WO2022156798A1 - 一种抗体及其用途 - Google Patents

一种抗体及其用途 Download PDF

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WO2022156798A1
WO2022156798A1 PCT/CN2022/073460 CN2022073460W WO2022156798A1 WO 2022156798 A1 WO2022156798 A1 WO 2022156798A1 CN 2022073460 W CN2022073460 W CN 2022073460W WO 2022156798 A1 WO2022156798 A1 WO 2022156798A1
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seq
sequence
chain variable
variable region
sequence seq
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French (fr)
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柯潇
冯晓
雷刚
任鹏飞
李剑红
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成都康弘生物科技有限公司
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
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    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention relates to the field of biotechnology, in particular to an antibody that mainly binds to plasma kallikrein and its use.
  • Plasma kallikrein is a member of the serine protease family and was first discovered in mammalian plasma. It is encoded by a single gene (KLKB1) located on chromosome 4q35 and is mainly synthesized in the liver. PK is a key enzyme of the kallikrein-kinin system (KKS), which can act on high molecular weight kininogen (KH) to activate and release small molecule bradykinin (BK). Peptide receptors are involved in biological processes such as coagulation, fibrinolysis, complement activation, and inflammation.
  • plasma kallikrein is closely related to various diseases such as inflammatory diseases, tumors, cardiovascular diseases, kidney diseases, central nervous system diseases, retinopathy, and diabetic retinal diseases (Costa-Neto, C.M. et al. Participation of kallikrein -kinin system in different pathologies. Int. Immunopharmacol. 2008, 8, 135-142).
  • hereditary angioedema which is an autosomal dominant inheritance, is mainly due to the lack of C1-INH in patients, resulting in a weakened inhibition of plasma kallikrein and uncontrolled activation of the KKS system , release vasoactive substances, increase vascular permeability and cause typical swelling (Farkas, H. Orphan drugs for the treatment of hereditary angioedema. Expert Opinion on Orphan Drugs, 2015, 1, 141-156).
  • the KKS system was found to be overactivated, resulting in increased retinal vascular permeability and retinal thickening.
  • protein or polypeptide drugs with PK have entered the clinic or have been successfully marketed.
  • macromolecular plasma kallikrein inhibitors such as icalatide and Lanadelumab have been marketed for clinical use in the treatment of hereditary angioedema, with significant curative effects.
  • Bicycle's THR-149 a bicyclopeptide peptide ligand that specifically binds to plasma kallikrein, is in Phase II clinical studies for the treatment of diabetic macular edema.
  • the present invention provides an antibody or antigen-binding fragment that binds plasma kallikrein, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the following three complementarity determinations Region (CDR):
  • CDR1 contains the sequence X 1 X 2 X 3 X 4 X 5 , wherein X 1 is D or N, X 2 is T, I or Y, X 3 is Y or W, and X 4 is I or M , X 5 is F or H, or HCDR1 is TSGMGVS;
  • CDR2 contains the sequence Y 1 IY 2 PY 3 Y 4 Y 5 Y 6 Y 7 Y 8 Y 9 Y 10 Y 11 Y 12 Y 13 Y 14 Y 15 , wherein Y 1 R or A, Y 2 is D or Y, Y 3 is E or G, Y 4 is N or D, Y 5 is D, G or S,
  • the light chain variable region includes the following three complementarity determining regions (CDRs):
  • CDR1 contains the sequence L 1 ASL 2 DINL 3 YL 4 L 5 , wherein L 1 is E or K, L 2 is H or Q, and L 3 is N , K or S, L 4 is I or L, L 5 is A or S, or CDR1 is SATSIINSNYFH;
  • CDR2 contains the sequence M 1 M 2 M 3 M 4 LM 5 M 6 , wherein M 1 is Y, F or R, M 2 is T or A, M 3 is S or N, M 4 is T, S, R or N, M 5 is Q, H, V or A, M 6 is S, Q, D or P;
  • CDR3 contains a sequence LQYDN 1 LN 2 T, where N 1 is N or D, N 2 is F or W, or CDR3 is VQYDEFPLT or QQGSSLPRT.
  • the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the following three complementarity determining regions (CDRs):
  • CDR1 contains the sequence SEQ ID NO: Any one of 1-5
  • CDR2 contains any one of sequence SEQ ID NO: 6-11
  • CDR3 contains any one of sequence SEQ ID NO: 12-15
  • light chain variable region includes the following three complementarity determining regions (CDR) :
  • CDR1 contains any one of sequence SEQ ID NO: 16-21
  • CDR2 contains any one of sequence SEQ ID NO: 22-27
  • CDR3 contains any one of sequence SEQ ID NO: 28-31.
