activation of one or more immune cells. Examples of immunomodulators include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, celecoxib, diclofenac (dicolofenac), etodolac (etodolac), fenoprofen (fenoprofen), indomethacin (indomethacin), ketorolac (ketorolac), oxaprozin (oxaprozin), nabumetone (nabumetone), sulindac (sulindac), tolmetin (tolmetin), rofecoxib (rofecoxib), naproxen (naproxen), ketoprofen (ketoprofen) and nabumetone (nabumetone); steroids such as glucocorticoids, dexamethasone, cortisone, hydroxycodendron, methylprednisolone, prednisone, prednisolone, and triamcinolone acetonide; eicosanoids such as prostaglandins, thromboxanes and leukotrienes; topical steroids such as dithranol (anthralin), calcipotriol (calcipotriene), clobetasol (clobetasol) and tazarotene (tazarote); cytokines such as TGF beta, IFN alpha, IFN beta, IFN gamma, IL-2, IL-4, IL-10; cytokines, chemokines or receptor antagonists, including antibodies, soluble receptors and receptor-Fc fusions to: BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40L, CD, CD45, CD52, CD64, CD80, CD86, CD147, CD152, complement factor (C5, D) CTLA-4, eosinophil chemokine (eotaxin), fas, ICAM, ICOS, IFN a, ifnβ, ifnγ, IFNAR, igE, IL-1, IL-2R, IL-4, IL-5R, IL-6, I L-8, IL-9IL-12, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGF beta, TNF alpha, TNF beta, TNFR1, TNFR2, and T cell receptors, including etanercept) Adalimumab (/ ->) And infliximab (++>) The method comprises the steps of carrying out a first treatment on the surface of the Heterologous anti-lymphoglobulin; and other immunomodulatory molecules such as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotype antibodies to MHC binding peptides and MHC fragments, azathioprine (azathioprine), buconazole (brequar), bromocriptine (Bromocryptine), cyclophosphamide, cyclosporin A, D-penicillamine, deoxyspergualin (deoxyspergualin), FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitrile amides (e.g., leflunomide), methotrexate, minocycline, mizoribine (mizoribine), mycophenolate (mycophenolate mofetil), rapamycin, and sulfasalazine.
In some examples, the multi-specific growth factor trap constructs described herein are administered with one or more cytokines. Examples of cytokines include, but are not limited to, lymphokines, monokines, and traditional polypeptide hormones. Cytokines include interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 and IL-15; tumor necrosis factors, such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL).
In some examples, the multi-specific growth factor trap constructs described herein are administered with one or more cytokines or other agents that stimulate cells of the immune system and enhance the desired effector function. For example, agents that stimulate Natural Killer (NK) cells, including but not limited to IL-2, may be administered with the multi-specific growth factor trap constructs described herein. In another embodiment, a macrophage stimulating agent formulation including, but not limited to, C5a and a formyl peptide, such as N-formyl-methionyl-leucyl-phenylalanine (see, e.g., beigier-Bompadre et al (2003) Scan.J.Immunol.57:221-228) may be administered with the multispecific growth factor trap constructs described herein. Formulations that stimulate neutrophils, including but not limited to G-CSF and GM-CSF, may also be administered with the multi-specific growth factor trap constructs described herein. Formulations that promote migration of such immunostimulatory cytokines may be administered with the multispecific growth factor trap constructs described herein. Other agents include, but are not limited to, interferon gamma, IL-3, and IL-7, which may promote one or more effector functions. In some examples, the multi-specific growth factor trap constructs described herein are administered with one or more cytokines or other agents that inhibit effector cell function.
In some examples, the multi-specific growth factor trap constructs described herein are administered with one or more antibiotics, including but not limited to: aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycin (bambermycin), butirudin, dibekacin, gentamycin (gentamicin), kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin); aminocyclools (e.g., spectinomycin), aminophenols (e.g., azidothalamycins (azidamfenicol), chloramphenicol (chloramanthenol), florfenicol (florfenicol) and thiamphenicol)), ansamycins (e.g., li Fumi t (rifamide) and rifampin (rifampin)), carbapenems (e.g., imipenem (imipenem), meropenem (meropenem) and panipenem (panipeem)); cephalosporins (e.g. cefaclor), cefadroxil, cefamandole, ceftriaxone, cefazedone, cefazolin, ceftizopran, ceftizoxime, cefpiramide, cefpirome, cefprozil, cefuroxime, cefalexin, and cefradine); cephalosporins (e.g. cefbuperazone, cefoxitin, cefminox, cefmetazole and cefotetan); lincomides (e.g., clindamycin and lincomycin); macrolides (such as azithromycin (azithromycin), brefeldin (brefeldin) a, clarithromycin (clarithromycin), erythromycin (roxithromycin), roxithromycin (roxithromycin) and tobramycin); monolactones (e.g., aztreonam (aztreonam), card Lu Mona (carumonam), and tigemonam); mupirocin (); oxacephems (e.g., floxacef, latamoxef, and moxalatam); penicillins (e.g., ampicillin (amdinocillin), pimicillin (amdinocillin pivoxil), amoxicillin (amoxicillin), baccaracillin (bacampicillin), benicillin acid (benzylpenicillinic acid), sodium benzyl penicillin (benzylpenicillin sodium), epicicillin (epicicillin), fen Bei Xilin (fenbenicillin), fluxacillin (floxacillin), pencicillin (pencicillin), hydroiodic acid sandbicillin (penethamate hydriodide), pencicillin (pencicillin O-bennethamine), penicillin O, penicillin V benzoate, hydrabamillin V (penicillin V hydrabamine), penciclin (pencicillin), and non-nesilk (phenethicillin potassium)); polypeptides (e.g., bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin); quinolones (e.g., amifloxacin, ciprofloxacin, enoxacin, enrofloxacin, ofloxacin, fluroxacin, fluquindox, gatifloxacin, gemifloxacin, lattice Lei Pasha star, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, pirafloxacin, paxifloxacin, and tolvafloxacin); rifampin; streptogramins (e.g., quinupristin and dalfopritin); sulfonamides (e.g., sulfa and sulfamethoxazole); and tetracyclines (for example, chlortetracycline (chlortetracycline), norchlortetracycline hydrochloride (demeclocycline hydrochloride), norchlortetracycline (demethylcycline), doxycycline (doxycycline), duramycin (Duramycin), minocycline (minocycline), neomycin (neomycin), oxytetracycline (oxytetracycline), streptomycin (streptomycin), tetracycline (tetracycline), and vancomycin).
In some examples, the multi-specific growth factor trap constructs provided herein are administered with one or more antifungal agents, including, but not limited to, amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine (terbinafine), terconazole, and tioconazole.
In some examples, the multispecific growth factor trap constructs described herein are administered with one or more antiviral agents, including, but not limited to, protease inhibitors, reverse transcriptase inhibitors, and the like, including type I interferon, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir (adefovir), amantadine, amprenavir (amprenavir), clavudine (clevudine), enfuvirtide, entecavir (entecavir), foscarnet (foscarnet), ganciclovir (ganciclovir), idouride (idoxuridine), indinavir (indinavir), lopinavir (lopinavir), plica (pleconaril), ribavirin (ribavirin), rimantadine (ritonavir), ritonavir (ritonavir), saquinavir (fludrodine), and vidarabine (vidone).
The multi-specific growth factor trap constructs provided herein may be combined with other therapeutic regimens. For example, in one embodiment, a patient to be treated with a multi-specific growth factor trap construct provided herein may receive radiation therapy. Radiation therapy may be performed according to protocols commonly used in the art and known to those skilled in the art. Such therapies include, but are not limited to, cesium, iridium, iodine, or cobalt radiation. Radiation therapy may be whole body irradiation or may be directed locally to a specific site or tissue in or on the body surface, such as the lung, bladder or prostate. Radiation therapy may also include treatment with isotopically labeled molecules, such as antibodies. Examples of radioimmunotherapeutic agents include those under the trademark(Y-90 labeled anti-CD 20), -A. About.>(Y-90 labeled anti-CD 22) and +.>(I-131 labeled anti-CD 20) those formulations sold.
Typically, radiation therapy is administered in pulses over a period of about 1 to 2 weeks. However, radiation therapy may be performed for a longer period of time. For example, radiation therapy may be administered to a patient with head and neck cancer for about 6 to about 7 weeks. Optionally, radiation therapy may be administered in a single dose or in multiple sequential doses. The appropriate radiation therapy dosage for use herein can be determined empirically by the skilled medical practitioner. In some examples, the multispecific growth factor trap construct and optionally one or more other anti-cancer therapies are used to treat cancer cells ex vivo. Such ex vivo treatments are expected to be useful in bone marrow transplants, particularly autologous bone marrow transplants. For example, treatment of a cell or tissue containing cancer cells with a multi-specific growth factor trap construct and one or more anti-cancer therapies, as described herein, may be used to eliminate or substantially eliminate cancer cells prior to implantation in a recipient patient.
Furthermore, it is contemplated that the multispecific growth factor trap constructs provided herein can be administered to a patient or subject in combination with other therapeutic techniques, such as surgery or phototherapy.
For example, provided herein are methods of treating cancer by co-administering any of the multi-specific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein, and another anti-cancer agent. The anticancer agent may include radiation and/or chemotherapy agents. For example, the anticancer agent may be a tyrosine kinase inhibitor or an antibody. Exemplary anti-cancer agents include quinazoline kinase inhibitors, antisense or siRNA or other double stranded RNA molecules, antibodies that interact with HER family receptors, and antibodies conjugated to radionuclides or cytotoxins. Other exemplary anticancer agents include gefitinib, lapatinib, erlotinib, panitumumab, cetuximab, trastuzumab, imatinib, platinum complexes, or nucleoside analogs. Examples of cytotoxic or chemotherapeutic agents include, for example, taxanes (e.g., paclitaxel and docetaxel) and anthracyclines, doxorubicin/doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, mitomycin C, porphyrin mycin, 5-fluorouracil, 6-mercaptopurine, cytarabine, podophyllotoxin or podophyllotoxin derivatives, such as etoposide or etoposide, melphalan, vinblastine, vincristine, vindesine, vinblastine, maytansine, epothilone a or B, taxotere, paclitaxel, estramustine, cisplatin, compstatin (combretastatin) and the like, and cyclophosphamide. Any other anti-cancer antibodies and chemotherapeutic agents described elsewhere herein or known in the art are also contemplated for use in treating cancer in combination with the multi-specific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein.
In another example, provided herein is a method of treating Rheumatoid Arthritis (RA) by administering any of the multi-specific growth factor trap constructs, nucleic acid molecules, or pharmaceutical compositions provided herein in combination with another antirheumatic agent, such as an anti-TNF therapy. Can be combined with the polyteres provided hereinExamples of anti-TNF therapies used in combination with a specific growth factor trap construct, nucleic acid molecule or pharmaceutical composition include conventionally synthesized DMARDs, such as Methotrexate (MTX), hydroxychloroquine (HCQ;) Sulfasalazine (+)>) And leflunomide (>) The method comprises the steps of carrying out a first treatment on the surface of the Biological DMARDs, e.g. Abacalcet (+)>) Anakinra (/ -A)>) Rituximab (+)> ) Tositumumab (tositumumab,/-for)>) Corticosteroids (e.g., dexamethasone, methylprednisolone, prednisolone, prednisone, or triamcinolone), tofacitinib (>) And TNF inhibitors/anti-TNF agents, such as polyethylene glycol cetuximab (++>) Infliximab (++>) Adalimumab() Golimumab (+)>) And etanercept ()>). Combination therapies may also include immunotherapeutic agents such as cyclosporin, methotrexate, doxorubicin or cisplatin, as well as immunotoxins.
Also provided herein is a method of treating chronic inflammatory, autoimmune, neurodegenerative and/or demyelinating diseases as described elsewhere herein, particularly RA, by administering any of the multispecific growth factor trap constructs described herein and any of the TNFR1 antagonists, TNFR2 agonists or bispecific TNFR1 antagonist/TNFR 2 agonist constructs provided herein. Optionally, additional anti-TNF therapies, such as methotrexate, or any of the therapies described above or elsewhere herein or known in the art, may be any other therapies useful in the treatment of chronic inflammatory, autoimmune, neurodegenerative, and/or demyelinating diseases, such as immunosuppressants, anti-angiogenic agents, cardioprotective agents, antibodies, cytotoxic agents, anti-inflammatory agents, cytokines, growth inhibitors, chemotherapeutic agents, biological or non-biological disease-modifying antirheumatic drugs (DMARDs), infectious disease therapeutic agents (including antibodies), or other suitable therapeutic agents described herein or known in the art.
Angiogenesis plays a key role in the formation and maintenance of RA pannus. The multi-specific growth factor trap constructs provided herein may be used in combination with other therapies to modulate angiogenesis. For example, an angiogenesis inhibitor may be used in combination with the multi-specific growth factor trap constructs provided herein to treat RA. Exemplary angiogenesis inhibitors include, but are not limited to, angiostatin, anti-angiogenic antithrombin III, angiostatin, cartilage derived inhibitors, fibronectin fragments, IL-12, angiostatin, and other formulations known in the art and described elsewhere herein.
In some embodiments, the growth factor trap constructs provided herein are used in combination with a TNF blocker and/or other DMARDs, such as methotrexate, and compared to standard RA therapies. For example, the growth factor trap constructs provided herein may be combined with etanercept and/or methotrexate, e.g., suboptimal doses of etanercept and/or methotrexate. To assess efficacy, the combination may be an etanercept and/or methotrexate monotherapy, including optimal and suboptimal doses of etanercept and/or methotrexate. The growth factor trap construct allows for lower doses of other treatments, thereby reducing adverse or undesirable side effects. In other embodiments, the growth factor trap constructs provided herein may be combined with other anti-TNF therapies, such as adalimumab or infliximab (including suboptimal doses), with or without methotrexate (including suboptimal doses of methotrexate), and the therapeutic efficacy is compared to anti-TNF therapies alone with or without methotrexate. In yet another embodiment, the growth factor trap constructs provided herein may be combined with any of the TNFR1 antagonists, TNFR2 agonists, or multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs provided herein, and compared to standard RA therapies, e.g., etanercept, adalimumab, or infliximab treatment with or without methotrexate.
H. Assessment of TNFR1 antagonists and TNFR1 antagonist/TNFR 2 agonist construct Activity and efficacy
If or when necessary, the activity and efficacy of the constructs provided herein can be assessed using any in vivo and/or in vitro assay known to those of skill in the art to assess the nature of the construct and/or the suitability for treating a particular disease, disorder or condition. These assays may also be used to monitor treatment and/or predict treatment response or select a subject for treatment. Exemplary assays are described in the following sections.
In general, the antagonist constructs herein are non-competitive; they are typically constructs that lock the receptor in an inactive conformation, which, as described above, means that selecting for high affinity is less important than selecting for antagonist activity.
1. Disease Activity score (DAS 28)
The disease activity score for 28 joint counts (DAS 28; or disease activity score for 28 joints) is a measure of Rheumatoid Arthritis (RA) disease activity and is a simplification of the original DAS score, which requires the count of 44 joints. The 28 joints counted included proximal interphalangeal joints (10 joints), metacarpophalangeal joints (10 joints), wrist joints (2), elbow joints (2), shoulder joints (2), and knee joints (2). DAS28 is indicative of RA disease activity and response to treatment and is therefore used in clinical trials to evaluate RA therapeutics. DAS28 scores a range of 0-10 based on a count of 28 swollen and tender joints, with higher values indicating higher disease activity. In addition to counting the number of swollen and tender joints (28 total), DAS28 also included measuring Erythrocyte Sedimentation Rate (ESR) or C-reactive protein (CRP), which are acute phase reactants/blood markers of inflammation, and General Health (GH) assessments, representing the patient's self-assessment of disease activity, scored on the 100mm Visual Analog Scale (VAS), 0 for "no activity" and 100 for "highest activity possible. "DAS28 is typically combined with other disease severity measurements, such as pain and grip strength, and uses the Health Assessment Questionnaire (HAQ) to assess bodily functions.
To calculate DAS28 values using ESR or CRP levels, the following formulas are used, respectively:
DAS28(ESR)＝0.56x√(TJC28)+0.28x√(SJC28)+0.014x GH+0.70x ln(ESR)；
DAS28(CRP)＝0.56x√(TJC28)+0.28x√(SJC28)+0.014x GH+0.36x ln(CRP+1)+0.96；
TJC = tender joint count, SJC = swollen joint count.
The value <2.6 indicates remission, +.3.2 (> 2.6 but+.3.2) indicates low disease activity, >3.2 but+.5.1 indicates medium disease activity, greater than 5.1 indicates high disease activity (i.e., active disease). An improvement of >1.2 (i.e. a decrease in DAS28 fraction/value) indicates a good reaction/improvement; an improvement of >0.6 to ∈1.2 indicates a moderate reaction; DAS28 drop of 0.6 or less indicates no improvement (see, e.g., prevoo et al (1995) Arthritis & Rheumatism 38 (1): 44-48; wells et al (2009) Ann. Rheum. Dis.68: 954-960).
The therapeutic efficacy of the selective TNFR1 antagonists, TNFR2 agonists, and/or bispecific constructs comprising combinations thereof provided herein can be assessed by calculating DAS28 (ESR) or DAS28 (CRP) before, during, and after treatment.
2.Proteomic analysis and other proteomic tools for quantifying analytes
Proteomic assays (inc.; boulder, co.) are multiple, sensitive, quantitative and reproducible proteomic tools based on aptamers that can simultaneously measure the amount of more than 5,000 protein analytes in a sample, such as serum, plasma or cerebrospinal fluid, which is small in volume, e.g., 150 μl. Other biological matrices, such as cell culture supernatants, cell and tissue lysates, synovial fluid, bronchoalveolar and nasal lavage, can also be used. Since a broad range of protein targets can be quantified simultaneously +. >Assays have been optimized for the discovery of protein biomarkers and have been used to identify biomarker signatures associated with a variety of diseases, such as non-small cell lung cancer, alzheimer's disease, cardiovascular disease, and inflammatory bowel disease. By analyzing samples taken before and after the start of treatment with the TNFR1 antagonist, TNFR2 agonist and bispecific construct provided herein, < >>Assays can be used to determine protein characteristics of, for example, RA patients. In this way, the patient's response can be monitored at an early point in time during the treatment and throughout the course of the treatment.
The assay employs protein capture reagents, termed slow off-rate modified aptamers (in order toAptamer format) reagents, which are short, single-stranded DNA-based protein affinity reagents, are constructed from chemically modified nucleotides that mimic amino acid chains, have slow off-rates, and allow specific, high affinity binding to protein targets. The assay measures proteins in the native folded conformation (i.e., tertiary structure) and does not detect unfolded and denatured (i.e., inactive) proteins. For->Determination of->The protein binding step is followed by a series of partitioning and washing steps, whereby by converting each individual protein concentration into the corresponding +. >Reagent concentration (based onDNA signal) and then quantified by standard DNA detection techniques (e.g., microarray or qPCR). The assay takes advantage of the dual nature of the SOMAmer reagent, namely, the protein affinity binding reagent having a defined three-dimensional structure, and the unique nucleotide sequence that is recognized by a particular DNA hybridization probe.
The reagent is equipped with three tags and contains one fluorophore linked to biotin via a photocleavable linker. Briefly, for assays, a biological sample of interest is diluted, thenAfter that, the corresponding ++to pre-immobilized on Streptavidin (SA) coated beads>The reagent mixture is incubated together.The reagent binds to proteins in the biological sample and the beads are then washed to remove unbound proteins. Any nonspecific complexes formed have a rapid dissociation rate. Labelling with NHS-biotin reagent still homologous thereto +.>Reagent-bound proteins, and adding a polyanionic competition solution to break down any non-specific complexes. Protein- & lt- & gt>Complex and unbound (free)The reagent is released from the streptavidin beads by cleavage of the photocleavable linker using ultraviolet light. Then will contain all->Photocleavable eluate of reagents (some bound to biotin-labeled protein and some free) and binding biotinylated protein and biotinylated protein-/-for the reagent >The second streptavidin-coated bead of the complex is incubated and unbound material is removed by a subsequent washing step. In the final elution step, protein-bound +.>The agent is released from its cognate protein under denaturing conditions>Reagents are quantified by standard DNA quantification techniques, for example by hybridization to custom DNA microarrays and measurement of fluorophore tags. After normalization and calibration, the data are reported in Relative Fluorescence Units (RFU) and measured +.>The reagent signal correlates with the protein level found in the biological sample (see, e.g., gold et al (2010) PLoS ONE 5 (12): e15004; candia et al (2017) Sci. Reports 7:14248;Tanaka et al. (2018) Aging cell.17: e 12799).
3. Transcriptome analysis to predict responsiveness to treatment and select subjects likely to benefit from treatment
Conventional anti-TNF therapies, i.e., TNF blockers such as etanercept, infliximab, etc., experience about 30% of the non-response in RA patients. However, there are several clinical markers that can predict the efficacy of these anti-TNF therapies. Genes differentially expressed following anti-TNF treatment with etanercept have been analyzed using global transcriptome analysis to determine RNA expression profile in Peripheral Blood Mononuclear Cells (PBMCs). Similar transcriptome analyses can be performed to assess the efficacy of the TNFR1 antagonists and TNFR1 antagonist/TNFR 2 agonist constructs herein, and/or to assess the responsiveness of the patient to them.
In an exemplary protocol, blood samples are obtained from a patient before and after treatment, and PBMC are isolated using Ficoll density gradient, followed by assessment of CD3 using flow cytometry + 、CD14 + 、CD19 + And CD56 + A population of cells. Total RNA is then extracted, e.g., using QiagenKit, and using microarray analysis (e.g., using +.>Microarray technology) to analyze the expression profile of tens of thousands of known genes in PBMCs to identify the gene should beExpression profile of respondents/non-respondents. Gene expression profiles can be determined early in the treatment to rapidly identify those non-responders. For example, by using this method to identify reliable biomarkers to predict therapeutic effects of etanercept in RA patients, prediction accuracy was identified>89% gene pair and prediction accuracy>95% of gene triplets. These include, for example, genes involved in TNF signaling through the NF-kappa B pathway, genes involved in NF-kappa B independent signaling, and genes involved in regulating cellular and oxidative stress responses. For example, the identified gene triplets include TNFAIP3, which encodes TNFaIP-induced protein 3, a zinc finger protein, shown to inhibit NF-. Kappa.B activation; PDE4B, which encodes a cyclic nucleotide phosphodiesterase specific for cAMP involved in NF-. Kappa.B independent signaling; and RAPGEF1, which encodes Rap guanine nucleotide exchange factor 1, an activator of RAS signaling. Expression of all three genes was down-regulated in responders after etanercept administration for 3 days compared to non-responders. Other genes evaluated include CCL4, CXCR4, CCL3, PIGO, FSD1, RUNX1, LGALS13, PTPRD, IL1B, ADAM, and HCG4P6 (see, e.g., koczan et al (2008) Arthritis Research) &Therapy 10:R50)。
The expression level of a subset of genes of interest can be measured by quantitative real-time PCR (RT-PCR) using pre-designed primers and probes to verify the results obtained by microarray analysis. To calculate the change in gene expression of a selected gene, a ΔΔct method can be used whereby the threshold Cycle (CT) value of specific mRNA expression in the sample is normalized to the CT value of GAPDH mRNA in the sample, and the change in gene expression (ΔΔct) is defined by the difference in CT values before and after treatment (see, e.g., koczan et al (2008) Arthritis Research & therapeutic 10: r 50).
L929 cytotoxicity assay
TNFR1 mediated processes and cellular responses can be determined, for example, by assessing TNF-induced cell death using an L929 cytotoxicity assay, whereby TNFR1 antagonists inhibit TNF-induced cytotoxicity. Briefly, L929 mouse fibroblasts were plated on microtiter plates and incubated overnight with TNFR1 antagonist, 100pg/ml TNF, and 1mg/ml actinomycin D. Cell viability was measured by reading absorbance at 490nm after incubation with 3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS). TNFR1 antagonists reduced TNF-mediated cytotoxicity compared to controls using TNF alone, resulting in increased absorbance.
HeLa IL-8 assay
The activity of TNFR1 antagonists can be determined using a HeLa IL-8 assay in which the ability of the antagonists to neutralize TNF-induced IL-8 secretion in HeLa cells is assessed. Briefly, heLa cells were plated on microtiter plates overnight in the presence of varying concentrations of TNFR1 antagonist and 300pg/ml TNF. The supernatant was then aspirated and the concentration of IL-8 was measured using a sandwich ELISA. TNFR1 antagonist activity reduced secretion of IL-8 into the supernatant compared to a control administered TNF alone.
HUVEC assay
Human Umbilical Vein Endothelial Cell (HUVEC) assays can be used to determine the activity of the TNFR1 antagonists herein. Treatment of HUVECs with TNF results in upregulation of VCAM-1 expression on the cells, as can be determined by ELISA, for example. Because TNFR1 antagonists inhibit the effects of TNF, VCAM-1 expression in HUVEC is reduced in the presence of the antagonist. The level of inhibition of TNF-induced VCAM-1 expression by a TNFR1 antagonist is then determined by plotting the concentration of the antagonist against the percent inhibition of VCAM-1 expression.
According to this assay protocol, HUVECs were incubated overnight, then incubated with TNFR1 antagonists for 1 hour, then stimulated with TNF (1 ng/mL) for 23 hours. Cells incubated with medium only served as negative control and cells incubated with TNF only served as positive control. The cell culture supernatant was then aspirated, the cells were washed 3 times with ice-cold PBS and washed with ice-cold Tris-glycerol lysis buffer (40mM Tris,274mM NaCl,2% Triton-X-100, 20% glycerol, 50mM NaF,1mM Na 3 VO 4 1x protease inhibitor tablets per 10 ml) and then incubated on ice for 15 minutes. Cell lysates were then used in a VCAM-1 sandwich ELISA. To calculate the inhibition of TNF-induced VCAM-1 expression, the percent inhibition of maximum VCAM-1 expression (i.e., in the sunMeasured in sexual control) =
{100- [ (OD value of antagonist concentration)/(OD value of positive control) ] } x 100.
EC was then determined by plotting antagonist concentration versus percent inhibition 50 Values, for example, use available software such as GraphPad Prism software.
Quantification and assessment of Treg cell Activity
To determine the effect of TNFR1 antagonists and TNFR1 antagonist/TNFR 2 agonist constructs on tregs, the number of tregs can be quantified by isolating Peripheral Blood Mononuclear Cells (PBMCs) from blood samples before and after treatment as well as during treatment, e.g., by using Ficoll-Paque method followed by monoclonal antibodies (mabs) and paramagnetic beads, or other similar methods known in the art, to isolate CD4 + CD25 + Treg and CD4 + CD25 - Non-regulatory T cells. The number of each cell type can be quantified using flow cytometry and mAb immunostaining for CD4 and CD25 (for Treg) or CD4 and CTLA-4 (for non-regulatory T cells) (see, e.g., vigna-Prez et al (2005) Clin. Exp. Immunol.141 (2): 372-380).
CD4 + CD25 + Treg inhibits CD4 + CD25 - Proliferation of T cells. To test Treg activity in a patient receiving treatment, a cell proliferation assay may be used in which Treg and T cells are incubated with phytohemagglutinin (PHA to stimulate T cells) for 48 hours. 3H-TdR (tritiated thymidine) was added during the last 12 hours of incubation, and the cells were harvested and proliferation was measured using a liquid scintillation counter. Individually cultured CD4 + CD25 - T cells served as a control and the results were expressed as a cell proliferation Stimulation Index (SI) calculated as:
si= (cpm of PHA-containing cells) where cpm is the count per minute, determined by the radioactivity counted (see e.g. Vigna-purez et al (2005) clin.exp.immunol.141 (2): 372-380).
To test for immune responsiveness against mycobacterium tuberculosis (m.tuberculosis), PBMCs were cultured in complete medium in the presence of whole protein extracts of bacteria for 72 hours. 3H-TdR (tritiated thymidine) was added during the last 12 hours of incubation, and the cells were harvested and proliferation was measured using a liquid scintillation counter. As described above, the results are expressed in terms of stimulation index. For in vivo reactivity against Mycobacterium tuberculosis, standard PPD (purified protein derivatives) skin assays can be used (see, e.g., vigna-Prez et al (2005) Clin. Exp. Immunol.141 (2): 372-380).
Assessment of binding Properties of TNFR1 antagonist/TNFR 2 agonist constructs
Specific binding of an antibody or antibody fragment or multispecific construct, such as the constructs provided herein, to TNFR1 and/or TNFR2, e.g., human TNFR1 and/or TNFR2, can be assessed by any of a variety of known methods. Affinity can be quantified by various indicators, including achieving half maximal enhancement of TNFR1 and/or TNFR2 signaling in vitro (EC 50 ) The concentration of the desired TNFR1 antagonist, TNFR2 agonist or multispecific construct, and the equilibrium constant (KD) for dissociation of the antagonist-TNFR 1 and/or agonist-TNFR 2 complex. The equilibrium constant KD describes the interaction of TNFR1 or TNFR2 with a binding agent such as a construct (binding agent) provided herein, which is the chemical equilibrium constant of TNFR 1-binding agent construct complex or TNFR 2-binding agent complex dissociation reaction into solvent-separated TNFR1 or TNFR2 and binding agent molecules that do not interact with each other.
TNFR1 antagonists, TNFR2 agonists and multispecific constructs can also be identified by various in vitro binding assays. Can be used for determining KD or EC 50 Examples of experiments include, for example, surface plasmon resonance (SPR, e.g., BIAcore TM Analysis), isothermal titration calorimetry, fluorescence anisotropy, ELISA-based assays, and the like. ELISA is a particularly useful method of assaying antibody activity because such assays typically require a minimum concentration of antibody. One common signal analyzed in a typical ELISA assay is luminescence, which is typically the result of secondary antibody activity of a peroxidase coupled to a specific binding primary antibody (e.g., a TNFR1 antagonist or a TNFR1 agonist TNFR2 agonist bispecific construct provided herein).
The association and dissociation kinetics of the TNFR1 antagonist with TNFR1 or TNFR2 agonist with TNFR2 can be quantitatively identified, for example, by monitoring the rate of antibody-antigen complex formation according to a pre-program. For example, surface Plasmon Resonance (SPR) can be used to determine the rate constants for formation (kon) and dissociation (koff) of the antagonist-TNFR 1 or agonist-TNFR 2 complex. From these data, the equilibrium constant (KD) can be determined, as the equilibrium constant for such single molecule dissociation can be expressed as the ratio of koff to kon values. SPR is a technique that facilitates determining kinetic and thermodynamic parameters of receptor-antibody (or other binding agent) interactions, as the assay does not require modification of a module by the addition of chemical tags. In contrast, the receptor is typically immobilized on a solid metal surface that is pulsed with a solution of an increasing concentration of an antibody or binding agent (i.e., a TNFR1 antagonist or TNFR2 agonist, or a bispecific construct thereof). Antibody-receptor binding results in a deformation of the angle of reflection of incident light at the metal surface, and the change in refractive index over time as the antibody is introduced into the system can be matched to an established regression model known in the art to calculate the binding and dissociation rate constants for antibody-receptor interactions.
9. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays
Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays can be used to assess immune effector function/cytotoxicity of the TNFR1 antagonists, TNFR2 agonists, and Fc monomer or dimer-containing multispecific constructs provided herein. In general, the Fc portion is modified to eliminate or significantly reduce (to eliminate or reduce to tolerable levels) ADCC or ADCC and CDC effector function. Such assays are well known in the art (see, e.g., ying et al (2014) mAbs 6 (5): 1201-1210). For example, for an exemplary ADCC assay, mesothelin negative a431 or mesothelin positive H9 cells are incubated with a TNFR1 antagonist, TNFR2 agonist, or multispecific construct provided herein for 30 minutes, and then target cells are added to wells containing effector cells (e.g., PBMCs) at a ratio of 50:1 effector cells to target cells. After 24 hours of incubation, target cell lysis was measured using a CytoTox-ONE homogeneous membrane integrity assay (Promega) according to the manufacturer's protocol.
For the exemplary CDC assay, a431 and H9 cells were washed in serum-free RPMI and the density was adjusted to 1 million/mL in serum-free RPMI. mu.L of the cell suspension was then incubated with 50. Mu.L of TNFR1 antagonist, TNFR2 agonist or multispecific construct dilution in RPMI. The negative control contained 50. Mu.L of cell suspension and 50. Mu.L of RPMI, and the positive control contained target cells lysed with 1% Triton X-100, with a final volume of 150. Mu.L. Fresh human plasma was diluted in PBS (1:4) and clarified by centrifugation, then 50. Mu.L of diluted plasma was added to each cell/construct mixture and incubated in 96-well plates at 37℃to allow complement mediated cell lysis. After 3 hours of incubation, 100. Mu.L of supernatant was transferred to a whiteboard and 100. Mu.L of substrate from the Cytotox-ONE homogeneous membrane integrity assay kit (Promega) was added. The plates were then incubated at room temperature for 10 minutes and the fluorescent signal was read using a fluorometer with excitation wavelength of 530nm and emission wavelength of 590nm. CDC of target cells is expressed as a percentage of test sample to positive control.
10. Disease model
The selective TNFR1 antagonists, TNFR2 agonists, and multispecific constructs provided herein can be evaluated in any clinically relevant disease model known to those skilled in the art to determine their effects on autoimmunity and inflammation, as well as other diseases or disorders mediated by TNF or involved in its etiology. Exemplary disease models include, but ARE not limited to, collagen-induced arthritis (CIA), rheumatoid arthritis synovial mononuclear cell cultures, tg197 arthritis mouse model, Δare arthritis/IBD mouse model, mouse Dextran Sodium Sulfate (DSS) induced IBD model, and Experimental Autoimmune Encephalomyelitis (EAE) model for multiple sclerosis. Other models are known to those skilled in the art. See, e.g., malaviya et al (2017) Pharmacol Ther.180:90-98, which provides a number of models to test constructs for the treatment of inflammatory lung disease; feldmann et al (2020) Lancet 395:1407-1409, anti-TNF therapies, models and use of therapies for COVID-19; shi et al (2013) crit. Care 17 (6): R301, treatment of H1N1 with anti-TNF therapies, and viral infection models; to be used forAnd Orti-Casan et al (2019) front. Neurosci.13:49, which describes that activating TNFR2 for the treatment of alzheimer's disease is advantageous, demonstrating that the methods herein inhibit TNF blockers of TNFR1 and TNFR2 are problematic. The following is a non-exhaustive discussion of diseases, disorders, and conditions that can be treated with the constructs provided herein, as well as an exemplary model of each disease. These are exemplary; the skilled artisan can select an appropriate model for a particular construct and targeted disease, disorder, or condition. Since the anti-TNFR 1 and TNFR2 antagonist/agonist constructs provided herein are intended for targeting human TNFR1/TNFR2, they are expected to be poorly effective in reacting/interacting with TNFR1/TNFR2 from non-human, particularly non-primate animals. For testing, in a non-human model, such as a rodent model, the model is, for example, a transgenic model of human TNFR1 and human TNFR 2. They can be used as in vivo models of inflammatory and autoimmune diseases in the context of mouse TNFR1/2 knockout mice. Alternatively, human CD34 is transplanted + Severely immunocompromised mice (e.g., NOD/NSG mice) of stem cells can be used for this purpose. Alternatively, human rheumatoid arthritis synovial cells can be transplanted into immunodeficient mice, resulting in RA-like inflammation (see, e.g., schinnerling et al (2019) Front immunol.10:203 for a description of this model).
a. Collagen-induced arthritis (CIA)
Type II collagen-induced arthritis (CIA) can be induced in mice as a model of autoimmune inflammatory joint disease that is histologically similar to RA, characterized by inflammatory synovitis, pannus formation, and erosion of cartilage and bone. To induce CIA, bovine type II collagen (B-CII) was injected intradermally into the caudal root in the presence of complete freund's adjuvant. After 21 days, mice can be re-immunized using the same protocol. To examine the effects of the selective TNFR1 antagonists, TNFR2 agonists, and multispecific constructs provided herein, the selective TNFR1 antagonists, TNFR2 agonists, or multispecific constructs, or controls, can be administered intraperitoneally for 3 weeks after initial challenge with B-CII, or at the occurrence of signs of arthritis. Mice were sacrificed 7 weeks after primary immunization for histological analysis.
To assess the effect of the construct on establishing treatment of the disease, it may be administered daily for a total of 10 days after the occurrence of clinical arthritis in one or more limbs. The extent of swelling of the initially affected joint can be monitored by measuring the paw thickness using calipers. Serum may be drawn from mice for measurement of pro-inflammatory cytokines and chemokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), IL-1β, IL-6, IL-8, RANTES (CCL 5), and monocyte chemotactic protein 1 (MCP-1; also known as CCL 2).
In another example, primate models can be used for RA treatment. The response of tenderness and swollen joints (e.g., as measured by clinical arthritis scoring) can be monitored in subjects and controls treated with recombinant therapeutic TNFR1 antagonists, TNFR2 agonists, or bispecific constructs to assess efficacy and treatment.
b. Rheumatoid arthritis synovial mononuclear cell culture
Human Rheumatoid Arthritis (RA) synovial Monocytes (MNCs) expressing TNFR1 and TNFR2 can also be used to test the therapeutic efficacy of the constructs provided herein. RA synovial MNCs may be obtained from RA patients undergoing joint replacement surgery and cultured in vitro to assess RA synovial cytokine production and modulation. In the absence of exogenous stimuli, RA synovial MNC cultures spontaneously produce inflammatory cytokines and chemokines; antibody-mediated TNF neutralization and selective blocking of TNFR1 in these cultures (e.g., by constructs provided herein) inhibits pro-inflammatory cytokines and chemokines, such as GM-CSF, IL-10, IL-1β, IL-6, IL-8, RANTES (CCL 5), and MCP-1 (CCL 2).
In an exemplary assay, to prepare RA synovial MNC, RA synovial tissue was cut into small pieces, incubated with 5mg/ml collagenase a and 0.15mg/ml DNase in RPMI 1640 for 1 hour at 37 ℃, after which the digested tissue was passed through a 170 μm filter and washed 3 times with RPMI 1640 containing 100 units/ml streptomycin, 100 μg penicillin and 10% FCS. The unfiltered heterogeneous RA synovial MNC was then used. For in vitro cell culture, single cell suspension of RA synovium MNC is arranged on a 96-well flat bottom plate2×10 5 Individual cells/well) with 5% FCS at 37 ℃ and 5% CO in RPMI 1640 medium 2 Incubate for 2-5 days, with or without the TNFR1 antagonist, TNFR2 agonist or bispecific construct or control. The supernatant is then collected and used immediately, or stored at-20 ℃, for cytokine and chemokine ELISA analysis (see, e.g., schmidt et al (2013) archritis)&Rheumatism 65 (9): 2262-2273). Alternatively, cytokines in culture supernatants can be quantified by cytokine bead array analysis.
Tg197 mouse arthritis model
The Tg197 transgenic mouse strain is a model of erosive arthritis mice and a mature animal model of RA. Tg197 mice are human TNF transgenic C57BL/6 mice that overexpress human TNF and develop symmetric polyarthritis with pannus formation, bone destruction and cartilage damage, which are characteristic of human RA. In addition to showing the characteristics of chronic destructive joint disorders, this model also shows the characteristics of other inflammatory disorders such as spinal Arthritis, for example, adnexitis or bilateral sacroiliac Arthritis (Blulml et al (2010) architis) &Rheumatism 62 (6): 1608-1619). Tg197 mice develop arthritis with 100% exotic rate and provide a rapid in vivo model for evaluation of RA-targeted human therapeutics. For example, tg197 mouse model was used to evaluate infliximab (initially in the form ofSold), which is the first therapeutic effect successfully applied to clinical anti-TNF therapeutic drugs, and is recommended by the FDA for screening potential anti-RA therapeutic drugs.
