WO2011090762A1 - Heterodimer binding proteins and uses thereof - Google Patents

Heterodimer binding proteins and uses thereof Download PDF

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Publication number
WO2011090762A1
WO2011090762A1 PCT/US2010/062436 US2010062436W WO2011090762A1 WO 2011090762 A1 WO2011090762 A1 WO 2011090762A1 US 2010062436 W US2010062436 W US 2010062436W WO 2011090762 A1 WO2011090762 A1 WO 2011090762A1
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Prior art keywords
region
polypeptide
single chain
domain
heterodimer
Prior art date
Application number
PCT/US2010/062436
Other languages
French (fr)
Inventor
John W. Blankenship
Philip Tan
Original Assignee
Emergent Product Development Seattle, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority to AU2010343057A priority Critical patent/AU2010343057B2/en
Priority to NZ600820A priority patent/NZ600820A/en
Application filed by Emergent Product Development Seattle, Llc filed Critical Emergent Product Development Seattle, Llc
Priority to SI201031227A priority patent/SI2519543T1/en
Priority to DK10803713.6T priority patent/DK2519543T3/en
Priority to RS20160812A priority patent/RS55229B1/en
Priority to LTEP10803713.6T priority patent/LT2519543T/en
Priority to EA201290568A priority patent/EA023674B1/en
Priority to CA2784814A priority patent/CA2784814C/en
Priority to KR1020127019837A priority patent/KR20120125611A/en
Priority to CN2010800649252A priority patent/CN102958942A/en
Priority to BR112012016135A priority patent/BR112012016135A2/en
Priority to US13/519,727 priority patent/US20130129723A1/en
Priority to SG2012047411A priority patent/SG181952A1/en
Priority to EP10803713.6A priority patent/EP2519543B1/en
Priority to ES10803713.6T priority patent/ES2592385T3/en
Priority to JP2012547282A priority patent/JP5851419B2/en
Priority to MX2012007533A priority patent/MX341796B/en
Priority to MEP-2016-186A priority patent/ME02505B/en
Publication of WO2011090762A1 publication Critical patent/WO2011090762A1/en
Priority to IL220398A priority patent/IL220398A/en
Priority to HK12111376.7A priority patent/HK1170741A1/en
Priority to US14/640,989 priority patent/US20150274844A1/en
Priority to HRP20160819TT priority patent/HRP20160819T1/en
Priority to CY20161100903T priority patent/CY1118008T1/en
Priority to SM201600335T priority patent/SMT201600335B/en
Priority to US15/802,636 priority patent/US20180273642A1/en

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    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2818—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 immunoglobulin superfamily against CD28 or CD152
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Definitions