  • the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 1; CDR2 contains the sequence SEQ ID NO: 6; CDR3 contains the sequence SEQ ID NO: 12; the light chain variable region includes the following three complementarity determining regions (CDR): CDR1 contains the sequence SEQ ID NO: 16; CDR2 contains the sequence SEQ ID NO: 16; NO: 22; CDR3 contains the sequence SEQ ID NO: 28;
  • CDRs CDR1 contains the sequence SEQ ID NO: 1
  • CDR2 contains the sequence SEQ ID NO: 6
  • CDR3 contains the sequence SEQ ID NO: 12
  • the light chain variable region includes the following three complementarity determining regions (CDR): CDR1 contains the sequence SEQ ID NO: 16; CDR2 contains the sequence SEQ ID NO: 16; NO: 22; CDR3 contains
  • the heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 1; CDR2 contains the sequence SEQ ID NO: 7; CDR3 contains the sequence SEQ ID NO: 12;
  • the light chain variable region includes The following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 17; CDR2 contains the sequence SEQ ID NO: 23; CDR3 contains the sequence SEQ ID NO: 28;
  • the heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 2; CDR2 contains the sequence SEQ ID NO: 8; CDR3 contains the sequence SEQ ID NO: 13;
  • the light chain variable region includes The following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 18; CDR2 contains the sequence SEQ ID NO: 24; CDR3 contains the sequence SEQ ID NO: 29;
  • the heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 3; CDR2 contains the sequence SEQ ID NO: 9; CDR3 contains the sequence SEQ ID NO: 14;
  • the light chain variable region includes The following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 19; CDR2 contains the sequence SEQ ID NO: 25; CDR3 contains the sequence SEQ ID NO: 30;
  • the heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 4; CDR2 contains the sequence SEQ ID NO: 10; CDR3 contains the sequence SEQ ID NO: 12; The light chain variable region includes The following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO:20; CDR2 contains the sequence SEQ ID NO:26; CDR3 contains the sequence SEQ ID NO:29;
  • the heavy chain variable region includes the following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO: 5; CDR2 contains the sequence SEQ ID NO: 11; CDR3 contains the sequence SEQ ID NO: 15; The light chain variable region includes The following three complementarity determining regions (CDRs): CDR1 contains the sequence SEQ ID NO:21; CDR2 contains the sequence SEQ ID NO:27; CDR3 contains the sequence SEQ ID NO:31.
  • Antibodies or antigen-binding fragments provided by the present invention include murine antibodies, chimeric antibodies, humanized antibodies or fully humanized antibodies or antigen-binding fragments thereof.
  • the antibody or antigen-binding fragment provided by the present invention its heavy chain variable region sequence is selected from SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 44. Any one of SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56, the light chain variable region sequence is selected Any one of SEQ ID NO:34, SEQ ID NO:36-42, SEQ ID NO:45, SEQ ID NO:47-49, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:57 .
  • the antibody or antigen-binding fragment provided by the present invention its heavy chain variable region sequence is selected from SEQ ID NO: 35, and the light chain variable region sequence is selected from any one of SEQ ID NO: 36-42; or Its heavy chain variable region sequence is selected from SEQ ID NO: 44 or 46, and its light chain variable region sequence is SEQ ID NO: 45; or its heavy chain variable region sequence is selected from SEQ ID NO: 50 or SEQ ID NO: 32, the light chain variable region sequence is selected from any one of SEQ ID NOs: 47-49; or its heavy chain variable region sequence is SEQ ID NO: 33, and the light chain variable region sequence is SEQ ID NO: 34 or its heavy chain variable region sequence is SEQ ID NO: 43, and its light chain variable region sequence is SEQ ID NO: 42; or its heavy chain variable region sequence is SEQ ID NO: 51, and its light chain variable region sequence is SEQ ID NO: 51.
  • SEQ ID NO: 48 is SEQ ID NO: 48; or its heavy chain variable region sequence is SEQ ID NO: 52, and its light chain variable region sequence is SEQ ID NO: 53; or its heavy chain variable region sequence is SEQ ID NO: 54, The light chain variable region sequence is SEQ ID NO: 55; or the heavy chain variable region sequence is SEQ ID NO: 56, and the light chain variable region sequence is SEQ ID NO: 57.
  • the antigen-binding fragments of the present invention are preferably selected from Fab, Fab', Fv, scFv or (Fab') 2 fragments.
  • the present invention also provides some nucleic acid molecules encoding the above-mentioned antibodies or antigen-binding fragments; further provides an expression vector for expressing the nucleic acid; and further provides a host cell comprising the vector, the host cell comprising a prokaryotic cell or Eukaryotic cells, more preferably selected from yeast cells, mammalian cells (eg 293 cells or CHO cells).