Tg197 mice carry five copies of the human TNF gene construct, in which the 3 '-region containing the 3' -untranslated sequence and the 3 '-flanking sequence is exchanged with the 3' -region of the human β -globin gene. This gene construct was microinjected into mouse fertilized eggs, creating an in vivo model of deregulated TNF gene expression, as a set of highly conserved UA-rich sequences in the 3' -untranslated region of TNF mRNA are critical for mRNA stability and regulation of translation (see, e.g., keffer et al (1991) EMBO j.10 (13): 4025-4031).
d. Delta ARE mouse arthritis/IBD model
Mice deficient in AU-rich elements (AREs) in TNF mRNA (TnfΔAREs) overproduce TNF at 4-8 weeks of age and develop inflammatory bowel disease that is histopathologically similar to Crohn's disease. These mice also developed clinical symptoms of RA. The efficacy of the TNFR1 antagonists, TNFR2 agonists, and bispecific constructs provided herein can be assessed by assessing inhibition of crohn's disease-like pathology and arthritis in tnfΔare mice following intraperitoneal injection (see, e.g., U.S. patent No. 9,028,822).
e. Humanized TNF/TNFR2 mice
One limitation in developing therapeutic agents that target TNF/TNFR2 signaling pathways is the lack of preclinical animal models, as many human anti-TNF therapeutic agents do not interact with murine TNF or TNFR2, whereas human TNF can bind to and occupy murine TNFR1, but not TNFNR2. Humanized TNF/TNFR2 mice carrying functional human TNF-TNFR2 (hTNF-hTNFR 2) signaling modules can be used to evaluate therapeutic agents, such as agonist and antagonist antibody constructs of human TNF or human TNFR2, in various autoimmune models. Such TNF/TNFR2 double-humanized mice can be used, for example, to evaluate the TNFR2 agonist constructs provided herein.
Humanized TNF/TNFR2 mice can be produced as described by Atretkhany et al (2018) Proc.Natl. Acad.Sci.U.S. A.115 (51): 13051-13056. Briefly, human TNFR2 knock-in (hTNFR 2 KI) and human TNF knock-in (hTNFKI) mice were generated using standard genetic engineering techniques. hTNFKI mice in which the human TNF gene replaced the mouse TNF gene were then crossed with hTNFR2KI mice containing the humanized TNFR2 ligand binding portion and then interacted to produce double-humanized double homozygote hTNFKI x hTNFR2KI mice. To assess the role of TNFR2 signaling in specific cells, such as tregs, two LoxP sites were inserted within the hTNFR2 locus to allow conditional Cre-mediated extracellular partial elimination of TNFR 2. For Treg-specific deletions of TNFR2, these mice were crossed with FoxP3-Cre transgenic mice (see, e.g., atretkhanny et al (2018) proc.Natl. Acad. Sci.U.S.A.115 (51): 13051-13056).
I. Methods of producing nucleic acids encoding TNFR1 antagonist constructs and TNFR1 antagonist/TNFR 2 agonist constructs
The TNFR1 antagonist polypeptide, TNFR2 agonist polypeptide, and TNFR1 antagonist/TNFR 2 agonist polypeptide constructs provided herein are polypeptides that can be obtained by methods well known in the art for protein purification and recombinant protein expression, as well as recombinant antibody preparation. Constructs provided herein, including portions thereof such as linkers, are not polypeptides, and can be prepared by suitable chemical conjugation methods. The polypeptide portions may be produced by standard recombinant techniques, for example by expression in a suitable host (in bacteria if glycosylation is not required; eukaryotic cells such as HEK293 and CHO cells if glycosylated form is required). Active antibodies and antibody fragments have been produced in E.coli, but due to improper folding these antibodies and antibody fragments often suffer from aggregation and solubility problems, which can be solved by further mutagenesis of the coding sequence (see e.g.Kunz et al (2018) Sci Rep.8 (1): 7934).
The polypeptides may also be chemically synthesized. Fusion polypeptides may be synthesized by standard methods of recombinant production. The components of the various constructs discussed above may be synthesized separately and combined using standard methods to produce the construct.
Nucleic acids encoding the polypeptide constructs, or polypeptide portions thereof, including modified or variants, including truncated forms, may be prepared from the nucleic acids. Modified or variant polypeptides may be engineered from nucleic acids encoding wild-type polypeptides using standard recombinant DNA methods. For example, modified TNF polypeptides, such as TNF muteins, that selectively bind to TNFR1 or TNFR2 and/or selectively antagonize TNFR1 or selectively agonize TNFR2, can be engineered from wild-type TNF, such as by site-directed mutagenesis of the encoding DNA. Any method known to those skilled in the art may be used. The discussion and descriptions of the embodiments that follow are illustrative.
1. Isolation or preparation of nucleic acids encoding TNFR1 antagonists and TNRF2 agonist polypeptides
Any available method known in the art for cloning and isolating nucleic acid molecules can be used to clone or isolate nucleic acids encoding a TNFR1 antagonist polypeptide, a TNFR2 agonist polypeptide, and a TNFR1 antagonist/TNFR 2 agonist polypeptide construct. Such methods include Polymerase Chain Reaction (PCR) amplification of nucleic acids and library screening, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening. For example, when the polypeptide is produced recombinantly, any method known to those skilled in the art for identifying nucleic acids encoding the desired polypeptide may be used.
Nucleic acid molecules encoding the polypeptides herein can be synthetically produced, or can be readily isolated and sequenced as desired, using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding genes encoding the heavy and/or light chains of antibody fragments, such as single domain antibodies (dabs), scFv fragments, and Fab antibody fragments). For example, any cell source known to produce or express a TNFR1 antagonist or TNFR2 agonist antibody or fragment thereof can be used as a source of such DNA. In another example, once a DNA sequence encoding a TNFR1 antagonist or TNFR2 agonist antibody, or fragment thereof, is determined, a nucleic acid sequence can be constructed using genetic synthesis techniques.
Nucleic acid amplification methods can be used to isolate nucleic acid molecules encoding a desired polypeptide, including, for example, polymerase Chain Reaction (PCR) methods. Examples of such methods include the use of a Perkin-Elmer Cetus thermocycler and Taq polymerase (Gene Amp). Nucleic acid-containing materials can be used as starting materials from which the desired polypeptide-encoding nucleic acid molecule can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, liquid samples (e.g., blood, serum, and saliva), and samples from healthy and/or diseased subjects can be used in the amplification method. The source is typically from a human source and, if appropriate, may be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources. Nucleic acid libraries can also be used as a source of starting materials. Primers can be designed to amplify the desired polypeptide. For example, primers can be designed based on the expressed sequence that produces the desired polypeptide. Primers can be designed based on the back-translation of the polypeptide amino acid sequence. If desired, degenerate primers can be used for amplification. Oligonucleotide primers that hybridize to the 3 'and 5' terminal sequences of the desired sequence can be used as primers for amplifying sequences from a nucleic acid sample by PCR. Primers can be used to amplify the entire full-length polypeptide or truncated sequences thereof, e.g., nucleic acids encoding any of the TNFR1 antagonist and TNFR1 agonist polypeptides and TNFR1 antagonist/TNFR 2 agonist polypeptide constructs provided herein. Nucleic acid molecules produced by amplification can be sequenced and confirmed to encode the desired polypeptide or construct.
Mutagenesis techniques can be used to generate further modified forms of TNFR1 antagonists or TNFR2 agonist antibodies or fragments thereof and to generate modified forms of activity modulators, such as Fc and hinge regions, as well as linker moieties. The DNA may also be modified. For example, gene synthesis and conventional molecular biology techniques can be used to effect insertion, deletion, addition or substitution/substitution of nucleotides. Additional nucleotide sequences may be associated with the polypeptide-encoding nucleic acid molecule, including linker sequences comprising restriction endonuclease sites, in order to clone the synthetic gene into a vector, such as a protein expression vector, or a vector designed for amplification of the core polypeptide-encoding DNA sequence. Additional nucleotide sequences specifying functional DNA elements such as promoters, enhancers and IRES sequences may be operably linked to the polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to promote expression of a protein in a cell and secretion sequences designed to promote secretion of a protein, such as heterologous signal sequences. Such sequences are known to those skilled in the art. Additional nucleotide sequences, such as sequences specifying protein binding regions, may also be linked to the polypeptide-encoding nucleic acid molecule. Such regions include, but are not limited to, sequences that promote uptake of the polypeptide into a particular target cell, or otherwise alter or enhance the pharmacokinetics of the synthetic gene product.
Tags and/or other moieties may be added, for example, to aid in detection or affinity purification of the polypeptide. For example, additional nucleotide sequences, such as base sequences specifying epitope tags or other detectable labels, may also be linked to the polypeptide-encoding nucleic acid molecule. Examples of such sequences include nucleic acid sequences encoding SUMO tags or His tags or Flag tags.
It will be appreciated that any of the amino acid sequences provided herein can be reverse translated (also referred to as back-translated) using standard methods commonly used by those skilled in the art to generate corresponding coding nucleic acid sequences that can be cloned into vectors and expressed to produce constructs, including polypeptides, antibodies, and antibody fragments, provided herein. For example, there are several online tools available for converting protein sequences into coding DNA sequences, such as bioinformation. Org/sms2/rev_trans. bi ophp.org/minitools/protein_to_dna/demo.php; the vivo.colossate.edu/molkit/rtranslate/; ebi.ac. uk/Tools/st/emboss_backtraneq/; molbriol. Ru/eng/scripts/01_19.Html; and geneininfinity. Such reverse translated sequences may be inserted into any of the expression vectors provided herein to express and produce the provided antibodies or fragments. anti-TFR 1 and anti-TNFR 2 antibodies, such as TNFR1 antagonists and TNFR2 agonist constructs, may be expressed as full-length proteins or as proteins that are shorter than full length. For example, antibody fragments, such as, but not limited to, single domain antibodies (dabs), scFv fragments, and Fab fragments, can be expressed.
The identified and isolated nucleic acids can then be inserted into a suitable cloning vector. A wide variety of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, phages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or pBluescript vectors (Stratagene, la Jolla, CA). Insertion into a cloning vector may be accomplished, for example, by ligating a DNA fragment into a cloning vector having complementary sticky ends. Insertion can be achieved using the TOPO cloning vector (Invitrogen, carlsbad, CA).
If the complementary restriction sites for fragmenting the DNA are not present in the cloning vector, the ends of the DNA molecule may be enzymatically modified. Alternatively, any desired site can be created by ligating a nucleotide sequence (linker) to the end of the DNA; these ligated linkers may contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In another approach, the cleaved vector and polypeptide gene may be modified by homopolymeric tailing.
Recombinant molecules can be introduced into host cells by, for example, transformation, transfection, infection, electroporation, and sonication, to produce many copies of the gene sequence. In particular embodiments, transformation of a host cell with a recombinant DNA molecule incorporating an isolated polypeptide gene, cDNA, or synthetic DNA sequence is capable of producing multiple copies of the gene. Thus, the gene can be obtained in large amounts by culturing the transformant, isolating the recombinant DNA molecule from the transformant, and, if necessary, recovering the inserted gene from the isolated recombinant DNA.
For expression of antibodies and fragments thereof, nucleic acid molecules encoding the heavy chain of the antibody are typically cloned into vectors, and nucleic acid molecules encoding the light chain of the antibody are cloned into vectors. Methods for producing antibodies and portions thereof are well known (see, e.g., U.S. Pat. nos. 4,816,567, 6,331,415, and 7,923,221, and many other open-ended patents). The genes may be cloned into a single vector for dual expression thereof, or into separate vectors. If desired, the vector may also contain further sequences encoding additional constant or hinge regions to produce other antibody formats. The vector may be transfected and expressed in a host cell. Expression may be performed in any cellular expression system known to those of skill in the art. For example, host cells include cells that do not otherwise produce immunoglobulins to obtain synthesis of antibodies in the recombinant host cell. For example, host cells include, but are not limited to, simian COS cells, chinese Hamster Ovary (CHO) cells, such as CHO-DG44 (DHFR-) and FreeStyle TM CHO-S cells (Invitrogen), 293FS cells, HEK293 cells, NSO cells or other myeloma cells. Other expression vectors and host cells are described herein.
Constructs provided herein, including TNFR1 antagonists, TNFR2 agonists, and TNFR1 antagonist/TNFR 2 agonist constructs, can be produced or expressed as full length or less than full length constructs, including but not limited to antigen binding fragments, e.g., single domain antibodies (dAbs), fab ', fab hinge, F (ab') 2 Single chain Fv (scFv), scFv tandem, fv, dsFv, scFv hinge, scFv hinge (Δe), diabodies, fd and Fd' fragments. There are a variety of techniques available for the production of antibody fragments. Fragments can be obtained, for example, by proteolytic digestion of the intact antibody (see, e.g., morimoto and In ouye (1992) Journal of Biochemical and Biophysical Methods 24:107-117; brennan et al (1985) Science 229:81-83). Fragments may also be produced directly by recombinant host cells. For example, both dAb, fab, fv and scFv antibody fragments can be expressed and secreted in host cells (e.g., e.coli, CHO cells or HEK293 cells) to facilitate the production of these fragments in large quantities. F (ab') 2 Fragments may be produced by chemical conjugation of Fab' -SH fragments (see, e.g., carter et al (1992) Bio/Technology, 10:163-167), or they may be isolated directly from recombinant host cell cultures. In some examples, TNFR1 antagonist constructs include single domain antibodies (dAbs; described, for example, in International application publication Nos. WO 2004/058820, WO 2004/081026, WO 2005/035572, WO 2006/038027, WO 2007/049017, WO 2008/149720, WO 2008/14978, WO 2010/094720, WO 2011/006914, WO 2011/051217, WO 2012/172070, WO 2012/104322, and WO 2015/104322;Enever et al, (2015) Protein Engineering, design &Selection 28 (3): 59-66); U.S. application publication nos. 2006/0083747, 2010/0034831 and 2012/0107330; and U.S. patent nos. 9,028,817 and 9,028,822), single chain Fv fragments (scFv) (see, e.g., international application publication nos. WO 2017/174586 and WO 2008/113515; see also Richter, F.thesis, entitled "Evolution of the Antagonistic Tum or Necrosis Factor Receptor One-Specific Antibody ATROSAB," A. B "Stuttgart,2015; obtainable from pdfs.semmantischolar.org/d 8e7/8b87d76dce36225c1d 497939ef445cfaa8a.pdf), or Fab fragments (see for example international application publication nos. WO 2017/174586 and WO 2008/113515; see also Richte r, F.thesis, entitled "Evolution of the Antagonistic Tumor Necrosis Factor Receptor One-Specific Antibody ATROSAB,">Stuttgart,2015; available from pdfs. SemaThe ntiiscscho lar. Org/d8e7/8b87d76d 36225c1d 497939ef375cfaa8a. Pdf). dabs, fv and scFv fragments have a complete binding site but lack constant regions; they are therefore suitable for reducing non-specific binding during in vivo use. Dabs and scFv fusion proteins can be constructed to attach an effector protein (e.g., igG Fc) at the amino or carboxy terminus of the dAb or scFv. The antibody fragment may also be a linear antibody (see, e.g., U.S. Pat. No. 5,641,870). Such linear antibody fragments may be monospecific or bispecific. Other techniques for producing antibody fragments are known to those skilled in the art.
Upon expression, the antibody heavy and light chains or fragments thereof are paired by interchain disulfide bonds to form a full length antibody or fragment thereof. For example, for expression of full-length Ig, the sequence encoding the VH-CH 1-hinge-CH 2-CH3 may be cloned into a first expression vector and the sequence encoding the VL-CL domain may be cloned into a second expression vector. After co-expression, the full length heavy and light chains are linked to each other by disulfide bonds to generate full length antibodies. In another example, to produce a Fab, sequences encoding fragments comprising VH and CH1 regions may be cloned into a first expression vector, and sequences encoding VL-CL domains may be cloned into a second expression vector. After co-expression, the heavy chain pairs with the light chain to form Fab monomers. The sequences of the CH1, hinge, CH2 and/or CH3 regions of the various IgG subtypes are known to the person skilled in the art (see, for example, U.S. publication No. 2008/02480248028; see also SEQ ID NOS: 9, 11, 13 and 15). Likewise, the sequence of CL, lambda or kappa is also known (see, e.g., U.S. publication No. 2008/02480228; see also SEQ ID NOS: 17-22).
In addition to recombinant production, the TNFR1 antagonist polypeptides, TNFR2 agonist polypeptides, and TNFR1 antagonist/TNFR 2 agonist polypeptide constructs provided herein can be produced by direct peptide synthesis techniques using well-known solid phase techniques. In vitro protein synthesis may be performed using artificial techniques or by automation. Automated synthesis may be accomplished, for example, using a Applied Biosystems A peptide synthesizer (Perkin Elmer; foster City, calif.) according to manufacturer's instructions. Various fragments of the polypeptides may be chemically synthesized and combined, respectively, using chemical methods.
2. Production of mutant or modified nucleic acids and encoded polypeptides
The modifications provided herein may be made by standard recombinant DNA techniques, such as those conventional to those skilled in the art. Mutation of any one or more amino acids in the target protein or polypeptide can be accomplished using any method known in the art. Methods include standard site-directed mutagenesis of the encoding nucleic acid molecule (using, for example, a kit such as the QuikChange kit available from Stratagene), or solid phase polypeptide synthesis methods.
3. Vectors and cells
For recombinant expression of one or more desired polypeptides, such as any of the TNFR1 antagonist or TNFR2 agonist polypeptides described herein, or TNFR1 antagonist/TNFR 2 agonist polypeptide constructs, a nucleic acid molecule comprising all or a portion of a nucleotide sequence encoding the polypeptide may be inserted into a suitable expression vector, i.e., a vector comprising the elements necessary to transcribe and translate the inserted polypeptide coding sequence. Vectors containing nucleic acid molecules encoding the polypeptides are also provided. Following insertion of the nucleic acid molecule, the vector is typically used to transform a host cell, for example, to amplify the nucleic acid for replication and/or expression thereof. In such an example, a vector suitable for high level expression is used. In other cases, a vector is selected that is compatible with the display of the expressed polypeptide on the cell surface. The choice of carrier may depend on the desired application. Many expression vectors are available and known to those of skill in the art for expressing anti-TNFR 1 and anti-TNFR 2 antibodies or portions thereof, such as antigen-binding fragments. Such a selection is well within the skill level of those skilled in the art. In general, expression vectors may include a transcriptional promoter and optionally an enhancer, translational signals, and transcriptional and translational stop signals. Expression vectors for stable transformation typically have a selectable marker that allows selection and maintenance of the transformed cells. In some cases, a high copy number origin of replication may be used to amplify the copy number of the vector in the cell. The vector may also typically contain additional nucleotide sequences (e.g., his tag, flag tag) operably linked to the linked nucleic acid molecule. For antibody applications, the vector typically includes sequences encoding constant regions. Thus, antibodies or portions thereof may also be expressed as protein fusions. For example, fusion proteins can be produced to add additional functions to the polypeptide. Examples of fusion proteins include, but are not limited to, fusion signal sequences, epitope tags such as those used for localization, e.g., his6 tags or myc tags, or tags used for purification, e.g., GST tags, and/or sequences that direct protein secretion and/or membrane binding. Fusion proteins herein also include fusion of a TNFR1 antagonist and/or TNFR2 agonist with a modified Fc region, a hinge region of IgG, and/or a peptide linker, such as a GS linker.
A variety of host-vector systems are available for expressing protein coding sequences. These include, but are not limited to, mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, and other viruses); insect cell systems infected with viruses (e.g., baculoviruses); microorganisms containing yeast vectors, such as yeasts; and bacteria transformed with phage, DNA, plasmid DNA, or cosmid DNA. The choice between eukaryotic expression system and bacterial system depends on the desired post-translational modification, e.g. glycosylation. The strength and specificity of the expression elements of the vectors vary. Any of a variety of suitable transcription and translation elements may be used, depending on the host vector system used.
Any method known to those skilled in the art for inserting a DNA fragment into a vector can be used to construct an expression vector containing a nucleic acid molecule encoding a polypeptide, such as an antibody fragment or TNFR1 antagonist or TNFR2 agonist provided herein, and an appropriate transcription/translation control signal. These methods may include recombinant DNA and synthetic techniques in vitro, as well as recombinant techniques in vivo (gene recombination). Insertion into a cloning vector may be accomplished, for example, by ligating a DNA fragment into a cloning vector having complementary sticky ends. If the complementary restriction sites for fragmenting the DNA are not present in the cloning vector, the ends of the DNA molecule may be enzymatically modified. Alternatively, any desired site can be created by ligating a nucleotide sequence (linker) to the end of the DNA; these ligated linkers may contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.
For example, expression of the TNFR1 antagonists, TNFR2 agonists, and polypeptide constructs of TNFR1 antagonist/TNFR 2 agonist constructs herein can be controlled by any promoter/enhancer known in the art. Suitable bacterial promoters are well known in the art and are described below. Other promoters suitable for use in mammalian cells, yeast cells, and insect cells are well known in the art, some of which are exemplified below. The choice of promoter used to direct expression of the heterologous nucleic acid depends on the particular application. Promoters that may be used include, but are not limited to: eukaryotic expression vectors containing the SV40 early promoter (see, e.g., benoist and Chambon (1981) Nature 290:304-310), promoters in the 3' -long terminal repeat of Rous sarcoma virus (see, e.g., yamamoto et al (1980) Cell 22:787-797), herpes thymidine kinase promoters (see, e.g., wagner et al (1981) Proc.Natl. Acad.Sci.U.S. A.78:1441-1445), regulatory sequences for metallothionein genes (see, e.g., brinster et al (1982) Nature 296:39-42) and Cytomegalovirus (CMV) promoters; prokaryotic expression vectors, such as the beta-lactamase promoter (see, e.g., jay et al (1981) Proc.Natl. Acad. Sci.U.S. A.78:5543), or the tac promoter (see, e.g., deBoer et al (1983) Proc.Natl. Acad. Sci.U.S. A.80:21-25); see also "Useful Proteins from Recombinant Bacteria": in Scientific American242:79-94 (1980); plant expression vectors containing nopaline synthase promoters (see, e.g., herrara-Estrella et al (1984) Nature 303:209-213), cauliflower mosaic virus 35S RNA promoters (see, e.g., gardner et al (1981) Nucleic Acids Res.9 (12): 2871-2888), and promoters for the light synthase ribulose bisphosphate carboxylase (see, e.g., herrra-Estrella et al (1984) Nature 310:115-120); promoter elements from yeasts and other fungi, such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerate kinase promoter, the alkaline phosphatase promoter, and the following animal transcription control regions which are tissue-specific and have been used for transgenic animals: an elastase I gene control region which is active in pancreatic acinar cells (see, e.g., sweet et al (1984) Cell 38:639-646; ornitz et al (1986) Cold Spring Harbor Symp. Quant. Biol.50:399-409; macDonald (1987) Hepatology 7:425-515), an insulin gene control region which is active in pancreatic beta cells (see, e.g., hanahan et al (1985) Nature 315:115-122), an immunoglobulin gene control region which is active in lymphoid cells (see, e.g., grosschedel et al (1984) Cell 38:647-658; adams et al (1985) Nature 318:533-538;Alexander et al (1987) mol. Cell biol. 7:1436-1444), a mouse mammary tumor virus control region which is active in testes, mammary glands, lymphocytes and mast cells (see, e.g., hanahan et al (1985) Nature 315:115-122), a liver gene control region (see, e.g., a liver gene control region (1985) beta. 35-235. 5) which is active in liver-35 and (see, e.g., liver-35) and liver-35.g., a gene control region (1984) Cell 38:647-658; adams et al (1985) Nature 318-58; adams et al (1985) Nature 318-533-538;Alexander et al) (1987) a mouse mammary tumor virus control region (1987), it is active in bone marrow cells (see, e.g., magram et al (1985) Nature 315:338-340;Kollias et al (1986) Cell 46:89-94), myelin basic protein gene control region is active in oligodendrocytes of the brain (see, e.g., readhead et al (1987) Cell 48:703-712), actin light chain 2 gene control region is active in skeletal muscle (see, e.g., shani (1985) Nature 314:283-286), and gonadotrophin releasing hormone gene control region is active in gonadotrophin cells of the hypothalamus (see, e.g., mason et al (1986) Science 234:1372-1378).
Expression vectors typically contain transcriptional units or expression cassettes containing all the additional elements required for expression of the construct or portion thereof in a host cell. Typical expression cassettes contain a promoter operably linked to a nucleic acid encoding a construct, such as an antibody fragment, domain, derivative or homolog thereof or other polypeptide described herein (e.g., TNF muteins and fusion proteins), as well as signals required for efficient polyadenylation of the transcript, ribosome binding site and translation termination. Other elements of the expression cassette may include enhancers. In addition, the expression cassette typically contains a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be derived from the same gene as the promoter sequence or may be derived from a different gene. For example, the vector comprises a promoter operably linked to a nucleic acid encoding a desired polypeptide or domain, fragment, derivative or homolog thereof, one or more origins of replication, and optionally one or more selectable markers (e.g., an antibiotic resistance gene).
The expression system may have markers that provide gene amplification, such as thymidine kinase and dihydrofolate reductase. Expression systems which do not involve gene amplification are also suitable, for example, in insect cells using baculovirus vectors having a nucleic acid sequence encoding a polypeptide under the direction of a polyhedrin promoter or other strong baculovirus promoter.
For the purposes herein, vectors are provided which contain a nucleotide sequence encoding the Fc region of an IgG antibody, typically a modified Fc, operably linked to a nucleic acid encoding a TNFR1 antagonist or TNFR2 agonist polypeptide, which nucleic acid also encodes a linker therebetween, such as an IgG hinge sequence and/or a short peptide linker, such as a GS linker, including glycine-rich flexible linkers, such as (Gly 4 Ser) n Where n is a positive integer, such as an integer of 1-5 or greater, and other linkers as described herein or known to those of skill in the art. The carrier may include C H 1、C H 2. Hinge, C H 3 or C H 4 and/or C L One or all of the sequences in (a). Typically, for example for expression of Fab, the vector contains C H 1 or C L (kappa or lambda light chain). For example, V can be H -C H 1 and V L -C L The sequences are inserted into a suitable expression vector to express the Fab molecules. The sequence of the constant or hinge region is known to those skilled in the art (see, e.g., U.S. publication No. 2008/02480248028). Examples of such sequences are provided herein.
In general, the vector may be a plasmid, viral vector, or other vector known in the art for expressing the polypeptide in vivo or in vitro. For example, the constructs provided herein, such nucleic acids encoding TNFR1 antagonists and TNFR2 agonist polypeptide constructs, are expressed in mammalian cells, including, for example, chinese Hamster Ovary (CHO) cells.
Exemplary eukaryotic vectors include, for example, well known readily available vectors such as pCMV (Agilent Technologies), pcdna3.1 (Invitrogen (Thermo Fisher Scientific)), pCBL (from Creative BioLabs, see, e.g., fig. 1). Other eukaryotic vectors, such as any vector containing regulatory elements from eukaryotic viruses, may be used as eukaryotic expression vectors. These include, for example, SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Exemplary eukaryotic vectors include, for example, pMSG, pav009/a+, pMT010/a+, pmarneo-5, baculovirus pDSCE, and any other vector that allows expression of proteins under the direction of a CMV promoter, SV40 early promoter, SV40 late promoter metallothionein promoter, murine mammary tumor virus promoter, rous sarcoma virus promoter, polyhedra promoter, or other promoters that are shown to be efficiently expressed in eukaryotes.
Viral vectors, such as adenovirus, retrovirus or vaccinia virus vectors, may be used. In some examples, the vector is a defective or attenuated retrovirus or other viral vector (see, e.g., U.S. patent No. 4,980,286). For example, retroviral vectors can be used (see, e.g., miller et al (1993) meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete undesirable retroviral sequences that are packaged and integrated into the host cell DNA in the viral genome. In some examples, viruses equipped with nucleic acids encoding the polypeptides herein can facilitate their replication and transmission within target tissues. The virus may also be a lytic or non-lytic virus, wherein the virus replicates selectively under a tissue specific promoter. Co-expression of the polypeptide with the viral gene will facilitate viral transmission in vivo as the virus replicates.
For bacterial expression, vectors include the well-known and widely-spread vectors pBR322, pUC, pSKF, pET D and fusion vectors, such as MBP (Sigma-Aldrich), GST (Sigma-Aldrich) and LacZ-containing vectors. Exemplary plasmid vectors for transforming E.coli cells include, for example, pQE expression vectors (available fromValencia, calif.; see also byPublished literature describing the system). The pQE vector has a phage T5 promoter (recognized by E.coli RNA polymerase) and a double lac operator repression module, which provides for stringent regulation, high level expression of recombinant proteins in E.coli, a synthetic ribosome binding site (RBS II) for efficient translation, 6XHIS tag coding sequences, T0 and T1 transcription terminators, colE1 origin of replication and the beta-lactamase gene for imparting ampicillin resistance. The pQE vector allows for placement of a 6XHis tag at the N-or C-terminus of the recombinant protein. Such plasmids include pQE 32, pQE 30 and pQE 31, which provide multiple cloning sites for all three reading frames and provide for the expression of the N-terminal 6 XHis-tagged protein. Other exemplary plasmid vectors for transforming E.coli cells include, for example, pET expression vectors (see, e.g., U.S. Pat. No. 4,952,496; available from NOVAGEN, madison, wis.; see also the literature published by NOVAGEN describing the system). Such plasmids include pET 11a, which contains the T7lac promoter, T7 terminator, inducible E.coli lac operator and lac repressor gene; pET 12a-c containing a T7 promoter, a T7 terminator and an E.coli ompT secretion signal; pET 15b and pET19b (NOVAGEN, madison, wis.) containing a His-tagTM leader sequence for purification using a His column, and a thrombin cleavage site, T7-lac promoter region and T7 terminator allowing cleavage after purification on the column.
Cells containing the vectors are also provided. In general, any cell type that can be engineered to express heterologous DNA and that has a secretory pathway is suitable. Cells include eukaryotic cells and prokaryotic cells, and vectors are any suitable vectors for use therein. Typically, a cell is a cell capable of glycosylation of the encoded protein. Prokaryotic and eukaryotic cells containing the vectors are provided. Such cells include bacterial cells, yeast cells, fungal cells, archaeal cells, plant cells, insect cells and animal cells, in particular mammalian cells. The cells are used to produce the polypeptide by culturing the above cells under conditions in which the encoded polypeptide is expressed by the cells and recovering the expressed polypeptide. For purposes herein, for example, the polypeptide may be secreted into the culture medium.
The host cell line may be selected for its ability to regulate the expression of the inserted sequence or process the expressed protein in a desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing can affect folding and/or function of the polypeptide. Different host cells, such as but not limited to Chinese Hamster Ovary (CHO) cells like DG44, freeStyle TM CHO-S cells (Invitrogen), DXB11, CHO-K1), heLa, MCDK, HEK293 and WI38 cells have specific cellular and characteristic mechanisms for this post-translational activity and can be selected to ensure correct modification and processing of the introduced proteins. Typically, the cell is selected to be one that is capable of introducing N-linked glycosylation into the expressed polypeptide. Thus, eukaryotic cells containing the vector are provided. An example of a eukaryotic cell is a mammalian Chinese Hamster Ovary (CHO) cell. For example, dihydrofolate reductase (DHFR - ) Defective CHO cells, such as DG44 cells, are used to produce the polypeptides provided herein.
4. Expression of
The polypeptide constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, TNFR1 antagonist/TNFR 2 agonist multispecific constructs, and portions thereof, may be produced by any method known to those skilled in the art for protein production, including in vivo and in vitro methods, recombinant and synthetic, and chemical methods. The desired protein may be expressed in any organism suitable for producing the desired amount and form of the protein, for example, the amount and form of the protein desired for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as E.coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. The level of protein production by the expression host and the type of post-translational modification present on the expressed protein may vary. The choice of expression host may be based on these and other factors known to those skilled in the art; these include regulatory and safety considerations, production costs, and purification requirements and methods. Purification methods and assembly methods are well known to those skilled in the art.
Expression in eukaryotic hosts may include expression in yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris, insect cells such as Drosophila cells and Lepidoptera cells, plants and plant cells such as tobacco, maize, rice, algae and duckweed. Eukaryotic cells for expression also include mammalian cell lines, such as Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK 293) cells, or Baby Hamster Kidney (BHK) cells. Eukaryotic expression hosts also include production in transgenic animals, including, for example, production in serum, milk, and eggs.
Many expression vectors are available and known to those skilled in the art and can be used for expression of proteins. The choice of expression vector will be influenced by the choice of host expression system. In general, expression vectors may include a transcriptional promoter and optionally an enhancer, translational signals, and transcriptional and translational stop signals. Expression vectors for stable transformation typically have selectable markers that allow selection and maintenance of transformed cells. In some cases, the origin of replication can be used to amplify the copy number of the vector in the cell.
The TNFR1 antagonists, TNFR2 agonists and bispecific TNFR1 antagonist/TNFR 2 agonist constructs herein may also be expressed as protein fusions. For example, fusion proteins can be produced to add additional functions to the polypeptide. Examples of fusion proteins include, but are not limited to, fusions of signal sequences, such as tags for localization, e.g., his6 tags or myc tags, or tags for purification, e.g., GST fusions, and sequences for directing protein secretion and/or membrane binding. Fusion proteins also include fusion to the Fc region of IgG and linkers, such as the hinge sequence and/or glycine-serine (GS) peptide linkers of IgG. Alternatively, in some embodiments, the TNFR1 antagonist, TNFR2 agonist, and bispecific TNFR1 antagonist/TNFR 2 agonist constructs herein can also be fused to serum albumin.
For long-term, high-yield production of recombinant proteins, stable expression is required. For example, cell lines that stably express the polypeptide may be transformed with an expression vector containing a viral origin of replication or an endogenous expression element and a selectable marker gene. After introduction of the vector, the cells can be grown in enriched medium for 1-2 days and then reconverted to selective medium. The purpose of the selectable marker is to confer resistance to selection, the presence of which allows cell growth and recovery of the introduced sequence for successful expression. Tissue culture techniques suitable for the cell type may be used to proliferate resistant cells of the stably transformed cells.
Any number of selection systems may be used to recover the transformed cell lines. These include, but are not limited to, herpes Simplex Virus (HSV) Thymidine Kinase (TK) (see, e.g., wigler et al, (1977) Cell 11:223-232) and adenine phosphoribosyl transferase (APRT) (see, e.g., lowy, I.et al, (1980) Cell, 22:817-23) genes, which can be used for TK-or APRT-cells, respectively. Furthermore, antimetabolite, antibiotic or herbicide resistance may be used as a basis for selection. For example, dihydrofolate reductase (DHFR) which confers methotrexate resistance can be used (see, e.g., wigler et al (1980) Proc.Natl. Acad. Sci. U.S.A. 77:3567-70); npt, which confers resistance to the aminoglycoside neomycin and G-418 (see, e.g., colbere-Garapin et al (1981) J.mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and glufosinate acetyltransferase, respectively. Additional selectable genes have been described, such as trpB, which allows cells to use indole in place of tryptophan, or hisD, and others allow cells to use histidine in place of histidine (see, e.g., hartman, S.C. and R.C. Mulligan (1988) Proc.Natl. Acad.Sci.U.S.A.85:8047-8051). Visible markers such as, but not limited to, anthocyanin, beta-glucuronidase and its substrates, GUS and luciferase and its substrate luciferin can also be used to identify transformants and quantify the amount of transient or stable protein expression attributable to a particular vector system (see, e.g., rhodes et al (1995) Methods mol. Biol. 55:121-131).
a. Prokaryotic cells
Prokaryotes, particularly E.coli, provide a system for producing large quantities of proteins. Prokaryotic expression systems are commonly used to produce non-glycosylated products. Transformation protocols for E.coli are well known to those skilled in the art. The expression vector of E.coli may contain an inducible promoter; such promoters may be used to induce high levels of protein expression and to express proteins that exhibit some toxicity to the host cell. Examples of inducible promoters include, for example, the lac promoter, trp promoter, hybrid tac promoter, T7 and SP6 RNA promoters, and the temperature regulated lambda PL promoter.
The polypeptides and fusion protein constructs provided herein, e.g., any of the polypeptides and fusion protein constructs provided herein, can be expressed in the cytosolic environment of E.coli. The cytoplasm is a reducing environment, which for some molecules leads to the formation of insoluble inclusion bodies. Reducing agents, such as dithiothreitol and beta-mercaptoethanol, and denaturing agents, such as guanidine hydrochloride and urea, may be used to redissolve the proteins. Another approach is to express proteins in the periplasmic space of bacteria, which provide an oxidizing environment and chaperonin-like and disulfide isomerase enzymes, and can lead to the production of soluble proteins. Typically, the leader sequence is fused to the protein to be expressed, directing the protein into the periplasm. The leader sequence is then removed by the signal peptidase within the periplasm. Exemplary pathways that translocate expressed proteins into the periplasm are the Sec pathway, the SRP pathway, and the TAT pathway. Examples of periplasmic targeting leader sequences include pelB leader sequence, stII leader sequence and DsbA leader sequence from pectin lyase gene, and leader sequence from alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. Secretion of proteins allows for rapid and simple purification from culture supernatants. Proteins that are not secreted may be obtained from the periplasm by osmotic cleavage. Similar to cytoplasmic expression, in some cases proteins may become insoluble, and denaturing and reducing agents may be used to facilitate solubilization and refolding. The temperature of induction and growth also affects expression levels and solubility; temperatures between 25 ℃ and 37 ℃ are typically used. Typically, bacteria produce deglycosylated proteins. Thus, if glycosylation is required for the function of the protein, glycosylation can be added in vitro after purification from the host cell.
b. Yeast cells
Yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (Schizosaccharomyces pombe), yarrowia lipolytica (Yarrowia lipolytica), kluyveromyces lactis (Kluyveromyces lactis) and Pichia pastoris (Pichia pastoris) are well known expression hosts that can be used to produce proteins, such as any of the proteins described herein. Yeast may be transformed with episomal replication vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7 and GAL5, and metallothionein promoters, such as CUP1, AOX1 or other pichia or other yeast promoters. Expression vectors typically include selection markers, such as LEU2, TRP1, HIS3, and URA3, for selection and maintenance of transformed DNA. Proteins expressed in yeast are typically soluble. Co-expression with chaperones, such as Bip and protein disulfide isomerase, can increase expression levels and solubility. In addition, secretion signal peptide fusions can be used to direct secretion of proteins expressed in yeast, such as yeast mating type alpha factor secretion signals from Saccharomyces cerevisiae, as well as fusions with yeast cell surface proteins, such as Aga2p mating adhesion receptor, or A.adenine (Arxula adeninivorans) glucoamylase. Protease cleavage sites, such as those for Kex-2 proteases, can be engineered to remove fusion sequences from expressed polypeptides as they leave the secretory pathway. Yeast is also capable of glycosylation at the Asn-X-Ser/Thr motif.
c. Insect and insect cell
Insect cells, particularly expressed using baculovirus, can be used to express polypeptides, including antibodies or fragments thereof. Insect cells express high levels of proteins and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a limited host range, which increases safety and reduces regulatory problems for eukaryotic expression. Typically, expression vectors use promoters for high levels of expression, such as the polyhedrin promoter and the p10 promoter of baculovirus. Baculovirus systems include baculoviruses such as the alfalfa spodoptera littoralis nuclear polyhedrosis virus (AcNPV) and silkworm nuclear polyhedrosis virus (BmNPV), as well as insect cell lines such as Sf9 from spodoptera frugiperda (Spodoptera frugiperda), TN from spodoptera frugiperda (trichodesia ni), A7S from armyworm americana (Pseudaletia unipuncta) and DpN1 from sansevieria nigra (Danaus plexippus). For high level expression, the nucleotide sequence of the molecule to be expressed is fused directly downstream of the viral polyhedrin initiation codon. To generate baculovirus recombinants capable of expressing human antibodies, double expression transfer may be used, e.g., pAcUW51 PharMingen). Mammalian secretion signals are accurately processed in insect cells and can be used to secrete expressed proteins into the culture medium. The cell lines myxoplasma gondii (Pseudaletia unipuncta) (A7S) and Danaus plexippus (DpN) produced proteins with a glycosylation pattern similar to that of mammalian cell systems. Exemplary insect cells are those engineered to reduce immunogenicity, including those cells harboring a "mammalian" baculovirus expression vector and those lacking the FT3 enzyme.