  • the present disclosure generally provides polypeptide heterodimers, compositions thereof, and methods for making and using such polypeptide heterodimers. More specifically, the polypeptide heterodimers provided herein are formed, in part, via natural heterodimerization between an immunoglobulin CHI region and an immunoglobulin light chain constant region (CL). In addition, the polypeptide heterodimers provided herein comprise two or more binding domains that specifically bind one or more targets. Furthermore, both single chain polypeptides of the polypeptide heterodimers provided herein each comprise an Fc region portion (e.g., immunoglobulin CH2 and CH3 domains). Description of the Related Art
  • the process of signal transduction often involves receptor proteins that have extracellular domains, transmembrane domains, and intracellular domains.
  • cell surface receptor molecules often oligomerize or multimerize (also referred to as "cross-link") to effectively transmit a signal to the intracellular compartment of the cell.
  • the stimulation or blockade of this interaction between a receptor and a ligand or of the subsequent oligomerization or multimerization of receptors has important therapeutic implications for a wide variety of diseases.
  • Exemplary molecules useful in modulating receptor and ligand interactions include antibodies or molecules derived from antibodies.
  • an antibody or its derivative may function as a receptor antagonist that binds to a cell surface receptor and inactivates it by blocking the binding site of an activating ligand or preventing receptor dimerization or multimerization required for activation.
  • an antibody or its derivative may function as an agonist by binding to and cross-linking multiple membrane receptors, mimicking the function of a natural ligand.
  • a bispecific antibody derivative that may be used to direct cytotoxic agents or immune effector cells to target sites, such as tumors.
  • Bispecific antibodies are antibody-based molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen).
  • One use of bispecific antibodies has been to redirect cytotoxic immune effector cells for enhanced killing of tumor cells by antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • one arm of the bispecific antibody binds an antigen on the tumor cell, and the other binds a determinant expressed on effector cells.
  • the bispecific antibody By cross-linking tumor and effector cells, the bispecific antibody not only brings the effector cells within the proximity of the tumor cells but also simultaneously triggers their activation, leading to effective tumor cell-killing.
  • Bispecific antibodies have also been used to enrich chemo- or radiotherapeutic agents in tumor tissues to minimize detrimental effects to normal tissue.
  • one arm of the bispecific antibody binds an antigen expressed on the cell targeted for destruction, and the other arm delivers a chemotherapeutic drug, radioisotope, or toxin.
  • bispecific antibodies A major obstacle in the general development of bispecific antibodies has been the difficulty of producing materials of sufficient quality and quantity for both preclinical and clinical studies.
  • the main route to the production of bispecific antibodies was by co-expression of both the light chains and both the heavy chains of two parent antibodies of different specificities in a single cell.
  • the desired binding-competent bispecific antibodies are a minor product, and purification from the other products is very difficult.
  • Another traditional method for bispecific antibody production is chemical conjugation of two antibodies or their fragments having different specificities. However, this method is also complicated, and the chemical modification process may inactivate the antibody or promote aggregation. Because purification from undesired products remains difficult, the resulting low yield and poor quality of bispecific antibody make this process unsuitable for the large scale production required for clinical development.
  • Fusion of two scFv fragments together has also been used to generate bispecific proteins (e.g., BITE® antibodies by Micromet Inc., Bethesda, MD, U.S. Patent No. 7,635,472).
  • bispecific proteins e.g., BITE® antibodies by Micromet Inc., Bethesda, MD, U.S. Patent No. 7,635,472.
  • BITE® antibodies by Micromet Inc., Bethesda, MD, U.S. Patent No. 7,635,472
  • these proteins do not contain Fc regions, and thus do not allow manipulation of their activities via Fc regions.
  • these proteins are small ( ⁇ 55 kDa) and thus have relatively short half lives in serum.
  • the present disclosure provides polypeptide heterodimers formed between two different single chain polypeptides via natural heterodimerization of an immunoglobulin CHI region and an immunoglobulin light chain constant region (CL).
  • the present disclosure also provides nucleic acids, vectors, host cells and methods for making polypeptide heterodimers as well as methods for using such polypeptide heterodimers, such as in directed T cell activation, inhibiting solid malignancy growth, treating autoimmune or inflammatory conditions, or treating B-cell associated disorders or diseases.
  • the present disclosure provides a polypeptide heterodimer that comprises (a) a first single chain polypeptide (SCP-I) comprising from one to four binding domains that specifically bind from one to four targets, a hinge (H-I), an immunoglobulin heterodimerization domain (HD-I), and an Fc region portion (FRP-I); and (b) a second single chain polypeptide (SCP-II) comprising from zero to four binding domains that specifically bind from zero to four targets, a hinge (H-II), an immunoglobulin heterodimerization domain (HD-II), and an Fc region portion (FRP- II); wherein (i) the immunoglobulin HD-I and the immunoglobulin HD-II preferentially associate with each other to form a polypeptide heterodimer comprised of SCP-I and SCP-II, and (1) the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobul
  • the binding domains of the polypeptide heterodimers are single chain Fv (scFv) polypeptides.
  • the polypeptide heterodimer comprises two binding domains (BDl and BD2).
  • the two binding domains (BDl and BD2) are both on the first single chain polypeptide (SCP-I) and wherein the HD-I and FRP-I are disposed between BDl and BD2.
  • the first binding domain (BDl) is on the first single chain polypeptide (SCP-I) and the second binding domain (BD2) is on the second single chain polypeptide (SCP-II).
  • the first binding domain (BDl) may be amino terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) may amino terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
  • the first binding domain (BDl) may be amino terminal to the Fc region portion of the first single chain polypeptide (FRP-I)
  • the second binding domain (BD2) may be carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
  • first binding domain (BDl) may be carboxyl terminal to the Fc region portion of the first single chain polypeptide (FRP-I)
  • second binding domain (BD2) may be carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
  • the polypeptide heterodimer comprises three binding domains (BDl, BD2 and BD3).
  • the HD-I and FRP-I are disposed between BDl and BD2, and the third binding domain (BD3) is amino terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
  • the HD-I and FRP-I are disposed between BDl and BD2, and the third binding domain (BD3) is carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
  • the polypeptide heterodimer comprises four binding domains (BDl, BD2, BD3, and BD4).
  • the HD-I and FRP-I may be disposed between BDl and BD2, and the HD-II and FRP-II may be disposed between BD3 and BD4.
  • the polypeptide heterodimer comprises five to eight binding domains (e.g., 5, 6, 7 or 8 binding domains).
  • At least one of the binding domains of the polypeptide heterodimers provided herein specifically binds to, or is an antagonist of, TCRa, TCRP, CD3y, CD35, CD3s, CD28, CD79b, hyperIL-6, monoIL-10 , CD86, CD20, PSMA, CD 19, HLA-DR, Ron, c-Met, CEACAM-6, LIGHT, GITRL, CD40, PDL1, PDL2, HVEM, LTBR, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGF1R, EphA2, PDGFR, VEGFR1-4, Angiopoietin 2, CD64, CD32A, CD 16, CD71, TNFR1, TNFR2, TWEAKR, TACI, BAFF-R, BCMA, FAS, CD32B, CD21, CD22, CD30, CD33, CD37, CD38, CD70, TNFa, IL-6, hyperIL-6, IL-2,
  • At least one of the binding domains of the polypeptide heterodimer is an agonist of IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, TNFR1, TNFR2, DR4, DR5, TWEAKR, or FAS.
  • At least one binding domain specifically binds a TCR complex or a component thereof, and at least another binding domain specifically binds to PSMA, CD79b, CD 19, HLA-DR, CD20, RON, c- Met, or CEACAM-6.
  • at least one binding domain specifically binds to CD28, and at least another binding domain specifically binds to, or is an antagonist of, CD79b, hyperIL-6, PDL2, monoIL-10, CD86, LIGHT, GITRL, CD40, PDL1, HVEM, or LTBR.
  • At least one binding domain specifically binds to CD28, and at least another binding domain is an agonist of IL-10, HLA-G, HGF, IL-35, PD-1, or BTLA.
  • the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region.
  • the first CHI region is amino terminal to the Fc region portion of the first single chain polypeptide
  • the first CL region is amino terminal to the Fc region portion of the second single chain polypeptide.
  • the first CHI region is carboxyl terminal to the Fc region portion of the first single chain polypeptide
  • the first CL region is carboxyl terminal to the Fc region portion of the second single chain polypeptide.
  • the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region
  • the first single chain polypeptide further comprises a second CHI region and the second single chain polypeptide further comprises a second CL region
  • the second CHI region of the first single chain polypeptide and the second CL region of the second single chain polypeptide associate with each other in the polypeptide heterodimer.
  • the Fc region portion of the first single chain polypeptide may be disposed between the first and second CHI regions, and the Fc region portion of the second single chain polypeptide may be disposed between the first and second CL regions.
  • both the first and second CHI regions may be amino terminal to the Fc region portion of the first single chain polypeptide
  • both the first and second CL regions may be amino terminal to the Fc region portion of the second single chain polypeptide.
  • both the first and second CHI regions may be carboxyl terminal to the Fc region portion of the first single chain polypeptide
  • both the first and second CL regions may be carboxyl terminal to the Fc region portion of the second single chain polypeptide.
  • the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region
  • the first single chain polypeptide further comprises a second CL region and the second single chain polypeptide further comprises a second CHI region
  • the second CL region of the first single chain polypeptide and the second CHI region of the second single chain polypeptide associate with each other in the polypeptide heterodimer.
  • the first CHI region is amino terminal to the Fc region portion and the second CL region is carboxyl terminal to the Fc region portion; and in the second single chain polypeptide, the first CL region is amino terminal to the Fc region portion, and the second CHI region is carboxyl terminal to the Fc region portion.
  • the first CHI region is carboxyl terminal to the Fc region portion, and the second CL region is amino terminal to the Fc region portion; and in the second single chain polypeptide, the first CL region is carboxyl terminal to the Fc region portion, and the second CHI region is amino terminal to the Fc region portion.
  • both the first CHI region and the second CL regions are amino terminal to the Fc region portion, and the first CHI region is amino terminal to the second CL region; and in the second single chain polypeptide, both the first CL region and the second CHI region are amino terminal to the Fc region portion, and the first CL region is amino terminal to the second CHI region.
  • both the first CHI region and the second CL regions are amino terminal to the Fc region portion, and the second CL region is amino terminal to the first CHI region; and in the second single chain polypeptide, both the first CL region and the second CHI region are amino terminal to the Fc region portion, and the second CHI region is amino terminal to the first CL region.
  • both the first CHI region and the second CL regions are carboxyl terminal to the Fc region portion, and the first CHI region is amino terminal to the second CL region; and in the second single chain polypeptide, both the first CL region and the second CHI region are carboxyl terminal to the Fc region portion, and the first CL region is amino terminal to the second CHI region.
  • both the first CHI region and the second CL regions are carboxyl terminal to the Fc region portion, and the second CL region is amino terminal to the first CHI region; and in the second single chain polypeptide, both the first CL region and the second CHI region are carboxyl terminal to the Fc region portion, and the second CHI region is amino terminal to the first CL region.
  • the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CL region
  • the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CHI region.
  • the first CL region is amino terminal to the Fc region portion of the first single chain polypeptide
  • the first CHI region is amino terminal to the Fc region portion of the second single chain polypeptide.
  • the first CL region is carboxyl terminal to the Fc region portion of the first single chain polypeptide
  • the first CHI region is carboxyl terminal to the Fc region portion of the second single chain polypeptide.
  • the first single chain polypeptide further comprises a second CL region and the second single chain polypeptide further comprises a second CHI region, and the second CL region of the first single chain polypeptide and the second CHI region of the second single chain polypeptide associate with each other in the polypeptide heterodimer.
  • the Fc region portion of the first single chain polypeptide may be disposed between the first and second CL regions, and the Fc region portion of the second single chain polypeptide may be disposed between the first and second CHI regions.
  • both the first and second CL regions may be amino terminal to the Fc region portion of the first single chain polypeptide
  • both the first and second CHI regions may be amino terminal to the Fc region portion of the second single chain polypeptide.
  • both the first and second CL regions may be carboxyl terminal to the Fc region portion of the first single chain polypeptide
  • both the first and second CHI regions may be carboxyl terminal to the Fc region portion of the second single chain polypeptide.
  • the first CL region is a CK region. In other embodiments, the first CL region is a region.
  • the second CL region is a CK region. In other embodiments, the second CL region is a C region.
  • the CK region is a wild type human immunoglobulin CK region.
  • the CK region is an altered human immunoglobulin CK region with one or more amino acids of a wild type human CK region substituted at N29, N30, Q52, V55, T56, T56, S68, or T70.
  • the one or more amino acid substitutions may be selected from Ala (A), Arg (R), Trp (W), Tyr (Y), Glu (E), Gin (Q), Lys (K), Asp (D), Met (M), Ser (S), and Phe (F).
  • the CHI region is an altered human immunoglobulin CHI region comprising an amino acid substitution by which Val (V) at position 68 is substituted by Lys (K), Arg (R) or His (H), and wherein the Ck region is an altered human immunoglobulin Ck region comprising an amino acid substitution by which Leu (L) at position 29 is substituted by Asp (D) or Glu (E).
  • the CHI region is an altered human immunoglobulin CHI region comprising an amino acid substitution by which Val (V) at position 68 is changed to Asp (D) or Glu (E), and wherein the Ck region is an altered human immunoglobulin Ck region comprising an amino acid substitution by which Leu (L) at position 29 is changed to Lys (K), Arg (R) or His (H).
  • the region is a wild type human immunoglobulin region.
  • the first CHI region or the second CHI region when present is a wild type human immunoglobulin CHI region, such as a wild type human IgGl CHI region.
  • the first CHI region or the second CHI region when present is an altered human immunoglobulin CHI region, such as an altered human IgGl CHI region, with the cysteine of a wild type human immunoglobulin CHI region that is involved in forming a disulfide bond with a wild type human immunoglobulin CL region deleted or substituted.
  • the CK region is an altered human immunoglobulin Ck region, such as an with the cysteine residue of a wild type human CK region that is involved in forming a disulfide bond with a wild type human immunoglobulin CHI region deleted or substituted.
  • the region is an altered human immunoglobulin region with the cysteine residue of a wild type human region that is involved in forming a disulfide bond with a wild type human immunoglobulin CHI region deleted or substituted.
  • the first CHI region and the second CHI region when present is a polypeptide comprising SEQ ID NO: l 14, 844 or 845.
  • the Ck region when present is selected from any one of the polypeptides comprising SEQ ID NOS: 141-178, 202, and 838-843.
  • the region when present is a polypeptide comprising SEQ ID NO: 140.
  • the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH2 domain, such as an IgGl CH2 domain or an IgG2, IgG3, IgG4, IgAl, IgA2, or IgD CH2 domain.
  • the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH3 domain, such as an IgGl CH3 domain or an IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE or IgM CH3 domain.
  • the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH2 domain and an immunoglobulin CH3 domain, such as are IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD CH2 and CH3 domains.
  • the Fc region portion comprises an immunoglobulin CH3 domain that is immediately amino terminal to an immunoglobulin heterodimerization domain (e.g., a CHI domain or a Ck domain)
  • the immunoglobulin CH3 domain is linked to the CHI domain immediately carboxyl terminal to the immunoglobulin CH3 domain in one single chain polypeptide via a peptide comprising SEQ ID NOS:846, 847, 848, or 849; and the immunoglobulin CH3 domain is linked to the Ck domain immediately carboxyl terminal to the immunoglobulin CH3 domain in the other single chain polypeptide via a peptide comprising SEQ ID NOs:846, 850, 951 , or 852.
  • the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second chain polypeptide (FRP-II) comprise IgM or IgE CH3 and CH4 domains.
  • the CH2 domain may be an altered human IgGl , IgG2, IgG3, or IgG4 CH2 domain.
  • exemplary altered human IgGl , IgG2, IgG3 or IgG4 CH2 domain include those that comprise (a) an amino acid substitution at position 297 and at least one additional substitution or deletion at positions 234 to 238; (b) one or more amino acid mutations at positions 234-238 and at least one substitution at position 253, 310, 318, 320, 322, or 331 ; or (c) an amino acid substitution at the asparagine of position 297, one or more substitutions or deletions at positions 234 to 238, and at least one substitution at position 253, 310, 318, 320, 322, or 331.
  • Another exemplary CH2 domain is an altered human IgGl CH2 domain that comprises amino acid substitutions at positions L234, L235, G237, E318, K320 and K322.
  • the CH3 domain of the first single chain polypeptide is an altered human IgGl , IgG2, IgG3, or IgG4 CH3 domain that comprises a T366W
  • the CH3 domain of the second single chain polypeptide is an altered human IgGl , IgG2, IgG3 or IgG4 CH3 domain that comprises a Y407A substitution.
  • the CH3 domain of the first single chain polypeptide is an altered human IgGl , IgG2, IgG3, or IgG4 CH3 domain that comprises a T366Y substitution
  • the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a Y407T substitution.
  • the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a T366W substitution
  • the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises T366S, L368A and Y407V substitutions.
  • the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a Y407A substitution
  • the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366W substitution.
  • the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a Y407T substitution
  • the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366Y substitution.
  • the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises T366S, L368A and Y407W substitutions
  • the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366W substitution.
  • the hinge of both the first and second single chain polypeptides is an immunoglobulin hinge region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, or IgE hinge.
  • the immunoglobulin hinge is a wild type immunoglobulin hinge.
  • the immunoglobulin hinge is an altered immunoglobulin hinge selected from SEQ ID NOS:232, 234, 240, 664-673, 675 and 676.
  • the hinge region is (a) at the amino terminal to the Fc region portion, (b) disposed between the binding domain and the immunoglobulin heterodimerization domain, (c) disposed between the immunoglobulin heterodimerization domain and the Fc region portion, (d) at the amino terminus of the first or second single chain polypeptide, (e) disposed between the Fc region portion and a binding domain, or (f) at the carboxyl terminus of the first or second single chain polypeptide.
  • At least one of the first and second single chain polypeptide hinges is a C-type lectin hinge region, such as an NKg2A or NKg2D peptide, or a derivative thereof.
  • the hinges of the first and second single chain polypeptides are identical. In certain other embodiments, the hinges of the first and second single chain polypeptides are different. In certain embodiments, the first single chain polypeptide comprises a binding domain that specifically binds a TCR complex or a component thereof, and the second single chain polypeptide comprises a binding domain that specifically binds CD 19, CD79b, HLA-DR or CD20.
  • the first single chain polypeptide comprises a binding domain that specifically binds CD28
  • the second single chain polypeptide comprises a binding domain (a) that specifically binds CD79b, hyperIL-6, or CD86 or (b) that comprises a PDL ectodomain or a monoIL-10.
  • the first single chain polypeptide comprises a binding domain that specifically binds c-Met
  • the second single chain polypeptide comprises a binding domain that specifically binds RON
  • the first and second single chain polypeptides comprise SEQ ID NOS: SEQ ID NOS:2 and 4, SEQ ID NOS:6 and 8, SEQ ID NOS: 10 and 12, SEQ ID NOS: 14 and 16, SEQ ID NOS: 18 and 20, SEQ ID NOS:20 and 22, SEQ ID NOS:20 and 24, SEQ ID NOS:30 and 32, SEQ ID NOS:29 and 31, SEQ ID NOS:29 and 32, SEQ ID NOS:30 and 72, SEQ ID NOS:53 and 72, SEQ ID NOS:54 and 72, SEQ ID NOS:55 and 72, SEQ ID NOS:70 and 72, SEQ ID NOS:71 and 72, SEQ ID NOS:63 and 56, SEQ ID NOS:64 and 57, SEQ ID NOS:65 and 60, SEQ ID NOS:66 and 58, SEQ ID NOS:67 and 59, SEQ ID NOS:68 and 61, SEQ ID NOS:69 and 62, SEQ ID NOS:
  • the present disclosure provides a composition comprising a polypeptide heterodimer provided herein and a pharmaceutically acceptable excipient.
  • the present disclosure provides an expression vector capable of expressing a polypeptide heterodimer provided herein, comprising a first polynucleotide encoding the first single chain polypeptide and a second polynucleotide encoding the second single chain polypeptide.
  • the present disclosure provides a host cell comprising the above expression vector. In another aspect, the present disclosure provides a host cell comprising first and second expression vectors capable of expressing the first and second single chain polypeptides, respectively, of the polypeptide heterodimer provided herein.
  • the present disclosure provides a method for making a polypeptide heterodimer, comprising (a) culturing a host cell provided herein under conditions suitable to express first and second single chain polypeptides, and (b) optionally isolating or purifying the heterodimers formed from the first and second single chain polypeptides from the culture.
  • the present disclosure provides a method for directing T cell activation, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, or a combination thereof, and a second binding domain that specifically binds a different target, for instance, a tumor-specific antigen or other antigen of choice at a site or cell where T cell activation is desired.
  • a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, or a combination thereof, and a second binding domain that specifically binds a different target, for instance, a tumor-specific antigen or other antigen of choice at a site or cell where T cell activation is desired.
  • the present disclosure provides a method for inhibiting growth, metastatis, or metastatic growth of a malignancy, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, c-Met, RON, or a combination thereof.
  • the method further comprises administering to a patient in need thereof a chemotherapeutic agent or ionizing radiation.
  • the present disclosure provides a method for treating an autoimmune or inflammatory condition, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRp, CD3y, CD35, CD3s, or CD28.
  • the present disclosure provides a method for treating a B-cell associated disorder or disease comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, or CD3s, and a second binding domain that specifically binds to CD 19, CD20, CD79b or HLA-DR.
  • the methods for using the polypeptide heterodimers provided herein may further comprise administering to a patient in need thereof a second active agent, such as a second polypeptide heterodimer, a monoclonal antibody, or an immunoglobulin-derived fusion protein.
  • a second active agent such as a second polypeptide heterodimer, a monoclonal antibody, or an immunoglobulin-derived fusion protein.
  • Figure 1 is a schematic representation of bivalent anti-CD28 polypeptide heterodimer X0172 (left) and SDS-PAGE analysis of X0172 (right).
  • NR stands for “Non-Reduced”
  • Red stands for “Reduced.”
  • Figure 2 shows that both a monovalent (X0124) and a bivalent (X0172) anti-CD28 polypeptide heterodimer synergize with a suboptimal concentration of PMA in stimulating purified human T cells when compared to an anti-CD28 scFv (2E12 scFv) but less than a bivalent anti-CD28 SMIP protein (2E12 SMIP).
  • Figure 3 shows that bivalent polypeptide heterodimer X0172 binds to CD4 + T cells better than 2E12 scFv and monovalent polypeptide heterodimer X0124.
  • Figure 4 shows cation exchange chromatography of polypeptide heterodimers X0251, X0252 and X0253 (left) and SDS-PAGE electrophoresis analysis of the same polypeptide heterodimers under non-reduced (NR) and reduced (Red) conditions (right).
  • Figure 5 shows the mass spectra of polypeptide heterodimer X0252, which demonstrates that the heterodimer is the predominant species.
  • Figure 6 shows SDS-PAGE electrophoresis analysis of polypeptide heterodimers X0283 and X0284 under non-reduced (NR) and reduced (Red) conditions (left) and cation exchange chromatography analysis of polypeptide heterodimer X0283.
  • FIG. 7 shows direct binding to CD86 by polypeptide heterodimer
  • X0283 as examined by BIACORE® analysis, with response units (Ru) plotted against time (left) and a schematic representation of X0283.
  • FIGS 8A and 8B show binding of bispecific anti-RON and anti-CD3 constructs (polypeptide heterodimer S0268 and Scorpion protein S0266) to MDA-MB- 453 cells (A) and to isolated T cells (B).
  • Figures 9 A and 9B show specificity of binding to (A) Reel (CD19 + , CD20 ) cells or (B) Jurkat (CD3 ) cells by bispecific heterodimers having either anti- CD ⁇ and anti-CD3 binding domains (TSC020) or having anti-CD20 and anti-CD3 binding domains (TSC021).
  • Figures 10A-10D show proliferation of CD4+ and CD8+ T-cells in response to bispecific polypeptide heterodimers TSC054, TSC078, TSC079, and bscl9x3 (TSC036) with (A and B) Daudi (CD19 + ) cells or (C and D) MDA-MB-453 (CD 19 ) cells.
  • FIGS 11A and 11B shows T-cell directed cytotoxicity induced by bispecific polypeptide heterodimers TSC054, TSC078, TSC079, and S0268 in a chromium ( 51 Cr) release assay with (A) Daudi (RON " , CD19 + ) cells or (B) BxPC-3 (RON + , CD 19 ) cells.
  • Figure 12 shows target-dependent T-cell proliferation induced by a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSC 165, TSC 166, TSC 167, TSC168 and TSCIOO at concentrations from 0.1 pM to 10000 pM.
  • Figure 13 shows target-dependent T-cell proliferation induced by a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSC127 and TS 165 with bscl9x3 BiTE as a control at concentrations from 0.001 pM to 1000 pM.
  • Figure 14 shows target-dependent redirected T-cell cytotoxicity on a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSCI OO, TSC165, TC166, TSC 167, and TSC 168.
  • TSCI OO target-dependent redirected T-cell cytotoxicity on a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSCI OO, TSC165, TC166, TSC 167, and TSC 168.
  • the present disclosure provides polypeptide heterodimers formed between two different single chain polypeptides via natural heterodimerization of an immunoglobulin CHI region and an immunoglobulin light chain constant region (CL).
  • the polypeptide heterodimer has two or more binding domains that specifically bind one or more targets (e.g., an antigen, a receptor, or a ligand).
  • targets e.g., an antigen, a receptor, or a ligand
  • both chains of a heterodimer each further comprise an Fc region portion (e.g., immunoglobulin CH2 and/or CH3 domains).
  • the present disclosure also provides nucleic acids, vectors, host cells and methods for making polypeptide heterodimers.
  • the heterodimerization technology described herein has one or more of the following advantages: (1) potential for minimal immunogenicity of the polypeptide heterodimers because the dimers are formed via natural heterodimerization of an immunoglobulin CHI region and an immunoglobulin CL region; (2) efficient production and purification of polypeptide heterodimers of the present disclosure is possible by co-expressing the two different single chain polypeptides, as shown in the examples; (3) the ability to mediate Fc effector functions (e.g., CDC, ADCC, ADCP), which can be modulated up or down by mutagenesis, and a longer serum half life because each chain of a polypeptide heterodimer according to the present disclosure has an Fc region portion (e.g., immunoglobulin CH2 and CH3 domains); and (4) polypeptide heterodimers of the present disclosure have a size that is typically smaller than an antibody molecule, which can allow for better tissue penetration, such as into a solid malignancy.
  • Fc effector functions e.g.
  • the polypeptide heterodimers provided herein are useful to direct therapeutic agents or immune effector cells to target cells.
  • the polypeptide heterodimers may comprise a binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRP, CD3y, CD35, and CD3s) and another binding domain that specifically binds a second different target, such as an oncology target (e.g., c-Met, RON, CEACAM-6, and PSMA) or a B- cell target (e.g., CD 19, CD79b, HLA-DR and CD20).
  • an oncology target e.g., c-Met, RON, CEACAM-6, and PSMA
  • B- cell target e.g., CD 19, CD79b, HLA-DR and CD20.
  • the binding domain specific for the second different target may have a higher affinity for its target than the affinity of the binding domain for the TCR complex or component thereof, such as CD3.
  • Such polypeptide heterodimers will preferably bind to the oncology target or the B-cell target first and subsequently recruit T cells to the tumor or cancer cells expressing the oncology target or B-cell target and are useful in inhibiting growth, metastasis or metastatic growth of a malignancy (including B-cell cancers). Additional uses of polypeptide heterodimers provided herein include directed T cell activation and treating autoimmune or inflammatory conditions.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • “about” means ⁇ 20% of the indicated range, value, sequence, or structure, unless otherwise indicated.
  • the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated or dictated by its context. The use of the alternative (e.g., "or”) should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the terms “include” and “comprise” are used synonymously.
  • a protein "consists essentially of several domains (e.g., a binding domain that specifically binds a target, a hinge, an immunoglobulin heterodimerization domain, and an Fc region portion) if the other portions of the protein (e.g., amino acids at the amino- or carboxy-terminus or between two domains), in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as more than 40%, 30%, 25%, 20%, 15%, 10%, or 5%) the activities of various domains (e.g., the target binding affinity of the binding domain, the activities of the Fc region portion, and the capability of the immunoglobulin heterodimerization domain in facilitating heterodimerization).
  • domains e.g., the target binding affinity of the binding domain, the activities of the Fc region portion,
  • a protein e.g., a single chain polypeptide
  • polypeptide heterodimer refers to a dimer formed from two different single chain polypeptides. This term does not include an antibody formed from four single chain polypeptides (i.e., two light chains and two heavy chains).
  • a “dimer” refers to a biological entity that consists of two subunits associated with each other via one or more forms of intramolecular forces, including covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions), and is stable under appropriate conditions (e.g., under physiological conditions, in an aqueous solution suitable for expressing, purifying, and/or storing recombinant proteins, or under conditions for non-denaturing and/or non-reducing electrophoresis).
  • covalent bonds e.g., disulfide bonds
  • other interactions e.g., electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions
  • a “single chain polypeptide” is a single, linear and contiguous arrangement of covalently linked amino acids. It does not include two polypeptide chains that link together in a non-linear fashion, such as via an interchain disulfide bond (e.g. , a half immunoglobulin molecule in which a light chain links with a heavy chain via a disulfide bond).
  • a single chain polypeptide may have or form one or more intrachain disulfide bonds.
  • immunoglobulin heterodimerization domain refers to an immunoglobulin domain of a single chain polypeptide that preferentially interacts or associates with a different immunoglobulin domain of another single chain polypeptide wherein the interaction of the different heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second single chain polypeptides (i.e., the formation of a dimer between two different single chain polypeptides, which is also referred to as a "heterodimer").
  • heterodimerization domains substantially contributes to or efficiently promotes" the heterodimerization of first and second single chain polypeptides if there is a statistically significant reduction in the dimerization between the first and second single chain polypeptides in the absence of the heterodimerization domain of the first single chain polypeptide (HD-I) and/or the heterodimerization domain of the second single chain polypeptide (HD-II).
  • the first and second single chain polypeptides when the first and second single chain polypeptides are co-expressed, at least 60%, at least about 60%) to about 70%>, at least about 70%> to about 80%>, at least about 80%> to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second single chain polypeptides form heterodimers with each other.
  • immunoglobulin heterodimerization domains of the present disclosure include an immunoglobulin CHI region, an immunoglobulin CL region (e.g., CK or C isotypes), or derivatives thereof, including wild type immunoglobulin CHI and CL regions and altered (or mutated) immunoglobulin CHI and CL regions, as provided herein.
  • immunoglobulin CHI region an immunoglobulin CL region (e.g., CK or C isotypes)
  • derivatives thereof including wild type immunoglobulin CHI and CL regions and altered (or mutated) immunoglobulin CHI and CL regions, as provided herein.
  • binding domain refers to a protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a target (e.g., CD3, TCR, CD28, c-Met, RON).
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest.
  • Exemplary binding domains include single chain antibody variable regions (e.g. , domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g. , c-Met, RON), or ligands (e.g. , cytokines, chemokines).
  • assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore analysis.
  • a binding domain and a fusion protein thereof "specifically binds" a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M "1 , while not significantly binding other components present in a test sample.
  • Binding domains (or fusion proteins thereof) may be classified as “high affinity” binding domains (or fusion proteins thereof) and "low affinity” binding domains (or fusion proteins thereof).
  • “High affinity” binding domains refer to those binding domains with a K a of at least 10 7 M “1 , at least 10 8 M “1 , at least 10 9 M “1 , at least 10 10 M “1 , at least 10 11 M “1 , at least 10 12 M “1 , or at least 10 13 M “1 .
  • “Low affinity” binding domains refer to those binding domains with a K a of up to 10 7 M “1 , up to 10 6 M “1 , up to 10 5 M “1 .
  • affinity may be defined as an equilibrium dissociation constant (IQ) of a particular binding interaction with units of M (e.g. , 10 "5 M to 10 "13 M).
  • binding domain polypeptides and single chain polyepeptides can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
  • T cell receptor is a molecule found on the surface of T cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It consists of a disulfide-linked heterodimer of the highly variable a and ⁇ chains in most T cells. In other T cells, an alternative receptor made up of variable ⁇ and ⁇ chains is expressed.
  • MHC major histocompatibility complex
  • Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see, Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway et al., Immunobiology: The Immune System in Health and Disease, 4 th Ed., Current Biology Publications, pl48, 149, and 172, 1999).
  • TCR as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
  • CD3 is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999).
  • the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3 ⁇ chains.
  • the CD3y, CD35, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains.
  • CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
  • TCR complex refers to a complex formed by the association of CD3 with TCR.
  • a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3 ⁇ chains, a TCRa chain, and a TCRP chain.
  • a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3 ⁇ chains, a TCRy chain, and a TCR5 chain.
  • a component of a TCR complex refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CD3y, CD35, CD3s or C Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRp, CD3y, CD35, and two CD3s chains).
  • Antibodies are known to have variable regions, a hinge region, and constant domains. Immunoglobulin structure and function are reviewed, for example, in Harlow et ah, Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
  • variable binding region refers to the variable binding region from an antibody light and heavy chain, respectively.
  • the variable binding regions are made up of discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • CL refers to an "immunoglobulin light chain constant region” or a "light chain constant region,” i.e., a constant region from an antibody light heavy chain.
  • CH refers to an "immunoglobulin heavy chain constant region" or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM).
  • a "Fab” fragment antigen binding is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond.
  • an Fc region constant domain portion or “Fc region portion” refers to the heavy chain constant region segment of the Fc fragment (the “fragment crystallizable” region or Fc region) from an antibody, which can include one or more constant domains, such as CH2, CH3, CH4, or any combination thereof.
  • the Fc region is responsible for the effector functions of an immunoglobulin, such as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (e.g., CD 16, CD32, FcRn), greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer (see Capon et al., Nature, 337:525 (1989)).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • complement fixation binding to Fc receptors (e.g., CD 16, CD32, FcRn), greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer (see Capon et al., Nature, 337
  • antibodies have a hinge sequence that is typically situated between the Fab and Fc region (but a lower section of the hinge may include an amino- terminal portion of the Fc region).
  • an immunoglobulin hinge acts as a flexible spacer to allow the Fab portion to move freely in space.
  • hinges are structurally diverse, varying in both sequence and length between immunoglobulin classes and even among subclasses.
  • a human IgGl hinge region is freely flexible, which allows the Fab fragments to rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges.
  • a human IgG2 hinge is relatively short and contains a rigid poly-proline double helix stabilized by four inter-heavy chain disulfide bridges, which restricts the flexibility.
  • a human IgG3 hinge differs from the other subclasses by its unique extended hinge region (about four times as long as the IgGl hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix and providing greater flexibility because the Fab fragments are relatively far away from the Fc fragment.
  • a human IgG4 hinge is shorter than IgGl but has the same length as IgG2, and its flexibility is intermediate between that of IgGl and IgG2.
  • an IgG hinge domain can be functionally and structurally subdivided into three regions: the upper, the core or middle, and the lower hinge regions (Shin et al., Immunological Reviews 130:87 (1992)).
  • Exemplary upper hinge regions include EPKSCDKTHT (SEQ ID NO:227) as found in IgGl, ER CCVE (SEQ ID NO:211) as found in IgG2, ELKTPLGDTT HT (SEQ ID NO:245) or EPKSCDTPPP (SEQ ID NO:246) as found in IgG3, and ESKYGPP (SEQ ID NO:247) as found in IgG4.
  • Exemplary middle or core hinge regions include CPPCP (SEQ ID NO:228) as found in IgGl and IgG2, CPRCP (SEQ ID NO:248) as found in IgG3, and CPSCP (SEQ ID NO:249) as found in IgG4. While IgGl, IgG2, and IgG4 antibodies each appear to have a single upper and middle hinge, IgG3 has four in tandem - one being ELKTPLGDTTHTCPRCP (SEQ ID NO:250) and three being EPKSCDTPPP CPRCP (SEQ ID NO:251).
  • IgA and IgD antibodies appear to lack an IgG-like core region, and IgD appears to have two upper hinge regions in tandem (see SEQ ID NOS:222 and 252).
  • Exemplary wild type upper hinge regions found in IgAl and IgA2 antibodies are set forth in SEQ ID NOS:215 and 216.
  • IgE and IgM antibodies in contrast, lack a typical hinge region and instead have a CH2 domain with hinge-like properties.
  • Exemplary wild-type CH2 upper hinge-like sequences of IgE and IgM are set forth in SEQ ID NO:253 (VCSRDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTINITWLEDG
  • DSTKKCA DSTKKCA
  • SEQ ID NO:254 VIAELPPKVSVFVPPRDGFFGNPRKSKLIC QATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTI KESDWLGQSMFTCRVDHRGLTFQQNASSMCVP
  • a "hinge region” or a “hinge” refers to (a) an immunoglobulin hinge region (made up of, for example, upper and core regions) or a functional variant thereof, including wild type and altered immunoglobulin hinges, (b) a lectin interdomain region or a functional variant thereof, (c) a cluster of differentiation (CD) molecule stalk region or a functional variant thereof, or (d) a portion of a cell surface receptor (interdomain region) that connects immunoglobulin V-like or immunoglobulin C-like domains.
  • a wild type immunoglobulin hinge region refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CHI and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CHI and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.
  • a wild type immunoglobulin hinge region sequence is human, and may comprises a human IgG hinge region.
  • Exemplary human wild type immunoglobulin hinge regions are set forth in SEQ ID NOS:215 (IgAl hinge), 216 (IgA2 hinge), 217 (IgD hinge), 667 (IgGl hinge), 219 (IgG2 hinge), 220 (IgG3 hinge) and 221 (IgG4 hinge).
  • altered wild type immunoglobulin hinge region refers to (a) a wild type immunoglobulin hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%>, 15%>, 10%>, or 5%> amino acid substitutions or deletions), or (b) a portion of a wild type immunoglobulin hinge region that has a length of about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to about 120 amino acids (for instance, having a length of about 10 to about 40 amino acids or about 15 to about 30 amino acids or about 15 to about 20 amino acids or about 20 to about 25 amino acids), has up to about 30% amino acid changes (e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutions or deletions or a combination thereof), and has an IgG core hinge region as set forth in SEQ ID NOS:
  • a “peptide linker” refers to an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions.
  • a linker is comprised of five to about 35 amino acids, for instance, about 15 to about 25 amino acids.
  • “Junction amino acids” or “junction amino acid residues” refer to one or more (e.g., about 2-10) amino acid residues between two adjacent regions or domains of a single chain polypeptide, such as between a hinge and an adjacent Fc region portion or between a hinge and an adjacent binding domain or between a peptide linker that links two immunoglobulin variable domains and an adjacent immunoglobulin variable domain. Junction amino acids may result from the construct design of a single chain polypeptide (e.g. , amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a single chain polypeptide).
  • a "linker between CH3 and CHI or CL” refers to one or more (e.g., about 2-12) amino acid residues between the C- terminus of a CH3 domain (e.g. , a wild type CH3 or a mutated CH3) and the N-terminus of a CHI domain or CL domain (e.g. , Ck).
  • wild type immunoglobulin region or wild type immunoglobulin domain refers to a naturally occurring immunoglobulin region or domain (e.g., a naturally occurring VL, VH, hinge, CL, CHI , CH2, CH3, or CH4) from various immunoglobulin classes or subclasses (including, for example, IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, and IgM) and from various species (including, for example, human, sheep, mouse, rat, and other mammals).
  • a naturally occurring immunoglobulin region or domain e.g., a naturally occurring VL, VH, hinge, CL, CHI , CH2, CH3, or CH4 from various immunoglobulin classes or subclasses (including, for example, IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, and IgM) and from various species (including, for example
  • Exemplary wild type human CHI regions are set forth in SEQ ID NOS: l 14, 186-192 and 194, wild type human CK region in SEQ ID NO: 1 12, wild type human C regions in SEQ ID NO: 1 13 and 224-226, wild type human CH2 domains in SEQ ID NOS: 1 15, 195-201 and 203, wild type human CH3 domains in SEQ ID NOS: 1 16, 204-210 and 212, and wild type human CH4 domains in SEQ ID NO:213 and 214.
  • an “altered immunoglobulin region,” “altered immunoglobulin domain,” “mutated immunoglobulin domain,” or the like refers to an immunoglobulin region with a sequence identity to a wild type immunoglobulin region or domain (e.g. , a wild type VL, VH, hinge, CL, CHI , CH2, CH3, or CH4) of at least 75% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).
  • a wild type immunoglobulin region or domain e.g., a wild type VL, VH, hinge, CL, CHI , CH2, CH3, or CH4 of at least 75% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).
  • an "altered immunoglobulin CHI region” or “altered CHI region” refers to a CHI region with a sequence identity to a wild type immunoglobulin CHI region (e.g. , a human CHI) of at least 75% (e.g. , 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%).
  • an "altered immunoglobulin CH2 domain” or “altered CH2 domain” refers to a CH2 domain with a sequence identity to a wild type immunoglobulin CH2 region (e.g.
  • sequence identity refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the percentage sequence identity values are generated by the NCBI BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.
  • an altered immunoglobulin domain only contains conservative amino acid substitutions of a wild type immunoglobulin domain. In certain other embodiments, an altered immunoglobulin domain only contains non- conservative amino acid substitutions of a wild type immunoglobulin domain. In yet other embodiments, an altered immunoglobulin domain contains both conservative and non-conservative amino acid substitutions.
  • a "conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).
  • a conservative substitution includes a leucine to serine substitution.
  • the term "derivative” refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g., by glycosylation, alkylation, acylation, ester formation, or amide formation.
  • a “derivative” differs from an "analogue” in that a parent polypeptide may be the starting material to generate a "derivative,” whereas the parent polypeptide may not necessarily be used as the starting material to generate an “analogue.”
  • a derivative may have different chemical, biological or physical properties of the parent polypeptide. For example, a derivative may be more hydrophilic or it may have altered reactivity (e.g., a CDR having an amino acid change that alters its affinity for a target) as compared to the parent polypeptide.
  • a position of an amino acid residue in a variable region of an immunoglobulin molecule is numbered according to the Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5 th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)), and a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94).
  • a "receptor” is a protein molecule present in the plasma membrane or in the cytoplasm of a cell to which a signal molecule (i.e., a ligand, such as a hormone, a neurotransmitter, a toxin, a cytokine) may attach.
  • a signal molecule i.e., a ligand, such as a hormone, a neurotransmitter, a toxin, a cytokine
  • the binding of the signal molecule to the receptor results in a conformational change of the receptor, which ordinarily initiates a cellular response.
  • some ligands merely block receptors without inducing any response (e.g., antagonists).
  • Some receptor proteins are peripheral membrane proteins.
  • Many hormone and neurotransmitter receptors are transmembrane proteins that are embedded in the phospholipid bilayer of cell membranes, and another major class of receptors are intracellular proteins such as those for steroid and intracrine peptide hormone receptors.
  • B-cell associated disorder or disease or "a disease or disorder associated with aberrant B-cell activity” refers to a disease or disorder associated with (e.g., causing or resulting from) aberrant B-cell activity or activity that deviates from the normal, proper, or expected course.
  • a B-cell associated disorder or disease may include inappropriate proliferation of B-cells that have damaged or defective DNA or other cellular components.
  • Aberrant B-cell activity may include cell proliferation characterized by inappropriately high levels of B-cell division, inappropriately low levels of B-cell apoptosis, or both.
  • diseases may have, for example, single or multiple local abnormal proliferations of B-cells, groups of B-cells or tissue(s), whether cancerous or non-cancerous, benign or malignant.
  • a B-cell associated disorder or disease may also include aberrant antibody production, such as production of autoantibodies, or overproduction of antibodies more desirable when produced at normal levels. It is also contemplated herein that aberrant B-cell activity may occur in certain subpopulations of B-cells and not in other subpopulations, or may include inappropriate stimulation of T-cells, such as by inappropriate antigen presentation to T-cells or by other B-cell pathways.
  • B-cell associated disorders or diseases include a B-cell malignancy or B-cell cancer (e.g., B-cell lymphoma, B-cell leukemia or B-cell myeloma), a disease characterized by autoantibody production (e.g., autoimmune diseases) or inflammation or a disease characterized by inappropriate T-cell stimulation caused by inappropriate B-cell antigen presentation to T-cells or caused by other pathways involving B-cells.
  • B-cell malignancy or B-cell cancer e.g., B-cell lymphoma, B-cell leukemia or B-cell myeloma
  • a disease characterized by autoantibody production e.g., autoimmune diseases
  • inflammation e.g., autoimmune diseases
  • Treatment refers to either a therapeutic treatment or prophylactic/preventative treatment.
  • a treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment may delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases.
  • a “therapeutically effective amount (or dose)” or “effective amount (or dose)” of a specific binding molecule or compound refers to that amount of the compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formulation or concurrently in separate formulations).
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce allergic or other serious adverse reactions when administered using routes well known in the art.
  • a “patient in need” refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a polypeptide heterodimer or a composition thereof provided herein.
  • immunoglobulin-derived fusion protein refers to a fusion protein that comprises at least one immunoglobulin region, such as a VL, VH, CL, CHI, CH2, CH3, and CH4 domain.
  • the immunoglobulin region may be a wild type immunoglobulin region or an altered immunoglobulin region.
  • immunoglobulin-derived fusion proteins include single chain variable antibody fragment (scFv) (see, e.g., Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-83, 1988), SMIPTM proteins (see, U.S. Patent Publication Nos.
  • the present disclosure provides a polypeptide heterodimer formed by the association of two different single chain polypeptides.
  • the first single chain polypeptide (SCP-I) comprises, consists essentially of, or consists of from one to four binding domains that specifically bind from one to four targets, a hinge (H-I), an immunoglobulin heterodimerization domain (HD-I), and an Fc region portion (FRP-I)
  • the second single chain polypeptide (SCP-II) comprises, consists essentially of, or consists of from zero to four binding domains that specifically bind from zero to four targets, a hinge (H-II), an immunoglobulin heterodimerization domain (HD-II), and an Fc region portion (FRP-II), provided that the polypeptide heterodimer comprises at least two binding domains that specifically bind to one or more targets, for instance, at least two different targets.
  • the H-I and H-II may have the same sequence, but may be different.
  • the HD-I may comprise an immunoglobulin CHI region, and the HD-II may comprise an immunoglobulin CL region.
  • the HD-I may comprise an immunoglobulin CL region, and the HD-II may comprise an immunoglobulin CHI region.
  • the FRP-I and FRP-II may have the same sequence, but may be different. The individual components of the polypeptide heterodimers of the present disclosure are described in detail herein.
  • the polypeptide heterodimer of the present disclosure comprises two single chain polypeptides: One single chain polypeptide comprises from one to four binding domains that specifically bind from one to four targets, and the other single chain polypeptide of the polypeptide heterodimer comprises from zero to four binding domains that bind from zero to four targets.
  • the total number of binding domains of the polypeptide heterodimer ranges from about two to eight, and the total number of different targets that the binding domains bind ranges from about one to eight, for instance, from two to eight, two to four, two to three or two targets.
  • a single chain polypeptide of a polypeptide heterodimer comprises a single binding domain
  • the binding domain may be located either amino or carboxyl terminal to the Fc region portion of the single chain polypeptide.
  • a single chain polypeptide comprising two binding domains may have one binding domain located amino terminal and the other carboxyl terminal to the Fc region portion of the single chain polypeptide, or both binding domains may be amino terminal or both carboxyl terminal to the Fc region portion.
  • a single chain polypeptide may comprise three binding domains wherein (a) two binding domains are amino terminal on different single chain proteins and the third binding domain is carboxyl terminal to the Fc region portion on either SCP-I or SCP-II, (b) two binding domains are carboxyl terminal on different single chain proteins and the third binding domain is amino terminal to the Fc region portion on either SCP-I or SCP-II.
  • a polypeptide heterodimer may comprise four binding domains, wherein two binding domains are located amino terminal to the Fc region portion on different chains and the other two binding domains are located carboxyl terminal to the Fc region portion on different chains.
  • two binding domains may be linked to each other in tandem and located on either SCP-I or SCP-II or both, depending on the number of binding domains present - the tandem stacking is used when five to eight binding domains combined are present in SCP-I and SCP-II.
  • Binding of a target by a binding domain modulates the interaction between the target (e.g., a receptor or a ligand) and another molecule.
  • the binding of a target (e.g., a receptor) by a binding domain stimulates certain functions of the target (e.g., signal transduction) or brings different targets closer together for a biological effect (e.g., directing T cells to a tumor which in turn activates the T cells).
  • the binding of a target by a binding domain blocks the interaction between the target and another molecule and thus interferes, reduces or eliminates certain functions of the target.
  • a target molecule which is specifically bound by a binding domain contained in a polypeptide heterodimer of the present disclosure, may be found on or in association with a cell of interest ("target cell").
  • target cells include cancer cells, cells associated with an autoimmune disease or disorder or with an inflammatory disease or disorder, B-cells and T-cells.
  • a target molecule may also not be associated with a cell.
  • Exemplary target molecules not associated with a cell include soluble proteins, secreted proteins, deposited proteins, and extracellular structural (matrix) proteins.
  • binding domains of polypeptide heterodimers of the present disclosure specifically bind a target selected from T-cell targets, tumor antigens, B-cell targets, pro-inflammatory cytokines or chemokines, pro-oncogenic cytokines or growth factors, angiogenic agents, Fc receptors, transferrin receptor, receptor tyrosine kinases (RTKs), TNFSFRs, or any combination thereof.
  • a target selected from T-cell targets, tumor antigens, B-cell targets, pro-inflammatory cytokines or chemokines, pro-oncogenic cytokines or growth factors, angiogenic agents, Fc receptors, transferrin receptor, receptor tyrosine kinases (RTKs), TNFSFRs, or any combination thereof.
  • RTKs receptor tyrosine kinases
  • a binding domain of a polypeptide heterodimer of the present disclosure specifically binds a T-cell target, such as a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3y, CD35, and CD3s), CD28, PD-1 , HVEM, BTLA, CD80, CD86, GITR, and TGFBR1.
  • a TCR complex or a component thereof e.g., TCRa, TCRp, CD3y, CD35, and CD3s
  • CD28 e.g., TCRa, TCRp, CD3y, CD35, and CD3s
  • CD28 e.g., CD28, PD-1 , HVEM, BTLA, CD80, CD86, GITR, and TGFBR1.
  • a binding domain of polypeptide heterodimer of the present disclosure specifically binds a TCR complex or a component thereof.
  • a binding domain specifically binds to an individual human CD3 chain (e.g., human CD3y chain, human CD35 chain, and human CD3s chain) or a combination of two or more of the individual human CD3 chains (e.g., a complex of human CD3y and human CD3s or a complex of human CD35 and human CD3s).
  • a binding domain specifically binds to a human CD3s chain.
  • a binding domain specifically binds to one or more of human TCRa, human TCRP, or a heterodimer formed from human TCRa and human TCRp.
  • a binding domain of the present disclosure binds to a complex formed from one or more human CD3 chains with one or more human TCR chains, such as a complex formed from a human CD3y chain, a human CD35 chain, or a human CD3s chain with a human TCRa chain or a human TCRP chain.
  • a polypeptide heterodimer of the present disclosure may also bind a tumor target.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a tumor target, including RON, c-Met, CEACAM-6, PSMA, EpCAM, CEA, PCTA-1, STEAP-1, PSCA, ALCAM (CD166), EphA2, CD151, CA-125, MUC-1, MAGE-1, TROP2, IGF1R, CD44v6, CD151, TGFBR2, GHRHR, GHR, IL-6R, gpl30, TNFR2, OSMRp, Patched-1, Frizzled, Robol, ⁇ , CD80, CD81, CD86, CCR5, HER-3, HER-4, EGFR, CEA, MUC2, MUC3, MUC4, MUC5 AC , MUC5 b , MUC7, phCG, Lewis-Y, CD33, CD30, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA,
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a B-cell target, such as CD 19, CD20, CD21, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, HLA-DR, or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a pro-inflammatory cytokine or chemokine, such as TNFa, IL-6, hyperIL-6, IL-2, IL-1, IL-8, IP-10, IFNy, IFNa, RANKL, FASL, TGFp, IL7, IL10, IL17A/F, TWEAK, CSF2, IGF1, IGF2 or BLyS/APRIL, or any combination thereof.
  • a pro-inflammatory cytokine or chemokine such as TNFa, IL-6, hyperIL-6, IL-2, IL-1, IL-8, IP-10, IFNy, IFNa, RANKL, FASL, TGFp, IL7, IL10, IL17A/F, TWEAK, CSF2, IGF1, IGF2 or BLyS/APRIL, or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a pro-oncogenic cytokine or growth factor, such as HGF, MSP, EGF (including epiregulin, herregulin, ⁇ -regulin, neuregulin), HIF- ⁇ , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, IL-6, hyperIL-6, IL- 8, Wnt, sHH, TGFp, PDGF, or any combination thereof.
  • a pro-oncogenic cytokine or growth factor such as HGF, MSP, EGF (including epiregulin, herregulin, ⁇ -regulin, neuregulin), HIF- ⁇ , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, IL-6, hyperIL-6, IL- 8, Wnt, sHH, TGFp, PDGF, or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind an angiogenic agent, such as PDGFR, VEGFR1 -4, NRP 1 , Angiopoietin 2, c-Met or any combination thereof.
  • an angiogenic agent such as PDGFR, VEGFR1 -4, NRP 1 , Angiopoietin 2, c-Met or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind an Fc receptor, such as CD64, CD32A, CD32B, CD 16, FcRn, or any combination thereof.
  • a binding domain of a polypeptide heterodimer of the present disclosure specifically binds a transferrin receptor, such as CD71.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically binds a receptor tyrosine kinase, such as EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGFIR, EphA2, c-Met, RON, or any combination thereof.
  • a receptor tyrosine kinase such as EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGFIR, EphA2, c-Met, RON, or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically binds a TNFSFR, such as TNFR1, TNFR2, TWEAKR, TACI, BAFF-R, BCMA, FAS, OX40, GITR, 4-1-BB, LTbetaR, HVEM, RANK, or any combination thereof.
  • a TNFSFR such as TNFR1, TNFR2, TWEAKR, TACI, BAFF-R, BCMA, FAS, OX40, GITR, 4-1-BB, LTbetaR, HVEM, RANK, or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind hyperIL-6, IL-10, LIGHT, CD40, PDL1, PDL2, or any combination thereof.
  • one or more binding domains of a polypeptide heterodimer of the present disclosure are an agonist of one of the following molecules: IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, TNFR1, TNFR2, DR4, DR5, TWEAKR, FAS, or any combination thereof.
  • a binding domain may be any peptide that specifically binds a target of interest.
  • Sources of binding domains include antibody variable regions from various species (which can be formatted as antibodies, sFvs, scFvs, Fabs, or soluble VH domain or domain antibodies), including human, rodent, avian, and ovine. Additional sources of binding domains include variable regions of antibodies from other species, such as camelid (from camels, dromedaries, or llamas; Ghahroudi et al. (1997) FEBS Letters 414(3):521-526; Vincke et al. (2009) Journal of Biological Chemistry (2009) 284:3273- 3284; Hamers-Casterman et al.
  • these antibodies can apparently form antigen- binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies”) (Jespers et al. (2004) Nature Biotechnology 22: 1161-1165; Cortez-Retamozo et al. (2004) Cancer Research 64:2853-2857; Baral et al. (2006) Nature Medicine 12:580-584, and Barthelemy et al. (2008) Journal of Biological Chemistry 283:3639-3654).
  • Exemplary anti-CD3 antibodies from which the binding domain of this disclosure may be derived include Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172: 1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herald et al. (2003) J. Clin. Invest. 11 :409), visilizumab (Carpenter et al.
  • An exemplary anti-TCR antibody is H57 monoclonal antibody (Lavasani et al. (2007) Scandinavian Journal of Immunology 65:39-47).
  • An alternative source of binding domains of this disclosure includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as fibrinogen domains ⁇ see, e.g., Shoesl et al. (1985) Science 230: 1388), Kunitz domains ⁇ see, e.g., US Patent No. 6,423,498), ankyrin repeat proteins (Binz et al. (2003) Journal of Molecular Biology 332:489-503 and Binz et al. (2004) Nature Biotechnology 22(5):575-582), fibronectin binding domains (Richards et al. (2003) Journal of Molecular Biology 326: 1475-1488; Parker et al.
  • Binding domains of this disclosure can be generated as described herein or by a variety of methods known in the art ⁇ see, e.g., U.S. Patent Nos. 6,291,161 and 6,291,158). For example, binding domains of this disclosure may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest ⁇ see Hoet et al. (2005) Nature Biotechnol. 23:344).
  • mice HUMAB MOUSE®, TC MOUSETM, KM-MOUSE ® , llamas, chicken, rats, hamsters, rabbits, etc.
  • mice HUMAB MOUSE®, TC MOUSETM, KM-MOUSE ® , llamas, chicken, rats, hamsters, rabbits, etc.
  • a polypeptide heterodimer comprises a CD86 binding domain, such as a CTLA4 ectodomain, a CD28 ectodomain, or an immunoglobulin variable region binding domain (such as a scFv) specific for CD86 ⁇ e.g., from monoclonal antibodies 3D1 or FUN1).
  • a CD86 binding domain such as a CTLA4 ectodomain, a CD28 ectodomain, or an immunoglobulin variable region binding domain (such as a scFv) specific for CD86 ⁇ e.g., from monoclonal antibodies 3D1 or FUN1).
  • a scFv immunoglobulin variable region binding domain
  • less than an entire ectodomain is used.
  • domains within the CTLA4 ectodomain that bind CD86 and prevent binding of CD86 to CD28 can be used.
  • the CD86 binding domain can block binding of CD86 to CD28 and thereby downregulate T-cell activation.
  • a polypeptide heterodimer comprises an IL-10 agonist, such as IL-10, monoIL-10, or a functional region thereof.
  • IL-10 refers to an IL-10 molecule having a short linker (GGGSGG, SEQ ID NO:760) that separates the two subdomains of the molecule (amino and carboxyl end domains) so that these subdomains can form an intramolecular dimer.
  • a polypeptide heterodimer comprises an HLA-G agonist, such as an HLA-G5, an HLA-Gl, an HLA-G mutein, or a functional region thereof; an ectodomain of an HLA-G5, an HLA-Gl or an HLA-G mutein; or an immunoglobulin variable region binding domain (such as an scFv) specific for ILT2, ILT4 or KIR2DL4.
  • HLA-G agonist such as an HLA-G5, an HLA-Gl, an HLA-G mutein, or a functional region thereof
  • an ectodomain of an HLA-G5, an HLA-Gl or an HLA-G mutein or an immunoglobulin variable region binding domain (such as an scFv) specific for ILT2, ILT4 or KIR2DL4.
  • a polypeptide heterodimer comprises an HGF agonist, such as an HGF or a sub-domain thereof.
  • a polypeptide heterodimer comprises an IL35 agonist, such as an immunoglobulin variable region binding domain (such as an scFv) specific for IL35R or IL35, or a functional region thereof.
  • an IL35 agonist such as an immunoglobulin variable region binding domain (such as an scFv) specific for IL35R or IL35, or a functional region thereof.
  • a polypeptide heterodimer comprises a LIGHT antagonist, such as an immunoglobulin variable region binding domain (such as an scFv) specific for LIGHT, or a HVEM ectodomain or a functional region thereof.
  • a polypeptide heterodimer comprises a PD-1 agonist, such as an immunoglobulin variable region binding domain (such as an scFv) specific for PD-1, or a PD-1 ligand (e.g. PD-L1 or PD-L2) or a functional region thereof.
  • a polypeptide heterodimer comprises a BTLA agonist, such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for BTLA, or a HVEM ectodomain or a functional region thereof.
  • a BTLA agonist such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for BTLA, or a HVEM ectodomain or a functional region thereof.
  • a polypeptide heterodimer comprises a GITRL antagonist, such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for GITRL, or a GITR ectodomain, soluble GITR, or a functional region thereof.
  • a GITRL antagonist such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for GITRL, or a GITR ectodomain, soluble GITR, or a functional region thereof.
  • a polypeptide heterodimer comprises a CD40 antagonist, such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for CD40.
  • a CD40 antagonist such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for CD40.
  • a binding domain is a single chain Fv fragment
  • VH and V L domains are human.
  • Exemplary VH regions include the VH region of 2E12 (anti-CD28) scFv as set forth in SEQ ID NO: 106, the VH region of P2C2 (anti-CD79b) scFv as set forth in SEQ ID NO: 184, the VH region of 5D5 (anti-c- Met) scFv as set forth in SEQ ID NO:258, the VH region of A2 (anti-hyperIL-6) scFv as set forth in SEQ ID NO: 80, the VH region of 3D1 (anti-CD86) scFv as set forth in SEQ ID NO:92, the VH region of MET021 (anti-c-Met) scFv as set forth in SEQ ID NO: 100, the VH region of G19-4 (anti-CD3) scFv as set forth in SEQ ID NO:
  • VL domains are the VL region of 2E12 (anti-CD28) scFv as set forth in SEQ ID NO: 107, the VL region of P2C2 (anti-CD79b) scFv as set forth in SEQ ID NO: 182, the VL region of 5D5 (anti-c-Met) scFv as set forth in SEQ ID NO:259, the VL region of A2 (anti-hyper IL-6) scFv as set forth in SEQ ID NO:84, the VL region of 3D1 (anti-CD86) scFv as set forth in SEQ ID NO:96, the VL region of MET021 (anti-c-Met) scFv as set forth in SEQ ID NO: 101, the VL region of G 19-4 (anti-CD3) scFv as set forth in SEQ ID NO: 104, the VL region of HD37 (anti-CD19) scFv as set forth in SEQ ID NO: 1 19,
  • a binding domain is a single chain Fv fragment (scFv) that comprises VH and VL domains specific for a TCR complex or a component thereof.
  • the VH and VL domains are human or humanized VH and VL domains.
  • Exemplary VH domains include BC3 (anti-CD3) VH, OKT3 (anti- CD3) V H , H57 (anti-TCR) V H , and 2C 1 1 (anti-CD3) V H domains as set forth in SEQ ID NOS:301 , 303, 313, and 317, respectively.
  • Further exemplary VH domains include Cris-7 (anti-CD3) VH domains, such as those set forth in SEQ ID NOS:327 and 331- 333.
  • V L domains are BC3 (anti-CD3) V L , OKT3 (anti-CD3) V L , H57 (anti- TCR) V L , and 2C1 1 (anti-CD3) V L domains as set forth in SEQ ID NOS:302, 304, 315 and 318, respectively.
  • Further exemplary VL domains include Cris-7 (anti-CD3) VL domains, such as those set forth in SEQ ID NOS:328, 329 and 330.
  • a binding domain comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR comprises zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g., c-Met, RON, CD28, CD79b, CD38, TCRa, TCRp, hyper IL-6, CD86, CD 19, and HLA-DR) and such mutant or derivative still binds its target.
  • a target of interest e.g., c-Met, RON, CD28, CD79b, CD38, TCRa, TCRp, hyper IL-6, CD86, CD 19, and
  • a binding domain VH or VL region of the present disclosure can be derived from or based on a VH or VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non- conservative amino acid substitutions), or a combination of the above -noted changes, when compared with the VH or VL of a known monoclonal antibody.
  • one or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non- conservative amino acid substitutions), or
  • the insertion(s), deletion(s) or substitution(s) may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided that a binding domain containing the modified VH or VL region can still specifically bind its target with an affinity about the same as the wild type binding domain.
  • VH and VL domains may be arranged in either orientation (i.e., from amino-terminus to carboxyl terminus, VH-VL or VL-VH) and may be joined by an amino acid sequence (e.g., having a length of about five to about 35 amino acids) capable of providing a spacer function such that the two sub-binding domains can interact to form a functional binding domain.
  • an amino acid sequence e.g., having a length of about five to about 35 amino acids
  • an amino acid sequence that joins the VH and VL domains includes those belonging to the (Gly n Ser) family, such as (Gly 3 Ser) n (Gly 4 Ser)i, (Gly 3 Ser)i(Gly 4 Ser) n , (Gly 3 Ser) n (Gly 4 Ser) n , or (Gly 4 Ser) n , wherein n is an integer of 1 to 5.
  • the linker is GGGGSGGGGS GGGGS (SEQ ID NO: 183) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:108).
  • An additional exemplary linker is GGGGSGGGGSGGGGAS (SEQ ID NO:739).
  • these (Gly n Ser)-based linkers are used to link the VH and VL domains in a binding domain, but are not used to link a binding domain to an immunoglobulin heterodimerization domain or to an Fc region portion.
  • binding domains specific for CD28 include a 2E12 scFv as set forth in SEQ ID NO: 109
  • binding domains specific for CD79b include a P2C2 scFv as set forth in SEQ ID NO: 185
  • binding domains specific for c-Met include a 5D5 scFv as set forth in SEQ ID NO:257
  • binding domains specific for RON include a 4C04 scFv as set forth in SEQ ID NO:261 and a 11H09 scFv as set forth in SEQ ID NO:265
  • binding domains specific for hyperIL-6 include an A2 scFv as set forth in SEQ ID NO: 86
  • binding domains specific for CD86 include a 3D1 scFv as set forth in SEQ ID NO:98
  • binding domains specific for HLA-DR include an M0042 scFv as set forth in SEQ ID NO: 120
  • binding domains specific for CD3 include a G19-4
  • nucleotide sequences encoding the A2 (anti-hyperIL6) and 3D1 (anti-CD86) scFv's are set forth in SEQ ID NOS:85 and 97, respectively.
  • Exemplary binding domains that bind a TCR complex or a component thereof include a BMA031 scFv as set forth in SEQ ID NO:830 and other scFv's as set forth in SEQ ID NOS:310, 311, 312, 319, and 334-340.
  • Additional exemplary binding domains include a PDL2 ectodomain as set forth in SEQ ID NO: 88 and monoIL-10 as set forth in SEQ ID NO: 90.
  • the nucleotide sequences encoding the PDL2 ectodomain and monoIL-10 are set forth in SEQ ID NOS:87 and 89, respectively.
  • the light chain amino acid sequence of the 4C04 (anti-RON) scFv is set forth in SEQ ID NO:602, and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:604-606, respectively.
  • the heavy chain amino acid sequence of the 4C04 (anti- RON) scFv is set forth in SEQ ID NO:603, and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:607-609, respectively.
  • the light chain amino acid sequence of the 1 1H09 (anti-RON) scFv is set forth in SEQ ID NO:610, and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:612-614, respectively.
  • the heavy chain amino acid sequence of the 1 1H09 (anti- RON) scFv is set forth in SEQ ID NO:61 1 , and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:615-617, respectively.
  • a binding domain may be located either at amino terminal or carboxyl terminal to the Fc region portion of a single chain polypeptide of the present disclosure. In certain embodiments, the binding domain is located at the amino terminus of a single chain polypeptide. In certain other embodiments, the binding domain is located at the carboxyl terminus of a single chain polypeptide. In certain other embodiments, a binding domain is located at both the amino and carboxyl terminus of a single chain polypeptide.
  • a polypeptide heterodimer of the present disclosure comprises an immunoglobulin heterodimerization domain in each polypeptide chain.
  • the immunoglobulin heterodimerization domains in the two single chain polypeptides of a polypeptide heterodimer are different from each other and thus may be differentially modified to facilitate heterodimerization of both chains and to minimize homodimerization of either chain.
  • immunoglobulin heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting polypeptide heterodimers.
  • immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides include immunoglobulin CHI and CL domains, for instance, human CHI and CL domains.
  • an immunoglobulin heterodimerization domain is a wild type CHI region, such as a wild type IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, or IgM CHI region.
  • an immunoglobulin heterodimerization domain is a wild type human IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, or IgM CHI region as set forth in SEQ ID NOS: 1 14, 186-192 and 194, respectively.
  • an immunoglobulin heterodimerization domain is a wild type human IgGl CHI region as set forth in SEQ ID NO: 1 14.
  • an immunoglobulin heterodimerization domain is an altered immunoglobulin CHI region, such as an altered IgGl , IgG2, IgG3, IgG4, IgAl , IgA2 IgD, IgE, or IgM CHI region.
  • an immunoglobulin heterodimerization domain is an altered human IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, or IgM CHI region.
  • a cysteine residue of a wild type CHI region e.g.
  • a human CHI involved in forming a disulfide bond with a wild type immunoglobulin CL domain (e.g., a human CL) is deleted or substituted in the altered immunoglobulin CHI region such that a disulfide bond is not formed between the altered CHI region and the wild type CL domain.
  • a wild type immunoglobulin CL domain e.g., a human CL
  • an immunoglobulin heterodimerization domain is a wild type CL domain, such as a wild type CK domain or a wild type C domain.
  • an immunoglobulin heterodimerization domain is a wild type human CK or human domain as set forth in SEQ ID NOS: l 12 and 1 13, respectively.
  • an immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or domain, for instance, an altered human CK or human domain.
  • a cysteine residue of a wild type CL domain (e.g. , a human CL) involved in forming a disulfide bond with a wild type immunoglobulin CHI region (e.g. , a human CHI) is deleted or substituted in the altered immunoglobulin CL domain.
  • Such altered CL domains may further comprise an amino acid deletion at their amino termini.
  • An exemplary CK domain is set forth in SEQ ID NO: 141 , in which the first arginine and the last cysteine of the wild type human Ck domain are both deleted.
  • only the last cysteine of the wild type human Ck domain is deleted in the altered Ck domain because the first arginine deleted from the wild type human Ck domain may be provided by a linker that has an arginine at its carboxyl terminus and links the amino terminus of the altered Ck domain with another domain (e.g. , an Fc region portion).
  • An exemplary domain is set forth in SEQ ID NO: 140, in which the first arginine of a wild type human domain is deleted and the cysteine involved in forming a disulfide bond with a cysteine in a CHI region is substituted by a serine.
  • an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions, as compared to a wild type CK domain, at positions that may be involved in forming the interchain-hydrogen bond network at a CK-CK interface.
  • an immunoglobulin heterodimerization domain is an altered human CK domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 that are substituted with a different amino acid. The numbering of the amino acids is based on their positions in the altered human CK sequence as set forth in SEQ ID NO: 141.
  • an immunoglobulin heterodimerization domain is an altered human CK domain having one, two, three or four amino acid substitutions at positions N29, N30, V55, or T70.
  • the amino acid used as a substitute at the above- noted positions may be an alanine, or an amino acid residue with a bulk side chain moiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, or lysine.
  • Additional amino acid residues that may be used to substitute amino acid residues of the wild type human Ck sequence at the above noted positions include aspartate, methionine, serine and phenyalanine.
  • Exemplary altered human CK domains are set forth in SEQ ID NOS: 142-178.
  • Altered human CK domains are those that facilitate heterodimerization with a CHI region, but minimize homodimerization with another CK domain.
  • Representative altered human CK domains are set forth in SEQ ID NOS: 160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30M V55A T70
  • both the immunoglobulin heterodimerization domains (i.e., immunoglobulin CHI and CL domains) of a polypeptide heterodimer have mutations so that the resulting immunoglobulin heterodimerization domains form salt bridges (i.e., ionic interactions) between the amino acid residues at the mutated sites.
  • the immunoglobulin heterodimerization domains of a polypeptide heterodimer may be a mutated CHI domain in combination with a mutated Ck domain.
  • valine at position 68 (V68) of the wild type human CHI domain is substituted by an amino acid residue having a negative charge (e.g., aspartate or glutamate), whereas leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g., lysine, arginine or histidine).
  • a negative charge e.g., aspartate or glutamate
  • leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g., lysine, arginine or histidine).
  • V68 of the wild type CHI may be substituted by an amino acid residue having a positive charge
  • L29 of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted may be substituted by an amino acid residue having a negative charge
  • Exemplary mutated CHI sequences in which V68 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:844 and 845.
  • Exemplary mutated Ck sequences in which L29 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:842 and 843.
  • Positions other than V68 of human CHI domain and L29 of human Ck domain may be substituted with amino acids having opposite charges to produce ionic interactions between the amino acids in addition or alternative to the mutations in V68 of CHI domain and L29 of Ck domain.
  • Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CHl-Ck pair to identify amino acid residues at the CHl-Ck interface, and further identifying suitable positions among the amino acid residues at the CHl-Ck interface using a set of criteria (e.g. , propensity to engage in ionic interactions, proximity to a potential partner residue, etc.).
  • polypeptide heterodimers of the present disclosure contain only one pair of immunoglobulin heterodimerization domains.
  • a first chain of a polypeptide heterodimer may comprise a CHI region as an immunoglobulin heterodimerization domain, while a second chain may comprise a CL domain (e.g. , a CK or CX) as an immunoglobulin heterodimerization domain.
  • a first chain may comprise a CL region (e.g. , a CK or CX) as an immunoglobulin heterodimerization domain
  • a second chain may comprise a CHI region as an immunoglobulin heterodimerization domain.
  • the immunoglobulin heterodimerization domains of the first and second chains are capable of associating to form a polypeptide heterodimer of this disclosure.
  • polypeptide heterodimers of the present disclosure may have two pairs of immunoglobulin heterodimerization domains.
  • a first chain of a polypeptide heterodimer may comprise two CHI regions, while a second chain may have two CL domains that associate with the two CHI regions in the first chain.
  • a first chain may comprise two CL domains, while a second chain may have two CHI regions that associate with the two CL domains in the first chain.
  • a first chain polypeptide comprises a CHI region and a CL domain
  • a second chain polypeptide comprises a CL domain and a CHI region that associate with the CHI region and the CL domain, respectively, of the first chain polypeptide.
  • the immunoglobulin heterodimerization domain of each chain may be located amino terminal to the Fc region portion of that chain.
  • the immunoglobulin heterodimerization domain in each chain may be located carboxyl terminal to the Fc region portion of that chain.
  • both immunoglobulin heterodimerization domains in each chain may be located amino terminal to the Fc region portion of that chain.
  • both immunoglobulin heterodimerization domains in each chain may be located carboxyl terminal to the Fc region portion of that chain.
  • one immunoglobulin heterodimerization domain in each chain may be located amino terminal to the Fc region portion of that chain, while the other immunoglobulin heterodimerization domain of each chain may be located carboxyl terminal to the Fc region portion of that chain.
  • the Fc region portion is interposed between the two immunoglobulin heterodimerization domains of each chain.
  • polypeptide heterodimers of the present disclosure comprise an Fc region constant domain portion (also referred to as an Fc region portion) in each polypeptide chain.
  • Fc region portion slows clearance of the heterodimers from circulation after administration to a subject.
  • the Fc region portion further enables relatively easy modulation of heterodimer polypeptide effector functions (e.g., ADCC, ADCP, CDC, complement fixation and binding to Fc receptors), which can either be increased or decreased depending on the disease being treated, as known in the art and described herein.
  • an Fc region portion of polypeptide heterodimers of the present disclosure will be capable of mediating one or more of these effector functions.
  • An Fc region portion present in single chain polypeptides that form part of the polypeptide heterodimers of the present disclosure may comprise a CH2 domain, a CH3 domain, a CH4 domain or any combination thereof.
  • an Fc region portion may comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, or two CH4 domains.
  • a CH2 domain that may form an Fc region portion of a single chain polypeptide of a heterodimer of the present disclosure may be a wild type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain thereof from certain immunoglobulin classes or subclasses (e.g., IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD) and from various species (including human, mouse, rat, and other mammals).
  • immunoglobulin classes or subclasses e.g., IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD
  • a CH2 domain is a wild type human immunoglobulin CH2 domain, such as wild type CH2 domains of human IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD, as set forth in SEQ ID NOS: 1 15, 199-201 and 195- 197, respectively.
  • the CH2 domain is a wild type human IgGl CH2 domain as set forth in SEQ ID NO: 1 15.
  • a CH2 domain is an altered immunoglobulin
  • CH2 region e.g., an altered human IgGl CH2 domain
  • an amino acid substitution at the asparagine of position 297 e.g., asparagine to alanine.
  • Such an amino acid substitution reduces or eliminates glycosylation at this site and abrogates efficient Fc binding to FcyR and C lq.
  • the sequence of an altered human IgGl CH2 domain with an Asn to Ala substitution at position 297 is set forth in SEQ ID NO:324.
  • a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human IgGl CH2 domain) that comprises at least one substitution or deletion at positions 234 to 238.
  • an immunoglobulin CH2 region can comprise a substitution at position 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234, 235 and 237, positions 234, 236 and 238, positions 234, 235, 237, and 238, positions 236-238, or any other combination of two, three, four, or five amino acids at positions 234-238.
  • an altered CH2 region may comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, for instance, at one of position 236 or position 237 while the other position is substituted.
  • the above -noted mutation(s) decrease or eliminate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide heterodimer that comprises the altered CH2 domain.
  • the amino acid residues at one or more of positions 234-238 has been replaced with one or more alanine residues.
  • only one of the amino acid residues at positions 234-238 have been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g., alanine or serine).
  • a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human IgGl CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331.
  • an immunoglobulin CH2 region can comprise a substitution at position 253, 310, 318, 320, 322, or 331 , positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322, and 331.
  • the above-noted mutation(s) decrease or eliminate the complement-dependent cytotoxicity (CDC) of a polypeptide heterodimer that comprises the altered CH2 domain.
  • CDC complement-dependent cytotoxicity
  • an altered CH2 region in addition to the amino acid substitution at position 297, can further comprise one or more (e.g., two, three, four, or five) additional substitutions at positions 234-238.
  • an immunoglobulin CH2 region can comprise a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297.
  • an altered CH2 region may comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, such as at position 236 or position 237.
  • the additional mutation(s) decreases or eliminates the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide heterodimer that comprises the altered CH2 domain.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues.
  • only one of the amino acid residues at positions 234-238 has been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g., alanine or serine).
  • a mutated CH2 region in addition to one or more (e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region (e.g., an altered human IgGl CH2 domain) in a fusion protein of the present disclosure may contain one or more (e.g., 2, 3, 4, 5, or 6) additional amino acid substitutions (e.g., substituted with alanine) at one or more positions involved in complement fixation (e.g., at positions 1253, H310, E318, K320, K322, or P331).
  • additional amino acid substitutions e.g., substituted with alanine
  • mutated immunoglobulin CH2 regions include human IgGl , IgG2, IgG4 and mouse IgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320 and 322.
  • An exemplary mutated immunoglobulin CH2 region is mouse IGHG2c CH2 region with alanine substitutions at L234, L235, G237, E318, K320, and K322 (SEQ ID NO:314).
  • an altered CH2 region in addition to the amino acid substitution at position 297 and the additional deletion(s) or substitution(s) at positions 234-238, an altered CH2 region (e.g., an altered human IgGl CH2 domain) can further comprise one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331.
  • an immunoglobulin CH2 region can comprise a (1) substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions 1253, H310, E318, K320, K322, and P331 , such as one, two, three substitutions at positions E318, K320 and K322.
  • the amino acids at the above-noted positions may be substituted by alanine or serine.
  • an immunoglobulin CH2 region polypeptide comprises: (i) an amino acid substitution at the asparagines of position 297 and one amino acid substitution at position 234, 235, 236 or 237; (ii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at two of positions 234- 237; (iii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution at the asparagine of position 297, amino acid substitutions at positions 234, 235 and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and 322.
  • Exemplary altered immunoglobulin CH2 regions with amino acid substitutions at the asparagine of position 297 include: human IgGl CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO:325), human IgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO:326), human IgG4 CH2 region with alanine substitutions at F234, L235, G237 and N297 and a deletion of G236 (SEQ ID NO:322), human IgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NO:343), human IgG4 CH2 region with alanine substitutions at L235 and N297 (SEQ ID NO:344), human IgG4 CH2 region with alanine substitutions at G236 and N297 (SEQ ID NO:345), and human IgG
  • an altered CH2 region may contain one or more additional amino acid substitutions at one or more positions other than the above-noted positions.
  • Such amino acid substitutions may be conservative or non-conservative amino acid substitutions.
  • P233 may be changed to E233 in an altered IgG2 CH2 region (see, e.g., SEQ ID NO:326).
  • the altered CH2 region may contain one or more amino acid insertions, deletions, or both.
  • the insertion(s), deletion(s) or substitution(s) may anywhere in an immunoglobulin CH2 region, such as at the N- or C- terminus of a wild type immunoglobulin CH2 region resulting from linking the CH2 region with another region (e.g., a binding domain or an immunoglobulin heterodimerization domain) via a hinge.
  • an altered CH2 region in a polypeptide heterodimer of the present disclosure comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%), at least 98%>, at least 99% identical to a wild type immunoglobulin CH2 region, such as the CH2 region of wild type human IgGl, IgG2, or IgG4, or mouse IgG2a (e.g., IGHG2c).
  • a wild type immunoglobulin CH2 region such as the CH2 region of wild type human IgGl, IgG2, or IgG4, or mouse IgG2a (e.g., IGHG2c).
  • An altered immunoglobulin CH2 region in a polypeptide heterodimer of the present disclosure may be derived from a CH2 region of various immunoglobulin isotypes, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and IgD, from various species (including human, mouse, rat, and other mammals).
  • an altered immunoglobulin CH2 region in a fusion protein of the present disclosure may be derived from a CH2 region of human IgGl, IgG2 or IgG4, or mouse IgG2a (e.g., IGHG2c), whose sequences are set forth in SEQ ID NOS: l 15, 199, 201 and 320.
  • an altered CH2 domain is a human IgGl CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e., a human IgGl CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:595), and optionally an N297 mutation (e.g., to alanine).
  • an altered CH2 domain is a human IgGl CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320 and 322 (i.e., a human IgGl CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO:596), and optionally an N297 mutation (e.g., to alanine).
  • an altered CH2 domain is an altered human IgGl CH2 domain with mutations known in the art that enhance immunological activities such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.
  • the CH3 domain that may form an Fc region portion of a single chain polypeptide of a heterodimer of the present disclosure may be a wild type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain thereof from certain immunoglobulin classes or subclasses (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM) of various species (including human, mouse, rat, and other mammals).
  • immunoglobulin classes or subclasses e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM
  • a CH3 domain is a wild type human immunoglobulin CH3 domain, such as wild type CH3 domains of human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM as set forth in SEQ ID NOS:l 16, 208-210, 204-207, and 212, respectively.
  • the CH3 domain is a wild type human IgGl CH3 domain as set forth in SEQ ID NO: 116.
  • a CH3 domain is an altered human immunoglobulin CH3 domain, such as an altered CH3 domain based on or derived from a wild-type CH3 domain of human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • an altered CH3 domain may be a human IgGl CH3 domain with one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering). The mutations in such positions may be involved in complement fixation.
  • an altered CH3 domain may be a human IgGl CH3 domain but with one or two amino acid substitutions at position F405 or Y407.
  • an altered CH3 domain may be an altered human IgGl CH3 domain with its last lysine deleted.
  • the sequence of this altered CH3 domain is set forth in SEQ ID NO:761.
  • a polypeptide heterodimer comprises a CH3 pair that comprises so called "knobs-into-holes" mutations (see, Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et ah, Protein Engineering 9:617-21, 1966). More specifically, mutations may be introduced into each of the two CH3 domains so that the steric complementarity required for CH3/CH3 association obligates these two CH3 domains to pair with each other.
  • a CH3 domain in one single chain polypeptide of a polypeptide heterodimer may contain a T366W mutation (a "knob” mutation, which substitutes a small amino acid with a larger one), and a CH3 domain in the other single chain polypeptide of the polypeptide heterodimer may contain a Y407A mutation (a "hole” mutation, which substitutes a large amino acid with a smaller one).
  • Other exemplary knobs-into-holes mutations include (1) a T366Y mutation in one CH3 domain and a Y407T in the other CH3 domain, and (2) a T366W mutation in one CH3 domain and T366S, L368A and Y407V mutations in the other CH3 domain.
  • the CH4 domain that may form an Fc region portion of a single chain polypeptide of a heterodimer of the present disclosure may be a wild type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain thereof from IgE or IgM molecules.
  • the CH4 domain is a wild type human immunoglobulin CH4 domain, such as wild type CH4 domains of human IgE and IgM molecules as set forth in SEQ ID NOS:213 and 214, respectively.
  • a CH4 domain is an altered human immunoglobulin CH4 domain, such as an altered CH4 domain based on or derived from a CH4 domain of human IgE or IgM molecules, which have mutations that increase or decrease an immunological activity known to be associated with an IgE or IgM Fc region.
  • an Fc region constant domain portion in heterodimers of the present disclosure comprises a combination of CH2, CH3 or CH4 domains (i.e., more than one constant sub-domain selected from CH2, CH3 and CH4).
  • the Fc region portion may comprise CH2 and CH3 domains or CH3 and CH4 domains.
  • the Fc region portion may comprise two CH3 domains and no CH2 or CH4 domains (i.e., only two or more CH3).
  • the multiple constant sub-domains that form an Fc region portion may be based on or derived from the same immunoglobulin molecule, or the same class or subclass immunoglobulin molecules.
  • the Fc region portion is an IgG CH2CH3 (e.g., IgGl CH2CH3, IgG2 CH2CH3, and IgG4 CH2CH3) and may be a human (e.g., human IgGl, IgG2, and IgG4) CH2CH3.
  • the Fc region portion comprises (1) wild type human IgGl CH2 and CH3 domains, (2) human IgGl CH2 with N297A substitution (i.e., CH2(N297A)) and wild type human IgGl CH3, or (3) human IgGl CH2(N297A) and an altered human IgGl CH3 with the last lysine deleted.
  • the multiple constant sub-domains may be based on or derived from different immunoglobulin molecules, or different classes or subclasses immunoglobulin molecules.
  • an Fc region portion comprises both human IgM CH3 domain and human IgGl CH3 domain.
  • the multiple constant sub-domains that form an Fc region portion may be directly linked together or may be linked to each other via one or more (e.g., about 2-10) amino acids.
  • Fc region portions are set forth in SEQ ID NOS:305-309,
  • the Fc region portions of both single chain polypeptides of a polypeptide heterodimer are identical to each other.
  • the Fc region portion of one single chain polypeptide of a polypeptide heterodimer is different from the Fc region portion of the other single chain polypeptide of the heterodimer.
  • one Fc region portion may contain a CH3 domain with a "knob" mutation, whereas the other Fc region portion may contain a CH3 domain with a "hole” mutation.
  • a hinge region contained in a single chain polypeptide of a polypeptide heterodimer according to the present disclosure may be located (a) immediately amino terminal to an Fc region portion (e.g., depending on the isotype, amino terminal to a CH2 domain wherein the Fc region portion is a CH2CH3, or amino terminal to a CH3 domain wherein the Fc region portion is a CH3CH4), (b) interposed between and connecting a binding domain (e.g., scFv) and an immunoglobulin heterodimerization domain, (c) interposed between and connecting an immunoglobulin heterodimerization domain and an Fc region portion (e.g., wherein the Fc region portion is a CH2CH3 or a CH3CH4, depending on the isotype or isotypes), (d) interposed between and connecting an Fc region portion and a binding domain, (e) at the amino terminus of the single chain polypeptide, or (f) at the carboxyl terminus of the single chain polypeptid
  • the single chain polypeptide comprising a hinge region as described herein will be capable of associating with a different single chain fusion polypeptide to form a polypeptide heterodimer provided herein, and the polypeptide heterodimer formed will contain a binding domain that retains its target specificity or its specific target binding affinity.
  • a hinge present in a single chain polypeptide that forms a polypeptide heterodimer with another single chain polypeptide may be an immunoglobulin hinge region, such as a wild type immunoglobulin hinge region or an altered immunoglobulin hinge region thereof.
  • a hinge is a wild type human immunoglobulin hinge region (e.g., human immunoglobulin hinge regions as set forth in SEQ ID NOS:215-221).
  • one or more amino acid residues may be added at the amino- or carboxy- terminus of a wild type immunoglobulin hinge region as part of a fusion protein construct design.
  • additional junction amino acid residues at the hinge amino-terminus can be "RT,” “RSS,” “TG,” or “T”
  • at the hinge carboxy-terminus can be "SG”
  • a hinge deletion can be combined with an addition, such as ⁇ with "SG” added at the carboxyl terminus.
  • a hinge is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues (e.g., serine or alanine).
  • a hinge may be an altered immunoglobulin hinge based on or derived from a wild type human IgGl hinge as set forth in SEQ ID NO:667, which from amino terminus to carboxyl terminus comprises the upper hinge region (EPKSCDKTHT, SEQ ID NO:227) and the core hinge region (CPPCP, SEQ ID NO:228).
  • Exemplary altered immunoglobulin hinges include an immunoglobulin human IgGl hinge region having one, two or three cysteine residues found in a wild type human IgGl hinge substituted by one, two or three different amino acid residues (e.g., serine or alanine).
  • An altered immunoglobulin hinge may additionally have a proline substituted with another amino acid (e.g., serine or alanine).
  • the above-described altered human IgGl hinge may additionally have a proline located carboxyl terminal to the three cysteines of wild type human IgGl hinge region substituted by another amino acid residue (e.g., serine, alanine).
  • the prolines of the core hinge region are not substituted.
  • Exemplary altered immunoglobulin hinges are set forth in SEQ ID NOS: 229-240, 255, 664-677, and 748-759.
  • An example of an altered IgGl hinge is an altered human IgGl hinge in which the first cysteine is substituted by serine.
  • the sequence of this altered IgGl hinge is set forth in SEQ ID NO:664, and is referred to as the " human IgGl SCC-P hinge" or "SCC-P hinge.”
  • one or more amino acid residues e.g., "RT,” "RSS,” or “T” may be added at the amino-or carboxy-terminus of a mutated immunoglobulin hinge region as part of a fusion protein construct design.
  • a hinge polypeptide comprises or is a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin hinge region, such as a wild type human IgGl hinge, a wild type human IgG2 hinge, or a wild type human IgG4 hinge.
  • a hinge present in a single chain polypeptide that forms a polypeptide heterodimer with another single chain polypeptide may be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild type immunoglobulin hinge or an altered immunoglobulin hinge).
  • immunoglobulin hinges may be used on or near the carboxyl end (e.g., located carboxyl terminal to Fc region portions) of the single chain polypeptides that form the polypeptide heterodimers.
  • hinges include peptides of about five to about 150 amino acids of the interdomain or stalk region of type II C-lectins or CD molecules, for instance, peptides of about eight to 25 amino acids and peptides of about seven to 18 amino acids, and derivatives thereof.
  • the "interdomain or stalk region" of a type II C-lectin or CD molecule refers to the portion of the extracellular domain of the type II C-lectin or CD molecule that is located between the C-type lectin- like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the transmembrane domain.
  • C-type lectin- like domain C-type lectin- like domain
  • the extracellular domain corresponds to amino acid residues 34-179
  • the CTLD corresponds to amino acid residues 61-176.
  • the interdomain or stalk region of the human CD94 molecule includes amino acid residues 34-60, which is found between the membrane and the CTLD (see Boyington et al., Immunity 10:75, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:753, 1992; and Figdor et al, Nature Rev. Immunol. 2:77, 2002).
  • These type II C-lectin or CD molecules may also have from six to 10 junction amino acids between the stalk region and the transmembrane region or the CTLD.
  • the 233 amino acid human NKG2A protein GenBank Accession No.
  • P26715.1 has a transmembrane domain ranging from amino acids 71-93 and an extracellular domain ranging from amino acids 94-233.
  • the CTLD is comprised of amino acids 119-231, and the stalk region comprises amino acids 99-116, which is flanked by junctions of five and two amino acids.
  • Other type II C-lectin or CD molecules, as well as their extracellular ligand-bind domains, interdomain or stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos.
  • a "derivative" of an interdomain or stalk region, or fragment thereof, of a type II C-lectin or CD molecule includes an about an eight to about 150 amino acid sequence in which one, two, or three amino acids of the stalk region of a wild type type II C-lectin or CD molecule have a deletion, insertion, substitution, or any combination thereof, for instance, the one or more changes are substitutions or the one or more mutations include only one deletion.
  • a derivative of an interdomain or stalk region is more resistant to proteolytic cleavage as compared to the wild-type interdomain or stalk region sequence, such as those derived from about eight to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94.
  • interdomain or stalk region hinges have seven to 18 amino acids and can form an a-helical coiled coil structure.
  • interdomain or stalk region hinges contain 0, 1, 2, 3, or 4 cysteines.
  • Exemplary interdomain or stalk region hinges are peptide fragments of the interdomain or stalk regions, such as ten to 150 amino acid fragments from the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D, as set forth in SEQ ID NOS :241-244, 716 and 601. Additional exemplary stalk region or interdomain hinges include those as set forth in SEQ ID NOS:78, 734-737, 742-747, and 766-790.
  • hinges that can be used in single chain polypeptides of polypeptide heterodimers are from portions of cell surface receptors (interdomain regions) that connect immunoglobulin V-like or immunoglobulin C-like domains. Regions between Ig V-like domains where the cell surface receptor contains multiple Ig V-like domains in tandem and between Ig C-like domains where the cell surface receptor contains multiple tandem Ig C-like regions are also contemplated as hinges useful in single chain polypeptides of polypeptide heterodimers.
  • hinge sequences comprised of cell surface receptor interdomain regions may further contain a naturally occurring or added motif, such as an IgG core hinge sequence that confers one or more disulfide bonds to stabilize the polypeptide heterodimer formation. Examples of hinges include interdomain regions between the Ig V-like and Ig C-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD 150, CD 166, and CD244.
  • hinge sequences have about 5 to 150 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60 amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 amino acids.
  • the hinge may be primarily flexible, but may also provide more rigid characteristics or may contain primarily a- helical structure with minimal ⁇ -sheet structure.
  • the lengths or the sequences of the hinges may affect the binding affinities of the binding domains to which the hinges are directly or indirectly (via another region or domain, such as an immunoglobulin heterodimerization domain) connected as well as one or more activities of the Fc region portions to which the hinges are directly or indirectly connected (see, Examples 9 and 10).
  • hinge sequences are stable in plasma and serum and are resistant to proteolytic cleavage.
  • the first lysine in the IgGl upper hinge region may be mutated to minimize proteolytic cleavage, for instance, the lysine may be substituted with methionine, threonine, alanine or glycine, or is deleted (see, e.g., SEQ ID NOS:379-434, which may include junction amino acids at the amino terminus such as RT).
  • hinge sequences may contain a naturally occurring or added motif such as an immunoglobulin hinge core structure CPPCP (SEQ ID NO:228) that confers the capacity to form a disulfide bond or multiple disulfide bonds to stabilize the carboxy-terminus of a molecule.
  • hinge sequences may contain one or more glycosylation sites.
  • Exemplary hinges including altered immunoglobulin hinges, are set forth in SEQ ID NOS:379-434, 618-749, and 763-791.
  • a single chain polypeptide of a polypeptide heterodimer according to the present disclosure comprises more than one hinge.
  • a single chain polypeptide having two binding domains, one of which at the amino terminus and the other at the carboxyl terminus may have two hinges.
  • One hinge may be directly or indirectly (e.g., via an immunoglobulin heterodimerization domain) connected to the binding domain at or near the amino terminus, and the other hinge may be connected (e.g., directly connected) to the other binding domain at or near the carboxyl terminus.
  • even if a single chain polypeptide has only one binding domain it may have more than one hinge, for example, at its amino or carboxyl terminus.
  • Such a hinge may interact with a corresponding hinge in the other chain of the heterodimer, such as forming one or more interchain disulfide bonds, to facilitate or enhance the heterodimerization of the two chains.
  • a hinge (H-I) of a SCP-I of a polypeptide heterodimer "corresponds to" a hinge (H-II) of a SCP-II of the heterodimer when H-I and H-II are located on the same end of the Fc region portion of their respective single chain polypeptide.
  • a polypeptide heterodimer may comprise the following two single chain polypeptides: A first chain polypeptide from amino to carboxyl terminus comprises a first binding domain, CHI, hinge, CH2, and CH3, and a second chain polypeptide from amino to carboxyl terminus comprises a CK, first hinge, CH2, CH3, second hinge, and a second binding domain.
  • the hinge in the first chain would be regarded as "corresponding" to the first hinge of the second chain because both are amino terminal to the Fc region portions to which they are connected.
  • a hinge may be present to link the binding domain with another portion of the single chain polypeptide (e.g. , an Fc region portion or an immunoglobulin heterodimerization domain).
  • a hinge is a non-immunoglobulin hinge (i.e., a hinge not based on or derived from a wild type immunoglobulin hinge) and may be a stalk region of a type II C-lectin or CD molecule, an interdomain region that connect IgV-like or IgC-like domains of a cell surface receptor, or a derivative or functional variant thereof.
  • Exemplary carboxyl terminal hinges sometimes referred to as "back- end" hinges, includes those set forth in SEQ ID NOS:78, 734-737, 742-747, and 766- 790.
  • a hinge of one single chain polypeptide of a polypeptide heterodimer is identical to a corresponding hinge of the other single chain polypeptide of the heterodimer.
  • a hinge of one chain is different from that of the other chain (in their length or sequence). The different hinges in the different chains allow different manipulation of the binding affinities of the binding domains to which the hinges are connected, so that the heterodimer is able to preferentially bind to the target of one binding domain over the target of the other binding domain.
  • a polypeptide heterodimer has a CD3 or TCR binding domain in one chain and a tumor antigen binding domain in another chain.
  • the heterodimer may recruit CD3 + T cells to tumor cells carrying the tumor antigen, which in turn may damage or destroy the tumor cells.
  • a single chain polypeptide that forms a heterodimer with another single chain polypeptide may contain one or more additional domains or regions.
  • additional regions may be a leader sequence (also referred to as "signal peptide") at the amino-terminus for secretion of an expressed single chain polypeptide.
  • leader peptides of this disclosure include natural leader sequences or others, such as those as set forth in SEQ ID NOS: 1 10 and 1 1 1.
  • Additional regions may also be sequences at the carboxy-terminus for identifying or purifying single chain polypeptides (e.g., epitope tags for detection or purification, such as a histidine tag, biotin, a FLAG® epitope, or any combination thereof).
  • epitope tags for detection or purification, such as a histidine tag, biotin, a FLAG® epitope, or any combination thereof.
  • junction amino acids or “junction amino acid residues” having a length of 1 to about 10 amino acids (e.g., about 2 to 5 amino acids), which may be resulted from use of specific expression systems or construct design for the single chain polypeptides of the present disclosure.
  • Such additional amino acid residues may be present at the amino or carboxyl terminus or between various regions or domains of a single chain polypeptide, such as between a binding domain and an immunoglobulin heterodimerization domain, between an immunoglobulin heterodimerization domain and a hinge, between a hinge and an Fc region portion, between domains of an Fc region portion (e.g., between CH2 and CH3 domains or between two CH3 domains), between a binding domain and a hinge, between an Fc region portion and an immunoglobulin heterodimerization domain, or between a variable domain and a linker.
  • a single chain polypeptide such as between a binding domain and an immunoglobulin heterodimerization domain, between an immunoglobulin heterodimerization domain and a hinge, between a hinge and an Fc region portion, between a variable domain and a linker.
  • junction amino acids amino- terminal to a hinge include RDQ (SEQ ID NO:598), RT, SS, SASS (SEQ ID NO:599) and SSS (SEQ ID NO: 600).
  • Exemplary junction amino acids carboxy-terminal to a hinge include amino acids SG. Additional exemplary junction amino acids include SR.
  • junction amino acids are present between an Fc region portion that comprises CH2 and CH3 domains and an immunoglobulin heterodimerization domain (CHI or CL). These junction amino acids are also referred to as a "linker between CH3 and CHI or CL" if they are present between the C- terminus of CH3 and the N-terminus of CHI or CL. Such a linker may be about 2-12 amino acids in length.
  • the Fc region portion comprises human IgGl CH2 and CH3 domains in which the C-terminal lysine residue of human IgGl CH3 is deleted.
  • Exemplary linkers between CH3 and CHI include those set forth in SEQ ID NO:847-849.
  • linkers between CH3 and Ck include those set forth in SEQ ID NOS:850-852 (in which the carboxyl terminal arginine in the linkers may alternatively be regarded as the first arginine of Ck).
  • the presence of such linkers or linker pairs e.g., SEQ ID NO:847 as a CH3-CH1 linker in one single chain polypeptide of a heterodimer and SEQ ID NO:850 as a CH3-Ck linker in the other single chain polypeptide of the heterodimer; SEQ ID NO:848 as a CH3- CHI linker and SEQ ID NO:851 as a CH3-Ck linker; and SEQ ID NO:849 as a CH3- CH1 linker and SEQ ID NO:852 as a CH3-Ck linker) improves the production of heterodimer compared the presence of a reference linker as set forth in SEQ ID NO: 846 (in which the last lysine of CH
  • an immunoglobulin Fc region (e.g., CH2, CH3, and/or CH4 regions) of a polypeptide heterodimer of the present disclosure may have an altered glycosylation pattern relative to an immunoglobulin reference sequence.
  • any of a variety of genetic techniques may be employed to alter one or more particular amino acid residues that form a glycosylation site (see Co et al. (1993) Mol. Immunol. 30: 1361; Jacquemon et al. (2006) J. Thromb. Haemost. 4: 1047; Schuster et al. (2005) Cancer Res. 65:7934; Warnock et al. (2005) Biotechnol. Bioeng.
  • the host cells producing polypeptide heterodimers of this disclosure may be engineered to produce an altered glycosylation pattern.
  • One method known in the art provides altered glycosylation in the form of bisected, non-fucosylated variants that increase ADCC. The variants result from expression in a host cell containing an oligosaccharide-modifying enzyme.
  • the POTELLIGENT® technology of BioWa/Kyowa Hakko is contemplated to reduce the fucose content of glycosylated molecules according to this disclosure.
  • a CHO host cell for recombinant immunoglobulin production modifies the glycosylation pattern of the immunoglobulin Fc region, through production of GDP-fucose.
  • glycosylation pattern of polypeptide heterodimers of this disclosure is altered.
  • a variety of glycosidase and/or mannosidase inhibitors provide one or more of desired effects of increasing ADCC activity, increasing Fc receptor binding, and altering glycosylation pattern.
  • cells expressing polypeptide heterodimers of the instant disclosure are grown in a culture medium comprising a carbohydrate modifier at a concentration that increases the ADCC of immunoglycoprotein molecules produced by said host cell, wherein said carbohydrate modifier is at a concentration of less than 800 ⁇ .
  • the cells expressing these polypeptide heterodimers are grown in a culture medium comprising castanospermine or kifunensine, for instance, castanospermine at a concentration of 100-800 ⁇ , such as 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , or 800 ⁇ .
  • a culture medium comprising castanospermine or kifunensine, for instance, castanospermine at a concentration of 100-800 ⁇ , such as 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , or 800 ⁇ .
  • Methods for altering glycosylation with a carbohydrate modifier such as castanospermine are provided in U.S. Patent No. 7,846,434 or PCT Publication No. WO 2008/052030.
  • two single chain polypeptides are designed so that the immunoglobulin heterodimerization domain of the first single chain polypeptide is properly aligned and interacts with the immunoglobulin heterodimerization domain of the second single chain polypeptide.
  • the heterodimer may comprise a second immunoglobulin heterodimerization domain pair to facilitate or enhance the heterodimerization of the two chains.
  • an Fc region portion in addition to the interaction between the two immunoglobulin heterodimerization domains, an Fc region portion (e.g., a CH3 domain) in the first chain may interact with an Fc region in the second chain to enhance heterodimerization (e.g., via interaction between two wild type CH3 domains or between a CH3 domain pair with "knobs-into-holes" mutations).
  • the hinge in the first chain may interact with the hinge in the second chain (e.g., the same altered human IgGl hinge as set forth in SEQ ID NO:664) to form, for example, disulfide bonds, which may further strengthen the interaction between the first and second single chain polypeptides to form a polypeptide heterodimer of the present disclosure.
  • the first and second chain may comprise a second hinge pair (e.g. , at the carboxyl termini of the two chains) to further enhance the interactions between the two chains.
  • a polypeptide heterodimer of the present disclosure comprises two binding domains (BDl and BD2), wherein the binding domains bind to two different target molecules.
  • the two binding domains (BD l and BD2) are both on the SCP-I with the immunoglobulin heterodimerization domain (HD-I) and the Fc region portion (FRP-1) disposed between BDl and BD2.
  • the first binding domain (BDl) is on the SCP-I
  • the second binding domain (BD2) is on the SCP-II.
  • both BDl and BD2 are amino terminal to the Fc region portion of the SCP-I and SCP- II, respectively.
  • BDl is amino terminal to the Fc region portion of the SCP-I and BD2 is carboxyl terminal to the Fc region portion of the SCP- II. In certain other embodiments, BDl is carboxyl terminal to the Fc region portion of the SCP-I and BD2 is amino terminal to the Fc region portion of the SCP-II. In certain other embodiments, both BDl and BD2 are carboxyl terminal to the Fc region portion of the SCP-I and SCP-II, respectively.
  • a polypeptide heterodimer comprises three binding domains (BDl , BD2 and BD3), wherein the binding domains bind to two or three different target molecules.
  • BDl , BD2, and BD3 each bind a different target, or BDl and BD2 bind a first target while BD3 binds a second target, or BDl and BD3 bind a first target while BD2 binds a second target, or BD2 and BD3 bind a first target while BDl binds a second target.
  • the immunoglobulin heterodimerization domain and the Fc region portion are disposed between BDl and BD2 on SCP-I, and BD3 is amino terminal to the Fc region portion of the SCP-II. In further embodiments, the immunoglobulin heterodimerization domain and the Fc region portion are disposed between BDl and BD2 on SCP-II, and BD3 is carboxyl terminal to the Fc region portion of the SCP-I.
  • a polypeptide heterodimer comprises four binding domains (BDl , BD2, BD3 and BD4), wherein the binding domains bind to two to four different target molecules.
  • the immunoglobulin heterodimerization domain and Fc region portion of the SCP-I are disposed between BDl and BD2, and the immunoglobulin heterodimerization domain and Fc region portion of SCP-II are disposed between BD3 and BD4.
  • a polypeptide heterodimer comprises five binding domains (BD l , BD2, BD3, BD4 and BD5), wherein the binding domains bind to two to four different target molecules.
  • SCP-I comprises three binding domains (BDl , BD2 and BD3), and SCP-II comprises two binding domains (BD4 and BD5).
  • BDl and BD2 may be, for example, linked in tandem to each other (either directly or via a peptide linker of about two to eight amino acids) at the amino (or carboxyl) terminus of SCP-I with BD3 located at the carboxyl (or amino) terminus of SCP-I or SCP-II, wherein BD4 and BD5 are on SCP-II if BD3 is on SCP-I, or BD4 or BD5 are on SCP-I if BD3 is on SCP-II.
  • the polypeptide heterodimer comprises six binding domains (BD1-BD6).
  • SCP-I and SCP-II each comprise three binding domains (e.g., BD1-BD3 on SCP-I, and BD4-BD6 on SCP-II).
  • BDl and BD2 may be linked in tandem and located at the SCP-I amino (or carboxyl) terminus, and BD3 may be located at the SCP-I carboxyl (or amino) terminus.
  • BD4 and BD5 may be linked in tandem and located at the SCP-II amino (or carboxyl) terminus, and BD6 may be located at the SCP-II carboxyl (or amino) terminus.
  • the first single chain polypeptide (SCP-I) comprises four binding domains (BD1-BD4) and the second single chain polypeptide (SCP-II) comprises two binding domains (BD5 and BD6).
  • BD 1 and BD2 may be linked in tandem and located at or near amino terminus of SCP-I
  • BD3 and BD4 may be linked in tandem and located at or near carboxyl terminus of SCP-I
  • BD5 and BD6 may be located at or near the amino and carboxyl termini of SCP-II, respectively.
  • the polypeptide heterodimer comprises seven binding domains (BD 1-BD7).
  • SCP-I may comprise four binding domains (BD1-BD4), and SCP-II may comprise the other three binding domains (BD5- BD7).
  • BD1 and BD2 may be linked in tandem and located at or near the amino terminus of SCP-I
  • BD3 and BD4 may be linked in tandem and located at or near the carboxyl terminus of SCP-II
  • BD5 and BD6 may be linked in tandem and located at or near the amino (or carboxyl) terminus of SCP-II
  • BD7 may be located at or near the carboxyl (or amino) terminus of SCP-II.
  • the polypeptide heterodimer comprises eight binding domains (BD1-BD8).
  • the first and second single chain polypeptides may each comprise four binding domains.
  • two binding domains may be located at or near the amino terminus, and the other two binding domains located at or near the carboxyl terminus.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CL region (e.g., CK, CX), a hinge, and an Fc region portion.
  • a CL region e.g., CK, CX
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, and a CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a second CHI region; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a second CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a second CHI region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CL region, a second CL region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, and a second CHI region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CL region and a second CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
  • a polypeptide heterodimer is formed from the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a second CHI region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a second CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CHI region, a hinge, and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, and a CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, and a second CL region; and a second single chain polypeptide comprising a second binding domain, a CHI region, a hinge, an Fc region portion, and a second CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a second CL region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CHI region, a second CHI region, a hinge, and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, and a second CL region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CHI region, and a second CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second CHI region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a second CL region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a second CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second CHI region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a second binding domain, a CHI region, a hinge, an Fc region portion, and a CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a CL region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CL region, a CHI region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a CHI region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CHI region, a CL region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region and a CL region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CL region and a CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region and a CHI region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CHI region and a CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a CL region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a CHI region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a CHI region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CL region, a hinge, and an Fc region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CHI region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge and an Fc region portion; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge and a first binding domain; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a second hinge; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a second hinge.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain; and the second single chain polypeptide comprising a hinge, an Fc region portion, and a CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge and a first binding domain; and the second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, and a CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, and a second hinge; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CL region and a second hinge.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge an Fc region portion, a CL region, a second hinge and a second binding domain; and the second single chain polypeptide comprising a CL region, a hinge, an Fc region portion and a CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region, a CHI region, a second hinge and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CL, a second hinge and a first binding domain; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second CHI region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, and a second CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, and a second CHI region; and a second single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second CL region, a second hinge, and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a second CHI region; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second CL region, a second hinge and a second binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a second CL region.
  • Exemplary embodiments (51) to (60) are also provided below in which three or four binding domains are included in each heterodimer. Additional binding domains may be included to make polypeptide heterodimers that comprise five to eight binding domains according to the present disclosure in view of the general description provided herein.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a CL region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge and a third binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second hinge and a second binding domain; and the second single chain polypeptide comprising a third binding domain, a CHI region, a hinge and an Fc region portion.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second hinge and a second binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge and a third binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge and a second binding domain; and the second single chain polypeptide comprising a third binding domain, a hinge, an Fc region portion and a CL region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge, and a third binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a hinge, an Fc region portion and a CHI region.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge, and a third binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus: a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a CL region, a hinge, an Fc region portion, a second hinge and a fourth binding domain.
  • a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a hinge, an Fc region portion, a CL region, a second hinge and a fourth binding domain.
  • a polypeptide heterodimer of the present disclosure comprises the following two single chain polypeptides: a first single chain polypeptide comprising a first binding domain (BD1) that specifically binds to a T cell target (e.g., a TCR complex or a component thereof, including TCRa, TCRP, CD3y, CD35, and CD3s), a hinge that is an SCC-P IgGl hinge, an Fc region portion that is a wild type or altered human IgGl CH2CH3, and a CL region that is wild type or an altered human Ck region with N30Y V55A T70E (YAE) substitutions; and a second single chain polypeptide comprising binding domain (BD2) that specifically binds to a B-cell target (e.g.
  • a tumor or cancer antigen e.g., RON, c-Met, EpCAM, CEACAM-6, PSMA
  • a hinge that is also an SCC-P IgGl hinge, an Fc region portion that is a wild type or altered human IgGl CH2CH3, and a CHI region that is human CHI region.
  • a polypeptide heterodimer of embodiments (1) to (32) may further comprise a third binding domain (BD3) that is identical to BDl or BD2, and is linked to a single chain polypeptide via a second hinge (e.g., linker H75 or H68 as set forth in SEQ ID NOS:742 and 78, respectively).
  • a polypeptide heterodimer of embodiment of embodiments (1) to (32) may further comprise third and fourth binding domains (BD3) and (BD4) that each are identical or different to BDl or BD2 and linked to the single chain polypeptide(s) via a second hinge (e.g., linker H75 or H68 as set forth in SEQ ID NOS:742 and 78).
  • the polypeptide heterodimers of the present disclosure can be engineered to have binding domains with different affinities to target specific cell types. For example, it may be desirable for a polypeptide heterodimer with a binding domain for a TCR complex or a component thereof and another binding domain for a tumor antigen (or a B-cell target) to preferentially bind tumor cells having the tumor antigen (or B-cells carrying the B-cell target) with a higher affinity so that the polypeptide heterodimer will bind the tumor cells (or the B-cells) first and then recruit T cells via its TCR/CD3 binding domain to the tumor site or cells.
  • Differential binding affinities may be achieved by, for example, choosing a binding domain for one target with a higher binding affinity than the other binding domain has for its target or by including multiple binding domains for one target on a polypeptide heterodimer and a single binding domain or fewer binding domains for the second or other targets.
  • using different hinges e.g., using hinges of different lengths
  • binding domains may need to be located at an appropriate distance from each other so that their interactions with their targets will produce a desirable effect.
  • a polypeptide heterodimer comprising a binding domain for a TCR complex or a component thereof in a single chain polypeptide and another binding domain for a tumor antigen or a B-cell target in a second single chain polypeptide may have both binding domains at the amino or carboxyl termini of their corresponding chains so that they are in physical proximity to each other in the polypeptide heterodimer.
  • Exemplary heterodimers may be formed from single chain polypeptide pairs described herein. If sequence identification numbers noted herein contain signal peptide sequences (e.g., the first 20 amino acids), such signal peptide sequences are not part of the mature single chain polypeptides that form the exemplary polypeptide heterodimers and thus should be considered excluded.
  • Exemplary single chain polypeptides are set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 29-32, 53-72, 74, 810-826, 859-864, and 874-882.
  • heterodimers may be formed from the following single chain polypeptide pairs: SEQ ID NOS:2 and 4, SEQ ID NOS:6 and 8, SEQ ID NOS: 10 and 12, SEQ ID NOS: 14 and 16, SEQ ID NOS: 18 and 20, SEQ ID NOS:20 and 22, SEQ ID NOS:20 and 24, SEQ ID NOS:30 and 32, SEQ ID NOS:29 and 31 , SEQ ID NOS:29 and 32, SEQ ID NOS:30 and 72, SEQ ID NOS:53 and 72, SEQ ID NOS:54 and 72, SEQ ID NOS:55 and 72, SEQ ID NOS:70 and 72, SEQ ID NOS:71 and 72, SEQ ID NOS:63 and 56, SEQ ID NOS:64 and 57, SEQ ID NOS:65 and 60, SEQ ID NOS:66 and 58, SEQ ID NOS:67 and 59, SEQ ID NOS:68 and 61 , SEQ ID NOS:69 and 62, SEQ ID NOS:
  • the present disclosure also provides isolated nucleic acid (used interchangeably with "polynucleotide”) molecules that encode single chain polypeptides provided herein.
  • isolated nucleic acid molecules (either with or without a nucleotide sequence encoding a signal peptide sequence) are set forth in SEQ ID NO: 1
  • ID NOS: l 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25-28, 33-52 and 792-808.
  • vectors that comprise nucleic acid sequence encoding single chain polypeptides provided herein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • exemplary vectors include plasmids, yeast artificial chromosomes, and viral genomes. Certain vectors can autonomously replicate in a host cell, while other vectors can be integrated into the genome of a host cell and thereby are replicated with the host genome.
  • the vectors may be recombinant expression vectors.
  • “Recombinant expression vectors” or “expression vectors” refer to vectors that contain nucleic acid sequences that are operatively linked to an expression control sequence (e.g., a promoter) and are thus capable of directing the expression of those sequences.
  • Promoter sequences useful in expression vectors provided herein can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. In certain embodiments, the promoters are inducible promoters.
  • a vector is an expression vector that comprises a nucleic acid sequence encoding a first single chain polypeptide of a polypeptide heterodimer provided herein. In certain other embodiments, a vector is an expression vector that comprises a nucleic acid sequence encoding a second single chain polypeptide of a polypeptide heterodimer provided herein.
  • a vector is an expression vector that comprises nucleic acid sequences encoding both first and second single chain polypeptides of a polypeptide heterodimer.
  • the promoter for the nucleic acid sequence encoding the first single chain polypeptide may be the same as the promoter for the nucleic acid encoding the second single chain polypeptide.
  • the promoter for the nucleic acid sequence encoding the first single chain polypeptide may be different from the promoter for the nucleic acid encoding the second single chain polypeptide so that the expression level of the first and second single chain polypeptides may be differentially modulated to maximum heterodimerization of the first and second single chain polypeptides.
  • one or both the promoters for the nucleic acid encoding the first and second single chain polypeptides are inducible promoters.
  • the present disclosure also provides a host cell transformed or transfected with, or otherwise containing, any of the nucleic acids or vectors provided herein.
  • exemplary host cells include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see US Patent Application Publication No.
  • COS cells such as COS-7
  • W138 BHK, HepG2, 3T3, RIN, MDCK, A549, PC 12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomyces cerevisiae cells, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or members of the Streptomycete family.
  • a host cell comprises a first expression vector containing a nucleic acid encoding a first single chain polypeptide and a second expression vector containing a nucleic acid encoding a second single chain polypeptide.
  • a host cell comprises an expression vector containing a nucleic acid encoding both first and second single chain polypeptides.
  • the disclosure also includes a method of producing polypeptide heterodimers described herein.
  • the method comprises culturing a host cell that comprises nucleic acids encoding both the first and second single chain polypeptides under conditions suitable to express the polypeptides, and optionally isolating or purifying the heterodimers formed from the first and second single chain polypeptides from the culture.
  • the nucleic acid encoding the first single chain polypeptide and the nucleic acid encoding the second single chain polypeptide may be present in a single expression vector in the host cell or in two different expression vectors in the host cells. In the latter case, the ratio between the two expression vectors may be controlled to maximize heterodimerization of the first and second single chain polypeptides.
  • the present disclosure provides purified polypeptide heterodimers as described herein.
  • purified refers to a composition, isolatable from other components, wherein the polypeptide heterodimer is enriched to any degree relative to its naturally obtainable state.
  • substantially purified polypeptide heterodimers refers to a polypeptide heterodimer composition in which the polypeptide heterodimer forms the major component of the composition, such as constituting at least about 50%, such as at least about 60%, about 70%>, about 80%>, about 90%), about 95%, about 99%, of the polypeptides, by weight, in the composition.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the polypeptide and non-polypeptide fractions. Further purification using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) is frequently desired. Analytical methods particularly suited to the preparation of a pure fusion protein are ion-exchange chromatography, size exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Particularly efficient methods of purifying peptides are fast protein liquid chromatography and HPLC. Various methods for quantifying the degree of purification are known to those of skill in the art in light of the present disclosure. These include, for example, assessing the amount of polypeptide heterodimers in a fraction by SDS/PAGE analysis and HPLC as illustrated in the examples provided herein.
  • the method for making polypeptide heterodimers provided herein is advantageous over a method for first expressing and purifying separately individual single chain polypeptides and then incubating purified individual single chain polypeptides together to form polypeptide heterodimers.
  • certain single chain polypeptides e.g., certain polypeptides containing only CHI regions as their immunoglobulin heterodimerization domains
  • separate expression and purification of individual single chain polypeptides followed by combining the purified individual single chain polypeptides involve more steps than coexpressing both single chain polypeptides followed by purifying resulting polypeptide heterodimers and generally less efficient.
  • the present disclosure also provides pharmaceutical compositions and unit dose forms that comprise the polypeptide heterodimers as well as methods for using the polypeptide heterodimers, the pharmaceutical compositions and unit dose forms.
  • compositions of polypeptide heterodimers of this disclosure generally comprise a polypeptide heterodimer provided herein in combination with a pharmaceutically acceptable excipient, including pharmaceutically acceptable carriers and diluents.
  • pharmaceutically acceptable excipients will be nontoxic to recipients at the dosages and concentrations employed. They are well known in the pharmaceutical art and described, for example, in Rowe et al., Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5 th Ed., 2006.
  • Pharmaceutically acceptable carriers for therapeutic use are also well known in the pharmaceutical art, and are described, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro (Ed.) 1985).
  • Exemplary pharmaceutically acceptable carriers include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes and the like may be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may also be used.
  • compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates ⁇ e.g., glucose, sucrose, dextrins), chelating agents (e.g., EDTA), glutathione and other stabilizers and excipients.
  • diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates ⁇ e.g., glucose, sucrose, dextrins), chelating agents (e.g., EDTA), glutathione and other stabilizers and excipients.
  • Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary diluents.
  • the product may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as
  • the present disclosure also provides a method for treating a disease or disorder associated with, for example, excessive receptor-mediated signal transduction, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer comprising a binding domain that specifically binds a receptor, such as a receptor tyrosine kinase, including EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGF1R, c-Met, RON, and EphA2.
  • a receptor tyrosine kinase including EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGF1R, c-Met, RON, and EphA2.
  • Exemplary diseases or disorders associated with excess receptor- mediated signal transduction include cancer (e.g., solid malignancy and hematologic malignancy), autoimmune or inflammatory diseases or conditions, sepsis resulting from bacterial infection, and viral infection.
  • the present disclosure provides a method for directing T cell activation, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s or a combination thereof, and a second binding domain that specifically binds a different target, for instance, a tumor-specific antigen or other antigen of choice at a site or cell where T cell activation is desired.
  • a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s or a combination thereof, and a second binding domain that specifically binds a different target, for instance, a tumor-specific antigen or other antigen of choice at a site or cell where T cell activation is desired.
  • the present disclosure provides a method for inhibiting growth, metastasis or metastatic growth of a malignancy (e.g. , a solid malignancy or a hematologic malignancy), comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer provided herein or a composition thereof.
  • a malignancy e.g. , a solid malignancy or a hematologic malignancy
  • cancers including solid malignancy and hematologic malignancy, are amenable to the compositions and methods disclosed herein.
  • Types of cancer that may be treated include, but are not limited to: adenocarcinoma of the breast, prostate, pancreas, colon and rectum; all forms of bronchogenic carcinoma of the lung (including squamous cell carcinoma, adenocarcinoma, small cell lung cancer and non- small cell lung cancer); myeloid; melanoma; hepatoma; neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoid heart disease; and carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronch
  • cancers include: histiocytic disorders; leukemia; histiocytosis malignant; Hodgkin's disease; immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma; odontoma; teratoma;
  • cancers are also contemplated as amenable to treatment: adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma;
  • the types of cancers that may be treated also include, but are not limited to, angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing; angiomatosis; glomangioma; hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms; ner
  • B-cell cancers including B-cell lymphomas [such as various forms of Hodgkin's disease, non-Hodgkins lymphoma (NHL) or central nervous system lymphomas], leukemias [such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myoblastic leukemia] and myelomas (such as multiple myeloma).
  • B-cell lymphomas such as various forms of Hodgkin's disease, non-Hodgkins lymphoma (NHL) or central nervous system lymphomas
  • leukemias such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myoblastic leukemia
  • myelomas such as multiple myeloma
  • Additional B cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, B-cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post- transplant lymphoproliferative disorder.
  • MALT mucosa-associated lymphoid tissue
  • MALT mu
  • polypeptide heterodimers useful for inhibiting growth of a solid malignancy or metastasis or metastatic growth of a solid malignancy or a hematologic malignancy include those that specifically bind to a tumor or cancer antigen and a T cell target.
  • Such heterodimers are typically designed to have a higher binding affinity to the tumor or cancer antigen (e.g., at least about 5, 10, 15, 20, 25, 50, 75 or 100 folds higher) than to the T cell target so that they preferentially bind to the tumor or cancer antigen first and subsequently bind to the T cell target, which in turn recruit and activate T cells to damage or destroy the tumor or cancer cells carrying the tumor or cancer antigen on their surface.
  • Exemplary tumor or cancer antigens and T cell targets include those provided above in the section describing binding domains of polypeptide heterodimers.
  • a T cell target can be CD3 or PSMA.
  • polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy, or by directed T cell activation comprise at least one binding domain that specifically binds an oncology target (including a tumor or cell antigen, such as RON, c-Met, CEACAM-6, or PSMA) and another binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3y, CD35, and CD3s).
  • an oncology target including a tumor or cell antigen, such as RON, c-Met, CEACAM-6, or PSMA
  • TCR complex or a component thereof e.g., TCRa, TCRp, CD3y, CD35, and CD3s.
  • polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise at least one binding domain that specifically binds CD28 and another binding domain that specifically binds CD79b.
  • Such polypeptide heterodimers may further comprise a binding domain that comprises a PDL2 ectodomain, a binding domain that specifically binds hyperIL-6, or both binding domains.
  • polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise at least one binding domain that specifically binds RON and another binding domain that specifically binds c-Met.
  • polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise binding domains that specifically bind one or more of the following receptor tyrosine kinases: c- Met, RON, EGFR, EGFRvIII, Her2, ErbB3, ErbB4, and IGF1R, EphA2.
  • the heterodimers may further comprise one or more binding domains that specifically bind to one or more of the following antiangiogenic agents: PDGFR, VEGFR1-4, and angiopoietin 2.
  • the above heterodimers may further comprise one or more binding domains that specifically bind to one or more the following Fc receptors to increase targeting of cytotoxic effector function: CD64, CD32A and CD 16.
  • the above heterodimers may further comprise a binding domain that specifically binds the transferrin receptor (CD71) to enable degradation of the receptors.
  • polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise binding domains that are agonists of two or more of the following TNFSFRs: TNFR1 , TNFR2,
  • polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise binding domains that specifically bind two or more of the following pro-oncogenic cytokines or growth factors: HGF, MSP, EGF (including epiregulin, herregulin, ⁇ -regulin, neuregulin), HIF- ⁇ , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, IL-6, hyperIL-6, IL-
  • polypeptide heterodimers useful for inhibiting growth of a solid malignancy or metastasis or metastatic growth of a solid malignancy include those that specifically bind to, for example, EGFR, ErbB3, ErbB4, c-Met, RON,
  • EphA2 IGF1R, VEGFR1 , VEGFR2, VEGFR3, CD44v6, CD151 , EpCAM,
  • CEACAM6 TGFBR2, , GHRHR, GHR, IL-6R, gpl30, TNFR2, PD1 , TWEAK-R,
  • Polypeptide heterodimers useful for inhibiting metastasis or metastatic growth of a hematologic malignancy include those that specifically bind to, for example, EGFR, ErbB3, c-Met, RON, EphA2, IGF1R, TGFBR2, IL-6R, gpl 30,
  • TNFR2 TNFR2, PD1 , OSMRP, LTpR, CD 19, CD80, CD81 , or CD86.
  • the present disclosure provides a method for treating an autoimmune or inflammatory disease, disorder or condition, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer provided herein or a composition thereof.
  • Exemplary autoimmune or inflammatory diseases, disorders or conditions that may be treated by the fusion proteins and compositions and unit dose forms thereof include, and are not limited to, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), diabetes mellitus (e.g., type I diabetes), dermatomyositis, polymyositis, pernicious anaemia, primary biliary cirrhosis, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, lupus nephritis, neuropsychiatric lupus, multiple sclerosis (MS), myasthenia grav
  • Exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprises one or more binding domain that specifically binds CD28, and one, two, or three of the following additional binding domains: a binding domain that comprises PDL2 ectodomain, a binding domain that comprises monoIL-10, and a binding domain that specifically binds CD86.
  • a polypeptide heterodimer may comprise a binding domain that specifically binds CD28 and one to three binding domains that specifically bind CD86.
  • Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise one or more binding domains that specifically bind CD28 and one or more binding domains that are an IL-10 agonist (e.g., monoIL-10), an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
  • an IL-10 agonist e.g., monoIL-10
  • polypeptide heterodimers may comprise two or more binding domains selected from an IL-10 agonist (e.g., monoIL-10), an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
  • an IL-10 agonist e.g., monoIL-10
  • HLA-G agonist e.g., monoIL-10
  • HGF agonist e.g., HGF
  • IL-35 agonist e.g., IL-35
  • a PD-1 agonist e.g., a BTLA agonist
  • LIGHT antagonist e.g., BTLA agonist
  • GITRL antagonist e.g., BTLA agonist
  • Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise one or more binding domains that specifically bind CD32B and one or more binding domains that are an IL-10 agonist (e.g., monoIL-10), an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
  • an IL-10 agonist e.g., monoIL-10
  • Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise binding domains that specifically bind one or more of the following TNFSFRs: TNFR1 , TNFR2, HVEW, LTpR, TWEAKR, TACI, BAFF-R, BCMA, or FAS.
  • Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise binding domains that specifically bind two or more of the following pro-inflammatory cytokines/chemokines, such as TNFa, IL-6, IL-2, IL-1 , IL-8, IP- 10, IFNy, IFNa, RANKL, FASL, TGFp, IL7, IL10, IL17A/F, TWEAK, CSF2, IGF1 , IGF2 or BLyS/APRIL.
  • pro-inflammatory cytokines/chemokines such as TNFa, IL-6, IL-2, IL-1 , IL-8, IP- 10, IFNy, IFNa, RANKL, FASL, TGFp, IL7, IL10, IL17A/F, TWEAK, CSF2, IGF1 , IGF2 or BLyS/APRIL.
  • Polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition include those that specifically bind to, for example, TGFBR2, IL-6R, gpl30, TNFR1 , TNFR2, PD1 , HVEM, OX40, CD40, CD137, TWEAK-R, LTpR, LIFRp, OSMRp, CD3, TCRa, TCRp, CD19, CD28, CD80, CD81 , CD86, TLR7, TLR9, or any combination thereof.
  • the present disclosure provides a method for treating a B-cell associated disorder or disease that comprises administering to a patient in need thereof (e.g. , a patient having or suspected of having a B-cell associated disorder or disease) an effective amount of a polypeptide heterodimer provided herein, such as those that specifically bind a B cell target.
  • Polypeptide heterodimers useful in treating a B-cell associated disorder or disease may comprise binding domains that specifically bind one or more B-cell targets, such as CD79b, CD19, HLA-DR, CD20, CD21 , CD22, CD30, CD33, CD37, CD38, or CD70.
  • the polypeptide heterodimers may further comprise a binding domain that specifically binds a T-cell target, such as a TCR complex or a component thereof, including TCRa, TCRp, CD3y, CD35, or CD3s.
  • Polypeptide heterodimers useful in treating a B-cell associated disorder or disease may comprise a binding domain that specifically bind a B-cell target, such as CD79b, CD19, HLA-DR, CD20, CD21, CD22, CD30, CD33, CD37, CD38, and CD70, and a binding domain that specifically binds CD64, CD32A, or CD 16 to increase targeting of cytotoxic effector function.
  • a B-cell target such as CD79b, CD19, HLA-DR, CD20, CD21, CD22, CD30, CD33, CD37, CD38, and CD70
  • CD64, CD32A, or CD 16 to increase targeting of cytotoxic effector function.
  • polypeptide heterodimers or compositions thereof of the present disclosure may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection, or any combination thereof.
  • the polypeptide heterodimers or compositions thereof are administered parenterally.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site is contemplated as well.
  • the invention includes administering polypeptide heterodimers or compositions thereof by intravenous injection.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.01 mg/kg and 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10-50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg) body weight (which can be administered as a single dose, daily, weekly, monthly, or at any appropriate interval) of active ingredient may be administered depending on the potency of a polypeptide heterodimer of this disclosure.
  • an amount between 0.01 mg/kg and 1000 mg/kg e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10-50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg
  • body weight which can be administered
  • polypeptide heterodimers or compositions thereof in combination with a second agent.
  • a second agent may be one accepted in the art as a standard treatment for a particular disease state or disorder, such as in cancer, inflammation, autoimmunity, and infection.
  • Exemplary second agents contemplated include polypeptide heterodimers that bind to targets different from those that primary polypeptide heterodimers bind, polyclonal antibodies, monoclonal antibodies, immunoglobulin-derived fusion proteins, chemotherapeutics, ionizing radiation, steroids, NSAIDs, anti-infective agents, or other active and ancillary agents, or any combination thereof.
  • a polypeptide heterodimer and a second agent act synergistically.
  • these two compounds interact such that the combined effect of the compounds is greater than the sum of the individual effects of each compound when administered alone (see, e.g., Berenbaum, Pharmacol. Rev. 41 :93, 1989).
  • a polypeptide heterodimer and a second agent act additively.
  • these two compounds interact such that the combined effect of the compounds is the same as the sum of the individual effects of each compound when administered alone.
  • Second agents useful in combination with polypeptide heterodimers or compositions thereof provided herein may be steroids, NSAIDs, mTOR inhibitors (e.g., rapamycin (sirolimus), temsirolimus, deforolimus, everolimus, zotarolimus, curcumin, farnesylthiosalicylic acid), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), antimetabolites (e.g., mycophenolic acid, mycophenolate mofetil), polyclonal antibodies (e.g., anti-thymocyte globulin), monoclonal antibodies (e.g., daclizumab, basiliximab), and CTLA4-Ig fusion proteins (e.g., abatacept or belatacept).
  • mTOR inhibitors e.g., rapamycin (sirolimus), temsirolimus, deforolimus, everolimus, zo
  • Second agents useful for inhibiting growth of a solid malignancy, inhibiting metastasis or metastatic growth of a solid malignancy, or treating or ameliorating a hematologic malignancy include chemotherapeutic agents, ionizing radiation, and other anti-cancer drugs.
  • chemotherapeutic agents contemplated as further therapeutic agents include alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil); bifunctional chemotherapeutics (e.g., bendamustine); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU)); ethyleneimines and methyl-melamines (e.g., triethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), and hexamethylmelamine (HMM, altretamine)); alkyl sulfonates (e.g., buslfan); and triazines (e.g., dacabazine (DTIC)); antimetabolites, such as folic acid analogues (e.g., methotrexate, trimetrexate, and
  • second agents useful for inhibiting growth metastasis or metastatic growth of a malignancy include polypeptide heterodimers according to the present disclosure that bind to cancer cell targets other than the target that the first polypeptide heterodimer binds.
  • second agents useful for such treatments include polyclonal antibodies, monoclonal antibodies, and immunoglobulin-derived fusion proteins that bind to cancer cell targets. Exemplary cancer cell targets are provided above in the context of describing targets of polypeptide heterodimers useful for the above-noted treatment.
  • Immunosuppressive agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) for the treatment of arthritis, or biologic response modifiers.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs disease-modifying antirheumatic drugs
  • Compositions in the DMARD description are also useful in the treatment of many other autoimmune diseases aside from rheumatoid arthritis.
  • Exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as Vioxx and Celebrex, and sialylates.
  • Exemplary analgesics are chosen from the group consisting of acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride.
  • Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.
  • Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g. etanercept (Enbrel), adalimumab (Humira) and infliximab (Remicade)), chemokine inhibitors and adhesion molecule inhibitors.
  • the biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules.
  • Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofm) and intramuscular) and minocycline.
  • Additional second agents useful for treating an autoimmune or inflammatory disease, disorder or condition may be a polyclonal or monoclonal antibody, an immunoglobulin-derived fusion protein (e.g., scFv, SMIPTM, PIMS, SCORPIONTM fusion proteins), or a polypeptide heterodimer according to the present disclosure that specifically bind a target associated with such a disease, disorder or condition.
  • an immunoglobulin-derived fusion protein e.g., scFv, SMIPTM, PIMS, SCORPIONTM fusion proteins
  • examples of such targets are provided above in the context of targets of polypeptide heterodimers of the present disclosure useful in the above-noted treatment.
  • binding molecule composition and the second active agent may be given simultaneously in the same formulation.
  • the second agents may be administered in a separate formulation but concurrently (i.e., given within less than one hour of each other).
  • the second active agent may be administered prior to administration of a polypeptide heterodimer or a composition thereof.
  • Prior administration refers to administration of the second active agent at least one hour prior to treatment with the polypeptide heterodimer or the composition thereof.
  • the active agent may be administered subsequent to administration of the binding molecule composition. Subsequent administration is meant to describe administration at least one hour after the administration of the polypeptide heterodimer or the composition thereof.
  • This disclosure contemplates a dosage unit comprising a pharmaceutical composition of this disclosure.
  • dosage units include, for example, a single-dose or a multi-dose vial or syringe, including a two-compartment vial or syringe, one comprising the pharmaceutical composition of this disclosure in lyophilized form and the other a diluent for reconstitution.
  • a multi-dose dosage unit can also be, e.g., a bag or tube for connection to an intravenous infusion device.
  • This disclosure also contemplates a kit comprising a pharmaceutical composition of this disclosure in unit dose, or multi-dose, container, e.g., a vial, and a set of instructions for administering the composition to patients suffering a disorder such as a disorder described above.
  • Polypeptide heterodimer X0172 was made by co-expressing single chain polypeptides X0130 (2E12 CHI CH2 CH3 Ck) and X0168 (Ck CH2 CH3 CHI H68 2E12).
  • Single chain polypeptide X0130 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and altered human Ck (without the first Arg or the last Cys).
  • the nucleic acid and amino acid sequences of XI 30 are set forth in SEQ ID NOS: l and 2, respectively.
  • Single chain polypeptide X0168 comprises, from its amino to carboxyl terminus: altered human Ck (without the first Arg or the last Cys), human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, human IgGl CHI, H68 linker, and 2E12 (anti-CD28) scFv.
  • the nucleic acid and amino acid sequences of X0168 are set forth in SEQ ID NOS:3 and 4, respectively.
  • the amino acid sequence of H68 linker is set forth in SEQ ID NO:78.
  • polypeptide heterodimer X0124 was made by co-expressing single chain polypeptide X0112 (2E12 CHI CH2 CH3) and X0113 (Ck CH2 CH3).
  • Single chain polypeptide X0112 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2, and human IgGl CH3.
  • the nucleic acid and amino acid sequences of X0112 are set forth in SEQ ID NOS:5 and 6, respectively.
  • Single chain polypeptide X0113 comprises, from its amino to carboxyl terminus: altered human Ck (without the first Arg or the last Cys), human IgGl SCC-P hinge, human IgGl CH2, and human IgGl CH3.
  • the nucleic acid and amino acid sequence of X0113 are set forth in SEQ ID NOS:7 and 8, respectively.
  • HEK293 cells were suspended at a cell concentration of 0.5xl0 6 cells/ml in Freestyle 293 expression medium (Gibco). For a large transfection, 250 ml of cells were used, but for a small transfection, 60 ml of cells were used. On the transfection day, 320 ul of 293fectin reagent (Invitrogen) was mixed with 8 ml of media. At the same time, 250 ug of DNA for each of the two chains were also mixed with 8 ml of media and incubated for 5 minutes.
  • Freestyle 293 expression medium Gibco
  • 293fectin reagent Invitrogen
  • the DNA-293fectin mixture was added to the 250ml of 293 cells and returned to the shaker at 37°C and shaken at a speed of 120 RPM.
  • a fourth of the DNA, 293fectin and media were used.
  • Protein A affinity chromatography was used to purify the proteins. 2 mL of packed protein A agarose (Repligen) was added to a Biorad column (Econo-column chromatography column, size 2.5 x 10 cm), washed extensively with PBS (lOx column volume) and the supernatants were loaded, washed with PBS again and eluted with 3 column volumes of Pierce IgG elution buffer. Proteins were then dialyzed extensively against PBS. Proteins were then concentrated using Amicon centrifugal filter devices to a final volume of around 0.5 mL.
  • Protein L affinity chromatography or cation exchange chromatography were used.
  • protein A purified Polypeptide heterodimer was passed over a Protein L agarose column that had been pre- equilibrated with PBS, washed with PBS (lOx column volume) and then eluted with Pierce IgG elution buffer. Proteins were then dialyzed against PBS extensively and concentrated using Amicon centrifugal filter devices to a final volume of around 0.5 mL.
  • Proteins purified were analyzed on a 10 % SDS-PAGE gel using Invitrogen's X-cell Surelock gel box.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • RPMI media Gibco- Invitrogen, Carlsbad, CA
  • CD4+ T-cells were then enriched from the PBMC using negative selection with a MACS CD4+ T-cell Isolation Kit (Miltenyi Biotec, Auburn, CA). The enriched (>95%) CD4+ T-cells were then resuspended at a concentration of lxl 0 6 cell/ml in complete RPMI/10% FCS.
  • Test reagents were prepared at 40 ug/ml (yielding a final concentration of 10 ⁇ g/ml) in complete RPMI/10% FCS and added in 50 ⁇ /well to flat-bottom 96-well plates (BD Falcon, San Jose, CA). PMA (Phorbol 12 myristate 13-acetate; A.G. Scientific, Inc., San Diego, CA) in complete RPMI/10% FCS was added in 50 ul/well at 4 ng/ml (final concentration of 1 ng/ml).
  • T-cells in complete RPMI/10% FCS were added at a concentration of 5 x 10 4 cells/well in a 50 ⁇ volume, and finally an appropriate amount of complete RPMI/10% FCS was added to each well (typically 50 ul) to bring the final volume to 200 ul/well.
  • the cells were treated with the test samples +/- PMA and incubated for 72 hours at 37°C in 5% C0 2 .
  • One microliter of tritiated thymidine (Amersham Biosciences, Pisctaway, NJ) in a 1 :50 dilution of complete RPMI/10% FCS (50 ul/well) was added to the wells for the last 6 hours of culture.
  • Plates were harvested onto a Unifilter-96, GF/C microplate (Perkin Elmer, Boston, MA) with a Packard Filtermate Harvester (Perkin Elmer, Boston, MA). Numbers are expressed as cpm and are the mean of replicate samples.
  • FIG. 2 shows that Polypeptide heterodimer X0172 has a differential property compared to 2E12 SMIP M0039 (SEQ ID NO:77). It did not synergize with PMA as well as the SMIP. This bivalent polypeptide heterodimer has a property closer to the monovalent polypeptide heterodimer, X0124, rather than the bivalent SMIP.
  • Figure 3 shows that bivalent polypeptide heterodimer X0172 bound to CD4 + cells better than 2E12 scFv (SEQ ID NO: 109) and monovalent polypeptide heterodimer X0124.
  • Bivalent polypeptide heterodimer X0251 was made by co-expressing single chain polypeptides X0244 (Ck(YAE) CH2(N297A) CH3 H68 2E12) and X0245 (2E12 CHI CH2(N297A) CH3).
  • Single chain polypeptide X0244 comprises, from its amino to carboxyl terminus: human Ck(YAE) (i.e., human Ck without the first Arg or last Cys but with N30Y, V55A, and T70E substitutions), human IgGl SCC-P hinge, human IgGl CH2 (N297A) (i.e., human IgGl CH2 with a N297A substitution), human IgGl CH3, H68 linker, and 2E12 (anti-CD28) scFv.
  • human Ck(YAE) i.e., human Ck without the first Arg or last Cys but with N30Y, V55A, and T70E substitutions
  • human IgGl SCC-P hinge human IgGl CH2 (N297A) (i.e., human IgGl CH2 with a N297A substitution)
  • human IgGl CH3, H68 linker i.e., human IgGl CH3, H
  • Single chain polypeptide X0245 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2 (N297A), and human IgGl CH3.
  • the nucleotide and amino acid sequences of X0245 are set forth in SEQ ID NOS: l 1 and 12, respectively.
  • Trivalent, bispecific polypeptide heterodimer X0252 was made by co- expressing single chain polypeptides X0246 (P2C2 Ck(YAE) CH2(N297A) CH3) and X0247 (2E12 CHI CH2(N297A) CH3 H68 2E12).
  • Single chain polypeptide X0246 comprises, from its amino to carboxyl terminus: P2C2 (anti-CD79b) scFv, human Ck (YAE), human IgGl SCC-P hinge, human IgGl CH2 (N297A), and human IgGl CH3.
  • the nucleotide and amino acid sequences of X0246 are set forth in SEQ ID NOS: 13 and 14, respectively.
  • Single chain polypeptide X0247 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 2E12 (anti- CD28) scFv.
  • the nucleotide and amino acid sequences of X0247 are set forth in SEQ ID NOS: 15 and 16, respectively.
  • Tetravalent, trispecific polypeptide heterodimer X0253 was made by co- expressing single chain polypeptides X0248 (P2C2 CK(YAE) CH2(N297A) CH3 H68 A2) and X0249 (PDL2 ECD CHI CH2(N297A) CH3 H68 2E12).
  • Single chain polypeptide X0248 comprises, from its amino to carboxyl terminus: P2C2 (anti-CD79b) scFv, human Ck (YAE), human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and A2 (anti-hyperIL-6) scFv.
  • the nucleotide and amino acid sequences of X0248 are set forth in SEQ ID NOS: 17 and 18, respectively.
  • Single chain polypeptide X0249 comprises, from its amino to carboxyl terminus: PDL2 ECD (i.e., PDL2 ectodomain), human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 2E12 (anti-CD28) scFv.
  • PDL2 ECD i.e., PDL2 ectodomain
  • human IgGl CHI human IgGl SCC-P hinge
  • human IgGl CH2 N297A
  • human IgGl CH3, H68 linker and 2E12 (anti-CD28) scFv.
  • the nucleotide and amino acid sequences of X0249 are set forth in SEQ ID NOS: 19 and 20, respectively.
  • Another tetravalent, tetraspecific polypeptide heterodimer X0283 was made by co-expressing single chain polypeptides X0249 (PDL2 ECD CHI CH2(N297A) CH3 H68 2E12) and X0281 (monoIL-10 Ck(YAE) CH2(N297A) CH3 H68 3D1).
  • Single chain polypeptide X0281 comprises, from its amino to carboxyl terminus: monoIL-10, human Ck(YAE), human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 3Dl(anti-CD86) scFv.
  • the nucleotide and amino acid sequences of X0281 are set forth in SEQ ID NOS:21 and 22.
  • polypeptide heterodimer X0283 As a control for polypeptide heterodimer X0283, tetravalent heterodimer, polypeptide heterodimer X0284, was made by co-expressing single chain polypeptides X0249 (PDL2 ECD CHI CH2(N297A) CH3 H68 2E12) and X0282 (monoIL-10 Ck CH2(N297A) CH3 H68 3D1).
  • Single chain polypeptide X0282 is identical to X0281 except that it does not contain N30Y V55A T70E substitutions in the human Ck sequence.
  • single chain polypeptide X0282 comprises, from its amino to carboxyl terminus: monoIL-10, altered human Ck (without the first Arg or the last Cys), human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 3Dl(anti-CD86) scFv.
  • monoIL-10 monoIL-10
  • human Ck without the first Arg or the last Cys
  • human IgGl SCC-P hinge human IgGl CH2 (N297A)
  • human IgGl CH3, H68 linker human IgGl CH3, H68 linker
  • 3Dl(anti-CD86) scFv 3Dl(anti-CD86) scFv.
  • polypeptide heterodimer molecules were expressed and purified according to Example 1. Purified proteins were analyzed using SDS-PAGE electrophoresis under reduced and non-reduced conditions. Size exclusion chromatography was performed on an AKTA Explorer FPLC (Pharmacia Biotech) using a Superdex200 10/300 GL column. Some proteins were analyzed by electrospray mass spectrometry using an Agilent 6120 TOF ES/MS.
  • SPR Surface plasmon resonance
  • a bivalent polypeptide heterodimer with anti-RON binding domains (ORN151) and two bispecific polypeptide heterodimers comprising anti-RON and anti- cMet binding domains (ORN152 and ORN153) were made.
  • Bivalent polypeptide heterodimer ORN151 comprises single chain polypeptides ORN145 (4C04 CH2 CH3 CHI) and ORN148 (11H09 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide ORN145 comprises from its amino to carboxyl terminus: 4C04 (anti-RON) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI .
  • the nucleotide and amino acid sequences of ORN145 are set forth in SEQ ID NOS:26 and 30, respectively.
  • ORN148 comprises from its amino to carboxyl terminus: 11H09 (anti- RON) scFv, human IgGl SCC-P hinge, human CH2, human CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of ORN148 are set forth in SEQ ID NOS:28 and 32, respectively.
  • Bispecific (c-Met, RON) polypeptide heterodimer ORN152 comprises single chain polypeptides ORN116 (MET021 CH2 CH3 CHI) and ORN146 (4C04 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide ORN116 comprises from its amino to carboxyl terminus: MET021 (anti-c-Met) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI .
  • the nucleotide and amino acid sequences of ORN116 are set forth in SEQ ID NOS:25 and 29, respectively.
  • Single chain polypeptide ORN146 comprises from its amino to carboxyl terminus: 4C04 (anti- RON) scFv, human IgGl SCC-P hinge, human CH2, human CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of ORN146 are set forth in SEQ ID NOS:27 and 31, respectively.
  • Bispecific (c-Met, RON) polypeptide heterodimer ORN153 comprises single chain polypeptides ORN116 (MET021 CH2 CH3 CHI) and ORN148 (11H09 CH2 CH3 Ck(YAE)).
  • polypeptide heterodimers S0268, S0269, and TSC020 to TSC030 polypeptide heterodimers S0268, S0269, and TSC020 to TSC030.
  • Polypeptide heterodimer S0268 comprises single chain polypeptides OR 145 (4C04 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC019 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2, human CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC019 are set forth in SEQ ID NOS:52 and 72, respectively.
  • Polypeptide heterodimer S0269 comprises single chain polypeptides ORN160 (5D5 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide OR 160 comprises from its amino to carboxyl terminus: 5D5 (anti-c-Met) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of ORN160 are set forth in SEQ ID NOS:33 and 53, respectively.
  • Bivalent (CD 19) polypeptide heterodimer TSC020 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSCOOl comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSCOOl are set forth in SEQ ID NOS:34 and 54, respectively.
  • Bispecific (CD20, CD3) polypeptide heterodimer TSC021 comprises single chain polypeptides TSC002 (2H7 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC002 comprises from its amino to carboxyl terminus: 2H7 (anti-CD20) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC002 are set forth in SEQ ID NOS:35 and 55, respectively.
  • Bispecific (CD79b, CD3) polypeptide heterodimer TSC022 comprises single chain polypeptides TSC017 (P2C2 H2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC017 comprises from its amino to carboxyl terminus: P2C2 (anti-CD79b) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC017 are set forth in SEQ ID NOS:50 and 70, respectively.
  • Bispecific (HLA-DR, CD3) polypeptide heterodimer TSC023 comprises single chain polypeptides TSC018 (M0042 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC018 comprises from its amino to carboxyl terminus: M0042 (anti-HLA-DR) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC018 are set forth in SEQ ID NOS:51 and 71, respectively.
  • Bivalent polypeptide heterodimer TSC024 comprises single chain polypeptides TSCOIO (HD37-"IgAl hinge”-CH2 CH3 CHI) and TSC003 (G19-4- "IgAl hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSCOIO comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgAl hinge (PSTPPTPSPSTPPTPSCPPCP, SEQ ID NO:752), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSCOIO are set forth in SEQ ID NOS:43 and 63, respectively.
  • Single chain polypeptide TSC003 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgAl hinge (SEQ ID NO:752), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC003 are set forth in SEQ ID NOS:36 and 56, respectively.
  • Bivalent polypeptide heterodimer TSC025 comprises single chain polypeptides TSC011 (HD37-"IgA2 hinge”-CH2 CH3 CHI) and TSC004 (G19-4- "IgA2 hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC011 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgA2 hinge (PPPPPCPPCP, SEQ ID NO:748), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC011 are set forth in SEQ ID NOS:44 and 64, respectively.
  • Single chain polypeptide TSC004 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgA2 hinge (SEQ ID NO:748), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC004 are set forth in SEQ ID NOS:37 and 57, respectively.
  • Bivalent polypeptide heterodimer TSC026 comprises single chain polypeptides TSC012 (HD37-"IgGl hinge”-CH2 CH3 CHI) and TSC007 (G19-4- "IgGl hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC012 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgGl hinge (EPKSSDKTHTSPPSPCPPCP, SEQ ID NO:750), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC012 are set forth in SEQ ID NOS:45 and 65, respectively.
  • Single chain polypeptide TSC007 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgGl hinge (SEQ ID NO:750), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC007 are set forth in SEQ ID NOS:40 and 60, respectively.
  • Bivalent polypeptide heterodimer TSC027 comprises single chain polypeptides TSC013 (HD37-"IgG3 hinge"-CH2 CH3 CHI) and TSC005 (G19-4- "IgG3 hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC013 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgG3 hinge (EPKSSDTPPPSPRSPCPPCP, SEQ ID NO:751), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC013 are set forth in SEQ ID NOS:46 and 66, respectively.
  • Single chain polypeptide TSC005 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgG3 hinge (SEQ ID NO:751), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC005 are set forth in SEQ ID NOS:38 and 58, respectively.
  • Bivalent polypeptide heterodimer TSC028 comprises single chain polypeptides TSC014 (HD37-"IgD hinge”-CH2 CH3 CHI) and TSC006 (G19-4-"IgD hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC014 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, altered human IgD hinge (ESPKAQASSVPTAQPQAEGSLAKATTAPATTR TCPPCP, SEQ ID NO:754), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • TSC014 The nucleotide and amino acid sequences of TSC014 are set forth in SEQ ID NOS:47 and 67, respectively.
  • Single chain polypeptide TSC006 comprises from its amino to carboxyl terminus: G19- 4 (anti-CD3) scFv, altered human IgD hinge (SEQ ID NO:754), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC006 are set forth in SEQ ID NOS:39 and 59, respectively.
  • Bivalent polypeptide heterodimer TSC029 comprises single chain polypeptides TSC015 (HD37-"IgE CH2 hinge”-CH2 CH3 CHI) and TSC008 (G19-4- "IgE CH2 hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC015 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgE CH2 hinge (SEQ ID NO:757), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC014 are set forth in SEQ ID NOS:48 and 68, respectively.
  • Single chain polypeptide TSC008 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgE CH2 hinge (SEQ ID NO:757), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC008 are set forth in SEQ ID NOS:41 and 61, respectively.
  • Bivalent polypeptide heterodimer TSC030 comprises single chain polypeptides TSC016 (HD37-"IgM CH2 hinge”-CH2 CH3 CHI) and TSC009 (G19-4- "IgM CH2 hinge”-CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC016 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgM CH2 hinge (SEQ ID NO:759), human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC015 are set forth in SEQ ID NOS:49 and 69, respectively.
  • Single chain polypeptide TSC009 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgM CH2 hinge (SEQ ID NO:759), human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC009 are set forth in SEQ ID NOS:42 and 62, respectively.
  • Bispecific polypeptide heterodimers S0268 (RON, CD3) and S0269 (CD3, c-Met) were expressed according to Example 1. The following expression levels were obtained: 2.2 ⁇ g protein / mL of culture for S0268 and 2.7 ⁇ g / mL for S0269.
  • bispecific polypeptide heterodimer molecules To compare the effectiveness of bispecific polypeptide heterodimer molecules at targeting a tumor cell antigen and T-cells, the on-cell binding characteristics of an anti-RON (4C04 binding domain) x anti-CD3 (G19-4 binding domain) polypeptide heterodimer, S0268 (as described in Example 4), with a different bispecific scaffold (SCORPIONTM protein) containing the same binding domains, S0266, were compared. Additionally, the on-cell binding characteristics of two bispecific polypeptide heterodimers, TSC020 and TSC021 (as described in Example 4), targeting different B-cell antigens (CD 19 for TSC020 and CD20 for TSC021 ) as well as a T-cell antigen (CD3) were compared.
  • TSC020 and TSC021 as described in Example 4
  • B-cell antigens CD 19 for TSC020 and CD20 for TSC021
  • CD3 T-cell antigen
  • nucleotide and amino acid sequences of SCORPION protein S0266 are set forth in SEQ ID NOS:73 and 74, respectively.
  • Nucleotide and amino acid sequences of the single chain polypeptides making up the bispecific polypeptide heterodimers S0268, TSC020, and TSC021 are set forth in SEQ ID NOS:26 and 52 (nucleotide), 30 and 72 (amino acid); 34 and 52 (nucleotide), 54 and 72 (amino acid); and 35 and 52 (nucleotide), 55 and 72 (amino acid), respectively (see also Example 4).
  • Transient transfection in human 293 cells produced 6.9 ⁇ g protein / mL of culture for S0266; 2.3 ⁇ g / mL of culture for S0268; 3.0 ⁇ g / mL of culture for TSC020; and 3.2 ⁇ g / mL of culture for TSC021.
  • MDA-MB-453 (RON+) breast carcinoma cells, Reel (CD19 + , CD20 + ) mantle cell lymphoma cells, and Jurkat (CD3 ) T cell leukemia cells were obtained from ATCC (Manassas, VA), and cultured according to the provided protocol.
  • T-cells were isolated from donor PBMCs using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany). Non T-cells were separated from PBMCs by being indirectly magnetically labeled with biotin-conjugated monoclonal antibodies and anti-biotin magnetic microbeads. These cells were then depleted by retaining them in a column surrounded by a magnetic field. The T-cells were not retained in the column and were collected in the flow through.
  • Binding was assessed by incubating 5x 10 5 T cells or target (MDA-MB- 453, Reel, Jurkat) cells for 30 minutes at 4°C with serially diluted bispecific molecules S0266 (aRON aCD3 SCORPIONTM protein) or S0268 (aRON aCD3 polypeptide heterodimer) (for MDA-MB-453 cells and isolated T cells); or TSC020 (aCD19 x aCD3) or TSC021 (aCD20 x aCD3) for Reel and Jurkat cells, in concentrations from 100 nM to 0.1 nM.
  • S0266 aRON aCD3 SCORPIONTM protein
  • S0268 aRON aCD3 polypeptide heterodimer
  • the cells were washed three times and then incubated with goat anti-human IgG-FITC (1 :200 dilution) for another 30 minutes at 4°C. The cells were then washed again three times, fixed in 1% paraformaldehyde and read on a FACS- Calibur instrument.
  • Bivalent polypeptide heterodimer TSC046 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC039 (BMA031 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSCOOl comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSCOOl are set forth in SEQ ID NOS:34 and 54, respectively.
  • Single chain polypeptide TSC039 comprises from its amino to carboxyl terminus: BMA031 (anti-TCR) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • BMA031 anti-TCR
  • scFv human IgGl SCC-P hinge
  • the nucleotide and amino acid sequences of TSC039 are set forth in SEQ ID NOS:793 and 811, respectively.
  • Bivalent polypeptide heterodimer TSC047 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC041 (CRIS7 CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC041 comprises from its amino to carboxyl terminus: CRIS7 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC041 are set forth in SEQ ID NOS:794 and 812, respectively.
  • Bivalent polypeptide heterodimer TSC048 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC043 (OKT3-M CH2 CH3 Ck(YAE)).
  • Single chain polypeptide TSC043 comprises from its amino to carboxyl terminus: OKT3-M (Micromet variant anti-CD3, see also US 7,635,472) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC043 are set forth in SEQ ID NOs:795 and 813, respectively.
  • Bivalent polypeptide heterodimer TSC054 comprises single chian polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC049 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null) (i.e., human IgGl CH2 with L234A, L235A, G237A, E318A, K320A, and K322A substitutions), human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC049 are set forth in SEQ ID NOS:796 and 814, respectively.
  • Single chain polypeptide TSC053 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null) (i.e., human IgGl CH2 with L234A, L235A, G237A, E318A, K320A, and K322A substitutions), human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC053 are set forth in SEQ ID NOS:800 and 818, respectively.
  • Bivalent polypeptide heterodimer TSC055 comprises single chain polypeptides TSC050 (2H7 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC050 comprises from its amino to carboxyl terminus: 2H7 (anti-CD20) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC050 are set forth in SEQ ID NOS:797 and 815, respectively.
  • Bivalent polypeptide heterodimer TSC056 comprises single chain polypeptides TSC051 (P2C2 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC051 comprises from its amino to carboxyl terminus: P2C2 (anti-CD79) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC051 are set forth in SEQ ID NOS:798 and 816, respectively.
  • Bivalent polypeptide heterodimer TSC057 comprises single chain polypeptides TSC052 (5D5 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC052 comprises from its amino to carboxyl terminus: 5D5 (anti-cMet) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC052 are set forth in SEQ ID NOS:799 and 817, respectively.
  • Bivalent polypeptide heterodimer TSC078 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC076 (OKT3 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC076 comprises from its amino to carboxyl terminus: OKT3 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC076 are set forth in SEQ ID NOS:802 and 820, respectively.
  • Bivalent polypeptide heterodimer TSC079 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC077 (Nuvion CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC077 comprises from its amino to carboxyl terminus: Nuvion (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC077 are set forth in SEQ ID NOS:803 and 821, respectively.
  • Trivalent polypeptide heterodimer TSC080 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC064 (G19-4 CH2 CH3 Ck(YAE) H75 Met021).
  • Single chain polypeptide TSC064 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, human Ck(YAE), H75 linker, and Met021 (anti-c-Met) scFv (with the three serine residues at the C-temrinus deleted).
  • the nucleotide and amino acid sequences of TSC064 are set forth in SEQ ID NOS:801 and 819, respectively.
  • Bivalent polypeptide heterodimer TSC099 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC097 (4C04 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC097 comprises from its amino to carboxyl terminus: 4C04 (anti-RON) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC097 are set forth in SEQ ID NOS:808 and 826, respectively.
  • Bivalent polypeptide heterodimer TSCIOO comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC093 (CRIS7 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC093 comprises from its amino to carboxyl terminus: CRIS7 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC093 are set forth in SEQ ID NOS:804 and 822, respectively.
  • Bivalent polypeptide heterodimer TSC101 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC094 (OKT3- M CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC094 comprises from its amino to carboxyl terminus: OKT3-M (Micromet variant anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC094 are set forth in SEQ ID NOS:805 and 823, respectively.
  • Bivalent polypeptide heterodimer TSC102 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC095 (BMA031 CH2(ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC095 comprises from its amino to carboxyl terminus: BMA031 (anti-TCR) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, human Ck(YAE).
  • the nucleotide and amino acid sequences of TSC095 are set forth in SEQ ID NOS:806 and 824, respectively.
  • polypeptide heterodimer molecules were expressed and purified according to Example 1.
  • bispecific polypeptide heterodimer molecules To compare the effectiveness of different bispecific polypeptide heterodimer molecules at inducing target-dependent T-cell activation and proliferation, three different bispecific molecules (TSC054, TSC078, and TSC079 as described in Example 6) with a common anti-CD 19 binding domain (HD37) and three different anti- CD3 binding domains (G19-4 for TSC054, OKT3 for TSC078, HuM291 for TSC079) were compared.
  • a Bispecific T-Cell Engaging molecule BiTE
  • bscl9x3 As a positive control, a Bispecific T-Cell Engaging molecule (BiTE) known as bscl9x3 was also prepared (see U.S. Patent 7,635,472).
  • the nucleotide and amino acid sequences for bscl9x3 are set forth in SEQ ID NOS:809 and 827, respectively.
  • Transient transfection in human 293 cells produced 2.33 ug/mL protein for TSC054, 0.67 ug/mL protein for TSC078, and 3.5 ug/mL for TSC079.
  • Daudi Burkitt's lymphoma cells CD19 +
  • MDA-MB-453 CD 19 " breast carcinoma cells were obtained from ATCC (Manassas, VA) and cultured according to the provided protocol.
  • Peripheral blood mononuclear cells PBMC
  • T cells were additionally isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • cells were labeled with antibodies for flow cytometric analysis. Cells were labeled and washed in their original plates to minimize cell losses during transfers, and all labeling was done in saline buffer with 0.2% bovine serum albumin.
  • saline buffer with 0.2% bovine serum albumin.
  • cells were pre-incubated with lOOug/ml human IgG at room temperature for 15 min. Subsequently, cells were incubated with a mixture (total volume 50 ul) of the following dye-labeled antibodies: CD5-PE, CD4-APC, CD8-Pacific Blue, CD25-PE-Cy7, as well as 7- Amino Actinomycin D (7AAD hereafter) for 40 min.
  • bispecific polypeptide heterodimer molecules were compared in a chromium ( 51 Cr) release assay.
  • Three different bispecific molecules (TSC054, TSC078, TSC079, as described in Example 6) with a common anti-CD 19 binding domain (HD37) and three different anti-CD3 binding domains (G19-4 for TSC054, OKT3 for TSC078, HuM291 for TSC079) were tested alongside a fourth bispecific molecule (S0268, described in Example 4) with an anti-RON binding domain (4C04) and an anti-CD3 binding domain (G19-4).
  • Transient transfection in human 293 cells produced about 2.33 ⁇ g/mL protein for TSC054, about 0.67 ⁇ g/mL protein for TSC078, and about 3.5 ⁇ g/mL protein for TSC079.
  • Daudi Burkitt's lymphoma cells (CD 19+, RON-) and BxPC-3 cells (CD 19-, RON+) were obtained from ATCC (Manassas, VA) and cultured according to the provided protocol.
  • Peripheral blood mononuclear cells (PBMC) were isolated from human blood using standard ficoll gradients. The isolated cells were washed in saline buffer. T cells were additionally isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
  • Cytotoxicity was assessed by a 51 Cr release assay. Approximately 5x 10 6 Daudi or BxPC-3 cells were treated with 0.3 mCi of 51 Cr and incubated for 75 minutes at 37°C. After 75 minutes, cells were washed 3 times with media (RPMI + 10% FBS) and resuspended in 1 1.5 mL of media. From this suspension, 50 ⁇ , was dispensed per well into 96 well U-bottom plates (approximately 20,000 cells/well). Concentrations of bispecific molecules ranging from 10 nM to 0.1 pM were added to the target (Daudi, BxPC-3) cells, bringing the total volume to 100 (jIJwell. Target cells were incubated at room temperature for 15 minutes.
  • T-cells approximately 200,000 were added to bring the T-cell to target cell ratio to 10: 1. 50 ⁇ . of 0.8% NP- 40 was added to a control well containing target cells, left for 15 minutes, then 100 of media was added to provide a total lysis control.
  • bispecific polypeptide heterodimer molecules with altered hinge sequences at inducing target-dependent T-cell activation and proliferation
  • six different bispecific heterodimers (TSCIOO, TSC127, TSC165, TSC166, TSC167 and TSC168 with a common anti-CD 19 binding domain (HD37), a common anti-CD3 binding domain (Cris7) and five different hinge constructs (IgGl SCC-P hinge for TSCIOO and TSC127, IgA2 hinge without the first Val residue linked to a human IgGl core hinge for TSC165, IgGl SSS-P hinge linked to a human IgGl core hinge for TSC166, a portion of mutated IgG3 hinge linked to a IgGl core hinge for TSC167, and the IgM CH2 domain without the first Val linked to a IgGl core hinge for TSC168) were compared.
  • bispecific heterodimer TSCIOO is as described in
  • Bispecific heterodimer TSC127 comprises single chain polypeptides
  • TSC125 (Cris7 CH2(ADCC/CDC null) CH3 CHI) and TSC096 (HD37 CH2(ADCC/CDC null) CH3 Ck(YAE).
  • Single chain polypeptide TSC125 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC125 are set forth in SEQ ID NOS:865 and 874, respectively.
  • Single chain polypeptide TSC096 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC096 are set forth in SEQ ID NOS:807 and 825, respectively.
  • Bispecific heterodimer TSC165 comprises single chain polypeptides TSC157 (Cris7 IgA2UH CH2 (ADCC/CDC null) CH3 CHI) and TSC161 (HD37 IgA2UH CH2 (ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC157 comprises from its amino to carboxyl terminus: humanized Cris7( anti-CD3) scFv, human IgA2 hinge without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • TSC157 The nucleotide and amino acid sequences of TSC157 are set forth in SEQ ID NOS:866 and 875, respectively.
  • Single chain polypeptide TSC161 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, human IgA2 hinge without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC161 are set forth in SEQ ID NOS:870 and 879, respectively.
  • the amino acid sequence of the hinge used in TSC157 and TSC161 is set forth in SEQ ID NO:748.
  • Bispecific heterodimer TSC166 comprises single chain polypeptide TSC158 (Cris7 IgGlminiUH CH2 (ADCC/CDC null) CH3 CHI) and TSC162 (HD37 IgGlminiUH CH2 (ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC158 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SSC-P hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • TSC158 The nucleotide and amino acid sequences of TSC158 are set forth in SEQ ID NOS:867 and 876, respectively.
  • Single chain polypeptide TSC162 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SSC-P hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC162 are set forth in SEQ ID NOS:871 and 880.
  • the amino acid sequence of the hinge used in TSC158 and TSC162 is set forth in SEQ ID NO:750.
  • Bispecific heterodimer TSC167 comprises single chain polypeptide
  • TSC159 (Cris7 IgG3UH CH2 (ADCC/CDC null) CH3 CHI) and TSC163 (HD37 IgG3UH CH2 (ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC159 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, a portion of mutated human IgG3 hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • the nucleotide and amino acid sequences of TSC159 are set forth in SEQ ID NOS:868 and 877, respectively.
  • Single chain polypeptide TSC163 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, a portion of mutated human IgG3 hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC163 are set forth in SEQ ID NOS:872 and 881.
  • the amino acid sequence of the hinge used in TSC159 and TSC163 is set forth in SEQ ID NO:751.
  • Bispecific heterodimer TSC168 comprises single chain polypeptide TSC160 (Cris7 IgMCH2UH CH2 (ADCC/CDC null) CH3 CHI) and TSC164 (HD37 IgMCH2UH CH2 (ADCC/CDC null) CH3 Ck(YAE)).
  • Single chain polypeptide TSC160 comprises from its amino to carboxyl terminus: humanized Cris7( anti-CD3) scFv, human IgM CH2 without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI .
  • TSC160 The nucleotide and amino acid sequences of TSC160 are set forth in SEQ ID NOS:869 and 878, respectively.
  • Single chain polypeptide TSC163 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, human IgM CH2 without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC164 are set forth in SEQ ID NOS:873 and 882.
  • the amino acid sequence of the hinge used in TSC160 and TSC164 is set forth in SEQ ID NO:759.
  • bscl9x3 a Bispecific T-Cell Engaging molecule (BiTE) known as bscl9x3 was also prepared (see U.S. Patent 7,635,472).
  • the nucleotide and amino acid sequences for bscl9x3 are set forth in SEQ ID NOS:809 and 827, respectively.
  • Transient transfection in human 293 cells produced 3.2 ug protein / mL of culture for TSCIOO, 6.1 ug protein / mL of culture for TSC127, 4.8 ug protein / mL of culture for TSC165, 6.2 ug protein / mL of culture for TSC166, 6.4 ug protein / mL of culture for TSC167, and 6.4 ug/mL protein for TSC168.
  • Daudi Burkitt's lymphoma cells CD19 +
  • C4-2 CD 19 " prostate carcinoma cells were obtained from ATCC (Manassas, VA) and MD Anderson Cancer Center (Houston, TX) and cultured according to the provided protocol.
  • T cells were isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
  • CFSE-labeled T cells were plated in U-bottom 96-well plates at 100,000 cells/well, with 10,000 tumor cells/well, to achieve a T cell to tumor cell ratio of 10: 1. Concentrations of test molecules ranging from 5 nM to 0.005 pM were added to the cell mixtures in a total of 200 ul/well in RPMI 1640 media supplemented with 10% human or bovine serum, sodium pyruvate and non-essential amino acids. Plates were incubated at 37°C, 5% C0 2 in humidified incubators. After 3 days, cells were labeled with antibodies for flow cytometric analysis.
  • Cells were labeled and washed in their original plates to minimize cell losses during transfers, and all labeling was done in saline buffer with 0.2% bovine serum albumin.
  • First, cells were pre-incubated with 100 ug/ml human IgG at room temperature for 15 min. Subsequently, cells were incubated with a mixture (total volume 50 ul) of the following dye-labeled antibodies: CD5-PE, CD4-APC, CD8- Pacific Blue, CD25-PE-Cy7, as well as 7-Amino Actinomycin D (7AAD hereafter) for 40 minutes.
  • TSC165 the molecule containing the shorter IgA2 hinge
  • TSCIOO standard IgGl hinge
  • TSC166, TSC167, TSC168 molecules with longer hinges
  • bispecific molecules were compared in a chromium ( 51 Cr) release assay.
  • Five different bispecific molecules (TSCIOO, TSC165, TSC166, TSC167 and TSC168, as described in Example 9) with a common anti-CD 19 binding domain (HD37), a common anti-CD3 binding domain (Cris7) and five different hinge constructs (IgGl SCC-P hinge for TSCIOO and TSC127, IgA2 hinge without the first Val linked to a human IgGl core hinge for TSC165, IgGl SSS-P hinge linked to a human IgGl core hinge for TSC166, a portion of mutated IgG3 hinge linked to a human IgGl core hinge for TSC167, and the IgM CH2 domain without the first Val linked to a human IgGl core hinge for TSC168) were compared.
  • Daudi Burkitt's lymphoma cells (CD 19+, RON-) were obtained from
  • T cells were isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
  • Cytotoxicity was assessed by a 51 Cr release assay. Approximately 5x 10 6 Daudi cells were treated with 0.3 mCi of 51 Cr and incubated for 75 minutes at 37°C. After 75 minutes, cells were washed 3 times with media (RPMI + 10% FBS) and resuspended in 11.5 mL of media. From this suspension, 50 ⁇ , was dispensed per well into 96 well U-bottom plates (approximately 20,000 cells/well). Concentrations of bispecific molecules ranging from 10 nM to 0.1 pM were added to the target (Daudi) cells, bringing the total volume to 100 (jIJwell. Target cells were incubated at room temperature for 15 minutes.
  • T-cells approximately 200,000 were added to bring the T-cell to target cell ratio to 10: 1.
  • 50 ⁇ , of 0.8%> NP-40 was added to a control well containing target cells, left for 15 minutes, then 100 of media was added to provide a total lysis control.
  • Bispecific heterodimer TSC151 comprises single chain polypeptides
  • TSC145 and TSC148 Single chain polypeptide TSC145 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human IgGl CHI .
  • the human CH3 and human IgGl CHI are linked by a linker having the sequence GGGSS (SEQ ID NO:847).
  • the nucleotide and amino acid sequences of TSC145 are set forth in SEQ ID NOS:853 and 859, respectively.
  • Single chain polypeptide TSC148 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human Ck (YAE).
  • the human CH3 and human Ck (YAE) are linked with a linker having the sequence GGGSR (SEQ ID NO:850) in which R may alternatively be regarded as the first arginine of human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC148 are set forth in SEQ ID NOS:856 and 862, respectively.
  • Bispecific heterodimer TSC152 comprises single chain polypeptides TSC146 and TSC149.
  • Single chain polypeptide TSC146 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human IgGl CHI .
  • the human CH3 and human IgGl CHI are linked by a linker having the sequence SYSPNS (SEQ ID NO:848).
  • the nucleotide and amino acid sequences of TSC146 are set forth in SEQ ID NOS:854 and 860, respectively.
  • Single chain polypeptide TSC149 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human Ck (YAE).
  • the human CH3 and human Ck (YAE) are linked with a linker having the sequence SYSPNSR (SEQ ID NO: 851) in which R may alternatively be regarded as the first arginine of human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC149 are set forth in SEQ ID NOS:857 and 863, respectively.
  • Bispecific heterodimer TSC153 comprises single chain polypeptides TSC147 and TSC150.
  • Single chain polypeptide TSC147 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human IgGl CHI .
  • the human CH3 and human IgGl CHI are linked by a linker having the sequence SSLNTPNS (SEQ ID NO:849).
  • the nucleotide and amino acid sequences of TSC147 are set forth in SEQ ID NOS:855 and 861, respectively.
  • Single chain polypeptide TSC150 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human Ck (YAE).
  • the human CH3 and human Ck (YAE) are linked with a linker having the sequence SSLNTPNSR (SEQ ID NO: 852) in which R may alternatively be regarded as the first arginine of human Ck (YAE).
  • the nucleotide and amino acid sequences of TSC150 are set forth in SEQ ID NOS:858 and 864, respectively.