  • the same invention further provides light chain constant regions such as SEQ ID NO: 61 and its mutants; heavy chain constant regions such as Human IgG1, Human IgG2, Human IgG4, etc., wherein Human IgG1 is preferably SEQ ID NO: 58 and its mutants; Human IgG2 is preferably SEQ ID NO: 59 and its mutants; Human IgG4 is preferably SEQ ID NO: 60 and its mutants.
  • Human IgG1 is preferably SEQ ID NO: 58 and its mutants
  • Human IgG2 is preferably SEQ ID NO: 59 and its mutants
  • Human IgG4 is preferably SEQ ID NO: 60 and its mutants.
  • the light chain constant region of the antibody KH01-18 involved in the examples given in the present invention is SEQ ID NO: 61; the heavy chain constant regions of KH10 and KH12-14 are Human IgG1 (SEQ ID NO: 58), KH01-KH09, KH11 , The heavy chain constant region of KH15-18 is Human IgG4 (SEQ ID NO: 60), and KH19-21 is a murine antibody.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment, and a pharmaceutically acceptable excipient;
  • the pharmaceutical composition is preferably an intravitreal injection, a subretinal injection, and an intrachoroidal injection , intravenous injection preparations, intratumoral injection preparations or intramuscular injection preparations.
  • the present invention also provides the use of the body or antigen-binding fragment or pharmaceutical composition in the preparation of a medicament for preventing or treating plasma kallikrein or plasma kallikrein-related diseases in a subject;
  • Said disease associated with plasma kallikrein or plasma kallikrein is preferably edema, rheumatoid arthritis, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, diabetes, arterial or venous thrombosis, aortic aneurysm, osteoarthritis, vasculitis, pulmonary embolism, stroke, sepsis, systemic lupus erythematosus nephritis and burns, retinal disease;
  • the edema is preferably selected from Hereditary angioedema, cerebral edema or head trauma;
  • the retinal disease is preferably selected from diabetic macular edema, retinal vein
  • the MHL (Lanadelumab) mentioned in the present invention can be referred to in the prior art (eg patents WO2011085103A, WO2014113701A, WO2017100679A, etc.), and can be constructed and prepared according to conventional technical means in the art. definition
  • Binding or “specific binding” as used herein refers to the binding of an antibody to an epitope of an antigen (eg, human PPK) in an in vitro assay.
  • the affinity of binding is characterized by KD (association rate) or Kd (dissociation constant).
  • KD association rate
  • Kd dissociation constant
  • Epitope as used in the present invention means the portion of an antigen that specifically binds to an antibody.
  • Epitopes typically consist of chemically active (such as polar, non-polar, or hydrophobic) surface groups of moieties, such as amino acids or polysaccharide side chains, and can have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • Epitopes may consist of contiguous and/or discontinuous amino acids forming units of conformational space. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought together in three-dimensional space due to the folding of the protein molecule.
  • the "antibody” in the present invention refers to a binding protein comprising an antigen-binding site.
  • binding site or “antigen binding site” refers to the region of an antibody molecule to which a ligand actually binds.
  • antigen binding site comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL) or VH/VL pair and can be derived from an intact antibody or antibody fragment such as a single chain Fv, VH domain and/or VL domain, Fab or (Fab) 2 .
  • each antigen binding site comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), and preferably consists of an antibody light chain variable domain Domain (VL) and antibody heavy chain variable domain (VH) pairs.
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain Domain
  • VH antibody heavy chain variable domain
  • Antibodies of the invention may comprise six complementarity determining regions (CDRs), of which three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL3) ).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Amino acid residues in the "complementarity determining regions” or “CDRs” are responsible for antigen binding.
  • “Framework” or "FR” regions are those variable domain regions other than the complementarity determining regions.
  • the variability of FR is lower than that of CDR, and there are four FR molecules, namely FR1, FR2, FR3 and FR4.
  • the light and heavy chains of antibodies include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus.
  • the CDRs on each chain are separated by the framework amino acids.
  • the four FR molecules are coiled to bring the CDR molecules close to each other.
  • the CDR3 of the heavy chain is the region most conducive to antigen binding.
  • Fab refers to an antibody comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL).
  • VH antibody heavy chain variable domain
  • CH1 antibody constant domain 1
  • VL antibody light chain variable domain
  • CL antibody light chain constant domain
  • the heavy and light chain domains are stabilized by disulfide bonds.
  • One Fab can form one antigen-binding site, and two “Fabs” can form "(Fab')2" through disulfide bonds, thus having two antigen-binding sites.
  • the "F(ab')2" fragment can be further reduced to form two Fab' fragments.
  • scFv refers to a polypeptide comprising an antibody heavy chain variable domain (VH) and a light chain variable domain (VL), the heavy chain and light chain domains being stabilized by short peptides, scFv is an antigen binding key areas.