Another expression system in insect cells is the use of stably transformed cells. Cell lines such as Schneider 2 (S2) and Kc cells (Drosophila melanogaster (Drosophila melanogaster)) and C7 cells (Aedes albopictus) can be used for expression. Drosophila metallothionein promoters can be used to induce high levels of expression in the presence of cadmium or copper heavy metal induction. Baculovirus immediate early gene promoter IE1 can be used to induce consistent expression levels. Typical expression vectors include pIE1-3 and pI31-4 transfer vectors (Novagen). Expression vectors are typically maintained through the use of selectable markers such as neomycin and hygromycin.
d. Mammalian expression cells
Mammalian expression systems can be used to express polypeptides, including the constructs herein, including the TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and fusions thereof, provided herein. The expression construct may be transferred to mammalian cells by viral infection (e.g., with adenovirus) or by direct DNA transfer (e.g., by use of liposomes, calcium phosphate, and DEAE-dextran) and by physical methods (e.g., electroporation and microinjection). Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translation initiation sequence (Kozak consensus sequence), and polyadenylation elements. An Internal Ribosome Entry Site (IRES) element can also be added to allow bicistronic expression with another gene (e.g., a selectable marker). Such vectors typically include a transcriptional promoter-enhancer for high levels of expression, such as the SV40 promoter-enhancer, the human Cytomegalovirus (CMV) promoter, and the long terminal repeat of the Rous Sarcoma Virus (RSV). These promoter-enhancers are active in many cell types. Promoter and enhancer regions of tissue and cell types may also be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulins, mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha-1 antitrypsin, beta-globin, myelin basic protein, myosin light chain 2, and gonadotropin releasing hormone control genes.
Selectable markers can be used to select and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), and Thymidine Kinase (TK). For example, expression may be performed in the presence of methotrexate to select for cells that express only the DHFR gene. Modified anti-TNFR antibodies and antigen-binding fragments thereof can be produced, for example, using the NEOR/G418 system, the dihydrofolate reductase (DHFR) system, or the Glutamine Synthetase (GS) system. The GS system uses a combined expression vector, such as pEE12/pEE6, to express both the heavy and light chains. Fusion with cell surface signaling molecules (e.g., TCR- ζ and Fc. Epsilon. RI- γ) can direct the expression of proteins whose cell surface is active.
Many cell lines are available for mammalian expression, including mouse, rat, human, monkey, chicken and hamster cells. Exemplary cell lines include, but are not limited to, BHK (e.g., BHK-21 cells), 293-F, CHO, CHO Express (CHOX; excelGene), balb/3T3, heLa, MT2, mouse NS0 (non-secretory) and other myeloma cell lines, hybridomas and heterospecies Hybridoma cell lines, lymphocytes, fibroblasts, sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines suitable for serum-free media are also available, which facilitate purification of secreted proteins from the cell culture medium. Examples include CHO-S cells [ ]Carlsbad, calif., cat# 11619-012) and serum-free EBNA-1 cell lines (see, e.g., pham et al (2003) Biotechnol. Bioeng. 84:332-342). Cell lines suitable for growth in specific media optimized for maximum expression may also be obtained. For example, DG44 CHO cells are suitable for suspension culture in chemically defined, animal product free medium.
e. Plants and methods of making the same
Transgenic plant cells and plants can be used to express polypeptides and proteins, such as any of the polypeptides and proteins described herein. Direct DNA transfer is typically used to transfer the expression construct to plants, for example, by microprojectile bombardment and PEG-mediated protoplast transfer, as well as agrobacterium-mediated transformation. Expression vectors may include promoter and enhancer sequences, transcription termination elements, and translation control elements. Expression vectors and transformation techniques typically vary between dicotyledonous plant hosts such as Arabidopsis and tobacco, and monocotyledonous plant hosts such as maize and rice. Examples of plant promoters for expression include, for example, the cauliflower mosaic virus promoter (CaMV 35S), the nopaline synthase promoter, the ribose diphosphate carboxylase promoter and ubiquitin (e.g., maize ubiquitin-1 (ubi-1)) and the UBQ3 promoter. Selection markers, such as hygromycin, phosphomannose isomerase, and neomycin phosphotransferase, are commonly used to facilitate selection and maintenance of transformed cells. The transformed plant cells can be maintained in culture as cells, aggregates (callus) or regenerated into whole plants. Transgenic plant cells may also include algae engineered to produce polypeptides. Since plants have a different glycosylation pattern than mammalian cells, this can affect the choice of proteins produced by these hosts.
5. Purification
Host cells transformed with a nucleic acid encoding a polypeptide construct provided herein can be cultured under conditions suitable for expression and recovery of the encoded protein from the cell culture. The proteins produced by recombinant cells are typically secreted, but may also be contained within the cell, depending on the sequence and/or vector used. As will be appreciated by those of skill in the art, expression vectors containing nucleic acid molecules encoding the polypeptides provided herein may be designed with signal sequences that facilitate direct secretion of the expressed polypeptides through prokaryotic or eukaryotic cell membranes.
The method of purifying a polypeptide from a host cell depends on the host cell and the expression system chosen. For secreted molecules, the protein is usually purified from the medium after removal of the cells. For intracellular expression, the cells may be lysed and the proteins may be purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting materials to prepare lysed cell extracts. In addition, transgenic animal production may include production of the polypeptide in collectable milk or eggs, and if desired, extraction and further purification of the protein using methods standard in the art.
The polypeptides, such as the TNFR1 antagonist construct, TNFR2 agonist construct, TNFR1 antagonist/TNFR 2 agonist bispecific construct and other constructs and components thereof provided herein, can be purified using protein purification techniques known to those of skill in the art. These include, but are not limited to, SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, column chromatography, HPLC, dialysis and ion exchange chromatography, such as anion exchange, and combinations thereof. Affinity purification techniques may also be used. The constructs herein can be purified using methods developed for purification of antibodies and antibody fragments. An example of a method for purifying antibodies and antibody fragments is a method comprising column chromatography, wherein a solid support column material is attached to protein G, a cell surface associated protein from streptococcus, which binds immunoglobulins with high affinity. Antibodies and antibody fragments can also be purified by methods including protein a chromatography, wherein protein a, a cell surface associated protein from staphylococcus aureus, binds immunoglobulins, such as IgG, with high affinity to a solid support column. Other immunoglobulin-binding bacterial proteins useful for purification of antibodies and antibody fragments, including protein a/G, a recombinant fusion protein that combines IgG binding domains of protein a and protein G; and protein L, a surface protein from Peptostreptococcus (1988) J.Immunol.140 (4): 1194-1197;Kastern et al (1992) J.biol. Chem.267 (18): 12820-12825;Eliasson et al (1988) J.biol. Chem.263:4323-4327). The construct is substantially pure, which is typically at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure. Purity can be assessed by standard methods, for example by SDS-PAGE and Coomassie blue staining.
When antibodies and fragments thereof and related polypeptides are expressed in large amounts by transformed bacteria, the polypeptides may form insoluble aggregates, typically after promoter induction, although expression may be constitutive. Purification protocols for polypeptide inclusion bodies are known to those skilled in the art. For example, in one method, the cell suspension is centrifuged to pellet the inclusion bodies, and the pellet containing the inclusion bodies is resuspended in a buffer, such as 20mM Tris-HCl (pH 7.2), 1mM EDTA, 150mM NaCl, and 2% Triton-X100, which is a non-ionic detergent that does not damage the inclusion bodies but dissolves the contaminants. The washing step may be repeated to remove as much cell debris as possible. The remaining inclusion body pellet is resuspended in an appropriate buffer, e.g., 20mM sodium phosphate pH 6.8, 150mM NaCl. Other suitable buffers and alternative purification schemes are known or may be developed by those skilled in the art.
Other methods for purifying antibodies and fragments thereof and constructs provided herein may be used or developed. They can be purified from the bacterial periplasm. For polypeptides exported into the periplasm of the bacteria producing the polypeptide, the periplasmic fraction of the bacteria may be isolated by cold osmotic shock, among other methods known to those skilled in the art. For example, in one method, to isolate a recombinant polypeptide from the periplasm, bacterial cells are centrifuged to form a pellet. Re-suspending the precipitate in a suspension containing In 20% sucrose buffer. To lyse the cells, the bacteria were centrifuged and the pellet resuspended in ice-cold 5mM MgSO 4 And placed in an ice bath for about 10 minutes. The cell suspension was centrifuged and the supernatant was decanted and stored. The recombinant polypeptide present in the supernatant may be isolated from the host protein by standard isolation techniques well known to those skilled in the art. These methods include, but are not limited to, the following steps: solubility fractionation, size difference filtration and column chromatography.
Expression constructs can also be engineered to include nucleic acids encoding affinity tags, for example, for detecting or purifying the expressed product, which upon expression is operably linked to the encoded polypeptide. Affinity tags include, for example, small molecule ubiquitin-like modified protein (SUMO) tags, myc epitopes, GST fusions or His6 for affinity purification with SUMO, myc antibodies, glutathione resins and Ni resins, respectively. Nucleic acids encoding such tags may be linked to nucleic acids encoding the polypeptide constructs provided herein. The tag may facilitate purification and/or detection of the soluble protein. For example, a TNFR1 antagonist polypeptide construct, or portion thereof, may be expressed as a recombinant protein to which one or more additional polypeptide domains are added to facilitate protein purification. Purification-promoting domains include, but are not limited to, metal chelating peptides, such as histidine-tryptophan modules that allow purification on immobilized metals, protein a domains that allow purification on immobilized immunoglobulins, and domains for the flag extension/affinity purification system (Immunex corp., seattle, WA). Between the purification domain and the encoded expressed polypeptide, a nucleic acid encoding a cleavable linker sequence, e.g. a factor Xa cleavable recognition site (Ile-Glu-Gly-Arg, engineered to include an Nru I restriction site, see e.g. EP 92115607 a) or enterokinase (Invitrogen (Thermo Fisher Scientific), san Diego, CA), is included to facilitate purification. Exemplary expression vectors encode the expression of fusion proteins comprising a TNFR1 antagonist and/or TNFR2 agonist polypeptide and an enterokinase cleavage site. Small molecule ubiquitin-like modified protein (SUMO) tags facilitate purification on Immobilized Metal Ion Affinity Chromatography (IMIAC), and enterokinase cleavage sites provide cleavage sites for purification of polypeptides from fusion proteins.
Purity may be assessed by any method known in the art, including gel electrophoresis, orthogonal HPLC methods, staining, and spectrophotometry. The expressed and purified polypeptide may be analyzed using any assay or method known to those of skill in the art, such as any of the assays or methods described herein. These include assays based on physical and/or functional properties of the polypeptide, including, but not limited to, gel electrophoresis assays, immunoassays, binding assays, and assays of TNF-mediated TNFR1 and/or TNFR2 activity.
6. Other modifications
As described above, modified TNFR1 antagonist constructs, TNFR2 agonist constructs, bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and other constructs and components thereof are provided that include components (activity modulators) that alter pharmacological properties, including pharmacokinetic and pharmacodynamic properties. Portions of the constructs, such as TNFR1 inhibitor polypeptides and small molecules, and TNFR2 agonist polypeptides and small molecules, may be conjugated to a polymer or polymer portion, such as, but not limited to, a polyethylene glycol (PEG) portion or dextran, or Human Serum Albumin (HSA), or may be sialylated to reduce immunogenicity and/or increase half-life in serum and other body fluids. The polypeptide may also be linked to a purification tag, such as a His tag or SUMO sequence. Other modifications include, for example, glycosylation, carboxylation, hydroxylation, phosphorylation, or other known modifications. Glycosylation can be incorporated in vivo, using a suitable expression system, e.g., mammalian expression system, in vitro, or by a combination of in vivo and in vitro methods, wherein, for example, the polypeptide is expressed in prokaryotic cells and further modified in vitro using enzymatic transglycosylation. Other modifications may be made in vitro, or, for example, by producing the modified polypeptide in a suitable host for producing such modifications.
These modifications or activity modulators and modifications are performed and selected such that the modified polypeptide incorporates the modified function and retains at least a portion, typically at least 50%, 60%, 70%, 80%, 90% or 95% of the activity as compared to the non-fused or unmodified polypeptide, including 96%, 97%, 98%, 99% or more of the activity as compared to the non-fused or unmodified polypeptide. For example, the TNFR1 antagonist construct retains the ability to inhibit TNF-mediated signaling through TNFR 1.
The attachment of a polypeptide, such as a TNFR1 antagonist or TNFR2 agonist, to another polypeptide may be accomplished directly or indirectly through a linker. In one example, the linkage may be through a chemical linkage, such as through a heterobifunctional agent, or a thiol linkage, or other such linkage. Fusion can also be achieved by recombinant expression. Fusion of a polypeptide, such as a TNFR1 antagonist or TNFR2 agonist, to another polypeptide can be at the N-or C-terminus of the TNFR1 antagonist or TNFR2 agonist polypeptide. Non-limiting examples of polypeptides that can be used in fusion proteins with the TNFR1 antagonist or TNFR2 agonist polypeptides provided herein include, for example, GST (glutathione S-transferase) polypeptides, fc domains of immunoglobulin G, albumin, heterologous signal sequences, and combinations thereof.
The encoded construct may be produced by standard recombinant techniques. For example, DNA fragments encoding the different polypeptide portions may be ligated together in-frame according to conventional techniques, e.g., by ligation using blunt ends or staggered ends, restriction endonuclease digestion to provide suitable ends, appropriate filling of cohesive ends, alkaline phosphatase treatment to avoid undesired ligation and enzymatic ligation. The fusion gene may be synthesized by conventional techniques, including an automated DNA synthesizer. Alternatively, PCR amplification of the gene fragments may be performed using anchor primers that create complementary overhangs between two consecutive gene fragments, which may then be annealed and reamplified to create the chimeric gene. Many expression vectors are commercially available that already encode fusion moieties (e.g., GST polypeptides). Nucleic acids encoding polypeptides may be cloned into such expression vectors such that the fusion moiety is linked in-frame to the TNFR1 antagonists, TNFR2 agonists, and multispecific, e.g., bispecific constructs provided herein.
a. Pegylation
Polyethylene glycol (PEG) is used in biological materials, biotechnology and medicine; it is a biocompatible, non-toxic, water-soluble polymer that is generally non-immunogenic. In the field of drug delivery, PEG derivatives are used in covalent attachment (i.e. "PEGylation") to proteins to reduce immunogenicity, proteolysis and renal clearance, and to increase serum half-life and enhance solubility (see, e.g., zalipsky (1995) adv. Drug Del. Rev. 16:157-182). PEG has been linked to low molecular weight, relatively hydrophobic drugs to increase solubility, reduce toxicity, and alter biodistribution. Conjugation to linear or branched PEG moieties increases the molecular weight and hydrodynamic radius of the polypeptide and decreases glomerular filtration rate of the kidney. Typically, the pegylated drug is administered as a solution, for example by injection. In the constructs herein, the pegylated moiety and other such polymers may be part of the linker moiety of the construct.
One related application is the synthesis of crosslinked degradable PEG networks or formulations for drug delivery, as many of the same chemistries used to design degradable, soluble drug carriers can also be used to design degradable gels (see, e.g., sawhney et al (1993) Macromolecules 26:581-587). The macromolecular complexes can be formed by mixing solutions of two complementary polymers. Such complexes are stabilized by electrostatic interactions (polyanion-polycation) and/or hydrogen bonding (polyacid-polybasic) between the polymers involved and/or by hydrophobic interactions between the polymers in a medium (see, e.g., krupers et al (1996) Eur. Polym. J. 32:785-790). For example, mixing a polyacrylic acid (PAAc) and polyethylene oxide (PEO) solution under appropriate conditions results in the formation of a complex based primarily on hydrogen bonds. Dissociation of these complexes under physiological conditions has been used to deliver free (i.e., non-pegylated) drugs. Complexes of complementary polymers have been formed from homopolymers and copolymers.
Many pegylation reagents are known, as are pegylated therapeutic proteins. Such agents include, but are not limited to, N-hydroxysuccinimide (NHS) activated PEG, succinimidyl mPEG, mPEG 2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutyrate, mPEG succinimidyl propionate, mPEG succinimidyl butyrate, mPEG carboxymethyl 3-hydroxybutyrate succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl carbonate PEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEG butyraldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methyl ketone, mPEG "no-linker" maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG o-pyridyl thioester, mPEG o-pyridyl disulfide, fmoc-PEG-NHS, boc-PEG-NHS, vinyl sulfone PEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS and biotin PEG-NHS (see, for example, U.S. 62:7; and numerous U.S. patent applications, such as this patent No. 7, and 17, and numerous U.S. Pat. No. 7. J.S. 7. Of the same general, and USP.E.E.S. of the patent to the same.S. of the following. In one example, the polyethylene glycol has a molecular weight in the range of about 3kDa to about 50kDa, typically about 5kDa to about 30kDa. Covalent attachment of PEG to the drug (referred to as "pegylation") can be accomplished by known chemical synthesis techniques. For example, pegylation of a protein can be accomplished by reacting NHS-activated PEG with the protein under suitable reaction conditions.
Although many pegylation reactions have been described, those most commonly applicable to proteins impart directionality, use mild reaction conditions, and do not require extensive downstream processing to remove toxic catalysts or byproducts. For example, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl group, and thus its use limits some heterogeneity of the resulting PEG-protein product mixture. It is often necessary to activate the hydroxyl groups at the end of the polymer opposite the terminal methoxy groups to achieve efficient protein pegylation in order to make the derivatized PEG more susceptible to nucleophilic attack. The aggressive nucleophile is typically epsilon-amino of a lysyl residue, but other amines may also react (e.g., N-terminal alpha-amine or cyclic amine of histidine) if local conditions are favorable. In proteins containing a single lysine or cysteine, a more direct linkage is possible. PEG-maleimides can target the latter residues for thiol-specific modifications. Alternatively, PEG hydrazide can react with periodate oxidized proteins and react with NaCNBH 3 Reducing in the presence of a catalyst. More specifically, the pegylated CMP saccharides may be reacted with proteins in the presence of a suitable glycosyltransferase. One technique is the "PEGylation" technique, in which a number of polymeric molecules are combined with the one in question The polypeptide in question is conjugated. Using this technique, it is difficult for the immune system to recognize epitopes on the surface of polypeptides responsible for the formation of antibodies, thereby reducing the immune response. For polypeptides (i.e., drugs) that are introduced directly into the human circulatory system to produce a particular physiological effect, typical potential immune responses are IgG and/or IgM responses, whereas polypeptides inhaled through the respiratory system (i.e., industrial polypeptides) may elicit IgE responses (i.e., allergic reactions). One of the theories explaining the reduction of the immune response is that the polymer molecule shields the epitope on the surface of the polypeptide, which is responsible for the immune response leading to the formation of antibodies. Another theory, or at least some of the factors, is that the heavier the conjugate, the lower the immune response.
For example, the polypeptide constructs and polypeptide components provided herein are pegylated and a PEG moiety is conjugated to the polypeptide by covalent attachment. PEGylation techniques include, but are not limited to, the use of specialized linker and coupling chemistry (see, e.g., harris (2002) Adv. Drug Deliv. Rev. 54:459-476), the attachment of multiple PEG moieties to a single conjugation site (e.g., by using branched PEG; see, e.g., veronese et al., (2002) Bioorg. Med. Chem. Lett. 12:177-180), site-specific PEGylation and/or monopegylation (see, e.g., chapman et al., (1999) Nature Biotech. 17:780-783), and site-directed enzymatic PEGylation (see, e.g., sato (2002) Adv. Drug Deliv. Rev. 54:487-504). Methods and techniques described in the art can produce proteins with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 PEGs or PEG derivatives attached to a single protein molecule (see, e.g., U.S. patent publication No. 2006/0104968).
b. Albumination
The polypeptides provided herein, e.g., TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific TNFR1 antagonist/TNFR 2 agonist constructs, can be fused to albumin (i.e., "albumin") for human therapy, to Human Serum Albumin (HSA), to increase the half-life, stability, bioavailability, and distribution of the polypeptide, and/or to improve pharmacological properties, e.g., pharmacokinetics. Many products related to Human Serum Albumin (HSA) are approved for use as therapeutic agents, including as cancer therapeutic agents and for the treatment of type 2 diabetes (see, e.g., alQahtani et al (2019) Biomed and Pharmacotherapy 113:108750;Roscoe et al (2018) mol. Pharmaceuticals 151:15046-5047; strohl, W.R. (2015) Biodrugs 4:215-239). In some examples, the mature HSA protein lacking the signal sequence and the activating sequence is fused to a protein of interest. In some examples, serum albumin, such as Human Serum Albumin (HSA), is conjugated to the polypeptide. An exemplary HSA protein is shown in SEQ ID NO. 35.
Provided herein are fusions with HSA. These include fusion of HSA to the N-or C-terminus of a TNFR1 antagonist (e.g., dAb, scFv, fab or other antigen binding fragment, as provided herein) or a TNFR2 agonist (e.g., TNF mutein), typically via a short peptide linker, such as, but not limited to, a glycine-serine (GS) linker, such as (GSGS) n Or (GGGGS) n Where n=1-5 or 6. Exemplary TNFR1 antagonist-HSA fusions are shown in SEQ ID NOS: 709, 713, 717, 721, and 725.
c. Purification tag
In some examples, the TNFR1 antagonist construct, TNFR2 agonist construct, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist construct, and fusion protein provided herein can contain a tag for purification of the product. Exemplary tags for purification are described elsewhere herein. For example, an exemplary polypeptide herein may contain a SUMO or His sequence for purification. Typically, the label is a cleavable label.
The polypeptide constructs (including fusion proteins) provided herein can include a His purification tag, such as a 6xHis tag. The His-tagged polypeptide optionally may contain a fusion partner and/or a signal for expression and secretion. For example, an exemplary His-polypeptide fusion protein may contain one or more of the human immunoglobulin light chain kappa (kappa) leader signal peptide sequence (SEQ ID NO: 835), 6XHis tag (SEQ ID NO: 836), SUMO sequence (SEQ ID NO: 837), and HSA (SEQ ID NO: 35). In another example, an exemplary His-tagged polypeptide fusion protein may contain a human immunoglobulin light chain kappa (kappa) leader signal peptide sequence (SEQ ID NO: 835), a 6XHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837), and an IgG Fc (see, e.g., SEQ ID NO:10, 12, 14, 16, 27, and 30).
In some embodiments, the polypeptides and fusion proteins provided herein may include His-tag and/or SUMO sequences that accumulate in inclusion bodies. For example, the His-SUMO sequence set forth in SEQ ID NO 838 may be linked to any of the polypeptides or fusion proteins provided herein. The His-SUMO tagged polypeptide optionally may contain a fusion partner and/or a signal for expression and secretion. For example, his-SUMO-polypeptide fusion proteins may contain the human immunoglobulin light chain kappa (kappa) leader signal peptide sequence (SEQ ID NO: 835), the 6XHis tag (SEQ ID NO: 836), the SUMO sequence (SEQ ID NO: 837) and HSA (SEQ ID NO: 35). In another example, an exemplary His-SUMO-polypeptide fusion protein may contain a human immunoglobulin light chain kappa (kappa) leader signal peptide sequence (SEQ ID NO: 835), a 6XHis tag (SEQ ID NO: 836), a SUMO sequence (SEQ ID NO: 837), and an IgG Fc (see, e.g., SEQ ID NO:10, 12, 14, 16, 27, and 30).
7. Nucleic acid molecules and gene therapy
Nucleic acid molecules encoding polypeptide constructs as fusion proteins provided herein may be used in gene therapy, e.g., for expression in a gene therapy vector or for administration as a DNA or RNA construct. Among other things, some of the TNFR1 antagonists, TNFR2 agonists, and bispecific TNFR1 antagonist/TNFR 2 agonist constructs provided herein are provided as fusion proteins. Nucleic acid molecules encoding these constructs, vectors, and other delivery vehicles are provided herein. The nucleic acid molecule may encode a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any polypeptide or construct provided herein. In another embodiment, the nucleic acid molecules can include those having degenerate codon sequences encoding any of the polypeptides or constructs provided herein.
If desired, the nucleic acid molecule for gene therapy may be operably linked to regulatory sequences of the nucleic acid. These sequences include promoters, enhancers, signal sequences and other trafficking sequences, as well as other such regulatory sequences known to those of skill in the art. For vectors with specific tissue tropism, the regulatory sequences may be specific for such tissues, for example for the liver of long-term gene expression, or for the eye of any ophthalmic application. Exemplary promoters include inducible and constitutive promoters for expression in mammalian cells. Such promoters are those recognized in eukaryotic organisms such as mammalian subjects, including, but not limited to, CMV and SV40 promoters; adenovirus promoters, such as the E2 gene promoter, which are responsive to E7 oncoproteins; a PV promoter, such as the BPV p89 promoter responsive to PV E2 protein; and other suitable promoters.
The polypeptides provided herein may also be delivered to cells in a gene transfer vector. The transfer vector may also encode additional other therapeutic agents for the treatment of a disease or disorder, such as rheumatoid arthritis, or any other chronic inflammatory, autoimmune, neurodegenerative or demyelinating disease or disorder as described herein or known in the art by administration of the polypeptide. A transfer vector encoding a polypeptide provided herein can be used systemically by administering a nucleic acid molecule to a subject. For example, the transfer vector may be a viral vector, such as an adenovirus vector. Vectors encoding the polypeptides or constructs herein may also be incorporated into stem cells, and such stem cells may be administered to a subject, for example, by transplanting or implanting the stem cells at the treatment site. For example, mesenchymal Stem Cells (MSCs) may be engineered to express therapeutic polypeptides, and such MSCs may be implanted at the site of implantation for treatment.
Unlike the delivery of proteins, the nucleic acid may be administered in vivo, e.g., systemically, or by any other route, or ex vivo, e.g., by removal of cells, including lymphocytes, into which the nucleic acid is introduced and reintroduced into the host or compatible receptor.
The polypeptides may be delivered to cells and tissues by expression of the nucleic acid molecules. The polypeptides may be administered as nucleic acid molecules encoding the polypeptides, including ex vivo techniques and direct in vivo expression. Nucleic acids may be delivered to cells and tissues by any method known to those of skill in the art. The isolated nucleic acid sequence may be incorporated into a vector for further manipulation. Methods of administering polypeptides by expressing a coding nucleic acid molecule include administering a recombinant vector. The vector may be designed to remain episomal, for example by including an origin of replication, or may be designed to integrate into a chromosome in the cell. Nucleic acid molecules encoding the polypeptides provided herein may also be used in ex vivo gene expression therapies using non-viral vectors. For example, a cell may be engineered to express a polypeptide operably linked to a regulatory sequence or placed into a genomic position operably linked to a regulatory sequence. Such cells may then be administered locally or systemically to a subject, such as a patient in need of treatment.
The gene therapy vector may remain episomal, or may integrate into the chromosome of the subject being treated. The polypeptide may be expressed by a virus, which is administered to a subject in need of treatment. Viral vectors suitable for use in gene therapy include adenoviruses, adeno-associated viruses (AAV), retroviruses, lentiviruses, vaccinia viruses and other such viruses. Viral vectors may be used, including, for example, adenoviruses, adeno-associated viruses (AAV), poxviruses, herpesviruses, retroviruses, and other viruses designed for gene therapy. AAV vectors having altered tropism, such as hepatocyte tropism, may be used. AAV vectors consist of a tropism-conferring capsid and a nucleic acid encoding a polypeptide flanked by ITRs.
For example, adenovirus expression techniques are well known in the art, as are methods of production and administration of adenovirus. Adenovirus serotypes are available, for example, from the american type culture collection (ATCC, rockville, MD). The adenoviral vector can be used ex vivo, for example, by isolating cells from a patient in need of treatment and transducing with the adenoviral vector expressing the polypeptide. After a suitable incubation period, the transduced cells are administered to the subject locally and/or systemically. Alternatively, the polypeptide adenovirus particles are isolated and formulated in a pharmaceutically acceptable carrier to deliver a therapeutically effective amount to prevent, treat, or ameliorate a disease or disorder in a subject. Typically, adenovirus particles are present at a rate of 1 to 10 per kilogram of subject body weight 14 Individual particles, typically 10 per kilogram of subject body weight 6 Or 10 8 From particle to 10 12 Dose delivery of individual particles. In some cases, it is desirable to provide a nucleic acid source with a cell-targeting agent, e.g.An antibody specific for a cell surface membrane protein or a target cell, or a ligand for a receptor on a target cell. The polypeptides or constructs provided herein can also be targeted for delivery into specific cell types. For example, adenoviral vectors encoding the polypeptides or constructs provided herein can be used for stable expression in non-dividing cells such as hepatocytes (see, e.g., margaritis et al (2004) J.Clin. Invest. 113:1025-1031). In another example, viral or non-viral vectors encoding the polypeptides or constructs herein can be transduced into isolated cells for subsequent delivery. Other cell types for expression and delivery include, but are not limited to, fibroblasts and endothelial cells.
Nucleic acid molecules can be introduced into artificial chromosomes and other non-viral vectors. Artificial chromosomes, such as ACES (see Lindebaum et al, (2004) Nucleic Acids Res.32 (21): e 172), can be engineered to encode and express isoforms. Briefly, mammalian Artificial Chromosomes (MACs) provide a method for introducing large amounts of genetic information payloads into cells in autonomously replicating, non-integrated form. In MAC, artificial Chromosomal Expression (ACE) based on mammalian satellite DNA is unique, can be generated de novo in cell lines of different species, and is easily purified from the chromosome of the host cell. Purified mammalian ACE can then be reintroduced into various recipient cell lines in which it is stable for a long period of time using the ACE system without selective pressure. Using this approach, specific loading of one or both gene targets has been achieved in LMTK (-) and CHO cells.
Another approach to introducing nucleic acids encoding polypeptides is to employ a two-step gene replacement technique in yeast from the complete adenovirus genome cloned in the Yeast Artificial Chromosome (YAC) (Ad 2; ketner et al (1994) Proc.Natl. Acad.Sci.U.S.A.91:6186-6190) and plasmids containing adenovirus sequences targeting specific regions in YAC clones, expression cassettes for the gene of interest, and positive and negative selection markers. YACs are of particular interest because they allow for the incorporation of larger genes. Such methods can be used to construct adenovirus-based vectors with nucleic acids encoding any of the polypeptides or constructs described herein for transferring genes to mammalian cells or whole animals.
The nucleic acid may be encapsulated in a vector, such as a liposome, or introduced into a cell, such as a bacterial cell, particularly an attenuated bacterium, or introduced into a viral vector. For example, when liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis can be used to target and/or facilitate uptake, e.g., capsid proteins or fragments thereof are addictive to specific cell types, antibodies are addictive to proteins that internalize in the circulation, and proteins that target intracellular localization and enhance intracellular half-life.
For ex vivo and in vivo methods, a nucleic acid molecule encoding a polypeptide or construct herein is introduced into cells from a suitable donor or subject to be treated. The cells into which the nucleic acid may be introduced for therapeutic purposes include, for example, any desired useful cell type suitable for the disease or disorder to be treated, including but not limited to epithelial cells; endothelial cells; keratinocytes; fibroblasts; muscle cells; liver cells; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes and granulocytes; and various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, such as stem cells obtained from bone marrow, cord blood, peripheral blood, fetal liver, and other sources.
For ex vivo treatment, cells from a donor compatible with the subject to be treated or cells from the subject to be treated are removed, nucleic acid is introduced into these isolated cells, and the modified cells are administered to the subject. Treatment includes direct administration, such as encapsulation in a porous membrane implanted in a patient (see, e.g., U.S. Pat. nos. 4,892,538 and 5,283,187, each of which is incorporated herein by reference in its entirety). Techniques suitable for transferring nucleic acids into mammalian cells in vitro include electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation methods using liposomes and cationic lipids (e.g., DOTMA, DOPE, and DC-Chol). DNA delivery methods can be used to express the polypeptides or constructs provided herein in vivo. Such methods include liposome delivery and naked DNA delivery of nucleic acids, including local and systemic delivery, e.g., using electroporation, ultrasound, and calcium phosphate delivery. Other techniques include microinjection, cell fusion, chromosome-mediated gene transfer, minicell-mediated gene transfer, and spheroplast fusion.
In vivo expression of the polypeptides or constructs herein may be correlated with expression of other molecules. For example, expression of the polypeptide may be correlated with expression of a cytotoxic product, such as in an engineered virus or in a cytotoxic virus. Such viruses may target specific cell types as targets for therapeutic effects.
In vivo expression of a polypeptide or construct provided herein may include operably linking a polypeptide-encoding nucleic acid molecule to a particular regulatory sequence, such as a cell-specific or tissue-specific promoter. The polypeptides may also be expressed from vectors that specifically infect the target cell type and/or tissue and/or replicates therein. Inducible promoters can be used to selectively regulate polypeptides or to construct expression.
Nucleic acid molecules, such as naked nucleic acids, or vectors, artificial chromosomes, liposomes and other vectors, can be administered to a subject by systemic administration, topical administration, focal administration, and other routes of administration. When administered systemically and in vivo, the nucleic acid molecule or vector containing the nucleic acid molecule may be targeted to the cell. Administration may include intravenous administration and direct injection into tissue, such as directly into the liver, including methods of direct injection into a compartmentalized organ such as the liver or a portion thereof (see, e.g., U.S. patent No. 9,821,114).
Administration may also be direct, for example by administration of a vector or cell that is generally targeted to the cell or tissue. Cells for in vivo expression of a polypeptide or construct herein also include patient autologous cells. Such cells may be removed from the patient, introduced into a nucleic acid expressing the polypeptide, and then administered to the patient, for example, by injection or implantation.
J. Compositions, formulations and dosages
Provided are pharmaceutical compositions comprising any of the polypeptides and constructs provided herein, including a TNFR1 antagonist construct, a TNFR2 agonist construct, and a multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist construct, including a fusion protein, or a nucleic acid molecule encoding a polypeptide or construct, in a pharmaceutically acceptable carrier. Such compositions contain an amount of the polypeptide, construct, or nucleic acid that can be diluted to a therapeutically effective amount or formulated for direct administration without dilution. The specific concentration of the construct or nucleic acid will depend on various parameters within the skill of the skilled artisan, including, for example, the indication of the treatment, the construct or nucleic acid, the route of administration, and the regimen. Routes of administration include systemic and topical routes, oral, rectal, intravenous, intramuscular, subcutaneous, mucosal, intraperitoneal administration, and any suitable route known to the skilled artisan.
The amount selected, e.g., a therapeutically effective amount, as described above, depends on the various parameters of the constructs provided herein, including the TNFR1 antagonist construct, the TNFR2 agonist construct, and the multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist construct, including the fusion protein, or the nucleic acid molecule encoding the construct, which amounts are formulated in a suitable carrier for administration. The pharmaceutical compositions may be formulated in any conventional manner by admixing a selected amount of the construct or mixture thereof with one or more physiologically acceptable carriers or excipients. The pharmaceutical compositions are useful for therapeutic, prophylactic and/or diagnostic applications. The concentration of the active compound (i.e., the construct or nucleic acid) in the composition depends on a variety of factors, including the factors described above, as well as the absorption, inactivation, and excretion rates of the active compound, the regimen and amount of administration, the age and size of the subject, and other factors known to those of skill in the art.
Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art as suitable for use in a particular mode of administration. Pharmaceutical compositions comprising a therapeutically effective amount of a construct or nucleic acid molecule described herein may also be provided as a lyophilized powder which is reconstituted with, for example, sterile water immediately prior to administration.
1. Preparing
Pharmaceutical compositions containing any of the constructs and nucleic acids provided herein may be formulated in any conventional manner by mixing a selected amount of the active compound with one or more physiologically acceptable carriers or excipients. The choice of carrier or excipient is within the skill of the practitioner and may depend on a number of parameters. These parameters include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, topical, focal, or any other mode) and the condition being treated. Typically, the pharmaceutical composition comprises components that do not significantly impair the biological properties of the construct or nucleic acid or encoded polypeptide or enhance or improve the pharmacological properties thereof. The formulation may also be a complex formulation with other active agents for combination therapy.
The pharmaceutical compositions provided herein can be in a variety of forms, such as, but not limited to, solid, semi-solid, liquid, emulsion, powder, aqueous, and lyophilized forms. The pharmaceutical compositions provided herein may be formulated for single dose (direct) administration, or for dilution, or other regimens. The concentration of the compound in the formulation is effective for delivering an amount effective for the intended treatment after dilution or after mixing with another composition, or for direct administration, after administration. The composition may be formulated in an amount for direct administration in a single dose or in multiple doses. The compounds may be suspended in micronized or other suitable form, or may be derivatized to produce more soluble active products. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the compound in the carrier or vehicle chosen. The resulting mixtures are solutions, suspensions, emulsions, and other such mixtures, and may be formulated as non-aqueous or aqueous mixtures, creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, perfusates, sprays, suppositories, bandages, or any other formulation suitable for systemic, focal, or topical administration. For local internal administration, e.g., intramuscular, parenteral or intra-articular administration, the constructs and nucleic acids may be formulated as a solution suspension in an aqueous medium, e.g., isotonic buffered saline, or administered internally in combination with a biocompatible support or bioadhesive. An effective concentration is a concentration that is sufficient to ameliorate a targeted condition and can be determined empirically. To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed or otherwise admixed in a selected carrier at an effective concentration such that the targeted disorder is alleviated or ameliorated.
Typically, the pharmaceutically acceptable compositions are prepared according to regulatory or other agency approval and/or according to recognized pharmacopoeias for use in animals and humans. The pharmaceutical composition may include a carrier, such as a diluent, adjuvant, excipient, or excipient, for administration with the polypeptide. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil and sesame oil. When the pharmaceutical composition is administered intravenously, water is a typical carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition may contain active ingredients and diluents such as lactose, sucrose, dicalcium phosphate and carboxymethyl cellulose; lubricants, such as magnesium stearate, calcium stearate, and talc; and binding agents such as starch, natural gums such as gum arabic, gelatin, dextrose, molasses, polyvinylpyrrolidone, cellulose and derivatives thereof, povidone, crospovidone, and other such binding agents known to those skilled in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water and ethanol. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, and other such formulations. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, granules, and sustained release formulations. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic compound and a suitable powder base such as lactose or starch. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and other such formulations. Formulations for oral administration may also be suitably formulated with protease inhibitors, such as Bowman-Birk inhibitor, conjugated Bowman-Birk inhibitor, aprotinin and camostat. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" of e.w. martin. Such compositions will contain a therapeutically effective amount of the compound, typically in purified form, and a suitable amount of carrier, in order to provide the compound in a form suitable for administration to a subject or patient.
The pharmaceutical compositions provided herein may contain other additives including, for example, antioxidants, preservatives, antimicrobial agents, analgesics, binding agents, disintegrants, colorants, diluents, excipients, extenders, glidants, solubilizers, stabilizers, tonicity agents, excipients, viscosity agents, flavoring agents, emulsions, such as oil-in-water or water-in-oil emulsions, emulsifiers and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomers, carrageenan, carboxymethyl cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, octylphenol polyether-9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum and its derivatives, solvents and other ingredients, such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate and starch and the like (see generally, alminfuro R.Gennaon (2000) 3779, and/or the like), and may be formulated in such carriers and/or in suitable formulations, such carriers, lipid-degrading agents, and lipid-stabilizing compositions, in vivo, such as may be formulated in a subject.
The formulation should be suitable for the mode of administration. For example, the active compounds may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). The injectable composition may take the form of a suspension, solution or emulsion, such as in an oily or aqueous vehicle. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 4-butanediol. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including but not limited to synthetic mono-or diglycerides, fatty acids including oleic acid, naturally occurring vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil and other oils, or synthetic fatty carriers such as ethyl oleate. Buffers, preservatives, antioxidants and suitable ingredients may be incorporated as required or may be included in the formulation.