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Abstract

The present disclosure provides polypeptide heterodimers formed between two different single chain fusion polypeptides via natural heterodimerization of an immunoglobulin CH1 region and an immunoglobulin light chain constant region (CL). The polypeptide heterodimer comprises two or more binding domains that specifically bind one or more targets (e.g., a receptor). In addition, both chains of the heterodimer further comprise an Fc region portion. The present disclosure also provides nucleic acids, vectors, host cells and methods for making polypeptide heterodimers as well as methods for using such polypeptide heterodimers, such as in directing T cell activation, inhibiting solid malignancy growth, and treating autoimmune or inflammatory conditions.

Description

HETERODIMER BINDING PROTEINS AND USES THEREOF
CROSS-REFERENCE(S) TO RELATED APPLICATION(S)
This application claims the benefit under 35 U.S.C. § 1 19(e) of U.S. Provisional Patent Application No. 61/290,840, filed December 29, 2009, and U.S. Provisional Patent Application No. 61/365,266, filed July 16, 2010, each of which is herein incorporated by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 910180_422PC_SEQUENCE_LISTING.txt. The text file is 839 KB, was created on December 29, 2010, and is being submitted electronically via EFS-Web, concurrent with the filing of the specification.
BACKGROUND Technical Field
The present disclosure generally provides polypeptide heterodimers, compositions thereof, and methods for making and using such polypeptide heterodimers. More specifically, the polypeptide heterodimers provided herein are formed, in part, via natural heterodimerization between an immunoglobulin CHI region and an immunoglobulin light chain constant region (CL). In addition, the polypeptide heterodimers provided herein comprise two or more binding domains that specifically bind one or more targets. Furthermore, both single chain polypeptides of the polypeptide heterodimers provided herein each comprise an Fc region portion (e.g., immunoglobulin CH2 and CH3 domains). Description of the Related Art
The process of signal transduction often involves receptor proteins that have extracellular domains, transmembrane domains, and intracellular domains. During ligand binding, cell surface receptor molecules often oligomerize or multimerize (also referred to as "cross-link") to effectively transmit a signal to the intracellular compartment of the cell. The stimulation or blockade of this interaction between a receptor and a ligand or of the subsequent oligomerization or multimerization of receptors has important therapeutic implications for a wide variety of diseases. Exemplary molecules useful in modulating receptor and ligand interactions include antibodies or molecules derived from antibodies. For instance, an antibody or its derivative may function as a receptor antagonist that binds to a cell surface receptor and inactivates it by blocking the binding site of an activating ligand or preventing receptor dimerization or multimerization required for activation. In certain other cases, an antibody or its derivative may function as an agonist by binding to and cross-linking multiple membrane receptors, mimicking the function of a natural ligand. Another example is a bispecific antibody derivative that may be used to direct cytotoxic agents or immune effector cells to target sites, such as tumors.
Bispecific antibodies are antibody-based molecules that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen). One use of bispecific antibodies has been to redirect cytotoxic immune effector cells for enhanced killing of tumor cells by antibody dependent cellular cytotoxicity (ADCC). In this context, one arm of the bispecific antibody binds an antigen on the tumor cell, and the other binds a determinant expressed on effector cells. By cross-linking tumor and effector cells, the bispecific antibody not only brings the effector cells within the proximity of the tumor cells but also simultaneously triggers their activation, leading to effective tumor cell-killing. Bispecific antibodies have also been used to enrich chemo- or radiotherapeutic agents in tumor tissues to minimize detrimental effects to normal tissue. In this setting, one arm of the bispecific antibody binds an antigen expressed on the cell targeted for destruction, and the other arm delivers a chemotherapeutic drug, radioisotope, or toxin.
A major obstacle in the general development of bispecific antibodies has been the difficulty of producing materials of sufficient quality and quantity for both preclinical and clinical studies. Initially, the main route to the production of bispecific antibodies was by co-expression of both the light chains and both the heavy chains of two parent antibodies of different specificities in a single cell. However, the desired binding-competent bispecific antibodies are a minor product, and purification from the other products is very difficult. Another traditional method for bispecific antibody production is chemical conjugation of two antibodies or their fragments having different specificities. However, this method is also complicated, and the chemical modification process may inactivate the antibody or promote aggregation. Because purification from undesired products remains difficult, the resulting low yield and poor quality of bispecific antibody make this process unsuitable for the large scale production required for clinical development.
Recently, various heterodimerization techniques have been used to improve the production of bispecific antibodies. However, fusion of simple heterodimerization domains like the Jun/Fos coiled-coil to scFv domains lead to a mixture of homo- and heterodimers and need to be assembled by refolding (de Kruif and Logtenberg, J. Biol. Chem. 271 : 7630-4, 1996). Fusion of scFv fragments to whole antibodies was also used as a dimerization device (Coloma and Morrison, Nat. Biotechnol. 15 : 159-63, 1997). However, such fusion results in a large molecule with poor solid tissue penetration capabilities. Fusion of two scFv fragments together has also been used to generate bispecific proteins (e.g., BITE® antibodies by Micromet Inc., Bethesda, MD, U.S. Patent No. 7,635,472). However, such proteins do not contain Fc regions, and thus do not allow manipulation of their activities via Fc regions. In addition, these proteins are small (~55 kDa) and thus have relatively short half lives in serum.
To date, immunoglobulin fusion technology has not provided commercially viable heterodimeric proteins or methods for making them. Thus, there remains a need in the art for alternative multispecific heterodimeric proteins as well as efficient methods for producing the same.
BRIEF SUMMARY
The present disclosure provides polypeptide heterodimers formed between two different single chain polypeptides via natural heterodimerization of an immunoglobulin CHI region and an immunoglobulin light chain constant region (CL). The present disclosure also provides nucleic acids, vectors, host cells and methods for making polypeptide heterodimers as well as methods for using such polypeptide heterodimers, such as in directed T cell activation, inhibiting solid malignancy growth, treating autoimmune or inflammatory conditions, or treating B-cell associated disorders or diseases.
In one aspect, the present disclosure provides a polypeptide heterodimer that comprises (a) a first single chain polypeptide (SCP-I) comprising from one to four binding domains that specifically bind from one to four targets, a hinge (H-I), an immunoglobulin heterodimerization domain (HD-I), and an Fc region portion (FRP-I); and (b) a second single chain polypeptide (SCP-II) comprising from zero to four binding domains that specifically bind from zero to four targets, a hinge (H-II), an immunoglobulin heterodimerization domain (HD-II), and an Fc region portion (FRP- II); wherein (i) the immunoglobulin HD-I and the immunoglobulin HD-II preferentially associate with each other to form a polypeptide heterodimer comprised of SCP-I and SCP-II, and (1) the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CHI region, and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CL region, or (2) the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CL region, and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CHI region; and (ii) the Fc region portion of SCP-I and the Fc region portion of SCP-II comprise an immunoglobulin CH2 and CH3 domain of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, or any combination thereof; one or two immunoglobulin CH3 domains of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM, or any combination thereof; or an immunoglobulin CH3 and CH4 domain of IgE, IgM, or any combination thereof, provided that the polypeptide heterodimer comprises at least two binding domains that specifically bind a target, for instance, at least two different targets.
In certain embodiments, the binding domains of the polypeptide heterodimers are single chain Fv (scFv) polypeptides.
In certain embodiments, the polypeptide heterodimer comprises two binding domains (BDl and BD2). In one embodiment, the two binding domains (BDl and BD2) are both on the first single chain polypeptide (SCP-I) and wherein the HD-I and FRP-I are disposed between BDl and BD2. In another embodiment, the first binding domain (BDl) is on the first single chain polypeptide (SCP-I) and the second binding domain (BD2) is on the second single chain polypeptide (SCP-II). For example, the first binding domain (BDl) may be amino terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) may amino terminal to the Fc region portion of the second single chain polypeptide (FRP-II). Alternatively, the first binding domain (BDl) may be amino terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) may be carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II). Also alternatively, the first binding domain (BDl) may be carboxyl terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) may be carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
In certain embodiments, the polypeptide heterodimer comprises three binding domains (BDl, BD2 and BD3). In one embodiment, the HD-I and FRP-I are disposed between BDl and BD2, and the third binding domain (BD3) is amino terminal to the Fc region portion of the second single chain polypeptide (FRP-II). In an alternative embodiment, the HD-I and FRP-I are disposed between BDl and BD2, and the third binding domain (BD3) is carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II). In certain embodiments, the polypeptide heterodimer comprises four binding domains (BDl, BD2, BD3, and BD4). For instance, the HD-I and FRP-I may be disposed between BDl and BD2, and the HD-II and FRP-II may be disposed between BD3 and BD4.
In certain embodiments, the polypeptide heterodimer comprises five to eight binding domains (e.g., 5, 6, 7 or 8 binding domains).
In certain embodiments, at least one of the binding domains of the polypeptide heterodimers provided herein specifically binds to, or is an antagonist of, TCRa, TCRP, CD3y, CD35, CD3s, CD28, CD79b, hyperIL-6, monoIL-10 , CD86, CD20, PSMA, CD 19, HLA-DR, Ron, c-Met, CEACAM-6, LIGHT, GITRL, CD40, PDL1, PDL2, HVEM, LTBR, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGF1R, EphA2, PDGFR, VEGFR1-4, Angiopoietin 2, CD64, CD32A, CD 16, CD71, TNFR1, TNFR2, TWEAKR, TACI, BAFF-R, BCMA, FAS, CD32B, CD21, CD22, CD30, CD33, CD37, CD38, CD70, TNFa, IL-6, hyperIL-6, IL-2, IL-1, IL-7, IL-8, IL-17A/C, IP- 10, IFNy, IFNa, RANKL, FASL, TGFp, IL10, IL17A/F, CSF2, IGF1, IGF2, BLyS/APRIL, , HGF, MSP, EGF (including epiregulin, herregulin, β-regulin, neuregulin), HIF-l , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, Wnt, sHH, TGFp, PDGF, TWEAK, EpCAM, CEA, PCTA-1, STEAP- 1, PSCA, ALCAM (CD 166), EphA2, CD 151, CA-125, MUC-1, MAGE-1, TROP2, CCR5, HER-3, HER-4, EGFR, CEA, MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, phCG, Lewis-Y, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, CD63, IGF1R, CD151, TGFBR2, GHRHR, GHR, IL-6R, gpl30, TNFR2, OSMRp, Patched- 1, Frizzled, Robol, CD80, CD81, CD86, OX40, CD40, CD 137, LIFRp, TLR7 or TLR9.
In certain other embodiments, at least one of the binding domains of the polypeptide heterodimer is an agonist of IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, TNFR1, TNFR2, DR4, DR5, TWEAKR, or FAS.
In certain polypeptide heterodimers provided herein, at least one binding domain specifically binds a TCR complex or a component thereof, and at least another binding domain specifically binds to PSMA, CD79b, CD 19, HLA-DR, CD20, RON, c- Met, or CEACAM-6. In certain other polypeptide heterodimers provided herein, at least one binding domain specifically binds to CD28, and at least another binding domain specifically binds to, or is an antagonist of, CD79b, hyperIL-6, PDL2, monoIL-10, CD86, LIGHT, GITRL, CD40, PDL1, HVEM, or LTBR.
In certain other polypeptide heterodimers provided herein, at least one binding domain specifically binds to CD28, and at least another binding domain is an agonist of IL-10, HLA-G, HGF, IL-35, PD-1, or BTLA.
In certain embodiments, the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region. In one embodiment, the first CHI region is amino terminal to the Fc region portion of the first single chain polypeptide, and the first CL region is amino terminal to the Fc region portion of the second single chain polypeptide. In another embodiment, the first CHI region is carboxyl terminal to the Fc region portion of the first single chain polypeptide, and the first CL region is carboxyl terminal to the Fc region portion of the second single chain polypeptide.
In certain embodiments in which the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region, the first single chain polypeptide further comprises a second CHI region and the second single chain polypeptide further comprises a second CL region, and the second CHI region of the first single chain polypeptide and the second CL region of the second single chain polypeptide associate with each other in the polypeptide heterodimer. For instance, the Fc region portion of the first single chain polypeptide may be disposed between the first and second CHI regions, and the Fc region portion of the second single chain polypeptide may be disposed between the first and second CL regions. Alternatively, both the first and second CHI regions may be amino terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CL regions may be amino terminal to the Fc region portion of the second single chain polypeptide. Also alternatively, both the first and second CHI regions may be carboxyl terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CL regions may be carboxyl terminal to the Fc region portion of the second single chain polypeptide.
In certain other embodiments in which the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region, the first single chain polypeptide further comprises a second CL region and the second single chain polypeptide further comprises a second CHI region, and the second CL region of the first single chain polypeptide and the second CHI region of the second single chain polypeptide associate with each other in the polypeptide heterodimer. For instance, in one embodiment, in the first single chain polypeptide, the first CHI region is amino terminal to the Fc region portion and the second CL region is carboxyl terminal to the Fc region portion; and in the second single chain polypeptide, the first CL region is amino terminal to the Fc region portion, and the second CHI region is carboxyl terminal to the Fc region portion. In another embodiment, in the first single chain polypeptide, the first CHI region is carboxyl terminal to the Fc region portion, and the second CL region is amino terminal to the Fc region portion; and in the second single chain polypeptide, the first CL region is carboxyl terminal to the Fc region portion, and the second CHI region is amino terminal to the Fc region portion. In yet another embodiment, in the first single chain polypeptide, both the first CHI region and the second CL regions are amino terminal to the Fc region portion, and the first CHI region is amino terminal to the second CL region; and in the second single chain polypeptide, both the first CL region and the second CHI region are amino terminal to the Fc region portion, and the first CL region is amino terminal to the second CHI region. In yet another embodiment, in the first single chain polypeptide, both the first CHI region and the second CL regions are amino terminal to the Fc region portion, and the second CL region is amino terminal to the first CHI region; and in the second single chain polypeptide, both the first CL region and the second CHI region are amino terminal to the Fc region portion, and the second CHI region is amino terminal to the first CL region. In a further embodiment, in the first single chain polypeptide, both the first CHI region and the second CL regions are carboxyl terminal to the Fc region portion, and the first CHI region is amino terminal to the second CL region; and in the second single chain polypeptide, both the first CL region and the second CHI region are carboxyl terminal to the Fc region portion, and the first CL region is amino terminal to the second CHI region. In another embodiment, in the first single chain polypeptide, both the first CHI region and the second CL regions are carboxyl terminal to the Fc region portion, and the second CL region is amino terminal to the first CHI region; and in the second single chain polypeptide, both the first CL region and the second CHI region are carboxyl terminal to the Fc region portion, and the second CHI region is amino terminal to the first CL region. In certain embodiments, the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CL region, and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CHI region. In one embodiment, the first CL region is amino terminal to the Fc region portion of the first single chain polypeptide, and the first CHI region is amino terminal to the Fc region portion of the second single chain polypeptide. In another embodiment, the first CL region is carboxyl terminal to the Fc region portion of the first single chain polypeptide, and the first CHI region is carboxyl terminal to the Fc region portion of the second single chain polypeptide. In yet another embodiment, the first single chain polypeptide further comprises a second CL region and the second single chain polypeptide further comprises a second CHI region, and the second CL region of the first single chain polypeptide and the second CHI region of the second single chain polypeptide associate with each other in the polypeptide heterodimer. For instance, the Fc region portion of the first single chain polypeptide may be disposed between the first and second CL regions, and the Fc region portion of the second single chain polypeptide may be disposed between the first and second CHI regions. Alternatively, both the first and second CL regions may be amino terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CHI regions may be amino terminal to the Fc region portion of the second single chain polypeptide. Also alternatively, both the first and second CL regions may be carboxyl terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CHI regions may be carboxyl terminal to the Fc region portion of the second single chain polypeptide.
In certain embodiments, the first CL region is a CK region. In other embodiments, the first CL region is a region.
In certain embodiments, the second CL region is a CK region. In other embodiments, the second CL region is a C region.
In certain embodiments, the CK region is a wild type human immunoglobulin CK region. In certain other embodiments, the CK region is an altered human immunoglobulin CK region with one or more amino acids of a wild type human CK region substituted at N29, N30, Q52, V55, T56, T56, S68, or T70. For example, the one or more amino acid substitutions may be selected from Ala (A), Arg (R), Trp (W), Tyr (Y), Glu (E), Gin (Q), Lys (K), Asp (D), Met (M), Ser (S), and Phe (F).
In certain embodiments, the CHI region is an altered human immunoglobulin CHI region comprising an amino acid substitution by which Val (V) at position 68 is substituted by Lys (K), Arg (R) or His (H), and wherein the Ck region is an altered human immunoglobulin Ck region comprising an amino acid substitution by which Leu (L) at position 29 is substituted by Asp (D) or Glu (E). In certain other embodiments, the CHI region is an altered human immunoglobulin CHI region comprising an amino acid substitution by which Val (V) at position 68 is changed to Asp (D) or Glu (E), and wherein the Ck region is an altered human immunoglobulin Ck region comprising an amino acid substitution by which Leu (L) at position 29 is changed to Lys (K), Arg (R) or His (H).
In certain embodiments, the region is a wild type human immunoglobulin region.
In certain embodiments, the first CHI region or the second CHI region when present is a wild type human immunoglobulin CHI region, such as a wild type human IgGl CHI region.
In certain embodiments, the first CHI region or the second CHI region when present is an altered human immunoglobulin CHI region, such as an altered human IgGl CHI region, with the cysteine of a wild type human immunoglobulin CHI region that is involved in forming a disulfide bond with a wild type human immunoglobulin CL region deleted or substituted.
In certain embodiments, the CK region is an altered human immunoglobulin Ck region, such as an with the cysteine residue of a wild type human CK region that is involved in forming a disulfide bond with a wild type human immunoglobulin CHI region deleted or substituted.
In certain embodiments, the region is an altered human immunoglobulin region with the cysteine residue of a wild type human region that is involved in forming a disulfide bond with a wild type human immunoglobulin CHI region deleted or substituted.
In certain embodiments, the first CHI region and the second CHI region when present is a polypeptide comprising SEQ ID NO: l 14, 844 or 845.
In certain embodiments, the Ck region when present is selected from any one of the polypeptides comprising SEQ ID NOS: 141-178, 202, and 838-843.
In certain embodiments, the region when present is a polypeptide comprising SEQ ID NO: 140.
In certain embodiments, the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH2 domain, such as an IgGl CH2 domain or an IgG2, IgG3, IgG4, IgAl, IgA2, or IgD CH2 domain.
In certain embodiments, the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH3 domain, such as an IgGl CH3 domain or an IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE or IgM CH3 domain.
In certain embodiments, the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH2 domain and an immunoglobulin CH3 domain, such as are IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD CH2 and CH3 domains.
In some embodiments in which the Fc region portion comprises an immunoglobulin CH3 domain that is immediately amino terminal to an immunoglobulin heterodimerization domain (e.g., a CHI domain or a Ck domain), the immunoglobulin CH3 domain is linked to the CHI domain immediately carboxyl terminal to the immunoglobulin CH3 domain in one single chain polypeptide via a peptide comprising SEQ ID NOS:846, 847, 848, or 849; and the immunoglobulin CH3 domain is linked to the Ck domain immediately carboxyl terminal to the immunoglobulin CH3 domain in the other single chain polypeptide via a peptide comprising SEQ ID NOs:846, 850, 951 , or 852.
In certain embodiments, the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second chain polypeptide (FRP-II) comprise IgM or IgE CH3 and CH4 domains.
In certain embodiments in which the Fc region portion comprises an immunoglobulin CH2 domain, the CH2 domain may be an altered human IgGl , IgG2, IgG3, or IgG4 CH2 domain. Exemplary altered human IgGl , IgG2, IgG3 or IgG4 CH2 domain include those that comprise (a) an amino acid substitution at position 297 and at least one additional substitution or deletion at positions 234 to 238; (b) one or more amino acid mutations at positions 234-238 and at least one substitution at position 253, 310, 318, 320, 322, or 331 ; or (c) an amino acid substitution at the asparagine of position 297, one or more substitutions or deletions at positions 234 to 238, and at least one substitution at position 253, 310, 318, 320, 322, or 331. Another exemplary CH2 domain is an altered human IgGl CH2 domain that comprises amino acid substitutions at positions L234, L235, G237, E318, K320 and K322.
In certain embodiments, the CH3 domain of the first single chain polypeptide is an altered human IgGl , IgG2, IgG3, or IgG4 CH3 domain that comprises a T366W, and the CH3 domain of the second single chain polypeptide is an altered human IgGl , IgG2, IgG3 or IgG4 CH3 domain that comprises a Y407A substitution. In certain other embodiments, the CH3 domain of the first single chain polypeptide is an altered human IgGl , IgG2, IgG3, or IgG4 CH3 domain that comprises a T366Y substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a Y407T substitution. In certain other embodiments, the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a T366W substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises T366S, L368A and Y407V substitutions. In certain other embodiments, the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a Y407A substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366W substitution. In certain other embodiments, the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a Y407T substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366Y substitution. In certain other embodiments, the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises T366S, L368A and Y407W substitutions, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366W substitution.
In certain embodiments, the hinge of both the first and second single chain polypeptides is an immunoglobulin hinge region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, or IgE hinge. In certain embodiments, the immunoglobulin hinge is a wild type immunoglobulin hinge. In certain other embodiments, the immunoglobulin hinge is an altered immunoglobulin hinge selected from SEQ ID NOS:232, 234, 240, 664-673, 675 and 676.
In certain embodiments, the hinge region is (a) at the amino terminal to the Fc region portion, (b) disposed between the binding domain and the immunoglobulin heterodimerization domain, (c) disposed between the immunoglobulin heterodimerization domain and the Fc region portion, (d) at the amino terminus of the first or second single chain polypeptide, (e) disposed between the Fc region portion and a binding domain, or (f) at the carboxyl terminus of the first or second single chain polypeptide.
In certain embodiments, at least one of the first and second single chain polypeptide hinges is a C-type lectin hinge region, such as an NKg2A or NKg2D peptide, or a derivative thereof.
In certain embodiments, the hinges of the first and second single chain polypeptides are identical. In certain other embodiments, the hinges of the first and second single chain polypeptides are different. In certain embodiments, the first single chain polypeptide comprises a binding domain that specifically binds a TCR complex or a component thereof, and the second single chain polypeptide comprises a binding domain that specifically binds CD 19, CD79b, HLA-DR or CD20.
In certain embodiments, the first single chain polypeptide comprises a binding domain that specifically binds CD28, and the second single chain polypeptide comprises a binding domain (a) that specifically binds CD79b, hyperIL-6, or CD86 or (b) that comprises a PDL ectodomain or a monoIL-10.
In certain embodiments, the first single chain polypeptide comprises a binding domain that specifically binds c-Met, and the second single chain polypeptide comprises a binding domain that specifically binds RON.
In certain embodiments, the first and second single chain polypeptides comprise SEQ ID NOS: SEQ ID NOS:2 and 4, SEQ ID NOS:6 and 8, SEQ ID NOS: 10 and 12, SEQ ID NOS: 14 and 16, SEQ ID NOS: 18 and 20, SEQ ID NOS:20 and 22, SEQ ID NOS:20 and 24, SEQ ID NOS:30 and 32, SEQ ID NOS:29 and 31, SEQ ID NOS:29 and 32, SEQ ID NOS:30 and 72, SEQ ID NOS:53 and 72, SEQ ID NOS:54 and 72, SEQ ID NOS:55 and 72, SEQ ID NOS:70 and 72, SEQ ID NOS:71 and 72, SEQ ID NOS:63 and 56, SEQ ID NOS:64 and 57, SEQ ID NOS:65 and 60, SEQ ID NOS:66 and 58, SEQ ID NOS:67 and 59, SEQ ID NOS:68 and 61, SEQ ID NOS:69 and 62, SEQ ID NOS:54 and 811, SEQ ID NOS:54 and 812, SEQ ID NOS:54 and 813, SEQ ID NOS:814 and 818, SEQ ID NOS:815 and 818, SEQ ID NOS:816 and 818, SEQ ID NOS:817 and 818, SEQ ID NOS:814 and 820, SEQ ID NOS:814 and 821, SEQ ID NOS:54 and 819, SEQ ID NOS:814 and 826, SEQ ID NOS:814 and 822, SEQ ID NOS:814 and 823, SEQ ID NOS:814 and 824, SEQ ID NOS:859 and 862, SEQ ID NOS: 860 and 863, SEQ ID NOS: 861 and 864, SEQ ID NOS: 874 and 825, SEQ ID NOS:875 and 879, SEQ ID NOS:876 and 880, SEQ ID NOS:877 and 881, or SEQ ID NOS:878 and 882.
In another aspect, the present disclosure provides a composition comprising a polypeptide heterodimer provided herein and a pharmaceutically acceptable excipient.
In another aspect, the present disclosure provides an expression vector capable of expressing a polypeptide heterodimer provided herein, comprising a first polynucleotide encoding the first single chain polypeptide and a second polynucleotide encoding the second single chain polypeptide.
In another aspect, the present disclosure provides a host cell comprising the above expression vector. In another aspect, the present disclosure provides a host cell comprising first and second expression vectors capable of expressing the first and second single chain polypeptides, respectively, of the polypeptide heterodimer provided herein.
In another aspect, the present disclosure provides a method for making a polypeptide heterodimer, comprising (a) culturing a host cell provided herein under conditions suitable to express first and second single chain polypeptides, and (b) optionally isolating or purifying the heterodimers formed from the first and second single chain polypeptides from the culture.
In another aspect, the present disclosure provides a method for directing T cell activation, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, or a combination thereof, and a second binding domain that specifically binds a different target, for instance, a tumor-specific antigen or other antigen of choice at a site or cell where T cell activation is desired.
In another aspect, the present disclosure provides a method for inhibiting growth, metastatis, or metastatic growth of a malignancy, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, c-Met, RON, or a combination thereof. In certain embodiments, the method further comprises administering to a patient in need thereof a chemotherapeutic agent or ionizing radiation.
In another aspect, the present disclosure provides a method for treating an autoimmune or inflammatory condition, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRp, CD3y, CD35, CD3s, or CD28.
In another aspect, the present disclosure provides a method for treating a B-cell associated disorder or disease comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, or CD3s, and a second binding domain that specifically binds to CD 19, CD20, CD79b or HLA-DR.
In certain embodiments, the methods for using the polypeptide heterodimers provided herein may further comprise administering to a patient in need thereof a second active agent, such as a second polypeptide heterodimer, a monoclonal antibody, or an immunoglobulin-derived fusion protein. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of bivalent anti-CD28 polypeptide heterodimer X0172 (left) and SDS-PAGE analysis of X0172 (right). "NR" stands for "Non-Reduced," and "Red" stands for "Reduced."
Figure 2 shows that both a monovalent (X0124) and a bivalent (X0172) anti-CD28 polypeptide heterodimer synergize with a suboptimal concentration of PMA in stimulating purified human T cells when compared to an anti-CD28 scFv (2E12 scFv) but less than a bivalent anti-CD28 SMIP protein (2E12 SMIP).
Figure 3 shows that bivalent polypeptide heterodimer X0172 binds to CD4+ T cells better than 2E12 scFv and monovalent polypeptide heterodimer X0124.
Figure 4 shows cation exchange chromatography of polypeptide heterodimers X0251, X0252 and X0253 (left) and SDS-PAGE electrophoresis analysis of the same polypeptide heterodimers under non-reduced (NR) and reduced (Red) conditions (right).
Figure 5 shows the mass spectra of polypeptide heterodimer X0252, which demonstrates that the heterodimer is the predominant species.
Figure 6 shows SDS-PAGE electrophoresis analysis of polypeptide heterodimers X0283 and X0284 under non-reduced (NR) and reduced (Red) conditions (left) and cation exchange chromatography analysis of polypeptide heterodimer X0283.
Figure 7 shows direct binding to CD86 by polypeptide heterodimer
X0283 as examined by BIACORE® analysis, with response units (Ru) plotted against time (left) and a schematic representation of X0283.
Figures 8A and 8B show binding of bispecific anti-RON and anti-CD3 constructs (polypeptide heterodimer S0268 and Scorpion protein S0266) to MDA-MB- 453 cells (A) and to isolated T cells (B).
Figures 9 A and 9B show specificity of binding to (A) Reel (CD19+, CD20 ) cells or (B) Jurkat (CD3 ) cells by bispecific heterodimers having either anti- CD^ and anti-CD3 binding domains (TSC020) or having anti-CD20 and anti-CD3 binding domains (TSC021).
Figures 10A-10D show proliferation of CD4+ and CD8+ T-cells in response to bispecific polypeptide heterodimers TSC054, TSC078, TSC079, and bscl9x3 (TSC036) with (A and B) Daudi (CD19+) cells or (C and D) MDA-MB-453 (CD 19 ) cells.
Figures 11A and 11B shows T-cell directed cytotoxicity induced by bispecific polypeptide heterodimers TSC054, TSC078, TSC079, and S0268 in a chromium (51Cr) release assay with (A) Daudi (RON", CD19+) cells or (B) BxPC-3 (RON+, CD 19 ) cells. Figure 12 shows target-dependent T-cell proliferation induced by a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSC 165, TSC 166, TSC 167, TSC168 and TSCIOO at concentrations from 0.1 pM to 10000 pM.
Figure 13 shows target-dependent T-cell proliferation induced by a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSC127 and TS 165 with bscl9x3 BiTE as a control at concentrations from 0.001 pM to 1000 pM.
Figure 14 shows target-dependent redirected T-cell cytotoxicity on a CD 19+ cell line (Daudi) using bispecific polypeptide heterodimers TSCI OO, TSC165, TC166, TSC 167, and TSC 168. DETAILED DESCRIPTION
The present disclosure provides polypeptide heterodimers formed between two different single chain polypeptides via natural heterodimerization of an immunoglobulin CHI region and an immunoglobulin light chain constant region (CL). The polypeptide heterodimer has two or more binding domains that specifically bind one or more targets (e.g., an antigen, a receptor, or a ligand). In addition, both chains of a heterodimer each further comprise an Fc region portion (e.g., immunoglobulin CH2 and/or CH3 domains). The present disclosure also provides nucleic acids, vectors, host cells and methods for making polypeptide heterodimers.
The heterodimerization technology described herein has one or more of the following advantages: (1) potential for minimal immunogenicity of the polypeptide heterodimers because the dimers are formed via natural heterodimerization of an immunoglobulin CHI region and an immunoglobulin CL region; (2) efficient production and purification of polypeptide heterodimers of the present disclosure is possible by co-expressing the two different single chain polypeptides, as shown in the examples; (3) the ability to mediate Fc effector functions (e.g., CDC, ADCC, ADCP), which can be modulated up or down by mutagenesis, and a longer serum half life because each chain of a polypeptide heterodimer according to the present disclosure has an Fc region portion (e.g., immunoglobulin CH2 and CH3 domains); and (4) polypeptide heterodimers of the present disclosure have a size that is typically smaller than an antibody molecule, which can allow for better tissue penetration, such as into a solid malignancy.
The polypeptide heterodimers provided herein are useful to direct therapeutic agents or immune effector cells to target cells. For instance, in certain embodiments, the polypeptide heterodimers may comprise a binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRP, CD3y, CD35, and CD3s) and another binding domain that specifically binds a second different target, such as an oncology target (e.g., c-Met, RON, CEACAM-6, and PSMA) or a B- cell target (e.g., CD 19, CD79b, HLA-DR and CD20). The binding domain specific for the second different target may have a higher affinity for its target than the affinity of the binding domain for the TCR complex or component thereof, such as CD3. Such polypeptide heterodimers will preferably bind to the oncology target or the B-cell target first and subsequently recruit T cells to the tumor or cancer cells expressing the oncology target or B-cell target and are useful in inhibiting growth, metastasis or metastatic growth of a malignancy (including B-cell cancers). Additional uses of polypeptide heterodimers provided herein include directed T cell activation and treating autoimmune or inflammatory conditions.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents defines a term that contradicts that term's definition in the application, the definition that appears in this application controls.
In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. As used herein, "about" means ± 20% of the indicated range, value, sequence, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components unless otherwise indicated or dictated by its context. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously. In addition, it should be understood that the individual single chain polypeptides or heterodimers derived from various combinations of the structures and substituents (e.g., domains, regions, hinges, and linkers) described herein are disclosed by the present application to the same extent as if each single chain polypeptide or heterodimer were set forth individually. Thus, selection of particular components to form individual single chain polypeptides or heterodimers is within the scope of the present disclosure.
As used herein, a protein "consists essentially of several domains (e.g., a binding domain that specifically binds a target, a hinge, an immunoglobulin heterodimerization domain, and an Fc region portion) if the other portions of the protein (e.g., amino acids at the amino- or carboxy-terminus or between two domains), in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as more than 40%, 30%, 25%, 20%, 15%, 10%, or 5%) the activities of various domains (e.g., the target binding affinity of the binding domain, the activities of the Fc region portion, and the capability of the immunoglobulin heterodimerization domain in facilitating heterodimerization). In certain embodiments, a protein (e.g., a single chain polypeptide) consists essentially of a binding domain that specifically binds a target, an immunoglobulin heterodimerization domain, a hinge, and an Fc region portion may comprise junction amino acids at the amino- and/or carboxy-terminus of the protein and/or between two different domains (e.g., between the binding domain and the immunoglobulin heterodimerization domain, between the immunoglobulin heterodimerization domain and the hinge, and/or between the hinge and the Fc region portion).
A "polypeptide heterodimer" or "heterodimer," as used herein, refers to a dimer formed from two different single chain polypeptides. This term does not include an antibody formed from four single chain polypeptides (i.e., two light chains and two heavy chains). A "dimer" refers to a biological entity that consists of two subunits associated with each other via one or more forms of intramolecular forces, including covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonding, and hydrophobic interactions), and is stable under appropriate conditions (e.g., under physiological conditions, in an aqueous solution suitable for expressing, purifying, and/or storing recombinant proteins, or under conditions for non-denaturing and/or non-reducing electrophoresis).
A "single chain polypeptide" is a single, linear and contiguous arrangement of covalently linked amino acids. It does not include two polypeptide chains that link together in a non-linear fashion, such as via an interchain disulfide bond (e.g. , a half immunoglobulin molecule in which a light chain links with a heavy chain via a disulfide bond). In certain embodiments, a single chain polypeptide may have or form one or more intrachain disulfide bonds.
An "immunoglobulin heterodimerization domain," as used herein, refers to an immunoglobulin domain of a single chain polypeptide that preferentially interacts or associates with a different immunoglobulin domain of another single chain polypeptide wherein the interaction of the different heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second single chain polypeptides (i.e., the formation of a dimer between two different single chain polypeptides, which is also referred to as a "heterodimer"). The interaction(s) between heterodimerization domains "substantially contributes to or efficiently promotes" the heterodimerization of first and second single chain polypeptides if there is a statistically significant reduction in the dimerization between the first and second single chain polypeptides in the absence of the heterodimerization domain of the first single chain polypeptide (HD-I) and/or the heterodimerization domain of the second single chain polypeptide (HD-II). In certain embodiments, when the first and second single chain polypeptides are co-expressed, at least 60%, at least about 60%) to about 70%>, at least about 70%> to about 80%>, at least about 80%> to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second single chain polypeptides form heterodimers with each other. Representative immunoglobulin heterodimerization domains of the present disclosure include an immunoglobulin CHI region, an immunoglobulin CL region (e.g., CK or C isotypes), or derivatives thereof, including wild type immunoglobulin CHI and CL regions and altered (or mutated) immunoglobulin CHI and CL regions, as provided herein.
A "binding domain" or "binding region," as used herein, refers to a protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a target (e.g., CD3, TCR, CD28, c-Met, RON). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding domains include single chain antibody variable regions (e.g. , domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g. , c-Met, RON), or ligands (e.g. , cytokines, chemokines). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore analysis.
A binding domain and a fusion protein thereof "specifically binds" a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 M"1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) may be classified as "high affinity" binding domains (or fusion proteins thereof) and "low affinity" binding domains (or fusion proteins thereof). "High affinity" binding domains refer to those binding domains with a Ka of at least 107 M"1, at least 108 M"1, at least 109 M"1, at least 1010 M"1, at least 1011 M"1, at least 1012 M"1, or at least 1013 M"1. "Low affinity" binding domains refer to those binding domains with a Ka of up to 107 M"1, up to 106 M"1, up to 105 M"1. Alternatively, affinity may be defined as an equilibrium dissociation constant (IQ) of a particular binding interaction with units of M (e.g. , 10"5 M to 10"13 M). Affinities of binding domain polypeptides and single chain polyepeptides according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
"T cell receptor" (TCR) is a molecule found on the surface of T cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It consists of a disulfide-linked heterodimer of the highly variable a and β chains in most T cells. In other T cells, an alternative receptor made up of variable γ and δ chains is expressed. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see, Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway et al., Immunobiology: The Immune System in Health and Disease, 4th Ed., Current Biology Publications, pl48, 149, and 172, 1999). TCR as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
"CD3" is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999). In mammals, the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3ζ chains. The CD3y, CD35, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD35, and CD 3 ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζ chain has three. It is believed the ITAMs are important for the signaling capacity of a TCR complex. CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.
"TCR complex," as used herein, refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3ζ chains, a TCRa chain, and a TCRP chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3ζ chains, a TCRy chain, and a TCR5 chain. "A component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CD3y, CD35, CD3s or C Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRp, CD3y, CD35, and two CD3s chains).
Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. Antibodies are known to have variable regions, a hinge region, and constant domains. Immunoglobulin structure and function are reviewed, for example, in Harlow et ah, Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
For example, the terms "VL" and "VH" refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well-defined sub-regions known as "complementarity determining regions" (CDRs) and "framework regions" (FRs). The term "CL" refers to an "immunoglobulin light chain constant region" or a "light chain constant region," i.e., a constant region from an antibody light heavy chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or a "heavy chain constant region," which is further divisible, depending on the antibody isotype into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM). A "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond.
As used herein, "an Fc region constant domain portion" or "Fc region portion" refers to the heavy chain constant region segment of the Fc fragment (the "fragment crystallizable" region or Fc region) from an antibody, which can include one or more constant domains, such as CH2, CH3, CH4, or any combination thereof. By way of background, the Fc region is responsible for the effector functions of an immunoglobulin, such as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (e.g., CD 16, CD32, FcRn), greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer (see Capon et al., Nature, 337:525 (1989)).
In addition, antibodies have a hinge sequence that is typically situated between the Fab and Fc region (but a lower section of the hinge may include an amino- terminal portion of the Fc region). By way of background, an immunoglobulin hinge acts as a flexible spacer to allow the Fab portion to move freely in space. In contrast to the constant regions, hinges are structurally diverse, varying in both sequence and length between immunoglobulin classes and even among subclasses. For example, a human IgGl hinge region is freely flexible, which allows the Fab fragments to rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. By comparison, a human IgG2 hinge is relatively short and contains a rigid poly-proline double helix stabilized by four inter-heavy chain disulfide bridges, which restricts the flexibility. A human IgG3 hinge differs from the other subclasses by its unique extended hinge region (about four times as long as the IgGl hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix and providing greater flexibility because the Fab fragments are relatively far away from the Fc fragment. A human IgG4 hinge is shorter than IgGl but has the same length as IgG2, and its flexibility is intermediate between that of IgGl and IgG2.
According to crystallographic studies, an IgG hinge domain can be functionally and structurally subdivided into three regions: the upper, the core or middle, and the lower hinge regions (Shin et al., Immunological Reviews 130:87 (1992)). Exemplary upper hinge regions include EPKSCDKTHT (SEQ ID NO:227) as found in IgGl, ER CCVE (SEQ ID NO:211) as found in IgG2, ELKTPLGDTT HT (SEQ ID NO:245) or EPKSCDTPPP (SEQ ID NO:246) as found in IgG3, and ESKYGPP (SEQ ID NO:247) as found in IgG4. Exemplary middle or core hinge regions include CPPCP (SEQ ID NO:228) as found in IgGl and IgG2, CPRCP (SEQ ID NO:248) as found in IgG3, and CPSCP (SEQ ID NO:249) as found in IgG4. While IgGl, IgG2, and IgG4 antibodies each appear to have a single upper and middle hinge, IgG3 has four in tandem - one being ELKTPLGDTTHTCPRCP (SEQ ID NO:250) and three being EPKSCDTPPP CPRCP (SEQ ID NO:251).
IgA and IgD antibodies appear to lack an IgG-like core region, and IgD appears to have two upper hinge regions in tandem (see SEQ ID NOS:222 and 252). Exemplary wild type upper hinge regions found in IgAl and IgA2 antibodies are set forth in SEQ ID NOS:215 and 216.
IgE and IgM antibodies, in contrast, lack a typical hinge region and instead have a CH2 domain with hinge-like properties. Exemplary wild-type CH2 upper hinge-like sequences of IgE and IgM are set forth in SEQ ID NO:253 (VCSRDFTPPTVKILQSSSDGGGHFPPTIQLLCLVSGYTPGTINITWLEDG
QVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFE
DSTKKCA) and SEQ ID NO:254 (VIAELPPKVSVFVPPRDGFFGNPRKSKLIC QATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTI KESDWLGQSMFTCRVDHRGLTFQQNASSMCVP), respectively.
As used herein, a "hinge region" or a "hinge" refers to (a) an immunoglobulin hinge region (made up of, for example, upper and core regions) or a functional variant thereof, including wild type and altered immunoglobulin hinges, (b) a lectin interdomain region or a functional variant thereof, (c) a cluster of differentiation (CD) molecule stalk region or a functional variant thereof, or (d) a portion of a cell surface receptor (interdomain region) that connects immunoglobulin V-like or immunoglobulin C-like domains.
As used herein, a "wild type immunoglobulin hinge region" refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CHI and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CHI and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, a wild type immunoglobulin hinge region sequence is human, and may comprises a human IgG hinge region. Exemplary human wild type immunoglobulin hinge regions are set forth in SEQ ID NOS:215 (IgAl hinge), 216 (IgA2 hinge), 217 (IgD hinge), 667 (IgGl hinge), 219 (IgG2 hinge), 220 (IgG3 hinge) and 221 (IgG4 hinge).
An "altered wild type immunoglobulin hinge region" or "altered immunoglobulin hinge region" refers to (a) a wild type immunoglobulin hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%>, 15%>, 10%>, or 5%> amino acid substitutions or deletions), or (b) a portion of a wild type immunoglobulin hinge region that has a length of about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to about 120 amino acids (for instance, having a length of about 10 to about 40 amino acids or about 15 to about 30 amino acids or about 15 to about 20 amino acids or about 20 to about 25 amino acids), has up to about 30% amino acid changes (e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutions or deletions or a combination thereof), and has an IgG core hinge region as set forth in SEQ ID NOS:228, 248, or 249.
A "peptide linker" refers to an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In certain embodiments, a linker is comprised of five to about 35 amino acids, for instance, about 15 to about 25 amino acids. "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-10) amino acid residues between two adjacent regions or domains of a single chain polypeptide, such as between a hinge and an adjacent Fc region portion or between a hinge and an adjacent binding domain or between a peptide linker that links two immunoglobulin variable domains and an adjacent immunoglobulin variable domain. Junction amino acids may result from the construct design of a single chain polypeptide (e.g. , amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a single chain polypeptide).
A "linker between CH3 and CHI or CL" refers to one or more (e.g., about 2-12) amino acid residues between the C- terminus of a CH3 domain (e.g. , a wild type CH3 or a mutated CH3) and the N-terminus of a CHI domain or CL domain (e.g. , Ck).
A "wild type immunoglobulin region" or "wild type immunoglobulin domain" refers to a naturally occurring immunoglobulin region or domain (e.g., a naturally occurring VL, VH, hinge, CL, CHI , CH2, CH3, or CH4) from various immunoglobulin classes or subclasses (including, for example, IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, and IgM) and from various species (including, for example, human, sheep, mouse, rat, and other mammals). Exemplary wild type human CHI regions are set forth in SEQ ID NOS: l 14, 186-192 and 194, wild type human CK region in SEQ ID NO: 1 12, wild type human C regions in SEQ ID NO: 1 13 and 224-226, wild type human CH2 domains in SEQ ID NOS: 1 15, 195-201 and 203, wild type human CH3 domains in SEQ ID NOS: 1 16, 204-210 and 212, and wild type human CH4 domains in SEQ ID NO:213 and 214.
An "altered immunoglobulin region," "altered immunoglobulin domain," "mutated immunoglobulin domain," or the like, refers to an immunoglobulin region with a sequence identity to a wild type immunoglobulin region or domain (e.g. , a wild type VL, VH, hinge, CL, CHI , CH2, CH3, or CH4) of at least 75% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). For example, an "altered immunoglobulin CHI region" or "altered CHI region" refers to a CHI region with a sequence identity to a wild type immunoglobulin CHI region (e.g. , a human CHI) of at least 75% (e.g. , 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). Similarly, an "altered immunoglobulin CH2 domain" or "altered CH2 domain" refers to a CH2 domain with a sequence identity to a wild type immunoglobulin CH2 region (e.g. , a human CH2) of at least 75% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). "Sequence identity," as used herein, refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The percentage sequence identity values are generated by the NCBI BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.
In certain embodiments, an altered immunoglobulin domain only contains conservative amino acid substitutions of a wild type immunoglobulin domain. In certain other embodiments, an altered immunoglobulin domain only contains non- conservative amino acid substitutions of a wild type immunoglobulin domain. In yet other embodiments, an altered immunoglobulin domain contains both conservative and non-conservative amino acid substitutions.
A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8). In certain embodiments, a conservative substitution includes a leucine to serine substitution.
As used herein, the term "derivative" refers to a modification of one or more amino acid residues of a peptide by chemical or biological means, either with or without an enzyme, e.g., by glycosylation, alkylation, acylation, ester formation, or amide formation. Generally, a "derivative" differs from an "analogue" in that a parent polypeptide may be the starting material to generate a "derivative," whereas the parent polypeptide may not necessarily be used as the starting material to generate an "analogue." A derivative may have different chemical, biological or physical properties of the parent polypeptide. For example, a derivative may be more hydrophilic or it may have altered reactivity (e.g., a CDR having an amino acid change that alters its affinity for a target) as compared to the parent polypeptide.
As used herein, unless otherwise provided, a position of an amino acid residue in a variable region of an immunoglobulin molecule is numbered according to the Kabat numbering convention (Kabat, Sequences of Proteins of Immunological Interest, 5th ed. Bethesda, MD: Public Health Service, National Institutes of Health (1991)), and a position of an amino acid residue in a constant region of an immunoglobulin molecule is numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94).
A "receptor" is a protein molecule present in the plasma membrane or in the cytoplasm of a cell to which a signal molecule (i.e., a ligand, such as a hormone, a neurotransmitter, a toxin, a cytokine) may attach. The binding of the signal molecule to the receptor results in a conformational change of the receptor, which ordinarily initiates a cellular response. However, some ligands merely block receptors without inducing any response (e.g., antagonists). Some receptor proteins are peripheral membrane proteins. Many hormone and neurotransmitter receptors are transmembrane proteins that are embedded in the phospholipid bilayer of cell membranes, and another major class of receptors are intracellular proteins such as those for steroid and intracrine peptide hormone receptors.
"B-cell associated disorder or disease" or "a disease or disorder associated with aberrant B-cell activity" refers to a disease or disorder associated with (e.g., causing or resulting from) aberrant B-cell activity or activity that deviates from the normal, proper, or expected course. For example, a B-cell associated disorder or disease may include inappropriate proliferation of B-cells that have damaged or defective DNA or other cellular components. Aberrant B-cell activity may include cell proliferation characterized by inappropriately high levels of B-cell division, inappropriately low levels of B-cell apoptosis, or both. Such diseases may have, for example, single or multiple local abnormal proliferations of B-cells, groups of B-cells or tissue(s), whether cancerous or non-cancerous, benign or malignant. A B-cell associated disorder or disease may also include aberrant antibody production, such as production of autoantibodies, or overproduction of antibodies more desirable when produced at normal levels. It is also contemplated herein that aberrant B-cell activity may occur in certain subpopulations of B-cells and not in other subpopulations, or may include inappropriate stimulation of T-cells, such as by inappropriate antigen presentation to T-cells or by other B-cell pathways. Examples of B-cell associated disorders or diseases include a B-cell malignancy or B-cell cancer (e.g., B-cell lymphoma, B-cell leukemia or B-cell myeloma), a disease characterized by autoantibody production (e.g., autoimmune diseases) or inflammation or a disease characterized by inappropriate T-cell stimulation caused by inappropriate B-cell antigen presentation to T-cells or caused by other pathways involving B-cells.
"Treatment," "treating" or "ameliorating" refers to either a therapeutic treatment or prophylactic/preventative treatment. A treatment is therapeutic if at least one symptom of disease in an individual receiving treatment improves or a treatment may delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases.
A "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a specific binding molecule or compound refers to that amount of the compound sufficient to result in amelioration of one or more symptoms of the disease being treated in a statistically significant manner. When referring to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously (in the same formulation or concurrently in separate formulations).
The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or other serious adverse reactions when administered using routes well known in the art.
A "patient in need" refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a polypeptide heterodimer or a composition thereof provided herein.
The term "immunoglobulin-derived fusion protein," as used herein, refers to a fusion protein that comprises at least one immunoglobulin region, such as a VL, VH, CL, CHI, CH2, CH3, and CH4 domain. The immunoglobulin region may be a wild type immunoglobulin region or an altered immunoglobulin region. Exemplary immunoglobulin-derived fusion proteins include single chain variable antibody fragment (scFv) (see, e.g., Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-83, 1988), SMIP™ proteins (see, U.S. Patent Publication Nos. 2003/0133939, 2003/0118592, and 2005/0136049), PIMS proteins (see, PCT Application Publication No. WO 2009/023386), and multi-functional binding proteins (such as SCORPION™ and Xceptor proteins) (see, PCT Application Publication No. WO 2007/146968, U.S. Patent Application Publication No. 2006/0051844, and U.S. Patent No. 7,166,707).
Additional definitions are provided throughout the present disclosure. Polypeptide Heterodimers
In one aspect, the present disclosure provides a polypeptide heterodimer formed by the association of two different single chain polypeptides. The first single chain polypeptide (SCP-I) comprises, consists essentially of, or consists of from one to four binding domains that specifically bind from one to four targets, a hinge (H-I), an immunoglobulin heterodimerization domain (HD-I), and an Fc region portion (FRP-I), whereas the second single chain polypeptide (SCP-II) comprises, consists essentially of, or consists of from zero to four binding domains that specifically bind from zero to four targets, a hinge (H-II), an immunoglobulin heterodimerization domain (HD-II), and an Fc region portion (FRP-II), provided that the polypeptide heterodimer comprises at least two binding domains that specifically bind to one or more targets, for instance, at least two different targets. The H-I and H-II may have the same sequence, but may be different. The HD-I may comprise an immunoglobulin CHI region, and the HD-II may comprise an immunoglobulin CL region. Alternatively, the HD-I may comprise an immunoglobulin CL region, and the HD-II may comprise an immunoglobulin CHI region. The FRP-I and FRP-II may have the same sequence, but may be different. The individual components of the polypeptide heterodimers of the present disclosure are described in detail herein.
Binding Domains
As indicated above, the polypeptide heterodimer of the present disclosure comprises two single chain polypeptides: One single chain polypeptide comprises from one to four binding domains that specifically bind from one to four targets, and the other single chain polypeptide of the polypeptide heterodimer comprises from zero to four binding domains that bind from zero to four targets. The total number of binding domains of the polypeptide heterodimer ranges from about two to eight, and the total number of different targets that the binding domains bind ranges from about one to eight, for instance, from two to eight, two to four, two to three or two targets.
If a single chain polypeptide of a polypeptide heterodimer comprises a single binding domain, the binding domain may be located either amino or carboxyl terminal to the Fc region portion of the single chain polypeptide. For example, a single chain polypeptide comprising two binding domains may have one binding domain located amino terminal and the other carboxyl terminal to the Fc region portion of the single chain polypeptide, or both binding domains may be amino terminal or both carboxyl terminal to the Fc region portion. In another example, a single chain polypeptide may comprise three binding domains wherein (a) two binding domains are amino terminal on different single chain proteins and the third binding domain is carboxyl terminal to the Fc region portion on either SCP-I or SCP-II, (b) two binding domains are carboxyl terminal on different single chain proteins and the third binding domain is amino terminal to the Fc region portion on either SCP-I or SCP-II. In still a further example, a polypeptide heterodimer may comprise four binding domains, wherein two binding domains are located amino terminal to the Fc region portion on different chains and the other two binding domains are located carboxyl terminal to the Fc region portion on different chains. Alternatively, in any of these embodiments, two binding domains may be linked to each other in tandem and located on either SCP-I or SCP-II or both, depending on the number of binding domains present - the tandem stacking is used when five to eight binding domains combined are present in SCP-I and SCP-II.
Binding of a target by a binding domain modulates the interaction between the target (e.g., a receptor or a ligand) and another molecule. In certain embodiments, the binding of a target (e.g., a receptor) by a binding domain stimulates certain functions of the target (e.g., signal transduction) or brings different targets closer together for a biological effect (e.g., directing T cells to a tumor which in turn activates the T cells). In certain other embodiments, the binding of a target by a binding domain blocks the interaction between the target and another molecule and thus interferes, reduces or eliminates certain functions of the target.
A target molecule, which is specifically bound by a binding domain contained in a polypeptide heterodimer of the present disclosure, may be found on or in association with a cell of interest ("target cell"). Exemplary target cells include cancer cells, cells associated with an autoimmune disease or disorder or with an inflammatory disease or disorder, B-cells and T-cells. A target molecule may also not be associated with a cell. Exemplary target molecules not associated with a cell include soluble proteins, secreted proteins, deposited proteins, and extracellular structural (matrix) proteins.
In certain embodiments, binding domains of polypeptide heterodimers of the present disclosure specifically bind a target selected from T-cell targets, tumor antigens, B-cell targets, pro-inflammatory cytokines or chemokines, pro-oncogenic cytokines or growth factors, angiogenic agents, Fc receptors, transferrin receptor, receptor tyrosine kinases (RTKs), TNFSFRs, or any combination thereof. For instance, polypeptide heterodimers of the present disclosure specifically bind a T-cell target and a tumor target.
In certain embodiments, a binding domain of a polypeptide heterodimer of the present disclosure specifically binds a T-cell target, such as a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3y, CD35, and CD3s), CD28, PD-1 , HVEM, BTLA, CD80, CD86, GITR, and TGFBR1.
In certain embodiments, a binding domain of polypeptide heterodimer of the present disclosure specifically binds a TCR complex or a component thereof. For example, in certain embodiments, a binding domain specifically binds to an individual human CD3 chain (e.g., human CD3y chain, human CD35 chain, and human CD3s chain) or a combination of two or more of the individual human CD3 chains (e.g., a complex of human CD3y and human CD3s or a complex of human CD35 and human CD3s). In certain embodiments, a binding domain specifically binds to a human CD3s chain. In certain other embodiments, a binding domain specifically binds to one or more of human TCRa, human TCRP, or a heterodimer formed from human TCRa and human TCRp. In certain other embodiments, a binding domain of the present disclosure binds to a complex formed from one or more human CD3 chains with one or more human TCR chains, such as a complex formed from a human CD3y chain, a human CD35 chain, or a human CD3s chain with a human TCRa chain or a human TCRP chain. In any of these embodiments, a polypeptide heterodimer of the present disclosure may also bind a tumor target.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a tumor target, including RON, c-Met, CEACAM-6, PSMA, EpCAM, CEA, PCTA-1, STEAP-1, PSCA, ALCAM (CD166), EphA2, CD151, CA-125, MUC-1, MAGE-1, TROP2, IGF1R, CD44v6, CD151, TGFBR2, GHRHR, GHR, IL-6R, gpl30, TNFR2, OSMRp, Patched-1, Frizzled, Robol, ίΤβΤ, CD80, CD81, CD86, CCR5, HER-3, HER-4, EGFR, CEA, MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, phCG, Lewis-Y, CD33, CD30, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP-2, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, CD63, B7-H3, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a B-cell target, such as CD 19, CD20, CD21, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, HLA-DR, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a pro-inflammatory cytokine or chemokine, such as TNFa, IL-6, hyperIL-6, IL-2, IL-1, IL-8, IP-10, IFNy, IFNa, RANKL, FASL, TGFp, IL7, IL10, IL17A/F, TWEAK, CSF2, IGF1, IGF2 or BLyS/APRIL, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind a pro-oncogenic cytokine or growth factor, such as HGF, MSP, EGF (including epiregulin, herregulin, β-regulin, neuregulin), HIF-Ι , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, IL-6, hyperIL-6, IL- 8, Wnt, sHH, TGFp, PDGF, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind an angiogenic agent, such as PDGFR, VEGFR1 -4, NRP 1 , Angiopoietin 2, c-Met or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind an Fc receptor, such as CD64, CD32A, CD32B, CD 16, FcRn, or any combination thereof.
In certain embodiments, a binding domain of a polypeptide heterodimer of the present disclosure specifically binds a transferrin receptor, such as CD71.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically binds a receptor tyrosine kinase, such as EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGFIR, EphA2, c-Met, RON, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically binds a TNFSFR, such as TNFR1, TNFR2, TWEAKR, TACI, BAFF-R, BCMA, FAS, OX40, GITR, 4-1-BB, LTbetaR, HVEM, RANK, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure specifically bind hyperIL-6, IL-10, LIGHT, CD40, PDL1, PDL2, or any combination thereof.
In certain embodiments, one or more binding domains of a polypeptide heterodimer of the present disclosure are an agonist of one of the following molecules: IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, TNFR1, TNFR2, DR4, DR5, TWEAKR, FAS, or any combination thereof.
A binding domain may be any peptide that specifically binds a target of interest. Sources of binding domains include antibody variable regions from various species (which can be formatted as antibodies, sFvs, scFvs, Fabs, or soluble VH domain or domain antibodies), including human, rodent, avian, and ovine. Additional sources of binding domains include variable regions of antibodies from other species, such as camelid (from camels, dromedaries, or llamas; Ghahroudi et al. (1997) FEBS Letters 414(3):521-526; Vincke et al. (2009) Journal of Biological Chemistry (2009) 284:3273- 3284; Hamers-Casterman et al. (1993) Nature, 363:446 and Nguyen et al. (1998) J. Mol. Biol, 275:413), nurse sharks (Roux et al. (1998) Proc. Natl. Acad. Sci. (USA) 95: 11804), spotted ratfish (Nguyen et al. (2002) Immunogenetics, 54:39), or lamprey (Herrin et al, (2008) Proc. Natl. Acad. Sci. (USA) 105:2040-2045 and Alder et al. (2008) Nature Immunology 9:319-327). These antibodies can apparently form antigen- binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies") (Jespers et al. (2004) Nature Biotechnology 22: 1161-1165; Cortez-Retamozo et al. (2004) Cancer Research 64:2853-2857; Baral et al. (2006) Nature Medicine 12:580-584, and Barthelemy et al. (2008) Journal of Biological Chemistry 283:3639-3654).
Exemplary anti-CD3 antibodies from which the binding domain of this disclosure may be derived include Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172: 1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herald et al. (2003) J. Clin. Invest. 11 :409), visilizumab (Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766). An exemplary anti-TCR antibody is H57 monoclonal antibody (Lavasani et al. (2007) Scandinavian Journal of Immunology 65:39-47).
An alternative source of binding domains of this disclosure includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as fibrinogen domains {see, e.g., Weisel et al. (1985) Science 230: 1388), Kunitz domains {see, e.g., US Patent No. 6,423,498), ankyrin repeat proteins (Binz et al. (2003) Journal of Molecular Biology 332:489-503 and Binz et al. (2004) Nature Biotechnology 22(5):575-582), fibronectin binding domains (Richards et al. (2003) Journal of Molecular Biology 326: 1475-1488; Parker et al. (2005) Protein Engineering Design and Selection 18(9):435-444 and Hackel et al. (2008) Journal of Molecular Biology 381 : 1238-1252), cysteine-knot miniproteins (Vita et al. (1995) Proc. Natl. Acad. Sci. (USA) 92:6404-6408; Martin et al. (2002) Nature Biotechnology 21 :71-76 and Huang et al. (2005) Structure 13:755-768), tetratricopeptide repeat domains (Main et al. (2003) Structure 11 :497-508 and Cortajarena et al. (2008) ACS Chemical Biology 3: 161-166), leucine-rich repeat domains (Stumpp et al. (2003) Journal of Molecular Biology 332:471-487), lipocalin domains {see, e.g., WO 2006/095164, Beste et al. (1999) Proc. Natl. Acad. Sci. (USA) 96: 1898-1903 and Schonfeld et al. (2009) Proc. Natl. Acad. Sci. (USA) 106:8198-8203), V-like domains {see, e.g., US Patent Application Publication No. 2007/0065431), C-type lectin domains (Zelensky and Gready (2005) FEBS J. 272:6179; Beavil et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:753-757 and Sato et al. (2003) Proc. Natl. Acad. Sci. (USA) 100:7779-7784), mAb2 or FCAB™ {see, e.g., PCT Patent Application Publication Nos. WO 2007/098934; WO 2006/072620), or the like (Nord et al. (1995) Protein Engineering 8(6):601-608; Nord et al. (1997) Nature Biotechnology 15:772-777; Nord et al. (2001) European Journal of Biochemistry 268(15):4269-4277 and Binz et al. (2005) Nature Biotechnology 23: 1257-1268).
Binding domains of this disclosure can be generated as described herein or by a variety of methods known in the art {see, e.g., U.S. Patent Nos. 6,291,161 and 6,291,158). For example, binding domains of this disclosure may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest {see Hoet et al. (2005) Nature Biotechnol. 23:344). Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems {e.g., mice, HUMAB MOUSE®, TC MOUSE™, KM-MOUSE®, llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains of this disclosure.
In certain embodiments, a polypeptide heterodimer comprises a CD86 binding domain, such as a CTLA4 ectodomain, a CD28 ectodomain, or an immunoglobulin variable region binding domain (such as a scFv) specific for CD86 {e.g., from monoclonal antibodies 3D1 or FUN1). In some embodiments, less than an entire ectodomain is used. For example, domains within the CTLA4 ectodomain that bind CD86 and prevent binding of CD86 to CD28 can be used. The CD86 binding domain can block binding of CD86 to CD28 and thereby downregulate T-cell activation.
In certain embodiments, a polypeptide heterodimer comprises an IL-10 agonist, such as IL-10, monoIL-10, or a functional region thereof. "MonoIL-10" refers to an IL-10 molecule having a short linker (GGGSGG, SEQ ID NO:760) that separates the two subdomains of the molecule (amino and carboxyl end domains) so that these subdomains can form an intramolecular dimer.
In certain embodiments, a polypeptide heterodimer comprises an HLA-G agonist, such as an HLA-G5, an HLA-Gl, an HLA-G mutein, or a functional region thereof; an ectodomain of an HLA-G5, an HLA-Gl or an HLA-G mutein; or an immunoglobulin variable region binding domain (such as an scFv) specific for ILT2, ILT4 or KIR2DL4.
In certain embodiments, a polypeptide heterodimer comprises an HGF agonist, such as an HGF or a sub-domain thereof.
In certain embodiments, a polypeptide heterodimer comprises an IL35 agonist, such as an immunoglobulin variable region binding domain (such as an scFv) specific for IL35R or IL35, or a functional region thereof.
In certain embodiments, a polypeptide heterodimer comprises a LIGHT antagonist, such as an immunoglobulin variable region binding domain (such as an scFv) specific for LIGHT, or a HVEM ectodomain or a functional region thereof. In certain embodiments, a polypeptide heterodimer comprises a PD-1 agonist, such as an immunoglobulin variable region binding domain (such as an scFv) specific for PD-1, or a PD-1 ligand (e.g. PD-L1 or PD-L2) or a functional region thereof.
In certain embodiments, a polypeptide heterodimer comprises a BTLA agonist, such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for BTLA, or a HVEM ectodomain or a functional region thereof.
In certain embodiments, a polypeptide heterodimer comprises a GITRL antagonist, such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for GITRL, or a GITR ectodomain, soluble GITR, or a functional region thereof.
In certain embodiments, a polypeptide heterodimer comprises a CD40 antagonist, such as an immunoglobulin-like variable region binding domain (such as an scFv) specific for CD40.
In some embodiments, a binding domain is a single chain Fv fragment
(scFv) that comprises VH and VL regions specific for a target of interest. In certain embodiments, the VH and VL domains are human. Exemplary VH regions include the VH region of 2E12 (anti-CD28) scFv as set forth in SEQ ID NO: 106, the VH region of P2C2 (anti-CD79b) scFv as set forth in SEQ ID NO: 184, the VH region of 5D5 (anti-c- Met) scFv as set forth in SEQ ID NO:258, the VH region of A2 (anti-hyperIL-6) scFv as set forth in SEQ ID NO: 80, the VH region of 3D1 (anti-CD86) scFv as set forth in SEQ ID NO:92, the VH region of MET021 (anti-c-Met) scFv as set forth in SEQ ID NO: 100, the VH region of G19-4 (anti-CD3) scFv as set forth in SEQ ID NO: 103, the VH region of HD37 (anti-CD19) scFv as set forth in SEQ ID NO: l 17, the VH region of M0042 (anti-HLA-DR) scFv as set forth in SEQ ID NO: 121, the VH region of BMA031 (anti-TCR) scFv as set forth in SEQ ID NO:828, the VH region of OKT3-M (anti-CD3) scFv as set forth in SEQ ID NO: 831, and the VH region of HuM291 (anti- CD3) scFv as set forth in SEQ ID NO:835. The nucleotide sequences encoding the VH regions of the A2 (anti-hyperIL-6) and 3D1 (anti-CD86) scFv's are set forth in SEQ ID NOS:79 and 91, respectively.
Exemplary VL domains are the VL region of 2E12 (anti-CD28) scFv as set forth in SEQ ID NO: 107, the VL region of P2C2 (anti-CD79b) scFv as set forth in SEQ ID NO: 182, the VL region of 5D5 (anti-c-Met) scFv as set forth in SEQ ID NO:259, the VL region of A2 (anti-hyper IL-6) scFv as set forth in SEQ ID NO:84, the VL region of 3D1 (anti-CD86) scFv as set forth in SEQ ID NO:96, the VL region of MET021 (anti-c-Met) scFv as set forth in SEQ ID NO: 101, the VL region of G 19-4 (anti-CD3) scFv as set forth in SEQ ID NO: 104, the VL region of HD37 (anti-CD19) scFv as set forth in SEQ ID NO: 1 19, the VL region of M0042 (anti-HLA-DR) scFv as set forth in SEQ ID NO: 122, the VL region of BMA031 (anti-TCR) scFv as set forth in SEQ ID NO:829, the VL region of OKT3-M (anti-CD3) scFv as set forth in SEQ ID NO:833, and the VL region of HuM291 (anti-CD3) scFv as set forth in SEQ ID NO:836. The nucleotide sequences encoding the VL regions of the A2 (anti-hyperIL-6) and 3D1 (anti-CD86) scFv's are set forth in SEQ ID NOS:83 and 95, respectively.
In some embodiments, a binding domain is a single chain Fv fragment (scFv) that comprises VH and VL domains specific for a TCR complex or a component thereof. In certain embodiments, the VH and VL domains are human or humanized VH and VL domains. Exemplary VH domains include BC3 (anti-CD3) VH, OKT3 (anti- CD3) VH, H57 (anti-TCR) VH, and 2C 1 1 (anti-CD3) VH domains as set forth in SEQ ID NOS:301 , 303, 313, and 317, respectively. Further exemplary VH domains include Cris-7 (anti-CD3) VH domains, such as those set forth in SEQ ID NOS:327 and 331- 333. Exemplary VL domains are BC3 (anti-CD3) VL, OKT3 (anti-CD3) VL, H57 (anti- TCR) VL, and 2C1 1 (anti-CD3) VL domains as set forth in SEQ ID NOS:302, 304, 315 and 318, respectively. Further exemplary VL domains include Cris-7 (anti-CD3) VL domains, such as those set forth in SEQ ID NOS:328, 329 and 330.
In certain embodiments, a binding domain comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR comprises zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g., c-Met, RON, CD28, CD79b, CD38, TCRa, TCRp, hyper IL-6, CD86, CD 19, and HLA-DR) and such mutant or derivative still binds its target.
In certain embodiments, a binding domain VH or VL region of the present disclosure can be derived from or based on a VH or VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non- conservative amino acid substitutions), or a combination of the above -noted changes, when compared with the VH or VL of a known monoclonal antibody. The insertion(s), deletion(s) or substitution(s) may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided that a binding domain containing the modified VH or VL region can still specifically bind its target with an affinity about the same as the wild type binding domain.
The VH and VL domains may be arranged in either orientation (i.e., from amino-terminus to carboxyl terminus, VH-VL or VL-VH) and may be joined by an amino acid sequence (e.g., having a length of about five to about 35 amino acids) capable of providing a spacer function such that the two sub-binding domains can interact to form a functional binding domain. In certain embodiments, an amino acid sequence that joins the VH and VL domains (also referred to herein as a "linker") includes those belonging to the (GlynSer) family, such as (Gly3Ser)n(Gly4Ser)i, (Gly3Ser)i(Gly4Ser)n, (Gly3Ser)n(Gly4Ser)n, or (Gly4Ser)n, wherein n is an integer of 1 to 5. In certain embodiments, the linker is GGGGSGGGGS GGGGS (SEQ ID NO: 183) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:108). An additional exemplary linker is GGGGSGGGGSGGGGAS (SEQ ID NO:739). In certain embodiments, these (GlynSer)-based linkers are used to link the VH and VL domains in a binding domain, but are not used to link a binding domain to an immunoglobulin heterodimerization domain or to an Fc region portion.
Exemplary binding domains specific for CD28 include a 2E12 scFv as set forth in SEQ ID NO: 109, binding domains specific for CD79b include a P2C2 scFv as set forth in SEQ ID NO: 185; binding domains specific for c-Met include a 5D5 scFv as set forth in SEQ ID NO:257; binding domains specific for RON include a 4C04 scFv as set forth in SEQ ID NO:261 and a 11H09 scFv as set forth in SEQ ID NO:265; binding domains specific for hyperIL-6 include an A2 scFv as set forth in SEQ ID NO: 86; binding domains specific for CD86 include a 3D1 scFv as set forth in SEQ ID NO:98; binding domains specific for HLA-DR include an M0042 scFv as set forth in SEQ ID NO: 120; binding domains specific for CD3 include a G19-4 scFv as set forth in SEQ ID NO:102, an OKT3-M scFv as set forth in SEQ ID NO:834, and a HuM291 scFv as set forth in SEQ ID NO:837; binding domain specific for CD 19 include a H37 scFv as set forth in SEQ ID NO: 105; and binding domains specific for c-Met include an MET021 scFv as set forth in SEQ ID NO: 120 (with the light chain CDR1, CDR2, CDR3 and heavy chain CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS:296-298 and 464-466, respectively). The nucleotide sequences encoding the A2 (anti-hyperIL6) and 3D1 (anti-CD86) scFv's are set forth in SEQ ID NOS:85 and 97, respectively. Exemplary binding domains that bind a TCR complex or a component thereof include a BMA031 scFv as set forth in SEQ ID NO:830 and other scFv's as set forth in SEQ ID NOS:310, 311, 312, 319, and 334-340.
Additional exemplary binding domains include a PDL2 ectodomain as set forth in SEQ ID NO: 88 and monoIL-10 as set forth in SEQ ID NO: 90. The nucleotide sequences encoding the PDL2 ectodomain and monoIL-10 are set forth in SEQ ID NOS:87 and 89, respectively.
The light chain amino acid sequence of the 4C04 (anti-RON) scFv is set forth in SEQ ID NO:602, and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:604-606, respectively. The heavy chain amino acid sequence of the 4C04 (anti- RON) scFv is set forth in SEQ ID NO:603, and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:607-609, respectively.
The light chain amino acid sequence of the 1 1H09 (anti-RON) scFv is set forth in SEQ ID NO:610, and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:612-614, respectively. The heavy chain amino acid sequence of the 1 1H09 (anti- RON) scFv is set forth in SEQ ID NO:61 1 , and its CDR1 , CDR2, and CDR3 are set forth in SEQ ID NOS:615-617, respectively.
A binding domain may be located either at amino terminal or carboxyl terminal to the Fc region portion of a single chain polypeptide of the present disclosure. In certain embodiments, the binding domain is located at the amino terminus of a single chain polypeptide. In certain other embodiments, the binding domain is located at the carboxyl terminus of a single chain polypeptide. In certain other embodiments, a binding domain is located at both the amino and carboxyl terminus of a single chain polypeptide. Heterodimerization Domains
As indicated above, a polypeptide heterodimer of the present disclosure comprises an immunoglobulin heterodimerization domain in each polypeptide chain. The immunoglobulin heterodimerization domains in the two single chain polypeptides of a polypeptide heterodimer are different from each other and thus may be differentially modified to facilitate heterodimerization of both chains and to minimize homodimerization of either chain. As shown in the examples, immunoglobulin heterodimerization domains provided herein allow for efficient heterodimerization between different polypeptides and facilitate purification of the resulting polypeptide heterodimers.
As provided herein, immunoglobulin heterodimerization domains useful for promoting heterodimerization of two different single chain polypeptides (e.g., one short and one long) according to the present disclosure include immunoglobulin CHI and CL domains, for instance, human CHI and CL domains. In certain embodiments, an immunoglobulin heterodimerization domain is a wild type CHI region, such as a wild type IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, or IgM CHI region. In further embodiments, an immunoglobulin heterodimerization domain is a wild type human IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, or IgM CHI region as set forth in SEQ ID NOS: 1 14, 186-192 and 194, respectively. In certain embodiments, an immunoglobulin heterodimerization domain is a wild type human IgGl CHI region as set forth in SEQ ID NO: 1 14.
In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CHI region, such as an altered IgGl , IgG2, IgG3, IgG4, IgAl , IgA2 IgD, IgE, or IgM CHI region. In certain embodiments, an immunoglobulin heterodimerization domain is an altered human IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, IgD, IgE, or IgM CHI region. In still further embodiments, a cysteine residue of a wild type CHI region (e.g. , a human CHI) involved in forming a disulfide bond with a wild type immunoglobulin CL domain (e.g., a human CL) is deleted or substituted in the altered immunoglobulin CHI region such that a disulfide bond is not formed between the altered CHI region and the wild type CL domain.
In certain embodiments, an immunoglobulin heterodimerization domain is a wild type CL domain, such as a wild type CK domain or a wild type C domain. In particular embodiments, an immunoglobulin heterodimerization domain is a wild type human CK or human domain as set forth in SEQ ID NOS: l 12 and 1 13, respectively. In further embodiments, an immunoglobulin heterodimerization domain is an altered immunoglobulin CL domain, such as an altered CK or domain, for instance, an altered human CK or human domain.
In certain embodiments, a cysteine residue of a wild type CL domain (e.g. , a human CL) involved in forming a disulfide bond with a wild type immunoglobulin CHI region (e.g. , a human CHI) is deleted or substituted in the altered immunoglobulin CL domain. Such altered CL domains may further comprise an amino acid deletion at their amino termini. An exemplary CK domain is set forth in SEQ ID NO: 141 , in which the first arginine and the last cysteine of the wild type human Ck domain are both deleted. In certain embodiments, only the last cysteine of the wild type human Ck domain is deleted in the altered Ck domain because the first arginine deleted from the wild type human Ck domain may be provided by a linker that has an arginine at its carboxyl terminus and links the amino terminus of the altered Ck domain with another domain (e.g. , an Fc region portion). An exemplary domain is set forth in SEQ ID NO: 140, in which the first arginine of a wild type human domain is deleted and the cysteine involved in forming a disulfide bond with a cysteine in a CHI region is substituted by a serine.
In further embodiments, an immunoglobulin heterodimerization domain is an altered CK domain that contains one or more amino acid substitutions, as compared to a wild type CK domain, at positions that may be involved in forming the interchain-hydrogen bond network at a CK-CK interface. For example, in certain embodiments, an immunoglobulin heterodimerization domain is an altered human CK domain having one or more amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 that are substituted with a different amino acid. The numbering of the amino acids is based on their positions in the altered human CK sequence as set forth in SEQ ID NO: 141. In certain embodiments, an immunoglobulin heterodimerization domain is an altered human CK domain having one, two, three or four amino acid substitutions at positions N29, N30, V55, or T70. The amino acid used as a substitute at the above- noted positions may be an alanine, or an amino acid residue with a bulk side chain moiety such as arginine, tryptophan, tyrosine, glutamate, glutamine, or lysine. Additional amino acid residues that may be used to substitute amino acid residues of the wild type human Ck sequence at the above noted positions (e.g., N30) include aspartate, methionine, serine and phenyalanine. Exemplary altered human CK domains are set forth in SEQ ID NOS: 142-178. Altered human CK domains are those that facilitate heterodimerization with a CHI region, but minimize homodimerization with another CK domain. Representative altered human CK domains are set forth in SEQ ID NOS: 160 (N29W V55A T70A), 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30M V55A T70E), 840 (N30S V55A T70E), and 841 (N30F V55A T70E).
In certain embodiments, in addition to or alternative to the mutations in Ck domains described herein, both the immunoglobulin heterodimerization domains (i.e., immunoglobulin CHI and CL domains) of a polypeptide heterodimer have mutations so that the resulting immunoglobulin heterodimerization domains form salt bridges (i.e., ionic interactions) between the amino acid residues at the mutated sites. For example, the immunoglobulin heterodimerization domains of a polypeptide heterodimer may be a mutated CHI domain in combination with a mutated Ck domain. In the mutated CHI domain, valine at position 68 (V68) of the wild type human CHI domain is substituted by an amino acid residue having a negative charge (e.g., aspartate or glutamate), whereas leucine at position 29 (L29) of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted is substituted by an amino acid residue having a positive charge (e.g., lysine, arginine or histidine). The charge-charge interaction between the amino acid residue having a negative charge of the resulting mutated CHI domain and the amino acid residue having a positive charge of the resulting mutated Ck domain forms a salt bridge, which stabilizes the heterodimeric interface between the mutated CHI and Ck domains. Alternatively, V68 of the wild type CHI may be substituted by an amino acid residue having a positive charge, whereas L29 of a mutated human Ck domain in which the first arginine and the last cysteine have been deleted may be substituted by an amino acid residue having a negative charge. Exemplary mutated CHI sequences in which V68 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:844 and 845. Exemplary mutated Ck sequences in which L29 is substituted by an amino acid with either a negative or positive charge are set forth in SEQ ID NOS:842 and 843.
Positions other than V68 of human CHI domain and L29 of human Ck domain may be substituted with amino acids having opposite charges to produce ionic interactions between the amino acids in addition or alternative to the mutations in V68 of CHI domain and L29 of Ck domain. Such positions can be identified by any suitable method, including random mutagenesis, analysis of the crystal structure of the CHl-Ck pair to identify amino acid residues at the CHl-Ck interface, and further identifying suitable positions among the amino acid residues at the CHl-Ck interface using a set of criteria (e.g. , propensity to engage in ionic interactions, proximity to a potential partner residue, etc.).
In certain embodiments, polypeptide heterodimers of the present disclosure contain only one pair of immunoglobulin heterodimerization domains. For example, a first chain of a polypeptide heterodimer may comprise a CHI region as an immunoglobulin heterodimerization domain, while a second chain may comprise a CL domain (e.g. , a CK or CX) as an immunoglobulin heterodimerization domain. Alternatively, a first chain may comprise a CL region (e.g. , a CK or CX) as an immunoglobulin heterodimerization domain, while a second chain may comprise a CHI region as an immunoglobulin heterodimerization domain. As set forth herein, the immunoglobulin heterodimerization domains of the first and second chains are capable of associating to form a polypeptide heterodimer of this disclosure.
In certain other embodiments, polypeptide heterodimers of the present disclosure may have two pairs of immunoglobulin heterodimerization domains. For example, a first chain of a polypeptide heterodimer may comprise two CHI regions, while a second chain may have two CL domains that associate with the two CHI regions in the first chain. Alternatively, a first chain may comprise two CL domains, while a second chain may have two CHI regions that associate with the two CL domains in the first chain. In certain embodiments, a first chain polypeptide comprises a CHI region and a CL domain, while a second chain polypeptide comprises a CL domain and a CHI region that associate with the CHI region and the CL domain, respectively, of the first chain polypeptide. In the embodiments where a polypeptide heterodimer comprises only one heterodimerization pair (i.e., one immunoglobulin heterodimerization domain in each chain), the immunoglobulin heterodimerization domain of each chain may be located amino terminal to the Fc region portion of that chain. Alternatively, the immunoglobulin heterodimerization domain in each chain may be located carboxyl terminal to the Fc region portion of that chain.
In the embodiments where a polypeptide heterodimer comprises two heterodimerization pairs (i.e., two immunoglobulin heterodimerization domains in each chain), both immunoglobulin heterodimerization domains in each chain may be located amino terminal to the Fc region portion of that chain. Alternatively, both immunoglobulin heterodimerization domains in each chain may be located carboxyl terminal to the Fc region portion of that chain. In further embodiments, one immunoglobulin heterodimerization domain in each chain may be located amino terminal to the Fc region portion of that chain, while the other immunoglobulin heterodimerization domain of each chain may be located carboxyl terminal to the Fc region portion of that chain. In other words, in those embodiments, the Fc region portion is interposed between the two immunoglobulin heterodimerization domains of each chain.
Fc region portion