  • Fv refers to a polypeptide comprising an antibody heavy chain variable domain (VH) and light chain variable domain (VL) joined together by non-covalent bonds.
  • the binding molecules or antibodies of the invention also comprise immunoglobulin constant regions of one or more immunoglobulin species.
  • Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE isotypes, and in the case of IgG and IgA, their subtypes.
  • the antibody of the invention has the constant domain structure of an IgG-type antibody.
  • constant region refers to the totality of antibody domains other than variable regions.
  • the constant regions are not directly involved in antigen binding, but display different effector functions.
  • Antibodies are classified into the following classes according to the amino acid sequence of the constant region of the heavy chain: IgA, IgD, IgE, IgG and IgM, and wherein IgG and IgA are further subdivided into the following subclasses: IgG1, IgG2, IgG3, and IgG4 , IgA1 and IgA2.
  • the heavy chain constant regions corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively.
  • constant regions derived from human sources refer to human antibody heavy chain constant regions and/or kappa or lambda light chain constant regions of subclasses IgG1, IgG2, IgG3, or IgG4.
  • Antibodies or antigen-binding fragments according to the present invention also include binding molecules or antibodies with "conservative sequence modifications" ("variants").
  • variants This means nucleotide and amino acid sequence modifications that do not affect or alter the above-mentioned characteristics. Nucleotide or amino acid modifications can be made by techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A non-essential amino acid residue in an antibody or antigen-binding fragment may preferably be replaced by another amino acid residue from the same side chain family.
  • a "variant" antibody or antigen-binding fragment is one whose amino acid sequence has one or more amino acid additions, deletions, and/or substitutions compared to the "parent" amino acid sequence.
  • nucleotide or amino acid sequences of the antibodies or antigen-binding fragments of the present invention include "conservative sequence modifications” that have at least 80%, 85%, 90%, 95%, 98%, 99% of the parent nucleotide or amino acid. Sequences with % or more homology.
  • chimeric antibody refers to an antibody comprising antibody variable domains from one source or species, and at least a portion of the constant regions from another source or species, typically prepared by recombinant DNA techniques.
  • a chimeric antibody includes a murine variable region and a human constant region.
  • humanized antibody refers to a portion of the constant region of an antibody (ie, the CH and CL regions) or that the antibody is entirely encoded by human antibody genes. Humanized antibodies can greatly reduce the immune response of heterologous antibodies to the human body.
  • host cell refers to any cell line that can be engineered to produce antibodies according to the present invention.
  • HEK293 cells and CHO cells are used as host cells.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and include their progeny.
  • pharmaceutically acceptable excipient refers to any formulation or carrier medium capable of delivering an effective amount of the active molecule of the present invention, without interfering with the biological activity of the active molecule, and without toxic side effects to the host or patient, including any formulation or carrier medium in the art.
  • the excipient is suitable for intraocular, intravenous, intramuscular, subcutaneous, parenteral, intraarticular, etc. administration.
  • Fig. 1 is the fundus fluorescein sodium angiography picture of rhesus monkey in Example 6
  • FIG. 1 shows the results of retinal microvascular leakage in Example 6
  • Figure 3 is the standard chart of fundus fluorescein sodium angiography scoring in rhesus monkeys (score from 0 to 10 points based on the severity of retinal microvascular leakage, 0 points for FFA pictures in the normal saline group, 8 points for the CA-I group only , Incomplete light transmission, blurred fundus is 9 points, no light transmission (animals have no light perception) is 10 points)
  • Fig. 4 is the fundus fluorescein sodium angiogram of the rhesus monkey in Example 7
  • the CDR transplantation of the heavy and light chains of the antibody described in the present invention, the site mutation introduced by PCR and the screening of the mutation library are completed by conventional gene recombination technology and immunological technology based on antigen-antibody interaction.
  • the specific experimental method steps are such as ⁇ molecular cloning >> Third edition (Joseph Sambrook, Science Press, August 1, 2002) and similar laboratory manuals.
  • mice immunization and hybridoma screening BALB/C female mice (6-8 weeks old, purchased from Dashuo Laboratory Animal Co., Ltd.) were immunized by standard intraperitoneal immunization or plantar immunization, 10ug Antigen Human kallikrein (Enzyme research, product number: 3792A)/mouse, injected every 2 to 3 weeks, all mice were initially immunized with an equal volume of CFA (SIGMA, product number: SLBW7430) and antigen Humankallikrein (Enzyme research, product number: 3792A), mixed emulsification, booster immunization before sprint immunization (about the second immunization-fourth immunization) was immunized with IFA (SIGMA, Cat.