The active compounds, e.g., constructs and nucleic acids provided herein, may be formulated as the sole pharmaceutical active ingredient in the composition, or may be combined with other active ingredients. The active compound may be targeted for delivery, for example by conjugation to a targeting agent such as an antibody. Liposomal suspensions, including tissue-targeting liposomes, are also suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared by methods well known to those skilled in the art, such as those described in U.S. Pat. No. 4,522,811. Liposome delivery may also include sustained release formulations including drug matrices such as collagen gels and liposomes modified with fibronectin (see, e.g., weiner et al (1985) J.Pharm. Sci.74 (9): 922-925). The compositions provided herein may also contain one or more adjuvants that facilitate delivery, such as, but not limited to, inert carriers or colloidal dispersion systems. Representative and non-limiting examples of such inert carriers may be selected from the group consisting of water, isopropanol, gaseous fluorocarbons, ethanol, polyvinylpyrrolidone, propylene glycol, gel generating materials, stearyl alcohol, stearic acid, cetyl alcohol, sorbitan monooleate, methylcellulose, and suitable combinations of two or more thereof.
The active compound is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without adverse side effects on the subject being treated. The therapeutically effective concentration can be determined empirically by testing the compound in known in vitro and in vivo systems, such as the assays described herein. Determination of a therapeutically effective amount is within the ability of those skilled in the art. Therapeutically effective dosages may be determined using in vitro and in vivo methods described herein. Thus, when in a pharmaceutical formulation, the active compounds provided herein, or mixtures thereof, may be present in unit dosage form for administration.
Administration of TFNR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific constructs, and nucleic acids
The active compounds, including constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, including fusion proteins, and nucleic acid molecules encoding the constructs, may be administered by any suitable route. These routes include administration in vitro, ex vivo, or in vivo by contacting a mixture, such as a body fluid or other tissue sample, with an active compound provided herein. For example, when the compound is administered ex vivo, a bodily fluid of the subject, such as a vitreous fluid or a tissue sample, may be contacted with the polypeptide coated on a tube or filter, such as a tube or filter in a bypass machine. When administered in vivo, the active compounds may be administered by any suitable route, such as oral, nasal, pulmonary, parenteral, intravenous, intradermal, intravitreal, intraretinal, subretinal, periocular, subcutaneous, intra-articular, intracisternal, intraocular, intraventricular, intrathecal, intramuscular, intraperitoneal, intratracheal, rectal or topical, or by direct injection into an organ, and by any combination of any two or more thereof, in liquid, semi-liquid or solid form, and formulated in a manner suitable for each route of administration.
The route of administration corresponds to known methods, for example by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, local or by injection or infusion via a slow release system. Antibodies or fragments thereof are typically administered continuously by infusion or bolus injection. As described above, the active compounds provided herein can be prepared in admixture with a pharmaceutically acceptable carrier. The techniques for formulating and administering the compounds are known to those skilled in the art. Such therapeutic compositions may be administered intravenously or nasally or pulmonary, for example as a liquid or powder aerosol (lyophilized). The composition may also be administered parenterally or subcutaneously as desired. When administered systemically, the therapeutic composition should be sterile, pyrogen-free, and in a parenterally acceptable solution, with appropriate consideration of pH, isotonicity and stability, as well as other conditions known to those skilled in the art.
The therapeutic formulation may be administered in a number of conventional dosage forms. The dosage forms of the active compounds provided herein are prepared for storage or administration by mixing a compound of the desired purity with a physiologically acceptable carrier, excipient, or stabilizer. Such materials are non-toxic to recipients at the dosages and concentrations employed, and may include buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salt buffers; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides, such as polyarginine; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as those sold under the trademarks TWEEN and PLURONICS, and polyethylene glycol (PEG).
The pharmaceutical composition may contain a stabilizer. The stabilizer may be an amino acid, an amino acid derivative, an amine, a sugar, a polyol, a salt or a surfactant. In some examples, the stable complex formulation comprises a single stabilizer. In other examples, the stable co-formulation contains 2, 3, 4, 5, or 6 different stabilizers. For example, the stabilizer may be a sugar or a polyol, such as glycerol, sorbitol, mannitol, inositol, sucrose, or trehalose. In a specific example, the stabilizer is sucrose. In other examples, the stabilizer is trehalose. The sugar or polyol is at a concentration of about 100mM to 500mM, 100mM to 400mM, 100mM to 300mM, 100mM to 200mM, 200mM to 500mM, 200mM to 400mM, 200mM to 300mM, 250mM to 500mM, 250mM to 400mM, 250mM to 300mM, 300mM to 500mM, 300mM to 400mM, or 400mM to 500mM, inclusive.
In an example, the stabilizer may be a surfactant, which is polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer (poloxamer), and polysorbate. For example, the surfactant may be polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamers or polysorbates, such as poloxamer 188, polysorbate 20 and polysorbate 80. In a particular example, the stabilizer is polysorbate 80. The concentration of surfactant is between or about 0.005% to 1.0%, 0.01% to 0.5%, 0.01% to 0.1%, 0.01% to 0.05%, or 0.01% to 0.02% by weight (w/v) of the formulation, inclusive.
For in vivo administration, the formulation should be sterile. This is easily accomplished, for example, by filtration through a sterile filtration membrane before or after lyophilization and reconstitution. The formulations may be stored in lyophilized form or in solution. Other carriers may be used, such as naturally occurring vegetable oils, e.g., sesame oil, peanut oil or cottonseed oil, or synthetic fatty carriers, e.g., ethyl oleate, and the like. Buffers, preservatives, antioxidants and the like may be incorporated in accordance with accepted pharmaceutical practices.
Determination of the dosage is within the skill of the physician and may depend on the particular disorder, route of administration, and subject. Exemplary dosages include, for example, 0.1 to 100mg/kg, e.g., 1 to 10mg/kg, or an appropriate amount based on the body weight of the subject being treated; calculated as an average weight of about 70-75kg in a human subject. The polypeptide may be administered one or more times, e.g., two, three, four, or any number of times required to achieve a therapeutic effect. Multiple administrations may be achieved by any route or combination of routes, and may be administered once an hour, every 2 hours, every 3 hours, every 4 hours, or longer.
The active compound may be provided in the composition, for example, at a concentration of from or about 0.1 to 10mg/mL, for example, at least or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10mg/mL, or more. The volume of the solution may be or about 0.1 to 100mL or more, for example at least or about at least or 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100mL or more. In some examples, the active compound is provided in phosphate buffered saline. For example, the composition may be provided in a 50mL vial or other container containing 100mg polypeptide or fusion protein at a concentration of 2mg/mL in phosphate buffered saline.
The active compounds provided herein can be stored lyophilized and reconstituted in a suitable carrier prior to use. This technique has proven effective for conventional immunoglobulin and protein formulations and can use lyophilization and reconstitution techniques known in the art.
The active compounds provided herein may be provided as controlled or sustained release compositions. Polymeric materials for formulating pills and capsules that achieve controlled or sustained release are known in the art (see, e.g., controlled Drug Bioavailability, drug Product Design and Performance, smolen and Ball (eds.), wiley, new York (1984); see also Levy et al, (1985) Science 228:190;During et al (1989) ann.neurol.25:351; howard et al (1989) j. Neurosurg.71:105; U.S. patent nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463 and 5,128,326; and international patent application publication nos. WO 99/015154 and WO 99/020253). Examples of polymers used in the sustained release formulation include, but are not limited to, poly (2-hydroxy methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), polyglycolide (PLG), polyanhydrides, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In general, the polymers used in the sustained release formulations are inert, free of leachable impurities, stable in storage, sterile and biodegradable. The sustained release formulation may be produced using any technique known in the art for producing sustained release formulations.
The constructs and nucleic acids, and physiologically acceptable salt and solvate forms thereof, may be formulated for administration by inhalation (via the mouth or nose) or other route of administration, including, for example, oral, transdermal, pulmonary, parenteral or rectal administration. For inhalation administration, the active compound may be delivered from a pressurized package or nebulizer in the form of an aerosol spray by using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic compound and a suitable powder base such as lactose or starch.
For pulmonary administration, the construct may be delivered in aerosol spray form using a suitable propellant from a nebulizer, a turbo-nebulizer or a microprocessor-controlled metered dose oral inhaler. Typically, the particle size of the aerosol is small, for example in the range of 0.5 to 5 microns. In the case of formulating pharmaceutical compositions for pulmonary administration, detergent surfactants are typically not used. Pulmonary drug delivery is a promising non-invasive method of systemic administration. The lung represents an attractive drug delivery route, mainly due to the large absorption surface area, thin alveolar epithelium, wide blood vessels, immune liver first pass metabolism, and relatively low metabolic activity.
For oral administration, the pharmaceutical composition may take the form of, for example, tablets, pills, liquid suspensions, or capsules, with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose) by conventional means; fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid formulations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid formulations may be prepared by conventional methods with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); a non-aqueous carrier (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and a preservative (e.g., methyl or propyl parahydroxybenzoate, or sorbic acid). The formulations may also, if appropriate, contain buffer salts, flavouring agents, colouring agents and sweetening agents.
Formulations for oral administration may be formulated to control release of the active compound. For oral administration, the composition may take the form of tablets or lozenges formulated in a conventional manner. The active compounds may be formulated as long-acting formulations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compound may be formulated with a suitable polymeric or hydrophobic material (e.g., an emulsion in an acceptable oil) or ion exchange resin, or be a poorly soluble derivative, such as a poorly sparingly soluble salt.
The active compounds can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampules or multi-dose containers) with the addition of a preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder lyophilized form for reconstitution with a suitable carrier, such as sterile pyrogen-free water, prior to use.
The pharmaceutical compositions may be formulated for topical or topical use in the form of gels, creams and lotions, for example for topical application to the skin (transdermal) and mucous membranes (e.g. ocular), as well as for ophthalmic applications or for intracisternal or intraspinal applications. Such solutions, particularly those intended for ophthalmic use, may be formulated with suitable salts as 0.01% to 10% isotonic solutions and pH values of about 5-7. The compounds may be formulated as aerosols for topical administration, for example by inhalation (see, e.g., U.S. Pat. nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols for delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma).
The concentration of the active compound in the pharmaceutical composition depends on the absorption, inactivation, and excretion rates of the active compound, the dosing regimen and dosing amount, and other factors known to those of skill in the art. As further described herein, the dosages may be determined empirically by comparing the properties and activity. For example, the inhibition of TNF-mediated inflammatory signaling by TNFR1 by the constructs provided herein can be compared to traditional anti-TNF therapies (e.g., adalimumab).
The composition may be present in a package, kit or dispenser device, if desired, which may contain one or more unit dosage forms containing the active ingredient. In some examples, the composition may be coated on a device, such as a tube or filter in a bypass machine. The package for example contains a metal or plastic foil, such as a blister package. The package or dispenser device may be accompanied by instructions for administration. The active agent-containing composition may be packaged into an article of manufacture comprising a packaging material, the agents provided herein, and a label indicating the condition for which the agents are provided. The pharmaceutical composition may be packaged in unit dose form containing a quantity of the pharmaceutical composition for single or multi-dose administration. The packaged composition may contain a lyophilized powder of a pharmaceutical composition containing a construct provided herein, including a TNFR1 antagonist construct, a TNFR2 agonist construct, and a multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist construct, including a fusion protein, and a nucleic acid molecule encoding a construct provided herein, which may be reconstituted prior to administration (e.g., reconstituted with water or saline).
Articles of manufacture provided herein contain packaging materials. Packaging materials for packaging pharmaceutical products are well known to those skilled in the art (see, e.g., U.S. patent nos. 5,323,907, 5,052,558 and 5,033,252). Examples of pharmaceutical packaging materials include, but are not limited to, blister packages, bottles, tubes, inhalers (e.g., pressurized Metered Dose Inhalers (MDI), dry Powder Inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers), and other single breath liquid systems), pumps, pouches, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.
The active compounds, including constructs provided herein, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, including fusion proteins, as well as nucleic acid molecules encoding such constructs, pharmaceutical compositions, and combinations of active agents and other compositions, including other therapeutic agents, may be provided as kits. The kit may optionally include one or more ingredients, such as instructions for use, devices, additional reagents (e.g., sterile water or saline solution for diluting the composition and/or reconstituting the lyophilized protein), and components such as tubing, containers, and syringes for practicing the methods. An example kit may include a construct and encoding nucleic acid provided herein, and optionally may include instructions for use, a device for administering a compound to a subject, a device for detecting a compound in a subject, one or more devices for detecting the compound or a metabolite thereof in a sample obtained from a subject, and a device for administering an additional therapeutic agent to a subject. Optionally, the kit may include instructions for use. The instructions generally include a tangible representation describing the active compound and optionally other components included in the kit, as well as the method of administration, including determining the appropriate status of the subject, the appropriate dosage, the regimen of administration, and the method of administration. The instructions may also include instructions for monitoring the subject during the treatment.
Kits may also include pharmaceutical compositions described herein and articles of manufacture for diagnosis. For example, such kits may include items for measuring the concentration, amount, or activity of an active compound administered in a subject. Kits provided herein may also include a device for administering a compound. Any of a variety of devices known in the art for administering a drug to a subject may be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, and catheters. Generally, the device used for administration is compatible with the desired method of active agent administration.
3. Administration of nucleic acids encoding polypeptides (Gene therapy)
Pharmaceutical compositions include those comprising nucleic acid molecules encoding the polypeptide constructs provided herein. Rather than delivering the protein, the nucleic acid molecule may be administered in vivo, e.g., systemically or by other means or ex vivo, e.g., by removal of cells, including lymphocytes, introduction of the nucleic acid molecule therein, and reintroduction into the host or compatible receptor.
The polypeptide constructs, including TNFR1 antagonist constructs, TNFR2 agonist constructs, and multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, including fusion proteins, provided herein can be delivered to cells and tissues by nucleic acid expression. Nucleic acids may be delivered to cells and tissues by any method known to those of skill in the art. The isolated nucleic acid may be incorporated into a vector for further manipulation. As described above, methods of administering polypeptides by expression of encoding nucleic acid molecules include administering recombinant vectors. The vector may be designed to remain episomal, for example by including an origin of replication, or may be designed to integrate into a chromosome in the cell. The polypeptides may also be used in ex vivo gene expression therapies using non-viral vectors. Suitable gene therapy vectors and delivery methods are known to those skilled in the art and are discussed in the section above.
K. Therapeutic uses and methods of treatment
Pharmaceutical compositions such as those described above are prepared and administered to a subject suffering from a disease, disorder or condition that is amenable to treatment with constructs that inhibit and/or agonize TNFR1 and TNFR2, respectively. The dosage will depend on the particular disorder, disease or condition being treated, as well as the particular subject. Typical dosages are similar to known TNF blockers, such as Etanercept (Etanercept). For subjects, including humans and other animals, exemplary dosages range from about or 0.1mg/kg to 100mg/kg, such as 1mg/kg to about 30mg/kg, such as 5mg/kg to 25mg/kg. The dosage may be determined on the assumption that the average human weight is about 75 kg. The dosage may be adjusted for children, infants and smaller adults.
The TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and fusion proteins provided herein can be used for any purpose known to those of skill in the art using such molecules, including for the treatment of any of the diseases, disorders, and conditions described herein. For example, the TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins provided herein may be used for one or more therapeutic, diagnostic, industrial and/or research purposes. Methods of treatment provided herein include methods of therapeutic use of the TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins provided herein. For example, the TNFR1 antagonists described herein can be used to antagonize TNFR1, and/or to inhibit TNF binding to TNFR1, and/or to inhibit TNF-mediated proinflammatory signaling through TNFR 1. TNFR2 agonists are useful for agonizing TNFR2 to induce protective/anti-inflammatory TNFR2 signaling, and/or to induce immunosuppressive TNFR2 + Expansion, proliferation and activation of regulatory T cells (tregs). In some embodiments, a combination of a TNFR1 antagonist and a TNFR2 agonist, as described herein, or using a bispecific TNFR1 antagonist/TNFR 2 agonist construct provided herein, provides selective inhibition of pro-inflammatory TNFR1 activity while maintaining or increasing TNFR 2-related protective signaling and Treg immunosuppressive activity, which is beneficial in the treatment of chronic inflammatory and autoimmune diseases, as well as in the treatment of neurodegenerative and demyelinating diseases and disorders.
The TNFR1 antagonists, TNFR2 agonists, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins provided herein may have therapeutic activity alone or in combination with other agents. The TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins, and encoding nucleic acid molecules provided herein are useful in the treatment of any condition for which anti-TNF therapy (e.g., adalimumab, infliximab, etanercept and other formulations described herein and/or known in the art) or other disease-modifying antirheumatic drugs (DMARDs; e.g., methotrexate, hydroxychloroquine, sulfasalazine, leflunomide (leflunomide), abatacept (abatacept), anakinra), rituximab, tolizumab, infliximab, etanercept and other formulations described herein and/or known in the art) are used, including but not limited to chronic inflammatory and autoimmune diseases and conditions, as well as neurodegenerative and demyelinating diseases and conditions. For example, a subject administered a therapeutic molecule provided herein exhibits acute or chronic inflammation of the joints, skin, lung, and/or intestine, and/or have autoimmune disease, rheumatoid Arthritis (RA), psoriasis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA), spondyloarthropathies, ankylosing spondylitis, crohn's disease, ulcerative colitis, inflammatory Bowel Disease (IBD), uveitis, fibrotic disease, endometriosis, lupus, multiple sclerosis, congestive heart failure, cardiovascular disease, myocardial Infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute Respiratory Distress Syndrome (ARDS), severe Acute Respiratory Syndrome (SARS), COVID-19, influenza, inflammatory Bowel Disease (IBD) acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, alzheimer's disease, parkinson's disease, behcet's disease, dupuytren's disease, tumor necrosis factor receptor-related periodic syndrome (TRA PS), pancreatitis, type I diabetes, chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, emphysema, graft rejection, graft-versus-host disease (GvHD), respiratory diseases, pulmonary inflammation, pulmonary diseases and disorders, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, hereditary diseases with TNF/TNFR1 as a pathological medium, periodic fever syndrome, and cancer.
Constructs provided herein can generally result in subjects exhibiting reduced or lessened side effects upon administration compared to those observed following administration of anti-TNF therapies. Treatment of diseases and conditions with the polypeptides provided herein, e.g., TNFR1 antagonists, TNFR2 agonists, and bispecific constructs and fusion proteins thereof, can be accomplished by any suitable route of administration, including but not limited to infusion, subcutaneous injection, and inhalation, or intramuscular, intradermal, oral, topical, and transdermal routes of administration, using the suitable formulations described herein.
As discussed elsewhere herein, existing anti-TNF therapies, such as adalimumab, have immunosuppressive effects due to blocking TNF signaling by TNFR1 and TNFR2, and are associated with risks of adverse side effects, including, for example, increased sepsis and serious infection risk, such as listeriosis, tuberculosis recurrence, hepatitis b/c recurrence, herpes zoster recurrence, and invasive fungi and other opportunistic infections. anti-TNF drugs can also lead to severe exacerbation of congestive heart failure and can lead to drug-induced lupus, liver injury, psoriasis, sarcoidosis, and demyelinating Central Nervous System (CNS) diseases, as well as increased susceptibility to other autoimmune diseases as well as lymphomas and solid malignancies such as non-melanoma skin cancers. Depending on the anti-TNF drug, approximately 3-33% of patients receiving treatment do not respond to treatment, and up to 46% of patients cease responding, resulting in withdrawal or increased dose. anti-TNF therapies fail to treat neurodegenerative and central nervous system diseases such as alzheimer's disease, parkinson's disease, stroke, and Multiple Sclerosis (MS), which are associated with overexpression of TNF. Due to adverse effects associated with the use of anti-TNF drugs, some patients do not respond, patients with initial responses lack sustained responses, and other therapies are needed for treatment failure and/or exacerbation of neurodegenerative diseases such as MS. Such therapies are provided herein.
As described herein, selective inhibition of TNFR1 retains the potent anti-inflammatory and protective activity of TNFR2 signaling, results in fewer opportunistic infections and cancers, and retains TNF-induced Treg function. Previous selective TNFR1 antagonists have been immunogenic, including the formation of anti-drug antibodies (ADA), poor pharmacokinetics and pharmacodynamics, including, for example, short serum half-life, fast renal clearance, and/or poor binding affinity and potency. The therapies provided herein overcome the limitations associated with existing selective TNFR1 antagonists. Examples of therapeutic improvements using polypeptides provided herein, such as TNFR1 antagonists, include, but are not limited to, lower doses, fewer and/or less frequent administrations, reduced side effects, and increased therapeutic effects.
Thus, the selective TNFR1 antagonists, TNFR2 agonists, and multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins provided herein are associated with reduced side effects, can be administered at higher dosage regimens if necessary, and can improve efficacy and safety. Side effects that may be reduced, lessened or eliminated as compared to side effects observed by existing anti-TNF therapies such as adalimumab and other therapies described herein and/or known in the art include any undesirable non-therapeutic effects described herein or known in the art, such as, but not limited to, sepsis, severe infection, congestive heart failure/cardiotoxicity, antibody production, and the development or progression of cancer, autoimmune diseases and/or demyelinating Central Nervous System (CNS) diseases. In some examples, administration of a TNFR1 antagonist, bispecific construct, or fusion protein provided herein reduces the severity of one or more side effects relative to the severity of one or more side effects of anti-TNF therapy by at least or about 99%, at least or about 95%, at least or about 90%, at least or about 85%, at least or about 80%, at least or about 75%, at least or about 70%, at least or about 65%, at least or about 60%, at least or about 55%, at least or about 50%, at least or about 45%, at least or about 40%, at least or about 35%, at least or about 30%, at least or about 25%, at least or about 20%, at least or about 15%, or at least or about 10% as compared to the side effects caused by administration of an existing anti-TNF therapy, such as adalimumab.
Dosage levels and regimens may be determined based on known dosages and regimens, and if desired may be extrapolated from variations in the properties of the polypeptides and constructs provided herein, and/or may be empirically determined based on a variety of factors. These factors include, for example, the weight of the individual and its general health, age, sex and diet, as well as the activity of the particular compound used, the time of administration, the rate of excretion, drug combination, the severity and course of the disease, as well as the patient's predisposition to the disease and judgment of the attending physician. The active ingredient is typically combined with a pharmaceutically effective carrier. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form or multiple dosage forms can vary depending upon the host treated and the particular mode of administration.
After the patient's condition has been improved, a maintenance dose of the compound or composition may be administered, if necessary; and the dosage, dosage form, or frequency of administration, or combinations thereof, may be modified. In some cases, the subject may need to undergo long-term intermittent treatment according to any recurrence of disease symptoms or according to a predetermined dosage regimen.
This section provides constructs, exemplary uses and methods of administration comprising the polypeptides and encoding nucleic acid molecules provided herein. These described therapies are merely exemplary and do not limit the application of the molecules/constructs provided herein. It is within the skill of the attending physician to identify diseases or conditions treatable with the TNFR1 antagonists, TNFR2 agonists, bispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins provided herein and encoding nucleic acid molecules.
1. Treatment of chronic inflammatory/autoimmune diseases and conditions
As described herein, elevated levels or uncontrolled expression of TNF and deregulation of TNF signaling can lead to chronic inflammation, which can lead to the occurrence of autoimmune diseases and tissue damage. TNF signaling through TNFR1 is primarily pro-inflammatory and drives the development of chronic inflammatory and autoimmune diseases and disorders. For example, TNFR1 signaling is associated with the development of arthritis, inflammatory Bowel Disease (IBD), respiratory diseases, and the like, the production of osteoclasts that lead to local bone destruction, and cardiotoxic effects in TNF-induced heart failure and myocardial infarction models. Thus, selective blocking of TNFR1 is usefulFor the treatment of chronic inflammatory and autoimmune diseases and disorders. TNF signaling by TNFR2, which has a major anti-inflammatory effect, is associated with neuroprotection in the nerve, heart, gut and bone. The anti-inflammatory and protective effects of TNFR2 signaling have been demonstrated in, for example, experimental colitis, heart failure/heart disease, myocardial infarction, inflammatory arthritis, infectious diseases, pancreatic regeneration, stem cell proliferation, autoreactive T cell destruction, and regulation of osteoclastic production to maintain bone mass, prevent joint inflammation and erosive destruction. TNFR2 agonism also leads to immunosuppressive TNFR2 + Proliferation and expansion of tregs and promotion of Treg cell inhibitory activity, thereby eliminating autoreactive/effector T cells, preventing tissue destruction, and inhibiting inflammatory and autoimmune diseases and disorders. Thus, TNFR2 agonism is also useful in treating or alleviating the symptoms of chronic inflammatory and autoimmune diseases and disorders.
The TNFR1 antagonists, TNFR2 agonists, multispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and encoding nucleic acids provided herein are useful for treating or alleviating the symptoms of autoimmune/inflammatory diseases and disorders associated with elevated levels and deregulated TNF signaling. The constructs, fusion proteins, and nucleic acids provided herein are useful in the treatment of diseases, disorders, and conditions including, but not limited to, for example, arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, spinal arthritis), inflammatory bowel disease (e.g., crohn's disease and ulcerative colitis), uveitis, fibrotic diseases (e.g., dupuytren's disease), behcet's disease, endometriosis, lupus, ankylosing spondylitis, psoriasis, tumor necrosis factor receptor associated periodic syndromes (trap), cardiovascular disease, congestive heart failure, myocardial Infarction (MI), atherosclerosis, respiratory disease, asthma, cystic fibrosis, chronic Obstructive Pulmonary Disease (COPD), pancreatitis, type I diabetes, metabolic disease, cytokine release syndrome, infectious shock, sepsis, acute Respiratory Distress Syndrome (ARDS), severe acute respiratory syndrome (COVID), COVID-19, influenza, chronic bronchitis, pulmonary inflammation, idiopathic pulmonary fibrosis, graft rejection, anti-host disease (fr), infectious disease, ghd, infectious disease, TNF-specific disease, or inflammatory disease as a pathological medium, and the like.
TNF blockade can also reduce cytokine storms observed in certain viral infections, such as SARS virus and COVID-19 infection. This can prevent ventilator-dependent, multiple organ damage and death in patients with Severe Acute Respiratory Syndrome (SARS), such as those caused by SARS-CoV-2 and other SARS viruses/coronaviruses. TNF-induced viral syndrome (TIVS) is caused by TNF-driven cytokine storms, which involve not only lung injury but also multiple organ failure. TIVS is similar to SIRS (severe inflammatory respiratory syndrome), SARS (severe acute respiratory syndrome) and sepsis (caused by bacteria). These conditions affect lung function and also affect many other organs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, which causes the expression of COVID-19) in alveolar cells secreting type II surfactant in the lung by angiotensin converting enzyme into host cells. Severe covd-19 is associated with a major immune inflammatory response in large numbers of neutrophils, lymphocytes, macrophages and immune mediators. Death of covd-19 is primarily due to diffuse alveolar injury with pulmonary edema, hyaline membrane formation, and inflammatory infiltrates of interstitial mononuclear cells that coexist with early Adult Respiratory Distress Syndrome (ARDS). TNF is present in blood and diseased tissue of patients with COVID-19; TNF is involved in almost all acute inflammatory reactions and acts as an inflammatory amplifier. Thus, treatment with the constructs herein is provided.
2. Treatment of neurodegenerative and demyelinating diseases and disorders
As discussed elsewhere herein, some neurodegenerative and demyelinating diseases and disorders are associated with long-term elevated TNF levels in the Central Nervous System (CNS). Elevated TNF levels and TNF signaling through TNFR1 are associated with initiating and maintaining neuroinflammation, and promoting neuronal cell death, demyelination, and cognitive decline. For example, in Alzheimer's Disease (AD) patients, TNF promotes microglial activation, synaptic dysfunction, neuronal cell death, and accumulation of neurofibrotic plaques and tangles, and elevated levels of TNF inhibit phagocytosis of amyloid (aβ) in the brain of AD patients, which prevents microglial cells from efficiently removing plaques. In patients with Parkinson's Disease (PD), elevated TNF levels can lead to neuroinflammation and dopaminergic neuronal toxicity. Elevated levels of TNF and the polymorphism of the gene encoding TNFR1 are associated with demyelination and the development of demyelinating diseases such as Multiple Sclerosis (MS). Thus, selective blocking of TNF signaling by TNFR1 can be used to treat or ameliorate neurodegenerative and demyelinating diseases and disorders, as well as other disorders of the CNS.
TNF signaling through TNFR2 is associated with anti-inflammatory and neuroprotective effects. For example, TNF activation TNFR2 inhibits seizures, reduces cognitive dysfunction following brain injury, and promotes remyelination and survival of neurons. Proliferation, expansion and activation of immunosuppressive tregs also have neuroprotective effects after TNFR2 agonism. For example, TNFR2 signaling promotes Treg cell expansion and inhibitory activity in Experimental Autoimmune Encephalomyelitis (EAE), an animal model of inflammatory central nervous system demyelinating diseases such as multiple sclerosis. Accordingly, TNFR2 agonists are also useful in the treatment or alleviation of neurodegenerative and demyelinating diseases and disorders, as well as other disorders of the CNS.
The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein are useful for treating or ameliorating the symptoms of neurodegenerative and demyelinating diseases, as well as other central nervous system diseases and disorders, including, but not limited to, alzheimer's disease, parkinson's disease, multiple sclerosis, and stroke.
3. Treatment of cancer and other immunosuppressive diseases, disorders and conditions
As described herein, tumors are heavily immunosuppressive TNFR2 + Treg infiltration, which prevents tumor killing of CD8 + Proliferation of Cytotoxic T Lymphocytes (CTLs) (also known as effector T cells (teffs)) allows tumor growth. Antagonism of TNFR2 on lymphocytes in Tumor Microenvironment (TME) restores both types by eliminating tregsBalance between T cells and allow for activation and expansion of effector T cells, resulting in tumor cell lysis (see, e.g., vanamee et al (2017) Trends in Molecular Medicine (11): P1037-P1046).
TNFR2 is abundantly expressed on the surface of many types of human cancer cells including, for example, renal cell carcinoma, colon carcinoma, hodgkin's lymphoma, multiple myeloma, cutaneous non-hodgkin's lymphoma, and ovarian cancer. TNFR2 mutations in cancer are associated with gene replication and constitutive activation. Mouse bone Marrow Derived Suppressor Cells (MDSCs) also express TNFR2, whose suppression has been demonstrated to control metastasis in a mouse liver cancer model. Furthermore, immune checkpoint inhibitors lead to upregulation of tumor-invasive tregs by TNFR2, leading to tumor immune escape and resistance. Not all patients respond to immune checkpoint inhibitor treatment, patients may relapse, and severe autoimmune side effects are observed in checkpoint inhibitor treatment (see, e.g., vanamee et al (2017) Trends in Molecular Medicine (11): P1037-P1046). Thus, the blockade of TNFR2 can be used to treat certain types of cancers by directly killing tumor cells, i.e., by inhibiting immunosuppressive tregs, which allow effector T cells to proliferate, and by inhibiting MDSCs, which can prevent the formation of metastases. Because TNFR2 is also expressed on normal tissues (especially macrophages; see, e.g., proteoplas. Org/ensg00000028137-tnfrsf1 b/tissue), TNFR2 antagonists do not have ADCC activity, but have FcRn activity (or enhanced FcRn activity). Administration is personalized in that, as described herein, to qualify for treatment, the TNFR2 level of a patient's tumor must be significantly higher than that of adjacent normal tissue. For this reason, TNFR2 antagonists will be used with other therapies, particularly immunomodulating therapies, which would otherwise result in accumulation of regulatory T cells in the tumor.
Accordingly, the TNFR2 agonists, bispecific TNFR1 antagonist/TNFR 2 agonist constructs and fusion proteins provided herein are also useful in the treatment of solid cancers, hematological malignancies, and other hyperproliferative diseases and disorders, including, but not limited to, for example, renal cell carcinoma, colon cancer, hodgkin's lymphoma, multiple myeloma, cutaneous non-hodgkin's lymphoma, and ovarian cancer.
4. Combination therapy
Combination therapy includes administration of the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein in combination with another agent or treatment (including radiation therapy and surgery). The additional agent or therapy may be administered simultaneously, prior to, subsequent to, or intermittently with the treatment provided herein. They may be present in separate compositions or in co-formulations.
The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein can be administered prior to, after, intermittently, or concurrently with administration of one or more other therapeutic regimens or formulations, including, but not limited to, TNF antagonists/blockers, antibodies, cytotoxic agents, anti-inflammatory agents, cytokines, growth factors, growth inhibitors, cardioprotective agents, immunosuppressants, chemotherapeutic agents, biological or non-biological disease-modifying antirheumatic drugs (DMARDs), infectious disease treatment drugs (including antibodies), or other therapeutic agents. The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and nucleic acids provided herein can be administered as a first line therapy or a second line therapy to a patient who is not effective in anti-TNF therapy, whether acute or chronic. Exemplary anti-TNF therapies useful in combination therapies herein include, for example, conventional synthetic DMARDs, such as (common name and exemplary trademark): methotrexate (MTX), hydroxychloroquine (HCQ; ) Sulfasalazine (/ -)>) And leflunomide (>) The method comprises the steps of carrying out a first treatment on the surface of the Biological DMARDs, e.g. Abacalcet (+)>) Anakinra (/ -A)>) Rituximab (+)> ) Touzumab (atlizumab,)>) A corticosteroid (e.g., dexamethasone, methylprednisolone, prednisolone prednisone or triamcinolone), tofacitinib (++>) And TNF inhibitors/anti-TNF agents, e.g. polyethylene glycol cetuximab (++>) Infliximab (++>) Adalimumab (/ ->) Golimumab (+)>) And etanercept ()>). Combination therapies may also include immunotherapeutic agents such as cyclosporin, methotrexate, doxorubicin or cisplatin, as well as immunotoxins.
Examples of anti-inflammatory agents and formulations useful in combination therapy include non-steroidsAnti-inflammatory drugs (NSAIDs), including salicylates, such as aspirin, conventional NSAIDs, such as ibuprofen, naproxen, ketoprofen, nabumetone, piroxicam, diclofenac, or indomethacin, and Cox-2 selective inhibitors, such as celecoxib (under the trademarkSold) or roteoxin (under the trade mark +.>Sales). Other compounds useful in combination therapy include antimetabolites such as methotrexate and leflunomide; corticosteroids or other steroids such as cortisone, dexamethasone, or prednisone; analgesics, such as acetaminophen; aminosalicylates, such as mesalamine; and cytotoxic agents, such as azathioprine (under the trademark +. >Sold), cyclophosphamide (under the trademark +.>Sold) and cyclosporin a.
Other agents useful in combination therapy include biological response modifiers including, for example, anti-inflammatory cytokines such as IL-10; b cell targeting agents such as anti-CD 20 antibodies (e.g., rituximab); a compound targeting the T antigen; an adhesion molecule blocking agent; chemokine receptor antagonists; kinase inhibitors, such as inhibitors of Mitogen Activated Protein (MAP) kinase, c-Jun N-terminal kinase (JNK), or NFkB; and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands. Other formulations useful in combination therapy include immunosuppressants. Immunosuppressants may include, for example, tacrolimus or FK-506; mycophenolic acid; calcineurin inhibitors (CNIs); csA; and sirolimus, or other agents known to inhibit the immune system.
The polypeptides and constructs provided herein can also be used in combination with agents, such as anticoagulants, that are administered to treat and/or during the treatment of cardiovascular diseases. Exemplary anticoagulants include, but are not limited to, heparin, warfarin, acenitrocoumarin, benzindene, EDTA, citrate, oxalate, and direct thrombin inhibitors such as argatroban (argatroban), lepirudin (lepirudin), bivalirudin (bivalirudin), and simetrygatran.
The polypeptides and constructs provided herein can be administered with antibodies for the treatment of autoimmune or inflammatory diseases, transplant rejection, or GvHD. Examples of such antibodies include, but are not limited to, anti- α4β7 integrin antibodies, such as LDP-02; anti- β2 integrin antibodies, such as LDP-01; anti-complement (C5) antibodies, such as 5G1.1; anti-CD 2 antibodies, such as BTI-322 and MEDI-507; anti-CD 3 antibodies, such as OKT3 and SMART anti-CD 3; anti-CD 4 antibodies, such as IDEC-151, MDX-CD4, and OKT4A; an anti-CD 11a antibody; anti-CD 14 antibodies, such as IC14; an anti-CD 18 antibody; anti-CD 23 antibodies, such as IDEC 152; anti-CD 25 antibodies, such as daclizumab; anti-CD 40L antibodies, such as 5c8, lu Lizhu mab and IDEC-131; anti-CD 64 antibodies, such as MDX-33; anti-CD 80 antibodies, such as IDEC-114; anti-CD 147 antibodies, such as ABX-CBL; anti-E-selectin antibodies, such as CDP850; anti-gpIIb/IIIa antibodies, e.g.Abcixima; anti-ICAM-3 antibodies, such as ICM3; anti-ICE antibodies, such as VX-740; anti-fcγr1 antibodies, such as MDX-33; anti-IgE antibodies, such as rhuMAb-E25; anti-IL-4 antibodies, such as SB-240683; anti-IL-5 antibodies, such as SB-240563 and SCH55700; anti-IL-8 antibodies, such as ABX-IL8; an anti-interferon gamma antibody; anti-tnfα antibodies, such as CDP571, CDP870, D2E7, infliximab, and MAK-195F; and anti-VLA-4 antibodies, e.g. +. >. Examples of other Fc-containing molecules that may be co-administered to treat autoimmune or inflammatory diseases, transplant rejection, and GvHD include, but are not limited to, TNFR2-Fc fusion proteins->(etanercept)) And the IL-1TRAP of the Regeneron.
Examples of antibodies that may be co-administered to treat infectious diseases include, but are not limited to, anti-anthrax antibodies, such as ABthrax; anti-CMV antibodies, such as CytoGam and sevirumab; anti-Cryptosporidium antibodies, such as CryptoGAM and Sporidin-G; anti-helicobacter pylori antibodies, such as Pyloran; anti-hepatitis B antibodies, such as HepeX-B and Nabi-HB; anti-HIV antibodies, such as HRG-214; anti-RSV antibodies, such as panavizumab (felvizumab), HNK-20, palivizumab (palivizumab), and RespiGam; and anti-staphylococcal antibodies such as Aurexis, aurograb, BSYX-A110 and SE-Mabs.