As indicated herein, polypeptide heterodimers of the present disclosure comprise an Fc region constant domain portion (also referred to as an Fc region portion) in each polypeptide chain. The inclusion of an Fc region portion slows clearance of the heterodimers from circulation after administration to a subject. By mutations or other alterations, the Fc region portion further enables relatively easy modulation of heterodimer polypeptide effector functions (e.g., ADCC, ADCP, CDC, complement fixation and binding to Fc receptors), which can either be increased or decreased depending on the disease being treated, as known in the art and described herein. In certain embodiments, an Fc region portion of polypeptide heterodimers of the present disclosure will be capable of mediating one or more of these effector functions.
An Fc region portion present in single chain polypeptides that form part of the polypeptide heterodimers of the present disclosure may comprise a CH2 domain, a CH3 domain, a CH4 domain or any combination thereof. For example, an Fc region portion may comprise a CH2 domain, a CH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3 domains, a CH4 domain, or two CH4 domains.
A CH2 domain that may form an Fc region portion of a single chain polypeptide of a heterodimer of the present disclosure may be a wild type immunoglobulin CH2 domain or an altered immunoglobulin CH2 domain thereof from certain immunoglobulin classes or subclasses (e.g., IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD) and from various species (including human, mouse, rat, and other mammals).
In certain embodiments, a CH2 domain is a wild type human immunoglobulin CH2 domain, such as wild type CH2 domains of human IgGl , IgG2, IgG3, IgG4, IgAl , IgA2, or IgD, as set forth in SEQ ID NOS: 1 15, 199-201 and 195- 197, respectively. In certain embodiments, the CH2 domain is a wild type human IgGl CH2 domain as set forth in SEQ ID NO: 1 15.
In certain embodiments, a CH2 domain is an altered immunoglobulin
CH2 region (e.g., an altered human IgGl CH2 domain) that comprises an amino acid substitution at the asparagine of position 297 (e.g., asparagine to alanine). Such an amino acid substitution reduces or eliminates glycosylation at this site and abrogates efficient Fc binding to FcyR and C lq. The sequence of an altered human IgGl CH2 domain with an Asn to Ala substitution at position 297 is set forth in SEQ ID NO:324.
In certain embodiments, a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human IgGl CH2 domain) that comprises at least one substitution or deletion at positions 234 to 238. For example, an immunoglobulin CH2 region can comprise a substitution at position 234, 235, 236, 237 or 238, positions 234 and 235, positions 234 and 236, positions 234 and 237, positions 234 and 238, positions 234-236, positions 234, 235 and 237, positions 234, 236 and 238, positions 234, 235, 237, and 238, positions 236-238, or any other combination of two, three, four, or five amino acids at positions 234-238. In addition or alternatively, an altered CH2 region may comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, for instance, at one of position 236 or position 237 while the other position is substituted. The above -noted mutation(s) decrease or eliminate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide heterodimer that comprises the altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 has been replaced with one or more alanine residues. In further embodiments, only one of the amino acid residues at positions 234-238 have been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g., alanine or serine).
In certain other embodiments, a CH2 domain is an altered immunoglobulin CH2 region (e.g., an altered human IgGl CH2 domain) that comprises one or more amino acid substitutions at positions 253, 310, 318, 320, 322, and 331. For example, an immunoglobulin CH2 region can comprise a substitution at position 253, 310, 318, 320, 322, or 331 , positions 318 and 320, positions 318 and 322, positions 318, 320 and 322, or any other combination of two, three, four, five or six amino acids at positions 253, 310, 318, 320, 322, and 331. The above-noted mutation(s) decrease or eliminate the complement-dependent cytotoxicity (CDC) of a polypeptide heterodimer that comprises the altered CH2 domain.
In certain other embodiments, in addition to the amino acid substitution at position 297, an altered CH2 region (e.g., an altered human IgGl CH2 domain) can further comprise one or more (e.g., two, three, four, or five) additional substitutions at positions 234-238. For example, an immunoglobulin CH2 region can comprise a substitution at positions 234 and 297, positions 234, 235, and 297, positions 234, 236 and 297, positions 234-236 and 297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297, positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or any combination of two, three, four, or five amino acids at positions 234-238 in addition to position 297. In addition or alternatively, an altered CH2 region may comprise one or more (e.g., two, three, four or five) amino acid deletions at positions 234-238, such as at position 236 or position 237. The additional mutation(s) decreases or eliminates the antibody-dependent cell-mediated cytotoxicity (ADCC) activity or Fc receptor-binding capability of a polypeptide heterodimer that comprises the altered CH2 domain. In certain embodiments, the amino acid residues at one or more of positions 234-238 have been replaced with one or more alanine residues. In further embodiments, only one of the amino acid residues at positions 234-238 has been deleted while one or more of the remaining amino acids at positions 234-238 can be substituted with another amino acid (e.g., alanine or serine).
In certain embodiments, in addition to one or more (e.g., 2, 3, 4, or 5) amino acid substitutions at positions 234-238, a mutated CH2 region (e.g., an altered human IgGl CH2 domain) in a fusion protein of the present disclosure may contain one or more (e.g., 2, 3, 4, 5, or 6) additional amino acid substitutions (e.g., substituted with alanine) at one or more positions involved in complement fixation (e.g., at positions 1253, H310, E318, K320, K322, or P331). Examples of mutated immunoglobulin CH2 regions include human IgGl , IgG2, IgG4 and mouse IgG2a CH2 regions with alanine substitutions at positions 234, 235, 237 (if present), 318, 320 and 322. An exemplary mutated immunoglobulin CH2 region is mouse IGHG2c CH2 region with alanine substitutions at L234, L235, G237, E318, K320, and K322 (SEQ ID NO:314).
In still further embodiments, in addition to the amino acid substitution at position 297 and the additional deletion(s) or substitution(s) at positions 234-238, an altered CH2 region (e.g., an altered human IgGl CH2 domain) can further comprise one or more (e.g., two, three, four, five, or six) additional substitutions at positions 253, 310, 318, 320, 322, and 331. For example, an immunoglobulin CH2 region can comprise a (1) substitution at position 297, (2) one or more substitutions or deletions or a combination thereof at positions 234-238, and one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions at positions 1253, H310, E318, K320, K322, and P331 , such as one, two, three substitutions at positions E318, K320 and K322. The amino acids at the above-noted positions may be substituted by alanine or serine.
In certain embodiments, an immunoglobulin CH2 region polypeptide comprises: (i) an amino acid substitution at the asparagines of position 297 and one amino acid substitution at position 234, 235, 236 or 237; (ii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at two of positions 234- 237; (iii) an amino acid substitution at the asparagine of position 297 and amino acid substitutions at three of positions 234-237; (iv) an amino acid substitution at the asparagine of position 297, amino acid substitutions at positions 234, 235 and 237, and an amino acid deletion at position 236; (v) amino acid substitutions at three of positions 234-237 and amino acid substitutions at positions 318, 320 and 322; or (vi) amino acid substitutions at three of positions 234-237, an amino acid deletion at position 236, and amino acid substitutions at positions 318, 320 and 322.
Exemplary altered immunoglobulin CH2 regions with amino acid substitutions at the asparagine of position 297 include: human IgGl CH2 region with alanine substitutions at L234, L235, G237 and N297 and a deletion at G236 (SEQ ID NO:325), human IgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQ ID NO:326), human IgG4 CH2 region with alanine substitutions at F234, L235, G237 and N297 and a deletion of G236 (SEQ ID NO:322), human IgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NO:343), human IgG4 CH2 region with alanine substitutions at L235 and N297 (SEQ ID NO:344), human IgG4 CH2 region with alanine substitutions at G236 and N297 (SEQ ID NO:345), and human IgG4 CH2 region with alanine substitutions at G237 and N297 (SEQ ID NO:346).
In certain embodiments, in addition to the amino acid substitutions described above, an altered CH2 region (e.g., an altered human IgGl CH2 domain) may contain one or more additional amino acid substitutions at one or more positions other than the above-noted positions. Such amino acid substitutions may be conservative or non-conservative amino acid substitutions. For example, in certain embodiments, P233 may be changed to E233 in an altered IgG2 CH2 region (see, e.g., SEQ ID NO:326). In addition or alternatively, in certain embodiments, the altered CH2 region may contain one or more amino acid insertions, deletions, or both. The insertion(s), deletion(s) or substitution(s) may anywhere in an immunoglobulin CH2 region, such as at the N- or C- terminus of a wild type immunoglobulin CH2 region resulting from linking the CH2 region with another region (e.g., a binding domain or an immunoglobulin heterodimerization domain) via a hinge.
In certain embodiments, an altered CH2 region in a polypeptide heterodimer of the present disclosure comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%), at least 98%>, at least 99% identical to a wild type immunoglobulin CH2 region, such as the CH2 region of wild type human IgGl, IgG2, or IgG4, or mouse IgG2a (e.g., IGHG2c).
An altered immunoglobulin CH2 region in a polypeptide heterodimer of the present disclosure may be derived from a CH2 region of various immunoglobulin isotypes, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and IgD, from various species (including human, mouse, rat, and other mammals). In certain embodiments, an altered immunoglobulin CH2 region in a fusion protein of the present disclosure may be derived from a CH2 region of human IgGl, IgG2 or IgG4, or mouse IgG2a (e.g., IGHG2c), whose sequences are set forth in SEQ ID NOS: l 15, 199, 201 and 320.
In certain embodiments, an altered CH2 domain is a human IgGl CH2 domain with alanine substitutions at positions 235, 318, 320, and 322 (i.e., a human IgGl CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:595), and optionally an N297 mutation (e.g., to alanine). In certain other embodiments, an altered CH2 domain is a human IgGl CH2 domain with alanine substitutions at positions 234, 235, 237, 318, 320 and 322 (i.e., a human IgGl CH2 domain with L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ ID NO:596), and optionally an N297 mutation (e.g., to alanine).
In certain embodiments, an altered CH2 domain is an altered human IgGl CH2 domain with mutations known in the art that enhance immunological activities such as ADCC, ADCP, CDC, complement fixation, Fc receptor binding, or any combination thereof.
The CH3 domain that may form an Fc region portion of a single chain polypeptide of a heterodimer of the present disclosure may be a wild type immunoglobulin CH3 domain or an altered immunoglobulin CH3 domain thereof from certain immunoglobulin classes or subclasses (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM) of various species (including human, mouse, rat, and other mammals). In certain embodiments, a CH3 domain is a wild type human immunoglobulin CH3 domain, such as wild type CH3 domains of human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM as set forth in SEQ ID NOS:l 16, 208-210, 204-207, and 212, respectively. In certain embodiments, the CH3 domain is a wild type human IgGl CH3 domain as set forth in SEQ ID NO: 116. In certain embodiments, a CH3 domain is an altered human immunoglobulin CH3 domain, such as an altered CH3 domain based on or derived from a wild-type CH3 domain of human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM antibodies. For example, an altered CH3 domain may be a human IgGl CH3 domain with one or two mutations at positions H433 and N434 (positions are numbered according to EU numbering). The mutations in such positions may be involved in complement fixation. In certain other embodiments, an altered CH3 domain may be a human IgGl CH3 domain but with one or two amino acid substitutions at position F405 or Y407. The amino acids at such positions are involved in interacting with another CH3 domain. In certain embodiments, an altered CH3 domain may be an altered human IgGl CH3 domain with its last lysine deleted. The sequence of this altered CH3 domain is set forth in SEQ ID NO:761.
In certain embodiments, a polypeptide heterodimer comprises a CH3 pair that comprises so called "knobs-into-holes" mutations (see, Marvin and Zhu, Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et ah, Protein Engineering 9:617-21, 1966). More specifically, mutations may be introduced into each of the two CH3 domains so that the steric complementarity required for CH3/CH3 association obligates these two CH3 domains to pair with each other. For example, a CH3 domain in one single chain polypeptide of a polypeptide heterodimer may contain a T366W mutation (a "knob" mutation, which substitutes a small amino acid with a larger one), and a CH3 domain in the other single chain polypeptide of the polypeptide heterodimer may contain a Y407A mutation (a "hole" mutation, which substitutes a large amino acid with a smaller one). Other exemplary knobs-into-holes mutations include (1) a T366Y mutation in one CH3 domain and a Y407T in the other CH3 domain, and (2) a T366W mutation in one CH3 domain and T366S, L368A and Y407V mutations in the other CH3 domain.
The CH4 domain that may form an Fc region portion of a single chain polypeptide of a heterodimer of the present disclosure may be a wild type immunoglobulin CH4 domain or an altered immunoglobulin CH4 domain thereof from IgE or IgM molecules. In certain embodiments, the CH4 domain is a wild type human immunoglobulin CH4 domain, such as wild type CH4 domains of human IgE and IgM molecules as set forth in SEQ ID NOS:213 and 214, respectively. In certain embodiments, a CH4 domain is an altered human immunoglobulin CH4 domain, such as an altered CH4 domain based on or derived from a CH4 domain of human IgE or IgM molecules, which have mutations that increase or decrease an immunological activity known to be associated with an IgE or IgM Fc region.
In certain embodiments, an Fc region constant domain portion in heterodimers of the present disclosure comprises a combination of CH2, CH3 or CH4 domains (i.e., more than one constant sub-domain selected from CH2, CH3 and CH4). For example, the Fc region portion may comprise CH2 and CH3 domains or CH3 and CH4 domains. In certain other embodiments, the Fc region portion may comprise two CH3 domains and no CH2 or CH4 domains (i.e., only two or more CH3). The multiple constant sub-domains that form an Fc region portion may be based on or derived from the same immunoglobulin molecule, or the same class or subclass immunoglobulin molecules. In certain embodiments, the Fc region portion is an IgG CH2CH3 (e.g., IgGl CH2CH3, IgG2 CH2CH3, and IgG4 CH2CH3) and may be a human (e.g., human IgGl, IgG2, and IgG4) CH2CH3. For example, in certain embodiments, the Fc region portion comprises (1) wild type human IgGl CH2 and CH3 domains, (2) human IgGl CH2 with N297A substitution (i.e., CH2(N297A)) and wild type human IgGl CH3, or (3) human IgGl CH2(N297A) and an altered human IgGl CH3 with the last lysine deleted.
Alternatively, the multiple constant sub-domains may be based on or derived from different immunoglobulin molecules, or different classes or subclasses immunoglobulin molecules. For example, in certain embodiments, an Fc region portion comprises both human IgM CH3 domain and human IgGl CH3 domain. The multiple constant sub-domains that form an Fc region portion may be directly linked together or may be linked to each other via one or more (e.g., about 2-10) amino acids.
Exemplary Fc region portions are set forth in SEQ ID NOS:305-309,
321, 323, 341, 342, and 762.
In certain embodiments, the Fc region portions of both single chain polypeptides of a polypeptide heterodimer are identical to each other. In certain other embodiments, the Fc region portion of one single chain polypeptide of a polypeptide heterodimer is different from the Fc region portion of the other single chain polypeptide of the heterodimer. For example, one Fc region portion may contain a CH3 domain with a "knob" mutation, whereas the other Fc region portion may contain a CH3 domain with a "hole" mutation.
Hinge
A hinge region contained in a single chain polypeptide of a polypeptide heterodimer according to the present disclosure may be located (a) immediately amino terminal to an Fc region portion (e.g., depending on the isotype, amino terminal to a CH2 domain wherein the Fc region portion is a CH2CH3, or amino terminal to a CH3 domain wherein the Fc region portion is a CH3CH4), (b) interposed between and connecting a binding domain (e.g., scFv) and an immunoglobulin heterodimerization domain, (c) interposed between and connecting an immunoglobulin heterodimerization domain and an Fc region portion (e.g., wherein the Fc region portion is a CH2CH3 or a CH3CH4, depending on the isotype or isotypes), (d) interposed between and connecting an Fc region portion and a binding domain, (e) at the amino terminus of the single chain polypeptide, or (f) at the carboxyl terminus of the single chain polypeptide. The single chain polypeptide comprising a hinge region as described herein will be capable of associating with a different single chain fusion polypeptide to form a polypeptide heterodimer provided herein, and the polypeptide heterodimer formed will contain a binding domain that retains its target specificity or its specific target binding affinity.
In certain embodiments, a hinge present in a single chain polypeptide that forms a polypeptide heterodimer with another single chain polypeptide may be an immunoglobulin hinge region, such as a wild type immunoglobulin hinge region or an altered immunoglobulin hinge region thereof.
In certain embodiments, a hinge is a wild type human immunoglobulin hinge region (e.g., human immunoglobulin hinge regions as set forth in SEQ ID NOS:215-221). In certain other embodiments, one or more amino acid residues may be added at the amino- or carboxy- terminus of a wild type immunoglobulin hinge region as part of a fusion protein construct design. For example, additional junction amino acid residues at the hinge amino-terminus can be "RT," "RSS," "TG," or "T", or at the hinge carboxy-terminus can be "SG", or a hinge deletion can be combined with an addition, such as ΔΡ with "SG" added at the carboxyl terminus.
In certain embodiments, a hinge is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region is substituted with one or more other amino acid residues (e.g., serine or alanine). For example, a hinge may be an altered immunoglobulin hinge based on or derived from a wild type human IgGl hinge as set forth in SEQ ID NO:667, which from amino terminus to carboxyl terminus comprises the upper hinge region (EPKSCDKTHT, SEQ ID NO:227) and the core hinge region (CPPCP, SEQ ID NO:228). Exemplary altered immunoglobulin hinges include an immunoglobulin human IgGl hinge region having one, two or three cysteine residues found in a wild type human IgGl hinge substituted by one, two or three different amino acid residues (e.g., serine or alanine). An altered immunoglobulin hinge may additionally have a proline substituted with another amino acid (e.g., serine or alanine). For example, the above-described altered human IgGl hinge may additionally have a proline located carboxyl terminal to the three cysteines of wild type human IgGl hinge region substituted by another amino acid residue (e.g., serine, alanine). In one embodiment, the prolines of the core hinge region are not substituted. Exemplary altered immunoglobulin hinges are set forth in SEQ ID NOS: 229-240, 255, 664-677, and 748-759. An example of an altered IgGl hinge is an altered human IgGl hinge in which the first cysteine is substituted by serine. The sequence of this altered IgGl hinge is set forth in SEQ ID NO:664, and is referred to as the " human IgGl SCC-P hinge" or "SCC-P hinge." In certain embodiments, one or more amino acid residues (e.g., "RT," "RSS," or "T") may be added at the amino-or carboxy-terminus of a mutated immunoglobulin hinge region as part of a fusion protein construct design.
In certain embodiments, a hinge polypeptide comprises or is a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild type immunoglobulin hinge region, such as a wild type human IgGl hinge, a wild type human IgG2 hinge, or a wild type human IgG4 hinge.
In further embodiments, a hinge present in a single chain polypeptide that forms a polypeptide heterodimer with another single chain polypeptide may be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild type immunoglobulin hinge or an altered immunoglobulin hinge). These types of non- immunoglobulin based hinges may be used on or near the carboxyl end (e.g., located carboxyl terminal to Fc region portions) of the single chain polypeptides that form the polypeptide heterodimers. Examples for such hinges include peptides of about five to about 150 amino acids of the interdomain or stalk region of type II C-lectins or CD molecules, for instance, peptides of about eight to 25 amino acids and peptides of about seven to 18 amino acids, and derivatives thereof.
The "interdomain or stalk region" of a type II C-lectin or CD molecule refers to the portion of the extracellular domain of the type II C-lectin or CD molecule that is located between the C-type lectin- like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the transmembrane domain. For example, in the human CD94 molecule (GenBank Accession No. AAC50291.1, PRI November 30, 1995), the extracellular domain corresponds to amino acid residues 34-179, whereas the CTLD corresponds to amino acid residues 61-176. Accordingly, the interdomain or stalk region of the human CD94 molecule includes amino acid residues 34-60, which is found between the membrane and the CTLD (see Boyington et al., Immunity 10:75, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:753, 1992; and Figdor et al, Nature Rev. Immunol. 2:77, 2002). These type II C-lectin or CD molecules may also have from six to 10 junction amino acids between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1, PRI June 15, 2010) has a transmembrane domain ranging from amino acids 71-93 and an extracellular domain ranging from amino acids 94-233. The CTLD is comprised of amino acids 119-231, and the stalk region comprises amino acids 99-116, which is flanked by junctions of five and two amino acids. Other type II C-lectin or CD molecules, as well as their extracellular ligand-bind domains, interdomain or stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1, PRI July 15, 2006; NP 001773.1, PRI June 20, 1010; AAL65234.1, PRI January 17, 2002, and CAA04925.1, PRI November 14, 2006, for the sequences of human CD23, CD69, CD72, NKG2A and NKG2D and their descriptions, respectively).
A "derivative" of an interdomain or stalk region, or fragment thereof, of a type II C-lectin or CD molecule includes an about an eight to about 150 amino acid sequence in which one, two, or three amino acids of the stalk region of a wild type type II C-lectin or CD molecule have a deletion, insertion, substitution, or any combination thereof, for instance, the one or more changes are substitutions or the one or more mutations include only one deletion. In further embodiments, a derivative of an interdomain or stalk region is more resistant to proteolytic cleavage as compared to the wild-type interdomain or stalk region sequence, such as those derived from about eight to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94.
In certain embodiments, interdomain or stalk region hinges have seven to 18 amino acids and can form an a-helical coiled coil structure. In certain embodiments, interdomain or stalk region hinges contain 0, 1, 2, 3, or 4 cysteines. Exemplary interdomain or stalk region hinges are peptide fragments of the interdomain or stalk regions, such as ten to 150 amino acid fragments from the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D, as set forth in SEQ ID NOS :241-244, 716 and 601. Additional exemplary stalk region or interdomain hinges include those as set forth in SEQ ID NOS:78, 734-737, 742-747, and 766-790.
Alternative hinges that can be used in single chain polypeptides of polypeptide heterodimers are from portions of cell surface receptors (interdomain regions) that connect immunoglobulin V-like or immunoglobulin C-like domains. Regions between Ig V-like domains where the cell surface receptor contains multiple Ig V-like domains in tandem and between Ig C-like domains where the cell surface receptor contains multiple tandem Ig C-like regions are also contemplated as hinges useful in single chain polypeptides of polypeptide heterodimers. In certain embodiments, hinge sequences comprised of cell surface receptor interdomain regions may further contain a naturally occurring or added motif, such as an IgG core hinge sequence that confers one or more disulfide bonds to stabilize the polypeptide heterodimer formation. Examples of hinges include interdomain regions between the Ig V-like and Ig C-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD 150, CD 166, and CD244.
In certain embodiments, hinge sequences have about 5 to 150 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, 40 to 50 amino acids, 50 to 60 amino acids, 5 to 60 amino acids, 5 to 40 amino acids, 8 to 20 amino acids, or 10 to 15 amino acids. The hinge may be primarily flexible, but may also provide more rigid characteristics or may contain primarily a- helical structure with minimal β-sheet structure. The lengths or the sequences of the hinges may affect the binding affinities of the binding domains to which the hinges are directly or indirectly (via another region or domain, such as an immunoglobulin heterodimerization domain) connected as well as one or more activities of the Fc region portions to which the hinges are directly or indirectly connected (see, Examples 9 and 10).
In certain embodiments, hinge sequences are stable in plasma and serum and are resistant to proteolytic cleavage. The first lysine in the IgGl upper hinge region may be mutated to minimize proteolytic cleavage, for instance, the lysine may be substituted with methionine, threonine, alanine or glycine, or is deleted (see, e.g., SEQ ID NOS:379-434, which may include junction amino acids at the amino terminus such as RT).
In some embodiments, hinge sequences may contain a naturally occurring or added motif such as an immunoglobulin hinge core structure CPPCP (SEQ ID NO:228) that confers the capacity to form a disulfide bond or multiple disulfide bonds to stabilize the carboxy-terminus of a molecule. In other embodiments, hinge sequences may contain one or more glycosylation sites.
Exemplary hinges, including altered immunoglobulin hinges, are set forth in SEQ ID NOS:379-434, 618-749, and 763-791.
In certain embodiments, a single chain polypeptide of a polypeptide heterodimer according to the present disclosure comprises more than one hinge. For example, a single chain polypeptide having two binding domains, one of which at the amino terminus and the other at the carboxyl terminus, may have two hinges. One hinge may be directly or indirectly (e.g., via an immunoglobulin heterodimerization domain) connected to the binding domain at or near the amino terminus, and the other hinge may be connected (e.g., directly connected) to the other binding domain at or near the carboxyl terminus. In certain embodiments, even if a single chain polypeptide has only one binding domain, it may have more than one hinge, for example, at its amino or carboxyl terminus. Such a hinge may interact with a corresponding hinge in the other chain of the heterodimer, such as forming one or more interchain disulfide bonds, to facilitate or enhance the heterodimerization of the two chains. A hinge (H-I) of a SCP-I of a polypeptide heterodimer "corresponds to" a hinge (H-II) of a SCP-II of the heterodimer when H-I and H-II are located on the same end of the Fc region portion of their respective single chain polypeptide. For example, a polypeptide heterodimer may comprise the following two single chain polypeptides: A first chain polypeptide from amino to carboxyl terminus comprises a first binding domain, CHI, hinge, CH2, and CH3, and a second chain polypeptide from amino to carboxyl terminus comprises a CK, first hinge, CH2, CH3, second hinge, and a second binding domain. The hinge in the first chain would be regarded as "corresponding" to the first hinge of the second chain because both are amino terminal to the Fc region portions to which they are connected.
In certain embodiments where a single chain polypeptide of a polypeptide heterodimer comprises a binding domain at or near its carboxyl terminus, a hinge may be present to link the binding domain with another portion of the single chain polypeptide (e.g. , an Fc region portion or an immunoglobulin heterodimerization domain). In one embodiment, such a hinge is a non-immunoglobulin hinge (i.e., a hinge not based on or derived from a wild type immunoglobulin hinge) and may be a stalk region of a type II C-lectin or CD molecule, an interdomain region that connect IgV-like or IgC-like domains of a cell surface receptor, or a derivative or functional variant thereof. Exemplary carboxyl terminal hinges, sometimes referred to as "back- end" hinges, includes those set forth in SEQ ID NOS:78, 734-737, 742-747, and 766- 790.
In certain embodiments, a hinge of one single chain polypeptide of a polypeptide heterodimer is identical to a corresponding hinge of the other single chain polypeptide of the heterodimer. In certain other embodiments, a hinge of one chain is different from that of the other chain (in their length or sequence). The different hinges in the different chains allow different manipulation of the binding affinities of the binding domains to which the hinges are connected, so that the heterodimer is able to preferentially bind to the target of one binding domain over the target of the other binding domain. For example, in certain embodiments, a polypeptide heterodimer has a CD3 or TCR binding domain in one chain and a tumor antigen binding domain in another chain. Having two different hinges in the two chains may allow the heterodimer to bind to the tumor antigen first, and then to a CD3 or TCR molecule second. Thus, the heterodimer may recruit CD3+ T cells to tumor cells carrying the tumor antigen, which in turn may damage or destroy the tumor cells. Other components or modifications
In certain embodiments, a single chain polypeptide that forms a heterodimer with another single chain polypeptide may contain one or more additional domains or regions. Such additional regions may be a leader sequence (also referred to as "signal peptide") at the amino-terminus for secretion of an expressed single chain polypeptide. Exemplary leader peptides of this disclosure include natural leader sequences or others, such as those as set forth in SEQ ID NOS: 1 10 and 1 1 1.
Additional regions may also be sequences at the carboxy-terminus for identifying or purifying single chain polypeptides (e.g., epitope tags for detection or purification, such as a histidine tag, biotin, a FLAG® epitope, or any combination thereof).
Further optional regions may be additional amino acid residues (referred to as "junction amino acids" or "junction amino acid residues") having a length of 1 to about 10 amino acids (e.g., about 2 to 5 amino acids), which may be resulted from use of specific expression systems or construct design for the single chain polypeptides of the present disclosure. Such additional amino acid residues (for instance, one, two, three, four or five additional amino acids) may be present at the amino or carboxyl terminus or between various regions or domains of a single chain polypeptide, such as between a binding domain and an immunoglobulin heterodimerization domain, between an immunoglobulin heterodimerization domain and a hinge, between a hinge and an Fc region portion, between domains of an Fc region portion (e.g., between CH2 and CH3 domains or between two CH3 domains), between a binding domain and a hinge, between an Fc region portion and an immunoglobulin heterodimerization domain, or between a variable domain and a linker. Exemplary junction amino acids amino- terminal to a hinge include RDQ (SEQ ID NO:598), RT, SS, SASS (SEQ ID NO:599) and SSS (SEQ ID NO: 600). Exemplary junction amino acids carboxy-terminal to a hinge include amino acids SG. Additional exemplary junction amino acids include SR.
In certain embodiments, junction amino acids are present between an Fc region portion that comprises CH2 and CH3 domains and an immunoglobulin heterodimerization domain (CHI or CL). These junction amino acids are also referred to as a "linker between CH3 and CHI or CL" if they are present between the C- terminus of CH3 and the N-terminus of CHI or CL. Such a linker may be about 2-12 amino acids in length. In certain embodiments, the Fc region portion comprises human IgGl CH2 and CH3 domains in which the C-terminal lysine residue of human IgGl CH3 is deleted. Exemplary linkers between CH3 and CHI include those set forth in SEQ ID NO:847-849. Exemplary linkers between CH3 and Ck include those set forth in SEQ ID NOS:850-852 (in which the carboxyl terminal arginine in the linkers may alternatively be regarded as the first arginine of Ck). In certain embodiments, the presence of such linkers or linker pairs (e.g., SEQ ID NO:847 as a CH3-CH1 linker in one single chain polypeptide of a heterodimer and SEQ ID NO:850 as a CH3-Ck linker in the other single chain polypeptide of the heterodimer; SEQ ID NO:848 as a CH3- CHI linker and SEQ ID NO:851 as a CH3-Ck linker; and SEQ ID NO:849 as a CH3- CH1 linker and SEQ ID NO:852 as a CH3-Ck linker) improves the production of heterodimer compared the presence of a reference linker as set forth in SEQ ID NO: 846 (in which the last lysine of CH3 is included as part of the linker) in both single chain polypeptides of a heterodimer.
In certain embodiments, an immunoglobulin Fc region (e.g., CH2, CH3, and/or CH4 regions) of a polypeptide heterodimer of the present disclosure may have an altered glycosylation pattern relative to an immunoglobulin reference sequence. For example, any of a variety of genetic techniques may be employed to alter one or more particular amino acid residues that form a glycosylation site (see Co et al. (1993) Mol. Immunol. 30: 1361; Jacquemon et al. (2006) J. Thromb. Haemost. 4: 1047; Schuster et al. (2005) Cancer Res. 65:7934; Warnock et al. (2005) Biotechnol. Bioeng. 92:831), such as N297 of the CH2 domain (EU numbering). Alternatively, the host cells producing polypeptide heterodimers of this disclosure may be engineered to produce an altered glycosylation pattern. One method known in the art, for example, provides altered glycosylation in the form of bisected, non-fucosylated variants that increase ADCC. The variants result from expression in a host cell containing an oligosaccharide-modifying enzyme. Alternatively, the POTELLIGENT® technology of BioWa/Kyowa Hakko is contemplated to reduce the fucose content of glycosylated molecules according to this disclosure. In one known method, a CHO host cell for recombinant immunoglobulin production is provided that modifies the glycosylation pattern of the immunoglobulin Fc region, through production of GDP-fucose.
Alternatively, chemical techniques are used to alter the glycosylation pattern of polypeptide heterodimers of this disclosure. For example, a variety of glycosidase and/or mannosidase inhibitors provide one or more of desired effects of increasing ADCC activity, increasing Fc receptor binding, and altering glycosylation pattern. In certain embodiments, cells expressing polypeptide heterodimers of the instant disclosure are grown in a culture medium comprising a carbohydrate modifier at a concentration that increases the ADCC of immunoglycoprotein molecules produced by said host cell, wherein said carbohydrate modifier is at a concentration of less than 800 μΜ. In one embodiment, the cells expressing these polypeptide heterodimers are grown in a culture medium comprising castanospermine or kifunensine, for instance, castanospermine at a concentration of 100-800 μΜ, such as 100 μΜ, 200 μΜ, 300 μΜ, 400 μΜ, 500 μΜ, 600 μΜ, 700 μΜ, or 800 μΜ. Methods for altering glycosylation with a carbohydrate modifier such as castanospermine are provided in U.S. Patent No. 7,846,434 or PCT Publication No. WO 2008/052030.
Structural Arrangements and Exemplary Heterodimers
To form a polypeptide heterodimer according to the present invention, two single chain polypeptides are designed so that the immunoglobulin heterodimerization domain of the first single chain polypeptide is properly aligned and interacts with the immunoglobulin heterodimerization domain of the second single chain polypeptide. In certain embodiments, the heterodimer may comprise a second immunoglobulin heterodimerization domain pair to facilitate or enhance the heterodimerization of the two chains. In certain embodiments, in addition to the interaction between the two immunoglobulin heterodimerization domains, an Fc region portion (e.g., a CH3 domain) in the first chain may interact with an Fc region in the second chain to enhance heterodimerization (e.g., via interaction between two wild type CH3 domains or between a CH3 domain pair with "knobs-into-holes" mutations). Moreover, in certain embodiments, the hinge in the first chain (e.g., an altered human IgGl hinge with two cysteine residues as set forth in SEQ ID NO: 664) may interact with the hinge in the second chain (e.g., the same altered human IgGl hinge as set forth in SEQ ID NO:664) to form, for example, disulfide bonds, which may further strengthen the interaction between the first and second single chain polypeptides to form a polypeptide heterodimer of the present disclosure. Furthermore, in certain embodiments, the first and second chain may comprise a second hinge pair (e.g. , at the carboxyl termini of the two chains) to further enhance the interactions between the two chains.
In certain embodiments, a polypeptide heterodimer of the present disclosure comprises two binding domains (BDl and BD2), wherein the binding domains bind to two different target molecules. In certain embodiments, the two binding domains (BD l and BD2) are both on the SCP-I with the immunoglobulin heterodimerization domain (HD-I) and the Fc region portion (FRP-1) disposed between BDl and BD2. In certain other embodiments, the first binding domain (BDl) is on the SCP-I and the second binding domain (BD2) is on the SCP-II. In certain embodiments, both BDl and BD2 are amino terminal to the Fc region portion of the SCP-I and SCP- II, respectively. In certain other embodiments, BDl is amino terminal to the Fc region portion of the SCP-I and BD2 is carboxyl terminal to the Fc region portion of the SCP- II. In certain other embodiments, BDl is carboxyl terminal to the Fc region portion of the SCP-I and BD2 is amino terminal to the Fc region portion of the SCP-II. In certain other embodiments, both BDl and BD2 are carboxyl terminal to the Fc region portion of the SCP-I and SCP-II, respectively.
In certain embodiments, a polypeptide heterodimer comprises three binding domains (BDl , BD2 and BD3), wherein the binding domains bind to two or three different target molecules. For example, BDl , BD2, and BD3 each bind a different target, or BDl and BD2 bind a first target while BD3 binds a second target, or BDl and BD3 bind a first target while BD2 binds a second target, or BD2 and BD3 bind a first target while BDl binds a second target. In certain embodiments, the immunoglobulin heterodimerization domain and the Fc region portion are disposed between BDl and BD2 on SCP-I, and BD3 is amino terminal to the Fc region portion of the SCP-II. In further embodiments, the immunoglobulin heterodimerization domain and the Fc region portion are disposed between BDl and BD2 on SCP-II, and BD3 is carboxyl terminal to the Fc region portion of the SCP-I.
In certain embodiments, a polypeptide heterodimer comprises four binding domains (BDl , BD2, BD3 and BD4), wherein the binding domains bind to two to four different target molecules. In certain embodiments, the immunoglobulin heterodimerization domain and Fc region portion of the SCP-I are disposed between BDl and BD2, and the immunoglobulin heterodimerization domain and Fc region portion of SCP-II are disposed between BD3 and BD4.
In certain embodiments, a polypeptide heterodimer comprises five binding domains (BD l , BD2, BD3, BD4 and BD5), wherein the binding domains bind to two to four different target molecules. In certain embodiments, SCP-I comprises three binding domains (BDl , BD2 and BD3), and SCP-II comprises two binding domains (BD4 and BD5). In further embodiments, BDl and BD2 may be, for example, linked in tandem to each other (either directly or via a peptide linker of about two to eight amino acids) at the amino (or carboxyl) terminus of SCP-I with BD3 located at the carboxyl (or amino) terminus of SCP-I or SCP-II, wherein BD4 and BD5 are on SCP-II if BD3 is on SCP-I, or BD4 or BD5 are on SCP-I if BD3 is on SCP-II.
In still further embodiments, the polypeptide heterodimer comprises six binding domains (BD1-BD6). In certain embodiments, SCP-I and SCP-II each comprise three binding domains (e.g., BD1-BD3 on SCP-I, and BD4-BD6 on SCP-II). In such embodiments, for example, BDl and BD2 may be linked in tandem and located at the SCP-I amino (or carboxyl) terminus, and BD3 may be located at the SCP-I carboxyl (or amino) terminus. Similarly, BD4 and BD5 may be linked in tandem and located at the SCP-II amino (or carboxyl) terminus, and BD6 may be located at the SCP-II carboxyl (or amino) terminus. In certain other embodiments, the first single chain polypeptide (SCP-I) comprises four binding domains (BD1-BD4) and the second single chain polypeptide (SCP-II) comprises two binding domains (BD5 and BD6). In such embodiments, BD 1 and BD2 may be linked in tandem and located at or near amino terminus of SCP-I, BD3 and BD4 may be linked in tandem and located at or near carboxyl terminus of SCP-I, and BD5 and BD6 may be located at or near the amino and carboxyl termini of SCP-II, respectively.
In certain embodiments, the polypeptide heterodimer comprises seven binding domains (BD 1-BD7). In such embodiments, SCP-I may comprise four binding domains (BD1-BD4), and SCP-II may comprise the other three binding domains (BD5- BD7). For example, BD1 and BD2 may be linked in tandem and located at or near the amino terminus of SCP-I, and BD3 and BD4 may be linked in tandem and located at or near the carboxyl terminus of SCP-II. BD5 and BD6 may be linked in tandem and located at or near the amino (or carboxyl) terminus of SCP-II, and BD7 may be located at or near the carboxyl (or amino) terminus of SCP-II.
In certain embodiments, the polypeptide heterodimer comprises eight binding domains (BD1-BD8). In such embodiments, the first and second single chain polypeptides may each comprise four binding domains. In each chain, two binding domains may be located at or near the amino terminus, and the other two binding domains located at or near the carboxyl terminus.
To simplify the description of how various components can be arranged to make first and second single chain polypeptides that form polypeptide heterodimers of the present disclosure, arrangements in exemplary embodiments (1) to (50) are provided below in which only two binding domains are included in each heterodimer.
In embodiment (1), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CL region (e.g., CK, CX), a hinge, and an Fc region portion.
In embodiment (2), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, and a CL region.
In embodiment (3), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a second CHI region; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a second CL region.
In embodiment (4), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a second CHI region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CL region, a second CL region, a hinge and an Fc region portion.
In embodiment (5), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, and a second CHI region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CL region and a second CL region.
In embodiment (6), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
In embodiment (7), a polypeptide heterodimer is formed from the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain.
In embodiment (8), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second CL region, a second hinge, and a second binding domain.
In embodiment (9), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a second CHI region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a second CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain. In embodiment (10), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second CL region, a second hinge, and a second binding domain.
In embodiment (11), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CHI region, a hinge, and an Fc region portion.
In embodiment (12), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, and a CHI region.
In embodiment (13), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, and a second CL region; and a second single chain polypeptide comprising a second binding domain, a CHI region, a hinge, an Fc region portion, and a second CHI region.
In embodiment (14), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a second CL region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CHI region, a second CHI region, a hinge, and an Fc region portion.
In embodiment (15), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, and a second CL region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CHI region, and a second CHI region.
In embodiment (16), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
In embodiment (17), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain.
In embodiment (18), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second CHI region, a second hinge, and a second binding domain.
In embodiment (19), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a second CL region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a second CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
In embodiment (20), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second CHI region, a second hinge, and a second binding domain.
In embodiment (21), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a CHI region.
In embodiment (22), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a second binding domain, a CHI region, a hinge, an Fc region portion, and a CL region. In embodiment (23), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a CL region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CL region, a CHI region, a hinge and an Fc region portion.
In embodiment (24), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a CHI region, a hinge, and an Fc region portion; and a second single chain polypeptide comprising a second binding domain, a CHI region, a CL region, a hinge and an Fc region portion.
In embodiment (25), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region and a CL region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CL region and a CHI region.
In embodiment (26), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region and a CHI region; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CHI region and a CL region.
In embodiment (27), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain.
In embodiment (28), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain.
In embodiment (29), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a CL region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CL region, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
In embodiment (30), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a CL region, a CHI region, a hinge, an Fc region portion, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a CHI region, a CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain.
In embodiment (31), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a CL region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a CHI region, a second hinge, and a second binding domain.
In embodiment (32), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino terminus to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a CL region, a second hinge, and a second binding domain.
In embodiment (33), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CL region, a hinge, and an Fc region.
In embodiment (34), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CHI region, a hinge and an Fc region portion.
In embodiment (35), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge and an Fc region portion; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge and a second binding domain.
In embodiment (36), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge and a first binding domain; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge and an Fc region portion.
In embodiment (37), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a second hinge; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a second hinge.
In embodiment (38), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain; and the second single chain polypeptide comprising a hinge, an Fc region portion, and a CL region.
In embodiment (39), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain.
In embodiment (40), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, and a CHI region; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge and a second binding domain.
In embodiment (41), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge and a first binding domain; and the second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, and a CL region.
In embodiment (42), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, and a second hinge; and a second single chain polypeptide comprising a second binding domain, a hinge, an Fc region portion, a CL region and a second hinge.
In embodiment (43), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge an Fc region portion, a CL region, a second hinge and a second binding domain; and the second single chain polypeptide comprising a CL region, a hinge, an Fc region portion and a CHI region.
In embodiment (44), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region, a CHI region, a second hinge and a second binding domain.
In embodiment (45), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a CL region; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain.
In embodiment (46), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CL, a second hinge and a first binding domain; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a CHI region.
In embodiment (47), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second CHI region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, and a second CL region.
In embodiment (48), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, and a second CHI region; and a second single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second CL region, a second hinge, and a second binding domain.
In embodiment (49), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, and a second CHI region; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second CL region, a second hinge and a second binding domain. In embodiment (50), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a CHI region, a second hinge, and a first binding domain; and a second single chain polypeptide comprising a second binding domain, a CL region, a hinge, an Fc region portion, and a second CL region.
Exemplary embodiments (51) to (60) are also provided below in which three or four binding domains are included in each heterodimer. Additional binding domains may be included to make polypeptide heterodimers that comprise five to eight binding domains according to the present disclosure in view of the general description provided herein.
In embodiment (51), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a CL region, a hinge and an Fc region portion.
In embodiment (52), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a CL region, a hinge, an Fc region portion, a second hinge and a third binding domain.
In embodiment (53), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second hinge and a second binding domain; and the second single chain polypeptide comprising a third binding domain, a CHI region, a hinge and an Fc region portion.
In embodiment (54), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a CL region, a hinge, an Fc region portion, a second hinge and a second binding domain; and a second single chain polypeptide comprising a CHI region, a hinge, an Fc region portion, a second hinge and a third binding domain.
In embodiment (55), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge and a second binding domain; and the second single chain polypeptide comprising a third binding domain, a hinge, an Fc region portion and a CL region.
In embodiment (56), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CL region, a second hinge, and a third binding domain.
In embodiment (57), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a hinge, an Fc region portion and a CHI region.
In embodiment (58), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CL region, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a hinge, an Fc region portion, a CHI region, a second hinge, and a third binding domain.
In embodiment (59), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus: a first binding domain, a CHI region, a hinge, an Fc region portion, a second hinge, and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a CL region, a hinge, an Fc region portion, a second hinge and a fourth binding domain.
In embodiment (60), a polypeptide heterodimer comprises the following two single chain polypeptides: from amino to carboxyl terminus, a first single chain polypeptide comprising a first binding domain, a hinge, an Fc region portion, a CHI region, a second hinge and a second binding domain; and a second single chain polypeptide comprising a third binding domain, a hinge, an Fc region portion, a CL region, a second hinge and a fourth binding domain.
In embodiments (1) to (32), a polypeptide heterodimer of the present disclosure comprises the following two single chain polypeptides: a first single chain polypeptide comprising a first binding domain (BD1) that specifically binds to a T cell target (e.g., a TCR complex or a component thereof, including TCRa, TCRP, CD3y, CD35, and CD3s), a hinge that is an SCC-P IgGl hinge, an Fc region portion that is a wild type or altered human IgGl CH2CH3, and a CL region that is wild type or an altered human Ck region with N30Y V55A T70E (YAE) substitutions; and a second single chain polypeptide comprising binding domain (BD2) that specifically binds to a B-cell target (e.g. , CD 19, CD79b, HLA-DR, CD37, CD20) or a tumor or cancer antigen (e.g., RON, c-Met, EpCAM, CEACAM-6, PSMA), a hinge that is also an SCC-P IgGl hinge, an Fc region portion that is a wild type or altered human IgGl CH2CH3, and a CHI region that is human CHI region.
In further embodiments, a polypeptide heterodimer of embodiments (1) to (32) may further comprise a third binding domain (BD3) that is identical to BDl or BD2, and is linked to a single chain polypeptide via a second hinge (e.g., linker H75 or H68 as set forth in SEQ ID NOS:742 and 78, respectively). In still further embodiments, a polypeptide heterodimer of embodiment of embodiments (1) to (32) may further comprise third and fourth binding domains (BD3) and (BD4) that each are identical or different to BDl or BD2 and linked to the single chain polypeptide(s) via a second hinge (e.g., linker H75 or H68 as set forth in SEQ ID NOS:742 and 78).
In certain embodiments, the polypeptide heterodimers of the present disclosure can be engineered to have binding domains with different affinities to target specific cell types. For example, it may be desirable for a polypeptide heterodimer with a binding domain for a TCR complex or a component thereof and another binding domain for a tumor antigen (or a B-cell target) to preferentially bind tumor cells having the tumor antigen (or B-cells carrying the B-cell target) with a higher affinity so that the polypeptide heterodimer will bind the tumor cells (or the B-cells) first and then recruit T cells via its TCR/CD3 binding domain to the tumor site or cells. Differential binding affinities may be achieved by, for example, choosing a binding domain for one target with a higher binding affinity than the other binding domain has for its target or by including multiple binding domains for one target on a polypeptide heterodimer and a single binding domain or fewer binding domains for the second or other targets. In addition, using different hinges (e.g., using hinges of different lengths) may affect binding of one domain more than another or using different hinges for different binding domains in order to alter the binding activity of the binding domains.
In certain embodiments, multiple binding domains may need to be located at an appropriate distance from each other so that their interactions with their targets will produce a desirable effect. For example, in certain embodiments, a polypeptide heterodimer comprising a binding domain for a TCR complex or a component thereof in a single chain polypeptide and another binding domain for a tumor antigen or a B-cell target in a second single chain polypeptide may have both binding domains at the amino or carboxyl termini of their corresponding chains so that they are in physical proximity to each other in the polypeptide heterodimer.
Exemplary heterodimers may be formed from single chain polypeptide pairs described herein. If sequence identification numbers noted herein contain signal peptide sequences (e.g., the first 20 amino acids), such signal peptide sequences are not part of the mature single chain polypeptides that form the exemplary polypeptide heterodimers and thus should be considered excluded.
Exemplary single chain polypeptides are set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 29-32, 53-72, 74, 810-826, 859-864, and 874-882.
Exemplary heterodimers may be formed from the following single chain polypeptide pairs: SEQ ID NOS:2 and 4, SEQ ID NOS:6 and 8, SEQ ID NOS: 10 and 12, SEQ ID NOS: 14 and 16, SEQ ID NOS: 18 and 20, SEQ ID NOS:20 and 22, SEQ ID NOS:20 and 24, SEQ ID NOS:30 and 32, SEQ ID NOS:29 and 31 , SEQ ID NOS:29 and 32, SEQ ID NOS:30 and 72, SEQ ID NOS:53 and 72, SEQ ID NOS:54 and 72, SEQ ID NOS:55 and 72, SEQ ID NOS:70 and 72, SEQ ID NOS:71 and 72, SEQ ID NOS:63 and 56, SEQ ID NOS:64 and 57, SEQ ID NOS:65 and 60, SEQ ID NOS:66 and 58, SEQ ID NOS:67 and 59, SEQ ID NOS:68 and 61 , SEQ ID NOS:69 and 62, SEQ ID NOS:54 and 81 1 , SEQ ID NOS:54 and 812, SEQ ID NOS:54 and 813, SEQ ID NOS:814 and 818, SEQ ID NOS:815 and 818, SEQ ID NOS:816 and 818, SEQ ID NOS:817 and 818, SEQ ID NOS:814 and 820, SEQ ID NOS:814 and 821 , SEQ ID NOS:54 and 819, SEQ ID NOS:814 and 826, SEQ ID NOS:814 and 822, SEQ ID NOS:814 and 823, SEQ ID NOS:814 and 824, SEQ ID NOS:859 and 862, SEQ ID NOS:860 and 863, SEQ ID NOS:861 and 864, SEQ ID NOS:874 and 825, SEQ ID NOS:875 and 879, SEQ ID NOS:876 and 880, SEQ ID NOS:877 and 881 , or SEQ ID NOS:878 and 882.
Nucleic Acids, Vectors, Host Cells, and Methods for Making Heterodimers
In a related aspect, the present disclosure also provides isolated nucleic acid (used interchangeably with "polynucleotide") molecules that encode single chain polypeptides provided herein. Exemplary nucleic acid molecules (either with or without a nucleotide sequence encoding a signal peptide sequence) are set forth in SEQ
ID NOS: l , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25-28, 33-52 and 792-808.
The present disclosure also provides vectors that comprise nucleic acid sequence encoding single chain polypeptides provided herein. As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Exemplary vectors include plasmids, yeast artificial chromosomes, and viral genomes. Certain vectors can autonomously replicate in a host cell, while other vectors can be integrated into the genome of a host cell and thereby are replicated with the host genome.
In certain embodiments, the vectors may be recombinant expression vectors. "Recombinant expression vectors" or "expression vectors" refer to vectors that contain nucleic acid sequences that are operatively linked to an expression control sequence (e.g., a promoter) and are thus capable of directing the expression of those sequences.
Promoter sequences useful in expression vectors provided herein can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. In certain embodiments, the promoters are inducible promoters.
In certain embodiments, a vector is an expression vector that comprises a nucleic acid sequence encoding a first single chain polypeptide of a polypeptide heterodimer provided herein. In certain other embodiments, a vector is an expression vector that comprises a nucleic acid sequence encoding a second single chain polypeptide of a polypeptide heterodimer provided herein.
In certain embodiments, a vector is an expression vector that comprises nucleic acid sequences encoding both first and second single chain polypeptides of a polypeptide heterodimer. The promoter for the nucleic acid sequence encoding the first single chain polypeptide may be the same as the promoter for the nucleic acid encoding the second single chain polypeptide. Alternatively, the promoter for the nucleic acid sequence encoding the first single chain polypeptide may be different from the promoter for the nucleic acid encoding the second single chain polypeptide so that the expression level of the first and second single chain polypeptides may be differentially modulated to maximum heterodimerization of the first and second single chain polypeptides. In certain embodiments, one or both the promoters for the nucleic acid encoding the first and second single chain polypeptides are inducible promoters.
The present disclosure also provides a host cell transformed or transfected with, or otherwise containing, any of the nucleic acids or vectors provided herein. Exemplary host cells include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see US Patent Application Publication No. 2003/0115614), COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC 12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomyces cerevisiae cells, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or members of the Streptomycete family.
In certain embodiments, a host cell comprises a first expression vector containing a nucleic acid encoding a first single chain polypeptide and a second expression vector containing a nucleic acid encoding a second single chain polypeptide.
In certain other embodiments, a host cell comprises an expression vector containing a nucleic acid encoding both first and second single chain polypeptides.
The disclosure also includes a method of producing polypeptide heterodimers described herein. In certain embodiments, the method comprises culturing a host cell that comprises nucleic acids encoding both the first and second single chain polypeptides under conditions suitable to express the polypeptides, and optionally isolating or purifying the heterodimers formed from the first and second single chain polypeptides from the culture. The nucleic acid encoding the first single chain polypeptide and the nucleic acid encoding the second single chain polypeptide may be present in a single expression vector in the host cell or in two different expression vectors in the host cells. In the latter case, the ratio between the two expression vectors may be controlled to maximize heterodimerization of the first and second single chain polypeptides.
The present disclosure provides purified polypeptide heterodimers as described herein. The term "purified," as used herein, refers to a composition, isolatable from other components, wherein the polypeptide heterodimer is enriched to any degree relative to its naturally obtainable state. In certain embodiments, the present disclosure provides substantially purified polypeptide heterodimers as described herein. "Substantially purified" refers to a polypeptide heterodimer composition in which the polypeptide heterodimer forms the major component of the composition, such as constituting at least about 50%, such as at least about 60%, about 70%>, about 80%>, about 90%), about 95%, about 99%, of the polypeptides, by weight, in the composition.
Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the polypeptide and non-polypeptide fractions. Further purification using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) is frequently desired. Analytical methods particularly suited to the preparation of a pure fusion protein are ion-exchange chromatography, size exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Particularly efficient methods of purifying peptides are fast protein liquid chromatography and HPLC. Various methods for quantifying the degree of purification are known to those of skill in the art in light of the present disclosure. These include, for example, assessing the amount of polypeptide heterodimers in a fraction by SDS/PAGE analysis and HPLC as illustrated in the examples provided herein.
The method for making polypeptide heterodimers provided herein is advantageous over a method for first expressing and purifying separately individual single chain polypeptides and then incubating purified individual single chain polypeptides together to form polypeptide heterodimers. For example, certain single chain polypeptides (e.g., certain polypeptides containing only CHI regions as their immunoglobulin heterodimerization domains) are unstable when expressed alone. In addition, separate expression and purification of individual single chain polypeptides followed by combining the purified individual single chain polypeptides involve more steps than coexpressing both single chain polypeptides followed by purifying resulting polypeptide heterodimers and generally less efficient. Compositions and Methods for Using Heterodimers
In addition to polypeptide heterodimers, the present disclosure also provides pharmaceutical compositions and unit dose forms that comprise the polypeptide heterodimers as well as methods for using the polypeptide heterodimers, the pharmaceutical compositions and unit dose forms.
Compositions of polypeptide heterodimers of this disclosure generally comprise a polypeptide heterodimer provided herein in combination with a pharmaceutically acceptable excipient, including pharmaceutically acceptable carriers and diluents. Pharmaceutical acceptable excipients will be nontoxic to recipients at the dosages and concentrations employed. They are well known in the pharmaceutical art and described, for example, in Rowe et al., Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5th Ed., 2006.
Pharmaceutically acceptable carriers for therapeutic use are also well known in the pharmaceutical art, and are described, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro (Ed.) 1985). Exemplary pharmaceutically acceptable carriers include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes and the like may be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may also be used.
Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates {e.g., glucose, sucrose, dextrins), chelating agents (e.g., EDTA), glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary diluents. For instance, the product may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.
The present disclosure also provides a method for treating a disease or disorder associated with, for example, excessive receptor-mediated signal transduction, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer comprising a binding domain that specifically binds a receptor, such as a receptor tyrosine kinase, including EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGF1R, c-Met, RON, and EphA2.
Exemplary diseases or disorders associated with excess receptor- mediated signal transduction include cancer (e.g., solid malignancy and hematologic malignancy), autoimmune or inflammatory diseases or conditions, sepsis resulting from bacterial infection, and viral infection.
In one aspect, the present disclosure provides a method for directing T cell activation, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer that comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s or a combination thereof, and a second binding domain that specifically binds a different target, for instance, a tumor-specific antigen or other antigen of choice at a site or cell where T cell activation is desired.
In another aspect, the present disclosure provides a method for inhibiting growth, metastasis or metastatic growth of a malignancy (e.g. , a solid malignancy or a hematologic malignancy), comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer provided herein or a composition thereof.
A wide variety of cancers, including solid malignancy and hematologic malignancy, are amenable to the compositions and methods disclosed herein. Types of cancer that may be treated include, but are not limited to: adenocarcinoma of the breast, prostate, pancreas, colon and rectum; all forms of bronchogenic carcinoma of the lung (including squamous cell carcinoma, adenocarcinoma, small cell lung cancer and non- small cell lung cancer); myeloid; melanoma; hepatoma; neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoid heart disease; and carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell). Additional types of cancers that may be treated include: histiocytic disorders; leukemia; histiocytosis malignant; Hodgkin's disease; immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. Further, the following types of cancers are also contemplated as amenable to treatment: adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; paraganglioma nonchromaffin; and glioblastoma multiforme. The types of cancers that may be treated also include, but are not limited to, angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing; angiomatosis; glomangioma; hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervical dysplasia.
Additional exemplary cancers that are also amenable to the compositions and methods disclosed herein are B-cell cancers, including B-cell lymphomas [such as various forms of Hodgkin's disease, non-Hodgkins lymphoma (NHL) or central nervous system lymphomas], leukemias [such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myoblastic leukemia] and myelomas (such as multiple myeloma). Additional B cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, B-cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post- transplant lymphoproliferative disorder. In certain embodiments, polypeptide heterodimers useful for inhibiting growth of a solid malignancy or metastasis or metastatic growth of a solid malignancy or a hematologic malignancy include those that specifically bind to a tumor or cancer antigen and a T cell target. Such heterodimers are typically designed to have a higher binding affinity to the tumor or cancer antigen (e.g., at least about 5, 10, 15, 20, 25, 50, 75 or 100 folds higher) than to the T cell target so that they preferentially bind to the tumor or cancer antigen first and subsequently bind to the T cell target, which in turn recruit and activate T cells to damage or destroy the tumor or cancer cells carrying the tumor or cancer antigen on their surface. Exemplary tumor or cancer antigens and T cell targets include those provided above in the section describing binding domains of polypeptide heterodimers. For instance, a T cell target can be CD3 or PSMA.
In certain embodiments, polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy, or by directed T cell activation, comprise at least one binding domain that specifically binds an oncology target (including a tumor or cell antigen, such as RON, c-Met, CEACAM-6, or PSMA) and another binding domain that specifically binds a TCR complex or a component thereof (e.g., TCRa, TCRp, CD3y, CD35, and CD3s).
In certain other embodiments, polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise at least one binding domain that specifically binds CD28 and another binding domain that specifically binds CD79b. Such polypeptide heterodimers may further comprise a binding domain that comprises a PDL2 ectodomain, a binding domain that specifically binds hyperIL-6, or both binding domains.
In certain other embodiments, polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise at least one binding domain that specifically binds RON and another binding domain that specifically binds c-Met.
In certain other embodiments, polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise binding domains that specifically bind one or more of the following receptor tyrosine kinases: c- Met, RON, EGFR, EGFRvIII, Her2, ErbB3, ErbB4, and IGF1R, EphA2. In certain embodiments, the heterodimers may further comprise one or more binding domains that specifically bind to one or more of the following antiangiogenic agents: PDGFR, VEGFR1-4, and angiopoietin 2. In certain embodiments, the above heterodimers may further comprise one or more binding domains that specifically bind to one or more the following Fc receptors to increase targeting of cytotoxic effector function: CD64, CD32A and CD 16. In certain embodiments, the above heterodimers may further comprise a binding domain that specifically binds the transferrin receptor (CD71) to enable degradation of the receptors.
In certain other embodiments, polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise binding domains that are agonists of two or more of the following TNFSFRs: TNFR1 , TNFR2,
DR4, DR5, TWEAKR, and FAS.
In certain other embodiments, polypeptide heterodimers useful for inhibiting growth, metastasis, or metastatic growth of a malignancy comprise binding domains that specifically bind two or more of the following pro-oncogenic cytokines or growth factors: HGF, MSP, EGF (including epiregulin, herregulin, β-regulin, neuregulin), HIF-Ι , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, IL-6, hyperIL-6, IL-
8, Wnt, sHH, TGFP, or PDGF.
In certain embodiments, polypeptide heterodimers useful for inhibiting growth of a solid malignancy or metastasis or metastatic growth of a solid malignancy include those that specifically bind to, for example, EGFR, ErbB3, ErbB4, c-Met, RON,
EphA2, IGF1R, VEGFR1 , VEGFR2, VEGFR3, CD44v6, CD151 , EpCAM,
CEACAM6, TGFBR2, , GHRHR, GHR, IL-6R, gpl30, TNFR2, PD1 , TWEAK-R,
OSMRP, Patched- 1 , Frizzled, or Robol .
Polypeptide heterodimers useful for inhibiting metastasis or metastatic growth of a hematologic malignancy include those that specifically bind to, for example, EGFR, ErbB3, c-Met, RON, EphA2, IGF1R, TGFBR2, IL-6R, gpl 30,
TNFR2, PD1 , OSMRP, LTpR, CD 19, CD80, CD81 , or CD86.
In another aspect, the present disclosure provides a method for treating an autoimmune or inflammatory disease, disorder or condition, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer provided herein or a composition thereof.
Exemplary autoimmune or inflammatory diseases, disorders or conditions that may be treated by the fusion proteins and compositions and unit dose forms thereof include, and are not limited to, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), diabetes mellitus (e.g., type I diabetes), dermatomyositis, polymyositis, pernicious anaemia, primary biliary cirrhosis, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), autoimmune hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, lupus nephritis, neuropsychiatric lupus, multiple sclerosis (MS), myasthenia gravis, pemphigus vulgaris, asthma, psoriatic arthritis, rheumatoid arthritis, Sjogren's syndrome, temporal arteritis (also known as "giant cell arteritis"), autoimmune hemolytic anemia, Bullous pemphigoid, vasculitis, coeliac disease, chronic obstructive pulmonary disease, endometriosis, Hidradenitis suppurativa, interstitial cystitis, morphea, scleroderma, narcolepsy, neuromyotonia, vitiligo, and autoimmune inner ear disease.
Exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprises one or more binding domain that specifically binds CD28, and one, two, or three of the following additional binding domains: a binding domain that comprises PDL2 ectodomain, a binding domain that comprises monoIL-10, and a binding domain that specifically binds CD86. For example, a polypeptide heterodimer may comprise a binding domain that specifically binds CD28 and one to three binding domains that specifically bind CD86.
Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise one or more binding domains that specifically bind CD28 and one or more binding domains that are an IL-10 agonist (e.g., monoIL-10), an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist. Alternatively, polypeptide heterodimers may comprise two or more binding domains selected from an IL-10 agonist (e.g., monoIL-10), an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise one or more binding domains that specifically bind CD32B and one or more binding domains that are an IL-10 agonist (e.g., monoIL-10), an HLA-G agonist, an HGF agonist, an IL-35 agonist, a PD-1 agonist, a BTLA agonist, a LIGHT antagonist, a GITRL antagonist or a CD40 antagonist.
Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise binding domains that specifically bind one or more of the following TNFSFRs: TNFR1 , TNFR2, HVEW, LTpR, TWEAKR, TACI, BAFF-R, BCMA, or FAS.
Additional exemplary polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition may comprise binding domains that specifically bind two or more of the following pro-inflammatory cytokines/chemokines, such as TNFa, IL-6, IL-2, IL-1 , IL-8, IP- 10, IFNy, IFNa, RANKL, FASL, TGFp, IL7, IL10, IL17A/F, TWEAK, CSF2, IGF1 , IGF2 or BLyS/APRIL. Polypeptide heterodimers useful for treating an autoimmune or inflammatory disease, disorder or condition include those that specifically bind to, for example, TGFBR2, IL-6R, gpl30, TNFR1 , TNFR2, PD1 , HVEM, OX40, CD40, CD137, TWEAK-R, LTpR, LIFRp, OSMRp, CD3, TCRa, TCRp, CD19, CD28, CD80, CD81 , CD86, TLR7, TLR9, or any combination thereof.
In another aspect, the present disclosure provides a method for treating a B-cell associated disorder or disease that comprises administering to a patient in need thereof (e.g. , a patient having or suspected of having a B-cell associated disorder or disease) an effective amount of a polypeptide heterodimer provided herein, such as those that specifically bind a B cell target.
Polypeptide heterodimers useful in treating a B-cell associated disorder or disease may comprise binding domains that specifically bind one or more B-cell targets, such as CD79b, CD19, HLA-DR, CD20, CD21 , CD22, CD30, CD33, CD37, CD38, or CD70. The polypeptide heterodimers may further comprise a binding domain that specifically binds a T-cell target, such as a TCR complex or a component thereof, including TCRa, TCRp, CD3y, CD35, or CD3s.
Polypeptide heterodimers useful in treating a B-cell associated disorder or disease may comprise a binding domain that specifically bind a B-cell target, such as CD79b, CD19, HLA-DR, CD20, CD21, CD22, CD30, CD33, CD37, CD38, and CD70, and a binding domain that specifically binds CD64, CD32A, or CD 16 to increase targeting of cytotoxic effector function.
The polypeptide heterodimers or compositions thereof of the present disclosure may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection, or any combination thereof. In one embodiment, the polypeptide heterodimers or compositions thereof are administered parenterally. The term "parenteral," as used herein, includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site is contemplated as well. For instance, the invention includes administering polypeptide heterodimers or compositions thereof by intravenous injection.
The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment, concurrent medication, and other factors that those skilled in the medical arts will recognize. For example, an amount between 0.01 mg/kg and 1000 mg/kg (e.g., about 0.1 to 1 mg/kg, about 1 to 10 mg/kg, about 10-50 mg/kg, about 50-100 mg/kg, about 100-500 mg/kg, or about 500-1000 mg/kg) body weight (which can be administered as a single dose, daily, weekly, monthly, or at any appropriate interval) of active ingredient may be administered depending on the potency of a polypeptide heterodimer of this disclosure.
Also contemplated is the administration of polypeptide heterodimers or compositions thereof in combination with a second agent. A second agent may be one accepted in the art as a standard treatment for a particular disease state or disorder, such as in cancer, inflammation, autoimmunity, and infection. Exemplary second agents contemplated include polypeptide heterodimers that bind to targets different from those that primary polypeptide heterodimers bind, polyclonal antibodies, monoclonal antibodies, immunoglobulin-derived fusion proteins, chemotherapeutics, ionizing radiation, steroids, NSAIDs, anti-infective agents, or other active and ancillary agents, or any combination thereof.
In certain embodiments, a polypeptide heterodimer and a second agent act synergistically. In other words, these two compounds interact such that the combined effect of the compounds is greater than the sum of the individual effects of each compound when administered alone (see, e.g., Berenbaum, Pharmacol. Rev. 41 :93, 1989).
In certain other embodiments, a polypeptide heterodimer and a second agent act additively. In other words, these two compounds interact such that the combined effect of the compounds is the same as the sum of the individual effects of each compound when administered alone.
Second agents useful in combination with polypeptide heterodimers or compositions thereof provided herein may be steroids, NSAIDs, mTOR inhibitors (e.g., rapamycin (sirolimus), temsirolimus, deforolimus, everolimus, zotarolimus, curcumin, farnesylthiosalicylic acid), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), antimetabolites (e.g., mycophenolic acid, mycophenolate mofetil), polyclonal antibodies (e.g., anti-thymocyte globulin), monoclonal antibodies (e.g., daclizumab, basiliximab), and CTLA4-Ig fusion proteins (e.g., abatacept or belatacept).
Second agents useful for inhibiting growth of a solid malignancy, inhibiting metastasis or metastatic growth of a solid malignancy, or treating or ameliorating a hematologic malignancy include chemotherapeutic agents, ionizing radiation, and other anti-cancer drugs. Examples of chemotherapeutic agents contemplated as further therapeutic agents include alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil); bifunctional chemotherapeutics (e.g., bendamustine); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU)); ethyleneimines and methyl-melamines (e.g., triethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), and hexamethylmelamine (HMM, altretamine)); alkyl sulfonates (e.g., buslfan); and triazines (e.g., dacabazine (DTIC)); antimetabolites, such as folic acid analogues (e.g., methotrexate, trimetrexate, and pemetrexed (multi-targeted antifolate)); pyrimidine analogues (such as 5-fluorouracil (5-FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, and 2,2'- difluorodeoxycytidine); and purine analogues (e.g., 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, 2-chlorodeoxyadenosine (cladribine, 2-CdA)); Type I topoisomerase inhibitors such as camptothecin (CPT), topotecan, and irinotecan; natural products, such as epipodophylotoxins (e.g., etoposide and teniposide); and vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine); anti-tumor antibiotics such as actinomycin D, doxorubicin, and bleomycin; radiosensitizers such as 5- bromodeozyuridine, 5-iododeoxyuridine, and bromodeoxycytidine; platinum coordination complexes such as cisplatin, carboplatin, and oxaliplatin; substituted ureas, such as hydroxyurea; and methylhydrazine derivatives such as N-methylhydrazine (MIH) and procarbazine.
In certain embodiments, second agents useful for inhibiting growth metastasis or metastatic growth of a malignancy include polypeptide heterodimers according to the present disclosure that bind to cancer cell targets other than the target that the first polypeptide heterodimer binds. In certain other embodiments, second agents useful for such treatments include polyclonal antibodies, monoclonal antibodies, and immunoglobulin-derived fusion proteins that bind to cancer cell targets. Exemplary cancer cell targets are provided above in the context of describing targets of polypeptide heterodimers useful for the above-noted treatment.
Further therapeutic agents contemplated by this disclosure for treatment of autoimmune diseases are referred to as immunosuppressive agents, which act to suppress or mask the immune system of the individual being treated. Immunosuppressive agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) for the treatment of arthritis, or biologic response modifiers. Compositions in the DMARD description are also useful in the treatment of many other autoimmune diseases aside from rheumatoid arthritis.
Exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as Vioxx and Celebrex, and sialylates. Exemplary analgesics are chosen from the group consisting of acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g. etanercept (Enbrel), adalimumab (Humira) and infliximab (Remicade)), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofm) and intramuscular) and minocycline.
Additional second agents useful for treating an autoimmune or inflammatory disease, disorder or condition may be a polyclonal or monoclonal antibody, an immunoglobulin-derived fusion protein (e.g., scFv, SMIP™, PIMS, SCORPION™ fusion proteins), or a polypeptide heterodimer according to the present disclosure that specifically bind a target associated with such a disease, disorder or condition. Examples of such targets are provided above in the context of targets of polypeptide heterodimers of the present disclosure useful in the above-noted treatment.
It is contemplated the binding molecule composition and the second active agent may be given simultaneously in the same formulation. Alternatively, the second agents may be administered in a separate formulation but concurrently (i.e., given within less than one hour of each other).
In certain embodiments, the second active agent may be administered prior to administration of a polypeptide heterodimer or a composition thereof. Prior administration refers to administration of the second active agent at least one hour prior to treatment with the polypeptide heterodimer or the composition thereof. It is further contemplated that the active agent may be administered subsequent to administration of the binding molecule composition. Subsequent administration is meant to describe administration at least one hour after the administration of the polypeptide heterodimer or the composition thereof.
This disclosure contemplates a dosage unit comprising a pharmaceutical composition of this disclosure. Such dosage units include, for example, a single-dose or a multi-dose vial or syringe, including a two-compartment vial or syringe, one comprising the pharmaceutical composition of this disclosure in lyophilized form and the other a diluent for reconstitution. A multi-dose dosage unit can also be, e.g., a bag or tube for connection to an intravenous infusion device. This disclosure also contemplates a kit comprising a pharmaceutical composition of this disclosure in unit dose, or multi-dose, container, e.g., a vial, and a set of instructions for administering the composition to patients suffering a disorder such as a disorder described above.
EXAMPLES
EXAMPLE 1
BIVALENT POLYPEPTIDE HETERODIMER WITH
Two PAIRS OF HETERODIMERIZATION DOMAINS Polypeptide heterodimer X0172 was made by co-expressing single chain polypeptides X0130 (2E12 CHI CH2 CH3 Ck) and X0168 (Ck CH2 CH3 CHI H68 2E12). Single chain polypeptide X0130 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and altered human Ck (without the first Arg or the last Cys). The nucleic acid and amino acid sequences of XI 30 are set forth in SEQ ID NOS: l and 2, respectively. Single chain polypeptide X0168 comprises, from its amino to carboxyl terminus: altered human Ck (without the first Arg or the last Cys), human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, human IgGl CHI, H68 linker, and 2E12 (anti-CD28) scFv. The nucleic acid and amino acid sequences of X0168 are set forth in SEQ ID NOS:3 and 4, respectively. The amino acid sequence of H68 linker is set forth in SEQ ID NO:78.
For comparison, polypeptide heterodimer X0124 was made by co- expressing single chain polypeptide X0112 (2E12 CHI CH2 CH3) and X0113 (Ck CH2 CH3). Single chain polypeptide X0112 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2, and human IgGl CH3. The nucleic acid and amino acid sequences of X0112 are set forth in SEQ ID NOS:5 and 6, respectively. Single chain polypeptide X0113 comprises, from its amino to carboxyl terminus: altered human Ck (without the first Arg or the last Cys), human IgGl SCC-P hinge, human IgGl CH2, and human IgGl CH3. The nucleic acid and amino acid sequence of X0113 are set forth in SEQ ID NOS:7 and 8, respectively.
Expression
The day before transfection, HEK293 cells were suspended at a cell concentration of 0.5xl06 cells/ml in Freestyle 293 expression medium (Gibco). For a large transfection, 250 ml of cells were used, but for a small transfection, 60 ml of cells were used. On the transfection day, 320 ul of 293fectin reagent (Invitrogen) was mixed with 8 ml of media. At the same time, 250 ug of DNA for each of the two chains were also mixed with 8 ml of media and incubated for 5 minutes. After 15 minutes of incubation, the DNA-293fectin mixture was added to the 250ml of 293 cells and returned to the shaker at 37°C and shaken at a speed of 120 RPM. For the smaller transfection using 60 ml of cells, a fourth of the DNA, 293fectin and media were used.
Purification
Protein A affinity chromatography was used to purify the proteins. 2 mL of packed protein A agarose (Repligen) was added to a Biorad column (Econo-column chromatography column, size 2.5 x 10 cm), washed extensively with PBS (lOx column volume) and the supernatants were loaded, washed with PBS again and eluted with 3 column volumes of Pierce IgG elution buffer. Proteins were then dialyzed extensively against PBS. Proteins were then concentrated using Amicon centrifugal filter devices to a final volume of around 0.5 mL.
For second step purification, Protein L affinity chromatography or cation exchange chromatography were used. For Protein L purification, protein A purified Polypeptide heterodimer was passed over a Protein L agarose column that had been pre- equilibrated with PBS, washed with PBS (lOx column volume) and then eluted with Pierce IgG elution buffer. Proteins were then dialyzed against PBS extensively and concentrated using Amicon centrifugal filter devices to a final volume of around 0.5 mL.
Samples (200-300 ug) of previously affinity purified (Protein A or Protein L) Polypeptide heterodimer constructs were dialyzed into 20 mM MES, pH 6.0 (Buffer A) and loaded onto a MonoS 5/50 GL cation exchange column (GE Healthcare) at a flow rate of 2 mL/min, using an AKTA Explorer FPLC. The column was allowed to equilibrate for 5 column volumes (CV) and then run in a gradient format to a mixture of 50%:50% buffer A:buffer B (buffer B being 20 mM MES, 1 M NaCl, pH 6.0) over 20 CV. A following mixture of 100% buffer B was run for 5 CV to clean the column, and the system was run for another 5 CV at 100% buffer A to re-equilibrate prior to the next injection. Peaks were collected and analyzed by SDS-PAGE and electrospray mass spectrometry.
SDS-PAGE Analysis
Proteins purified were analyzed on a 10 % SDS-PAGE gel using Invitrogen's X-cell Surelock gel box.
Synergy with Suboptimal Concentration of PMA
Peripheral blood mononuclear cells (PBMC) from in-house donors were isolated from heparinized whole blood via centrifugation over Lymphocyte Separation Media (MP Biomedicals, Aurora, OH) and washed two times with RPMI media (Gibco- Invitrogen, Carlsbad, CA). CD4+ T-cells were then enriched from the PBMC using negative selection with a MACS CD4+ T-cell Isolation Kit (Miltenyi Biotec, Auburn, CA). The enriched (>95%) CD4+ T-cells were then resuspended at a concentration of lxl 06 cell/ml in complete RPMI/10% FCS. Test reagents were prepared at 40 ug/ml (yielding a final concentration of 10 μg/ml) in complete RPMI/10% FCS and added in 50 μΐ/well to flat-bottom 96-well plates (BD Falcon, San Jose, CA). PMA (Phorbol 12 myristate 13-acetate; A.G. Scientific, Inc., San Diego, CA) in complete RPMI/10% FCS was added in 50 ul/well at 4 ng/ml (final concentration of 1 ng/ml). Then T-cells in complete RPMI/10% FCS were added at a concentration of 5 x 104 cells/well in a 50 μΐ volume, and finally an appropriate amount of complete RPMI/10% FCS was added to each well (typically 50 ul) to bring the final volume to 200 ul/well. The cells were treated with the test samples +/- PMA and incubated for 72 hours at 37°C in 5% C02. One microliter of tritiated thymidine (Amersham Biosciences, Pisctaway, NJ) in a 1 :50 dilution of complete RPMI/10% FCS (50 ul/well) was added to the wells for the last 6 hours of culture. Plates were harvested onto a Unifilter-96, GF/C microplate (Perkin Elmer, Boston, MA) with a Packard Filtermate Harvester (Perkin Elmer, Boston, MA). Numbers are expressed as cpm and are the mean of replicate samples.
Figure 2 shows that Polypeptide heterodimer X0172 has a differential property compared to 2E12 SMIP M0039 (SEQ ID NO:77). It did not synergize with PMA as well as the SMIP. This bivalent polypeptide heterodimer has a property closer to the monovalent polypeptide heterodimer, X0124, rather than the bivalent SMIP.
Figure 3 shows that bivalent polypeptide heterodimer X0172 bound to CD4+ cells better than 2E12 scFv (SEQ ID NO: 109) and monovalent polypeptide heterodimer X0124.
EXAMPLE 2
BIVALENT, TRIVALENT, TETRAVALENT POLYPEPTIDE HETERODIMER USING
SINGLE HETERODIMERIZATION DOMAIN PAIR
Bivalent polypeptide heterodimer X0251 was made by co-expressing single chain polypeptides X0244 (Ck(YAE) CH2(N297A) CH3 H68 2E12) and X0245 (2E12 CHI CH2(N297A) CH3). Single chain polypeptide X0244 comprises, from its amino to carboxyl terminus: human Ck(YAE) (i.e., human Ck without the first Arg or last Cys but with N30Y, V55A, and T70E substitutions), human IgGl SCC-P hinge, human IgGl CH2 (N297A) (i.e., human IgGl CH2 with a N297A substitution), human IgGl CH3, H68 linker, and 2E12 (anti-CD28) scFv. The nucleotide and amino acid sequences of X0244 are set forth in SEQ ID NOS:9 and 10, respectively. Single chain polypeptide X0245 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2 (N297A), and human IgGl CH3. The nucleotide and amino acid sequences of X0245 are set forth in SEQ ID NOS: l 1 and 12, respectively.