  • mice were sprinted immunized three days before fusion, and sprinted before fusion
  • the antigen Human kallikrein used for immunization was prepared in PBS (20ug/mouse). The mice were killed by twisting their necks, and the mice were immersed in 70% ethanol for 5 min for disinfection. Extract the spleen and lymph nodes, grind the spleen and lymph nodes properly, and separate the obtained lymphoid B cells or spleen B cells with P3X63Ag8.653 myeloma cells (Cat. No.: CRL-1580, purchased from ATCC) or FO myeloma cells (Cat.
  • TCM31 purchased from the Chinese Academy of Sciences Cell Bank
  • TCM31 purchased from the Chinese Academy of Sciences Cell Bank
  • ELISA detection was performed. According to the detection results of ELISA, positive hybridoma cells were selected for expansion culture. After 2-3 days, the second detection (combining ELISA and functional activity) was performed, and the functionally active hybridoma cell lines were expanded and cultured.
  • RNA of hybridoma cells was extracted by Trizol method, and 5 ⁇ g of total RNA was taken for reverse transcription to obtain cDNA; then cDNA was used as a template, and 5'RACE technology was used for PCR amplification, and the PCR product was ligated into the restriction vector pcDNA3.4 The ligation products were transformed into competent Stellar Competent Cells by heat shock, colony PCR was performed, and a small amount of plasmids were extracted from the positive clones and sent for sequencing to obtain the variable region sequences of the light and heavy chains of hybridoma antibody genes.
  • Example 2 Construction of chimeric antibody: primers were designed according to the variable region gene sequence of mouse hybridoma antibody and the constant region of human light and heavy chains, and the gene sequence of light and heavy chain variable region of mouse hybridoma antibody was PCR amplified to obtain chimeric Antibody Q fragment; PCR amplification of human light chain constant region and human heavy chain constant region (hIgG4 or hIgG1) to obtain a chimeric antibody H fragment; using homologous recombination to connect the Q fragment and H fragment into the enzyme digestion vector at the same time pcDNA3.4.
  • primers were designed according to the variable region gene sequence of mouse hybridoma antibody and the constant region of human light and heavy chains, and the gene sequence of light and heavy chain variable region of mouse hybridoma antibody was PCR amplified to obtain chimeric Antibody Q fragment; PCR amplification of human light chain constant region and human heavy chain constant region (hIgG4 or hIgG1) to obtain a chimeric antibody H fragment; using homolog
  • the correctly sequenced plasmid was transfected into expiCHO cells or HEK293 cells for protein expression, the cells were cultured for about 7-10 days, and the cell supernatant was collected by centrifugation; the cell supernatant was affinity purified using an AKTA protein purifier to obtain a chimeric antibody protein. .
  • Example 3 Antibody Humanization: Since mouse-derived antibodies are likely to cause human HAMA reaction, in order to reduce the immunogenicity of mouse-derived antibodies in humans, it is necessary to carry out humanized antibody transformation on mouse-derived antibodies. In this example, traditional methods such as CDR transplantation, framework shuffling and key amino acid back-mutation design were used to humanize multiple candidate molecules. The specific method is to first compare the light and heavy chain CDR regions of the mouse-derived antibody into the FR region of the human germline gene with high sequence similarity through the NCBI database comparison, and obtain the humanized antibody variable region; The recombination method splices the constant regions of the light and heavy chains with the corresponding antibody variable regions respectively and connects them into the expression vector.
  • the correct antibody light and heavy chain expression plasmids are obtained by colony PCR screening and sequencing identification. Then, the correctly sequenced light and heavy chain plasmids were transfected into expiCHO or HEK293 cells for expression; 7-10 days later, the cell supernatant was affinity purified using an AKTA protein purifier to obtain humanized antibody proteins. The humanized antibodies were then subjected to activity assays.
  • the sequence comparison analysis of the preferred antibody molecule and the parent antibody molecule is carried out, and the amino acid molecules that have a greater impact on the activity are determined according to the amino acid position, polarity, molecular weight, spatial conformation, etc., and these amino acids are back mutated to further increase the antibody
  • the activity of the molecule is finally obtained by combinatorial screening of humanized antibody molecules.
  • OctetQKe refurb detector uses the OctetQKe refurb detector to detect the affinity of the candidate antibody with the antigen Human kallikrein (Enzyme research, product number: HPK1302), load the candidate antibody on the sensor, and carry out binding and dissociation reactions with different concentrations of the antigen.
  • the conditions are baseline time: 120s, loading Time: 300s, association time: 600s, dissociation time: 2000s, after the reaction was completed, the results were analyzed by Octet analysis software. The results are shown in Table 2.
  • the MHL (Lanadelumab) can be found in the prior art (eg, patents WO2011085103A, WO2014113701A, WO2017100679A, etc.), and can be constructed and prepared according to conventional technical means in the art.