In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein are administered with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide @) The method comprises the steps of carrying out a first treatment on the surface of the Alkyl sulfonates such as busulfan (busulfan), imperoshu (imposulfan) and piposulfan (piposulfan); androgens, such as carbosterone (calibretone), drotasone propionate (dromostanolone propionate), thioandrosterol (epiostanol), melandrane (mepistane), and testosterone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane) and trilostane (trilostane); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; antibiotics, e.g. aclacinomycins, actinomycins, anthracyclines (anthracyclines), diazoserines (azaserines), bleomycins, actinomycins (calicheamicins), calicheamicins (calicheamicins), carboxilins (carbamicins), carminomycin (carminomycin), carcinomycins (carminomycin), spinomycins (chromomycins), dactinomycin (dactinomycin), daunorubicins (daunorubicins), dithicins (detorubicins), 6-diazo-5-oxo-L-norleucine Doxorubicin (doxorubicin), epirubicin (epirubicin), doxorubicin (esoubicin), idarubicin (idarubicin), maculomycin (marcelomycin), mitomycin (mitomycins), mycophenolic acid (mycophenolic acid), nogamycin (nogamycin), olivomycin (olivanins), pepstatin (pepstatin), pofeomycin (porfirimomycin), puromycin (puromycin), clarithromycin (quemycin), rodobutycin (rodorubicin), streptozocin (streptozocin), tuberculin (tuberculin), ubenimustine (enomycin), zinostatin (zistatin) and zorubicin (zorubicin); antiestrogens including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), 4 (5) -imidazole inhibiting aromatase, 4-hydroxy tamoxifen, tamoxifen (trioxifene), ketoxifene (keoxifene), LY 117018, onapristone (onapristone), and toremifene (toremifene, farston); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as methotrexate (denopterin), methotrexate (methotrexate), pterin (pteroprerin), and trimetrexate (trimerexate); aziridines such as benzodopa (benzodepa), carboquone (carboquone), mettuyepa (meturedepa) and uratepa (uredepa); ethyleneimine and methyl melamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylol melamine; folic acid supplements, such as folinic acid (folinic acid); nitrogen mustards such as chlorambucil (chlorrambucil), chlornapazine (chloronaphazine), chlorphosphamide (chlorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine (mechlorethamine oxide hydrochloride), melphalan (melphalan), novobiocin (novemblic), phenyllactone (phenoterine), prednisone (prednimustine), trofosfamide (trofosfamide) and uracil mustard (uracilmustard); nitrosoureas, such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ranimustine (ranimustine); platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; proteins, e.g. arginine deiminase And asparaginase; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiominoprine (thiamiprine), and thioguanine (thioguanine); pyrimidine analogs such as, for example, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, execitabine, fluorouridine and 5-FU; taxanes (taxanes), e.g. paclitaxel ()>Bristol-Myers Squibb Oncology, prencton, N.J.) and docetaxel (/ -)>Rhone-Poulenc Rorer, antonny, france); topoisomerase inhibitors such as RFS 2000; thymidylate synthase inhibitors, such as Tomudex; additional chemotherapeutic agents, including acetylacetone (aceglatone); aldehyde phosphoramidate glycoside (aldophosphamide glycoside); an aminopentanonic acid (aminolevulinic acid); amsacrine (amacrine); armustine (bestabucil); bisantrene (bisantrene); edatraxate (edatrexate); ifosfamide (defosfamide); desmethylcolchicine (demecolcine); deaquinone (diaziquone); difluoromethyl ornithine (DMFO); efluoornithine (eflornithine); ammonium elide (elliptinium acetate); etoposide (etoglucid); gallium nitrate; hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mo Pi dipyridamole (mopidamol); nitrocline (nitrocrine); penstatin (penstatin); egg ammonia nitrogen mustard (phenol); pirarubicin (pirarubicin); podophylloic acid (podophyllinic acid); 2-ethyl hydrazide (ethyl hydrazide); procarbazine (procarbazine); polysaccharide K (PSK, krestin); raschig (razoxane); cilzopran (silzofiran); spirogermanium (spirogermanium); tenuazonic acid; trinquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; a urethane; vindesine (vindeline); dacarbazine (dacarbazine); mannomustine (mannomustine); mi Tuobu Luosol (mi) tobronitol); mitolactol (mitolactol); pipobromine (pipobroman); a gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil (chloramucil); gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine (mercaptopurine); methotrexate (methotrexate); etoposide (VP-16); ifosfamide (ifosfamide); mitomycin C; mitoxantrone (mitoxantrone); vincristine (vincristine); vinorelbine (vinorelbine); novibine (Navelbine); novanone (Novantrone); teniposide (teniposide); daunomycin (daunomycin); aminopterin (aminopterin); hilded (Xeloda); ibandronate (ibandronate); CPT-11; retinoic acid; epothilones (esperamicins); capecitabine (capecitabine); topoisomerase inhibitors such as irinotecan (irinotecan). Any of the above pharmaceutically acceptable salts, acids or derivatives may also be used.
The chemotherapeutic agent may be administered as a prodrug. Examples of prodrugs that may be administered with the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific such as bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, or optionally substituted phenylacetamide-containing prodrugs, as well as 5-fluorocytosine and other 5-fluorouridine prodrugs, for example, that may be converted to more active non-cytotoxic drugs. TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific such as bispecific TNFR1 antagonist/TNFR 2 agonist constructs may be provided as prodrugs, for example, by linking them to a targeting agent that targets a specific tissue or site of disease with an in vivo cleavable linker, thereby releasing the active form construct.
In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, such as bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein are administered with one or more antibiotics, including, but not limited to: aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycin, butirisin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromycin, ribostamycin, sisomicin, and spectinomycin), aminocyclools (e.g., spectinomycin), aminophenol antibiotics (e.g., azidothalamycins), chloramphenicol (chlororamhenicol), florfenicol (florfenicol), and thiamphenicol), ansamycin antibiotics (e.g., li Fumi t (paromycin), and penmycins (spinmycin)), and penmycins (e.g., pennisin), and panipenemin (panipenem); cephalosporins (e.g. cefaclor), cefadroxil, cefamandole, ceftriaxone, cefazedone, ceftizoxime, cefmetazole, cefpiramide, cefpirome, cefprozil, cefuroxime, cefalexin, and ceftetan); lincomides (e.g., clindamycin and lincomycin); macrolides (such as azithromycin (azithromycin), brefeldin (brefeldin) a, clarithromycin (clarithromycin), erythromycin (roxithromycin), roxithromycin (roxithromycin) and tobramycin); monolactones (e.g., aztreonam (aztreonam), card Lu Mona (carumonam), and tigemonam); mupirocin (mupirocin); oxacephems (e.g., floxacef, latamoxef, and moxalatam); penicillins (e.g., ampicillin (amdinocillin), pimicillin (amdinocillin pivoxil), amoxicillin (amoxicillin), baccaracillin (bacampicillin), benicillin acid (benzylpenicillinic acid), sodium benzyl penicillin (benzylpenicillin sodium), epicicillin (epicicillin), fen Bei Xilin (fenbenicillin), fluxacillin (floxacillin), pencicillin (pencicillin), hydroiodic acid sandbicillin (penethamate hydriodide), pencicillin (pencicillin O-bennethamine), penicillin O, penicillin V benzoate, hydrabamillin V (penicillin V hydrabamine), penciclin (pencicillin), and non-nesilk (phenethicillin potassium)); polypeptides (e.g., bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin); quinolones (e.g., amifloxacin, ciprofloxacin, enoxacin, enrofloxacin, ofloxacin, fluroxacin, fluquindox, gatifloxacin, gemifloxacin, lattice Lei Pasha star, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, pirafloxacin, paxifloxacin, and tolvafloxacin); rifampin; streptogramins (e.g., quinupristin and dalfopritin); sulfonamides (e.g., sulfa and sulfamethoxazole); and tetracyclines (for example, chlortetracycline (chlortetracycline), norchlortetracycline hydrochloride (demeclocycline hydrochloride), norchlortetracycline (demethylcycline), doxycycline (doxycycline), duramycin (Duramycin), minocycline (minocycline), neomycin (neomycin), oxytetracycline (oxytetracycline), streptomycin (streptomycin), tetracycline (tetracycline), and vancomycin).
In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein may be administered with one or more antifungal agents including, but not limited to, amphotericin (amphotericin) B, ciclopirox, clotrimazole, econazole, fluconazole (fluconazole), fluconazole, itraconazole (itraconazole), ketoconazole (ketoconazole), miconazole (miconazole), nystatin, terbinafine (terbizole), terbizole (terconazole), and tioconazole (tioconazole). In some examples, the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, and nucleic acids provided herein are administered with one or more antiviral agents, including but not limited to protease inhibitors, reverse transcriptase inhibitors, and the like, including type I interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir (acyclovir), adefovir (adefovir), amantadine (amantadine), amprenavir (amprenavir), clavulanavidine (clevidine), enfuvirtide (enfuvirtide), entecavir (entecavir), foscarnet (foscarnet), ganciclovir (ganciclovir), idoside (idoxidine), indinavir (lopinavir), lopinavir (plica), ribavirin (ribavirin), and fludrovir (fludrovir).
The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein can be administered in combination with a growth factor trap construct described below, and also in combination with any of the therapeutic anti-TNF agents and therapies described below, for combination therapy with a growth factor trap construct. The combination therapy may also include a growth factor trap construct provided herein.
Pharmaceutical compositions containing the TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein can be used to treat any of the diseases, disorders, and conditions described herein or known to those of skill in the art. The diseases, disorders and conditions include one or more chronic inflammatory, autoimmune, neurodegenerative or demyelinating diseases or disorders. Also provided are combinations of the polypeptides and constructs provided herein with another treatment or compound for treating chronic inflammatory, autoimmune, neurodegenerative or demyelinating diseases or disorders. The TNFR1 antagonist constructs, TNFR2 agonist constructs, multispecific, e.g., bispecific TNFR1 antagonist/TNFR 2 agonist constructs, fusion proteins, and nucleic acids provided herein, as well as additional agents, can be packaged as separate compositions for administration together, or sequentially or intermittently. Alternatively, they may be applied as a single composition, or two compositions applied as a single composition. The combination may be packaged as a kit, optionally with additional reagents, instructions for use, vials and other containers, syringes, and other items for use in therapy.
L. examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Expression and evaluation of candidate monovalent TNFR1 antagonist molecules
Expression and purification of anti-TNFR 1 molecules
The candidate monovalent TNFR1 antagonist molecule was expressed in mammalian cells under the control of a CMV promoter using the expression plasmid depicted in fig. 1 (where TE19080L is an inserted fragment). For expression of protein therapeutics, e.g., constructs herein, mammalian cells, e.g., chinese Hamster Ovary (CHO) or human embryonic kidney 293 (HEK 293) cells, are used to provide post-translational modifications, including glycosylation, which may be important for proper protein structure, function, and activity. Expression in bacterial, yeast or insect cells results in no glycosylation (for bacterial cells) or in a different glycosylation pattern (for yeast or insect cells) than expression in mammalian cells. Expression in bacteria can also lead to contamination of protein therapeutics with bacterial endotoxins, which can activate innate immune cells, complicate cell-based assays, and lead to pyrogenic effects upon in vivo administration.
UsingExpression plasmid construction and transient and stable cell line expression were performed as described by Vazquez-Lombardi et al (2018) Nat. Protoc.13 (1): 99-117. Five exemplary TNFR1 antagonist molecules were generated for initial evaluation. TNFR1 antagonist molecules comprise H398-derived scFv (SEQ ID NO: 678), containing one V of H398 L And a V H Domain, pass (GGGGS) 3 Peptide linkers are linked together; TNFR1 antagonist domain antibody (dAb) DOM1h-574-16 (SEQ ID NO: 57); TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58); fusion protein (SEQ ID NO: 704), containing dAb DOM1h-574-208 (SEQ ID NO: 54) as a TNFR1 antagonist from DMS5541 (described elsewhere herein), by (GGGGS) 3 The peptide linker was fused to an antiserum albumin dAb (albudAb) of DMS5541 (DOM 7h-11-3; SEQ ID NO: 52); and fusion proteins (SEQ ID NO: 705), comprising an anti-TNFR 1 dAb called DOM1h-131-206 (SEQ ID NO: 59), by (GGGGS) 3 The peptide linker was fused to an antisera albumin dAb (albudAb or DOM7h-11-3; SEQ ID NO: 52) of DMS 5541. Insertion (GGGGS) in fusion protein H398scFv and DOM1H-574-208 and DOM1H-131-206 3 Joints at V respectively H And V L The domains and between the two dabs provide greater flexibility and increase stability and resistance to denaturation of the molecule, improving the production process. The sequences of each of these TNFR1 antagonist molecules are provided in Table 12 below, some of which have different linker sequences (see also Enever et al, (2015) Protein Engineering, design&Selection 28 (3): 59-66, which describes dAbs and modifications thereof, can be used for further modification and addition of linkers and modifiers.
Table 12
Nanobodies and nanobody-containing constructs comprising two heavy chains selected from any of the sequences shown in SEQ ID NOS 53-83 and 503-671, such as SEQ ID NOS 57-59, and variants thereof having at least 95%, 96%, 97%, 98%, 99% sequence identity are also provided. Constructs comprising the same heavy chain are provided. These examples are TNFR1 dAbs called DOM1 h-131-206. Constructs comprising any of these dabs, such as those directly linked or more generally linked to human serum albumin by a linker such as a GS linker or provided as Fc fusions, or any other construct described herein, are also provided.
These and other such dabs and TNFR1 binding molecules can be modified to increase specificity for TNFR1 by eliminating any antagonistic activity for TNFR2, and/or to increase or add TNFR2 agonist activity, and/or can be modified to reduce or eliminate immunogenic epitopes, and/or can be linked to activity modulators, such as Fc units and modified Fc units/modified Fc dimers, and/or serum half-life extending moieties.
HEK293 cell lines are used for transient expression and after in vitro assessment of expressed antagonists and identification of molecules with desired properties, e.g. high affinity for TNFR1 (e.g. K d <50nM, or<10nM, or<5 nM), and potent inhibition of TNFR1 signaling (e.g., IC) 50 <50nM, or<10nM, or<5 nM), stable cell lines were prepared in derivatives of CHO cells. Typically, it is picomolar (pM) affinity, for example about 19pM affinity or 20pM, 15pM, 10pM, 5pM, 2pM or 1pM affinity.
In CHO DG44 cells (e.g., CHO-DG44 (DHFR) - ) And FreeStyle TM CHO-S cells, invitrogen) to generate a transfection library, which is screened to identify or select high expression clones. Non-polyhistidine or other purification tags are used. In contrast, the proteins used for screening were purified from serum-free medium by HPLC in combination with other well-known methods. The matrix for HPLC was Amsphere TM Protein A3 chromatography resin (JSR Life Sciences), or other similar resins following manufacturer's protocols. If the protein is not at least 95% pure as judged by size exclusion HPLC, further purification (e.g., ion exchange or hydrophobic chromatography) is performed.
Endotoxin removal (see, e.g., vazquez-Lombardi et al (2018) for an exemplary protocol). After protein purification, endotoxin levels are determined using a detection kit such as QCL-1000Endpoint Chromogenic LAL Assay Kit (Lonza). For the removal of the endotoxin, The theoretical pI of the purified protein is determined using a sequence analysis tool (e.g., exPASy ProtParam) and the pH low-endotoxin PBS buffer is adjusted to a pH below but near the theoretical pI of the purified protein. The protein samples were then dialyzed against at least 30 volumes of pH adjusted PBS at 4℃for at least 2 hours. An additional dialysis step was performed overnight, followed by a second dialysis for at least 2 hours on the next day. The sample was then purified using anion exchange affinity chromatography, retested to determine endotoxin levels, and the process repeated until acceptable endotoxin levels were reached. The size and purity of the protein product is determined by SDS-PAGE analysis or other suitable method. Alternatively, other methods may be used, such as Proteus Endotoxin Removal Kit and the accompanying manufacturer manual (BIORAD, see bio-rad-anti-I)Cams/static/uploads/ifu/pur030. Pdf). This step can be repeated until the desired level of endotoxin is reached, typically>0.5 endotoxin units/ml (less than or equal to 0.5 endotoxin units/ml).
Screening of purified proteins
Purified TNFR1 antagonist molecule candidates are screened to measure binding affinity to the extracellular domain of TNFR1 using methods described in detail above, or methods known in the art, such as immunoassays (e.g., ELISA), surface Plasmon Resonance (SPR), isothermal Titration Calorimetry (ITC), or other kinetic interaction assays known in the art. SPR can be performed using a variety of commercially available platforms, such as the BIAcore system (GE Healthcare Life Sciences). Exemplary assays are described, for example, in Lang et al (2015) J.biol Chem 291:5022-5037, wherein various assays for assessing binding affinity are described and compared. The selected candidates include those having K d Value of<Those of 5 nM.
TNFR1 antagonists are also screened to determine whether binding to TNFR1 is competitive or non-competitive with respect to TNF using methods known in the art such as SPR. If the inhibitor binds to a receptor (e.g., TNFR 1) and blocks binding of a ligand (e.g., TNF), for example by attachment to an active site, this is a competitive inhibition, as the inhibitor "competes" with the substrate for the enzyme; that is, only the inhibitor or substrate may be bound at a given time. In non-competitive inhibition, the inhibitor does not block ligand binding to the ligand binding site on the receptor. Instead, it attaches to another site and blocks the receptor from responding to the bound ligand. This inhibition is referred to as "non-competitive" because the inhibitor and substrate can be bound simultaneously. Thus, if the ligand is added to the receptor binding assay at a saturated concentration and it does not inhibit binding of the antibody, then the two molecules are independent and non-competitive. And vice versa. If both are competitive, increasing the antibody concentration will prevent TNF from binding to the receptor. This is effective both for cells and for binding assays that bind receptors or ligands on the surface. For exemplary assays, see, e.g., frey et al (2001) Current Protocols in Neuroscience, "Receptor Binding Techniques", available from doi.org/10.1002/0471142301.Ns0104s00.
For example, the ability of TNF to bind to human TNFR1 coated on a BIAcore chip was first determined. The TNFR1 surface was then saturated with TNFR1 antagonist molecules, followed by TNF injection and re-assessment of TNF binding to TNFR 1. If the binding is non-competitive, then it binds to TNFR 1; if competing, they interfere with each other. Binding of the antagonist to TNFR1 is non-competitive with respect to TNF if TNF binding is not affected, or only slightly reduced, and is considered competitive with respect to TNF if TNF binding is abrogated or significantly reduced. For purposes herein, competitive binders are selected.
The TNFR1 antagonist molecules are further screened to determine their ability to inhibit TNFR1 signaling in the presence of TNF on cells using methods known in the art, such as those described by McFarlane et al (2002) FEBS Lett.515 (1-3): 119-126, which results in activation of NFkB-luciferase expression (i.e., reporter assay). The cells used in these experiments do not express TNFR1 or TNFR2 (e.g., myeloma Cell lines AMO1, U266, and L363; see, e.g., rauert et al (2011) Cell Death Dis.2 (8): e 194), unless TNFR1 or TNFR2 is expressed Plasmid transfection of TNFR 2. Alternatively, a human cell line may be used in which the TNFR1 and/or TNFR2 genes are inactivated or knocked out using CRISPR vectors, antisense RNA expression, or other methods known in the art. TNFR1 from commercial sources (e.g., genoway and Synthesis) can also be used - And/or TNFR2 - A cell line. These cell lines can then be transfected specifically with TNFR1 and/or TNFR2 expression cassettes. For example, cells expressing TNFR1, TNFR2, or TNFR1 and TNFR2 can be used to evaluate the selectivity of an antagonist for TNFR1 and determine the efficacy of inhibiting TNF signaling by TNFR 1.
To determine the inhibition of TNFR1 signaling by TNFR1 antagonists, cells expressing human TNFR1 were transiently transfected with NF-. Kappa.B-luciferase reporter constructs using Lipofectamine and receptor stimulated luciferase transcription was measured 48 hours after transfection. Cells stably expressing TNFR1 and NF- κB-luciferase were grown at 1×10 5 The density of individual cells/ml medium was inoculated into 24-well plates and incubated until 80% confluence was reached (about 24 hours). The cells were then incubated with 50ng/ml TNF and varying concentrations of TNFR1 antagonist for 6 hours. By washing the cells twice with ice-cold PBS, 200. Mu.l of ice-cold lysis buffer (25 mM Tris-phosphate pH 7.8,8mM MgCl was added 2 1mM DTT, 1% Triton X-100, 15% glycerol) and incubated on ice for 5 minutes, whereby NF- κB stimulated luciferase activity was detected. The cell extract was then scraped into a 1.5ml Eppendorf tube, centrifuged to pellet the cell debris, and 100 μl of the supernatant was used to measure luciferase induction using a luminometer. IC is then calculated by plotting Relative Luminescence Units (RLU) versus TNFR1 antagonist concentration and using curve fitting software such as GraphPad Prism 50 . Other similar assays for assessing inhibition of TNFR1 signaling include assays measuring induction of phosphorylated-iκbα, which indicate activation of the classical NF- κb pathway in TNFR 1-expressing cells treated with TNF (see, e.g., rauert et al (2011) Cell de ath dis.2 (8): e 194).
Selecting a TNFR1 signal inhibition and IC that exhibits at least 80% as determined above 50 The value is approximately equal to Kd (i.e<5 nM) of TNFR1 antagonist candidate was used for further optimization.
Example 2
Optimization of selected candidate monovalent TNFR1 antagonist molecules
Optimization of affinity for TNFR1 and efficacy of TNFR1 signaling inhibition
For satisfying the above selection criteria (i.e., high affinity for TNFR1 and effective inhibition of TNFR1 signaling, K) d And IC 50 Value of<5 nM) was optimized to increase affinity for TNFR1 and efficacy of TNFR1 signaling inhibition. This is accomplished by a method comprising one or more of random mutagenesis, site-directed mutagenesis, molecular modeling and/or error-prone PCR to achieve K d And IC 50 Low value of<1nM, usually at least equal to or<100nM、<50 nM、<10 nM、<Or 5 nM. This can be achieved, for example, by the following method (see Tiller et al (2017) front immunol.8:986), where the amino acids most critical for binding are conserved, whereas V H The remaining amino acids of the domain were subjected to mutagenesis and phage libraries were prepared and selected for high affinity binding variants to TNFR 1. According to this method, phage display libraries for selecting such variants are generated. In the first step, the calculation and experimental alanine scanning mutagenesis identified sites in the Complementarity Determining Regions (CDRs) that allow mutagenesis while maintaining antigen binding. Next, based on natural antibody diversity, the most permissive CDR positions are mutated using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position. This mutagenesis approach results in antibody libraries having a large number of CDR mutated variants, including antibody domains with single mutations, as well as other antibody domains with tens of mutations. In the last step, the library displayed on the yeast surface (about 1000 ten thousand variants) was panned to identify CDR mutations with the most increased affinity.
Half-life extension
The optimized molecule is then linked to a half-life extending moiety, for example by fusion with an IgG Fc domain, particularly a modified Fc domain, or Human Serum Albumin (HSA), or by pegylation, as described in the detailed description above, to achieve an in vivo serum half-life of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days, for example 10-12 days in SCID mice. The modified molecules were then retested in the in vitro assay described above to ensure retention of high affinity binding to TNFR1 and effective inhibition of TNFR1 signaling. The production of successful candidates is amplified for further in vitro and in vivo assays.
In vitro assay
Phagocytosis assay
As discussed and described in the detailed description, existing TNF blockers that inhibit TNFR1 activity also inhibit TNFR2.TNF blockers have two black box warnings from regulatory authorities. The first is the susceptibility of anti-TNF (TNF blocker) treated patients to infection. These infections may involve various organ systems and sites due to bacteria, mycobacteria (e.g. tuberculosis), fungi (e.g. histoplasmosis, aspergillosis, candidiasis, coccidiosis, blastomycosis and pneumosporosis), viruses (e.g. hepatitis b) and other opportunistic pathogens (organisms that normally do not cause disease in healthy humans but cause severe disease when the immune system (resistance) of the human is weakened). Another published Black Box alert is related to childhood malignancy (see, e.g., online. Eporates. Com/u/10b 3301/Hum-ira/Black+Box+Warning). As discussed in the detailed description, this weakness is due to the complete blockage of TNFR1 and TNFR2 signaling when its ligand TNF is blocked. This inhibition of innate immunity to TNF is mediated by TNFR2 (see, e.g., ahmad et al (2018) front. Immunol. 9:2572), and is mediated primarily by transmembrane forms of TNF that preferentially activate TNFR2 (see, e.g., miller et al (2015) Journal of Immunology (195) (6): 2633-2647).
anti-TNF therapies, such as adalimumab (adalimumab), infliximab (infliximab) and etanercept (etanercept), have been shown to have an inhibitory effect on IFN-gamma induced phagosome maturation in phorbol myristate acetate differentiated human THP-1 cells. Adalimumab and infliximab, but not etanercept, inhibit phagosome maturation in primary human peripheral blood mononuclear cell-derived macrophages in the presence or absence of IFN- γ (see, e.g., harris et al (2008) j. Effect. Dis. 198:1842-1850). In view of the above, an advantage of certain TNFR1 inhibitor constructs is that TNFR2, and therefore macrophage, function is preserved; macrophages are important for clearing the organism of opportunistic infections. Opportunistic pathogens include tuberculosis-causing mycobacteria (see, e.g., fraga et al (2018) Curr. Issues mol. Biol. 25:169-198). In autoimmune diseases in patients receiving TNF blocker therapy, macrophage function is disrupted. TNFR 1-specific antagonists inhibit TNFR1 function only, thereby avoiding TNFR2 function required to disrupt normal macrophage function. These TNFR 1-specific antagonists can be identified by measuring the effect of anti-TNF on macrophage phagocytosis. Among the identified antagonists specific for TNFR1, some also stimulate TNFR2 function, thereby enhancing macrophage activity (TNFR 1 antagonists and TNFR2 agonists).
Determination of the Effect of TNFR1 antagonists on macrophage response to Mycobacterium tuberculosis infection
TNF plays an important role in mediating inflammatory host responses to a variety of pathogens, including mycobacterium tuberculosis; TNF also plays a role in the immunopathology of Tuberculosis (TB). Mycobacterial infection induces macrophages to secrete TNF. TNF enhances the ability of macrophages to phagocytose and kill mycobacteria. TNF also stimulates macrophage apoptosis, resulting in increased killing and presentation of mycobacterial antigens by dendritic cells. TNF is also required for granuloma formation and maintenance; in mice infected with long-term mycobacterium tuberculosis, neutralization of TNF can disrupt granulomatous integrity, exacerbating the infection and increasing mortality. TNF blockers, such as adalimumab and infliximab, increase susceptibility to infection by various pathogens including mycobacterium tuberculosis (m. Tuberculosis) and increase the risk of reactivation of latent tuberculosis by inhibiting phagosome maturation containing mycobacteria in human macrophages. Phagosome maturation (i.e., phagosome acidification and fusion with lysosomes) is critical for presenting mycobacterial antigens to T cells and initiating adaptive immune responses (see, e.g., harris et al (2008) j. Effect. Dis. 198:1842-1850).
To identify and/or characterize TNFR1 antagonists, the effect of TNFR1 antagonists provided herein on macrophage response to mycobacterium tuberculosis infection was evaluated. Phagosome maturation with mycobacteria in human macrophages was analyzed and compared to phagosome maturation in TNF blockers such as adalimumab using the method described by Harris et al (2008) j. Effect. Dis.198:1842-1850. TNFR1 antagonists that do not increase susceptibility to infection are of great interest compared to known TNF blockers such as adalimumab.
Preparation of THP-1 cells and monocyte-derived macrophages (MDMs)
Human THP-1 cells were cultured in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS; gibco). Cells were differentiated into macrophage-like cells by treatment with 100nmol/L Phorbol Myristate Acetate (PMA) for 24 hours, and then cultured in normal medium for 3 days. For the preparation of human monocyte-derived macrophages (MDM), in-Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood of healthy donors using density gradient centrifugation at 1077 (Sigma). Monocytes were isolated by adhesion to gelatin-coated dishes and cultured overnight in RPMI 1640 containing 5% human AB serum (Sigma). Adherent cells were removed with 10mmol/L EDTA in PBS and grown on 12-well plate coverslips for 10 days. THP-1 cells and MDM were treated at 2X 10 5 The concentration of individual cells/wells was grown on coverslips.
Preparation of Mycobacteria
Green Fluorescent Protein (GFP) labeled Mycobacterium bovis BCG (GFP-BCG) and attenuated Mycobacterium tuberculosis (M.tuberculosis) strain H37Ra and its toxic counterpart H37Rv were grown in Middlebrook 7H9 broth containing 0.5% Tween, 0.2% glycerol and 10% albumin-glucose-catalase supplement (BD). The mycobacteria were grown to log phase prior to use and resuspended in RPMI 1640 containing 10% FBS prior to infection. The Mycobacterium tuberculosis strain H37Ra was fluorescently labeled with PKH67 (Sigma) and strain H37Rv was labeled with fluorescein isothiocyanate (FITC, 1mg/mL; sigma) according to the manufacturer's protocol.
Determination of phagosome maturation
Mycobacteria can inhibit fusion of phagosome with lysosomes, preventing acidification and recruitment of lysosomal hydrolases. This blocking of phagosome maturation can be overcome by pre-treating macrophages with IFN-gamma. Treatment of mycobacterial infected cells with TNF (5 ng/mL) also enhanced phagosome acidification. To determine the effect of TNFR1 antagonists and TNF blockers on lysosomal fusion of mycobacterial phagosome to macrophages, PMA-differentiated THP-1 cells or human peripheral blood MDM were infected with GFP-BCG or PKH 67-labeled Mycobacterium tuberculosis strain H37Ra or FITC-labeled Mycobacterium tuberculosis strain H37Rv, with or without IFN-gamma treatment in the presence of TNFR1 antagonists or TNF blockers, and phagosome-lysosomal fusion was analyzed by confocal microscopy, using Red (LT) as a marker of acidified phagosome, CD63 and cathepsin D as phagolysosome markers. Inhibition of IFN-gamma-induced phagosome maturation/acidification by +.>Co-localization of Red, CD63 or cathepsin D-labeled mycobacteria. For example, a reduced co-localization percentage of LT, CD63 or cathepsin D-labeled mycobacteria compared to a control indicates inhibition of IFN- γ -induced phagosome acidification.
Prior to infection, a TNFR1 antagonist or TNF blocker (e.g., adalimumab, infliximab, etanercept, or otherwise; 10 μg/mL), with or without IFN- γ (200U/mL), is added to THP-1 cells or MDM for 24 hours. As controls, cells were treated with medium alone or 10. Mu.g/mL human IgG1 (Calbiochem) from an IgG1 producing myeloma patient. Cells were then infected with mycobacterium bovis GFP-BCG, PKH 67-labeled mycobacterium tuberculosis H37Ra or FITC-labeled mycobacterium tuberculosis H37Rv for 15 minutes, washed 3 times with PBS to remove unbound mycobacterium, and incubated for 2 hours. The multiplicity of infection (MOI) was recorded microscopically 15 minutes after infection of macrophages by acid fast bacterial staining. Approximately 70% of the cells are infected with 1-5 bacilli at MOI.
After 2 hours of incubation, cells were fixed in 2% paraformaldehyde for 20 minutes at Room Temperature (RT); for strain H37Rv, cells were fixed in 4% paraformaldehyde overnight. Cells were then permeabilized with 0.1% Triton X-100 in PBS and blocked at RT for 30 min with 1% bovine serum albumin and 1% goat serum in PBS. Cells were incubated with primary antibodies (1. Mu.g/mL mouse monoclonal antibody against CD63 (LAMP-3;Santa Cruz Biotechnology), or 10. Mu.g/mL mouse monoclonal antibody against cathepsin D (Calbiochem)) for 1 hour at RT, followed by incubation with secondary antibodies (4. Mu.g/mL Alexa Fluor 488 or 568-labeled goat anti-mouse IgG; invitrogen) for 1 hour at RT. Alternatively, the cells are contacted with the cells prior to fixationRed DND-99 (100 nmol/L; invitrogen) was incubated with the Mycobacteria for 60 minutes.Red DND-99 is a Red fluorescent dye used to label and track acidic organelles (e.g., acidified phagosomes) in living cells.
Coverslips are mounted on slides with fluorescent coverslips (Dako) and images of a laser scanning confocal microscope, e.g. Olympus FluoView, are recorded TM 1000 and Zeiss LSM 510 laser scanning confocal microscopes. Images were analyzed and prepared using appropriate software and Adobe Photoshop.
Measurement of TNF
THP-1 cells were prepared as described above and infected with BCG or Mycobacterium tuberculosis H37Ra, with or without IFN-gamma pretreatment. The level of immunoreactive TNF (secreted in response to mycobacterial infection) in the supernatant was measured using a commercial ELISA kit (R & D systems) according to the manufacturer's instructions.
Differential TNF blocking (e.g., adalimumab, infliximab or etanercept treatment) and specific TNFR1 inhibited regulatory T cell (Treg cell) assay and cytokine assay
Preservation of FoxP3 expression
TNF blockade (using adalimumab, rituximab, or etanercept) was compared to specific TNFR1 inhibition of FoxP3 promoter methylation as an alternative marker for functionally regulatory T cells. Transgenic mice constitutively expressing human TNFR1 (HuTNFR 1) were used to evaluate the different effects of TNF blocking on FoxP3 promoter methylation from specific TNFR1 inhibition. Transgenic mice are prepared by standard methods, and can be prepared by contractor service or any method, for example by Cyagen, genoway or Polygene.
This effect was evaluated in transgenic mice with collagen-induced arthritis (CIA), a widely used model of RA. Having a C57/BL6N.Q; mice with H-2q/HuTNFR1/Hunt background were prepared from either Genoway or Tacouc Labs. Mice will be immunized with bovine type II collagen emulsified in Complete Freund's Adjuvant (CFA) as described by Tseng et al ((2019) Proc. Natl. Acad. Sci. U.S. A.116:21666-21672). The determination of FoxP3 methylation was also performed as described in Tseng et al (2019) Proc.Natl. Acad.Sci.U.S.A.116:21666-21672). Regulatory T cells from TNF blocking treated mice expressed lower levels of FoxP3 than regulatory T cells with specific TNFR1 blocking, as determined by Median Fluorescence Intensity (MFI) and histogram of FoxP3 in cd4+cd25+ cells.
Specific inhibition of tnfr1 and TNF blockers in reserve regulatory T cells
The number of regulatory T cells in lymph nodes and spleen was compared after treatment of transgenic (C57/BL 6N.Q; H-2q/HuTNFR 1/Hunt) CIA mice with a TNF blocker and a TNFR1 specific inhibitor using the method described by McCann et al (2014) Arthris & Rheumatology 66 (10): 2728-2738).
3. Inflammatory cytokines up-regulated in TNF blockade and specific TNFR1 inhibition in collagen-induced arthritis
Transgenic mice (C57/BL 6N.Q; H-2q/HuTNFR 1/Hunt) with collagen-induced Arthritis (CIA) (see, e.g., mcCann et al (2014) Arthritis & Rheumatology 66 (10): 2728-2738) are treated with a TNF blocker and a TNFR 1-specific antagonist. Serum inflammatory cytokines (IFN-. Gamma., IL-12p70, IL-10, RANTES (CCL 5) were evaluated, see, e.g., mcCann et al (2014) Arthritis & Rheumatology 66 (10): 2728-2738). Antagonists that specifically block TNFR1 induce a significant reduction in one or more of IFN-gamma, IL-12p70, IL-10, or RANTES (CCL 5). This is due to spare TNFR2 function and regulatory T cell function in the spleen and lymph nodes.
In vivo assays
Studies were performed using humanized (HuTNFR 1/HuTNF) transgenic mice. The efficacy of TNFR1 antagonist molecules in various autoimmune disease models was evaluated. These models include the above models, which express human transgenes for TNFR1 and TNF. Alternative models of autoimmune diseases are known. For example, schinnerling et al (2019) front. Immunol 10:203) describe models in detail, including the RA model. To determine efficacy, specific TNFR1 antagonist constructs as well as other constructs provided herein were tested in more than one model. At least Rheumatoid Arthritis (RA), crohn's disease and multiple sclerosis (experimental autoimmune encephalitis) models (discussed in the detailed description) are included among the models.
Inflammatory bowel disease (IBD, including ulcerative colitis and crohn's disease) is the first indication of TNF blockers. A number of mouse models of these diseases are available and have been described by Mueller ((2002) Immunology 105 (1): 1-8). As discussed in the detailed description, autoimmune neurodegenerative diseases, including Multiple Sclerosis (MS) and alzheimer's disease, are important disease targets. Alzheimer's Disease (AD) is a major cause of dementia worldwide and is one of the most serious health problems for the elderly. It is estimated that 540 million americans suffer from AD, and this figure is expected to be tripled by 2050 if there is no medical breakthrough to stop, prevent or slow the disease (see e.g., chang et al (2017) j.cent.nerv.syst.dis.9: 1179573517709278). Evidence suggests that TNFR1 antagonist constructs and other constructs provided herein are therapeutic candidates because subjects who have undergone prolonged treatment with TNF blockers are less likely to suffer from this disease (see, e.g., chou et al (2016) CNS Drugs 30:1111). The data indicate that up-regulated TNF expression is associated with different neurodegenerative diseases and disorders, such as alzheimer's disease, parkinson's disease, stroke, and multiple sclerosis (see, e.g., mcCoy et al (2008) j. Neuroisframmation 5 (1): 45). However, existing TNF blockers appear to be ineffective in treating, ameliorating, preventing or slowing the progression of disease (see, e.g., tontarolo et al (2015) J. Neurochem. 135:109-124). As described herein, various evidence suggests that TNF blockers do not play a role in such indications due to the co-inhibition of TNFR1 and TNFR 2; TNFR2 has neuroprotective properties lost by TNF blocker treatment. Others have attempted to solve this problem with various forms of "TNFR1 inhibitors" or "TNFR2 agonists". None of these studies included cross-reactivity to determine whether TNFR1 or TNFR2 was selectively targeted, rather than as one of many epitopes that can be targeted in vivo.
This problem is solved herein. First, a family of anti-TNFR 1 antagonists was generated and tested in the above model, indicating that their effect was consistent with that expected. Then, they were proved to be selective using immunochemistry. Some contract research laboratories offer this service (e.g., sino Biological, inc. and LSBio).
As discussed in the detailed description, other autoimmune and chronic inflammatory disease states are associated with the presence of TNF. These include type II diabetes and endometriosis. Mouse models of these diseases are known and the HuTNFR1/HuTNF transgenic versions of these mice are used to demonstrate the efficacy of the specific anti-TNFR 1 antagonists provided herein.
Acute respiratory distress syndrome
Respiratory viral pathogens (e.g., influenza, SARS virus/coronavirus) infect respiratory epithelial cells, while tissue resident alveolar macrophages are the first responders to pulmonary viral infection. They achieve clearance by opsonizing phagocytosis of viral particles or infected apoptotic cells and release of excessive inflammatory cytokines and chemokines to initiate an immune response (see, e.g., herord et al (2015) Eur. Resp. J. 45:1463-1478). TNF blockers have been shown to prolong survival of influenza infected mice (see, e.g., shi et al (2013) crit. Care 17: r 301). TNF blockers have been used to treat SARS-Cov 2 (see, e.g., feldmann et al (2020) Lancet 395:1407-1409). As described in the detailed description, the known effect of TNF blockers is to reduce regulatory T cells (tregs), which is problematic because tregs are natural inhibitors of inflammation. Thus, as described and provided herein, inhibitors specific for TNFR1 that do not interact with TNFR2 or agonize TNFR2 are superior for the stated purpose. To test the constructs provided herein, mice were engineered to lack endogenous TNFR1 and express HuTNFR1/HuTNF. These mice will exhibit acute respiratory distress syndrome induced by HuTNF/HuTNFR1 following influenza infection (as described by Shi et al (2013) crit. Care 17: R301). A TNFR 1-specific antagonist construct that does not antagonize TNFR2 is administered. Efficacy is determined by any of several criteria:
1. Circulating inflammatory cytokines (e.g., IFN-gamma, IL-1α, IL1- β, and IL-17) significantly reduced relative to TNF blockers;
2. significantly faster recovery as measured by weight gain (see, e.g., shi et al (2013) crit. Care 17: r 301) compared to TNF blockers; and
3. significantly increased survival compared to TNF blockers (see, e.g., shi et al (2013) crit. Care 17: r 301).
Transgenic mice expressing human TNFR1 and human TNFR2 for use in disease models are produced by standard genetic engineering methods known in the art, such as those described by Atretkhany et al (2018) Proc.Natl. Acad.Sci.U.S.A.115 (51): 13051-13056. Specific in vivo assays for various diseases and conditions are as follows. For example, a humanized RA mouse model, such as a collagen-induced arthritis (CIA) model of RA, or any other RA animal model known in the art and/or described herein, is used to evaluate the therapeutic effect of the TNFR1 antagonist molecules provided herein and compare it to the therapeutic effect of an anti-TNF therapy, such as etanercept or adalimumab. The TNFR1 antagonist molecules or anti-TNF therapies provided herein, such as etanercept or adalimumab, are administered to the animals daily for a total of 10 days after the occurrence of clinical arthritis in one or more limbs. The extent of swelling of the initially affected joint was monitored by measuring the jaw thickness using calipers. Serum was taken from mice and used to measure pro-inflammatory cytokines and chemokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), IL-1β, IL-6, IL-8, RANTES (CCL 5), and monocyte chemotactic protein 1 (MCP-1; also known as CCL 2). The resolution of RA in the mouse model was then compared for mice administered with TNFR1 antagonist molecules and mice administered with etanercept or adalimumab.
The TNFR1 antagonist molecules provided herein were also tested in a humanized mouse model of Severe Acute Respiratory Syndrome (SARS) and virus-induced cytokine storm. For example, the SARS mouse model is obtained by administering different doses, e.g., 10 2 、10 3 、10 4 And 10 5 SARS-CoV infection of individual Plaque Forming Units (PFU) was generated by knocking-in (knock-in) mice with humanized hTNFR1 (or hTNFR1/hTNFR 2). The survival rate was assessed by administering the TNFR1 antagonist molecules to infected mice and compared to the survival rate of mice administered anti-TNF therapy, such as adalimumab.