Trivalent, bispecific polypeptide heterodimer X0252 was made by co- expressing single chain polypeptides X0246 (P2C2 Ck(YAE) CH2(N297A) CH3) and X0247 (2E12 CHI CH2(N297A) CH3 H68 2E12). Single chain polypeptide X0246 comprises, from its amino to carboxyl terminus: P2C2 (anti-CD79b) scFv, human Ck (YAE), human IgGl SCC-P hinge, human IgGl CH2 (N297A), and human IgGl CH3. The nucleotide and amino acid sequences of X0246 are set forth in SEQ ID NOS: 13 and 14, respectively. Single chain polypeptide X0247 comprises, from its amino to carboxyl terminus: 2E12 (anti-CD28) scFv, human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 2E12 (anti- CD28) scFv. The nucleotide and amino acid sequences of X0247 are set forth in SEQ ID NOS: 15 and 16, respectively.
Tetravalent, trispecific polypeptide heterodimer X0253 was made by co- expressing single chain polypeptides X0248 (P2C2 CK(YAE) CH2(N297A) CH3 H68 A2) and X0249 (PDL2 ECD CHI CH2(N297A) CH3 H68 2E12). Single chain polypeptide X0248 comprises, from its amino to carboxyl terminus: P2C2 (anti-CD79b) scFv, human Ck (YAE), human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and A2 (anti-hyperIL-6) scFv. The nucleotide and amino acid sequences of X0248 are set forth in SEQ ID NOS: 17 and 18, respectively. Single chain polypeptide X0249 comprises, from its amino to carboxyl terminus: PDL2 ECD (i.e., PDL2 ectodomain), human IgGl CHI, human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 2E12 (anti-CD28) scFv. The nucleotide and amino acid sequences of X0249 are set forth in SEQ ID NOS: 19 and 20, respectively.
Another tetravalent, tetraspecific polypeptide heterodimer X0283, was made by co-expressing single chain polypeptides X0249 (PDL2 ECD CHI CH2(N297A) CH3 H68 2E12) and X0281 (monoIL-10 Ck(YAE) CH2(N297A) CH3 H68 3D1). Single chain polypeptide X0281 comprises, from its amino to carboxyl terminus: monoIL-10, human Ck(YAE), human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 3Dl(anti-CD86) scFv. The nucleotide and amino acid sequences of X0281 are set forth in SEQ ID NOS:21 and 22.
As a control for polypeptide heterodimer X0283, tetravalent heterodimer, polypeptide heterodimer X0284, was made by co-expressing single chain polypeptides X0249 (PDL2 ECD CHI CH2(N297A) CH3 H68 2E12) and X0282 (monoIL-10 Ck CH2(N297A) CH3 H68 3D1). Single chain polypeptide X0282 is identical to X0281 except that it does not contain N30Y V55A T70E substitutions in the human Ck sequence. Thus, single chain polypeptide X0282 comprises, from its amino to carboxyl terminus: monoIL-10, altered human Ck (without the first Arg or the last Cys), human IgGl SCC-P hinge, human IgGl CH2 (N297A), human IgGl CH3, H68 linker, and 3Dl(anti-CD86) scFv. The nucleotide and amino acid sequences of X0282 are set forth in SEQ ID NOS:23 and 24, respectively.
The polypeptide heterodimer molecules were expressed and purified according to Example 1. Purified proteins were analyzed using SDS-PAGE electrophoresis under reduced and non-reduced conditions. Size exclusion chromatography was performed on an AKTA Explorer FPLC (Pharmacia Biotech) using a Superdex200 10/300 GL column. Some proteins were analyzed by electrospray mass spectrometry using an Agilent 6120 TOF ES/MS.
Surface plasmon resonance (SPR) measurements were performed on a Biacore T100 SPR using HBS-P+ (GE Healthcare) as a running buffer. Target was directly immobilized onto a CM5 chip using standard amine coupling chemistry (BIACORE® Amine Coupling Kit, GE Healthcare), with final immobilization levels between 800 and 1900 Ru (resonance units). Polypeptide heterodimer X0283 was injected at 25°C or 37°C for 150 seconds at a flow rate of 30 μΐ/min in a series of concentrations from 10 nM to 1 μΜ. Dissociation was monitored for 1200 seconds, and the surface was regenerated by injecting 50 mM NaOH for 60 seconds. Binding interactions with the surface were stable through at least 60 regeneration cycles. Data were analyzed using BiaEvaluation for the T100 software (version 2.0, GE Healthcare).
The SDS-PAGE electrophoresis and cation exchange chromatography analyses show that polypeptide heterodimers X0251, X0252 and X0253 were reasonably well expressed and purified (Figure 4). Mass spectrometry analysis of X0252 shows that the protein is mostly homogeneous with an undetectable amount of the homodimers (Figure 5). In addition, the SDS-PAGE electrophoresis and cation exchange chromatography analyses of polypeptide heterodimers X0283 and X0284 show that polypeptide heterodimer X0283 having the CHl/Ck(YAE) heterodimerization domains efficiently assembled into a heterodimer compared to control polypeptide heterodimer X0284 having the CHl/Ck heterodimerization domains, which formed homodimers as well (Figure 6). The Biocore analysis further shows that the 3D1 (anti-CD86) binding domain at the carboxyl terminus of single chain polypeptide X0281 monovalently bound to CD86 (Figure 7). EXAMPLE 3
POLYPEPTIDE HETERODIMERS WITH ANTI-RON AND
ANTI-C-MET BINDING DOMAINS
A bivalent polypeptide heterodimer with anti-RON binding domains (ORN151) and two bispecific polypeptide heterodimers comprising anti-RON and anti- cMet binding domains (ORN152 and ORN153) were made.
Bivalent polypeptide heterodimer ORN151 comprises single chain polypeptides ORN145 (4C04 CH2 CH3 CHI) and ORN148 (11H09 CH2 CH3 Ck(YAE)). Single chain polypeptide ORN145 comprises from its amino to carboxyl terminus: 4C04 (anti-RON) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI . The nucleotide and amino acid sequences of ORN145 are set forth in SEQ ID NOS:26 and 30, respectively. Single chain polypeptide ORN148 comprises from its amino to carboxyl terminus: 11H09 (anti- RON) scFv, human IgGl SCC-P hinge, human CH2, human CH3, and human Ck(YAE). The nucleotide and amino acid sequences of ORN148 are set forth in SEQ ID NOS:28 and 32, respectively.
Bispecific (c-Met, RON) polypeptide heterodimer ORN152 comprises single chain polypeptides ORN116 (MET021 CH2 CH3 CHI) and ORN146 (4C04 CH2 CH3 Ck(YAE)). Single chain polypeptide ORN116 comprises from its amino to carboxyl terminus: MET021 (anti-c-Met) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI . The nucleotide and amino acid sequences of ORN116 are set forth in SEQ ID NOS:25 and 29, respectively. Single chain polypeptide ORN146 comprises from its amino to carboxyl terminus: 4C04 (anti- RON) scFv, human IgGl SCC-P hinge, human CH2, human CH3, and human Ck(YAE). The nucleotide and amino acid sequences of ORN146 are set forth in SEQ ID NOS:27 and 31, respectively.
Bispecific (c-Met, RON) polypeptide heterodimer ORN153 comprises single chain polypeptides ORN116 (MET021 CH2 CH3 CHI) and ORN148 (11H09 CH2 CH3 Ck(YAE)).
Polypeptide heterodimers ORN151, ORN152 and ORN153 were expressed according to Example 1. The following expression levels were obtained: 1.9 μg protein / mL of culture for ORN151 , 3.1 μg / mL for ORN152, and 4.9 μg / mL for ORN153. EXAMPLE 4
POLYPEPTIDE HETERODIMERS WITH ANTI-CD3 BINDING DOMAINS
Several bispecific polypeptide heterodimers with an anti-CD3 binding domain were made: polypeptide heterodimers S0268, S0269, and TSC020 to TSC030. Polypeptide heterodimer S0268 comprises single chain polypeptides OR 145 (4C04 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)). Single chain polypeptide TSC019 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2, human CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC019 are set forth in SEQ ID NOS:52 and 72, respectively.
Bispecific (CD3, c-Met) Polypeptide heterodimer S0269 comprises single chain polypeptides ORN160 (5D5 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)). Single chain polypeptide OR 160 comprises from its amino to carboxyl terminus: 5D5 (anti-c-Met) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of ORN160 are set forth in SEQ ID NOS:33 and 53, respectively.
Bivalent (CD 19) polypeptide heterodimer TSC020 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)). Single chain polypeptide TSCOOl comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI . The nucleotide and amino acid sequences of TSCOOl are set forth in SEQ ID NOS:34 and 54, respectively.
Bispecific (CD20, CD3) polypeptide heterodimer TSC021 comprises single chain polypeptides TSC002 (2H7 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)). Single chain polypeptide TSC002 comprises from its amino to carboxyl terminus: 2H7 (anti-CD20) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI . The nucleotide and amino acid sequences of TSC002 are set forth in SEQ ID NOS:35 and 55, respectively.
Bispecific (CD79b, CD3) polypeptide heterodimer TSC022 comprises single chain polypeptides TSC017 (P2C2 H2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)). Single chain polypeptide TSC017 comprises from its amino to carboxyl terminus: P2C2 (anti-CD79b) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3 and human IgGl CHI . The nucleotide and amino acid sequences of TSC017 are set forth in SEQ ID NOS:50 and 70, respectively.
Bispecific (HLA-DR, CD3) polypeptide heterodimer TSC023 comprises single chain polypeptides TSC018 (M0042 CH2 CH3 CHI) and TSC019 (G19-4 CH2 CH3 Ck(YAE)). Single chain polypeptide TSC018 comprises from its amino to carboxyl terminus: M0042 (anti-HLA-DR) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC018 are set forth in SEQ ID NOS:51 and 71, respectively.
Bivalent polypeptide heterodimer TSC024 comprises single chain polypeptides TSCOIO (HD37-"IgAl hinge"-CH2 CH3 CHI) and TSC003 (G19-4- "IgAl hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSCOIO comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgAl hinge (PSTPPTPSPSTPPTPSPSCPPCP, SEQ ID NO:752), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSCOIO are set forth in SEQ ID NOS:43 and 63, respectively. Single chain polypeptide TSC003 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgAl hinge (SEQ ID NO:752), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC003 are set forth in SEQ ID NOS:36 and 56, respectively.
Bivalent polypeptide heterodimer TSC025 comprises single chain polypeptides TSC011 (HD37-"IgA2 hinge"-CH2 CH3 CHI) and TSC004 (G19-4- "IgA2 hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSC011 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgA2 hinge (PPPPPCPPCP, SEQ ID NO:748), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC011 are set forth in SEQ ID NOS:44 and 64, respectively. Single chain polypeptide TSC004 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgA2 hinge (SEQ ID NO:748), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC004 are set forth in SEQ ID NOS:37 and 57, respectively.
Bivalent polypeptide heterodimer TSC026 comprises single chain polypeptides TSC012 (HD37-"IgGl hinge"-CH2 CH3 CHI) and TSC007 (G19-4- "IgGl hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSC012 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgGl hinge (EPKSSDKTHTSPPSPCPPCP, SEQ ID NO:750), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC012 are set forth in SEQ ID NOS:45 and 65, respectively. Single chain polypeptide TSC007 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgGl hinge (SEQ ID NO:750), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC007 are set forth in SEQ ID NOS:40 and 60, respectively. Bivalent polypeptide heterodimer TSC027 comprises single chain polypeptides TSC013 (HD37-"IgG3 hinge"-CH2 CH3 CHI) and TSC005 (G19-4- "IgG3 hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSC013 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgG3 hinge (EPKSSDTPPPSPRSPCPPCP, SEQ ID NO:751), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC013 are set forth in SEQ ID NOS:46 and 66, respectively. Single chain polypeptide TSC005 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgG3 hinge (SEQ ID NO:751), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC005 are set forth in SEQ ID NOS:38 and 58, respectively.
Bivalent polypeptide heterodimer TSC028 comprises single chain polypeptides TSC014 (HD37-"IgD hinge"-CH2 CH3 CHI) and TSC006 (G19-4-"IgD hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSC014 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, altered human IgD hinge (ESPKAQASSVPTAQPQAEGSLAKATTAPATTR TCPPCP, SEQ ID NO:754), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC014 are set forth in SEQ ID NOS:47 and 67, respectively. Single chain polypeptide TSC006 comprises from its amino to carboxyl terminus: G19- 4 (anti-CD3) scFv, altered human IgD hinge (SEQ ID NO:754), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC006 are set forth in SEQ ID NOS:39 and 59, respectively.
Bivalent polypeptide heterodimer TSC029 comprises single chain polypeptides TSC015 (HD37-"IgE CH2 hinge"-CH2 CH3 CHI) and TSC008 (G19-4- "IgE CH2 hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSC015 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgE CH2 hinge (SEQ ID NO:757), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC014 are set forth in SEQ ID NOS:48 and 68, respectively. Single chain polypeptide TSC008 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgE CH2 hinge (SEQ ID NO:757), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC008 are set forth in SEQ ID NOS:41 and 61, respectively.
Bivalent polypeptide heterodimer TSC030 comprises single chain polypeptides TSC016 (HD37-"IgM CH2 hinge"-CH2 CH3 CHI) and TSC009 (G19-4- "IgM CH2 hinge"-CH2 CH3 Ck(YAE)). Single chain polypeptide TSC016 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, altered human IgM CH2 hinge (SEQ ID NO:759), human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC015 are set forth in SEQ ID NOS:49 and 69, respectively. Single chain polypeptide TSC009 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, altered human IgM CH2 hinge (SEQ ID NO:759), human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC009 are set forth in SEQ ID NOS:42 and 62, respectively.
Bispecific polypeptide heterodimers S0268 (RON, CD3) and S0269 (CD3, c-Met) were expressed according to Example 1. The following expression levels were obtained: 2.2 μg protein / mL of culture for S0268 and 2.7 μg / mL for S0269.
EXAMPLE 5
CELL BINDING OF BISPECIFIC POLYPEPTIDE HETERODIMERS
To compare the effectiveness of bispecific polypeptide heterodimer molecules at targeting a tumor cell antigen and T-cells, the on-cell binding characteristics of an anti-RON (4C04 binding domain) x anti-CD3 (G19-4 binding domain) polypeptide heterodimer, S0268 (as described in Example 4), with a different bispecific scaffold (SCORPION™ protein) containing the same binding domains, S0266, were compared. Additionally, the on-cell binding characteristics of two bispecific polypeptide heterodimers, TSC020 and TSC021 (as described in Example 4), targeting different B-cell antigens (CD 19 for TSC020 and CD20 for TSC021 ) as well as a T-cell antigen (CD3) were compared. The nucleotide and amino acid sequences of SCORPION protein S0266 are set forth in SEQ ID NOS:73 and 74, respectively. Nucleotide and amino acid sequences of the single chain polypeptides making up the bispecific polypeptide heterodimers S0268, TSC020, and TSC021 are set forth in SEQ ID NOS:26 and 52 (nucleotide), 30 and 72 (amino acid); 34 and 52 (nucleotide), 54 and 72 (amino acid); and 35 and 52 (nucleotide), 55 and 72 (amino acid), respectively (see also Example 4). Transient transfection in human 293 cells produced 6.9 μg protein / mL of culture for S0266; 2.3 μg / mL of culture for S0268; 3.0 μg / mL of culture for TSC020; and 3.2 μg / mL of culture for TSC021.
MDA-MB-453 (RON+) breast carcinoma cells, Reel (CD19+, CD20+) mantle cell lymphoma cells, and Jurkat (CD3 ) T cell leukemia cells were obtained from ATCC (Manassas, VA), and cultured according to the provided protocol. T-cells were isolated from donor PBMCs using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany). Non T-cells were separated from PBMCs by being indirectly magnetically labeled with biotin-conjugated monoclonal antibodies and anti-biotin magnetic microbeads. These cells were then depleted by retaining them in a column surrounded by a magnetic field. The T-cells were not retained in the column and were collected in the flow through.
Binding was assessed by incubating 5x 105 T cells or target (MDA-MB- 453, Reel, Jurkat) cells for 30 minutes at 4°C with serially diluted bispecific molecules S0266 (aRON aCD3 SCORPION™ protein) or S0268 (aRON aCD3 polypeptide heterodimer) (for MDA-MB-453 cells and isolated T cells); or TSC020 (aCD19 x aCD3) or TSC021 (aCD20 x aCD3) for Reel and Jurkat cells, in concentrations from 100 nM to 0.1 nM. The cells were washed three times and then incubated with goat anti-human IgG-FITC (1 :200 dilution) for another 30 minutes at 4°C. The cells were then washed again three times, fixed in 1% paraformaldehyde and read on a FACS- Calibur instrument.
Analysis of the FSC high, SSC high subset in FlowJo v7.5 (Tree Star, Inc, Ashland, OR) showed dose-dependent binding of bispecific molecules S0266 and S0268 to both MDA-MB-453 and isolated T-cells (Figures 8A and 8B). Unexpectedly, the S0268 polypeptide heterodimer bound with similar affinity to the comparable SCORPION™ molecule (S0266) on both MDA-MB-453 cells and T-cells, although it lacked the potential for any avidity. Higher saturation on both target cell types was also observed with the polypeptide heterodimer, which would be the case if the polypeptide heterodimer was binding at a higher stoichometry (1 :1 binding of polypeptide heterodimer to surface antigen) than the equivalent SCORPION™ (potential 1 :2 binding of the bivalent Scorpion to surface antigens). Comparison of the CD 19- targeting bispecific heterodimer (TSC020; HD37 and G19-4 binding domains) and the CD20-targeting bispecific heterodimer (TSC021; 2H7 and G19-4 binding domains) on Reel and Jurkat cells revealed that the TSC020 heterodimer had higher affinity for the Reel cells than TSC021 heterodimer (Figure 9A). However, both TSC020 and TSC021 heterodimers showed similar binding to CD3+ Jurkat cells, which is expected since both heterodimers possess the same anti-CD3 binding domain (G19-4) (Figure 9B).
EXAMPLE 6
ADDITIONAL BIVALENT & T IVALENT POLYPEPTIDE HETERODIMERS USING
SINGLE HETERODIMERIZATION DOMAIN PAIR
Additional multispecific heterodimers were made: TSC046, TSC047, TSC048, TSC054, TSC055, TSC056, TSC057, TSC078, TSC079, TSC080, TSC099, TSCIOO, TSC101, and TSC102.
Bivalent polypeptide heterodimer TSC046 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC039 (BMA031 CH2 CH3 Ck(YAE)). Single chain polypeptide TSCOOl comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSCOOl are set forth in SEQ ID NOS:34 and 54, respectively. Single chain polypeptide TSC039 comprises from its amino to carboxyl terminus: BMA031 (anti-TCR) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC039 are set forth in SEQ ID NOS:793 and 811, respectively.
Bivalent polypeptide heterodimer TSC047 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC041 (CRIS7 CH2 CH3 Ck(YAE)). Single chain polypeptide TSC041 comprises from its amino to carboxyl terminus: CRIS7 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC041 are set forth in SEQ ID NOS:794 and 812, respectively.
Bivalent polypeptide heterodimer TSC048 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC043 (OKT3-M CH2 CH3 Ck(YAE)). Single chain polypeptide TSC043 comprises from its amino to carboxyl terminus: OKT3-M (Micromet variant anti-CD3, see also US 7,635,472) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC043 are set forth in SEQ ID NOs:795 and 813, respectively.
Bivalent polypeptide heterodimer TSC054 comprises single chian polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC049 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null) (i.e., human IgGl CH2 with L234A, L235A, G237A, E318A, K320A, and K322A substitutions), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC049 are set forth in SEQ ID NOS:796 and 814, respectively. Single chain polypeptide TSC053 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null) (i.e., human IgGl CH2 with L234A, L235A, G237A, E318A, K320A, and K322A substitutions), human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC053 are set forth in SEQ ID NOS:800 and 818, respectively.
Bivalent polypeptide heterodimer TSC055 comprises single chain polypeptides TSC050 (2H7 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC050 comprises from its amino to carboxyl terminus: 2H7 (anti-CD20) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC050 are set forth in SEQ ID NOS:797 and 815, respectively.
Bivalent polypeptide heterodimer TSC056 comprises single chain polypeptides TSC051 (P2C2 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC051 comprises from its amino to carboxyl terminus: P2C2 (anti-CD79) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC051 are set forth in SEQ ID NOS:798 and 816, respectively.
Bivalent polypeptide heterodimer TSC057 comprises single chain polypeptides TSC052 (5D5 CH2(ADCC/CDC null) CH3 CHI) and TSC053 (G19-4 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC052 comprises from its amino to carboxyl terminus: 5D5 (anti-cMet) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC052 are set forth in SEQ ID NOS:799 and 817, respectively.
Bivalent polypeptide heterodimer TSC078 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC076 (OKT3 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC076 comprises from its amino to carboxyl terminus: OKT3 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC076 are set forth in SEQ ID NOS:802 and 820, respectively.
Bivalent polypeptide heterodimer TSC079 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC077 (Nuvion CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC077 comprises from its amino to carboxyl terminus: Nuvion (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC077 are set forth in SEQ ID NOS:803 and 821, respectively.
Trivalent polypeptide heterodimer TSC080 comprises single chain polypeptides TSCOOl (HD37 CH2 CH3 CHI) and TSC064 (G19-4 CH2 CH3 Ck(YAE) H75 Met021). Single chain polypeptide TSC064 comprises from its amino to carboxyl terminus: G19-4 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2, human IgGl CH3, human Ck(YAE), H75 linker, and Met021 (anti-c-Met) scFv (with the three serine residues at the C-temrinus deleted). The nucleotide and amino acid sequences of TSC064 are set forth in SEQ ID NOS:801 and 819, respectively.
Bivalent polypeptide heterodimer TSC099 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC097 (4C04 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC097 comprises from its amino to carboxyl terminus: 4C04 (anti-RON) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC097 are set forth in SEQ ID NOS:808 and 826, respectively.
Bivalent polypeptide heterodimer TSCIOO comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC093 (CRIS7 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC093 comprises from its amino to carboxyl terminus: CRIS7 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, and human Ck(YAE). The nucleotide and amino acid sequences of TSC093 are set forth in SEQ ID NOS:804 and 822, respectively.
Bivalent polypeptide heterodimer TSC101 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC094 (OKT3- M CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC094 comprises from its amino to carboxyl terminus: OKT3-M (Micromet variant anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, human Ck(YAE). The nucleotide and amino acid sequences of TSC094 are set forth in SEQ ID NOS:805 and 823, respectively.
Bivalent polypeptide heterodimer TSC102 comprises single chain polypeptides TSC049 (HD37 CH2(ADCC/CDC null) CH3 CHI) and TSC095 (BMA031 CH2(ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC095 comprises from its amino to carboxyl terminus: BMA031 (anti-TCR) scFv, human IgGl SCC-P hinge, human IgGl CH2(ADCC/CDC null), human IgGl CH3, human Ck(YAE). The nucleotide and amino acid sequences of TSC095 are set forth in SEQ ID NOS:806 and 824, respectively.
The polypeptide heterodimer molecules were expressed and purified according to Example 1.
EXAMPLE 7
TARGET -DEPENDENT T-CELL PROLIFERATION BY POLYPEPTIDE HETERODIMERS
To compare the effectiveness of different bispecific polypeptide heterodimer molecules at inducing target-dependent T-cell activation and proliferation, three different bispecific molecules (TSC054, TSC078, and TSC079 as described in Example 6) with a common anti-CD 19 binding domain (HD37) and three different anti- CD3 binding domains (G19-4 for TSC054, OKT3 for TSC078, HuM291 for TSC079) were compared. As a positive control, a Bispecific T-Cell Engaging molecule (BiTE) known as bscl9x3 was also prepared (see U.S. Patent 7,635,472). The nucleotide and amino acid sequences for bscl9x3 are set forth in SEQ ID NOS:809 and 827, respectively. Transient transfection in human 293 cells produced 2.33 ug/mL protein for TSC054, 0.67 ug/mL protein for TSC078, and 3.5 ug/mL for TSC079.
Daudi Burkitt's lymphoma cells (CD19+) and MDA-MB-453 (CD 19") breast carcinoma cells were obtained from ATCC (Manassas, VA) and cultured according to the provided protocol. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using standard ficoll gradients. The isolated cells were washed in saline buffer. T cells were additionally isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
Proliferation was assessed by labeling isolated PBMC or T cell populations with carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE- labeled PBMC or T cells were plated in U-bottom 96-well plates at 150,000 or 100,000 cells/well, respectively, with various numbers of tumor cells, to achieve T cell to tumor cell ratios of 10: 1 to 3: 1. Concentrations of test molecules ranging from 8 nM to 0.08 pM were added to the cell mixtures in a total of 200ul/well in RPMI 1640 media supplemented with 10% human or bovine serum, sodium pyruvate and non-essential amino acids. Plates were incubated at 37°C, 5% C02 in humidified incubators. After 3 days, cells were labeled with antibodies for flow cytometric analysis. Cells were labeled and washed in their original plates to minimize cell losses during transfers, and all labeling was done in saline buffer with 0.2% bovine serum albumin. First, cells were pre-incubated with lOOug/ml human IgG at room temperature for 15 min. Subsequently, cells were incubated with a mixture (total volume 50 ul) of the following dye-labeled antibodies: CD5-PE, CD4-APC, CD8-Pacific Blue, CD25-PE-Cy7, as well as 7- Amino Actinomycin D (7AAD hereafter) for 40 min. Plates were washed twice, resuspended in 80 to 120ul volumes and ran immediately in a BD LSRII flow cytometer to acquire 80% of the contents of each well. The sample files were analyzed using Flow Jo software to calculate the percentages and numbers of cells that had undergone at least one cell division, according to their CFSE profile, by gating sequentially on activated, live CD4+ or CD8+ T cells (7AAD-, CD5+ CD25+ CD4+ or 7AAD- CD5+ CD25+ CD8+, respectively). Mean values and standard deviations were calculated using Microsoft Excel software. Graphs were plotted using Microsoft Excel or Graphpad Prism.
Analysis of live CD4+ and CD8+ populations from Daudi cells or MDA- MB-453 cells treated with whole PBMCs (Figures 10A-10D) revealed a significant increase in both the total number of cells and percent proliferating cells in the presence of Daudi cells displaying the target CD 19 antigen (Figures 10A, 10B), but a lack of significant proliferation in the presence of MDA-MB-453 cells that lacked the CD 19 antigen (Figures IOC, 10D). Some proliferation was observed at a lower level with the MDA-MB-453 cells and whole PBMCs, since B-cells (CD 19+) were present in the PBMCs, but overall proliferation was greatly reduced in comparison. This selectivity was observed with isolated T-cells as well. Proliferation was higher for CD8+ T-cells than CD4+ T-cells in the presence of Daudi cells or MDA-MB-453 cells treated with PBMCs (Figure 10A -10D), and the proliferation induced by TSC078 (HD37xOKT3) was generally higher at all concentrations than the response induced by TSC054 (HD37xG19-4) or TSC079 (HD37xHuM291)(Figures 10A-10D). Induced proliferation of CD4+ T-cells was lower in all cases for TSC054, TSC078, and TSC079 than the BiTE bscl9x3 (Figure 10A), although TSC078 and TSC079 showed superior induction of proliferation of CD8+ cells at lower concentrations (e.g. 5 pM) than the BiTE molecule (Figure 10B). EXAMPLE 8
REDIRECTED T-CELL CYTOTOXICITY BY POLYPEPTIDE HETERODIMERS
To compare the effectiveness of different bispecific polypeptide heterodimer molecules at inducing target-dependent T-cell cytotoxicity, four different bispecific molecules were compared in a chromium (51Cr) release assay. Three different bispecific molecules (TSC054, TSC078, TSC079, as described in Example 6) with a common anti-CD 19 binding domain (HD37) and three different anti-CD3 binding domains (G19-4 for TSC054, OKT3 for TSC078, HuM291 for TSC079) were tested alongside a fourth bispecific molecule (S0268, described in Example 4) with an anti-RON binding domain (4C04) and an anti-CD3 binding domain (G19-4). Transient transfection in human 293 cells produced about 2.33 μg/mL protein for TSC054, about 0.67 μg/mL protein for TSC078, and about 3.5 μg/mL protein for TSC079.
Daudi Burkitt's lymphoma cells (CD 19+, RON-) and BxPC-3 cells (CD 19-, RON+) were obtained from ATCC (Manassas, VA) and cultured according to the provided protocol. Peripheral blood mononuclear cells (PBMC) were isolated from human blood using standard ficoll gradients. The isolated cells were washed in saline buffer. T cells were additionally isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
Cytotoxicity was assessed by a 51Cr release assay. Approximately 5x 106 Daudi or BxPC-3 cells were treated with 0.3 mCi of 51Cr and incubated for 75 minutes at 37°C. After 75 minutes, cells were washed 3 times with media (RPMI + 10% FBS) and resuspended in 1 1.5 mL of media. From this suspension, 50 μΐ, was dispensed per well into 96 well U-bottom plates (approximately 20,000 cells/well). Concentrations of bispecific molecules ranging from 10 nM to 0.1 pM were added to the target (Daudi, BxPC-3) cells, bringing the total volume to 100 (jIJwell. Target cells were incubated at room temperature for 15 minutes. Then 100 of isolated T-cells (approximately 200,000) were added to bring the T-cell to target cell ratio to 10: 1. 50 μΐ. of 0.8% NP- 40 was added to a control well containing target cells, left for 15 minutes, then 100 of media was added to provide a total lysis control.
Plates were incubated for 4 hours, spun at 1500 rpm for 3 minutes, and 25 μΐ^ of supernatant was transferred from each well to the corresponding well of a 96- well Luma sample plate. Sample plates were allowed to air dry in a chemical safety hood for 18 hours, and then radioactivity was read on a Topcount scintillation counter using a standard protocol.
Analysis of cytotoxicity data showed a lack of off-target cytotoxicity on the Daudi (RON-) cells from the anti-RON directed bispecific molecule S0268 (Figure 1 1 A). Similarly, there was a lack of direct cytotoxicity observed from treating Daudi cells with TSC054 in the absence of T-cells (Figure 1 1 A). However, strong T-cell directed cytotoxicity was observed with the Daudi cells in the presence of T-cells and an anti-CD 19 directed bispecific molecule (TSC054), reaching maximal lysis at a concentration between 10 and 100 pM (Figure 1 1 A). Similarly, using a second T-cell donor (Figure 1 1B), no off-target cytotoxicity of the BxPC-3 (CD 19-) cells was observed from the CD 19-directed bispecifics TSC054, TSC078, or TSC079, or the CD19-directed BiTE bscl9x3. The anti-RON directed S0268 bispecific molecule induced cytotoxicity in BxPC-3 (RON+) cells, reaching a maximum between 10 and 100 pM (Figure 1 IB).
EXAMPLE 9
MODULATION OF TARGET -DEPENDENT T-CELL PROLIFERATION BY POLYPEPTIDE
HETERODIMERS WITH ALTERED HINGE SEQUENCES
To compare the effectiveness of different bispecific polypeptide heterodimer molecules with altered hinge sequences at inducing target-dependent T-cell activation and proliferation, six different bispecific heterodimers (TSCIOO, TSC127, TSC165, TSC166, TSC167 and TSC168 with a common anti-CD 19 binding domain (HD37), a common anti-CD3 binding domain (Cris7) and five different hinge constructs (IgGl SCC-P hinge for TSCIOO and TSC127, IgA2 hinge without the first Val residue linked to a human IgGl core hinge for TSC165, IgGl SSS-P hinge linked to a human IgGl core hinge for TSC166, a portion of mutated IgG3 hinge linked to a IgGl core hinge for TSC167, and the IgM CH2 domain without the first Val linked to a IgGl core hinge for TSC168) were compared.
More specifically, bispecific heterodimer TSCIOO is as described in
Example 6.
Bispecific heterodimer TSC127 comprises single chain polypeptides
TSC125 (Cris7 CH2(ADCC/CDC null) CH3 CHI) and TSC096 (HD37 CH2(ADCC/CDC null) CH3 Ck(YAE). Single chain polypeptide TSC125 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC125 are set forth in SEQ ID NOS:865 and 874, respectively. Single chain polypeptide TSC096 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE). The nucleotide and amino acid sequences of TSC096 are set forth in SEQ ID NOS:807 and 825, respectively.
Bispecific heterodimer TSC165 comprises single chain polypeptides TSC157 (Cris7 IgA2UH CH2 (ADCC/CDC null) CH3 CHI) and TSC161 (HD37 IgA2UH CH2 (ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC157 comprises from its amino to carboxyl terminus: humanized Cris7( anti-CD3) scFv, human IgA2 hinge without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC157 are set forth in SEQ ID NOS:866 and 875, respectively. Single chain polypeptide TSC161 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, human IgA2 hinge without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE). The nucleotide and amino acid sequences of TSC161 are set forth in SEQ ID NOS:870 and 879, respectively. The amino acid sequence of the hinge used in TSC157 and TSC161 is set forth in SEQ ID NO:748.
Bispecific heterodimer TSC166 comprises single chain polypeptide TSC158 (Cris7 IgGlminiUH CH2 (ADCC/CDC null) CH3 CHI) and TSC162 (HD37 IgGlminiUH CH2 (ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC158 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SSC-P hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC158 are set forth in SEQ ID NOS:867 and 876, respectively. Single chain polypeptide TSC162 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SSC-P hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE). The nucleotide and amino acid sequences of TSC162 are set forth in SEQ ID NOS:871 and 880. The amino acid sequence of the hinge used in TSC158 and TSC162 is set forth in SEQ ID NO:750.
Bispecific heterodimer TSC167 comprises single chain polypeptide
TSC159 (Cris7 IgG3UH CH2 (ADCC/CDC null) CH3 CHI) and TSC163 (HD37 IgG3UH CH2 (ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC159 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, a portion of mutated human IgG3 hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC159 are set forth in SEQ ID NOS:868 and 877, respectively. Single chain polypeptide TSC163 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, a portion of mutated human IgG3 hinge linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE). The nucleotide and amino acid sequences of TSC163 are set forth in SEQ ID NOS:872 and 881. The amino acid sequence of the hinge used in TSC159 and TSC163 is set forth in SEQ ID NO:751.
Bispecific heterodimer TSC168 comprises single chain polypeptide TSC160 (Cris7 IgMCH2UH CH2 (ADCC/CDC null) CH3 CHI) and TSC164 (HD37 IgMCH2UH CH2 (ADCC/CDC null) CH3 Ck(YAE)). Single chain polypeptide TSC160 comprises from its amino to carboxyl terminus: humanized Cris7( anti-CD3) scFv, human IgM CH2 without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human IgGl CHI . The nucleotide and amino acid sequences of TSC160 are set forth in SEQ ID NOS:869 and 878, respectively. Single chain polypeptide TSC163 comprises from its amino to carboxyl terminus: HD37 (anti-CD 19) scFv, human IgM CH2 without the first Val linked with a human IgGl core hinge, human IgGl CH2 (ADCC/CDC null), human IgGl CH3, and human Ck (YAE). The nucleotide and amino acid sequences of TSC164 are set forth in SEQ ID NOS:873 and 882. The amino acid sequence of the hinge used in TSC160 and TSC164 is set forth in SEQ ID NO:759.
As a positive control, a Bispecific T-Cell Engaging molecule (BiTE) known as bscl9x3 was also prepared (see U.S. Patent 7,635,472). The nucleotide and amino acid sequences for bscl9x3 are set forth in SEQ ID NOS:809 and 827, respectively.
Transient transfection in human 293 cells produced 3.2 ug protein / mL of culture for TSCIOO, 6.1 ug protein / mL of culture for TSC127, 4.8 ug protein / mL of culture for TSC165, 6.2 ug protein / mL of culture for TSC166, 6.4 ug protein / mL of culture for TSC167, and 6.4 ug/mL protein for TSC168.
Daudi Burkitt's lymphoma cells (CD19+) and C4-2 (CD 19") prostate carcinoma cells were obtained from ATCC (Manassas, VA) and MD Anderson Cancer Center (Houston, TX) and cultured according to the provided protocol. T cells were isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
Proliferation was assessed by labeling isolated T cell populations with carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE-labeled T cells were plated in U-bottom 96-well plates at 100,000 cells/well, with 10,000 tumor cells/well, to achieve a T cell to tumor cell ratio of 10: 1. Concentrations of test molecules ranging from 5 nM to 0.005 pM were added to the cell mixtures in a total of 200 ul/well in RPMI 1640 media supplemented with 10% human or bovine serum, sodium pyruvate and non-essential amino acids. Plates were incubated at 37°C, 5% C02 in humidified incubators. After 3 days, cells were labeled with antibodies for flow cytometric analysis. Cells were labeled and washed in their original plates to minimize cell losses during transfers, and all labeling was done in saline buffer with 0.2% bovine serum albumin. First, cells were pre-incubated with 100 ug/ml human IgG at room temperature for 15 min. Subsequently, cells were incubated with a mixture (total volume 50 ul) of the following dye-labeled antibodies: CD5-PE, CD4-APC, CD8- Pacific Blue, CD25-PE-Cy7, as well as 7-Amino Actinomycin D (7AAD hereafter) for 40 minutes. Plates were washed twice, resuspended in 80 to 120 ul volumes and ran immediately in a BD LSRII flow cytometer to acquire 80% of the contents of each well. The sample files were analyzed using Flow Jo software to calculate the percentages and numbers of cells that had undergone at least one cell division, according to their CFSE profile, by gating sequentially on activated, live CD4+ or CD8+ T cells (7AAD-, CD5+ CD25+ CD4+ or 7AAD- CD5+ CD25+ CD8+, respectively). Mean values and standard deviations were calculated using Microsoft Excel software. Graphs were plotted using Microsoft Excel or Graphpad Prism.
Analysis of live CD4+ and CD8+ populations from Daudi cells or C4-2 cells treated with isolated T-cells revealed a significant increase in both the total number of cells and percent proliferating cells in the presence of Daudi cells displaying the target CD 19 antigen, but a lack of significant proliferation in the presence of C4-2 cells that lacked the CD19 antigen (Figure 12). Proliferation of CD8+ cells was very similar for bispecific molecules with the default IgGl hinge (TSCIOO) as well as those with longer hinges (TSC166, TSC167, TSC168). Slightly higher CD8+ proliferation at low concentrations was observed with the bispecific with the shorter IgA2 upper hinge (TSC165). Similar, but more marked differences were observed with proliferation of CD4+ cells, wherein the molecule containing the shorter IgA2 hinge (TSC165) showed higher proliferation at most concentrations than the standard IgGl hinge (TSCIOO), and molecules with longer hinges (TSC166, TSC167, TSC168) showed lower proliferation.
To confirm the differential activity of the IgA2 hinge, a second proliferation experiment was carried out with a titration to lower protein concentrations (Figure 13), comparing the bispecific featuring the IgA2 hinge (TSC165) to two different production lots of a bispecific featuring the default IgGl hinge (TSC127) and the bscl9x3 BiTE molecule. Similar to the previous experiment, less variation was observed with proliferation of CD8+ cells, with TSC165 showing comparable or greater induction of proliferation to bscl9x3, which in turn showed slightly higher or comparable proliferation to TSC127. Again, similar to the previous experiment, these trends repeated in a magnified fashion with the CD4+ cell proliferation, with TSC165 and bscl9x3 showing comparable proliferation, which in turn was noticeably greater than that of TSC127.
EXAMPLE 10
MODULATION OF REDIRECTED T-CELL CYTOTOXICITY BY POLYPEPTIDE HETERODIMERS
WITH ALTERED HINGE SEQUENCES
To compare the effectiveness of changing hinge composition in bispecific polypeptide heterodimer molecules on inducing target-dependent T-cell cytotoxicity, five different bispecific molecules were compared in a chromium (51Cr) release assay. Five different bispecific molecules (TSCIOO, TSC165, TSC166, TSC167 and TSC168, as described in Example 9) with a common anti-CD 19 binding domain (HD37), a common anti-CD3 binding domain (Cris7) and five different hinge constructs (IgGl SCC-P hinge for TSCIOO and TSC127, IgA2 hinge without the first Val linked to a human IgGl core hinge for TSC165, IgGl SSS-P hinge linked to a human IgGl core hinge for TSC166, a portion of mutated IgG3 hinge linked to a human IgGl core hinge for TSC167, and the IgM CH2 domain without the first Val linked to a human IgGl core hinge for TSC168) were compared.
Daudi Burkitt's lymphoma cells (CD 19+, RON-) were obtained from
ATCC (Manassas, VA) and cultured according to the provided protocol. T cells were isolated using a Pan T-cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) using the manufacturer's protocol (see also Example 5 for more information).
Cytotoxicity was assessed by a 51Cr release assay. Approximately 5x 106 Daudi cells were treated with 0.3 mCi of 51Cr and incubated for 75 minutes at 37°C. After 75 minutes, cells were washed 3 times with media (RPMI + 10% FBS) and resuspended in 11.5 mL of media. From this suspension, 50 μΐ, was dispensed per well into 96 well U-bottom plates (approximately 20,000 cells/well). Concentrations of bispecific molecules ranging from 10 nM to 0.1 pM were added to the target (Daudi) cells, bringing the total volume to 100 (jIJwell. Target cells were incubated at room temperature for 15 minutes. Then 100 of isolated T-cells (approximately 200,000) were added to bring the T-cell to target cell ratio to 10: 1. 50 μΐ, of 0.8%> NP-40 was added to a control well containing target cells, left for 15 minutes, then 100 of media was added to provide a total lysis control.
Plates were incubated for 4 hours, spun at 1500 rpm for 3 minutes, and
25 μί of supernatant was transferred from each well to the corresponding well of a 96- well Luma sample plate. Sample plates were allowed to air dry in a chemical safety hood for 18 hours, and then radioactivity was read on a Topcount scintillation counter using a standard protocol.
Analysis of cytotoxicity data showed strong T-cell directed cytotoxicity with the Daudi cells in the presence of T-cells and the anti-CD 19 directed bispecific molecules (TSCIOO - TSC168), reaching maximal lysis at a concentration between 5 and 50 pM (Figure 14). Similar to the trends observed in Example 9, the bispecific molecule with a shorter IgA2 upper hinge region (TSC165) showed comparable or greater cytotoxicity to the molecule with the standard IgGl upper hinge region (TSCIOO), whereas the molecules with longer upper hinge regions (TSC166, TSC167, TSC168) were less potent at inducing cytotoxicity.
EXAMPLE 11
BISPECIFIC HETE ODIME S WITH CH3-CH1 AND CH3-C LINKER VARIATIONS
The following bispecific heterodimers with CH3-CH1 and CH3-Ck linker variations were made:
Bispecific heterodimer TSC151 comprises single chain polypeptides
TSC145 and TSC148. Single chain polypeptide TSC145 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human IgGl CHI . The human CH3 and human IgGl CHI are linked by a linker having the sequence GGGSS (SEQ ID NO:847). The nucleotide and amino acid sequences of TSC145 are set forth in SEQ ID NOS:853 and 859, respectively. Single chain polypeptide TSC148 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human Ck (YAE). The human CH3 and human Ck (YAE) are linked with a linker having the sequence GGGSR (SEQ ID NO:850) in which R may alternatively be regarded as the first arginine of human Ck (YAE). The nucleotide and amino acid sequences of TSC148 are set forth in SEQ ID NOS:856 and 862, respectively.
Bispecific heterodimer TSC152 comprises single chain polypeptides TSC146 and TSC149. Single chain polypeptide TSC146 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human IgGl CHI . The human CH3 and human IgGl CHI are linked by a linker having the sequence SYSPNS (SEQ ID NO:848). The nucleotide and amino acid sequences of TSC146 are set forth in SEQ ID NOS:854 and 860, respectively. Single chain polypeptide TSC149 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human Ck (YAE). The human CH3 and human Ck (YAE) are linked with a linker having the sequence SYSPNSR (SEQ ID NO: 851) in which R may alternatively be regarded as the first arginine of human Ck (YAE). The nucleotide and amino acid sequences of TSC149 are set forth in SEQ ID NOS:857 and 863, respectively.
Bispecific heterodimer TSC153 comprises single chain polypeptides TSC147 and TSC150. Single chain polypeptide TSC147 comprises from its amino to carboxyl terminus: humanized Cris7 (anti-CD3) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human IgGl CHI . The human CH3 and human IgGl CHI are linked by a linker having the sequence SSLNTPNS (SEQ ID NO:849). The nucleotide and amino acid sequences of TSC147 are set forth in SEQ ID NOS:855 and 861, respectively. Single chain polypeptide TSC150 comprises from its amino to carboxyl terminus: HD37 (anti-CD19) scFv, human IgGl SCC-P hinge, human CH2 (ADCC/CDC null), human CH3, and human Ck (YAE). The human CH3 and human Ck (YAE) are linked with a linker having the sequence SSLNTPNSR (SEQ ID NO: 852) in which R may alternatively be regarded as the first arginine of human Ck (YAE). The nucleotide and amino acid sequences of TSC150 are set forth in SEQ ID NOS:858 and 864, respectively.
The above bispecific heterodimers were expressed according to Example
1. The following expression levels were obtained: 9.2 μg protein / ml of culture for TSC151, 11.2 μg protein / ml of culture for TSC152, and 14.7 μg protein / ml of culture for TSC153. In comparison, about 6 μg protein / ml of culture was obtained for heterodimers with a CH3-CH1 and CH3-Ck linker having the amino acid sequence KSR (SEQ ID NO:846). The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A polypeptide heterodimer, comprising:
(a) a first single chain polypeptide (SCP-I) comprising from one to four binding domains that specifically bind from one to four targets, a hinge (H-I), an immunoglobulin heterodimerization domain (HD-I), and an Fc region portion (FRP-I); and
(b) a second single chain polypeptide (SCP-II) comprising from zero to four binding domains that specifically bind from zero to four targets, a hinge (H-II), an immunoglobulin heterodimerization domain (HD-II), and an Fc region portion (FRP-
II);
wherein
(i) the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) preferentially associate with each other to form a polypeptide heterodimer comprised of the first single chain polypeptide (SCP-I) and the second single chain polypeptide (SCP-II), and
(1) the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CHI region, and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CL region, or
(2) the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CL region, and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CHI region; and
(ii) the Fc region portion of the first single chain polypeptide (FCP-I) and the Fc region portion of the second single chain polypeptide (FCP-II) each comprise an immunoglobulin CH2 and CH3 domain of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, or any combination thereof; an immunoglobulin CH3 domain of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM, or any combination thereof; or an immunoglobulin CH3 and CH4 domain of IgE, IgM, or a combination thereof,
provided that the polypeptide heterodimer comprises at least two binding domains that specifically bind at least two different targets.
2. The polypeptide heterodimer of claim 1, wherein the binding domains are single chain Fv (scFv) polypeptides.
3. The polypeptide heterodimer of claim 1, wherein the polypeptide heterodimer comprises two binding domains (BDl and BD2).
4. The polypeptide heterodimer of claim 3, wherein the two binding domains (BDl and BD2) are both on the first single chain polypeptide (SCP-I) and wherein the HD-I and FRP-I are disposed between BDl and BD2.
5. The polypeptide heterodimer of claim 3, wherein the first binding domain (BDl) is on the first single chain polypeptide (SCP-I) and the second binding domain (BD2) is on the second single chain polypeptide (SCP-II).
6. The polypeptide heterodimer of claim 5, wherein the first binding domain (BDl) is amino terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) is amino terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
7. The polypeptide heterodimer of claim 5, wherein the first binding domain (BDl) is amino terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) is carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
8. The polypeptide heterodimer of claim 5, wherein the first binding domain (BDl) is carboxyl terminal to the Fc region portion of the first single chain polypeptide (FRP-I), and the second binding domain (BD2) is carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
9. The polypeptide heterodimer of claim 1, wherein the polypeptide heterodimer comprises three binding domains (BDl, BD2 and BD3).
10. The polypeptide heterodimer of claim 9, wherein the HD-I and FRP-I are disposed between BDl and BD2, and the third binding domain (BD3) is amino terminal to the Fc region portion of the second single chain polypeptide (FRP-II).
11. The polypeptide heterodimer of claim 9, wherein the HD-I and FRP-I are disposed between BDl and BD2, and the third binding domain (BD3) is carboxyl terminal to the Fc region portion of the second single chain polypeptide (FRP- II).
12. The polypeptide heterodimer of claim 1, wherein the polypeptide heterodimer comprises four binding domains (BDl, BD2, BD3, and BD4).
13. The polypeptide heterodimer of claim 12, wherein the HD-I and FRP-I are disposed between BDl and BD2, and the HD-II and FRP-II are disposed between BD3 and BD4.
14. The polypeptide heterodimer of claim 1, wherein the polypeptide heterodimer comprises five to eight binding domains.
15. The polypeptide heterodimer of any one of claims 1 to 14, wherein at least one of the binding domains specifically binds to, or is an antagonist of, TCRa, TCRP, CD3y, CD35, CD3s, CD28, CD79b, hyperIL-6, monoIL-10 , CD86, CD20, PSMA, CD 19, HLA-DR, Ron, c-Met, CEACAM-6, LIGHT, GITRL, CD40, PDL1, PDL2, HVEM, LTBR, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, IGF1R, EphA2, PDGFR, VEGFR1-4, Angiopoietin 2, CD64, CD32A, CD 16, CD71, TNFR1, TNFR2, TWEAKR, TACI, BAFF-R, BCMA, FAS, CD32B, CD21, CD22, CD30, CD33, CD37, CD38, CD70, TNFa, IL-6, hyperIL-6, IL-2, IL-1, IL-7, IL-8, IL-17A/C, IP- 10, IFNy, IFNa, RANKL, FASL, TGFp, IL10, IL17A/F, CSF2, IGF1, IGF2, BLyS/APRIL, , HGF, MSP, EGF (including epiregulin, herregulin, β-regulin, neuregulin), HIF-l , VEGFA, VEGFB, VEGFC, VEGFD, TNFa, Wnt, sHH, TGFp, PDGF, TWEAK, EpCAM, CEA, PCTA-1, STEAP- 1, PSCA, ALCAM (CD 166), EphA2, CD 151, CA-125, MUC-1, MAGE-1, TROP2, CCR5, HER-3, HER-4, EGFR, CEA, MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, phCG, Lewis-Y, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, CD63, IGF1R, CD151, TGFBR2, GHRHR, GHR, IL-6R, gpl30, TNFR2, OSMRp, Patched- 1, Frizzled, Robol, CD80, CD81, CD86, OX40, CD40, CD 137, LIFRp, TLR7 or TLR9.
16. The polypeptide heterodimer of any one of claims 1 to 6, wherein at least one of the binding domains is an agonist of IL-10, HLA-G, HGF, IL-35, PD-1, BTLA, TNFR1, TNFR2, DR4, DR5, TWEAKR, or FAS.
17. The polypeptide heterodimer of any one of claims 1 to 6, wherein at least one binding domain specifically binds a TCR complex or a component thereof, and at least another binding domain specifically binds to PSMA, CD79b, CD 19, HLA- DR, CD20, RON, c-Met, or CEACAM-6.
18. The polypeptide heterodimer of any one of claims 1 to 6, wherein at least one binding domain specifically binds to CD28, and at least another binding domain specifically binds to, or is an antagonist of, CD79b, hyperIL-6, PDL2, monoIL- 10, CD86, LIGHT, GITRL, CD40, PDL1, HVEM, or LTBR.
19. The polypeptide heterodimer of any one of claim 1 to 6, wherein at least one binding domain specifically binds to CD28, and at least another binding domain is an agonist of IL-10, HLA-G, HGF, IL-35, PD-1, or BTLA.
20. The polypeptide heterodimer according to any one of claims 1 to 19 wherein the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises the first immunoglobulin CHI region and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises the first immunoglobulin CL region.
21. The polypeptide heterodimer of claim 20 wherein the first CHI region is amino terminal to the Fc region portion of the first single chain polypeptide, and the first CL region is amino terminal to the Fc region portion of the second single chain polypeptide.
22. The polypeptide heterodimer of claim 20 wherein the first CHI region is carboxyl terminal to the Fc region portion of the first single chain polypeptide, and the first CL region is carboxyl terminal to the Fc region portion of the second single chain polypeptide.
23. The polypeptide heterodimer of claim 20 wherein the first single chain polypeptide further comprises a second CHI region and the second single chain polypeptide further comprises a second CL region, and wherein the second CHI region of the first single chain polypeptide and the second CL region of the second single chain polypeptide associate with each other in the polypeptide heterodimer.
24. The polypeptide heterodimer of claim 23 wherein the Fc region portion of the first single chain polypeptide is disposed between the first and second CHI regions, and wherein the Fc region portion of the second single chain polypeptide is disposed between the first and second CL regions.
25. The polypeptide heterodimer of claim 23 wherein both the first and second CHI regions are amino terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CL regions are amino terminal to the Fc region portion of the second single chain polypeptide.
26. The polypeptide heterodimer of claim 23 wherein both the first and second CHI regions are carboxyl terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CL regions are carboxyl terminal to the Fc region portion of the second single chain polypeptide.
27. The polypeptide heterodimer according to any one of claims 1 to 19 wherein the immunoglobulin heterodimerization domain of the first single chain polypeptide (HD-I) comprises a first immunoglobulin CL region, and the immunoglobulin heterodimerization domain of the second single chain polypeptide (HD-II) comprises a first immunoglobulin CHI region.
28. The polypeptide heterodimer of claim 27 wherein the first CL region is amino terminal to the Fc region portion of the first single chain polypeptide, and the first CHI region is amino terminal to the Fc region portion of the second single chain polypeptide.
29. The polypeptide heterodimer of claim 27 wherein the first CL region is carboxyl terminal to the Fc region portion of the first single chain polypeptide, and the first CHI region is carboxyl terminal to the Fc region portion of the second single chain polypeptide.
30. The polypeptide heterodimer of claim 27 wherein the first single chain polypeptide further comprises a second CL region and the second single chain polypeptide further comprises a second CHI region, and wherein the second CL region of the first single chain polypeptide and the second CHI region of the second single chain polypeptide associate with each other in the polypeptide heterodimer.
31. The polypeptide heterodimer of claim 30 wherein the Fc region portion of the first single chain polypeptide is disposed between the first and second CL regions, and wherein the Fc region portion of the second single chain polypeptide is disposed between the first and second CHI regions.
32. The polypeptide heterodimer of claim 30, wherein both the first and second CL regions are amino terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CHI regions are amino terminal to the Fc region portion of the second single chain polypeptide.
33. The polypeptide heterodimer of claim 30, wherein both the first and second CL regions are carboxyl terminal to the Fc region portion of the first single chain polypeptide, and both the first and second CHI regions are carboxyl terminal to the Fc region portion of the second single chain polypeptide.
34. The polypeptide heterodimer of claim 20 wherein the first single chain polypeptide further comprises a second CL region and the second single chain polypeptide further comprises a second CHI region, and wherein the second CL region of the first single chain polypeptide and the second CHI region of the second single chain polypeptide associate with each other in the polypeptide heterodimer.
35. The polypeptide heterodimer of claim 34 wherein
(a) in the first single chain polypeptide, the first CHI region is amino terminal to the Fc region portion, and the second CL region is carboxyl terminal to the Fc region portion, and
(b) in the second single chain polypeptide, the first CL region is amino terminal to the Fc region portion, and the second CHI region is carboxyl terminal to the Fc region portion.
36. The polypeptide heterodimer of claim 34 wherein
(a) in the first single chain polypeptide, the first CHI region is carboxyl terminal to the Fc region portion, and the second CL region is amino terminal to the Fc region portion, and (b) in the second single chain polypeptide, the first CL region is carboxyl terminal to the Fc region portion, and the second CHI region is amino terminal to the Fc region portion.
37. The polypeptide heterodimer of claim 34 wherein
(a) in the first single chain polypeptide, both the first CHI region and the second CL regions are amino terminal to the Fc region portion, and the first CHI region is amino terminal to the second CL region, and
(b) in the second single chain polypeptide, both the first CL region and the second CHI region are amino terminal to the Fc region portion, and the first CL region is amino terminal to the second CHI region.
38. The polypeptide heterodimer of claim 34 wherein
(a) in the first single chain polypeptide, both the first CHI region and the second CL regions are amino terminal to the Fc region portion, and the second CL region is amino terminal to the first CHI region, and
(b) in the second single chain polypeptide, both the first CL region and the second CHI region are amino terminal to the Fc region portion, and the second CHI region is amino terminal to the first CL region.
39. The polypeptide heterodimer of claim 34 wherein
(a) in the first single chain polypeptide, both the first CHI region and the second CL regions are carboxyl terminal to the Fc region portion, and the first CHI region is amino terminal to the second CL region, and
(b) in the second single chain polypeptide, both the first CL region and the second CHI region are carboxyl terminal to the Fc region portion, and the first CL region is amino terminal to the second CHI region.
40. The polypeptide heterodimer of claim 34 wherein
(a) in the first single chain polypeptide, both the first CHI region and the second CL regions are carboxyl terminal to the Fc region portion, and the second CL region is amino terminal to the first CHI region, and
(b) in the second single chain polypeptide, both the first CL region and the second CHI region are carboxyl terminal to the Fc region portion, and the second CHI region is amino terminal to the first CL region.
I l l
41. The polypeptide heterodimer of any one of claims 1 to 40 wherein the first CL region is a CK region.
42. The polypeptide heterodimer of any one of claims 1 to 40 wherein the first CL region is a region.
43. The polypeptide heterodimer of any one of claims 24 to 27 and 31 to 41 , wherein the second CL region is a CK region.
44. The polypeptide heterodimer of any one of claims 23 to 26 and 30 to 40, wherein the second CL region is a C region.
45. The polypeptide heterodimer of claim 41 or claim 43, wherein the CK region is a wild type human immunoglobulin CK region.
46. The polypeptide heterodimer of claim 41 or claim 43, wherein the CK region is an altered human immunoglobulin CK region with one or more amino acids of a wild type human CK region substituted at N29, N30, Q52, V55, T56, T56, S68, or T70.
47. The polypeptide heterodimer of claim 46, wherein the one or more amino acid substitutions are selected from Ala (A), Arg (R), Trp (W), Tyr (Y), Glu (E), Gin (Q), Lys (K), Asp (D), Met (M), Ser (S), and Phe (F).
48. The polypeptide heterodimer of claim 41 or claim 43, wherein the CHI region is an altered human immunoglobulin CHI region comprising an amino acid substitution by which Val (V) at position 68 is substituted by Lys (K), Arg (R) or His (H), and wherein the Ck region is an altered human immunoglobulin Ck region comprising an amino acid substitution by which Leu (L) at position 29 is substituted by Asp (D) or Glu (E).
49. The polypeptide heterodimer of claim 41 or claim 43, wherein the CHI region is an altered human immunoglobulin CHI region comprising an amino acid substitution by which Val (V) at position 68 is changed to Asp (D) or Glu (E), and wherein the Ck region is an altered human immunoglobulin Ck region comprising an amino acid substitution by which Leu (L) at position 29 is changed to Lys (K), Arg (R) or His (H).
50. The polypeptide heterodimer of claim 42 or claim 44, wherein the region is a wild type human immunoglobulin region.
51. The polypeptide heterodimer of any one of claims 1 to 50, wherein the first CHI region or the second CHI region when present is a wild type human immunoglobulin CHI region.
52. The polypeptide heterodimer of any one of claims 1 to 50, wherein the first CHI region or the second CHI region when present is a wild type human IgGl CHI region.
53. The polypeptide heterodimer of any one of claims 1 to 50, wherein the first CHI region or the second CHI region when present is an altered human immunoglobulin CHI region with the cysteine of a wild type human immunoglobulin CHI region that is involved in forming a disulfide bond with a wild type human immunoglobulin CL region deleted or substituted.
54. The polypeptide heterodimer of any one of claims 1 to 50, wherein the first CHI region or the second CHI region when present is an altered human IgGl CHI region with the cysteine of a wild type human immunoglobulin CHI region that is involved in forming a disulfide bond with a wild type human immunoglobulin CL region deleted or substituted.
55. The polypeptide heterodimer of claim 41 or 43, wherein the CK region is an altered human immunoglobulin Ck region with the cysteine residue of a wild type human CK region that is involved in forming a disulfide bond with a wild type human immunoglobulin CHI region deleted or substituted.
56. The polypeptide heterodimer of claim 42 or claim 44, wherein the region is an altered human immunoglobulin region with the cysteine residue of a wild type human region that is involved in forming a disulfide bond with a wild type human immunoglobulin CHI region deleted or substituted.
57. The polypeptide heterodimer of claim 53, wherein the first CHI region and the second CHI region when present is a polypeptide comprising SEQ ID NO: 114, 844 or 845.
58. The polypeptide heterodimer of claim 46 or 55, wherein the Ck region is selected from any one of the polypeptides comprising SEQ ID NOS: 141-178, 202, and 838-843.
59. The polypeptide heterodimer of claim 50 or 56, wherein the region is a polypeptide comprising SEQ ID NO: 140.
60. The polypeptide heterodimer of any one of claims 1 to 59, wherein the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH2 domain.
61. The polypeptide heterodimer of claim 60, wherein the immunoglobulin CH2 domain is an IgGl CH2 domain.
62. The polypeptide heterodimer of claim 60, wherein the immunoglobulin CH2 domain is an IgG2, IgG3, IgG4, IgAl, IgA2, or IgD CH2 domain.
63. The polypeptide heterodimer of any one of claims 1 to 59, wherein the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH3 domain.
64. The polypeptide heterodimer of claim 63, wherein the immunoglobulin CH3 domain is an IgGl CH3 domain.
65. The polypeptide heterodimer of claim 63, wherein the immunoglobulin CH3 domain is an IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE or IgM CH3 domain.
66. The polypeptide heterodimer of any one of claims 1 to 65, wherein the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second single chain polypeptide (FRP-II) each comprise an immunoglobulin CH2 domain and an immunoglobulin CH3 domain.
67. The polypeptide heterodimer of claim 66, wherein
(i) the immunoglobulin CH3 domain is linked to a CHI domain immediately carboxyl terminal to the immunoglobulin CH3 domain in one single chain polypeptide via a peptide comprising SEQ ID NOS:846, 847, 848, or 849, and
(ii) the immunoglobulin CH3 domain is linked to a Ck domain immediately carboxyl terminal to the immunoglobulin CH3 domain in the other single chain polypeptide via a peptide comprising SEQ ID NOs:846, 850, 951, or 852.
68. The polypeptide heterodimer of claim 66, wherein the immunoglobulin CH2 and CH3 domains are IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, or IgD CH2 and CH3 domains.
69. The polypeptide heterodimer of any one of claims 1 to 59, wherein the Fc region portion of the first single chain polypeptide (FRP-I) and the Fc region portion of the second chain polypeptide (FRP-II) comprise IgM or IgE CH3 and CH4 domains.
70. The polypeptide heterodimer of any one of claims 60 to 62 and 66 to 68, wherein the CH2 domain is an altered human IgGl, IgG2, IgG3, or IgG4 CH2 domain that comprises an amino acid substitution at position 297 and at least one additional substitution or deletion at positions 234 to 238.
71. The polypeptide heterodimer of any one of claims 60 to 62 and 66 to 68, wherein the CH2 domain is an altered human IgGl, IgG2, IgG3, or IgG4 CH2 domain that comprises one or more amino acid mutations at positions 234-238 and at least one substitution at position 253, 310, 318, 320, 322, or 331.
72. The polypeptide heterodimer of any one of claims 60 to 62 and 66 to 68, wherein the CH2 domain is an altered human IgGl, IgG2, IgG3, or IgG4 CH2 domain that comprises an amino acid substitution at the asparagine of position 297, one or more substitutions or deletions at positions 234 to 238, and at least one substitution at position 253, 310, 318, 320, 322, or 331.
73. The polypeptide heterodimer of any one of claims 60 to 62 and 66 and 68, wherein the CH2 domain is an altered human IgGl CH2 domain that comprises amino acid substitutions at positions L234, L235, G237, E318, K320 and K322.
74. The polypeptide heterodimer of any one of claims 63 to 67, wherein
(a) the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a T366W, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a Y407A substitution,
(b) the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a T366Y substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a Y407T substitution,
(c) the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a T366W substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises T366S, L368A and Y407V substitutions,
(d) the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a Y407A substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366W substitution,
(e) the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises a Y407T substitution, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366Y substitution, or
(f) the CH3 domain of the first single chain polypeptide is an altered human IgGl, IgG2, IgG3, or IgG4 CH3 domain that comprises T366S, L368A and Y407W substitutions, and the CH3 domain of the second single chain polypeptide is an altered human IgGl, IgG2, IgG3 or IgG4 CH3 domain that comprises a T366W substitution.
75. The polypeptide heterodimer of any one of claims 1 to 74 wherein the hinge of both the first and second single chain polypeptides is an immunoglobulin hinge region.
76. The polypeptide heterodimer of claim 75 wherein the immunoglobulin hinge is an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, or IgE hinge.
77. The polypeptide heterodimer of claim 75 wherein the immunoglobulin hinge is a wild type immunoglobulin hinge.
78. The polypeptide heterodimer of claim 75 wherein the immunoglobulin hinge is an altered immunoglobulin hinge selected from SEQ ID NOS:232, 234, 240, 664-673, 675 and 676.
79. The polypeptide heterodimer of any one of claims 75 to 78 wherein the hinge region is
(a) at the amino terminal to the Fc region portion,
(b) disposed between the binding domain and the immunoglobulin heterodimerization domain,
(c) disposed between the immunoglobulin heterodimerization domain and the Fc region portion, or
(d) at the amino terminus of the first or second single chain polypeptide.
80. The polypeptide heterodimer of any one of claims 1 to 74 wherein at least one of the first and second single chain polypeptide hinges is a C-type lectin hinge region.
81. The polypeptide heterodimer of claim 80 wherein the C-type lectin hinge region is an NKg2A or NKg2D peptide, or a derivative thereof.
82. The polypeptide heterodimer of claim 80 or claim 81 wherein the hinge region is disposed between the Fc region portion and a binding domain, or at the carboxyl terminus of the first or second single chain polypeptide.
83. The polypeptide heterodimer of any one of claims 1 to 82 wherein the hinges of the first and second single chain polypeptides are identical.
84. The polypeptide heterodimer of any one of claims 1 to 82 wherein the hinges of the first and second single chain polypeptides are different.
85. The heterodimer of any one of claims 1-14 and 20-84, wherein the first single chain polypeptide comprises a binding domain that specifically binds a TCR complex or a component thereof, and the second single chain polypeptide comprises a binding domain that specifically binds CD 19, CD79b, HLA-DR or CD20.
86. The heterodimer of any one of claims 1-14 and 20-84, wherein the first single chain polypeptide comprises a binding domain that specifically binds CD28, and the second single chain polypeptide comprises a binding domain (a) that specifically binds CD79b, hyperIL-6, or CD86 or (b) that comprises a PDL ectodomain or a monoIL-10.
87. The heterodimer of any one of claims 1-14 and 20-84, wherein the first single chain polypeptide comprises a binding domain that specifically binds c- Met, and the second single chain polypeptide comprises a binding domain that specifically binds RON.
88. The heterodimer of claim 1, wherein the first and second single chain polypeptides comprise SEQ ID NOS: SEQ ID NOS:2 and 4, SEQ ID NOS:6 and 8, SEQ ID NOS: 10 and 12, SEQ ID NOS: 14 and 16, SEQ ID NOS: 18 and 20, SEQ ID NOS:20 and 22, SEQ ID NOS:20 and 24, SEQ ID NOS:30 and 32, SEQ ID NOS:29 and 31, SEQ ID NOS:29 and 32, SEQ ID NOS:30 and 72, SEQ ID NOS:53 and 72, SEQ ID NOS:54 and 72, SEQ ID NOS:55 and 72, SEQ ID NOS:70 and 72, SEQ ID NOS:71 and 72, SEQ ID NOS:63 and 56, SEQ ID NOS:64 and 57, SEQ ID NOS:65 and 60, SEQ ID NOS:66 and 58, SEQ ID NOS:67 and 59, SEQ ID NOS:68 and 61, SEQ ID NOS:69 and 62, SEQ ID NOS:54 and 811, SEQ ID NOS:54 and 812, SEQ ID NOS:54 and 813, SEQ ID NOS:814 and 818, SEQ ID NOS:815 and 818, SEQ ID NOS:816 and 818, SEQ ID NOS:817 and 818, SEQ ID NOS:814 and 820, SEQ ID NOS:814 and 821, SEQ ID NOS:54 and 819, SEQ ID NOS:814 and 826, SEQ ID NOS:814 and 822, SEQ ID NOS: 814 and 823, SEQ ID NOS:814 and 824, SEQ ID NOS:859 and 862, SEQ ID NOS:860 and 863, SEQ ID NOS:861 and 864, SEQ ID NOS:874 and 825, SEQ ID NOS:875 and 879, SEQ ID NOS:876 and 880, SEQ ID NOS:877 and 881, or SEQ ID NOS:878 and 882.
89. A composition comprising a polypeptide heterodimer of any one of claims 1 to 88 and a pharmaceutically acceptable excipient.
90. An expression vector capable of expressing the polypeptide heterodimer of any one of claims 1 to 88, comprising a first polynucleotide encoding the first single chain polypeptide and a second polynucleotide encoding the second single chain polypeptide.
91. A host cell comprising the expression vector of claim 90.
92. A host cell comprising first and second expression vectors capable of expressing the first and second single chain polypeptides, respectively, of the polypeptide heterodimer of any one of claims 1 to 88.
93. A method for making a polypeptide heterodimer, comprising
(a) culturing a host cell of claim 91 or claim 92 under conditions suitable to express first and second single chain polypeptides, and
(b) optionally isolating or purifying the heterodimers formed from the first and second single chain polypeptides from the culture.
94. A method for directing T cell activation, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer according to any one of claims 1 to 88, wherein the polypeptide heterodimer comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s or a combination thereof, and a second binding domain that specifically binds a different target.
95. A method for inhibiting growth, metastasis or metastatic growth of a malignancy, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer according to any one of claims 1 to 88, wherein the polypeptide heterodimer comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, c-Met, or Ron.
96. A method for treating an autoimmune or inflammatory condition, comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer according to any one of claims 1 to 88, wherein the polypeptide heterodimer comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, CD3s, or CD28.
97. A method for treating a B-cell associated disorder or disease comprising administering to a patient in need thereof an effective amount of a polypeptide heterodimer according to any one of claims 1 to 88, wherein the polypeptide heterodimer comprises a binding domain that specifically binds TCRa, TCRP, CD3y, CD35, or CD3s, and a second binding domain that specifically binds to CD 19, CD20, CD79b or HLA-DR.
98. The method of claim 95, further comprising administering to a patient in need thereof a chemotherapeutic agent or ionizing radiation.
99. The method of any one of claims 94 to 97, further comprising administering to a patient in need thereof a second active agent.
100. The method of claim 99, wherein the second active agent is a second polypeptide heterodimer according to any one of claims 1 to 88.
101. The method of claim 99, wherein the second active agent is a monoclonal antibody or an immunoglobulin-derived fusion protein.
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Cited By (187)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2011108714A1 (en) * 2010-03-04 2013-06-27 中外製薬株式会社 Antibody constant region variants
US8524234B2 (en) 2011-11-03 2013-09-03 Tolera Therapeutics, Inc Antibody for selective inhibition of T-cell responses
WO2014008391A1 (en) * 2012-07-05 2014-01-09 Genentech, Inc. Expression and secretion system
US8632774B2 (en) 2008-07-02 2014-01-21 Emergent Product Development Seattle, Llc Antagonists of IL-6
WO2014047231A1 (en) * 2012-09-21 2014-03-27 Regeneron Pharmaceuticals, Inc. Anti-cd3 antibodies, bispecific antigen-binding molecules that bind cd3 and cd20, and uses thereof
US8722049B2 (en) 2011-11-03 2014-05-13 Tolera Therapeutics, Inc. Antibody and methods for selective inhibition of T-cell responses
US8772459B2 (en) 2009-12-02 2014-07-08 Imaginab, Inc. J591 minibodies and Cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use
JP2014524234A (en) * 2011-07-29 2014-09-22 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Switch costimulatory receptors
WO2014177460A1 (en) 2013-04-29 2014-11-06 F. Hoffmann-La Roche Ag Human fcrn-binding modified antibodies and methods of use
WO2014177461A1 (en) 2013-04-29 2014-11-06 F. Hoffmann-La Roche Ag Fcrn-binding abolished anti-igf-1r antibodies and their use in the treatment of vascular eye diseases
US8940298B2 (en) 2007-09-04 2015-01-27 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection
US8940871B2 (en) 2006-03-20 2015-01-27 The Regents Of The University Of California Engineered anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting
US8951737B2 (en) 1996-05-06 2015-02-10 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
CN104487587A (en) * 2012-04-20 2015-04-01 新兴产品开发西雅图有限公司 Cd3 binding polypeptides
WO2015101588A1 (en) 2014-01-06 2015-07-09 F. Hoffmann-La Roche Ag Monovalent blood brain barrier shuttle modules
WO2015101586A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
WO2015107026A1 (en) 2014-01-15 2015-07-23 F. Hoffmann-La Roche Ag Fc-region variants with modified fcrn- and maintained protein a-binding properties
JP2015528003A (en) * 2012-07-13 2015-09-24 ザイムワークス，インコーポレイテッド Bispecific asymmetric heterodimer comprising anti-CD3 construct
WO2015150446A1 (en) 2014-04-02 2015-10-08 F. Hoffmann-La Roche Ag Method for detecting multispecific antibody light chain mispairing
WO2015150447A1 (en) 2014-04-02 2015-10-08 F. Hoffmann-La Roche Ag Multispecific antibodies
RU2577226C2 (en) * 2014-04-10 2016-03-10 Общество с ограниченной ответственностью, "Международный биотехнологический центр "Генериум" ("МБЦ "Генериум") Methods for making bispecific antibodies against cd3*cd19 in flexybody format in mammalian cells
US9346887B2 (en) 2010-03-12 2016-05-24 Immunogen, Inc. CD37-binding molecules and immunoconjugates thereof
JPWO2014054804A1 (en) * 2012-10-05 2016-08-25 協和発酵キリン株式会社 Heterodimeric protein composition
US9447189B2 (en) 2011-04-01 2016-09-20 Immunogen, Inc. CD37-binding molecules and immunoconjugates thereof
WO2016154585A1 (en) 2015-03-26 2016-09-29 Charles Sentman Anti-mica antigen binding fragments, fusion molecules, cells which express and methods of using
US9493564B2 (en) 2008-10-02 2016-11-15 Aptevo Research And Development Llc CD86 antagonist multi-target binding proteins
US9611323B2 (en) 2010-11-30 2017-04-04 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses therefor
EP3150637A1 (en) 2015-10-02 2017-04-05 F. Hoffmann-La Roche AG Multispecific antibodies
WO2017055395A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xrob04 bispecific t cell activating antigen binding molecules
WO2017055391A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules binding mesothelin and cd3
WO2017055388A2 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules
WO2017055389A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific anti-ceaxcd3 t cell activating antigen binding molecules
WO2017055399A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Cellular based fret assay for the determination of simultaneous binding
WO2017055393A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xtim-3 bispecific t cell activating antigen binding molecules
WO2017055392A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xcd44v6 bispecific t cell activating antigen binding molecules
WO2017055404A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific antibodies specific for pd1 and tim3
WO2017055314A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific anti-cd19xcd3 t cell activating antigen binding molecules
WO2017055385A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xgd2 bispecific t cell activating antigen binding molecules
US9670269B2 (en) 2006-03-31 2017-06-06 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
EP3178848A1 (en) 2015-12-09 2017-06-14 F. Hoffmann-La Roche AG Type ii anti-cd20 antibody for reducing formation of anti-drug antibodies
US9688762B2 (en) 2007-09-26 2017-06-27 Chugai Sciyaku Kabushiki Kaisha Modified antibody constant region
JP2017127305A (en) * 2011-04-22 2017-07-27 アプティーボ リサーチ アンド デベロップメント エルエルシー Prostate-specific membrane antigen binding protein and related composition and method
WO2017191101A1 (en) 2016-05-02 2017-11-09 F. Hoffmann-La Roche Ag The contorsbody - a single chain target binder
US9815897B2 (en) 2013-05-02 2017-11-14 Anaptysbio, Inc. Antibodies directed against programmed death-1 (PD-1)
US9828429B2 (en) 2007-09-26 2017-11-28 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substitution in CDR
EP3252078A1 (en) 2016-06-02 2017-12-06 F. Hoffmann-La Roche AG Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer
WO2018007314A1 (en) 2016-07-04 2018-01-11 F. Hoffmann-La Roche Ag Novel antibody format
US9890204B2 (en) 2009-04-07 2018-02-13 Hoffmann-La Roche Inc. Trivalent, bispecific antibodies
WO2018038684A1 (en) * 2016-08-26 2018-03-01 Agency For Science, Technology And Research Macrophage stimulating protein receptor (or ron - recepteur d' origine nantais) antibodies and uses thereof
US9914776B2 (en) 2014-08-04 2018-03-13 Hoffmann-La Roche Inc. Bispecific T cell activating antigen binding molecules
WO2018060301A1 (en) 2016-09-30 2018-04-05 F. Hoffmann-La Roche Ag Bispecific antibodies against cd3
WO2018005226A3 (en) * 2016-06-22 2018-04-05 Alkermes, Inc. Compositions and methods for modulating il-10 immunostimulatory and anti-inflammatory properties
US9982036B2 (en) 2011-02-28 2018-05-29 Hoffmann-La Roche Inc. Dual FC antigen binding proteins
CN108137671A (en) * 2015-10-10 2018-06-08 中国科学院广州生物医药与健康研究院 The soluble PDL-1 molecules of high-affinity
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
CN108367004A (en) * 2015-09-21 2018-08-03 阿帕特夫研究和发展有限公司 CD3 combination polypeptides
US10066018B2 (en) 2009-03-19 2018-09-04 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US10087250B2 (en) 2012-10-08 2018-10-02 Roche Glycart Ag Fc-free antibodies comprising two fab-fragments and methods of use
WO2018184966A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Antibodies binding to steap-1
WO2018185043A1 (en) 2017-04-05 2018-10-11 F. Hoffmann-La Roche Ag Bispecific antibodies specifically binding to pd1 and lag3
WO2018184965A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of il-2 with an anti-pd-1 and tim-3 bispecific antibody
WO2018184964A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of an anti-pd-1 antibody with a mutant il-2 or with il-15
WO2018189220A1 (en) 2017-04-13 2018-10-18 F. Hoffmann-La Roche Ag An interleukin-2 immunoconjugate, a cd40 agonist, and optionally a pd-1 axis binding antagonist for use in methods of treating cancer
US10106600B2 (en) 2010-03-26 2018-10-23 Roche Glycart Ag Bispecific antibodies
US10138293B2 (en) 2007-12-21 2018-11-27 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
WO2018220099A1 (en) 2017-06-02 2018-12-06 F. Hoffmann-La Roche Ag Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer
US10150808B2 (en) 2009-09-24 2018-12-11 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant regions
US10155815B2 (en) 2013-02-26 2018-12-18 Roche Glycart Ag Bispecific T cell activating antigen binding molecules
US10174124B2 (en) 2013-12-17 2019-01-08 Genentech, Inc. Anti-CD3 antibodies and methods of use
US10253091B2 (en) 2009-03-19 2019-04-09 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US10251952B2 (en) 2014-06-26 2019-04-09 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibody brain shuttles and use thereof
WO2019077092A1 (en) 2017-10-20 2019-04-25 F. Hoffmann-La Roche Ag Method for generating multispecific antibodies from monospecific antibodies
WO2019086362A1 (en) 2017-10-30 2019-05-09 F. Hoffmann-La Roche Ag Method for in vivo generation of multispecific antibodies from monospecific antibodies
WO2019086394A1 (en) 2017-11-01 2019-05-09 F. Hoffmann-La Roche Ag The compbody - a multivalent target binder
WO2019086395A1 (en) 2017-11-01 2019-05-09 F. Hoffmann-La Roche Ag Trifab-contorsbody
US10323094B2 (en) 2015-06-16 2019-06-18 Genentech, Inc. Humanized and affinity matured antibodies to FcRH5 and methods of use
US10323099B2 (en) 2013-10-11 2019-06-18 Hoffmann-La Roche Inc. Multispecific domain exchanged common variable light chain antibodies
WO2019122052A2 (en) 2017-12-21 2019-06-27 F. Hoffmann-La Roche Ag Antibodies binding to hla-a2/wt1
WO2019154890A1 (en) 2018-02-09 2019-08-15 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2019154776A1 (en) 2018-02-06 2019-08-15 F. Hoffmann-La Roche Ag Treatment of ophthalmologic diseases
US10435479B2 (en) 2014-05-10 2019-10-08 Sorrento Therapeutics, Inc. Chemically-locked bispecific antibodies
CN110327458A (en) * 2019-07-09 2019-10-15 上海交通大学医学院 Autocrine VEGFB is in T cell metabolism and the application in function and immunotherapy of tumors
WO2019202041A1 (en) 2018-04-18 2019-10-24 F. Hoffmann-La Roche Ag Multispecific antibodies and use thereof
US10465000B2 (en) 2013-12-20 2019-11-05 Hoffmann-La Roche Inc. Humanized anti-Tau(pS422) antibodies and methods of use
US10501545B2 (en) 2015-06-16 2019-12-10 Genentech, Inc. Anti-CLL-1 antibodies and methods of use
US10517969B2 (en) 2009-02-17 2019-12-31 Cornell University Methods and kits for diagnosis of cancer and prediction of therapeutic value
US10544220B2 (en) 2015-01-08 2020-01-28 Genmab A/S Bispecific antibodies against CD3 and CD20
US10550193B2 (en) 2014-03-19 2020-02-04 Regeneron Pharmaceuticals, Inc. Methods and antibody compositions for tumor treatment
EP3611188A1 (en) 2014-11-06 2020-02-19 F. Hoffmann-La Roche AG Fc-region variants with modified fcrn-binding and methods of use
US10596257B2 (en) 2016-01-08 2020-03-24 Hoffmann-La Roche Inc. Methods of treating CEA-positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD3 bispecific antibodies
US10611825B2 (en) 2011-02-28 2020-04-07 Hoffmann La-Roche Inc. Monovalent antigen binding proteins
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
WO2020089051A1 (en) 2018-10-29 2020-05-07 F. Hoffmann-La Roche Ag Antibody formulation
US10662244B2 (en) 2014-11-17 2020-05-26 Regeneron Pharmaceuticals, Inc. Methods for tumor treatment using CD3XCD20 bispecific antibody
WO2020127619A1 (en) 2018-12-21 2020-06-25 F. Hoffmann-La Roche Ag Antibodies binding to cd3
WO2020136060A1 (en) 2018-12-28 2020-07-02 F. Hoffmann-La Roche Ag A peptide-mhc-i-antibody fusion protein for therapeutic use in a patient with amplified immune response
WO2020180398A1 (en) * 2019-01-14 2020-09-10 The Regents Of The University Of California Compositions and methods for modulating cellular internalization
US10781262B2 (en) 2014-11-20 2020-09-22 Hoffmann-La Roche Inc. Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists
US10822402B2 (en) 2015-06-24 2020-11-03 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibodies and methods of use
US10881741B2 (en) 2014-12-19 2021-01-05 Alkermes, Inc. Single chain Fc fusion proteins
WO2021001289A1 (en) 2019-07-02 2021-01-07 F. Hoffmann-La Roche Ag Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody
WO2021009146A1 (en) 2019-07-15 2021-01-21 F. Hoffmann-La Roche Ag Antibodies binding to nkg2d
WO2021018925A1 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021018859A2 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021058804A1 (en) * 2019-09-25 2021-04-01 Universität Stuttgart Binding modules comprising modified ehd2 domains
US11007267B2 (en) 2015-06-16 2021-05-18 Genentech, Inc. Anti-CD3 antibodies and methods of use
US11013801B2 (en) 2015-12-09 2021-05-25 Hoffmann-La Roche Inc. Treatment method
WO2021122733A1 (en) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Bispecific anti-ccl2 antibodies
WO2021122875A1 (en) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Antibodies binding to hla-a2/mage-a4
US11046784B2 (en) 2006-03-31 2021-06-29 Chugai Seiyaku Kabushiki Kaisha Methods for controlling blood pharmacokinetics of antibodies
EP3842453A1 (en) 2014-11-06 2021-06-30 F. Hoffmann-La Roche AG Fc-region variants with modified fcrn- and protein a-binding properties
US11084877B2 (en) 2014-09-12 2021-08-10 Genentech, Inc. Anti-CLL-1 antibodies and immunoconjugates
US11104740B2 (en) 2015-08-28 2021-08-31 Debiopharm International, S.A. Antibodies and assays for detection of CD37
EP3878866A1 (en) 2013-04-29 2021-09-15 F. Hoffmann-La Roche AG Fc-receptor binding modified asymmetric antibodies and methods of use
US11124576B2 (en) 2013-09-27 2021-09-21 Chungai Seiyaku Kabushiki Kaisha Method for producing polypeptide heteromultimer
US11142587B2 (en) 2015-04-01 2021-10-12 Chugai Seiyaku Kabushiki Kaisha Method for producing polypeptide hetero-oligomer
US11142577B2 (en) 2014-09-12 2021-10-12 The Board Of Trustees Of The Leland Stanford Junior University WNT signaling agonist molecules
WO2021209402A2 (en) 2020-04-15 2021-10-21 F. Hoffmann-La Roche Ag Immunoconjugates
US11155624B2 (en) 2016-11-01 2021-10-26 Anaptysbio, Inc. Antibodies directed against programmed death-1 (PD-1)
WO2021255146A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and cea
WO2021255138A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Immune activating fc domain binding molecules
WO2021255155A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and cd19
WO2021255142A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3
WO2021255143A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and folr1
US11242390B2 (en) 2016-03-22 2022-02-08 Hoffmann-La Roche Inc. Protease-activated T cell bispecific molecules
US11254744B2 (en) 2015-08-07 2022-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies
US11278629B2 (en) 2016-11-02 2022-03-22 Debiopharm International, S.A. Methods for improving anti-CD37 immunoconjugate therapy
US11286300B2 (en) 2015-10-01 2022-03-29 Hoffmann-La Roche Inc. Humanized anti-human CD19 antibodies and methods of use
WO2022129120A1 (en) 2020-12-17 2022-06-23 F. Hoffmann-La Roche Ag Anti-hla-g antibodies and use thereof
US11385233B2 (en) 2015-03-05 2022-07-12 Autolus Limited Methods of depleting malignant T-cells
WO2022148853A1 (en) 2021-01-11 2022-07-14 F. Hoffmann-La Roche Ag Immunoconjugates
WO2022148732A1 (en) 2021-01-06 2022-07-14 F. Hoffmann-La Roche Ag Combination therapy employing a pd1-lag3 bispecific antibody and a cd20 t cell bispecific antibody
US11395796B2 (en) 2015-06-08 2022-07-26 Debiopharm International, S.A. Anti-CD37 immunoconjugate and anti-CD20 antibody combinations
US11407830B2 (en) 2017-01-09 2022-08-09 Tesaro, Inc. Methods of treating cancer with anti-PD-1 antibodies
US11459404B2 (en) 2013-02-26 2022-10-04 Roche Glycart Ag Bispecific T cell activating antigen binding molecules
US11466094B2 (en) 2016-11-15 2022-10-11 Genentech, Inc. Dosing for treatment with anti-CD20/anti-CD3 bispecific antibodies
WO2022228705A1 (en) 2021-04-30 2022-11-03 F. Hoffmann-La Roche Ag Dosing for combination treatment with anti-cd20/anti-cd3 bispecific antibody and anti-cd79b antibody drug conjugate
WO2022228706A1 (en) 2021-04-30 2022-11-03 F. Hoffmann-La Roche Ag Dosing for treatment with anti-cd20/anti-cd3 bispecific antibody
WO2022263501A1 (en) 2021-06-18 2022-12-22 F. Hoffmann-La Roche Ag Bispecific anti-ccl2 antibodies
WO2022263507A1 (en) 2021-06-17 2022-12-22 Boehringer Ingelheim International Gmbh Novel tri-specific binding molecules
US11535679B2 (en) 2020-09-10 2022-12-27 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating follicular lymphoma
WO2023279092A2 (en) 2021-07-02 2023-01-05 Genentech, Inc. Methods and compositions for treating cancer
US11548952B2 (en) 2020-09-10 2023-01-10 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma
WO2023001884A1 (en) 2021-07-22 2023-01-26 F. Hoffmann-La Roche Ag Heterodimeric fc domain antibodies
WO2023010095A1 (en) 2021-07-28 2023-02-02 F. Hoffmann-La Roche Ag Methods and compositions for treating cancer
US11590223B2 (en) 2018-08-31 2023-02-28 Regeneron Pharmaceuticals, Inc. Dosing strategy that mitigates cytokine release syndrome for therapeutic antibodies
US11608383B2 (en) 2020-09-10 2023-03-21 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating follicular lymphoma
WO2023062048A1 (en) 2021-10-14 2023-04-20 F. Hoffmann-La Roche Ag Alternative pd1-il7v immunoconjugates for the treatment of cancer
WO2023062050A1 (en) 2021-10-14 2023-04-20 F. Hoffmann-La Roche Ag New interleukin-7 immunoconjugates
US11639397B2 (en) 2011-08-23 2023-05-02 Roche Glycart Ag Bispecific antibodies specific for T-cell activating antigens and a tumor antigen and methods of use
US11649262B2 (en) 2015-12-28 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for promoting efficiency of purification of Fc region-containing polypeptide
WO2023094282A1 (en) 2021-11-25 2023-06-01 F. Hoffmann-La Roche Ag Quantification of low amounts of antibody sideproducts
US11667723B2 (en) 2020-08-17 2023-06-06 Utc Therapeutics (Shanghai) Co., Ltd. Lymphocytes-antigen presenting cells co-stimulators and uses thereof
US11673968B2 (en) 2014-06-26 2023-06-13 Hoffmann-La Roche Inc. Anti-BRDU antibodies and methods of use
WO2023104938A1 (en) 2021-12-10 2023-06-15 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and plap
US11746150B2 (en) 2017-12-19 2023-09-05 Surrozen Operating, Inc. Anti-LRP5/6 antibodies and methods of use
US20230279154A1 (en) * 2013-01-14 2023-09-07 Xencor, Inc. Novel heterodimeric proteins
WO2023180353A1 (en) 2022-03-23 2023-09-28 F. Hoffmann-La Roche Ag Combination treatment of an anti-cd20/anti-cd3 bispecific antibody and chemotherapy
US11773171B2 (en) 2017-12-19 2023-10-03 Surrozen Operating, Inc. WNT surrogate molecules and uses thereof
WO2023198727A1 (en) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Pharmaceutical compositions of anti-cd20/anti-cd3 bispecific antibodies and methods of use
US11820809B2 (en) 2016-08-26 2023-11-21 Sanofi Multispecific antibodies facilitating selective light chain pairing
US11845805B2 (en) 2020-09-10 2023-12-19 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma
US11845799B2 (en) 2019-12-13 2023-12-19 Genentech, Inc. Anti-Ly6G6D antibodies and methods of use
US11858995B2 (en) 2020-09-10 2024-01-02 Genmab A/S Bispecific antibodies against CD3 and CD20 for treating chronic lymphocytic leukemia
US11866498B2 (en) 2018-02-08 2024-01-09 Genentech, Inc. Bispecific antigen-binding molecules and methods of use
US11885806B2 (en) 2014-03-05 2024-01-30 Autolus Limited Method for depleting malignant T-cells
WO2024079015A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and imids
WO2024079009A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and proteasome inhibitors
WO2024079010A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and cd38 antibodies
US11981737B2 (en) 2016-11-18 2024-05-14 Hoffmann-La Roche Inc. Anti-HLA-G antibodies and use thereof
WO2024100170A1 (en) 2022-11-11 2024-05-16 F. Hoffmann-La Roche Ag Antibodies binding to hla-a*02/foxp3
WO2024104933A1 (en) 2022-11-15 2024-05-23 F. Hoffmann-La Roche Ag Antigen binding molecules
US12006368B2 (en) 2017-12-19 2024-06-11 Surrozen Operating, Inc. Anti-frizzled antibodies and methods of use
WO2024153722A1 (en) 2023-01-20 2024-07-25 F. Hoffmann-La Roche Ag Immunoconjugates
WO2024156672A1 (en) 2023-01-25 2024-08-02 F. Hoffmann-La Roche Ag Antibodies binding to csf1r and cd3
US12054495B2 (en) 2019-11-25 2024-08-06 Alkermes, Inc. Substituted macrocyclic compounds and related methods of treatment
WO2024163494A1 (en) 2023-01-31 2024-08-08 F. Hoffmann-La Roche Ag Methods and compositions for treating non-small cell lung cancer and triple-negative breast cancer
WO2024163009A1 (en) 2023-01-31 2024-08-08 Genentech, Inc. Methods and compositions for treating urothelial bladder cancer
WO2024184287A1 (en) 2023-03-06 2024-09-12 F. Hoffmann-La Roche Ag Combination therapy of an anti-egfrviii/anti-cd3 antibody and an tumor-targeted 4-1bb agonist
WO2024188965A1 (en) 2023-03-13 2024-09-19 F. Hoffmann-La Roche Ag Combination therapy employing a pd1-lag3 bispecific antibody and an hla-g t cell bispecific antibody
US12103982B2 (en) 2014-11-20 2024-10-01 Hoffmann-La Roche Inc. T cell activating bispecific antigen binding molecules
WO2024208777A1 (en) 2023-04-03 2024-10-10 F. Hoffmann-La Roche Ag All-in-one agonistic antibodies
WO2024208776A1 (en) 2023-04-03 2024-10-10 F. Hoffmann-La Roche Ag Agonistic split antibodies
US12139553B2 (en) 2014-11-20 2024-11-12 Hoffmann-La Roche Inc. T cell activating bispecific antigen binding molecules