  • PK can specifically cleave the H-Pro-Phe-Arg-AMC substrate from the C-terminus of arginine, thereby releasing AMC (7-amino-4-methylcoumarin), which can emit fluorescence (excitation light). 360nm, emission light 480nm), detecting the change of the fluorescence value of AMC can reflect the level of PK activity, so as to achieve the purpose of detecting the biological activity of PK inhibitors.
  • the PK was then diluted to 250ng/ml with detection buffer for use, and the H-Pro-Phe-Arg-AMC substrate was diluted to 1000 ⁇ M with detection buffer for use.
  • Embodiment 6 Human plasma activity detection method
  • the first is antibody sample preparation.
  • Detection parameters excitation light 360nm, emission light 480nm, read every 60s for a total of 10min. After normalizing the enzyme reaction rate (slope) with the concentration (nM), a four-parameter curve fitting was performed to calculate the IC50 (nM). The results are shown in Table 3.
  • CA-I carbonic anhydrase I
  • the increased expression of CA-I can cause changes in the intravitreal microenvironment, thereby stimulating the activation of the KKS signaling pathway, resulting in increased retinal vascular permeability, resulting in retinal vascular leakage and macular edema (DME) in patients.
  • Intravitreal injection of carbonic anhydrase in rhesus monkeys (4 years old, 3.2-4.9Kg) was 0.4mg-0.55mg/eye, and the more serious microvascular leakage was observed by fluorescein fundus angiography (FFA) at 30min.
  • FFA fluorescein fundus angiography
  • test drug group (KH02) (500 ⁇ g/eye) was injected into the vitreous body 30 minutes after administration, and carbonic anhydrase CA-I was injected into the vitreous body to create a model, and the drug was detected and observed by fundus fluorescein sodium angiography (FFA) 30 minutes after the modeling.
  • FFA fundus fluorescein sodium angiography
  • the experimental fundus fluorescein sodium angiography picture is shown in Figure 1, and the fundus leakage is shown in Figure 2.
  • the medicament of the present invention can effectively inhibit leakage.
  • Fundus fluorescein sodium angiography scoring standard the severity of retinal microvascular leakage was scored from 0 to 10, the FFA picture in the normal saline group was 0 points, and the CA-I group was only given 8 points, incomplete light transmission, fundus 9 points for ambiguity and 10 points for inability to transmit light (animals have no light perception), see Figure 3 for the specific scoring criteria.
  • the test drug (KH02) 100 ⁇ g/eye was injected into the vitreous body 30 minutes after administration, and carbonic anhydrase CA-I was injected into the vitreous body to create a model, and the first detection and observation of the drug was performed by fundus fluorescein sodium angiography (FFA) 30 minutes after the model was established.
  • FFA fundus fluorescein sodium angiography