Viral-induced cytokine storm mouse models include, for example, lymphocytic choriomeningitis virus (LCMV) -induced Cytokine Storm Syndrome (CSS) models. LCMV-induced CSS mouse model was obtained by combining 2×10 5 LCMV-Armstrong of PFU was intraperitoneally administered to perforin defects (Prf -/- ) Or Prf-Tmem178 double knockout mice. To deplete monocytes/macrophages, 100 μl of chlorophosphonate liposomes were intravenously injected into Prf two days prior to LCMV infection and 48 and 96 hours after infection -/- In mice. Alternatively, 1mg of neutralizing anti-CSF 1 antibody (clone 5a1, bioxcell) was administered 2 days prior to infection, and 0.5mg of antibody was administered 48 and 96 hours later. Animals were bled by submandibular venipuncture at days 3 and 8 post-infection to measure serum cytokines (see, e.g., mahajan et al (2019) j.autoimmunimun.100:62-74).
Example 3
Identification and removal of immunogenic sequences
As described herein, immunogenic sequences in protein therapeutics, such as B-cell and/or T-cell epitopes, can negatively affect the activity, efficacy, and in vivo half-life of the therapeutic, for example, by forming anti-drug antibodies (ADA) that neutralize the therapeutic and/or accelerate its clearance from the body. Immunogenic sequences also are detrimental to the safety and tolerability of protein therapies, as they can induce adverse immune responses, leading to clinical complications such as delayed infusion-like allergic reactions, allergies, and in some cases life-threatening autoimmunity. Thus, candidate protein therapeutics are screened for immunogenicity and the identified immunogenic sequences are removed/replaced, e.g., by mutagenesis, to improve the in vivo efficacy and safety of the therapeutics and ensure successful transformation into the clinic from preclinical studies.
The immunogenic sequences are identified using methods such as those described in the detailed description, or known to those of skill in the art, including methods using computer immunogenicity prediction tools and in vitro immunogenicity testing, and the like. For example, linear B cell epitopes are predicted as described elsewhere herein using, for example, ABCPred, APCPred, BCPREDs, bepiPred, LBtope, bcePred, EPMLR, BEST, COBEpro and SVMTriP or any other available computer linear B cell epitope prediction tool described herein and/or known to those of skill in the art. Conformational B cell epitopes are predicted using, for example, CEP, discoTope, BEpro, elliPro, SEPPA, CBTOPE, EPITOPIA, EPCES, EPSVR, EPMeta, PEASE, epiPred, 3DEX, PEPOP, PEPOP 2.0 and EpiSearch or any other available computational mechanism as described herein and/or known to those of skill in the art, such as a B cell epitope tool. T cell epitopes are predicted using, for example, epiMatrix, janusMatrix, IEDB, SYFPEITHI, MHC Thread, MHCPred, MHCPred 2.0, epiJen, netMHC, netCTL, nHLAPred, SVMHC, proPred, MMBPred, protean 3D and Bimas or any other available computer T cell epitope prediction tool described herein and/or known in the art. The following is an exemplary analysis of the human TNFR1 antagonist DMS5541 sequence (SEQ ID NO: 38) directed against an immunogenic linear B cell epitope.
DMS5541 analysis of immunogenic linear B cell epitopes
The sequence of human TNFR1 antagonist DMS5541 (SEQ ID NO: 38) was analyzed for potential immunogenicity using the SVMTRIP algorithm to detect linear B cell epitopes within the molecule. The algorithm identified three possible epitopes in the sequence of DMS5541, as shown in table 13 below. The results indicate that an epitope of sequence AVKGRFTISRDNSKNTLYLQ having residues 63-82 corresponding to SEQ ID NO. 38 has a high probability of immunogenicity. The three epitopes identified were then tested for immunogenicity in an in vitro B cell assay. Alanine scans were performed on any sequences positive for immunogenicity. Positive amino acids/sequences were modified by substituting each amino acid in the sequence with an alanine residue one by one until the immunogenic epitope was destroyed. This results in safer and more potent TNFR1 antagonists.
As a positive control, the SVMTriP algorithm was also used to predict the known high immunogenicity of adalimumab; administration of adalimumab without methotrexate is immunogenic in about 50% of patients (see e.g. Ducourau et al (2020) RMD Open 6:e001047). The SVMTriP algorithm identified at least ten possible epitopes in the adalimus Shan Kangchong chain, four of which were highly probable and therefore of good relevance to clinical data, and validated the use of this procedure for predicting immunogenicity.
Table 13: b cell epitopes in DMS5541 sequences predicted by SVMTrip algorithm
The SVMTRIP analysis of the DMS5541 is supported by a second algorithm, ABCPred, which is used to predict immunogenicity within the DMS5541 sequence. All three B cell epitopes predicted by SVMTriP are also included in the epitope prediction results of abcpin. For example, as shown in Table 14 below, epitopes AVKGRFTIRSRDNSKNT, TGRWVPFEYWGQGTLV and STDIQMTQSPSSSLSAS (see the residue positions in SEQ ID NO:38 for each epitope in the following tables) each contain sequences that overlap with the three epitopes identified by SVMTRIP.
Table 14: b cell epitopes in DMS5541 sequences predicted by ABCPred prediction server
Example 4
Exemplary TNFR1 antagonist constructs containing human TNFR1 antagonist antibody fragment (dAb, scFv, fab)
Provided herein are TNFR1 antagonist constructs that selectively inhibit TNFR1 but not TNFR 2. To avoid clustering of TNFR1 receptors that agonize TNFR1, the TNFR1 antagonists are monomeric and monovalent. The TNFR1 antagonist comprises a human single domain antibody (dAb) specific for TNFR 1. dabs contain a variable region heavy chain (V H ) Variable region light chain (V) L ) A domain. For example, a dAb comprises any dAb whose amino acid sequence is set forth in any of SEQ ID NOS.54-672, or a dAb having at least or at least about 90% or 95% sequence identity to a dAb set forth in any of SEQ ID NOS.54-672, which retains binding affinity for TNFR 1. Alternatively, the TNFR1 antagonist comprises an scFv, fab or other antigen-binding fragment, such as those derived from a human TNFR1 antagonist antibody, such as H398 or ATROSAB. For example, a TNFR1 antagonist comprises an H398-derived scFv shown in SEQ ID NO 677 or 678; or ATROSAB derived scFv as set forth in any one of SEQ ID NOs 673-676; or ATROSAB derived Fab fragments light and heavy chain as shown in SEQ ID NOS 679 and 680 (FabATR) respectively or SEQ ID NOS 681 or 682 (Fab 13.7) respectively; or an scFv or Fab fragment having at least or at least about 90% or 95% sequence identity to any of the scFv shown in SEQ ID nos. 673-678, or Fab light and heavy chains shown in SEQ ID nos. 679 and 680 (fabtr), respectively, or SEQ ID nos. 681 or 682 (Fab 13.7), respectively, and retaining affinity for TNFR 1.
The TNFR1 antagonist is fused to a serum half-life extender such as IgG Fc, particularly modified Fc, to eliminate or reduce ADCC, ADCP and/or CDC, human Serum Albumin (HSA) and/or poly (ethylene) glycol (PEG) molecules. For example, the C-terminus of human anti-TNFR 1 dAb, scFV, fab or other antigen-binding fragment is fused to the N-terminus of the Fc region of a human IgG1 or IgG4 antibody via a linker. An IgG1 Fc region, such as that derived from trastuzumab (see SEQ ID NO: 27), or an IgG4 Fc region, such as that derived from nivolumab (see SEQ ID NO: 30), is used. When the Fc is derived from trastuzumab, the linker comprises a hinge of trastuzumabA portion of the chain sequence, a sequence containing amino acid residues SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26), or when the Fc is derived from nivolumab, a nivolumab hinge sequence, a sequence containing amino acid residues ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), or portions thereof that provide flexibility or other structural properties. To confer protease resistance and increase the flexibility of the fusion protein, the SCDKTH or ESKYGPPCPPCP hinge sequence is replaced with a short glycine-serine (GS) peptide linker, e.g., (GSGS) or (GGGGS) n (see, e.g., residues 199-202 and 116-120 of SEQ ID NO:707, respectively), wherein n=1-5 or 1-6, or other combinations of Gly and Ser residues, e.g., GGGGSGGGGSGGGGS (e.g., residues 116-130 or SEQ ID NO: 707). In other embodiments, the C-terminus of the human anti-TNFR 1 dAb, scFv, fab or other antigen-binding fragment is linked to a GS linker, and the GS linker is linked to all or a portion of the trastuzumab or nivolumab hinge sequence sufficient to provide flexibility, which is linked to the N-terminus of the respective Fc region. In some embodiments, the second Fc subunit is linked to the first Fc subunit to increase the serum half-life and stability of the molecule. Because there are two Fc regions, any resulting construct is not a fusion protein because it contains one discontinuous Fc region. In some embodiments, the N-terminus of the human TNFR1 antagonistic dAb, scFV, fab or other antigen-binding fragment is fused to the C-terminus of the serum half-life extender via a linker, as described above.
Also provided herein are TNFR1 antagonist fusion proteins comprising an anti-TNFR 1 dAb, scFv, fab or other antigen-binding fragment fused to Human Serum Albumin (HSA) by a short peptide linker, such as (GSGS) n Or (GGGGS) n Where n=1-5 or 6, e.g. ggggsggggsgggggs.
Also provided herein are TNFR1 antagonist molecules comprising an anti-TNFR 1 dAb, scFv, fab or other antigen-binding fragment linked to a PEG molecule of at least 30kDa in size.
These constructs can be modified to reduce or eliminate immunogenicity, as described herein. The TNFR1 antagonist dAb, scFv, fab or other antigen-binding fragment is analyzed by in silico, in vitro, and/or in vivo methods to predict or identify immunogenic sequences. Based on the identification of immunogenic sequences, e.g., B-cell and/or T-cell epitopes, the identified sequences are modified by mutagenesis, e.g., by alanine scanning as described elsewhere herein, to deimmunize/remove or replace the immunogenic sequences.
The following are exemplary constructs for the TNFR1 antagonist fusion proteins described and provided herein. In all embodiments of the Fc comprising trastuzumab or nivolumab, the Fc region is optionally modified to reduce or eliminate immune effector functions, including ADCC, ADCP and CDC, and is also optionally modified to enhance binding to FcRn, increase the serum half-life of the fusion protein, and optionally replace or otherwise modify or eliminate immunogenic sequences.
The Fc modifications that reduce or eliminate immune effector function are summarized in table 9 above, and the Fc modifications that enhance FcRn binding are summarized in table 7 above. Any one or combination of such modifications is included in the Fc region of the fusion proteins provided herein. All of the exemplary constructs provided herein were also prepared with TNFR1 antagonists at the C-terminus, but not the N-terminus, of the fusion protein.
1a) H398 scFv-SCDKTH-trastuzumab Fc
Provided herein are human TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to a portion of the trastuzumab hinge sequence, which contains the sequence of at least the amino acid residue SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26), and fused to the N-terminus of the trastuzumab Fc region (residues 234-450 corresponding to SEQ ID NO: 26; see also SEQ ID NO: 27). H398 The scFv-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 706): QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRSCDKTHAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced with the complete sequence of the trastuzumab hinge region, which contains or has the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues thereof.
1b) H398 scFv-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to a GGGGSGGGGSGGS peptide linker, which was fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). H398 The scFv-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 707): QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g. (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker described herein or known in the art.
1c) H398 scFv-GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to a GGGGSGGGGSGGS peptide linker, which was fused to a portion of a trastuzumab hinge sequence comprising at least the amino acid residue SCDKTH sequence (corresponding to residues 222-227 of SEQ ID NO: 26), which was fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 6; see also SEQ ID NO: 27). H398 The scFv-GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 708):
QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGH
AYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTV
SSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHW
YVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTH
VPYTFGGGTKLEIKRGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence, up to a complete sequence substitution by the trastuzumab hinge region, contains at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues of the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
1d)H398 scFv-GGGGSGGGGSGGGGS-HSA
Provided herein are TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to a GGGGSGGGGSGGS peptide linker fused to the N-terminus of Human Serum Albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). H398 The scFv-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 709):
QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGH
AYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTV
SSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHW
YVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTH
VPYTFGGGTKLEIKRGGGGSGGGGSGGGGSRGVFRRDAHKSEVAHRFKDLGEENFK
ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTV
ATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEET
FLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGK
ASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECC
HGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPS
LAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC
CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ
VSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRV
TKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVEL
VKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1e)H398 scFv-GGGGSGGGGSGGGGS-PEG 30kDa
Provided herein are TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to a GGGGSGGGGSGGGGS peptide linker covalently linked to a PEG molecule of 30 kDa.
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the PEG molecule can have a molecular weight equal to about 30kDa or greater than 30kDa, such as 35kDa, 40kDa, 45kDa, or 50 kDa.
1f) DOM1 h-574-16-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to a portion of the trastuzumab hinge sequence containing at least the amino acid residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) and fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOMlh-574-16-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 710): EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence may be replaced up to the complete sequence of the trastuzumab hinge region, which contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues thereof.
1g) DOM1 h-574-16-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminal of DOM1h-574-16 is fused to a GGGGSGGGGSGGGGS peptide linker, which is fused to the N-terminal of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-574-16-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 711):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGD
RTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQG
TLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1h) DOM1 h-574-16-GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to a GGGGSGGGGSGGGGS peptide linker fused to a portion of the hinge sequence of trastuzumab containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) fused to the N-terminus of trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-574-16-GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 712): EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced by a complete sequence up to the trastuzumab hinge region, which contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues thereof.
1i)DOM1h-574-16-GGGGSGGGGSGGGGS-HSA
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of Human Serum Albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). The DOM1h-574-16-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 713): EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker described herein or known in the art.
1j)DOM1h-574-16-GGGGSGGGGSGGGGS-PEG 30kDa
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to a GGGGSGGGGSGGGGS peptide linker, which was covalently linked to a PEG molecule of 30kDa in size.
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the PEG molecule may have a molecular weight of greater than 30kDa, such as 35kDa, 40kDa, 45kDa, or 50kDa.
1k) DOM1 h-549-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a portion of a trastuzumab hinge sequence containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) and fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-549-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 714): EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced with the complete sequence of the trastuzumab hinge region, which contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues thereof.
1 l) DOM1 h-549-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the Fc region of trastuzumab (residues 234-450 corresponding to SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-549-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 715): EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 m) DOM1 h-549-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a GGGGSGGGGSGGGGS peptide linker which was fused to a portion of the trastuzumab hinge sequence containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) which was fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-549-GGGGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 716):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTT
TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGT
LVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTTH hinge sequence is replaced by a portion of the trastuzumab hinge region containing at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues up to the complete sequence containing the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
1n)DOM1h-549-GGGGSGGGGSGGGGS-HSA
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a GGGGSGGGGSGGGGS peptide linker which was fused to the N-terminus of Human Serum Albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). The DOM1h-549-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 717):
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTT
TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGT
LVTVSSGGGGSGGGGSGGGGSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQ
YLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGE
MADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI
ARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRL
KCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECA
DDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVES
KDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHE
CYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVE
VSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKA
TKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1o)DOMlh-549-GGGGSGGGGSGGGGS-PEG 30kDa
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a GGGGSGGGGSGGGGS peptide linker covalently linked to a PEG molecule of 30kDa in size.
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker described herein or known in the art. Alternatively or additionally, the PEG molecule may have a molecular weight of greater than 30kDa, such as 35kDa, 40kDa, 45kDa, or 50kDa.
1 p) DOM1 h-574-208-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a portion of the trastuzumab hinge sequence containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) and fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-574-208-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 718): EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced with a portion comprising at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues of the trastuzumab hinge region up to the complete region, comprising the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
1 q) DOM1 h-574-208-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-574-208-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 719):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTAD
RTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQ
GTLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 r) DOM1 h-574-208-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a GGGGSGGGGSGGGGS peptide linker which was fused to a portion of the hinge sequence of trastuzumab containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) and fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-574-208-GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 720): EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced with a portion comprising at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues of the trastuzumab hinge region up to the complete sequence, which comprises the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
1s)DOM1h-574-208-GGGGSGGGGSGGGGS-HSA
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of Human Serum Albumin (HSA) without a signal peptide (corresponding to residues 19-609 of SEQ ID NO: 35). The DOM1h-574-208-GGGGSGGGGSGGGGS-HSA fusion protein has the following sequence (SEQ ID NO: 721): EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1t)DOM1h-574-208-GGGGSGGGGSGGGGS-PEG 30kDa
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a GGGGSGGGGSGGGGS peptide linker, which was covalently linked to a PEG molecule of 30kDa in size.
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the PEG molecule may have a molecular weight of at least or greater than 30kDa, such as 35kDa, 40kDa, 45kDa, or 50kDa.
1 u) DOM1 h-131-206-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 was fused to a portion of the trastuzumab hinge sequence containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) and fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-131-206-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 722): EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced with a portion comprising at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues of the trastuzumab hinge region up to the complete sequence, which comprises the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
1 v) DOM1 h-131-206-GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of the Fc region of trastuzumab (residues 234-450 corresponding to SEQ ID NO: 26; SEQ ID NO: 27). The DOM1 h-131-206-GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 723):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQ
DPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQG
TLVTVSSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 w) DOM1 h-131-206-GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 was fused to a GGGGSGGGGSGGGGS peptide linker which was fused to a portion of the hinge sequence of trastuzumab containing at least the residue sequence SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) and fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). The DOM1 h-131-206-GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 724):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQ
DPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQG
TLVTVSSGGGGSGGGGSGGGGSSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the GGGGSGGGGSGGGGS linker is replaced by another Gly-Ser (GS) linker, e.g., a (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced with a portion or up to the complete sequence comprising at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues of the trastuzumab hinge region, which comprises the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
1 z) H398 scFv-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are human TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), which was fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). H398 The scFv-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 726): QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or part of a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) is used.
1 aa) H398 scFv-GGGGSGGGGSGGGGS-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. The C-terminus of H398 scFv (SEQ ID NO: 678) was fused to a GGGGSGGGGSGGS peptide linker, which was fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). H398 The scFv-GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 727): QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGHAYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 ab) H398 scFv-GGGGSGGGGSGGS-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising scFv derived from human TNFR1 antagonist antibody H398. The scFv contains V of H398 L And V H Domain, pass (GGGGS) 3 Peptide linkers are linked together. H398 scFv (SEQ ID NO: 678)The C-terminus is fused to a GGGGSGGGGSGGGGS peptide linker fused to a hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). H398 The scFv-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 728):
QVQLQESGAELARPGASVKLSCKASGYTFTDFYINWVKQRTGQGLEWIGEIYPYSGH
AYYNEKFKAKATLTADKSSSTAFMQLNSLTSEDSAVYFCVRWDFLDYWGQGTTLTV
SSGGGGSGGGGSGGGGSDIVMTQSPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHW
YVQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTH
VPYTFGGGTKLEIKRGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR
WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, all or a portion of a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223:29 of SEQ ID NO) is used.
1 ac) DOM1 h-574-16-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to the hinge sequence of nivolumab, containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), and to the N-terminal region of nivolumab Fc (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). DOM1 h-574-16-ESKYGPPCPPCP-Nawuzumab Fc fusion protein had the following sequence (SEQ ID NO: 729): EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or a portion of a nivolumab hinge sequence containing at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) is used.
1 ad) DOM1 h-574-16-GGGGSGGGGSGGGGGGS-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-574-16-GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 730): EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 ae) DOM1 h-574-16-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-16 (SEQ ID NO: 57). The C-terminus of DOM1h-574-16 was fused to a GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-574-16-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 731): EVQLLESGGGLVQPGGSLRLSCAASGFTFVKYSMGWVRQAPGKGPEWVSQISNTGDRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWEPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues comprising at least a portion of the nivolumab hinge sequence (containing ESKYGPPCPPCP hinge sequence (corresponding to residues including 212-223 of SEQ ID NO: 29)) up to the complete sequence.
1 af) DOM1 h-549-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) and fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). DOM1 h-549-ESKYGPPCPPCP-Nawuzumab Fc fusion protein had the following sequence (SEQ ID NO: 732): EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or a portion of the nivolumab hinge sequence is included that contains at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
1 ag) DOM1 h-549-GGGGSGGGGSGGGGS-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30.) DOM1 h-549-GGGGSGGGGSGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 733): EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 ah) DOM1 h-549-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a human TNFR1 antagonist dAb DOM1h-549 (SEQ ID NO: 58). The C-terminus of DOM1h-549 was fused to a GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-549-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 734): EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
In some embodiments, the GGGGSGGGGSGGGGS linker is replaced with a GS linker, e.g., a (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, all or a portion of the nivolumab hinge sequence is included that contains at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
1 ai) DOM1 h-574-208-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) and to the N-terminal region of the Fc of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). DOM1 h-574-208-ESKYGPPCPPCP-Nawuzumab Fc fusion protein had the following sequence (SEQ ID NO: 735):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTAD
RTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQ
GTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ
KSLSLSLGK
alternatively, all or a portion of the nivolumab hinge sequence is included that contains at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
1 aj) DOM1 h-574-208-GGGGSGGGGSGGGGS-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-574-208-GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 736):
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTAD
RTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQ
GTLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH
YTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
1 ak) DOM1 h-574-208-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-574-208 (SEQ ID NO: 54). The C-terminus of DOM1h-574-208 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to a hinge sequence containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) of nal Wu Liyou mab, which was fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-574-208-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 737): EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linker (GSGS) n Or (GGGGS) n Linker substitution, wherein n=1, 2, 3, 4 or 5, or any other suitable short peptide linker as described herein or as known in the art. Alternatively or additionally, all or a portion of the nivolumab hinge sequence is included that contains at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
1 al) DOM1 h-131-206-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 was fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) and fused to the N-terminal region of the Fc of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). DOM1 h-131-206-ESKYGPPCPPCP-Nawuzumab Fc fusion protein had the following sequence (SEQ ID NO: 738):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQ
DPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQG
TLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK
SLSLSLGK
alternatively, all or a portion of the nivolumab hinge sequence is included that contains at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
1 am) DOM1 h-131-206-GGGGSGGGGSGGGGGGS-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-131-206-GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 739):
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQ
DPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQG
TLVTVSSGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLGK
alternatively, the GGGGSGGGGSGGGGS linker is replaced by a (GSGS) n or (GGGGS) n linker, wherein n = 1, 2, 3, 4 or 5, or any other suitable short peptide linker as described herein or known in the art.
1 an) DOM1 h-131-206-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising human TNFR1 antagonist dAb DOM1h-131-206 (SEQ ID NO: 59). The C-terminus of DOM1h-131-206 was fused to a GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). The DOM1 h-131-206-GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 740): EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linker (GSGS) n Or (GGGGS) n Linker substitution, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, all or a portion of the nivolumab hinge sequence is included that contains at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
Vhh (nanobody) linked to HSA, in particular in its single chain form, comprising dabs, e.g. DOM1h-131-206, is also synthesized. An exemplary Vhh construct was evaluated for binding and inhibition of TNFR 1. These evaluations are described in the examples below.
Example 5
Exemplary TNFR1 antagonist constructs containing human TNFR1 antagonist TNF muteins
Provided herein are TNFR1 antagonists that selectively inhibit TNFR1 but not TNFR 2. To avoid clustering of TNFR1 receptors that agonize TNFR1, the TNFR1 antagonist is monovalent. TNFR1 antagonists may contain a dominant negative TNF inhibitor of signaling failure (DN-TNF), also known as a TNF mutein, which is an engineered variant of TNF with one or more mutations that abrogate signaling through TNFR 1. For example, a TNF mutein may contain one or more mutations that confer selectivity for TNFR1 over TNFR 2. TNFR 1-selective TNF mutations include any one or more of the following mutations: L29S, L29G, L29Y, R31E, R31N, R32Y, R32W, S86T, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, E146R, V1M, R31C, C69V, Y87H, C101A, A145R, V1M/R31C/C69V/Y87H/C101A/A145R, I97T, I97T/A145R, A84S, V85T, Q88N, T89Q, and A84S/V85T/S86T/Y87H/Q88N/T89Q and combinations thereof, reference is made to the sequence of soluble TNF (solTNF) as shown in SEQ ID NO 2.
For example, a TNFR1 antagonist may contain a TNF mutein having the mutation R32W/S86T (SEQ ID NO: 685), V1M/R31C/C69V/Y87H/C101A/A145R (SEQ ID NO:701; as in XPro 1595), A84S/V85T/S86T/Y87H/Q88N/T89Q (SEQ ID NO:703; as in R1 antTNF) or I97T/A145R (SEQ ID NO:702; as in XENP 345).
The TNFR1 antagonist is fused to a serum half-life extender such as IgG Fc. For example, the C-terminus of a human TNFR1 antagonistic TNF mutein is fused via a linker to the N-terminus of the Fc region of a human IgG1 or IgG4 antibody. An IgG1 Fc region, such as an IgG1 Fc derived from trastuzumab (see SEQ ID NO: 27), or an IgG4 Fc region, such as an IgG4 derived from nivolumab (see SEQ ID NO: 30), is used. The linker may contain all or part of the hinge sequence of trastuzumab when Fc is derived from trastuzumab, at least residues SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26), or the nivolumab hinge sequence ESKYGPPCPPCP (residues 212-223 corresponding to SEQ ID NO: 29) or a portion thereof when Fc is derived from nivolumab. To confer protease resistance and increase the flexibility of the fusion protein, the SCDKTH or ESKYGPPCPPCP hinge sequence or a portion thereof is replaced with a short glycine-serine (GS) peptide linker, e.g., (GSGS) n Or (GGGGS) n Wherein n=1-5, e.g. GGGGSGGGGSGGGGS. In an alternative embodiment, the C-terminus of the human anti-TNFR 1 TNF mutein is linked to the GS linker and the GS linker is linked to all or part of the trastuzumab or nivolumab hinge sequence sufficient to provide flexibility, which is linked to the N-terminus of the corresponding Fc region. In some embodiments, the second Fc subunit is linked to the first Fc region, which may increase the serum half-life and stability of the molecule. The resulting construct is not a fusion protein.
The following are exemplary constructs of TNFR1 antagonist fusion proteins containing TNFR 1-selective antagonistic TNF muteins as described and provided herein. In all embodiments of the trastuzumab-containing Fc or the nivolumab Fc, the Fc region is optionally modified to reduce or eliminate immune effector functions, including ADCC, ADCP and CDC, and is also optionally modified to enhance binding to FcRn, increasing the serum half-life of the fusion protein. The Fc modifications that reduce or eliminate immune effector function are summarized in table 9, and the Fc modifications that enhance FcRn binding are summarized in table 7. Any one or combination of such modifications is included in the Fc region of the fusion proteins provided herein.
2a) TNF (R32W/S86T) -SCDKTH-trastuzumab Fc
Provided herein is a human TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having the mutation R32W/S86T with reference to the soluble TNF sequence as set forth in SEQ ID NO. 2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) was fused to all or part of the hinge sequence of trastuzumab containing at least the residue SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26) fused to the N-terminus of the trastuzumab Fc region (residues 234-450 corresponding to SEQ ID NO: 26; see SEQ ID NO: 27). TNF (R32W/S86T) -SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 741): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced with at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues of the trastuzumab hinge region comprising the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26) up to the complete sequence.
2b) TNF (R32W/S86T) -GGGGSGGGGSGGGGS-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein having the mutation R32W/S86T, referenced to the sequence of soluble TNF, as shown in SEQ ID No. 2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (R32W/S86T) -GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 742):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGL
YLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGS
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linker is replaced with Gly-Ser linker, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2c) TNF (R32W/S86T) -GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein having the mutation R32W/S86T, referenced to the sequence of soluble TNF, as shown in SEQ ID No. 2. The C-terminus of TNF (R32W/S86T) mutein (SEQ ID NO: 685) was fused to a GGGGSGGGGSGGGGS peptide linker fused to a part of the trastuzumab hinge sequence containing at least the residue SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (R32W/S86T) -GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 743):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGL
YLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGS
SCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, GGGGSGGGGSGGGGS linker is replaced with Gly-Ser linker, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced by the complete sequence of the trastuzumab hinge region, contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or a portion thereof containing at least 5,6, 7, 8, 9, 10 or 11 consecutive residues.
2d) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -SCDKTH-trastuzumab Fc
The present invention provides human TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein having mutations V1M, R31C, C69V, Y87H, C101A and A145R, referenced to the soluble TNF sequence, as shown in SEQ ID NO. 2. The C-terminus of TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) was fused to a portion of the trastuzumab hinge sequence, containing at least residue SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 744):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGL
YLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALSCDKTHAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced by the complete sequence of the trastuzumab hinge region, contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or a portion thereof containing at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues.
2e) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-trastuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations V1M, R31C, C69V, Y87H, C A and A145R, referenced to the soluble TNF sequence as set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) was fused to a GGGGSGGGGSGGGGS peptide linker, which was fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 745):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGL
YLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2f) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-SC DKTH-trastuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations V1M, R31C, C69V, Y87H, C A and A145R, referenced to the soluble TNF sequence as set forth in SEQ ID NO: 2. The C-terminus of TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) was fused to a GGGGSGGGGSGGGGS peptide linker fused to a portion of the hinge sequence of trastuzumab, containing at least residue SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26) fused to the N-terminus of the Fc region of trastuzumab (residues 234-450 corresponding to SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 746):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGL
YLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
SCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linker is replaced with Gly-Ser linker, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced by a portion comprising at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues up to the complete sequence of the trastuzumab hinge region comprising the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
2g) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -SCDKTH-trastuzumab Fc
The present invention provides a human TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations A84S, V85T, S86T, Y H, Q88N and T89Q with reference to the soluble TNF sequence as shown in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) was fused to a portion of the trastuzumab hinge sequence, containing at least residue SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 747): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced with a portion comprising at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues up to the complete sequence of the trastuzumab hinge region comprising the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26).
2h) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-trastuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations A84S, V85T, S86T, Y H, Q N and T89Q with reference to the soluble TNF sequence as shown in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 748):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLY
LIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWY
EPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linker is replaced with Gly-Ser linker, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2i) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-SCD KTH-trastuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations A84S, V85T, S86T, Y H, Q N and T89Q with reference to the soluble TNF sequence as shown in SEQ ID NO: 2. The C-terminus of TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) was fused to a GGGGSGGGGSGGGGS peptide linker fused to a portion of the hinge sequence of trastuzumab, containing at least residue SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26) fused to the N-terminus of the Fc region of trastuzumab (residues 234-450 corresponding to SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 749):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLY
LIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWY
EPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSS
CDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linker is replaced with Gly-Ser linker, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced by the complete sequence of the trastuzumab hinge region, which contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or which contains a portion of at least 5,6, 7, 8, 9, 10 or 11 consecutive residues.
2j) TNF (I97T/A145R) -SCDKTH-trastuzumab Fc
Provided herein are human TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein having the mutation I97T/a145R, referenced to the sequence of soluble TNF, as shown in SEQ ID No. 2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) was fused to all or part of the hinge sequence of trastuzumab, containing at least the residue SCDKTH (residues 222-227 corresponding to SEQ ID NO: 26) fused to the N-terminus of the Fc region of trastuzumab (residues 234-450 corresponding to SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (I97T/A145R) -SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 750): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALSCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, the SCDKTH hinge sequence is replaced by the complete sequence of the trastuzumab hinge region, which contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or which contains a portion of at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues.
2k) TNF (I97T/A145R) -GGGGSGGGGSGGGGS-trastuzumab Fc
The invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having the mutation I97T/A145R with reference to the sequence of soluble TNF as shown in SEQ ID NO. 2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the Fc region of trastuzumab (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (I97T/A145R) -GGGGSGGGGSGGGGS-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 751):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLY
LIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2 l) TNF (I97T/A145R) -GGGGSGGGGSGGGGS-SCDKTH-trastuzumab Fc
The invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having the mutation I97T/A145R with reference to the sequence of soluble TNF as shown in SEQ ID NO. 2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) was fused to a GGGGSGGGGSGGGGS peptide linker fused to a part of the trastuzumab hinge sequence containing at least the residue SCDKTH (corresponding to residues 222-227 of SEQ ID NO: 26) fused to the N-terminus of the trastuzumab Fc region (corresponding to residues 234-450 of SEQ ID NO: 26; see also SEQ ID NO: 27). TNF (I97T/A145R) -GGGGSGGGGSGGS-SCDKTH-trastuzumab Fc fusion protein has the following sequence (SEQ ID NO: 752):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLY
LIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
SCDKTHAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, the SCDKTH hinge sequence is replaced by the complete sequence of the trastuzumab hinge region, which contains the sequence EPKSCDKTHTCPPCP (corresponding to residues 219-233 of SEQ ID NO: 26), or which contains a portion of at least 5,6, 7, 8, 9, 10 or 11 consecutive residues.
2 m) TNF (R32W/S86T) -ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are human TNFR1 antagonist fusion protein constructs comprising a TNFR1 selective antagonist TNF mutein having the mutation R32W/S86T, referenced to the sequence of soluble TNF, as shown in SEQ ID No. 2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) was fused to the hinge sequence of nivolumab, containing residues ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), and fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (R32W/S86T) -ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 753): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or part of the nivolumab hinge sequence is included, containing at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
2 n) TNF (R32W/S86T) -GGGGSGGGGSGGGGS-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein having the mutation R32W/S86T, referenced to the sequence of soluble TNF, as shown in SEQ ID No. 2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) was fused to a GGGGSGGGGSGGS peptide linker fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (R32W/S86T) -GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 754): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2 o) TNF (R32W/S86T) -GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein mutated to R32W/S86T, referenced to the sequence of soluble TNF, as set forth in SEQ ID No. 2. The C-terminus of the TNF (R32W/S86T) mutein (SEQ ID NO: 685) is fused to a GGGGSGGGGSGGS peptide linker fused to the hinge sequence of nivolumab, containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (R32W/S86T) -GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 755): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRWANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVTYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, a portion of the nivolumab hinge sequence is included, containing at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
2 p) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -ESKYGPPCPPCP-Nawuzumab Fc
The invention provides a human TNFR1 antagonist fusion protein, which contains a TNFR1 selective antagonist TNF mutein mutated into V1M, R31C, C69V, Y87H, C A and A145R, and the sequence of soluble TNF is referenced as shown in SEQ ID NO. 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) was fused to the hinge sequence of nivolumab, which contained the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 756): MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or part of the nivolumab hinge sequence is included, containing at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29).
2 q) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-Nawuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations V1M, R31C, C69V, Y87H, C A and A145R, referenced to the soluble TNF sequence as set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 757):
MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGL
YLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
alternatively, GGGGSGGGGSGGGGS linkers are replaced with Gly-Ser linkers, e.g., (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2R) TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-ES KYGPPCPPCP-Nawuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations V1M, R31C, C69V, Y87H, C A and A145R, referenced to the soluble TNF sequence as set forth in SEQ ID NO: 2. The C-terminus of the TNF (V1M/R31C/C69V/Y87H/C101A/A145R) mutein (SEQ ID NO: 701) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab, containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (V1M/R31C/C69V/Y87H/C101A/A145R) -GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 758): MRSSSRTPSDKPVAHVVANPQAEGQLQWLNCRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGVPSTHVLLTHTISRIAVSHQTKVNLLSAIKSPAQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g. (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, all or a portion of a nivolumab hinge sequence comprising at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223:29 of SEQ ID NO) is included.
2S) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -ESKYGPPCPPCP-Nawuzumab Fc
The present invention provides a human TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations A84S, V85T, S86T, Y H, Q88N and T89Q with reference to the soluble TNF sequence as shown in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) was fused to the hinge sequence of nivolumab, which contained the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 759): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or a portion of the nivolumab hinge sequence comprising at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) is included.
2T) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-Nawuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations A84S, V85T, S86T, Y H, Q N and T89Q with reference to the soluble TNF sequence as shown in SEQ ID NO: 2. The C-terminus of the TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the Nawuzumab Fc region (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 760): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g. (GSGS) n Or (GGGGS) n A linker wherein n=1, 2,3. 4 or 5, or any other suitable short peptide linker as described herein or known in the art.
2 u) TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-ESK YGPPCPPCP-Nawuzumab Fc
The present invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having mutations A84S, V85T, S86T, Y H, Q N and T89Q with reference to the soluble TNF sequence as shown in SEQ ID NO: 2. The C-terminus of TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) mutein (SEQ ID NO: 703) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab, containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (A84S/V85T/S86T/Y87H/Q88N/T89Q) -GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 761): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRISTTHNQKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g. (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, all or a portion of a nivolumab hinge sequence comprising at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) is included.
2 v) TNF (I97T/A145R) -ESKYGPPCPPCP-Nawuzumab Fc
Provided herein are human TNFR1 antagonist fusion proteins comprising a TNFR1 selective antagonist TNF mutein having the mutation I97T/a145R, referenced to the sequence of soluble TNF, as shown in SEQ ID No. 2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) was fused to the hinge sequence of nivolumab, containing sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29), and fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (I97T/A145R) -ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 762): VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, all or a portion of the nivolumab hinge sequence comprising at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues of the ESKYGPPCPPCP hinge sequence (corresponding to residues 212-223 of SEQ ID NO: 29) is included.
2 w) TNF (I97T/A145R) -GGGGSGGGGSGGGGS-Nawuzumab Fc
The invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having the mutation I97T/A145R with reference to the sequence of soluble TNF as shown in SEQ ID NO. 2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (I97T/A145R) -GGGGSGGGGSGGGGS-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 763):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLY
LIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g. (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art.
2 x) TNF (I97T/A145R) -GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc
The invention provides a TNFR1 antagonist fusion protein comprising a TNFR1 selective antagonist TNF mutein having the mutation I97T/A145R with reference to the sequence of soluble TNF as shown in SEQ ID NO. 2. The C-terminus of the TNF (I97T/A145R) mutein (SEQ ID NO: 702) was fused to a GGGGSGGGGSGGGGS peptide linker fused to the hinge sequence of nivolumab, containing the sequence ESKYGPPCPPCP (corresponding to residues 212-223 of SEQ ID NO: 29) fused to the N-terminus of the Fc region of nivolumab (corresponding to residues 224-440 of SEQ ID NO: 29; see also SEQ ID NO: 30). TNF (I97T/A145R) -GGGGSGGGGSGGGGS-ESKYGPPCPPCP-Nawuzumab Fc fusion protein has the following sequence (SEQ ID NO: 764):
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLY
LIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSATKSPCQRETPEGAEAKPW
YEPIYLGGVFQLEKGDRLSAEINRPDYLDFRESGQVYFGIIALGGGGSGGGGSGGGGS
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN
WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Alternatively, GGGGSGGGGSGGGGS linker is replaced by Gly-Ser linker, e.g. (GSGS) n Or (GGGGS) n A linker, wherein n=1, 2, 3, 4, or 5, or any other suitable short peptide linker as described herein or known in the art. Alternatively or additionally, it includes a polypeptide comprising a ESKYGPPCPPCP hinge sequence (corresponding to residue SEQ ID NO:29Base 212-223) all or a portion of the nivolumab hinge sequence of at least 5, 6, 7, 8, 9, 10, or 11 consecutive residues.
Example 6
Presentation in the context of antibodies presents difficulties in the context of inhibiting target receptors. The bivalent antibody induces dimerization of the target receptor, which may lead to its activation. While this may be desirable in some circumstances, it is highly undesirable for TNFR1 inhibitors. Even brief activation of TNFR1 causes cytokine storm and significant toxicity. Thus, monovalent inhibitors are needed. To achieve this, monovalent constructs are provided herein (see description of the text and example 5 above). This example illustrates the activity and nature of an exemplary construct, which is an N-terminal fusion protein of a single-stranded dAb with human serum albumin.
This example provides an exemplary nanobody. Nanobodies are Vhh domain-containing proteins, comprising only the heavy chain, and do not require synergy of the light chain, just as in the case of humans and mice (Harmsen and De Haard, appl Microbiol biotechnol.77:13-22 (2007)). Because they are single chain, grafted CDRs, e.g., in the form of antibodies, must be presented as fusion proteins because of their short half-life as small proteins (-13-15 KDa).