Families Citing this family (343)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9493578B2 (en) 2009-09-02 2016-11-15 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
JP2013533746A (en) 2010-07-06 2013-08-29 アベオ ファーマシューティカルズ， インコーポレイテッド Anti-RON antibody
EP2598530A2 (en) 2010-07-29 2013-06-05 Xencor, Inc. Antibodies with modified isoelectric points
LT3124483T (en) 2010-11-10 2019-09-25 Genentech, Inc. Pyrazole aminopyrimidine derivatives as lrrk2 modulators
WO2013012733A1 (en) * 2011-07-15 2013-01-24 Biogen Idec Ma Inc. Heterodimeric fc regions, binding molecules comprising same, and methods relating thereto
US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
BR112014012005A2 (en) 2011-11-21 2017-12-19 Genentech Inc compositions, methods, pharmaceutical formulation and article
SI2794905T1 (en) * 2011-12-20 2020-08-31 Medimmune, Llc Modified polypeptides for bispecific antibody scaffolds
EP2836512B1 (en) * 2012-04-11 2018-10-24 F.Hoffmann-La Roche Ag Improved antibody light chains
KR102089526B1 (en) 2012-05-10 2020-03-17 바이오아트라, 엘엘씨 Multi-specific monoclonal antibodies
US9701759B2 (en) 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
US10487155B2 (en) 2013-01-14 2019-11-26 Xencor, Inc. Heterodimeric proteins
US9605084B2 (en) 2013-03-15 2017-03-28 Xencor, Inc. Heterodimeric proteins
US11053316B2 (en) 2013-01-14 2021-07-06 Xencor, Inc. Optimized antibody variable regions
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
US10131710B2 (en) 2013-01-14 2018-11-20 Xencor, Inc. Optimized antibody variable regions
WO2014113510A1 (en) 2013-01-15 2014-07-24 Xencor, Inc. Rapid clearance of antigen complexes using novel antibodies
US10047163B2 (en) * 2013-02-08 2018-08-14 Abbvie Stemcentrx Llc Multispecific constructs
KR102132246B1 (en) 2013-02-15 2020-07-09 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 Chimeric Antigen Receptor and Methods of Use Thereof
US10106624B2 (en) 2013-03-15 2018-10-23 Xencor, Inc. Heterodimeric proteins
US10519242B2 (en) 2013-03-15 2019-12-31 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
WO2014145907A1 (en) 2013-03-15 2014-09-18 Xencor, Inc. Targeting t cells with heterodimeric proteins
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
UY35468A (en) 2013-03-16 2014-10-31 Novartis Ag CANCER TREATMENT USING AN ANTI-CD19 CHEMERIC ANTIGEN RECEIVER
BR112015029224A2 (en) 2013-05-22 2017-09-19 Metabolic Eng Laboratories Co Ltd TNF-ALPHA/CXCL10 DUAL TARGET ANTIBODY, TRANSFORMANT, METHOD OF PRODUCTION OF A TNF-ALPHA/CXCL10 DUAL TARGET ANTIBODY, PHARMACEUTICAL COMPOSITION AND METHOD OF PREVENTION OR TREATMENT OF AN IMMUNOLOGICAL DISEASE
WO2015006749A2 (en) * 2013-07-12 2015-01-15 Zymeworks Inc. Bispecific cd3 and cd19 antigen binding contructs
WO2015066413A1 (en) 2013-11-01 2015-05-07 Novartis Ag Oxazolidinone hydroxamic acid compounds for the treatment of bacterial infections
AU2014348657A1 (en) 2013-11-13 2016-05-19 Novartis Ag mTOR inhibitors for enhancing the immune response
CN106459989B (en) 2013-12-19 2023-02-17 诺华股份有限公司 Human mesothelin chimeric antigen receptor and uses thereof
MX2016009050A (en) * 2014-01-15 2016-12-09 Zymeworks Inc Bi-specific cd3 and cd19 antigen-binding constructs.
JO3517B1 (en) 2014-01-17 2020-07-05 Novartis Ag N-azaspirocycloalkane substituted n-heteroaryl compounds and compositions for inhibiting the activity of shp2
JOP20200094A1 (en) 2014-01-24 2017-06-16 Dana Farber Cancer Inst Inc Antibody molecules to pd-1 and uses thereof
JOP20200096A1 (en) 2014-01-31 2017-06-16 Children’S Medical Center Corp Antibody molecules to tim-3 and uses thereof
DK3105246T3 (en) * 2014-02-10 2021-06-14 Merck Patent Gmbh Targeted TGF-beta inhibition
ES2758173T3 (en) * 2014-03-05 2020-05-04 Ucl Business Ltd Chimeric antigen receptor (CAR) with antigen-binding domains for the constant region of beta T lymphocyte receptors
MY187246A (en) 2014-03-14 2021-09-14 Novartis Ag Antibody molecules to lag-3 and uses thereof
US9907791B2 (en) 2014-03-14 2018-03-06 University Of Utah Research Foundation Ron inhibitors for use in preventing and treating bone loss
CN106163547A (en) 2014-03-15 2016-11-23 诺华股份有限公司 Use Chimeric antigen receptor treatment cancer
UA121383C2 (en) 2014-03-24 2020-05-25 Новартіс Аг Monobactam organic compounds for the treatment of bacterial infections
WO2015149077A1 (en) 2014-03-28 2015-10-01 Xencor, Inc. Bispecific antibodies that bind to cd38 and cd3
RU2021134101A (en) 2014-04-03 2022-03-22 АйДжиЭм БАЙОСАЙЕНСИЗ, ИНК. MODIFIED J-CHAIN
IL293603B2 (en) 2014-04-07 2024-03-01 Novartis Ag Treatment of cancer using anti-cd19 chimeric antigen receptor
US20170218091A1 (en) * 2014-07-03 2017-08-03 Abbvie Inc. Monovalent binding proteins
SG11201700476VA (en) 2014-07-21 2017-02-27 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
WO2016014530A1 (en) 2014-07-21 2016-01-28 Novartis Ag Combinations of low, immune enhancing. doses of mtor inhibitors and cars
US11542488B2 (en) 2014-07-21 2023-01-03 Novartis Ag Sortase synthesized chimeric antigen receptors
KR102594343B1 (en) 2014-07-21 2023-10-26 노파르티스 아게 Treatment of cancer using a cd33 chimeric antigen receptor
EP3660042B1 (en) 2014-07-31 2023-01-11 Novartis AG Subset-optimized chimeric antigen receptor-containing t-cells
EP2982692A1 (en) 2014-08-04 2016-02-10 EngMab AG Bispecific antibodies against CD3epsilon and BCMA
WO2016020836A1 (en) 2014-08-06 2016-02-11 Novartis Ag Quinolone derivatives as antibacterials
US10851149B2 (en) 2014-08-14 2020-12-01 The Trustees Of The University Of Pennsylvania Treatment of cancer using GFR α-4 chimeric antigen receptor
CN107108744B (en) 2014-08-19 2020-09-25 诺华股份有限公司 anti-CD 123 Chimeric Antigen Receptor (CAR) for cancer therapy
ES2771926T3 (en) 2014-09-13 2020-07-07 Novartis Ag Combination therapies
AU2015317608B2 (en) 2014-09-17 2021-03-11 Novartis Ag Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
JP2017535528A (en) 2014-10-03 2017-11-30 ノバルティス アーゲー Combination therapy
WO2016055432A2 (en) * 2014-10-08 2016-04-14 F. Hoffmann-La Roche Ag Combination therapy of bispecific antibodies specific for fap and dr5 and chemotherapeutic agents
MA41044A (en) 2014-10-08 2017-08-15 Novartis Ag COMPOSITIONS AND METHODS OF USE FOR INCREASED IMMUNE RESPONSE AND CANCER TREATMENT
WO2016057705A1 (en) 2014-10-08 2016-04-14 Novartis Ag Biomarkers predictive of therapeutic responsiveness to chimeric antigen receptor therapy and uses thereof
US11952421B2 (en) 2014-10-09 2024-04-09 Bristol-Myers Squibb Company Bispecific antibodies against CD3EPSILON and ROR1
MA40035A (en) 2014-10-14 2016-04-21 Dana Farber Cancer Inst Inc PD-L1 ANTIBODY MOLECULES AND THEIR USES
EP3209769B1 (en) * 2014-10-24 2020-08-05 The Board of Trustees of the Leland Stanford Junior University Compositions and methods for inducing phagocytosis of mhc class i positive cells and countering anti-cd47/sirpa resistance
WO2016065245A1 (en) 2014-10-24 2016-04-28 Incept, Llc Extra luminal scaffold
EP3023437A1 (en) 2014-11-20 2016-05-25 EngMab AG Bispecific antibodies against CD3epsilon and BCMA
US10259887B2 (en) 2014-11-26 2019-04-16 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
KR20170084327A (en) 2014-11-26 2017-07-19 젠코어 인코포레이티드 Heterodimeric antibodies that bind cd3 and cd38
CU24597B1 (en) 2014-11-26 2022-05-11 Xencor Inc HETERODIMERIC BIESPECIFIC ANTIBODIES THAT BIND CD3 AND CD20
US20180334490A1 (en) 2014-12-03 2018-11-22 Qilong H. Wu Methods for b cell preconditioning in car therapy
PE20171044A1 (en) 2014-12-16 2017-07-19 Novartis Ag ISOXAZOLE HYDROXAMIC ACID COMPOUNDS AS LpxC INHIBITORS
EP3233918A1 (en) 2014-12-19 2017-10-25 Novartis AG Combination therapies
EP3237449A2 (en) 2014-12-22 2017-11-01 Xencor, Inc. Trispecific antibodies
SG11201705293WA (en) 2014-12-29 2017-07-28 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
SG10202001779UA (en) 2015-01-20 2020-04-29 Igm Biosciences Inc Tumor necrosis factor (tnf) superfamily receptor binding molecules and uses thereof
WO2016126608A1 (en) 2015-02-02 2016-08-11 Novartis Ag Car-expressing cells against multiple tumor antigens and uses thereof
WO2016124661A1 (en) * 2015-02-04 2016-08-11 Universität Zürich Use of hla-b27 homodimers for cancer treatment
CA2978324A1 (en) 2015-03-04 2016-09-09 Igm Biosciences, Inc. Cd20 binding molecules and uses thereof
US10227411B2 (en) 2015-03-05 2019-03-12 Xencor, Inc. Modulation of T cells with bispecific antibodies and FC fusions
EP3268038B1 (en) * 2015-03-09 2021-05-05 Agensys, Inc. Antibody drug conjugates (adc) that bind to flt3 proteins
PE20171448A1 (en) 2015-03-10 2017-10-02 Aduro Biotech Inc COMPOSITIONS AND METHODS FOR ACTIVATING SIGNALING DEPENDENT ON THE INTERFERON GENE STIMULATOR
US9752199B2 (en) 2015-03-31 2017-09-05 Fundamental Solutions Corporation Biosensor system for the rapid detection of analytes
KR102540406B1 (en) * 2015-03-31 2023-06-09 펀다멘탈 솔류션스 코포레이션 Biosensor system for rapid detection of analytes
EP3280795B1 (en) 2015-04-07 2021-03-24 Novartis AG Combination of chimeric antigen receptor therapy and amino pyrimidine derivatives
BR112017021500A2 (en) 2015-04-08 2018-09-25 Novartis Ag cd20 therapies, cd22 therapies, and combination therapies with a cell expressing (car) cd19 chimeric antigen receptor
WO2016168595A1 (en) 2015-04-17 2016-10-20 Barrett David Maxwell Methods for improving the efficacy and expansion of chimeric antigen receptor-expressing cells
US12128069B2 (en) 2015-04-23 2024-10-29 The Trustees Of The University Of Pennsylvania Treatment of cancer using chimeric antigen receptor and protein kinase a blocker
IL274151B (en) 2015-05-21 2022-07-01 Harpoon Therapeutics Inc Trispecific binding proteins and methods of use
WO2017008169A1 (en) 2015-07-15 2017-01-19 Zymeworks Inc. Drug-conjugated bi-specific antigen-binding constructs
CN109476722A (en) 2015-07-21 2019-03-15 诺华股份有限公司 The method of the effect of for improving immunocyte and expansion
EP3328418A1 (en) 2015-07-29 2018-06-06 Novartis AG Combination therapies comprising antibody molecules to pd-1
DK3317301T3 (en) 2015-07-29 2021-06-28 Immutep Sas COMBINATION THERAPIES INCLUDING ANTIBODY MOLECULES AGAINST LAYER-3
US20180207273A1 (en) 2015-07-29 2018-07-26 Novartis Ag Combination therapies comprising antibody molecules to tim-3
CN108780084B (en) 2015-09-03 2022-07-22 诺华股份有限公司 Biomarkers predictive of cytokine release syndrome
DK3356401T3 (en) 2015-09-30 2020-09-07 Igm Biosciences Inc BINDING MOLECULES WITH MODIFIED J-CHAIN
EP3824903A1 (en) 2015-09-30 2021-05-26 IGM Biosciences Inc. Binding molecules with modified j-chain
WO2017072210A1 (en) 2015-10-29 2017-05-04 F. Hoffmann-La Roche Ag Anti-variant fc-region antibodies and methods of use
HRP20220436T1 (en) 2015-11-03 2022-05-27 Janssen Biotech, Inc. Antibodies specifically binding pd-1 and their uses
US11623957B2 (en) 2015-12-07 2023-04-11 Xencor, Inc. Heterodimeric antibodies that bind CD3 and PSMA
CN106883297B (en) * 2015-12-16 2019-12-13 苏州康宁杰瑞生物科技有限公司 CH3 domain-based heterodimer molecule, preparation method and application thereof
WO2017106656A1 (en) 2015-12-17 2017-06-22 Novartis Ag Antibody molecules to pd-1 and uses thereof
US20170198040A1 (en) 2015-12-18 2017-07-13 Novartis Ag ANTIBODIES TARGETING CD32b AND METHODS OF USE THEREOF
US11413340B2 (en) 2015-12-22 2022-08-16 Novartis Ag Mesothelin chimeric antigen receptor (CAR) and antibody against PD-L1 inhibitor for combined use in anticancer therapy
CA3009852A1 (en) 2015-12-28 2017-07-06 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
CN116334113A (en) 2016-01-08 2023-06-27 加利福尼亚大学董事会 Conditionally active heterodimeric polypeptides and methods of use thereof
US10889643B2 (en) 2016-01-11 2021-01-12 Universität Zürich Immune-stimulating humanized monoclonal antibodies against human interleukin-2, and fusion proteins thereof
US20200270265A1 (en) 2016-02-19 2020-08-27 Novartis Ag Tetracyclic pyridone compounds as antivirals
KR20180118175A (en) 2016-03-04 2018-10-30 노파르티스 아게 Cells expressing multiple chimeric antigen receptor (CAR) molecules and their uses
EP3432927A4 (en) 2016-03-24 2019-11-20 Gensun Biopharma Inc. Trispecific inhibitors for cancer treatment
JP7005508B2 (en) 2016-03-24 2022-01-21 ノバルティス アーゲー Alkynyl nucleoside analogs as inhibitors of human rhinovirus
AR108377A1 (en) * 2016-05-06 2018-08-15 Medimmune Llc BISPECIFIC UNION PROTEINS AND ITS USES
CN107365387B (en) * 2016-05-12 2022-03-15 阿思科力(苏州)生物科技有限公司 Bispecific antigen binding construct, preparation method and application thereof
US11339225B2 (en) 2016-05-12 2022-05-24 Asclepius (Suzhou) Technology Company Group, Co., Ltd. Bispecific antigen-binding construct and preparation method and use thereof
EA201892693A1 (en) 2016-05-20 2019-04-30 Харпун Терапьютикс, Инк. PROTEINS CONTAINING A SINGLE-BANDY VARIABLE FRAGMENT, CONNECTING CD3
SG11201810327XA (en) 2016-05-20 2018-12-28 Harpoon Therapeutics Inc Single domain serum albumin binding protein
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
RU2022104399A (en) 2016-06-14 2022-05-05 Ксенкор, Инк. BISPECIFIC ANTIBODIES-CHECKPOINT INHIBITORS
AU2017283768B2 (en) 2016-06-14 2019-05-23 Novartis Ag Crystalline form of (R)-4-(5-(cyclopropylethynyl)isoxazol-3-yl)-N-hydroxy-2-methyl-2-(methylsulfonyl)butanamide as an antibacterial agent
WO2017216685A1 (en) 2016-06-16 2017-12-21 Novartis Ag Pentacyclic pyridone compounds as antivirals
WO2017216686A1 (en) 2016-06-16 2017-12-21 Novartis Ag 8,9-fused 2-oxo-6,7-dihydropyrido-isoquinoline compounds as antivirals
MX2018016227A (en) 2016-06-24 2019-07-08 Infinity Pharmaceuticals Inc Combination therapies.
CN116063545A (en) 2016-06-28 2023-05-05 Xencor股份有限公司 Heterodimeric antibodies that bind somatostatin receptor 2
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
US11365252B2 (en) 2016-07-20 2022-06-21 University Of Utah Research Foundation CD229 CAR T cells and methods of use thereof
JP7010439B2 (en) 2016-08-10 2022-01-26 ウニヴェルズィテート チューリッヒ MHC class Ia open conformer
US10793632B2 (en) 2016-08-30 2020-10-06 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
TW201811788A (en) 2016-09-09 2018-04-01 瑞士商諾華公司 Polycyclic pyridone compounds as antivirals
US11077178B2 (en) 2016-09-21 2021-08-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptor (CAR) that targets chemokine receptor CCR4 and its use
BR112019005333A2 (en) 2016-09-21 2019-06-11 Aptevo Research And Development Llc cd123 binding proteins and related compositions and methods
JOP20190061A1 (en) 2016-09-28 2019-03-26 Novartis Ag Beta-lactamase inhibitors
EP3519820B1 (en) 2016-09-30 2020-12-09 H. Hoffnabb-La Roche Ag Spr-based dual-binding assay for the functional analysis of multispecific molecules
MX2019003886A (en) 2016-10-07 2019-08-05 Novartis Ag Chimeric antigen receptors for the treatment of cancer.
CN110214152A (en) * 2016-10-14 2019-09-06 丹娜法伯癌症研究所公司 Modularization tetramer bispecific antibody platform
BR112019007281A2 (en) 2016-10-14 2019-07-09 Xencor Inc heterodimeric protein, nucleic acid and expression vector compositions, expression vector, host cell, and methods for producing heterodimeric protein and for treating cancer in a patient
TW201819380A (en) 2016-10-18 2018-06-01 瑞士商諾華公司 Fused tetracyclic pyridone compounds as antivirals
KR20190087539A (en) * 2016-11-23 2019-07-24 하푼 테라퓨틱스, 인크. PSMA-targeted triple specific proteins and methods of use
CN110198955A (en) 2016-11-23 2019-09-03 哈普恩治疗公司 Prostate-specific membrane antigen conjugated protein
ES2961666T3 (en) 2016-12-03 2024-03-13 Juno Therapeutics Inc Methods to determine CAR-T cell dosage
SG10201913491PA (en) 2016-12-22 2020-03-30 Amgen Inc Benzisothiazole, isothiazolo[3,4-b]pyridine, quinazoline, phthalazine, pyrido[2,3-d]pyridazine and pyrido[2,3-d]pyrimidine derivatives as kras g12c inhibitors for treating lung, pancreatic or colorectal cancer
US10613083B2 (en) 2016-12-22 2020-04-07 Fundamental Solutions Corporation Universal biosensor system for analyte detection
CN109071656B (en) 2017-01-05 2021-05-18 璟尚生物制药公司 Checkpoint modulator antagonists
WO2018140845A2 (en) * 2017-01-27 2018-08-02 Duke University Bi-specific antibodies to cd64 and a disease antigen
CN106832002A (en) * 2017-02-16 2017-06-13 上海科医联创生物科技有限公司 The fusion protein and its related application of a kind of targeting PD 1
WO2018160754A2 (en) 2017-02-28 2018-09-07 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
WO2018162517A1 (en) 2017-03-10 2018-09-13 F. Hoffmann-La Roche Ag Method for producing multispecific antibodies
MX2019011858A (en) 2017-04-03 2019-11-21 Oncologie Inc Methods for treating cancer using ps-targeting antibodies with immuno-oncology agents.
AR111419A1 (en) 2017-04-27 2019-07-10 Novartis Ag INDAZOL PIRIDONA FUSIONED COMPOUNDS AS ANTIVIRALS
EP3615068A1 (en) 2017-04-28 2020-03-04 Novartis AG Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor
EP3615055A1 (en) 2017-04-28 2020-03-04 Novartis AG Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor
UY37695A (en) 2017-04-28 2018-11-30 Novartis Ag BIS 2’-5’-RR- (3’F-A) (3’F-A) CYCLE DINUCLEOTIDE COMPOUND AND USES OF THE SAME
AR111658A1 (en) 2017-05-05 2019-08-07 Novartis Ag 2-TRICYCLINAL CHINOLINONES AS ANTIBACTERIAL AGENTS
CA3063362A1 (en) 2017-05-12 2018-11-15 Harpoon Therapeutics, Inc. Msln targeting trispecific proteins and methods of use
BR112019023855B1 (en) 2017-05-12 2021-11-30 Harpoon Therapeutics, Inc MESOTHELIN BINDING PROTEINS
JOP20190272A1 (en) 2017-05-22 2019-11-21 Amgen Inc Kras g12c inhibitors and methods of using the same
US10676516B2 (en) 2017-05-24 2020-06-09 Pandion Therapeutics, Inc. Targeted immunotolerance
US20200181274A1 (en) 2017-06-01 2020-06-11 Novartis Ag Bispecific antibodies that bind cd 123 cd3
WO2018223004A1 (en) 2017-06-01 2018-12-06 Xencor, Inc. Bispecific antibodies that bind cd20 and cd3
EP3630132A1 (en) 2017-06-02 2020-04-08 Juno Therapeutics, Inc. Articles of manufacture and methods for treatment using adoptive cell therapy
EP3634584B1 (en) 2017-06-09 2024-09-18 Providence Health & Services - Oregon Tumor-infiltrating t-cells for use in the treatment of cancer
CA3067602A1 (en) 2017-06-29 2019-01-03 Juno Therapeutics, Inc. Mouse model for assessing toxicities associated with immunotherapies
MA49517A (en) 2017-06-30 2020-05-06 Xencor Inc TARGETED HETERODIMERIC FC FUSION PROTEINS CONTAINING IL-15 / IL-15RA AND AREAS OF ANTIGEN BINDING
AR112603A1 (en) 2017-07-10 2019-11-20 Lilly Co Eli BIS SPECIFIC ANTIBODIES CONTROL POINT INHIBITORS
US11905328B2 (en) 2017-07-14 2024-02-20 Immatics Biotechnologies Gmbh Dual specificity polypeptide molecule
CA3070272A1 (en) 2017-07-20 2019-01-24 Aptevo Research And Development Llc Antigen binding proteins binding to 5t4 and 4-1bb and related compositions and methods
EP3679040B1 (en) 2017-09-08 2022-08-03 Amgen Inc. Inhibitors of kras g12c and methods of using the same
CN111094356B (en) * 2017-09-25 2022-11-01 苏州丁孚靶点生物技术有限公司 Protein heterodimer and application thereof
CA3078969A1 (en) 2017-10-13 2019-04-18 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
CN111465612B (en) 2017-10-13 2024-08-27 哈普恩治疗公司 B cell maturation antigen binding proteins
BR112020007710A2 (en) 2017-10-25 2020-10-20 Novartis Ag methods to produce cells that express chimeric antigen receptor
CN109721657B (en) * 2017-10-27 2021-11-02 北京比洋生物技术有限公司 Fusion protein for blocking PD-1/PD-L1 signal transduction pathway and activating T cells and application thereof
TWI829658B (en) 2017-11-01 2024-01-21 瑞士商赫孚孟拉羅股份公司 Bispecific 2+1 contorsbodies
CA3082010A1 (en) 2017-11-01 2019-05-09 Juno Therapeutics, Inc. Chimeric antigen receptors specific for b-cell maturation antigen (bcma)
CN111511370A (en) 2017-11-01 2020-08-07 朱诺治疗学股份有限公司 Antibodies and chimeric antigen receptors specific for B cell maturation antigens
US12031975B2 (en) 2017-11-01 2024-07-09 Juno Therapeutics, Inc. Methods of assessing or monitoring a response to a cell therapy
CN111246884A (en) 2017-11-01 2020-06-05 豪夫迈·罗氏有限公司 Novel antigen binding molecules comprising trimers of TNF family ligands
US10981992B2 (en) 2017-11-08 2021-04-20 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
KR20200085828A (en) 2017-11-08 2020-07-15 젠코어 인코포레이티드 Bispecific and monospecific antibodies using novel anti-PD-1 sequences
EP3710455A1 (en) 2017-11-17 2020-09-23 Novartis AG Novel dihydroisoxazole compounds and their use for the treatment of hepatitis b
CN111989106A (en) 2017-12-01 2020-11-24 朱诺治疗学股份有限公司 Methods of administering and regulating genetically engineered cells
US10174092B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
KR20200110745A (en) 2017-12-15 2020-09-25 주노 쎄러퓨티크스 인코퍼레이티드 Anti-CCT5 binding molecule and method of use thereof
EP3728302A1 (en) 2017-12-19 2020-10-28 Xencor, Inc. Engineered il-2 fc fusion proteins
JP2021507906A (en) 2017-12-20 2021-02-25 ノバルティス アーゲー Fusion tricyclic pyrazolo-dihydropyrazinyl-pyridone compound as an antiviral agent
EP3728327A1 (en) 2017-12-22 2020-10-28 F. Hoffmann-La Roche AG Depletion of light chain mispaired antibody variants by hydrophobic interaction chromatography
CN109970856B (en) 2017-12-27 2022-08-23 信达生物制药(苏州)有限公司 anti-LAG-3 antibodies and uses thereof
WO2019129137A1 (en) 2017-12-27 2019-07-04 信达生物制药(苏州)有限公司 Anti-lag-3 antibody and uses thereof
EP3735272A4 (en) * 2018-01-05 2021-09-22 Biograph 55, Inc. Compositions and methods for cancer immunotherapy
CN111801331A (en) 2018-02-28 2020-10-20 诺华股份有限公司 Indole-2-carbonyl compounds and their use for the treatment of hepatitis b
US10982006B2 (en) 2018-04-04 2021-04-20 Xencor, Inc. Heterodimeric antibodies that bind fibroblast activation protein
US11524991B2 (en) 2018-04-18 2022-12-13 Xencor, Inc. PD-1 targeted heterodimeric fusion proteins containing IL-15/IL-15Ra Fc-fusion proteins and PD-1 antigen binding domains and uses thereof
JP2021520829A (en) 2018-04-18 2021-08-26 ゼンコア インコーポレイテッド TIM-3 targeted heterodimer fusion protein containing IL-15 / IL-15RA Fc fusion protein and TIM-3 antigen binding domain
EP3784351A1 (en) 2018-04-27 2021-03-03 Novartis AG Car t cell therapies with enhanced efficacy
US20210396739A1 (en) 2018-05-01 2021-12-23 Novartis Ag Biomarkers for evaluating car-t cells to predict clinical outcome
AU2019262599B2 (en) 2018-05-04 2023-10-12 Amgen Inc. KRAS G12C inhibitors and methods of using the same
CA3098574A1 (en) 2018-05-04 2019-11-07 Amgen Inc. Kras g12c inhibitors and methods of using the same
CA3099045A1 (en) 2018-05-10 2019-11-14 Amgen Inc. Kras g12c inhibitors for the treatment of cancer
WO2019227003A1 (en) 2018-05-25 2019-11-28 Novartis Ag Combination therapy with chimeric antigen receptor (car) therapies
EP3802611A2 (en) 2018-06-01 2021-04-14 Novartis AG Binding molecules against bcma and uses thereof
EP3801617A1 (en) 2018-06-01 2021-04-14 Novartis Ag Dosing of a bispecific antibody that bind cd123 and cd3
MX2020012731A (en) 2018-06-01 2021-02-22 Amgen Inc Kras g12c inhibitors and methods of using the same.
WO2019241157A1 (en) 2018-06-11 2019-12-19 Amgen Inc. Kras g12c inhibitors for treating cancer
JP7369719B2 (en) 2018-06-12 2023-10-26 アムジエン・インコーポレーテツド KRas G12C inhibitors and methods of using the same
SG11202011830SA (en) 2018-06-13 2020-12-30 Novartis Ag Bcma chimeric antigen receptors and uses thereof
CA3103975A1 (en) * 2018-06-21 2019-12-26 Shattuck Labs, Inc. Heterodimeric proteins and uses thereof
EP3816291A4 (en) 2018-06-26 2022-03-16 Kyowa Kirin Co., Ltd. Antibody binding to chondroitin sulfate proteoglycan-5
CA3105000A1 (en) 2018-06-26 2020-01-02 Kyowa Kirin Co., Ltd. Antibody binding to cell adhesion molecule 3
EP3813880A4 (en) 2018-06-29 2022-07-13 Gensun Biopharma Inc. Antitumor immune checkpoint regulator antagonists
CN112639083A (en) 2018-08-31 2021-04-09 诺华股份有限公司 Method for producing cells expressing chimeric antigen receptor
US20210171909A1 (en) 2018-08-31 2021-06-10 Novartis Ag Methods of making chimeric antigen receptor?expressing cells
CA3107137A1 (en) 2018-09-07 2020-03-12 Pfizer Inc. Anti-avb8 antibodies and compositions and uses thereof
CN112996789A (en) 2018-09-12 2021-06-18 诺华股份有限公司 Antiviral pyridopyrazinedione compounds
IL297931B1 (en) 2018-09-25 2024-11-01 Harpoon Therapeutics Inc Dll3 binding proteins and methods of use
WO2020069409A1 (en) 2018-09-28 2020-04-02 Novartis Ag Cd19 chimeric antigen receptor (car) and cd22 car combination therapies
US20220047633A1 (en) 2018-09-28 2022-02-17 Novartis Ag Cd22 chimeric antigen receptor (car) therapies
AU2019350592B2 (en) 2018-09-29 2024-09-26 Novartis Ag Process of manufacture of a compound for inhibiting the activity of SHP2
US11358999B2 (en) 2018-10-03 2022-06-14 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
PE20211058A1 (en) 2018-11-01 2021-06-07 Juno Therapeutics Inc SPECIFIC CHEMERIC ANTIGEN RECEPTORS FOR MEMBER D OF GROUP 5 OF CLASS C OF THE RECEPTOR COUPLED TO PROTEIN G (GPRC5D)
AU2019372331A1 (en) 2018-11-01 2021-05-27 Juno Therapeutics, Inc. Methods for treatment using chimeric antigen receptors specific for B-cell maturation antigen
MX2021005734A (en) 2018-11-16 2021-09-10 Juno Therapeutics Inc Methods of dosing engineered t cells for the treatment of b cell malignancies.
JP7516029B2 (en) 2018-11-16 2024-07-16 アムジエン・インコーポレーテツド Improved synthesis of key intermediates for KRAS G12C inhibitor compounds
MA55136A (en) 2018-11-19 2022-02-23 Amgen Inc G12C KRAS INHIBITORS AND METHODS OF USE THEREOF
JP7377679B2 (en) 2018-11-19 2023-11-10 アムジエン・インコーポレーテツド Combination therapy comprising a KRASG12C inhibitor and one or more additional pharmaceutically active agents for the treatment of cancer
CN113710256A (en) 2018-11-30 2021-11-26 朱诺治疗学股份有限公司 Methods of treatment using adoptive cell therapy
JP2022513708A (en) 2018-12-05 2022-02-09 モルフォシス・アーゲー Multispecific antigen-binding molecule
AU2019403488A1 (en) 2018-12-20 2021-06-24 Amgen Inc. KIF18A inhibitors
MA54546A (en) 2018-12-20 2022-03-30 Amgen Inc HETEROARYL AMIDES USEFUL AS KIF18A INHIBITORS
AU2019401495A1 (en) 2018-12-20 2021-06-24 Amgen Inc. Heteroaryl amides useful as KIF18A inhibitors
EA202191730A1 (en) 2018-12-20 2021-08-24 Эмджен Инк. KIF18A INHIBITORS
JP2022515424A (en) 2018-12-24 2022-02-18 サノフイ Multispecific binding protein with mutated fab domain
CN111378045B (en) * 2018-12-28 2022-08-02 长春金赛药业有限责任公司 Bivalent and bispecific antibody, preparation method thereof, encoding gene, host cell and composition
WO2020160050A1 (en) 2019-01-29 2020-08-06 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for receptor tyrosine kinase like orphan receptor 1 (ror1)
CN114173875A (en) 2019-03-01 2022-03-11 Xencor股份有限公司 Heterodimeric antibodies that bind ENPP3 and CD3
JP2022522778A (en) 2019-03-01 2022-04-20 レボリューション メディシンズ インコーポレイテッド Bicyclic heterocyclyl compounds and their use
SG11202109036WA (en) 2019-03-01 2021-09-29 Revolution Medicines Inc Bicyclic heteroaryl compounds and uses thereof
JP7249432B2 (en) 2019-03-29 2023-03-30 エフ． ホフマン－ラ ロシュ アーゲー SPR-based binding assays for functional analysis of multivalent molecules
WO2020200944A1 (en) 2019-03-29 2020-10-08 F. Hoffmann-La Roche Ag Method for generating avid-binding multispecific antibodies
EP3946382A1 (en) * 2019-04-04 2022-02-09 UMC Utrecht Holding B.V. Modified immune receptor constructs
US20220168389A1 (en) 2019-04-12 2022-06-02 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
WO2020219742A1 (en) 2019-04-24 2020-10-29 Novartis Ag Compositions and methods for selective protein degradation
WO2020216879A1 (en) 2019-04-25 2020-10-29 F. Hoffmann-La Roche Ag Therapeutic multispecific polypeptides activated by polypeptide chain exchange
KR20220005031A (en) 2019-04-25 2022-01-12 에프. 호프만-라 로슈 아게 Production of Antibody-Derived Polypeptides by Polypeptide Chain Exchange
JP2022530034A (en) 2019-04-25 2022-06-27 エフ．ホフマン－ラ ロシュ アーゲー Activateable therapeutic multispecific polypeptide with extended half-life
WO2020229378A1 (en) 2019-05-13 2020-11-19 F. Hoffmann-La Roche Ag Interference-suppressed pharmacokinetic immunoassay
EP3738593A1 (en) 2019-05-14 2020-11-18 Amgen, Inc Dosing of kras inhibitor for treatment of cancers
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
AU2020279240A1 (en) 2019-05-20 2021-12-23 Pandion Operations, Inc. MAdCAM targeted immunotolerance
PE20220504A1 (en) 2019-05-21 2022-04-07 Amgen Inc SOLID STATE FORMS
AU2020282736A1 (en) * 2019-05-29 2021-10-28 Cue Biopharma, Inc. Multimeric T-cell modulatory polypeptides and methods of use thereof
WO2020254357A1 (en) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Method for the generation of a protein expressing cell by targeted integration using cre mrna
JP7450647B2 (en) 2019-06-26 2024-03-15 エフ． ホフマン－ラ ロシュ アーゲー Mammalian cell line with SIRT-1 gene knockout
US10851157B2 (en) 2019-07-01 2020-12-01 Gensun Biopharma, Inc. Antagonists targeting the TGF-β pathway
CN114302880A (en) 2019-08-02 2022-04-08 美国安进公司 KIF18A inhibitors
WO2021026101A1 (en) 2019-08-02 2021-02-11 Amgen Inc. Kif18a inhibitors
US20220372018A1 (en) 2019-08-02 2022-11-24 Amgen Inc. Kif18a inhibitors
EP4007753A1 (en) 2019-08-02 2022-06-08 Amgen Inc. Kif18a inhibitors
CA3150762A1 (en) 2019-08-12 2021-02-18 Aptevo Research And Development Llc 4-1 bb and ox40 binding proteins and related compositions and methods, antibodies against 4-1 bb, antibodies against ox40
US11752197B2 (en) 2019-08-12 2023-09-12 Regeneron Pharmaceuticals, Inc. Macrophage stimulating 1 receptor (MST1R) variants and uses thereof
AU2020353055B2 (en) 2019-09-26 2024-03-07 Gilead Sciences, Inc. Antiviral pyrazolopyridinone compounds
WO2021073626A1 (en) * 2019-10-17 2021-04-22 北京门罗生物科技有限公司 Chimeric antigen receptor and t cells expressing chimeric antigen receptors therein
US20240190862A1 (en) 2019-10-24 2024-06-13 Amgen Inc. Epoxyamides as kras g12c and kras g12d inhibitors and methods of using the same
MX2022005357A (en) 2019-11-04 2022-06-02 Revolution Medicines Inc Ras inhibitors.
CR20220243A (en) 2019-11-04 2022-08-04 Revolution Medicines Inc Ras inhibitors
PE20221278A1 (en) 2019-11-04 2022-09-05 Revolution Medicines Inc RAS INHIBITORS
KR20220100903A (en) 2019-11-08 2022-07-18 레볼루션 메디슨즈, 인크. Bicyclic heteroaryl compounds and uses thereof
EP4058453A1 (en) 2019-11-14 2022-09-21 Amgen Inc. Improved synthesis of kras g12c inhibitor compound
AR120456A1 (en) 2019-11-14 2022-02-16 Amgen Inc ENHANCED SYNTHESIS OF KRAS G12C INHIBITOR COMPOUND
AR120566A1 (en) 2019-11-26 2022-02-23 Novartis Ag CHIMERIC ANTIGEN RECEPTORS AND THEIR USES
US11975026B2 (en) 2019-11-26 2024-05-07 Novartis Ag CD19 and CD22 chimeric antigen receptors and uses thereof
WO2021108683A1 (en) 2019-11-27 2021-06-03 Revolution Medicines, Inc. Covalent ras inhibitors and uses thereof
US11913945B2 (en) 2020-01-02 2024-02-27 Hoffmann-La Roche Inc. Method for determining the amount of a therapeutic antibody in the brain
EP4087611A1 (en) 2020-01-07 2022-11-16 Revolution Medicines, Inc. Shp2 inhibitor dosing and methods of treating cancer
CN115279406A (en) 2020-01-13 2022-11-01 阿帕特夫研究和发展有限公司 Formulations of protein therapeutics
CN115666639A (en) 2020-01-13 2023-01-31 阿帕特夫研究和发展有限公司 Methods and compositions for preventing adsorption of therapeutic proteins to drug delivery system components
WO2021163618A1 (en) 2020-02-14 2021-08-19 Novartis Ag Method of predicting response to chimeric antigen receptor therapy
CA3170833A1 (en) 2020-02-21 2021-08-26 Harpoon Therapeutics, Inc. Flt3 binding proteins and methods of use
WO2021168079A1 (en) 2020-02-21 2021-08-26 Pandion Operations, Inc. Tissue targeted immunotolerance with a cd39 effector
CA3173737A1 (en) 2020-02-27 2021-09-02 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
WO2021171264A1 (en) 2020-02-28 2021-09-02 Novartis Ag Dosing of a bispecific antibody that binds cd123 and cd3
BR112022020333A2 (en) 2020-04-10 2022-11-22 Juno Therapeutics Inc METHODS AND USES RELATED TO CELL THERAPY DESIGNED WITH A CHIMERIC ANTIGEN RECEPTOR THAT TARGETS B CELL MATURATION ANTIGEN
JP2023524068A (en) 2020-04-30 2023-06-08 ブリストル－マイヤーズ スクイブ カンパニー Methods of treating cytokine-related adverse events
EP4149524A1 (en) 2020-05-11 2023-03-22 F. Hoffmann-La Roche AG Combination therapy with modified pbmcs and an immunoconjugate
US11919956B2 (en) 2020-05-14 2024-03-05 Xencor, Inc. Heterodimeric antibodies that bind prostate specific membrane antigen (PSMA) and CD3
JP2023530109A (en) 2020-06-11 2023-07-13 プロヴェンション・バイオ・インコーポレイテッド Methods and compositions for preventing type 1 diabetes
CN111675763B (en) * 2020-06-18 2024-02-09 美国德州精准药靶有限公司 anti-MET and RON bispecific antibody, and preparation and application of antibody-drug conjugate thereof
CA3183032A1 (en) 2020-06-18 2021-12-23 Mallika Singh Methods for delaying, preventing, and treating acquired resistance to ras inhibitors
US20230340079A1 (en) 2020-07-08 2023-10-26 Astrazeneca Ab Methods of improving protein expression
CN116368155A (en) * 2020-08-19 2023-06-30 潘迪恩运营公司 Multi-paratope anti-PD-1 antibodies and uses thereof
EP4200332A1 (en) 2020-08-19 2023-06-28 Xencor, Inc. Anti-cd28 and/or anti-b7h3 compositions
WO2022042576A1 (en) * 2020-08-27 2022-03-03 盛禾(中国)生物制药有限公司 Multifunctional fusion protein and use thereof
AU2021344830A1 (en) 2020-09-03 2023-04-06 Revolution Medicines, Inc. Use of SOS1 inhibitors to treat malignancies with SHP2 mutations
JP2023541916A (en) 2020-09-15 2023-10-04 レボリューション メディシンズ インコーポレイテッド Indole derivatives as RAS inhibitors in the treatment of cancer
WO2022063877A1 (en) 2020-09-24 2022-03-31 F. Hoffmann-La Roche Ag Mammalian cell lines with gene knockout
WO2022100613A1 (en) 2020-11-10 2022-05-19 上海齐鲁制药研究中心有限公司 Bispecific antibody for claudin 18a2 and cd3 and application of bispecific antibody
AU2021378316A1 (en) 2020-11-13 2023-06-01 Novartis Ag Combination therapies with chimeric antigen receptor (car)-expressing cells
US20240301086A1 (en) 2020-12-01 2024-09-12 Aptevo Research And Development Llc Tumor-associated antigens and cd3-binding proteins, related compositions, and methods
TW202229354A (en) * 2020-12-03 2022-08-01 大陸商江蘇恆瑞醫藥股份有限公司 Multispecific antigen binding protein
EP4259149A1 (en) 2020-12-08 2023-10-18 Infinity Pharmaceuticals, Inc. Eganelisib for use in the treatment of pd-l1 negative cancer
CN116601175A (en) 2020-12-18 2023-08-15 豪夫迈·罗氏有限公司 Precursor proteins and kits for targeted therapy
JP2024501662A (en) 2020-12-22 2024-01-15 エフ． ホフマン－ラ ロシュ アーゲー Oligonucleotide targeting XBP1
WO2022140427A1 (en) 2020-12-22 2022-06-30 Qilu Regor Therapeutics Inc. Sos1 inhibitors and uses thereof
JP2024508746A (en) 2021-02-17 2024-02-28 アプティーボ リサーチ アンド デベロップメント エルエルシー Compositions and methods of use comprising 4-1BB and OX40 binding proteins
EP4295154A1 (en) 2021-02-18 2023-12-27 F. Hoffmann-La Roche AG Method for resolving complex, multistep antibody interactions
KR20230156079A (en) 2021-03-09 2023-11-13 젠코어 인코포레이티드 Heterodimeric antibody binding to CD3 and CLDN6
JP2024509274A (en) 2021-03-10 2024-02-29 ゼンコア インコーポレイテッド Heterodimeric antibody that binds to CD3 and GPC3
JP2024514077A (en) 2021-03-31 2024-03-28 江▲蘇▼恒瑞医▲薬▼股▲フン▼有限公司 Truncated TACI polypeptides and fusion proteins thereof and uses
CR20230558A (en) 2021-05-05 2024-01-24 Revolution Medicines Inc Ras inhibitors for the treatment of cancer
MX2023012902A (en) 2021-05-05 2023-11-08 Immatics Biotechnologies Gmbh Antigen binding proteins specifically binding prame.
CN117500811A (en) 2021-05-05 2024-02-02 锐新医药公司 Covalent RAS inhibitors and uses thereof
AU2022270116A1 (en) 2021-05-05 2023-12-21 Revolution Medicines, Inc. Ras inhibitors
TW202306990A (en) 2021-05-12 2023-02-16 大陸商江蘇恆瑞醫藥股份有限公司 Antigen-binding molecule that specifically binds to rankl and ngf and the medicinal use thereof
MX2023013307A (en) 2021-05-14 2023-12-04 Jiangsu Hengrui Pharmaceuticals Co Ltd Antigen-binding molecule.
EP4341291A1 (en) 2021-05-21 2024-03-27 Aptevo Research and Development LLC Dosing regimens for protein therapeutics
WO2022254337A1 (en) 2021-06-01 2022-12-08 Novartis Ag Cd19 and cd22 chimeric antigen receptors and uses thereof
KR20240049794A (en) 2021-06-07 2024-04-17 프로비던스 헬스 앤드 서비시즈 - 오레곤 CXCR5, PD-1, and ICOS expressing tumor-reactive CD4 T cells and uses thereof
AU2022309554A1 (en) 2021-07-14 2024-02-22 Jiangsu Hengrui Pharmaceuticals Co., Ltd. Antigen-binding molecule specifically binding to hgfr and egfr, and pharmaceutical use thereof
JP2024527020A (en) 2021-07-27 2024-07-19 モルフォシス・アーゲー Combination of antigen-binding molecules
CA3231180A1 (en) 2021-09-08 2023-03-16 Redona Therapeutics, Inc. Papd5 and/or papd7 inhibiting 4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives
TW202323277A (en) 2021-09-23 2023-06-16 大陸商江蘇恆瑞醫藥股份有限公司 Anti-klb antibodies and uses
TW202323304A (en) 2021-09-30 2023-06-16 大陸商江蘇恆瑞醫藥股份有限公司 Anti-il23 antibody fusion protein and uses thereof
AR127308A1 (en) 2021-10-08 2024-01-10 Revolution Medicines Inc RAS INHIBITORS
WO2023114954A1 (en) 2021-12-17 2023-06-22 Genzyme Corporation Pyrazolopyrazine compounds as shp2 inhibitors
CN118541385A (en) 2022-01-13 2024-08-23 阿斯利康(瑞典)有限公司 Methods for improving protein expression
TW202337902A (en) 2022-02-07 2023-10-01 大陸商江蘇恆瑞醫藥股份有限公司 Antigen-binding molecules that specifically bind psma and cd3 and the pharmaceutical use thereof
CN118510815A (en) 2022-02-11 2024-08-16 江苏恒瑞医药股份有限公司 Immunoconjugates and uses thereof
EP4227307A1 (en) 2022-02-11 2023-08-16 Genzyme Corporation Pyrazolopyrazine compounds as shp2 inhibitors
TW202342474A (en) 2022-02-14 2023-11-01 美商基利科學股份有限公司 Antiviral pyrazolopyridinone compounds
WO2023172940A1 (en) 2022-03-08 2023-09-14 Revolution Medicines, Inc. Methods for treating immune refractory lung cancer
CN118660910A (en) 2022-03-14 2024-09-17 江苏恒瑞医药股份有限公司 Antigen binding molecules that specifically bind GPRC5D and CD3 and medical uses thereof
TW202406571A (en) 2022-04-13 2024-02-16 美商建南德克公司 Pharmaceutical compositions of mosunetuzumab and methods of use
WO2023202967A1 (en) 2022-04-19 2023-10-26 F. Hoffmann-La Roche Ag Improved production cells
WO2023232961A1 (en) 2022-06-03 2023-12-07 F. Hoffmann-La Roche Ag Improved production cells
WO2023240263A1 (en) 2022-06-10 2023-12-14 Revolution Medicines, Inc. Macrocyclic ras inhibitors
WO2023250400A1 (en) 2022-06-22 2023-12-28 Juno Therapeutics, Inc. Treatment methods for second line therapy of cd19-targeted car t cells
US20240041929A1 (en) 2022-08-05 2024-02-08 Juno Therapeutics, Inc. Chimeric antigen receptors specific for gprc5d and bcma
WO2024079069A1 (en) 2022-10-12 2024-04-18 F. Hoffmann-La Roche Ag Method for classifying cells
WO2024081916A1 (en) 2022-10-14 2024-04-18 Black Diamond Therapeutics, Inc. Methods of treating cancers using isoquinoline or 6-aza-quinoline derivatives
WO2024104988A1 (en) 2022-11-15 2024-05-23 F. Hoffmann-La Roche Ag Recombinant binding proteins with activatable effector domain
WO2024129778A2 (en) 2022-12-13 2024-06-20 Juno Therapeutics, Inc. Chimeric antigen receptors specific for baff-r and cd19 and methods and uses thereof
WO2024148328A2 (en) 2023-01-06 2024-07-11 Aptevo Research And Development Llc Bispecific pd-l1 and cd40 binding molecules and uses thereof
WO2024206858A1 (en) 2023-03-30 2024-10-03 Revolution Medicines, Inc. Compositions for inducing ras gtp hydrolysis and uses thereof
WO2024206820A1 (en) * 2023-03-30 2024-10-03 Dna Twopointo, Inc. Fc engineering for heterodimeric antibody format
WO2024211663A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024211712A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024216048A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of ras inhibitors, compositions containing the same, and methods of use thereof
WO2024216016A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of a ras inhibitor