  • Molecular inhibitory effect on CA-I-induced retinal vascular leakage CA-I was used to build the model again after 14 days, and the second FFA test was performed 30 minutes after the model was established. It will be divided into drug group (2 rhesus monkeys, 4 eyes), control group as normal saline group and CA-I group.
  • the fundus fluorescein sodium angiography picture is shown in Figure 4.
  • the medicament of the present invention can significantly inhibit leakage.

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Abstract

提供了结合血浆激肽释放酶的抗体，以及这些抗体在制备预防或治疗血浆激肽释放酶或血浆前激肽释放酶相关疾病的药物中的用途。

Description

一种抗体及其用途 技术领域
本发明涉及生物技术领域，具体涉及主要结合血浆激肽释放酶的抗体及其用途。
背景技术
血浆激肽释放酶(plasma kallikrein,PK)属丝氨酸蛋白酶家族的一员，最早是在哺乳动物的血浆中被发现。它由位于4q35染色体上单个基因(KLKB1)编码，主要在肝脏中合成。PK是激肽释放酶-激肽系统(KKS)的一个关键酶，可作用于高分子量激肽原(KH)，从而使其活化释放小分子缓激肽(BK)，进而通过作用于缓激肽受体参与凝血、纤维蛋白溶解、补体激活以及炎症发生等生物学过程。近年来，随着对血浆激肽释放酶的基因学、分子学和药理学的研究更加深入，人们对其生理和病理角色有了深入的认识。研究表明，血浆激肽释放酶与炎症性疾病、肿瘤、心血管疾病、肾病、中枢神经系统疾病、视网膜病以及糖尿病视网膜疾病等多种疾病密切相关(Costa-Neto,C.M.et al.Participation of kallikrein-kinin system in different pathologies.Int.Immunopharmacol.2008,8,135-142)。例如遗传性血管水肿(HAE)，它是一种常染色体显性遗传性，主要是由于患者体内C1-INH缺乏，导致其对血浆激肽释放酶的抑制作用减弱，不受控制的激活KKS系统，释放血管活性物质，使血管通透性增加引起典型的肿胀(Farkas,H.Orphan drugs for the treatment of hereditary angioedema.Expert Opinion on Orphan Drugs,2015,1,141-156)。又例如，在糖尿病黄斑水肿患者眼部玻璃体中，发现其KKS系统过度激活，导致视网膜血管通透性增加和视网膜增厚。近年来已公开多项资料表明血浆激肽释放酶抑制剂可以降低视网膜血管通透性，用于治疗糖尿病视网膜疾病和糖尿病黄斑水肿(Feener,E.P.Plasma kallikrein and diabetic macular edema.Curr.Diab.Rep.2010,10,270-275；Liu J.et al.Plasma kallikrein-kinin system and diabetic retinopathy.Biol.Chem.2013,394,319-328)。
目前已有PK的蛋白或多肽类药物进入临床或者成功上市。例如，目前已有艾卡拉肽和Lanadelumab等大分子血浆激肽释放酶抑制剂上市在临床上用于治疗遗传性血管水肿，疗效显著。Bicycle公司的THR-149，其通过一种特异性结合血浆激肽释放酶的双环肽肽配体用于治疗糖尿病性黄斑水肿，处于临床II期研究阶段。
发明内容
本发明提供一种结合血浆激肽释放酶的抗体或抗原结合片段，所述抗体或抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列X 1X 2X 3X 4X 5，其中，X 1为D或N，X 2为T、I或Y，X 3为Y或W，X 4为I或M，X 5为F或H，或者HCDR1为TSGMGVS；CDR2含有序列 Y 1IY 2PY 3Y 4Y 5Y 6Y 7Y 8Y 9Y 10Y 11Y 12Y 13Y 14Y 15，其中Y 1为R或A，Y 2为D或Y，Y 3为E或G，Y 4为N或D，Y 5为D、G或S，Y 6为N或D，Y 7为T或I，Y 8为K、V或S，Y 9为Y或F，Y 10为D或N，Y 11为P或Q，Y 12为K或R，Y 13为I或F，Y 14为Q或K，Y 15为G或D，或者CDR2为HIYWDDDKRYNPSLKS；CDR3含有序列Z 1GZ 2Z 3Z 4Z 5Y，其中Z 1为G或A，Z 2为G或S，Z 3为L或I，Z 4为F或P，Z 5为A或S，或者CDR3为PHYYAFDGFGY或ERAYYRYDEDFDY；
其中轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列L 1ASL 2DINL 3YL 4L 5，其中L 1为E或K，L 2为H或Q，L 3为N、K或S，L 4为I或L，L 5为A或S，或者CDR1为SATSIINSNYFH；CDR2含有序列M 1M 2M 3M 4LM 5M 6，其中M 1为Y、F或R，M 2为T或A，M 3为S或N，M 4为T、S、R或N，M 5为Q、H、V或A，M 6为S、Q、D或P；CDR3含有序列LQYDN 1LN 2T，其中N 1为N或D，N 2为F或W，或者CDR3为VQYDEFPLT或QQGSSLPRT。
进一步的，本发明所述的抗体或抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:1-5中任一个，CDR2含有序列SEQ ID NO:6-11中任一个，CDR3含有序列SEQ ID NO:12-15中任一个；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:16-21中任一个，CDR2含有序列SEQ ID NO:22-27中任一个，CDR3含有序列SEQ ID NO:28-31中任一个。
进一步的，本发明所述的抗体或抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:1；CDR2含有序列SEQ ID NO:6；CDR3含有序列SEQ ID NO:12；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:16；CDR2含有序列SEQ ID NO:22；CDR3含有序列SEQ ID NO:28；