Phage libraries were prepared using the method described by Sabir et al (((2014) Comptes Rendus Biologies 337:244-249). Phages that bind to tumor necrosis factor receptor-1 (TNFR 1) with high affinity were recovered and tested for their ability to bind to TNFR1, as well as their ability to compete with human tumor necrosis factor-alpha (TNF-alpha) for binding to TNFR1, as described in U.S. published application No. US 20140112929.
Nucleic acids encoding each of the single-chain-containing Vhh antibodies were expressed in CHO cells (see Sokolowska-Wedzina et al (2014) Protein Expression and Purification 99:50-57 for description of expression vectors) and were all purified by HPLC chromatography. Sample 1 was a control anti-TNFR 1 antibody (H398 from ThermoFisher) and Vhh1-4 was a Vhh antibody containing a dAb. The sequences (see SEQ ID NOS: 54, 1478, 58 and 59) are as follows:
Vhh-1：
EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSS
Vhh-2：
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYRMHWVRQAPGKSLEWVSSIDTRGSSTYYADPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAVTMFSPFFDYWGQGTLV
Vhh-3：
EVQLLESGGGLVQPGGSLRLSCAASGFTFVDYEMHWVRQAPGKGLEWVSSISESGTTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRFSASTFDYWGQGTLVTVSS
Vhh-4：EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSS
the highlighted and underlined cysteine (C) in Vhh-4 forms a loop with the corresponding cysteine in the other Vhh chain, whereby Vhh is a constrained polypeptide. The proline at amino acid residue 14 can be replaced with alanine.
Expression cassettes encoding Vhh domain antibody fragments were prepared and expressed and purified by HPLC chromatography. The following table shows the expression levels of each of the tested Vhh antibodies. There are two molecules that require His-tags for purification. The results are shown in the following table:
Table 15: expression and yield results of Vhh 1-4
Project Description of the invention Label (Label) Harvesting Yield of products
Sample 1 Anti-human TNFRSF1A therapeutic antibody scFv fragment (H398) His 146μg Abt.1g/L
Vhh-1 Recombinant human anti-TNFRSF 1A single domain antibodies Without any means for 500μg 30mg/L
Vhh-2 Recombinant human anti-TNFRSF 1A single domain antibodies His 1000μg 49.4mg/L
Vhh-3 Tandem scFV bispecific antibodies Without any means for 500μg 42mg/L
Vhh-4 Tandem scFV bispecific antibody (SEQ ID NO: 1475) Without any means for 500μg 51mg/L
* TNF receptor superfamily member 1A antibody H398 (ThermoFisher; H398 comprises SEQ ID NO: 678).
Each of the Vhh 1-4 was then tested in a binding study using the Surface Plasmon Resonance (SPR) method (Sciences GL. Biacore Assay handbook general Electric Company (2012); and Richter et al (2019) MAbs 11:166-177). Competition assays for Vhh inhibition of TNF-a binding to the extracellular domain of TNFR1 were also performed as described in Richter et al (2019). Inhibition of TNF-induced VCAM or IL8 expression was performed as described in Lin et al, (2015) J Biomed Sci 22:53 and Sonnier et al (2010) Journal of Gastrointestinal Surgery 14:1592-1599, respectively, which are comparable assays.
The results are summarized in the following table. The results show that Vhh-4 has a very high affinity for the extracellular domain of TNFR-1 (6.6X10) -13 M) is selected from the group consisting of; it was 100% competitive for TNF binding to TNFR1 (IC 50 About 1 nM), and is the most effective in inhibiting TNF-induced synthesis of VCAM-1 (0.3 nM) among the 4 candidates. Of these Vhh dAb antibodies tested, vhh-4 performed best, and constructs containing Vhh-4 and HSA were prepared.
The following sequence represents a construct containing Vhh-4 (SEQ ID NO: 1475): the dAb moiety is residues 20-138 of SEQ ID NO:1475, linked to human serum albumin (HSA; residues 148-732 of SEQ ID NO: 1475) by a Gly-Ser linker (residues 139-147 of SEQ ID NO: 1475).
EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSS…HSA…
Table: comparing binding and competition assays (results for control antibodies are not shown)
* The amount of antibody recovered was very small
Constructs containing Vhh-4 antibodies linked to HSA were shown to have activity as TNFR1 antagonists. It blocked TNFR1 as indicated by a significant decrease in IL-6 and IL-8 gene expression in LPS-stimulated THP1 cells. Will be 3X 10 5 THP1 cells were treated with 5. Mu.g and 20. Mu.g and 0. Mu.g of the construct as a control for 30 min, and then stimulated with LPS (10 ng/ml) for 4 hours. RNA samples were collected and used with tubesThe housekeeping gene, xanthine-guanine phosphoribosyl transferase (HPRT), was used as an internal control for qPCR analysis to analyze L-6 and IL-8 gene expression. The results show that both doses significantly reduced IL-6 and IL-8 expression (n=3, mean ± SEM;. P) <0.05，**p<0.01，***p<0.001)。
Since modifications will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the appended claims.

Claims (286)

1. A construct which is a tumor necrosis factor receptor 1 (TNFR 1) antagonist construct of formula 1:
(TNFR 1 inhibitor) n -a joint p - (activity modulating agent) q Wherein:
n and q are each an integer and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
TNFR1 inhibitors are molecules that bind TNFR1 to inhibit (antagonize) TNFR1 activity;
an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and
the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media.
2. The construct according to claim 1, wherein the linker is selected from the group consisting of a chemical linker, a polypeptide linker, and combinations thereof.
3. A construct according to claim 1 or claim 2, which is a fusion protein.
4. A construct according to any one of claims 1 to 3, wherein the linker comprises a plurality of linker components.
5. A construct according to any one of claims 1 to 4, wherein the TNFR1 inhibitor comprises a domain antibody (dAb).
6. Construct according to any one of claims 1 to 5, wherein if the TNFR1 inhibitor is a domain antibody (dAb), the activity modulator is not an unmodified single Fc region and is not a human serum albumin antibody.
7. The construct according to any one of claims 1 to 6, wherein the TNFR1 inhibitor inhibits TNFR1 signaling.
8. Construct according to any one of claims 1 to 7, wherein said activity modulator increases the serum half-life of said construct.
9. Construct according to any one of claims 1 to 8, wherein said activity modulator is albumin or an Fc modified to have reduced or no ADCC activity and/or reduced or no CDC activity.
10. Construct according to any of claims 1 to 9, wherein said TNFR1 inhibitor inhibits TNFR1 activity but does not antagonize tumor necrosis factor receptor 2 (TNFR 2) activity.
11. The construct according to claim 10, wherein the TNFR1 inhibitor inhibits TNFR1 signaling.
12. A construct which is a multispecific construct comprising a TNFR1 inhibitor and a Treg amplicon, wherein the bispecific construct interacts with two different target receptors or antigens or epitopes on the receptors.
13. Construct according to claim 12, which is bispecific for TFNR1 and Treg amplicons.
14. Construct according to claim 12 or claim 13, wherein said Treg expansion is a TNFR2 agonist.
15. Construct according to any of claims 12 to 14, comprising a linker to provide flexibility, to increase solubility or to reduce or reduce steric hindrance or van der waals interactions.
16. Construct according to any of claims 12 to 15, further comprising an activity modulator to alter the activity of the construct.
17. Construct according to any one of claims 12 to 16, having formula 2:
(TNFR 1 inhibitor) n - (activity modulating agent) r1 - (linker- (L)) p - (activity modulating agent) r2 - (TNFR 2 agonists) q Or (b)
(TNFR 1 inhibitor) n - (activity modulating agent) r1 - (joint (L)) p - (activity modulating agent) r2 - (Treg amplificates) q ，
Wherein:
n=1, 2 or 3, p=1, 2 or 3, q=0, 1 or 2, r1 and r2 are each independently 0, 1 or 2; and
the components may be in the order specified or any other order, so long as the construct interacts with TNFR1 and TNFR2 to antagonize TNFR1 and agonize TNFR2, or has Treg amplicon activity.
18. Construct according to any of claims 1 to 17, wherein said TNFR1 inhibitor moiety inhibits tnfα binding to TNFR1 and/or inhibits signaling.
19. A construct of formula 3a or 3 b:
(TNFR 2 agonist or Treg amplificates) n -a joint p - (activity modulating agent) q Of formula 3a, or
(Activity-controlling Agents) q -a joint p - (TNFR 2 agonist or Treg amplimer) n Formula 3b, wherein:
n and q are each an integer and are each independently 1, 2 or 3; p is 0, 1, 2 or 3;
an activity modulator is a moiety that alters the pharmacological properties of the construct;
the TNFR2 agonist interacts with TNFR2 resulting in TNFR2 activity;
treg amplicons, including TNFR2 agonists, and are molecules that lead to an increase in Treg cells; and
the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with the receptor; and/or altering the solubility of the construct.
20. Construct according to any of claims 1 to 19, wherein said activity modulator is an Fc region or a modified Fc region or a short FcRnBP; and
the linker comprises a hinge region, or is a linker comprising G and S residues.
21. Construct according to any of claims 1 to 20, wherein said linker has the sequence shown in any of SEQ ID NOs 812-834 or is a PEG moiety linker.
22. Construct according to any one of claims 1 to 21, comprising an activity modulator, which is a modified Fc region or peptide that increases the serum half-life of the construct.
23. Construct according to any one of claims 1 to 22, comprising an Fc region or an Fc dimer.
24. Construct according to any one of claims 1 to 23, comprising an Fc region modified to have reduced ADCC and/or CDC activity.
25. A construct according to claim 24, wherein the Fc is modified to have reduced ADCC activity or no ADCC activity.
26. Construct according to any one of claims 1 to 25, wherein:
the TNFR1 inhibitor is any inhibitor defined in the sequence listing, listed below, or known in the art;
the Treg amplicons are any known in the art, are TNFR2 agonists, or any Treg amplicons shown in the sequence listing, listed below, or known in the art;
the linker is a sequence listing or any linker listed below or known in the art; and
the activity modulator is any activity modulator listed in the sequence listing, known in the art, and/or listed below.
27. A construct which is a TNFR1 antagonist construct comprising a TNFR1 inhibitor which is a single chain antibody, or antigen-binding portion thereof, which specifically targets and inhibits TNFR1, but does not antagonize TNFR2, thereby preventing transient activation of TNFR1 by receptor clustering.
28. A construct according to claim 27, wherein said antibody or antigen binding portion thereof comprises modifications that improve the pharmacological properties and/or structure of said construct.
29. Construct according to any one of claims 1 to 28, which agonizes TNFR2 signaling thereby increasing expression of regulatory T cells (tregs).
30. Construct according to any of claims 27 to 29, wherein said single chain antibody inhibits TNFR1 by inhibiting TNFR1 signaling.
31. A TNFR1 antagonist construct according to any one of claims 27 to 30, wherein the antibody portion or antigen-binding portion of the construct inhibits binding of tnfα to TNFR1.
32. Construct according to any of claims 27 to 30, wherein the antibody or antigen binding portion of the construct does not inhibit tnfα binding to TNFR1, but does inhibit TNFR1 signaling.
33. Construct according to any of claims 28 to 32, wherein said pharmacological property is an increased serum half-life.
34. Construct according to any of claims 27 to 33, wherein said TNFR1 construct comprises an Fc modified to eliminate ADCC and/or CDC activity.
35. Construct according to any of claims 27 to 34, wherein said TNFR1 construct comprises an Fc dimer.
36. A construct according to claim 35, wherein one Fc monomer comprises a recess and the other Fc monomer comprises a projection to form a heterodimer.
37. The construct according to claim 35, wherein:
the convex mutation is selected from the group consisting of S354C, T366Y, T366W and T394W of EU numbering; and
the concave mutation is selected from the group consisting of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V, EU numbering, whereby the Fc monomer forms a heterodimer.
38. Construct according to any one of claims 1 to 37, comprising Fc, wherein said Fc is from trastuzumab.
39. A construct according to claim 38, comprising a linker which is a hinge region from an Fc region.
40. The construct of claim 39, wherein the hinge region is from trastuzumab and is linked to the Fc region.
41. The construct according to any one of claims 27 to 40, comprising a linker linked to an anti-TNFR 1 antagonist antibody, or antigen-binding portion thereof.
42. The construct according to claim 27 to 41, comprising a linker linked to the anti-TNFR 1 antagonist antibody or antigen-binding portion thereof, directly or through a hinge region to an Fc region.
43. A construct according to any one of claims 1 to 42, comprising an Fc region or a modified Fc region comprising the amino acid sequence shown in any one of SEQ ID NOs 10, 12, 14, 16, 27, 30, 1469 and 1470.
44. The construct according to any one of claims 1 to 43, wherein said construct comprises a hinge region linked to an Fc portion.
45. The construct according to any one of claims 1 to 44, wherein the construct binds to neonatal Fc receptor (FcRn).
46. The construct of claim 45, wherein:
the TNFR1 construct comprises a short FcRn binding peptide (FcRnBP); and
the short FcRn binding peptide (FcRnBP) provides for the interaction of the construct with FcRn and contains 6-25 or 10-20 amino acid residues.
47. The TNFR1 antagonist construct according to claim 46 wherein said FcRnBP contains 12-20 residues or 15 residues or 16 residues.
48. A TNFR1 antagonist construct according to claim 46 or claim 47 wherein said FcRn binding peptide (FcRnBP) comprises a peptide set forth in any one of SEQ ID NOs 48-51.
49. The TNFR1 antagonist construct according to any one of claims 46-48 wherein said FcRn binding peptide (FcRnBP) consists of a peptide set forth in any one of SEQ ID NOs 48-51.
50. The construct according to any one of claims 1 to 49, wherein the TNFR1 construct comprises an Fc heterodimer, wherein one Fc monomer comprises a recess and the other comprises a protrusion, whereby the resulting Fc dimer is a heterodimer.
51. The construct according to any one of claims 1 to 50, which is a TNFR1 antagonist construct comprising:
TNFR1 inhibitors;
fc dimers; and
treg amplicons, wherein:
the Fc dimer comprises two complementary Fc monomers;
the TNFR1 inhibitor is linked to one of the Fc monomers, and the Treg expansion is linked to another Fc monomer.
52. The construct of claim 51, wherein said Treg expansion is a TNFR2 agonist.
53. The construct of claim 51 or claim 52, further comprising a second Treg expansion linked to the same Fc monomer as the TNFR1 inhibitor, wherein the first and second Treg expansion are the same or different.
54. The construct of claim 53, wherein the second Treg expansion is a TNFR2 agonist.
55. The construct of claim 53, wherein the Treg amplicons are identical.
56. The construct according to any one of claims 51 to 55, wherein the TNFR1 inhibitor inhibits or blocks TNFR1 signaling.
57. The construct according to any one of claims 51 to 56, wherein the TNFR1 inhibitor binds TNFR1 and blocks or inhibits tnfa binding and TNFR1 signaling.
58. The construct according to any one of claims 51 to 56, wherein the TNFR1 inhibitor binds TNFR1, does not interfere with tnfα binding, and blocks or inhibits TNFR1 signaling.
59. Construct according to any one of claims 51 to 58, wherein said Treg expansion is a TNFR2 agonist.
60. The construct of claim 59, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling.
61. Construct according to any one of claims 51 to 60, wherein said Treg expansion is a TNFR2 agonist, which is a Fab of scFv, VHH single domain antibodies or TNFR2 agonist monoclonal antibodies.
62. The construct according to any one of claims 1 to 61, comprising all or a portion of trastuzumab and dimerized by fusion of the N-terminus with the C-terminus of trastuzumab.
63. Construct according to any one of claims 7 to 16, wherein said Treg expansion is a TNFR2 agonist, which is a small molecule, or a nucleic acid aptamer, or a peptide aptamer.
64. The construct according to any one of claims 1 to 63, which is a TNFR2 agonist construct of formula 3:
(Treg amplificates) n -a joint p - (activity modulating agent) q Of formula 3a, or
(Activity-controlling Agents) q -a joint p - (Treg amplificates) n Formula 3b, wherein:
n and q are each an integer and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and
the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media.
65. Construct according to any one of claims 51 to 64, wherein said Treg expansion is a TNFR2 agonist.
66. The construct of claim 65, wherein the TNFR2 agonist stimulates or induces TNFR2 signaling.
67. Construct according to any one of claims 51 to 66, wherein said Treg expansion is a TNFR2 agonist, which is a Fab of scFv, VHH single domain antibodies or TNFR2 agonist monoclonal antibodies.
68. The construct according to claim 67, which dimerizes by fusion of the N-terminus with the C-terminus of trastuzumab.
69. The TNFR1 antagonist construct of any one of claims 51 to 68, wherein said Treg expansion is a TNFR2 agonist, which is a small molecule, or a nucleic acid, or a peptide aptamer.
70. A construct comprising a TNFR1 inhibitor moiety linked to one or more Treg amplicons by a central PEG linker, or comprising at least two identical or different TNFR1 inhibitors, or comprising two identical or different Treg amplicons.
71. The construct according to claim 70, comprising a branched PEG moiety linking the TNFR1 inhibitor and one or more Treg amplicons.
72. A construct according to claim 70 or claim 71 having a structure selected from formulae 4A to 4D:
formula 4A:
n is 1 to 5;
R 1 is H or CH 3 Or CH 2 CH 3 Or other C1-C5 alkyl groups;
is a TNFR1 inhibitor (TNFR 1 antagonist);
is a Treg amplicon; or (b)
Formula 4B:
is a TNFR1 inhibitor (TNFR 1 antagonist);
is a Treg amplicon;
n is 1 to 5; or (b)
Formula 4C:
is a TNFR1 inhibitor (TNFR 1 antagonist), or is a Treg amplicon; and
n is 1 to 5; or (b)
Formula 4D:
wherein the method comprises the steps of
Each of which isAre identical or different and are each independently selected from a TNFR1 inhibitor (TNFR 1 antagonist) and a TNFR2 agonist;
the activity modulator is optional and may be attached to any suitable site in the molecule; and
n is 1 to 5.
73. The construct according to any one of claims 70 to 72, wherein said Treg expansion is a TNFR2 agonist.
74. A construct according to any one of claims 1 to 73 comprising an activity modulator, wherein:
the activity modulator is an Fc region, or an Fc region comprising a hinge region or other linker; and
the Fc region or Fc region having a hinge region is an Fc modified to reduce or eliminate ADCC and/or CDC activity.
75. The construct according to claim 74, wherein the Fc or modified Fc is an IgG Fc or an IgG1 or IgG4 Fc.
76. A construct according to any one of claims 1 to 75 which binds to neonatal Fc receptor (FcRn).
77. The construct according to claim 76, wherein:
the construct comprises a short FcRn binding peptide (FcRnBP); and
the short FcRn binding peptide (FcRnBP) provides for interaction of the construct with FcRn and contains 6-25, e.g., 10-20 amino acid residues.
78. The construct according to claim 77, wherein the FcRnBP contains 12-20 residues or 15 residues or 16 residues.
79. The TNFR1 antagonist construct according to claim 78, wherein said FcRn binding peptide (FcRnBP) comprises a peptide set forth in any one of SEQ ID NOs 48-51.
80. The TNFR1 antagonist construct according to claim 78, wherein said FcRn binding peptide (FcRnBP) consists of a peptide set forth in any one of SEQ ID NOs 48-51.
81. A construct according to any one of claims 1 to 80, which is a TNFR1 antagonist construct comprising:
a) A TNFR 1-selective TNFR1 inhibitor moiety;
b) Optionally one or more linkers; and
c) Optionally a half-life extending moiety, wherein
The antagonist construct comprises at least one of b) and c).
82. The construct of claim 81, wherein said TNFR 1-selective antagonist selectively binds to and inhibits TNFR1 signaling, and does not bind to and does not inhibit TNFR2 signaling.
83. The construct according to claim 81 or claim 82, wherein the TNFR1 inhibitor is a selective antagonist comprising an antigen-binding fragment that selectively binds to and inhibits TNFR1 signaling, and does not bind to and does not inhibit TNFR2 signaling.
84. The construct of claim 83, wherein the antigen binding fragment that selectively binds to and inhibits TNFR1 signaling and does not bind to and does not inhibit TNFR2 signaling comprises a domain antibody (dAb), scFv, or Fab fragment.
85. The construct according to any one of claims 1 to 84, wherein the TNFR1 inhibitor comprises an antigen-binding fragment of a human anti-TNFR 1 antagonist monoclonal antibody.
86. The construct of claim 85, wherein the human anti-TNFR 1 antagonist monoclonal antibody is H398 comprising SEQ ID No. 678, or ATROSAB, or an antigen binding portion thereof, or a sequence having at least 95% sequence identity to SEQ ID No. 31 or 32 or 673 or 678, or an antigen binding portion thereof that binds TNFR 1.
87. A construct according to any of claims 1 to 86, wherein the TNFR1 inhibitor comprises a domain antibody (dAb), or antigen-binding portion thereof, or comprises the amino acid sequence depicted in any of SEQ ID NOs 52-672, or a sequence having at least 95% sequence identity thereto that retains the activity of a TNFR1 inhibitor.
88. Construct according to any of claims 1 to 87, wherein the TNFR1 inhibitor comprises the scFv shown in any of SEQ ID nos. 673-678 or variants of these polypeptides having at least 90% or 95% sequence identity thereto, which retain the activity of the TNFR1 inhibitor.
89. Construct according to any of claims 1 to 88, wherein the TNFR1 inhibitor comprises a Fab as shown in any of SEQ ID NOs 679-682 or a sequence having at least 90% or 95% sequence identity thereto which retains TNFR1 inhibitor activity or binding activity.
90. The construct according to any one of claims 1 to 89, wherein the TNFR1 inhibitor comprises a nanobody whose sequence is shown as SEQ ID No. 683 or 684 or a sequence having at least 90% or 95% sequence identity thereto that retains TNFR1 inhibitor activity or binding activity.
91. The construct according to any one of claims 1 to 90, wherein the TNFR1 inhibitor comprises a dominant negative tumor necrosis factor (DN-TNF) or TNF mutein.
92. The construct of claim 91, wherein said DN-TNF or TNF mutein is a soluble TNF molecule comprising one or more amino acid substitutions which confer selective inhibition of TNFR1 and is selected from the group consisting of:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, A84S/V85T/S86T/Y87H/Q88N/T89Q, reference is made to the soluble TNF sequence shown in SEQ ID NO 2.
93. A construct according to claim 91 or claim 92, wherein the TNFR1 inhibitor TNF mutein comprises the residue sequence set forth in any one of SEQ ID NOs 701-703, or a sequence having at least 90% or 95% or at least about 90% or 95% sequence identity to the residue sequence set forth in any one of SEQ ID NOs 701-703, or a fragment thereof retaining TNFR1 inhibitor activity.
94. Construct according to any of claims 1 to 93, comprising a linker, wherein the linker comprises all or part of the hinge sequence of trastuzumab, SCDKTH corresponding to residues 222-227 of SEQ ID No. 26 or up to the entire sequence of the hinge region of trastuzumab, which contains or has the sequence epkscdkthtcpp (corresponding to residues 219-233 of SEQ ID No. 26), or at least 5, 6, 7, 8, 9, 10 or 11 consecutive residues thereof, or residues 212-223 of residue ESKYGPPCPPCP of SEQ ID No. 29, or a sequence of at least 98% or 99% sequence identity thereto of the linker.
95. The construct according to any one of claims 1 to 94 comprising a linker, wherein the linker comprises the sequence SCDKTH, corresponding to residues 222-227 of SEQ ID No. 26.
96. Construct according to any of claims 1 to 95, comprising a linker, wherein the linker comprises a glycine-serine (GS) linker.
97. A construct according to claim 96, wherein the GS linker is selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n The method comprises the steps of carrying out a first treatment on the surface of the Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS.
98. Construct according to any of claims 1 to 97, comprising a linker, wherein the linker comprises all or part of the hinge sequence of the GS linker and trastuzumab, corresponding to residue epkscdkthtcpcp (219-233 of SEQ ID NO: 26).
99. Construct according to any of claims 1 to 98, wherein the linker comprises a GS linker and comprises the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID No. 31.
100. Construct according to any of claims 1 to 99, wherein said linker comprises all or part of the hinge sequence of the GS linker and nivolumab, corresponding to residues 212-223 of SEQ ID No. 29.
101. Construct according to any one of claims 1 to 100, comprising an activity modulator, wherein the activity modulator is a half-life extending moiety which is an IgG Fc, a polyethylene glycol (PEG) molecule or Human Serum Albumin (HSA).
102. The construct according to claim 101, wherein said IgG Fc is IgG1 or IgG4 Fc.
103. The construct according to claim 101 or claim 102, wherein said IgG1 Fc is the Fc of trastuzumab as shown in SEQ ID No. 27, or an amino acid sequence having at least 95% sequence identity thereto.
104. The construct according to claim 101 or claim 102, wherein said IgG4 Fc is the Fc of nivolumab shown in SEQ ID No. 30, or an amino acid sequence having at least 95% sequence identity thereto.
105. The construct according to any of claims 101 to 104, wherein said IgG1 Fc is the Fc of human IgG1 shown in SEQ ID No. 10.
106. Construct according to any of claims 101 to 104, wherein said IgG4 Fc is the Fc of human IgG4 shown in SEQ ID No. 16.
107. A construct according to any one of claims 1 to 106 comprising a TNFR1 inhibitor, wherein said TNFR1 inhibitor is monovalent.
108. Construct according to any of claims 81 to 107, comprising a linker, wherein said linker comprises (Gly 4 Ser) 3 。
109. Construct according to any of claims 81 to 107, comprising a linker, wherein said linker comprises (Gly 4 Ser) 3 And SCDKTH (residues 217-222 of SEQ ID NO: 31).
110. Construct according to any of claims 1 to 109, comprising a linker, wherein the linker comprises (Gly 4 Ser) 3 And a hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26.
111. Construct according to any of claims 1 to 109, comprising a linker, wherein the linker comprises (Gly 4 Ser) 3 And a hinge sequence of Nawuzumab, pairResidues 212-223 corresponding to SEQ ID NO. 29.
112. The construct according to any one of claims 1 to 111, comprising the residue sequence set forth in any one of SEQ ID NOs 704 to 764, or a construct that inhibits TNFR1 and has a sequence having at least or at least about 95% sequence identity to the residue sequence set forth in any one of SEQ ID NOs 704 to 764.
113. Construct according to any of claims 1 to 112, which is a TNFR1 antagonist construct, wherein:
the TNFR1 construct comprises a short FcRn binding peptide (FcRnBP); and
the short FcRn binding peptide (FcRnBP) provides for the interaction of the construct with FcRn and contains 6-25, e.g., 10-20, amino acid residues.
114. A construct according to claim 113, wherein the FcRnBP contains 12-20 residues or 15 residues or 16 residues.
115. A construct according to claim 113, wherein said FcRn binding peptide (FcRnBP) comprises a peptide of any one of SEQ ID NOs 48 to 51.
116. A TNFR1 antagonist construct according to claim 113, wherein said FcRn binding peptide (FcRnBP) consists of a peptide shown in any one of SEQ ID NOs 48-51.
117. A construct according to any one of claims 1 to 116 comprising:
a) Domain antibodies that inhibit TNFR 1;
b) A linker that increases flexibility, reduces steric effects, or increases solubility; and
c) Half-life extending moieties.
118. The construct according to claim 117, wherein the half-life extending moiety is not a human serum albumin antibody and is not an unmodified Fc.
119. A construct according to any one of claims 117 or 118 which is a TNFR1 antagonist comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) Half-life extending moiety, which is an IgG Fc.
120. The TNFR1 antagonist construct of any one of claims 117-119, wherein:
the GS linker is (GGGGS) 3 The method comprises the steps of carrying out a first treatment on the surface of the And
the IgG Fc is the Fc of trastuzumab or the Fc of nivolumab.
121. Construct according to any one of claims 1 to 120, which is a TNFR1 antagonist construct comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) A linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; and
c) Half-life extending moiety, which is an IgG Fc.
122. A construct according to claim 121, wherein:
the linker comprises all or part of the hinge sequence of trastuzumab; and
the IgG Fc is that of trastuzumab.
123. A construct according to claim 121, wherein:
The linker comprises all or part of the hinge sequence of nivolumab; and
the IgG Fc is that of nivolumab.
124. Construct according to any one of claims 1 to 123, which is a TNFR1 antagonist construct comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) A second linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; and
d) Half-life extending moiety, which is an IgG Fc.
125. A construct according to claim 124, wherein:
the GS linker is (GGGGS) 3 ；
The second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
the IgG Fc is that of trastuzumab.
126. A construct according to claim 124, wherein:
the GS linker is (GGGGS) 3 ；
The second linker comprises all or part of the hinge sequence of nivolumab; and
the IgG Fc is that of nivolumab.
127. A construct which is a TNFR1 agonist comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) Half-life extending moiety, which is a PEG molecule.
128. A construct according to claim 127, wherein the GS linker is (GGGGS) 3 。
129. A construct according to claim 127 or claim 128 wherein the PEG molecule has a molecular weight of 30kDa or greater.
130. A construct according to any one of claims 1 to 129 which is a TNFR1 agonist construct comprising:
a) A domain antibody (dAb) as shown in any one of SEQ ID NO:52-672, or an scFv as shown in any one of SEQ ID NO:673-678, or a Fab as shown in any one of SEQ ID NO:679-682, or a nanobody as shown in SEQ ID NO:683 or 684, or a TNF mutein as shown in any one of SEQ ID NO: 685-703;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n, wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) A half-life extending moiety that is human serum albumin.
131. A construct according to claim 130, wherein the GS linker is (GGGGS) 3 。
132. Construct according to any of claims 1 to 131, which is a TNFR1 antagonist, wherein the construct is optimized to eliminate an immunogenic sequence or immunogenic epitope.
133. The construct according to any one of claims 1 to 132, wherein said IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the male and female;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) One or more modifications that reduce or eliminate immune effector function.
134. The TNFR1 antagonist construct of claim 133, wherein:
the convex mutation is selected from S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering.
135. A TNFR1 antagonist construct according to claim 133 or claim 134, wherein the one or more modifications that increase or enhance FcRn recycling are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering.
136. The TNFR1 antagonist construct of any one of claims 133 to 135, wherein said immune effector function is selected from one or more of the following: complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cell-mediated phagocytosis (ADCP).
137. The TNFR1 antagonist construct of claim 136, wherein the one or more modifications for reducing or eliminating immune effector function are selected from one or more of the following:
in IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG4, L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A are numbered according to EU.
138. A construct according to any one of claims 1 to 137 which is a TNFR1 antagonist or multispecific or comprises a central PEG linker moiety, and which construct comprises a modified Fc region.
139. The construct according to claim 138, wherein the Fc region is a modified IgG Fc and the modified IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that increase or enhance one or more immune effector functions, wherein:
the one or more immune effector functions are selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
140. A construct according to any one of claims 1 to 139 comprising an Fc region comprising an IgG1 Fc comprising one or more modifications to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib.
141. A TNFR1 antagonist construct according to claim 140, wherein said modification that increases binding to fcyriib is selected from one or more of the following: S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
142. A construct which is a Treg amplicon construct comprising:
a) Treg amplicons;
b) A linker, wherein the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with a receptor and/or increases the solubility of the construct in an aqueous medium; and
c) An activity modulator, wherein the activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator.
143. A construct according to claim 142 or any one of claims 1 to 141 comprising a Treg amplicon, wherein the Treg amplicon is a TNFR2 agonist.
144. The construct according to claim 142 or claim 143, further comprising a TNFR 1-inhibitor.
145. A construct according to claim 143 or claim 144, wherein the TNFR2 agonist is a TNFR2 selective agonist.
146. A construct according to any one of claims 1 to 145 which is a TNFR2 agonist construct comprising:
a) TNFR2 agonists;
b) A linker, wherein the linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with a receptor and/or increases the solubility of the construct in an aqueous medium; and
c) An activity modulator, wherein the activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator.
147. A construct according to claim 146, wherein said TNFR2 agonist is a TNFR2 selective agonist.
148. A construct according to any of claims 142 to 147 comprising an activity modulator, wherein the activity modulator is a half-life extending moiety.
149. The construct according to any of claims 142 to 148, wherein the TNFR2 agonist selectively activates or antagonizes TNFR2 and does not activate or antagonize TNFR1.
150. A construct according to any one of claims 1 to 149 comprising a TNFR2 agonist, wherein the TNFR2 agonist binds to one or more epitopes within TNFR 2.
151. The construct according to claim 150, wherein the TNFR2 is human TNFR2.
152. The construct according to claim 151, wherein the epitope is selected from one or more of the epitopes comprising or consisting of the amino acid sequences shown in SEQ ID NOs 839-865, 1202 and 1204.
153. The construct according to any of claims 149 to 152, wherein said TNFR2 agonist comprises an agonist human anti-TNFR 2 antibody or an antigen-binding fragment of a humanized anti-TNFR 2 antibody, or an antigen-binding portion thereof, or a single chain form thereof.
154. The construct according to claim 153, wherein said agonist anti-TNFR 2 antibody is selected from MR2-1 (also known as ab8161; U.S. patent No. 9,821,010) or MAB2261 (U.S. patent No. 9,821,010).
155. A construct according to any of claims 149 to 154, wherein the TNFR2 agonist is an antigen-binding fragment selected from a dAb, scFv or Fab fragment.
156. A construct according to any of claims 149 to 155 wherein the TNFR2 agonist is a TNFR2 selective agonist.
157. A construct according to any one of claims 1 to 156 comprising a TNFR2 agonist, wherein said TNFR2 agonist comprises a TNFR2 agonist TNF mutein.
158. The construct of claim 157, wherein the TNFR2 mutant protein is a soluble TNF variant comprising one or more TNFR2 selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, and combinations of any of the foregoing, refer to SEQ ID NO:2.
159. The construct of claim 157, wherein the TNFR2 agonist is a TNF mutein comprising the mutation D143N/a 145R.
160. The construct according to any of claims 142 to 159, wherein said linker comprises all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID No. 26 or comprises all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID No. 29.
161. The construct according to any one of claims 1 to 160 comprising a linker, wherein the linker comprises the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID No. 31.
162. A construct according to any of claims 161 comprising a linker, wherein said linker comprises a glycine-serine (GS) linker.
163. A construct according to claim 162, wherein the GS linker is selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS.
164. A construct according to any of claims 146 to 163, wherein the linker comprises all or part of the hinge sequence of the GS linker and trastuzumab corresponding to residues 219-233 of SEQ ID No. 26.
165. The construct of any of claims 146 to 163, wherein the linker comprises the GS linker and the sequence SCDKTH, corresponding to residues 217-222 of SEQ ID No. 31.
166. A construct according to any of claims 146 to 163, wherein the linker comprises all or part of the hinge sequence of GS linker and nivolumab corresponding to residues 212-223 of SEQ ID No. 29.
167. The construct according to any one of claims 1 to 166, comprising a half-life extending moiety, wherein the half-life extending moiety is an IgG Fc, a polyethylene glycol (PEG) molecule, or Human Serum Albumin (HSA).
168. The construct according to claim 167, wherein said IgG Fc is IgG1 or IgG4 Fc.
169. The construct according to claim 168, wherein said IgG1 Fc is the Fc of trastuzumab as shown in SEQ ID No. 27.
170. The construct according to claim 168, wherein said IgG4 Fc is that of nivolumab shown in SEQ ID No. 30.
171. The construct according to claim 168, wherein said IgG1 Fc is the Fc of human IgG1 shown in SEQ ID No. 10.
172. The construct according to claim 168, wherein said IgG4 Fc is the Fc of human IgG4 shown in SEQ ID No. 16.
173. A construct according to any of claims 146 to 172, wherein said TNFR2 agonist is monovalent.
174. A construct according to any one of claims 1 to 173 which is a TNFR2 agonist construct wherein the TNFR2 agonist is bivalent.
175. A construct according to any one of claims 1 to 173 which is a TNFR2 agonist construct wherein the TNFR2 is trivalent.
176. Construct according to any of claims 1 to 175, which is a TNFR2 agonist construct, wherein said linker comprises (Gly 4 Ser) 3 。
177. Construct according to any of claims 1 to 176, which is a TNFR2 agonist construct TNFR2, wherein said linker comprises (Gly 4 Ser) 3 And SCDKTH (residues 217-222 of SEQ ID NO: 31).
178. Construct according to any of claims 1 to 177, which is a TNFR2 agonist construct, wherein said linker comprises (Gly 4 Ser) 3 And a hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26.
179. Construct according to any of claims 1 to 177, which is a TNFR2 agonist construct, wherein said linker comprises (Gly 4 Ser) 3 And a hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO. 29.
180. A construct according to any one of claims 1 to 179 comprising an activity modulator that is a half-life extending moiety.
181. The construct according to claim 180, wherein said half-life extending moiety is PEG.
182. The construct according to claim 180, wherein said PEG has a molecular weight of at least or at least about 30 kDa.
183. The construct of claim 180, wherein the half-life extending moiety is Human Serum Albumin (HSA).
184. A construct according to any one of claims 1 to 183 which is a TNFR2 agonist construct comprising:
a) A TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and
c) The activity modulator, which is a half-life extending moiety, is an IgG Fc.
185. A construct according to claim 184, wherein:
the GS linker is (GGGGS) 3 The method comprises the steps of carrying out a first treatment on the surface of the And
the IgG Fc is the Fc of trastuzumab or the Fc of nivolumab.
186. A construct according to any one of claims 1 to 185 which is a TNFR2 agonist construct comprising:
a) A TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204;
b) A linker selected from the group consisting of all or part of a hinge sequence of trastuzumab and all or part of a hinge sequence of nivolumab; and
c) The activity modulator, which is a half-life extending moiety, is an IgG Fc.
187. A construct according to claim 186, wherein:
The linker comprises all or part of the hinge sequence of trastuzumab; and
the IgG Fc is that of trastuzumab.
188. A construct according to claim 187, wherein:
the linker comprises all or part of the hinge sequence of nivolumab; and
the IgG Fc is that of nivolumab.
189. Construct according to any of claims 1 to 183, which is a TNFR2 construct comprising:
a) A TNFR2 agonist that binds to one or more epitopes within human TNFR2 selected from the group consisting of the epitopes set forth in SEQ ID NOs 839-865, 1202 and 1204;
b) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS;
c) A second linker selected from the group consisting of all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; and
d) The activity modulator, which is a half-life extending moiety, is an IgG Fc.
190. A construct according to claim 189, wherein:
the GS linker is (GGGGS) 3 ；
The second linker comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
The IgG Fc is that of trastuzumab.
191. A construct according to claim 189, wherein:
the GS linker is (GGGGS) 3 ；
The second linker comprises all or part of the hinge sequence of nivolumab; and
the IgG Fc is that of nivolumab.
192. A construct according to any one of claims 1 to 191 which is a TNFR2 agonist construct comprising:
a) A TNFR2 agonist comprising an antigen-binding fragment of an agonist human anti-TNFR 2 antibody selected from MR2-1 or MAB 2261;
b) A joint, comprising:
i) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10;
(Gly 3 Ser) n wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5;
GSGGSSGG；GSSSGSGSGSSG；GSSSGSGSGSSGG；GGSSGG；GGSSGGSGGSSSG；
GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and
c) An activity modulator that is a half-life extending moiety selected from the group consisting of IgG1 or IgG4 Fc, a PEG molecule, and Human Serum Albumin (HSA), wherein:
the IgG1 Fc is the Fc of human IgG1 shown in SEQ ID NO. 10 or the Fc of trastuzumab shown in SEQ ID NO. 27; and
the PEG molecules have a molecular weight of at least or at least about 30 kDa.
193. A construct according to any one of claims 1 to 192 which is or comprises a TNFR2 agonist construct comprising:
a) A TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, reference to SEQ ID NO:2;
b) A joint, comprising:
i) GS linker selected from (GlySer) n Wherein n=1-10；(GlySer 2 )；(Gly 4 Ser) n Wherein n=1-10;
(Gly 3 Ser) n wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5;
GSGGSSGG；GSSSGSGSGSSG；GSSSGSGSGSSGG；GGSSGG；GGSSGGSGGSSSG；
GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab or all or part of the hinge sequence of nivolumab; and
c) An activity modulator that is a half-life extending moiety selected from the group consisting of IgG1 or IgG4 Fc, a PEG molecule, and Human Serum Albumin (HSA), wherein:
the IgG1 Fc is the Fc of human IgG1 shown in SEQ ID NO. 10 or the Fc of trastuzumab shown in SEQ ID NO. 27; and
the PEG molecules have a molecular weight of at least or at least about 30 kDa.