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO1997009433A1 (en) 1995-09-06 1997-03-13 Icos Corporation Cell-cycle checkpoint genes
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
WO2002002781A1 (en) * 2000-06-30 2002-01-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Heterodimeric fusion proteins
US6423498B1 (en) 1994-01-11 2002-07-23 Dyax Corp. Variegated Kunitz domain peptide library and uses thereof
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US20030118592A1 (en) 2001-01-17 2003-06-26 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US20030133939A1 (en) 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US6809185B1 (en) * 1998-01-23 2004-10-26 Vlaams Interuniversitair Instituut Voor Biotechnologie Multipurpose antibody derivatives
WO2005017148A1 (en) * 2003-07-26 2005-02-24 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US20060051844A1 (en) 2004-09-03 2006-03-09 Heavner George A Human EPO mimetic hinge core mimetibodies, compositions, methods and uses
WO2006072620A1 (en) 2005-01-05 2006-07-13 F-Star Biotechnologische Forschungs- Und Entwicklungsges.M.B.H. Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions
WO2006074399A2 (en) * 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
WO2006095164A1 (en) 2005-03-08 2006-09-14 Ares Trading S.A. Lipocalin protein
US20070014794A1 (en) * 1995-03-01 2007-01-18 Genentech, Inc. Method for making heteromultimeric polypeptides
US7166707B2 (en) 1998-10-23 2007-01-23 Amgen Inc. Modified peptides as therapeutic agents
US20070065431A1 (en) 1998-03-06 2007-03-22 Gregory Coia V-like domain binding molecules
WO2007048037A2 (en) * 2005-10-21 2007-04-26 Amgen Inc. METHODS FOR GENERATING MONOVALENT IgG
WO2007098934A1 (en) 2006-03-03 2007-09-07 F-Star Biotechnologische Forschungs- Und Entwicklungsges.M.B.H. Bispecific molecule binding tlr9 and cd32 and comprising a t cell epitope for treatment of allergies
WO2007146968A2 (en) 2006-06-12 2007-12-21 Trubion Pharmaceuticals, Inc. Single-chain multivalent binding proteins with effector function
WO2008052030A2 (en) 2006-10-24 2008-05-02 Trubion Pharmaceuticals, Inc. A method for increasing antibody-dependent cytotoxicity with castanospermine
WO2009023386A2 (en) 2007-07-06 2009-02-19 Trubion Pharmaceuticals, Inc. Binding peptides having a c-terminally disposed specific binding domain
WO2009080251A1 (en) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
WO2009080254A1 (en) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
US7635472B2 (en) 2003-05-31 2009-12-22 Micromet Ag Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
WO2010014629A1 (en) * 2008-07-28 2010-02-04 Trubion Pharmaceuticals, Inc. Multi-specific binding proteins targeting b cell disorders
WO2010034441A1 (en) * 2008-09-26 2010-04-01 F. Hoffmann-La Roche Ag Bispecific anti-egfr/anti-igf-1r antibodies
WO2010040508A1 (en) * 2008-10-08 2010-04-15 F. Hoffmann-La Roche Ag Bispecific anti-vegf/anti-ang-2 antibodies
WO2010042904A2 (en) * 2008-10-10 2010-04-15 Trubion Pharmaceuticals, Inc. Tcr complex immunotherapeutics
WO2010136172A1 (en) * 2009-05-27 2010-12-02 F. Hoffmann-La Roche Ag Tri- or tetraspecific antibodies
US7846434B2 (en) 2006-10-24 2010-12-07 Trubion Pharmaceuticals, Inc. Materials and methods for improved immunoglycoproteins