194. A construct according to any one of claims 1 to 193 which is or comprises a TNFR2 agonist construct comprising:
a) TNFR2 TNF muteins comprising the mutation D143N/A145R;
b)(GGGGS) 3 a joint; and
c) An activity modulator, which is a half-life extending moiety, is the Fc of trastuzumab or the Fc of nivolumab.
195. A construct according to any one of claims 1 to 194, which is a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b)(GGGGS) 3 a linker and a second linker comprising the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31); and
c) An activity modulator, which is a half-life extending moiety, is the Fc of trastuzumab.
196. A construct which is a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b)(GGGGS) 3 a linker and a second linker comprising all or part of the hinge sequence of nivolumab; and
c) An activity modulator, which is a half-life extending moiety, is the Fc of nivolumab.
197. A construct according to any one of claims 1 to 196 which is a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b) A linker comprising all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID No. 26; and
c) A half-life extending moiety that is an Fc of trastuzumab.
198. A construct according to any one of claims 1 to 197 which is or comprises a TNFR2 agonist construct comprising:
a) A TNFR2 selective TNF mutein comprising the mutation D143N/A145R;
b) A linker comprising all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID No. 29; and
c) An activity modulator, which is a half-life extending moiety, is the Fc of nivolumab.
199. A construct according to any one of claims 1 to 198 which is a TNFR1 antagonist construct or a TNFR2 agonist construct or both, wherein the IgG Fc is a monomer or dimer.
200. A construct according to any one of claims 1 to 199, comprising a dAb.
201. A construct according to claim 200, comprising a single or double chain (nanobody) of Vhh comprising a dAb.
202. The construct of claim 201, comprising HSA linked to the dAb directly or via a linker.
203. The construct according to claim 202, wherein said linker is a Gly-Ser (GS) linker and/or said HSA is attached to the C-terminus of said dAb via a linker or directly.
204. The construct according to any one of claims 1 to 203, comprising residues 20-732 of SEQ ID No. 1475, which is dAb Dom1h-131-206 of SEQ ID No. 59, linked to HSA by a linker, or a construct having at least 95%, 96%, 97%, 98%,99% sequence identity to the construct of SEQ ID No. 1475 and having TNFR1 antagonist activity.
205. A construct according to claim 200 or 203 comprising the dabs shown in any one of SEQ ID NOs 52-83, 503-672, 1478 and 1479 and variants thereof having at least 95%, 96%, 97%, 98%,99% sequence identity thereto, whereby said construct has TNFR1 antagonist activity.
206. A construct according to claim 205, wherein said dAb has the sequence as shown in any one of SEQ ID NOs 57 to 59 and variants thereof having at least 95% sequence identity thereto, whereby said construct has TNFR1 antagonist activity.
207. A construct according to claim 206, wherein the dAb is specified as DOM1h-131-206 of SEQ ID No. 59 and variants thereof having TNFR1 antagonist activity.
208. A construct according to any of claims 200 to 207, wherein the sequence of the dAb or the essential part of the construct for administration to a human is humanised.
209. A construct according to any one of claims 1 to 208 comprising a TNFR2 agonist or being a TNFR2 agonist construct.
210. A construct according to any one of claims 1 to 208 which is or also is or comprises a TNFR2 agonist construct wherein the TNFR2 agonist is modified to eliminate an amino acid sequence or epitope which is immunogenic in the subject to be treated.
211. A construct according to claim 200, wherein said subject is a human.
212. A construct according to any of claims 189 to 211 wherein said TNFR2 agonist is a TNFR2 selective agonist.
213. A construct according to any one of claims 1 to 212, which is a TNFR2 agonist construct and comprises a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the male and female;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) One or more modifications that reduce or eliminate immune effector function selected from one or more of Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cell-mediated phagocytosis (ADCP).
214. A construct according to claim 213, wherein:
a) The one or more modifications introducing the bulge and the recess are selected from:
one or more of the following mutations: S354C, T366Y, T366W and T394W according to EU numbering; and
one or more concave mutations selected from the group consisting of: Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering whereby Fc forms a dimer;
b) The one or more modifications that increase or enhance FcRn recycling are selected from one or more of the following: T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) The one or more modifications that reduce or eliminate immune effector function are selected from one or more of the following:
in IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG 4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A according to EU numbering.
215. A construct according to any one of claims 1 to 213, which is a TNFR2 agonist construct comprising a modified IgG Fc, wherein said IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering;
c) One or more modifications that increase or enhance immune effector function, wherein:
The immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
216. A construct according to any one of claims 1 to 215, which is or comprises a TNFR2 agonist construct comprising a modified IgG1 Fc, wherein said Fc is modified to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib.
217. A construct according to claim 216, wherein the modification that increases binding to fcyriib is selected from one or more of: according to EU numbering S267E, N297A, L328F, L351S, T366R, L368H, P395K, S267E/L328F and L351S/T366R/L368H/P395K.
218. Construct according to any one of claims 1 to 217, which is or comprises a TNFR2 agonist construct and selectively activates or agonizes TNFR2 but does not activate and does not antagonize TNFR1, comprising:
a) TNFR2 agonists;
b) One or more joints; and
c) An activity modulator, which is a half-life extending moiety, wherein:
the TNFR2 agonist construct is a fusion protein comprising a single-chain TNFR 2-selective TNF mutein trimer fused to a multimerization domain, and comprises the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or (b)
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III),
wherein MD are identical or different multimerization domains; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers that may be the same or different.
219. A construct according to claim 218, wherein said TNF mutein comprises one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, reference to SEQ ID NO:2.
220. A construct according to claim 218, wherein said TNF mutein comprises the TNFR2 selective mutation D143N/a145R.
221. Construct according to any of claims 218 to 220, wherein the multimerization domain is selected from EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139;SEQ ID NO:805 of SEQ ID NO: 804), or the trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806).
222. The TNFR2 agonist construct of any one of claims 218 to 221, wherein the multimerization domain is an IgG1 Fc or an IgG4 Fc and wherein the IgG1 Fc or IgG4 Fc is also a half-life extending moiety.
223. The TNFR2 agonist construct according to any one of claims 218 to 222, wherein the L1, L2 and/or L3 linker is independently selected from (GGGGS) n Wherein n=1-5, or all or part of the TNF stalk region (SEQ ID NO: 812).
224. The TNFR2 agonist construct of any one of claims 218 to 223, wherein the junction between the TNFR2 agonist and the half-life extending moiety is:
GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or (b)
A linker selected from the group consisting of a full or partial hinge sequence of trastuzumab and a full or partial hinge sequence of nivolumab; or alternatively
A combination thereof.
225. The TNFR2 agonist construct according to any one of claims 218 to 224, wherein the half-life extending moiety is selected from the group consisting of:
IgG1 Fc which is the Fc of human IgG1 shown in SEQ ID NO. 10 or the Fc of trastuzumab shown in SEQ ID NO. 27;
IgG4 Fc, which is the Fc of human IgG4 shown in SEQ ID NO. 16, or the Fc of nivolumab shown in SEQ ID NO. 30;
a PEG molecule having a size of at least or at least about 30kDa; and
human Serum Albumin (HSA).
226. Construct according to any one of claims 1 to 217, which is or comprises a TNFR2 agonist construct comprising:
a) The construct has the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III),
wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, wherein:
i) The MD is selected from the group consisting of EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139 of SEQ ID NO: 804; SEQ ID NO: 805), or a trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806);
ii) L1, L2 and L3 are each (GGGGS) n Wherein n=1-5, or all or part of the TNF stem region (SEQ ID NO: 812), or mixtures thereof; and
iii) The TNF mutein comprises the TNFR2 selective mutation D143N/A145R;
b) A half-life extending moiety selected from the group consisting of:
IgG1 Fc which is the Fc of human IgG1 shown in SEQ ID NO. 10 or the Fc of trastuzumab shown in SEQ ID NO. 27;
IgG4 Fc, which is the Fc of human IgG4 shown in SEQ ID NO. 16, or the Fc of nivolumab shown in SEQ ID NO. 30;
a PEG molecule having a size of at least or at least about 30kDa; and
human Serum Albumin (HSA); and
c) A linker between a TNFR 2-selective agonist and a half-life extending moiety, wherein the linker comprises:
GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Which is provided withN=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or (b)
A linker selected from the group consisting of all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; or alternatively
A combination thereof.
227. Construct according to any of claims 1 to 217, which is a TNFR2 agonist construct comprising the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III),
wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, wherein:
i) MD is selected from IgG1 Fc or IgG4 Fc;
II) L2 and L3 in formula II and L1 and L2 in formula III are each independently (GGGGS) n Wherein n=1-5, or all or part of the TNF stem region (SEQ ID NO: 812), or a combination thereof;
iii) L1 in formula II and L3 in formula III are each independently selected from:
GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10; (Gly) 3 Ser) n Wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5; GSGGSSGG; GSSSGSGSGSSG; GSSSGSGSGSSGG; GGSSGG; GGSSGGSGGSSSG; GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; or (b)
A linker selected from the group consisting of all or part of the hinge sequence of trastuzumab and all or part of the hinge sequence of nivolumab; or alternatively
A combination thereof; and
iv) the TNF mutein comprises the TNFR2 selective mutation D143N/A145R.
228. The construct according to claim 227, wherein the MD is selected from the group consisting of:
IgG1 Fc which is the Fc of human IgG1 shown in SEQ ID NO. 10 or the Fc of trastuzumab shown in SEQ ID NO. 27; or alternatively
IgG4 Fc, which is the Fc of human IgG4 shown in SEQ ID NO. 16, or the Fc of nivolumab shown in SEQ ID NO. 30.
229. The construct according to claim 227 or claim 228, wherein said MD is an IgG1 Fc of trastuzumab and the linker between said MD and adjacent TNF muteins is all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID No. 26.
230. The construct according to claim 227 or claim 228, wherein said MD is an IgG1Fc of trastuzumab and the linker between said MD and adjacent TNF muteins comprises the sequence SCDKTH (residues 217-222 of SEQ ID NO: 31).
231. The construct according to any of claims 227 to 230, wherein said MD is an IgG1Fc of trastuzumab and the linker between said MD and adjacent TNF muteins comprises (Gly 4 Ser) 3 And a hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26.
232. The construct of claim 227 or claim 228, wherein said MD is an IgG1Fc of trastuzumab and the linker between said MD and adjacent TNF muteins comprises (Gly 4 Ser) 3 And SCDKTH (residues 222-227 of SEQ ID NO: 31).
233. The construct according to claim 227 or claim 228, wherein said MD is IgG4Fc of nivolumab and the linker between said MD and adjacent TNF muteins comprises all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID No. 29.
234. The construct of claim 227 or claim 228, wherein said MD is IgG4Fc of nivolumab and the linker between said MD and adjacent TNF muteins comprises (Gly 4 Ser) 3 And all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO. 29.
235. A construct according to any of claims 1 to 234 which is an agonist construct wherein the TNFR2 agonist is modified to eliminate an immunogenic sequence or epitope which is immunogenic in the subject.
236. A construct according to claim 235, wherein said subject is a human.
237. A construct according to any one of claims 1 to 236, which is a TNFR2 agonist construct and comprises a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that reduce or eliminate immune effector function, wherein:
the immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that reduce or eliminate immune effector function are selected from one or more of the following:
in IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG 4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A according to EU numbering.
238. A construct according to any one of claims 1 to 237, which is a TNFR2 agonist construct comprising a modified IgG Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
according to EU numbering, T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y; and
c) One or more modifications that increase or enhance immune effector function, wherein:
the immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
239. A TNFR2 agonist construct according to any one of claims 1 to 238 which is a TNFR2 agonist construct comprising an IgG1 Fc modified to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib.
240. A TNFR2 agonist construct according to claim 239, wherein the modification that increases binding to fcyriib is selected from one or more of S267E, N297A, L328F, L S, T366R, L368 38395K, S267E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
241. A construct according to any one of claims 1 to 240 which is a multispecific TNFR1 inhibitor/TNFR 2 agonist construct and has the formula:
(TNFR 1 inhibitor) n Joint (L) p - (TNFR 2 agonists) q (formula I), or
(TNFR 1 inhibitor) n Joint (L) p - (TNFR 2 agonists) q Or (b)
(TNFR 1 inhibitor) n - (TNFR 2 agonists) q Joint (L) p Or (b)
(TNFR 2 agonist) q - (TNFR 1 inhibitors) n Joint (L) p Or (b)
Any of the above comprising an optional activity modulator, wherein:
n=1 or 2, p=1, 2 or 3, and q=1 or 2;
the TNFR1 inhibitor interacts with TNFR1 to inhibit its activity;
an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and
The linker increases the solubility of the construct, or increases the flexibility of the construct, or alters the steric effect of the construct.
242. A construct according to any one of claims 1 to 241 which is a multispecific TNFR1 inhibitor/TNFR 2 agonist construct, wherein:
the TNFR1 inhibitor selectively inhibits or antagonizes TNFR1 signaling and does not inhibit or antagonize TNFR2 signaling;
the TNFR1 inhibitor does not interfere with activation or agonism of TNFR 2;
the TNFR2 agonist selectively activates or agonizes TNFR2 signaling and does not activate or agonize TNFR1 signaling; and
the TNFR2 agonist does not interfere with the inhibition or antagonism of TNFR 1.
243. A construct according to claim 241 or claim 242, wherein:
a) The TNFR1 inhibitor is selected from the group consisting of:
i) An antigen binding fragment of a human anti-TNFR 1 antagonist monoclonal antibody selected from H398 or ATROSAB or a polypeptide having a sequence with at least 95% sequence identity thereto; or alternatively
ii) a domain antibody (dAb) as shown in any one of SEQ ID NOS.52-672, or an scFv as shown in any one of SEQ ID NOS.673-678, or a Fab as shown in any one of SEQ ID NOS.679-682, or a nanobody as shown in SEQ ID NOS.683 or 684, or a TNF mutein as shown in any one of SEQ ID NOS.701-703, or a polypeptide having a sequence of at least 95% sequence identity to any of the foregoing polypeptides and being a TNFR1 inhibitor; or alternatively
iii) A dominant negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule having one or more amino acid substitutions that confer selective inhibition of TNFR1 and selected from the group consisting of:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF shown in SEQ ID NO 2;
b) The linker is selected from:
i) GS linker selected from (GlySer) n Wherein n=1-10; (GlySer) 2 )；(Gly 4 Ser) n Wherein n=1-10;
(Gly 3 Ser) n wherein n=1-5; (SerGly) 4 ) n Wherein n=1-5; (GlySerSerGly) n Wherein n=1-5;
GSGGSSGG；GSSSGSGSGSSG；GSSSGSGSGSSGG；GGSSGG；GGSSGGSGGSSSG；
GSSSGSGSGGSSSGSGSG; GGSSGGSSGGGSSGGSSG; and GSSSGS; and/or
ii) all or part of the hinge sequence of trastuzumab corresponding to residues 219-233 of SEQ ID NO. 26 or all or part of the hinge sequence of nivolumab corresponding to residues 212-223 of SEQ ID NO. 29; and
iii) IgG1 or IgG4 Fc, wherein:
IgG1 Fc is selected from the group consisting of the IgG1 Fc of human IgG1 shown in SEQ ID NO. 10, and the IgG1 Fc of trastuzumab shown in SEQ ID NO. 27;
IgG4 Fc is selected from human IgG4 Fc shown in SEQ ID NO. 16, or Nawuzumab IgG4 Fc shown in SEQ ID NO. 30; and
optionally, the Fc comprises one or more modifications to introduce a bulge recess, and/or to increase or enhance neonatal Fc receptor (FcRn) recycling, and/or to reduce or eliminate immune effector function; and
c) The TNFR2 agonist is selected from the group consisting of:
i) An antigen binding fragment that binds to one or more epitopes within human TNFR2, said epitopes selected from the group consisting of SEQ ID NOs:
839-865, 1202 and 1204; or alternatively
ii) an antigen binding fragment of an agonistic human anti-TNFR 2 antibody selected from MR2-1 or MAB 2261; or alternatively
iii) A TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V,
A145R，A145H，A145K，A145F，A145W，E146Q，E146H，E146K，E146N，
D143N/A145R，A145R/S147T，Q88N/T89S/A145S/E146A/S147D，
Q88N/A145I/E146G/S147D，A145H/E146S/S147D，A145H/S147D，
L29V/A145D/E146D/S147D，A145N/E146D/S147D，A145T/E146S/S147D，
A145Q/E146D/S147D，A145T/E146D/S147D，A145D/E146G/S147D，A145D/S147D，
A145K/E146D/S147T，A145R/E146T/S147D，A145R/S147T，E146D/S147D，
D143V/F144L/A145S, S95C/G148C, and D143V/A145S, with reference to SEQ ID NO. 2; or (b)
iv) Single-chain TNFR 2-selective TNF mutein trimer comprising the mutation D143N/A145R, wherein
The TNF mutein consists of (GGGGS) n Or all or part of the TNF stem region (SEQ ID NO: 812) where n=1-5; or alternatively
v) a TNFR 2-selective agonist comprising the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III);
wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, and wherein:
the MD is selected from EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139;SEQ ID NO:805 of SEQ ID NO: 804), or the trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806);
l1, L2 and L3 are each (GGGGS) n Wherein n=1-5, or all or part of the stem region of TNF (SEQ ID NO: 812), or mixtures thereof; and
TNF muteins comprise the TNFR 2-selective mutation D143N/A145R.
244. A construct according to claim 241 or claim 242 which is a multispecific TNFR1 antagonist/TNFR 2 agonist construct wherein:
a) The TNFR1 inhibitor comprises a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) The joint comprises (GGGGS) 3 An Fc comprising a polypeptide of sequence SCDKTH (residues 222-227 of SEQ ID NO: 26) and trastuzumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, reference to SEQ ID NO:2.
245. A construct according to claim 241 or claim 242, wherein:
a) The TNFR1 inhibitor comprises a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) The joint comprises (GGGGS) 3 All or part of the hinge sequence of nivolumab and Fc of nivolumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, reference to SEQ ID NO:2.
246. A construct according to claim 241 or claim 242, wherein:
a) The TNFR1 inhibitor comprises a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) The joint comprises (GGGGS) 3 And Fc of trastuzumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, reference to SEQ ID NO:2.
247. A construct according to claim 241 or claim 242, wherein:
a) The TNFR1 inhibitor comprises a domain antibody (dAb) as set forth in any one of SEQ ID NOS: 52-672, or an scFv as set forth in any one of SEQ ID NOS: 673-678, or a Fab as set forth in any one of SEQ ID NOS: 679-682, or a nanobody as set forth in SEQ ID NOS: 683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS: 701-703, or a sequence having at least or at least about 95% sequence identity thereto;
b) The joint comprises (GGGGS) 3 And Fc of nivolumab; and
c) The TNFR2 agonist comprises a TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V, A145R, A145H, A145K, A145F, A145W, E146Q, E146H, E146K, E146N, D143N/A145R, A145R/S147T, Q88N/T89S/A145S/E146A/S147D, Q88N/A145I/E146G/S147D, A145H/E146S/S147D, A145H/S147D, L29V/A145D/E146D/S147D, A145N/E146D/S147D, A145T/E146S/S147D, A145Q/E146D/S147D, A145T/E146D/S147D, A145D/E146G/S147D, A145D/S147D, A145K/E146D/S147T, A145R/E146T/S147D, A145R/S147T, E146D/S147D, D143V/F144L/A145S, S95C/G148C, and D143V/A145S, and any combination of the foregoing mutations, refer to SEQ ID NO:2.
248. The construct according to any of claims 241 to 247, comprising a modified Fc, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the male and female;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling; and
c) One or more modifications that reduce or eliminate immune effector function.
249. A construct according to claim 248, wherein said Fc comprises modifications that introduce a bulge recess:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W of EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V of EU numbering.
250. A construct according to claim 248, wherein the Fc comprises modifications that increase or enhance FcRn recycling selected from one or more of:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering.
251. A construct according to claim 248, wherein said Fc comprises modifications to immune effector functions selected from one or more of Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cell-mediated phagocytosis (ADCP).
252. A construct according to claim 248, comprising one or more modifications that reduce or eliminate immune effector function selected from one or more of the following:
in IgG 1: L235E, L234A/L235A, L234E/L235F/P331S, L234F/L235E/P331S, L234A/L235A/P329G, L234A/L235A/G237A/P238S/H268A/A330S/P331S, G236R/L328R, G237A, E318A, D265A, E233P, N297A, N297Q, N297D, N297G, N297G/D265A, A330L, D270A, P329A, P331A, K322A, V264A, and F241A according to EU numbering; and
In IgG 4: L235E, F234A/L235A, S228P/L235E, and S228P/F234A/L235A according to EU numbering.
253. The construct according to any one of claims 241 to 247, wherein the IgG Fc comprises one or more of the following modifications:
a) Introducing one or more modifications of the bulge and the recess, wherein:
the convex mutation is selected from one or more of S354C, T366Y, T366W and T394W according to EU numbering; and
the concave mutation is selected from one or more of Y349C, T366S, L368A, F405A, Y407T, Y407A and Y407V according to EU numbering;
b) One or more modifications that increase or enhance neonatal Fc receptor (FcRn) recycling, wherein the modifications are selected from one or more of the following:
T250Q, T250R, M252F, M252W, M252Y, S254T, T256D, T256E, T256Q, V259I, V308F, E380A, M428L, H433K, N434F, N434A, N434W, N434S, N434Y, Y436H, M252Y/T256Q, M252F/T256D, M252Y/S254T/T256E, H433K/N434F/Y436H, N434F/Y436H, T250Q/M428L, T250R/M428L, M428L/N434S, V259I/V308F/M428L, E294del/T307P/N434Y, and T256N/A378V/S383N/N434Y according to EU numbering; and
c) One or more modifications that increase or enhance immune effector function, wherein:
the immune effector function is selected from one or more of CDC, ADCC and ADCP; and
the one or more modifications that increase or enhance immune effector function are selected from one or more of the following:
in IgG 1: S239D, I332E, S239D/I332E, S239D/A330L/I332E, S298A/E333A/K334A according to EU numbering; F243L/R292P/Y300L/V305I/P396L; L235V/F243L/R292P/Y300L/P396L; F243L/R292P/Y300L; L234Y/G236W/S298A in the first heavy chain and S239D/A330L/I332E in the second heavy chain; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A in the first heavy chain and D270E/K326D/A330M/K334E in the second heavy chain; A327Q/P329A; D265A/S267A/H268A/D270A/K326A/S337A; T256A/K290A/S298A/E333A/K334A; G236A; G236A/I332E; G236A/S239D/I332E; G236A/S239D/A330L/I332E; introducing a biantennary glycan at residue N297; introduction of a defucosylated glycan at residue N297; K326W; K326A; E333A; K326A/E333A; K326W/E333S; K326M/E333S; K222W/T223W; K222W/T223W/H224W; D221W/K222W; C220D/D221C; C220D/D221C/K222W/T223W; H268F/S324T; S267E; H268F; S324T; S267E/H268F/S324T; G236A/I332E/S267E/H268F/S324T; E345R; and E345R/E430G/S440Y.
254. A construct according to any of claims 241 to 253, wherein said construct comprises IgG1 Fc modified to increase binding to an inhibitory fcγ receptor (fcγr) fcγriib.
255. A construct according to claim 254, wherein the modification to increase binding to fcyriib is selected from one or more of S267E, N297A, L328F, L351S, T366R, L368H, P395K, S E/L328F and L351S/T366R/L368H/P395K according to EU numbering.
256. A construct according to any one of claims 1 to 255 which is a multispecific TNFR1 antagonist/TNFR 2 agonist, wherein:
the TNFR1 antagonist is monovalent; and
the TNFR2 agonist is monovalent.
257. A construct according to any one of claims 1 to 255 which is a multispecific TNFR1 antagonist/TNFR 2 agonist, wherein:
the TNFR1 antagonist is monovalent; and
the TNFR2 agonist is bivalent.
258. The construct according to any of claims 241 to 257, which is a multispecific TNFR1 antagonist/TNFR 2 agonist, wherein:
a) The TNFR1 antagonist is selected from the group consisting of:
i) An antigen binding fragment of a human anti-TNFR 1 antagonist monoclonal antibody selected from H398 or ATROSAB; or alternatively
ii) a domain antibody (dAb) as set forth in any one of SEQ ID NOS.52-672, or an scFv as set forth in any one of SEQ ID NOS.673-678, or a Fab as set forth in any one of SEQ ID NOS.679-682, or a nanobody as set forth in SEQ ID NOS.683 or 684, or a TNF mutein as set forth in any one of SEQ ID NOS.701-703, or a sequence having at least or at least about 95% sequence identity thereto; or alternatively
iii) A dominant negative tumor necrosis factor (DN-TNF) or TNF mutein comprising a soluble TNF molecule having one or more amino acid substitutions that confer selective inhibition of TNFR1 and selected from the group consisting of:
V1M, L29S, L29G, L29Y, R31C, R31E, R31N, R32Y, R32W, C69V, A84S, V85T, S86T, Y87H, Q88N, T89Q, I97T, C101A, A145R, E146R, L29S/R32W, L29S/S86T, R32W/S86T, L29S/R32W/S86T, R31N/R32T, R31E/S86T, R31N/R32T/S86T, I97T/A145R, V1M/R31C/C69V/Y87H/C101A/A145R, and A84S/V85T/S86T/Y87H/Q88N/T89Q, with reference to the sequence of soluble TNF shown in SEQ ID NO 2;
b) The linker is a branched PEG molecule having a size of at least or at least about 30kDa; and
c) The TNFR2 agonist is selected from the group consisting of:
i) An antigen binding fragment that binds to one or more epitopes within human TNFR2, said epitopes selected from the group consisting of SEQ ID NOs:
839-865, 1202 and 1204; or alternatively
ii) an antigen binding fragment of an agonistic human anti-TNFR 2 antibody selected from MR2-1 or MAB 2261; or alternatively
iii) A TNFR 2-selective TNF mutein that is a soluble TNF variant comprising one or more TNFR 2-selective mutations selected from the group consisting of: K65W, D143Y, D143F, D143N, D143E, D143W, D143V,
A145R，A145H，A145K，A145F，A145W，E146Q，E146H，E146K，E146N，
D143N/A145R，A145R/S147T，Q88N/T89S/A145S/E146A/S147D，
Q88N/A145I/E146G/S147D，A145H/E146S/S147D，A145H/S147D，
L29V/A145D/E146D/S147D，A145N/E146D/S147D，A145T/E146S/S147D，
A145Q/E146D/S147D，A145T/E146D/S147D，A145D/E146G/S147D，A145D/S147D，
A145K/E146D/S147T，A145R/E146T/S147D，A145R/S147T，E146D/S147D，
D143V/F144L/A145S, S95C/G148C, and D143V/A145S, with reference to SEQ ID NO. 2; or (b)
iv) a single-chain TNFR 2-selective TNF mutein trimer comprising the mutation D143N/A145R, wherein said TNF mutein consists of (GGGGS) n Or all or part of the TNF stem region (SEQ ID NO: 812) is ligated, wherein
n=1-5; or alternatively
v) a TNFR 2-selective agonist comprising the formula:
MD-L1-TNFut-L2-TNFut-L3-TNFut (formula II); or alternatively
TNFut-L1-TNFut-L2-TNFut-L3-MD (formula III);
wherein MD is a multimerization domain; TNFut is a TNFR 2-selective TNF mutein; and L1, L2 and L3 are linkers, which may be the same or different, and wherein:
the MD is selected from EHD2 (SEQ ID NO: 808), MHD2 (SEQ ID NO: 811), the trimerization domain of chicken tenascin C (TNC) (residues 110-139;SEQ ID NO:805 of SEQ ID NO: 804), or the trimerization domain of human TNC (residues 110-139,SEQ ID NO:807 of SEQ ID NO: 806);
l1, L2 and L3 are each (GGGGS) n Wherein n=1-5, or all or part of the TNF stalk region (SEQ ID NO: 812), or mixtures thereofThe method comprises the steps of carrying out a first treatment on the surface of the And
The TNF muteins comprise the TNFR2 selective mutation D143N/A145R.
259. A construct according to claim 258, wherein each of said TNFR1 antagonist and TNFR2 agonist is monovalent.
260. A construct according to claim 258, wherein said TNFR1 antagonist is monovalent and said TNFR2 agonist is bivalent.
261. A construct according to any one of claims 1 to 260 which is a multispecific TNFR1 antagonist/TNFR 2 agonist for use in the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder whose etiology is characterized by TNF overexpression or deregulation of TNFR1 signaling.
262. Use of a construct according to any one of claims 1 to 260, which is a multispecific TNFR1 antagonist/TNFR 2 agonist, for the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder whose etiology is characterized by TNF overexpression or deregulation of TNFR1 signaling.
263. A composition comprising the construct of any one of claims 1 to 260 in a pharmaceutically acceptable carrier or vehicle.
264. A composition according to claim 263, for use in the treatment of a chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder whose etiology is characterized by TNF overexpression or TNFR1 signaling imbalance.
265. A construct according to any one of claims 1 to 261 or the use of claim 262, or a composition of claim 263 or claim 264, wherein said chronic inflammatory, autoimmune, neurodegenerative, demyelinating or respiratory disease or disorder, or a disease, condition or disorder whose etiology is characterized by TNF overexpression or TNFR1 signaling is selected from the group consisting of:
rheumatoid Arthritis (RA), psoriasis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA), spinal arthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, inflammatory Bowel Disease (IBD), uveitis, fibrotic disease, endometriosis, lupus, multiple Sclerosis (MS), congestive heart failure, cardiovascular disease, myocardial Infarction (MI), atherosclerosis, metabolic disease, cytokine release syndrome, septic shock, sepsis, acute Respiratory Distress Syndrome (ARDS), severe Acute Respiratory Syndrome (SARS), SARS-CoV-2, influenza, acute and chronic neurodegenerative diseases, demyelinating diseases and disorders, stroke, alzheimer's disease, parkinson's disease, behcet's disease, dupuytren's disease, tumor necrosis factor receptor-related periodic syndrome (trap), pancreatitis, type I diabetes, chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, emphysema, graft rejection, graft-versus-host disease (GvHD), pulmonary inflammation, pulmonary diseases and conditions, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, acute fulminant viral or bacterial infections, pneumonia, genetic diseases with TNF/TNFR1 as the causative pathological medium, periodic fever syndrome or cancer.
266. A construct according to any one of claims 1 to 261 or the use of claim 262, or a composition of claim 263 or claim 264, for use in the treatment of rheumatoid arthritis.
267. Use of a construct according to any one of claims 1 to 261 or of claim 262 or of a composition according to claim 263 or claim 264 for the treatment of rheumatoid arthritis.
268. A construct which is a TNFR2 antagonist construct comprising a TNFR2 antagonist and optionally a linker and optionally an activity modulator.
269. A construct according to claim 268, comprising formula 5:
(TNFR 2 antagonists )n -a joint p - (activity modulating agent) q Or (b)
Joint p - (activity modulating agent) q - (TNFR 2 antagonists) n Wherein:
n and q are each an integer and are each independently 1, 2 or 3;
p is 0, 1, 2 or 3;
a TNFR2 antagonist is a molecule that interacts with TNFR2 to inhibit (antagonize) its TNFR2 activity, thereby inhibiting Treg proliferation and/or inducing death thereof, and may also inhibit proliferation and induce death of tumor cells expressing TNFR 2;
an activity modulator is a moiety that modulates or alters the activity or pharmacological properties of a construct compared to the construct in the absence of the activity modulator; and
The linker increases the flexibility of the construct and/or mitigates or reduces the steric effect of the construct or its interaction with the receptor and/or increases the solubility of the construct in aqueous media.
270. A construct according to claim 268 or claim 269, wherein the activity modulator and linker are each as defined and described for the construct of any one of claims 1 to 250.
271. A construct according to any of claims 268 to 270, wherein the TNFR2 antagonist:
reducing and/or inhibiting proliferation of bone Marrow Derived Suppressor Cells (MDSCs); and/or
Inducing apoptosis within MDSCs by binding to TNFR2 expressed on the surface of MDSCs present in the tumor microenvironment; and/or
By inhibiting Treg expansion and activity, expansion of T effector cells, including cytotoxic cd8+ T cells, is induced.
272. Construct according to any of claims 268 to 271, wherein the TNRF2 antagonist is an antibody, antigen binding fragment thereof or a single chain antibody which binds to an epitope within human TNFR2 containing one or more of residues KCRPG (corresponding to residues 142-146 of SEQ ID NO: 4) or larger epitopes containing for example residues 130-149, 137-144 or 142-149 or at least 5 consecutive or non-consecutive residues within these epitopes and does not bind to an epitope containing residue KCSPG (corresponding to residues 56-60 of SEQ ID NO: 4); or binding to the TNFR2 epitope PECLSCGS (corresponding to residues 91-98 of SEQ ID NO: 4), RICTCRPG (corresponding to residues 116-123 of SEQ ID NO: 4), CAPLRCR (corresponding to residues 137-144 of SEQ ID NO: 4), LRKCRPGFGVA (corresponding to residues 140-150 of SEQ ID NO: 4) and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or an epitope comprising at least 5 contiguous or non-contiguous residues within residues 75-128, 86-103, 111-128 or 150-190 of SEQ ID NO: 4.
273. The construct according to any of claims 268 to 272, wherein said antibody, fragment thereof or single chain form thereof binds to an epitope comprising one or more residues of the KCRPG sequence (SEQ ID NO: 840) with an affinity that is at least 10-fold higher than the affinity of the same antibody or antigen binding fragment for a peptide comprising the KCSPG sequence of human TNFR2 (SEQ ID NO: 839).
274. A construct according to any of claims 268 to 273, wherein the TNFR2 antagonist is an antibody or fragment or single chain form of an antibody selected from the group consisting of:
TNFRAB1 (see SEQ ID NOs: 1212 and 1213, representing the heavy and light chain sequences of TNFRAB1, respectively), TNFRAB2, and TNFR2A3 (see, e.g., U.S. patent publication No. 2019/0144556 for descriptions of these antibodies);
antibodies and antibody fragments, and single chain versions thereof, which contain the CDR-H3 sequence of TNFRAB1 (QRVDGYSSYWYFDV; corresponding to residues 99-112 of SEQ ID NO: 1212), the CDR-H3 sequence of TNFRAB2 (ARDDGSYSPFDYWG; SEQ ID NO: 1217), or the CDR-H3 sequence of TNFR2A3 (ARDDGSYSPFDYFG; SEQ ID NO: 1223), or a CDR-H3 sequence having at least about 85% sequence identity thereto, e.g., TNFRAB1 specifically binds residues 130-149 of residue KCRPG containing TNFR2 with an affinity 40-fold higher than residues 48-67 of residue KCSPG containing TNFR 2.
275. The construct according to any of claims 268 to 274, wherein the TNFR2 antagonist binds to one or more epitopes in TNFR2 selected from the group consisting of:
epitope containing residues 137-144 (CAPLRKCR; SEQ ID NO: 851)
Epitopes comprising one or more residues within positions 80-86 (DSTYTQL; SEQ ID NO: 1247), 91-98 (PECLSCGS; SEQ ID NO: 1248) and/or 116-123 (RICTCRPG; SEQ ID NO: 1249) of human TNFR 2; and
TNFR2A3 epitope is selected from a first epitope comprising residues 140-150 (LRKCRPGFGVA; SEQ ID NO: 1463) of human TNFR2 and containing the KCRPG motif, and/or a second epitope comprising residues 159-171 (VVCKPCAPGTFSN; SEQ ID NO: 1464) of human TNFR 2.
276. A construct according to any of claims 268 to 275, wherein said TNFR2 antagonist is an antibody, fragment thereof, or single chain form thereof, comprising one or more of the following sequences: one or more CDR-H1 amino acids having a sequence as shown in any one of SEQ ID NO 1214, 1215 and 1231-1233, a CDR-H2 sequence as shown in any one of SEQ ID NO 1216, 1224 and 1230, a CDR-H3 sequence as shown in any one of SEQ ID NO 1217, 1223 and 1225-1229, and/or a CDR-H3 of TNFRAB1 corresponding to residues 99-112 of SEQ ID NO 1212; the CDR-L1 sequences shown in either of SEQ ID NOS 1218 and 1234-1236, and/or the CDR-L1 sequence of TNFRAB1 corresponding to residues 24-33 of SEQ ID NO 1213; the CDR-L2 sequences shown in any one of SEQ ID NOS 1219, 1220, 1237 and 1238, or the CDR-L2 sequence of TNFRAB1 corresponding to residues 49-55 of SEQ ID NO 1213; and/or CDR-L3 sequences as shown in any one of SEQ ID NOS 1221, 1222 and 1241 to 1244 or CDR-L3 sequences of TNFRAB1 corresponding to residues 88 to 96 of SEQ ID NO 1213; and/or replacing the CDR-H1 and CDR-H2 sequences of the human antibody heavy chain variable domain consensus sequence of SEQ ID NO. 1245 with corresponding CDR sequences of a phenotypically neutral, TNFR2 specific antibody and/or replacing the CDR-L1, CDR-L2 and CDR-L3 sequences of the human antibody light chain variable domain sequence of SEQ ID NO. 1246 with corresponding CDR sequences of a phenotypically neutral, TNFR2 specific antibody to produce a humanized antagonistic TNFR2 antibody.
277. The construct according to any of claims 268 to 275, wherein the TNFR2 antagonist specifically binds an epitope within TNFR2 as set forth in any of SEQ ID NOs 1247-1464.
278. The construct according to any of claims 268 to 277, wherein the TNFR2 antagonist specifically binds to one or more epitopes selected from the group consisting of:
(a) One or more epitopes within human TNFR2 that contain one or more of residues KCRPG corresponding to residues 142-146 of SEQ ID NO. 4, or larger epitopes containing residues 130-149, 137-144 or 142-149 or at least 5 consecutive or discontinuous residues within these epitopes, and that do not bind epitopes containing residues KCSPG corresponding to residues 56-60 of SEQ ID NO. 4; and/or
(b) One or more TNFR2 epitopes comprising an amino acid sequence comprising:
PECLSCGS corresponding to residues 91-98 of SEQ ID No. 4, and/or RICTCRPG corresponding to residues 116-123 of SEQ ID No. 4, and/or
CAPLRKCR corresponding to residues 137-144 of SEQ ID NO:4, and/or LRKCRPGFGVA corresponding to residues 140-150 of SEQ ID NO: 4), and/or VVCKPCAPGTFSN (corresponding to residues 159-171 of SEQ ID NO: 4), and/or
An epitope comprising at least 5 contiguous or non-contiguous residues within residues 75-128, 86-103, 111-128 or 150-190 of SEQ ID NO. 4.
279. The construct according to any of claims 268 to 278, comprising a TNFR2 antagonist which is a small molecule.
280. The construct of claim 279, wherein the TNFR2 antagonist is thalidomide or an analog thereof.
281. The construct of claim 280, wherein the thalidomide analogs are lenalidomide and pomalidomide.
282. A construct according to any one of claims 268 to 281, comprising a TNFR2 antagonist that reduces FoxP3 expression and inhibits the inhibitory activity of Treg.
283. A construct according to claim 282, wherein said TNFR2 antagonist is a histone deacetylase inhibitor, which reduces FoxP3 expression and inhibits the inhibitory activity of tregs.
284. A construct according to claim 282 or claim 283, wherein the inhibitor is panobinostat or cyclophosphamide or triptolide.
285. A construct according to any one of claims 268 to 282 for use in the treatment of infectious diseases and cancers that express TNFR 2.
286. A construct according to claim 285, wherein said cancer is a cancer selected from the group consisting of: t cell lymphomas such as hodgkin's lymphoma and cutaneous non-hodgkin's lymphoma, ovarian cancer, colon cancer, multiple myeloma, renal cell carcinoma, breast cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, and lung cancer.
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