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
JPS6023084B2 (en) 1979-07-11 1985-06-05 味の素株式会社 blood substitute
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
EP0206448B1 (en) 1985-06-19 1990-11-14 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
JP3101690B2 (en) * 1987-03-18 2000-10-23 エス・ビィ・２・インコーポレイテッド Modifications of or for denatured antibodies
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
DE10046960A1 (en) * 2000-09-22 2002-04-11 Roche Diagnostics Gmbh Process for the production of an active, heterodimeric AMW-RT in prokaryotic cells
PL364623A1 (en) 2001-01-17 2004-12-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
CA2550551C (en) * 2004-01-16 2013-10-29 Regeneron Pharmaceuticals, Inc. Fusion polypeptides capable of activating receptors
AU2005227326B2 (en) * 2004-03-24 2009-12-03 Xencor, Inc. Immunoglobulin variants outside the Fc region
CA2566647A1 (en) * 2004-05-13 2005-12-22 Imclone Systems Incorporated Inhibition of macrophage-stimulating protein receptor (ron)
WO2006020258A2 (en) * 2004-07-17 2006-02-23 Imclone Systems Incorporated Novel tetravalent bispecific antibody
WO2006112033A1 (en) 2005-04-15 2006-10-26 Hitachi, Ltd. Ac motor controller
CN1891716B (en) * 2005-07-08 2012-11-21 北京天广实生物技术股份有限公司 No mitogen activity anti CD3 small molecular antibody designing method
CN101432015A (en) * 2006-02-15 2009-05-13 英克隆系统公司 Functional antibodies
AU2007227292B2 (en) * 2006-03-17 2012-04-12 Biogen Ma Inc. Stabilized polypeptide compositions
BRPI0713000A8 (en) * 2006-06-12 2017-12-05 Trubion Pharmaceuticals Inc SINGLE CHAIN MULTIESPECIFIC BINDING PROTEIN, PHARMACEUTICAL COMPOSITION AND USE OF SAID SINGLE CHAIN MULTIESPECIFIC BINDING PROTEIN
AR069393A1 (en) * 2007-11-21 2010-01-20 Imclone Systems Inc INHIBITION OF THE RECEIVER FOR MACROFAGO STIMULATING PROTEIN (RON) AND METHODS FOR THE TREATMENT OF THE SAME
US8242247B2 (en) * 2007-12-21 2012-08-14 Hoffmann-La Roche Inc. Bivalent, bispecific antibodies
WO2009089004A1 (en) * 2008-01-07 2009-07-16 Amgen Inc. Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
KR20100106590A (en) * 2008-01-22 2010-10-01 바이오겐 아이덱 엠에이 인코포레이티드 Ron antibodies and uses thereof

Patent Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
US5468614A (en) 1990-01-24 1995-11-21 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US6423498B1 (en) 1994-01-11 2002-07-23 Dyax Corp. Variegated Kunitz domain peptide library and uses thereof
US20070014794A1 (en) * 1995-03-01 2007-01-18 Genentech, Inc. Method for making heteromultimeric polypeptides
WO1997009433A1 (en) 1995-09-06 1997-03-13 Icos Corporation Cell-cycle checkpoint genes
US6809185B1 (en) * 1998-01-23 2004-10-26 Vlaams Interuniversitair Instituut Voor Biotechnologie Multipurpose antibody derivatives
US20070065431A1 (en) 1998-03-06 2007-03-22 Gregory Coia V-like domain binding molecules
US7166707B2 (en) 1998-10-23 2007-01-23 Amgen Inc. Modified peptides as therapeutic agents
WO2002002781A1 (en) * 2000-06-30 2002-01-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Heterodimeric fusion proteins
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US20050136049A1 (en) 2001-01-17 2005-06-23 Ledbetter Jeffrey A. Binding constructs and methods for use thereof
US20030133939A1 (en) 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US20030118592A1 (en) 2001-01-17 2003-06-26 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US7635472B2 (en) 2003-05-31 2009-12-22 Micromet Ag Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
WO2005017148A1 (en) * 2003-07-26 2005-02-24 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US20060051844A1 (en) 2004-09-03 2006-03-09 Heavner George A Human EPO mimetic hinge core mimetibodies, compositions, methods and uses
WO2006072620A1 (en) 2005-01-05 2006-07-13 F-Star Biotechnologische Forschungs- Und Entwicklungsges.M.B.H. Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions
WO2006074399A2 (en) * 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
WO2006095164A1 (en) 2005-03-08 2006-09-14 Ares Trading S.A. Lipocalin protein
WO2007048037A2 (en) * 2005-10-21 2007-04-26 Amgen Inc. METHODS FOR GENERATING MONOVALENT IgG
WO2007098934A1 (en) 2006-03-03 2007-09-07 F-Star Biotechnologische Forschungs- Und Entwicklungsges.M.B.H. Bispecific molecule binding tlr9 and cd32 and comprising a t cell epitope for treatment of allergies
WO2007146968A2 (en) 2006-06-12 2007-12-21 Trubion Pharmaceuticals, Inc. Single-chain multivalent binding proteins with effector function
WO2008052030A2 (en) 2006-10-24 2008-05-02 Trubion Pharmaceuticals, Inc. A method for increasing antibody-dependent cytotoxicity with castanospermine
US7846434B2 (en) 2006-10-24 2010-12-07 Trubion Pharmaceuticals, Inc. Materials and methods for improved immunoglycoproteins
WO2009023386A2 (en) 2007-07-06 2009-02-19 Trubion Pharmaceuticals, Inc. Binding peptides having a c-terminally disposed specific binding domain
WO2009080254A1 (en) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
WO2009080251A1 (en) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
WO2010014629A1 (en) * 2008-07-28 2010-02-04 Trubion Pharmaceuticals, Inc. Multi-specific binding proteins targeting b cell disorders
WO2010034441A1 (en) * 2008-09-26 2010-04-01 F. Hoffmann-La Roche Ag Bispecific anti-egfr/anti-igf-1r antibodies
WO2010040508A1 (en) * 2008-10-08 2010-04-15 F. Hoffmann-La Roche Ag Bispecific anti-vegf/anti-ang-2 antibodies
WO2010042904A2 (en) * 2008-10-10 2010-04-15 Trubion Pharmaceuticals, Inc. Tcr complex immunotherapeutics
WO2010136172A1 (en) * 2009-05-27 2010-12-02 F. Hoffmann-La Roche Ag Tri- or tetraspecific antibodies

Non-Patent Citations (67)

* Cited by examiner, † Cited by third party
Title
"Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
"N. Engl. J. Med.", vol. 313, 1985, ORTHO MULTICENTER TRANSPLANT STUDY GROUP, pages: 337
"Remington's Phannaceutical Sciences", 1985, MACK PUBLISHING CO.
ABBAS; LICHTMAN: "Cellular and Molecular Immunology", 2003, SAUNDERS
ALDER ET AL., NATURE IMMUNOLOGY, vol. 9, 2008, pages 319 - 327
ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
ANASETTI ET AL., J. EXP. MED., vol. 172, 1990, pages 1691
BARAL ET AL., NATURE MEDICINE, vol. 12, 2006, pages 580 - 584
BARTHELEMY ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, 2008, pages 3639 - 3654
BEAVIL ET AL., PROC. NAT'L. ACAD. SCI. USA, vol. 89, 1992, pages 753
BEAVIL ET AL., PROC. NAT'L. ACAD. SCI., vol. 89, 1992, pages 753 - 757
BERENBAUM, PHARMACOL. REV., vol. 41, 1989, pages 93
BESTE ET AL., PROC. NAT'L. ACAD. SCI., vol. 96, 1999, pages 1898 - 1903
BINZ ET AL., JOURNAL OF MOLECULAR BIOLOGY, vol. 332, 2003, pages 489 - 503
BINZ ET AL., NATURE BIOTECHNOLOGY, vol. 22, no. 5, 2004, pages 575 - 582
BINZ ET AL., NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1257 - 1268
BOYINGTON ET AL., IMMUNITY, vol. 10, 1999, pages 75
CAPON ET AL., NATURE, vol. 337, 1989, pages 525
CARPENTER ET AL., BLOOD, vol. 99, 2002, pages 2712
CO ET AL., MOL. IMMUNOL., vol. 30, 1993, pages 1361
COLOMA; MORRISON, NAT. BIOTECHNOL., vol. 15, 1997, pages 159 - 63
CORTAJARENA ET AL., ACS CHEMICAL BIOLOGY, vol. 3, 2008, pages 161 - 166
CORTEZ-RETAMOZO ET AL., CANCER RESEARCH, vol. 64, 2004, pages 2853 - 2857
DE KRUIF; LOGTENBERG, J. BIOL. CHEM., vol. 271, 1996, pages 7630 - 4
FIGDOR ET AL., NATURE REV. IMMUNOL., vol. 2, 2002, pages 77
GHAHROUDI ET AL., FEBS LETTERS, vol. 414, no. 3, 1997, pages 521 - 526
HACKEL ET AL., JOURNAL OF MOLECULAR BIOLOGY, vol. 381, 2008, pages 1238 - 1252
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446
HEROLD ET AL., J. CLIN. INVEST., vol. 11, 2003, pages 409
HERRIN ET AL., PROC. NAT'L. ACAD. SCI., vol. 105, 2008, pages 2040 - 2045
HIRSCH ET AL., J. IMMUNOL., vol. 140, 1988, pages 3766
HOET ET AL., NATURE BIOTECHNOL., vol. 23, 2005, pages 344
HUANG ET AL., STRUCTURE, vol. 13, 2005, pages 755 - 768
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 83
JACQUEMON ET AL., J. THROMB. HAEMOST., vol. 4, 2006, pages 1047
JANEWAY ET AL.: "Immunobiology: The Immune System in Health and Disease", 1999, CURRENT BIOLOGY PUBLICATIONS, pages: 148,149,
JESPERS ET AL., NATURE BIOTECHNOLOGY, vol. 22, 2004, pages 1161 - 1165
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
LAVASANI ET AL., SCANDINAVIAN JOURNAL OF IMMUNOLOGY, vol. 65, 2007, pages 39 - 47
LEHNINGER: "Biochemistry", 1975, WORTH PUBLISHERS, INC., pages: 71 - 77
LEWIN: "Genes IV", 1990, OXFORD UNIVERSITY PRESS, NY AND CELL PRESS, pages: 8
MAIN ET AL., STRUCTURE, vol. 11, 2003, pages 497 - 508
MARTIN ET AL., NATURE BIOTECHNOLOGY, vol. 21, 2002, pages 71 - 76
MARVIN; ZHU, ACTA PHARMACOLOGICA SINICA, vol. 26, 2005, pages 649 - 58
NGUYEN ET AL., IMMUNOGENETICS, vol. 54, 2002, pages 39
NGUYEN ET AL., J. MOL. BIOL., vol. 275, 1998, pages 413
NORD ET AL., EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 268, no. 15, 2001, pages 4269 - 4277
NORD ET AL., NATURE BIOTECHNOLOGY, vol. 15, 1997, pages 772 - 777
NORD ET AL., PROTEIN ENGINEERING, vol. 8, no. 6, 1995, pages 601 - 608
PARKER ET AL., PROTEIN ENGINEERING DESIGN AND SELECTION, vol. 18, no. 9, 2005, pages 435 - 444
REINHERZ, E. L. ET AL.: "Leukocyte typing II.", 1986, SPRINGER VERLAG
RICHARDS ET AL., JOURNAL OF MOLECULAR BIOLOGY, vol. 326, 2003, pages 1475 - 1488
RIDGWAY ET AL., PROTEIN ENGINEERING, vol. 9, 1966, pages 617 - 21
ROUX ET AL., PROC. NAT'L. ACAD. SCI., vol. 95, 1998, pages 11804
ROWE ET AL.: "Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety", 2006
SATO ET AL., PROC. NAT'L. ACAD. SCI., vol. 100, 2003, pages 7779 - 7784
SCATCHARD ET AL., ANN. N.Y. ACAD. SCI., vol. 51, 1949, pages 660
SCHONFDD ET AL., PROC. NAT'L. ACAD. SCI., vol. 106, 2009, pages 8198 - 8203
SCHUSTER ET AL., CANCER RES., vol. 65, 2005, pages 7934
SHIN ET AL., IMMUNOLOGICAL REVIEWS, vol. 130, 1992, pages 87
STUMPP ET AL., JOURNAL OF MOLECULAR BIOLOGY, vol. 332, 2003, pages 471 - 487
VINCKE ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 284, 2009, pages 3273 - 3284
VITA ET AL., PROC. NAT'L. ACAD. SCI., vol. 92, 1995, pages 6404 - 6408
WARD, THERAP. IMMUNOL., vol. 2, 1995, pages 77 - 94
WARNOCK ET AL., BIOTECHNOL. BIOENG., vol. 92, 2005, pages 831
WEISEL ET AL., SCIENCE, vol. 230, 1985, pages 1388
ZELENSKY; GREADY, FEBS J., vol. 272, 2005, pages 6179

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8951737B2 (en) 1996-05-06 2015-02-10 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US11168344B2 (en) 2005-03-31 2021-11-09 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
US8940871B2 (en) 2006-03-20 2015-01-27 The Regents Of The University Of California Engineered anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting
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US8940298B2 (en) 2007-09-04 2015-01-27 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection
US9527919B2 (en) 2007-09-04 2016-12-27 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection
US9828429B2 (en) 2007-09-26 2017-11-28 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substitution in CDR
US9688762B2 (en) 2007-09-26 2017-06-27 Chugai Sciyaku Kabushiki Kaisha Modified antibody constant region
US11248053B2 (en) 2007-09-26 2022-02-15 Chugai Seiyaku Kabushiki Kaisha Method of modifying isoelectric point of antibody via amino acid substitution in CDR
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US11332533B2 (en) 2007-09-26 2022-05-17 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant region
US10927163B2 (en) 2007-12-21 2021-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US10138293B2 (en) 2007-12-21 2018-11-27 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US8632774B2 (en) 2008-07-02 2014-01-21 Emergent Product Development Seattle, Llc Antagonists of IL-6
US9493564B2 (en) 2008-10-02 2016-11-15 Aptevo Research And Development Llc CD86 antagonist multi-target binding proteins
US10517969B2 (en) 2009-02-17 2019-12-31 Cornell University Methods and kits for diagnosis of cancer and prediction of therapeutic value
US10066018B2 (en) 2009-03-19 2018-09-04 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US10253091B2 (en) 2009-03-19 2019-04-09 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variant
US11993642B2 (en) 2009-04-07 2024-05-28 Hoffmann-La Roche Inc. Trivalent, bispecific antibodies
US9890204B2 (en) 2009-04-07 2018-02-13 Hoffmann-La Roche Inc. Trivalent, bispecific antibodies
US11673945B2 (en) 2009-06-16 2023-06-13 Hoffmann-La Roche Inc. Bispecific antigen binding proteins
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
US10640555B2 (en) 2009-06-16 2020-05-05 Hoffmann-La Roche Inc. Bispecific antigen binding proteins
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US10150808B2 (en) 2009-09-24 2018-12-11 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant regions
US8772459B2 (en) 2009-12-02 2014-07-08 Imaginab, Inc. J591 minibodies and Cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use
US11180570B2 (en) 2009-12-02 2021-11-23 Imaginab, Inc. J591 minibodies and cys-diabodies for targeting human prostate specific membrane antigen (PSMA) and methods for their use
US10435458B2 (en) 2010-03-04 2019-10-08 Chugai Seiyaku Kabushiki Kaisha Antibody constant region variants with reduced Fcgammar binding
JPWO2011108714A1 (en) * 2010-03-04 2013-06-27 中外製薬株式会社 Antibody constant region variants
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US10202460B2 (en) 2010-03-12 2019-02-12 Debiopharm International, S.A. CD37-binding molecules and immunoconjugates thereof
US11466095B2 (en) 2010-03-12 2022-10-11 Debiopharm International S.A. CD37-binding molecules and immunoconjugates thereof
US9346887B2 (en) 2010-03-12 2016-05-24 Immunogen, Inc. CD37-binding molecules and immunoconjugates thereof
US10106600B2 (en) 2010-03-26 2018-10-23 Roche Glycart Ag Bispecific antibodies
US10941215B2 (en) 2010-11-30 2021-03-09 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses thereof
US9611323B2 (en) 2010-11-30 2017-04-04 Genentech, Inc. Low affinity blood brain barrier receptor antibodies and uses therefor
US10793621B2 (en) 2011-02-28 2020-10-06 Hoffmann-La Roche Inc. Nucleic acid encoding dual Fc antigen binding proteins
US9982036B2 (en) 2011-02-28 2018-05-29 Hoffmann-La Roche Inc. Dual FC antigen binding proteins
US10611825B2 (en) 2011-02-28 2020-04-07 Hoffmann La-Roche Inc. Monovalent antigen binding proteins
US10556958B2 (en) 2011-04-01 2020-02-11 Debiopharm International, S.A. CD37-binding molecules and immunoconjugates thereof
US9447189B2 (en) 2011-04-01 2016-09-20 Immunogen, Inc. CD37-binding molecules and immunoconjugates thereof
JP2019165734A (en) * 2011-04-22 2019-10-03 アプティーボ リサーチ アンド デベロップメント エルエルシー Prostate-specific membrane antigen binding protein and related composition and method
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JP2014524234A (en) * 2011-07-29 2014-09-22 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Switch costimulatory receptors
JP7000242B2 (en) 2011-07-29 2022-02-04 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Switch co-stimulation receptor
JP2018148896A (en) * 2011-07-29 2018-09-27 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Switch costimulatory receptors
US10981969B2 (en) 2011-07-29 2021-04-20 The Trustees Of The University Of Pennsylvania Switch costimulatory receptors
US11639397B2 (en) 2011-08-23 2023-05-02 Roche Glycart Ag Bispecific antibodies specific for T-cell activating antigens and a tumor antigen and methods of use
US8524234B2 (en) 2011-11-03 2013-09-03 Tolera Therapeutics, Inc Antibody for selective inhibition of T-cell responses
US8722049B2 (en) 2011-11-03 2014-05-13 Tolera Therapeutics, Inc. Antibody and methods for selective inhibition of T-cell responses
US8968739B2 (en) 2011-11-03 2015-03-03 Tolera Therapeutics, Inc. Antibody and methods for selective inhibition of T-cell responses
RU2673153C2 (en) * 2012-04-20 2018-11-22 АПТЕВО РИСЁРЧ ЭНД ДИВЕЛОПМЕНТ ЭлЭлСи Polypeptides binding to cd3
US10202452B2 (en) 2012-04-20 2019-02-12 Aptevo Research And Development Llc CD3 binding polypeptides
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US10633650B2 (en) 2012-07-05 2020-04-28 Genentech, Inc. Expression and secretion system
WO2014008391A1 (en) * 2012-07-05 2014-01-09 Genentech, Inc. Expression and secretion system
US9803191B2 (en) 2012-07-05 2017-10-31 Genentech, Inc. Expression and secretion system
EP2872170A4 (en) * 2012-07-13 2016-06-22 Zymeworks Inc Bispecific asymmetric heterodimers comprising anti-cd3 constructs
JP2015528003A (en) * 2012-07-13 2015-09-24 ザイムワークス，インコーポレイテッド Bispecific asymmetric heterodimer comprising anti-CD3 construct
US11155621B2 (en) 2012-09-21 2021-10-26 Regeneran Pharmaceuticals, Inc. Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof
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US9657102B2 (en) 2012-09-21 2017-05-23 Regeneron Pharmaceuticals, Inc. Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof
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WO2014047231A1 (en) * 2012-09-21 2014-03-27 Regeneron Pharmaceuticals, Inc. Anti-cd3 antibodies, bispecific antigen-binding molecules that bind cd3 and cd20, and uses thereof
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AU2013318059B2 (en) * 2012-09-21 2018-10-18 Regeneron Pharmaceuticals, Inc. Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof
US11072656B2 (en) 2012-09-21 2021-07-27 Regeneran Pharmaceuticals, Inc. Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof
US10494437B2 (en) 2012-10-05 2019-12-03 Kyowa Kirin Co., Ltd Heterodimer protein composition
JPWO2014054804A1 (en) * 2012-10-05 2016-08-25 協和発酵キリン株式会社 Heterodimeric protein composition
US10087250B2 (en) 2012-10-08 2018-10-02 Roche Glycart Ag Fc-free antibodies comprising two fab-fragments and methods of use
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US10781257B2 (en) 2013-02-26 2020-09-22 Roche GlyeArt AG Bispecific T cell activating antigen binding molecules
US10155815B2 (en) 2013-02-26 2018-12-18 Roche Glycart Ag Bispecific T cell activating antigen binding molecules
US11459404B2 (en) 2013-02-26 2022-10-04 Roche Glycart Ag Bispecific T cell activating antigen binding molecules
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US10738117B2 (en) 2013-05-02 2020-08-11 Anaptysbio, Inc. Antibodies directed against programmed death-1 (PD-1)
US9815897B2 (en) 2013-05-02 2017-11-14 Anaptysbio, Inc. Antibodies directed against programmed death-1 (PD-1)
US11124576B2 (en) 2013-09-27 2021-09-21 Chungai Seiyaku Kabushiki Kaisha Method for producing polypeptide heteromultimer
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US10640572B2 (en) 2013-12-17 2020-05-05 Genentech, Inc. Anti-CD3 antibodies and methods of use
US10865251B2 (en) 2013-12-17 2020-12-15 Genentech, Inc. Anti-CD3 antibodies and methods of use
US11732054B2 (en) 2013-12-17 2023-08-22 Genentech, Inc. Anti-CD3 antibodies and methods of use
US10174124B2 (en) 2013-12-17 2019-01-08 Genentech, Inc. Anti-CD3 antibodies and methods of use
US11530275B2 (en) 2013-12-17 2022-12-20 Genentech, Inc. Anti-CD3 antibodies and methods of use
US11186650B2 (en) 2013-12-17 2021-11-30 Genentech, Inc. Anti-CD3 antibodies and methods of use
US10465000B2 (en) 2013-12-20 2019-11-05 Hoffmann-La Roche Inc. Humanized anti-Tau(pS422) antibodies and methods of use
US10561737B2 (en) 2014-01-03 2020-02-18 Hoffmann-La Roche Inc. Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
WO2015101586A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
WO2015101588A1 (en) 2014-01-06 2015-07-09 F. Hoffmann-La Roche Ag Monovalent blood brain barrier shuttle modules
WO2015107026A1 (en) 2014-01-15 2015-07-23 F. Hoffmann-La Roche Ag Fc-region variants with modified fcrn- and maintained protein a-binding properties
EP3835318A1 (en) 2014-01-15 2021-06-16 F. Hoffmann-La Roche AG Fc-region variants with modified fcrn- and maintained protein a-binding properties
US11982672B2 (en) 2014-03-05 2024-05-14 Autolus Limited Methods
US11885807B2 (en) 2014-03-05 2024-01-30 Autolus Limited Method for depleting malignant T-cells
US11885806B2 (en) 2014-03-05 2024-01-30 Autolus Limited Method for depleting malignant T-cells
US11982673B2 (en) 2014-03-05 2024-05-14 Autolus Limited Methods
US10550193B2 (en) 2014-03-19 2020-02-04 Regeneron Pharmaceuticals, Inc. Methods and antibody compositions for tumor treatment
US11434300B2 (en) 2014-03-19 2022-09-06 Regeneron Pharmaceuticals, Inc. Methods and antibody compositions for tumor treatment
WO2015150447A1 (en) 2014-04-02 2015-10-08 F. Hoffmann-La Roche Ag Multispecific antibodies
EP3590968A1 (en) 2014-04-02 2020-01-08 F. Hoffmann-La Roche AG Multispecific antibodies
WO2015150446A1 (en) 2014-04-02 2015-10-08 F. Hoffmann-La Roche Ag Method for detecting multispecific antibody light chain mispairing
RU2577226C2 (en) * 2014-04-10 2016-03-10 Общество с ограниченной ответственностью, "Международный биотехнологический центр "Генериум" ("МБЦ "Генериум") Methods for making bispecific antibodies against cd3*cd19 in flexybody format in mammalian cells
US10435479B2 (en) 2014-05-10 2019-10-08 Sorrento Therapeutics, Inc. Chemically-locked bispecific antibodies
US11673968B2 (en) 2014-06-26 2023-06-13 Hoffmann-La Roche Inc. Anti-BRDU antibodies and methods of use
US10251952B2 (en) 2014-06-26 2019-04-09 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibody brain shuttles and use thereof
US11117965B2 (en) 2014-08-04 2021-09-14 Hoffmann-La Roche Inc. Bispecific T cell activating antigen binding molecules
US10611841B2 (en) 2014-08-04 2020-04-07 Hoffmann-La Roche Inc. Bispecific T cell activating antigen binding molecules
US10611840B2 (en) 2014-08-04 2020-04-07 Hoffman-La Roche Inc. Bispecific T cell activating antigen binding molecules
EP3608337A1 (en) 2014-08-04 2020-02-12 F. Hoffmann-La Roche AG Bispecific t cell activating antigen binding molecules
US9914776B2 (en) 2014-08-04 2018-03-13 Hoffmann-La Roche Inc. Bispecific T cell activating antigen binding molecules
US11084877B2 (en) 2014-09-12 2021-08-10 Genentech, Inc. Anti-CLL-1 antibodies and immunoconjugates
US11142577B2 (en) 2014-09-12 2021-10-12 The Board Of Trustees Of The Leland Stanford Junior University WNT signaling agonist molecules
EP3611188A1 (en) 2014-11-06 2020-02-19 F. Hoffmann-La Roche AG Fc-region variants with modified fcrn-binding and methods of use
EP3842453A1 (en) 2014-11-06 2021-06-30 F. Hoffmann-La Roche AG Fc-region variants with modified fcrn- and protein a-binding properties
US10662244B2 (en) 2014-11-17 2020-05-26 Regeneron Pharmaceuticals, Inc. Methods for tumor treatment using CD3XCD20 bispecific antibody
US11613587B2 (en) 2014-11-20 2023-03-28 Hoffmann-La Roche Inc. Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists
US12103982B2 (en) 2014-11-20 2024-10-01 Hoffmann-La Roche Inc. T cell activating bispecific antigen binding molecules
US10781262B2 (en) 2014-11-20 2020-09-22 Hoffmann-La Roche Inc. Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists
US12139553B2 (en) 2014-11-20 2024-11-12 Hoffmann-La Roche Inc. T cell activating bispecific antigen binding molecules
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
US11999801B2 (en) 2014-12-03 2024-06-04 Hoffman-La Roche Inc. Multispecific antibodies
US10881741B2 (en) 2014-12-19 2021-01-05 Alkermes, Inc. Single chain Fc fusion proteins
US10544220B2 (en) 2015-01-08 2020-01-28 Genmab A/S Bispecific antibodies against CD3 and CD20
US11385233B2 (en) 2015-03-05 2022-07-12 Autolus Limited Methods of depleting malignant T-cells
US11857571B2 (en) 2015-03-26 2024-01-02 The Trustees Of Dartmouth College Anti-mica antigen binding fragments, fusion molecules, cells which express and methods of using
EP3270937A4 (en) * 2015-03-26 2018-09-12 The Trustees Of Dartmouth College Anti-mica antigen binding fragments, fusion molecules, cells which express and methods of using
WO2016154585A1 (en) 2015-03-26 2016-09-29 Charles Sentman Anti-mica antigen binding fragments, fusion molecules, cells which express and methods of using
US10744157B2 (en) 2015-03-26 2020-08-18 The Trustees Of Dartmouth College Anti-MICA antigen binding fragments, fusion molecules, cells which express and methods of using
US11142587B2 (en) 2015-04-01 2021-10-12 Chugai Seiyaku Kabushiki Kaisha Method for producing polypeptide hetero-oligomer
US11395796B2 (en) 2015-06-08 2022-07-26 Debiopharm International, S.A. Anti-CD37 immunoconjugate and anti-CD20 antibody combinations
US10501545B2 (en) 2015-06-16 2019-12-10 Genentech, Inc. Anti-CLL-1 antibodies and methods of use
US11007267B2 (en) 2015-06-16 2021-05-18 Genentech, Inc. Anti-CD3 antibodies and methods of use
US11466087B2 (en) 2015-06-16 2022-10-11 Genentech, Inc. Anti-CLL-1 antibodies and methods of use
US11192950B2 (en) 2015-06-16 2021-12-07 Genentech, Inc. Humanized and affinity matured antibodies to FcRH5 and methods of use
US10323094B2 (en) 2015-06-16 2019-06-18 Genentech, Inc. Humanized and affinity matured antibodies to FcRH5 and methods of use
US12030947B2 (en) 2015-06-16 2024-07-09 Genentech, Inc. Humanized and affinity matured antibodies to FcRH5 and methods of use
US10822402B2 (en) 2015-06-24 2020-11-03 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibodies and methods of use
US11254744B2 (en) 2015-08-07 2022-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
US11104740B2 (en) 2015-08-28 2021-08-31 Debiopharm International, S.A. Antibodies and assays for detection of CD37
EP3352760A4 (en) * 2015-09-21 2019-03-06 Aptevo Research and Development LLC Cd3 binding polypeptides
IL258202B (en) * 2015-09-21 2022-10-01 Aptevo Res & Development Llc Cd3-binding polypeptides, method for their preparation, pharmaceutical compositions, nucleic acids, vectors and use of such polypeptides
CN108367004B (en) * 2015-09-21 2022-09-13 阿帕特夫研究和发展有限公司 CD3 binding polypeptides
CN108367004A (en) * 2015-09-21 2018-08-03 阿帕特夫研究和发展有限公司 CD3 combination polypeptides
IL258202B2 (en) * 2015-09-21 2023-02-01 Aptevo Res & Development Llc Cd3-binding polypeptides, method for their preparation, pharmaceutical compositions, nucleic acids, vectors and use of such polypeptides
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
US11286300B2 (en) 2015-10-01 2022-03-29 Hoffmann-La Roche Inc. Humanized anti-human CD19 antibodies and methods of use
WO2017055389A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific anti-ceaxcd3 t cell activating antigen binding molecules
EP3913000A1 (en) 2015-10-02 2021-11-24 F. Hoffmann-La Roche AG Bispecific anti-cd19xcd3 t cell activating antigen binding molecules
WO2017055388A2 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules
WO2017055392A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xcd44v6 bispecific t cell activating antigen binding molecules
WO2017055391A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules binding mesothelin and cd3
WO2017055395A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xrob04 bispecific t cell activating antigen binding molecules
WO2017055314A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific anti-cd19xcd3 t cell activating antigen binding molecules
WO2017055404A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific antibodies specific for pd1 and tim3
US10766967B2 (en) 2015-10-02 2020-09-08 Hoffmann-La Roche Inc. Bispecific T cell activating antigen binding molecules
EP3150637A1 (en) 2015-10-02 2017-04-05 F. Hoffmann-La Roche AG Multispecific antibodies
WO2017055393A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xtim-3 bispecific t cell activating antigen binding molecules
WO2017055399A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Cellular based fret assay for the determination of simultaneous binding
WO2017055385A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xgd2 bispecific t cell activating antigen binding molecules
CN108137671A (en) * 2015-10-10 2018-06-08 中国科学院广州生物医药与健康研究院 The soluble PDL-1 molecules of high-affinity
CN108137671B (en) * 2015-10-10 2021-07-09 中国科学院广州生物医药与健康研究院 Soluble PDL-1 molecules
US11013801B2 (en) 2015-12-09 2021-05-25 Hoffmann-La Roche Inc. Treatment method
EP3178848A1 (en) 2015-12-09 2017-06-14 F. Hoffmann-La Roche AG Type ii anti-cd20 antibody for reducing formation of anti-drug antibodies
EP4026848A1 (en) 2015-12-09 2022-07-13 F. Hoffmann-La Roche AG Type ii anti-cd20 antibody for reducing the cytokine release syndrome
US11649262B2 (en) 2015-12-28 2023-05-16 Chugai Seiyaku Kabushiki Kaisha Method for promoting efficiency of purification of Fc region-containing polypeptide
US10596257B2 (en) 2016-01-08 2020-03-24 Hoffmann-La Roche Inc. Methods of treating CEA-positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD3 bispecific antibodies
EP3862365A1 (en) 2016-01-08 2021-08-11 F. Hoffmann-La Roche AG Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
US11242390B2 (en) 2016-03-22 2022-02-08 Hoffmann-La Roche Inc. Protease-activated T cell bispecific molecules
WO2017191101A1 (en) 2016-05-02 2017-11-09 F. Hoffmann-La Roche Ag The contorsbody - a single chain target binder
EP3889175A1 (en) 2016-05-02 2021-10-06 F. Hoffmann-La Roche AG The contorsbody - a single chain target binder
EP3252078A1 (en) 2016-06-02 2017-12-06 F. Hoffmann-La Roche AG Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer
US10335459B2 (en) 2016-06-22 2019-07-02 Alkermes, Inc. Compositions for modulating IL-10 immunostimulatory and anti-inflammatory properties
WO2018005226A3 (en) * 2016-06-22 2018-04-05 Alkermes, Inc. Compositions and methods for modulating il-10 immunostimulatory and anti-inflammatory properties
US11534480B2 (en) 2016-06-22 2022-12-27 Alkermes, Inc. Compositions and methods for modulating IL-10 immunostimulatory and anti-inflammatory properties
WO2018007314A1 (en) 2016-07-04 2018-01-11 F. Hoffmann-La Roche Ag Novel antibody format
US11225515B2 (en) 2016-08-26 2022-01-18 Agency For Science, Technology And Research Macrophage stimulating protein receptor (or RON—recepteur d'Origine Nantais) antibodies and uses thereof
US11820809B2 (en) 2016-08-26 2023-11-21 Sanofi Multispecific antibodies facilitating selective light chain pairing
WO2018038684A1 (en) * 2016-08-26 2018-03-01 Agency For Science, Technology And Research Macrophage stimulating protein receptor (or ron - recepteur d' origine nantais) antibodies and uses thereof
WO2018060301A1 (en) 2016-09-30 2018-04-05 F. Hoffmann-La Roche Ag Bispecific antibodies against cd3
US10882918B2 (en) 2016-09-30 2021-01-05 Hoffmann-La Roche Inc. Bispecific T cell activating antigen binding molecules
US11155624B2 (en) 2016-11-01 2021-10-26 Anaptysbio, Inc. Antibodies directed against programmed death-1 (PD-1)
US11278629B2 (en) 2016-11-02 2022-03-22 Debiopharm International, S.A. Methods for improving anti-CD37 immunoconjugate therapy
US11466094B2 (en) 2016-11-15 2022-10-11 Genentech, Inc. Dosing for treatment with anti-CD20/anti-CD3 bispecific antibodies
US11981737B2 (en) 2016-11-18 2024-05-14 Hoffmann-La Roche Inc. Anti-HLA-G antibodies and use thereof
US11407830B2 (en) 2017-01-09 2022-08-09 Tesaro, Inc. Methods of treating cancer with anti-PD-1 antibodies
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies
WO2018184965A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of il-2 with an anti-pd-1 and tim-3 bispecific antibody
US11180571B2 (en) 2017-04-03 2021-11-23 Hoffmann-La Roche Inc. Antibodies binding to STEAP-1
US11685790B2 (en) 2017-04-03 2023-06-27 Hoffmann-La Roche Inc. Antibodies binding to STEAP-1
WO2018184966A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Antibodies binding to steap-1
US12129306B2 (en) 2017-04-03 2024-10-29 Hoffman-La Roche Inc. Antibodies binding to STEAP-1
WO2018184964A1 (en) 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of an anti-pd-1 antibody with a mutant il-2 or with il-15
WO2018185043A1 (en) 2017-04-05 2018-10-11 F. Hoffmann-La Roche Ag Bispecific antibodies specifically binding to pd1 and lag3
WO2018189220A1 (en) 2017-04-13 2018-10-18 F. Hoffmann-La Roche Ag An interleukin-2 immunoconjugate, a cd40 agonist, and optionally a pd-1 axis binding antagonist for use in methods of treating cancer
WO2018220099A1 (en) 2017-06-02 2018-12-06 F. Hoffmann-La Roche Ag Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer
WO2019077092A1 (en) 2017-10-20 2019-04-25 F. Hoffmann-La Roche Ag Method for generating multispecific antibodies from monospecific antibodies
WO2019086362A1 (en) 2017-10-30 2019-05-09 F. Hoffmann-La Roche Ag Method for in vivo generation of multispecific antibodies from monospecific antibodies
US12043651B2 (en) 2017-11-01 2024-07-23 Hoffmann-La Roche Inc. Compbody—a multivalent target binder
WO2019086394A1 (en) 2017-11-01 2019-05-09 F. Hoffmann-La Roche Ag The compbody - a multivalent target binder
WO2019086395A1 (en) 2017-11-01 2019-05-09 F. Hoffmann-La Roche Ag Trifab-contorsbody
US12006368B2 (en) 2017-12-19 2024-06-11 Surrozen Operating, Inc. Anti-frizzled antibodies and methods of use
US11746150B2 (en) 2017-12-19 2023-09-05 Surrozen Operating, Inc. Anti-LRP5/6 antibodies and methods of use
US11773171B2 (en) 2017-12-19 2023-10-03 Surrozen Operating, Inc. WNT surrogate molecules and uses thereof
US12098213B2 (en) 2017-12-21 2024-09-24 Hoffmann-La Roche Inc. Antibodies binding to HLA-A2/WT1
US11192957B2 (en) 2017-12-21 2021-12-07 Hoffmann-La Roche Inc. Antibodies binding to HLA-A2/WT1
WO2019122052A2 (en) 2017-12-21 2019-06-27 F. Hoffmann-La Roche Ag Antibodies binding to hla-a2/wt1
WO2019154776A1 (en) 2018-02-06 2019-08-15 F. Hoffmann-La Roche Ag Treatment of ophthalmologic diseases
US11866498B2 (en) 2018-02-08 2024-01-09 Genentech, Inc. Bispecific antigen-binding molecules and methods of use
WO2019154890A1 (en) 2018-02-09 2019-08-15 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2019202041A1 (en) 2018-04-18 2019-10-24 F. Hoffmann-La Roche Ag Multispecific antibodies and use thereof
US11590223B2 (en) 2018-08-31 2023-02-28 Regeneron Pharmaceuticals, Inc. Dosing strategy that mitigates cytokine release syndrome for therapeutic antibodies
WO2020089051A1 (en) 2018-10-29 2020-05-07 F. Hoffmann-La Roche Ag Antibody formulation
WO2020127619A1 (en) 2018-12-21 2020-06-25 F. Hoffmann-La Roche Ag Antibodies binding to cd3
WO2020136060A1 (en) 2018-12-28 2020-07-02 F. Hoffmann-La Roche Ag A peptide-mhc-i-antibody fusion protein for therapeutic use in a patient with amplified immune response
WO2020180398A1 (en) * 2019-01-14 2020-09-10 The Regents Of The University Of California Compositions and methods for modulating cellular internalization
US12144818B2 (en) 2019-05-29 2024-11-19 Debiopharm International, S.A. Method for treating cancer in a human patient by administering an anti-CD37 immunoconjugate using various dosing regimens
WO2021001289A1 (en) 2019-07-02 2021-01-07 F. Hoffmann-La Roche Ag Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody
CN110327458A (en) * 2019-07-09 2019-10-15 上海交通大学医学院 Autocrine VEGFB is in T cell metabolism and the application in function and immunotherapy of tumors
CN110327458B (en) * 2019-07-09 2022-02-25 上海交通大学医学院 Application of autocrine VEGFB in T cell metabolism and function and tumor immunotherapy
WO2021009146A1 (en) 2019-07-15 2021-01-21 F. Hoffmann-La Roche Ag Antibodies binding to nkg2d
WO2021018859A2 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021018925A1 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021058804A1 (en) * 2019-09-25 2021-04-01 Universität Stuttgart Binding modules comprising modified ehd2 domains
US12054495B2 (en) 2019-11-25 2024-08-06 Alkermes, Inc. Substituted macrocyclic compounds and related methods of treatment
US11845799B2 (en) 2019-12-13 2023-12-19 Genentech, Inc. Anti-Ly6G6D antibodies and methods of use
WO2021122733A1 (en) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Bispecific anti-ccl2 antibodies
WO2021122875A1 (en) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Antibodies binding to hla-a2/mage-a4
US12103967B2 (en) 2019-12-18 2024-10-01 Hoffmann-La Roche Inc. Bispecific anti-CCL2 antibodies
US11739142B2 (en) 2019-12-18 2023-08-29 Hoffmann-La Roche Inc. Bispecific anti-CCL2 antibodies
US11987632B2 (en) 2019-12-18 2024-05-21 Hoffmann-La Roche Inc. Antibodies binding to HLA-A2/MAGE-A4
WO2021209402A2 (en) 2020-04-15 2021-10-21 F. Hoffmann-La Roche Ag Immunoconjugates
US11780920B2 (en) 2020-06-19 2023-10-10 Hoffmann-La Roche Inc. Antibodies binding to CD3 and CD19
WO2021255143A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and folr1
WO2021255146A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and cea
WO2021255138A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Immune activating fc domain binding molecules
WO2021255155A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and cd19
WO2021255142A1 (en) 2020-06-19 2021-12-23 F. Hoffmann-La Roche Ag Antibodies binding to cd3
US11667723B2 (en) 2020-08-17 2023-06-06 Utc Therapeutics (Shanghai) Co., Ltd. Lymphocytes-antigen presenting cells co-stimulators and uses thereof
US11548952B2 (en) 2020-09-10 2023-01-10 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma
US11858995B2 (en) 2020-09-10 2024-01-02 Genmab A/S Bispecific antibodies against CD3 and CD20 for treating chronic lymphocytic leukemia
US11845805B2 (en) 2020-09-10 2023-12-19 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating diffuse large B-cell lymphoma
US11535679B2 (en) 2020-09-10 2022-12-27 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating follicular lymphoma
US11608383B2 (en) 2020-09-10 2023-03-21 Genmab A/S Bispecific antibody against CD3 and CD20 in combination therapy for treating follicular lymphoma
WO2022129120A1 (en) 2020-12-17 2022-06-23 F. Hoffmann-La Roche Ag Anti-hla-g antibodies and use thereof
WO2022148732A1 (en) 2021-01-06 2022-07-14 F. Hoffmann-La Roche Ag Combination therapy employing a pd1-lag3 bispecific antibody and a cd20 t cell bispecific antibody
WO2022148853A1 (en) 2021-01-11 2022-07-14 F. Hoffmann-La Roche Ag Immunoconjugates
WO2022228705A1 (en) 2021-04-30 2022-11-03 F. Hoffmann-La Roche Ag Dosing for combination treatment with anti-cd20/anti-cd3 bispecific antibody and anti-cd79b antibody drug conjugate
WO2022228706A1 (en) 2021-04-30 2022-11-03 F. Hoffmann-La Roche Ag Dosing for treatment with anti-cd20/anti-cd3 bispecific antibody
WO2022263507A1 (en) 2021-06-17 2022-12-22 Boehringer Ingelheim International Gmbh Novel tri-specific binding molecules
WO2022263501A1 (en) 2021-06-18 2022-12-22 F. Hoffmann-La Roche Ag Bispecific anti-ccl2 antibodies
WO2023279092A2 (en) 2021-07-02 2023-01-05 Genentech, Inc. Methods and compositions for treating cancer
WO2023001884A1 (en) 2021-07-22 2023-01-26 F. Hoffmann-La Roche Ag Heterodimeric fc domain antibodies
WO2023010095A1 (en) 2021-07-28 2023-02-02 F. Hoffmann-La Roche Ag Methods and compositions for treating cancer
WO2023062048A1 (en) 2021-10-14 2023-04-20 F. Hoffmann-La Roche Ag Alternative pd1-il7v immunoconjugates for the treatment of cancer
WO2023062050A1 (en) 2021-10-14 2023-04-20 F. Hoffmann-La Roche Ag New interleukin-7 immunoconjugates
WO2023094282A1 (en) 2021-11-25 2023-06-01 F. Hoffmann-La Roche Ag Quantification of low amounts of antibody sideproducts
WO2023104938A1 (en) 2021-12-10 2023-06-15 F. Hoffmann-La Roche Ag Antibodies binding to cd3 and plap
WO2023180353A1 (en) 2022-03-23 2023-09-28 F. Hoffmann-La Roche Ag Combination treatment of an anti-cd20/anti-cd3 bispecific antibody and chemotherapy
WO2023198727A1 (en) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Pharmaceutical compositions of anti-cd20/anti-cd3 bispecific antibodies and methods of use
WO2024079015A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and imids
WO2024079010A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and cd38 antibodies
WO2024079009A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and proteasome inhibitors
WO2024100170A1 (en) 2022-11-11 2024-05-16 F. Hoffmann-La Roche Ag Antibodies binding to hla-a*02/foxp3
WO2024104933A1 (en) 2022-11-15 2024-05-23 F. Hoffmann-La Roche Ag Antigen binding molecules
WO2024153722A1 (en) 2023-01-20 2024-07-25 F. Hoffmann-La Roche Ag Immunoconjugates
WO2024156672A1 (en) 2023-01-25 2024-08-02 F. Hoffmann-La Roche Ag Antibodies binding to csf1r and cd3
WO2024163009A1 (en) 2023-01-31 2024-08-08 Genentech, Inc. Methods and compositions for treating urothelial bladder cancer
WO2024163494A1 (en) 2023-01-31 2024-08-08 F. Hoffmann-La Roche Ag Methods and compositions for treating non-small cell lung cancer and triple-negative breast cancer
WO2024184287A1 (en) 2023-03-06 2024-09-12 F. Hoffmann-La Roche Ag Combination therapy of an anti-egfrviii/anti-cd3 antibody and an tumor-targeted 4-1bb agonist
WO2024188965A1 (en) 2023-03-13 2024-09-19 F. Hoffmann-La Roche Ag Combination therapy employing a pd1-lag3 bispecific antibody and an hla-g t cell bispecific antibody
WO2024208776A1 (en) 2023-04-03 2024-10-10 F. Hoffmann-La Roche Ag Agonistic split antibodies
WO2024208777A1 (en) 2023-04-03 2024-10-10 F. Hoffmann-La Roche Ag All-in-one agonistic antibodies

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JP2013515508A (en) 2013-05-09
LT2519543T (en) 2016-10-10
EP2519543A1 (en) 2012-11-07
KR20120125611A (en) 2012-11-16
CN103124743A (en) 2013-05-29
ES2592385T3 (en) 2016-11-29
BR112012016135A2 (en) 2017-03-07
US20130089554A1 (en) 2013-04-11
AU2010343057A2 (en) 2015-04-02
AU2010343049A2 (en) 2015-04-02
US20130095097A1 (en) 2013-04-18
JP5851419B2 (en) 2016-02-03
EA023674B1 (en) 2016-06-30
AU2010343057A1 (en) 2012-07-19
SG181952A1 (en) 2012-07-30
DK2519543T3 (en) 2016-09-26
PT2519543T (en) 2016-10-07
SMT201600335B (en) 2016-11-10
EP2519541A1 (en) 2012-11-07
HRP20160819T1 (en) 2016-08-12

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