或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:1；CDR2含有序列SEQ ID NO:7；CDR3含有序列SEQ ID NO:12；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:17；CDR2含有序列SEQ ID NO:23；CDR3含有序列SEQ ID NO:28；
或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:2；CDR2含有序列SEQ ID NO:8；CDR3含有序列SEQ ID NO:13；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:18；CDR2含有序列SEQ ID NO:24；CDR3含有序列SEQ ID NO:29；
或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:3；CDR2含有 序列SEQ ID NO:9；CDR3含有序列SEQ ID NO:14；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:19；CDR2含有序列SEQ ID NO:25；CDR3含有序列SEQ ID NO:30；
或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:4；CDR2含有序列SEQ ID NO:10；CDR3含有序列SEQ ID NO:12；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:20；CDR2含有序列SEQ ID NO:26；CDR3含有序列SEQ ID NO:29；
或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:5；CDR2含有序列SEQ ID NO:11；CDR3含有序列SEQ ID NO:15；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:21；CDR2含有序列SEQ ID NO:27；CDR3含有序列SEQ ID NO:31。
本发明提供的抗体或抗原结合片段包括鼠源抗体、嵌合抗体、人源化抗体或全人源化抗体或其抗原结合片段。
进一步的，本发明提供的抗体或抗原结合片段，其重链可变区序列选自SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：46、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：54或SEQ ID NO：56中的任一种，轻链可变区序列选自SEQ ID NO：34、SEQ ID NO：36-42、SEQ ID NO：45、SEQ ID NO：47-49、SEQ ID NO：53、SEQ ID NO：55或SEQ ID NO：57的任一种。
进一步的，本发明提供的抗体或抗原结合片段，其重链可变区序列选自SEQ ID NO：35，轻链可变区序列选自SEQ ID NO：36-42中的任一种；或其重链可变区序列选自SEQ ID NO：44或46，轻链可变区序列为SEQ ID NO：45；或其重链可变区序列选自SEQ ID NO：50或SEQ ID NO：32，轻链可变区序列选自SEQ ID NO：47-49中的任一种；或其重链可变区序列为SEQ ID NO：33，轻链可变区序列为SEQ ID NO：34；或其重链可变区序列为SEQ ID NO：43，轻链可变区序列为SEQ ID NO：42；或其重链可变区序列为SEQ ID NO：51，轻链可变区序列为SEQ ID NO：48；或其重链可变区序列为SEQ ID NO：52，轻链可变区序列为SEQ ID NO：53；或其重链可变区序列为SEQ ID NO：54，轻链可变区序列为SEQ ID NO：55；或其重链可变区序列为SEQ ID NO：56，轻链可变区序列为SEQ ID NO：57。
本发明所述抗原结合片段优选自Fab、Fab’、Fv、scFv或(Fab’) 2片段。
本发明还提供了一些核酸分子，其编码上述抗体或抗原结合片段；还进一步提供了表达所述核酸的表达载体；同时进一步提供了包含所述载体的宿主细胞，所述宿主细胞包含原核细胞或真核细胞，更优选的选自酵母细胞、哺乳动物细胞(例如293细胞或CHO细胞)。
同发明还进一步提供了轻链恒定区如SEQ ID NO：61及其突变体；重链恒定区如Human IgG1、Human IgG2、Human IgG4等，其中Human IgG1优选SEQ ID NO：58及其突变体；Human IgG2优选SEQ ID NO：59及其突变体；Human IgG4优选SEQ ID NO：60及其突变体。
本发明示例给出的抗体及抗原结合片段的序列对应关系见表1：
表1部分抗体及抗原结合片段的序列
产品 重链可变区 轻链可变区 产品 重链可变区 轻链可变区
KH01 SEQ ID NO:33 SEQ ID NO:34 KH12 SEQ ID NO:32 SEQ ID NO:47
KH02 SEQ ID NO:35 SEQ ID NO:36 KH13 SEQ ID NO:32 SEQ ID NO:48
KH03 SEQ ID NO:35 SEQ ID NO:37 KH14 SEQ ID NO:32 SEQ ID NO:49
KH04 SEQ ID NO:35 SEQ ID NO:38 KH15 SEQ ID NO:50 SEQ ID NO:47
KH05 SEQ ID NO:35 SEQ ID NO:39 KH16 SEQ ID NO:50 SEQ ID NO:48
KH06 SEQ ID NO:35 SEQ ID NO:40 KH17 SEQ ID NO:50 SEQ ID NO:49
KH07 SEQ ID NO:35 SEQ ID NO:41 KH18 SEQ ID NO:51 SEQ ID NO:48
KH08 SEQ ID NO:35 SEQ ID NO:42 KH19 SEQ ID NO:52 SEQ ID NO:53
KH09 SEQ ID NO:43 SEQ ID NO:42 KH20 SEQ ID NO:54 SEQ ID NO:55
KH10 SEQ ID NO:44 SEQ ID NO:45 KH21 SEQ ID NO:56 SEQ ID NO:57
KH11 SEQ ID NO:46 SEQ ID NO:45      
本发明给出示例中涉及的抗体KH01-18的轻链恒定区为SEQ ID NO：61；KH10和KH12-14的重链恒定区为Human IgG1(SEQ ID NO：58)，KH01-KH09、KH11、KH15-18的重链恒定区为Human IgG4(SEQ ID NO：60)，KH19-21为鼠源抗体。
本发明还提供了一种药物组合物，包含上述的抗体或抗原结合片段，和药学上可接受的赋形剂；所述药物组合物优选为玻璃体内注射剂、视网膜下注射制剂、脉络膜内注射制剂、静脉注射制剂、肿瘤内注射制剂或肌肉注射制剂。
本发明还提供了所述的体或抗原结合片段或药物组合物在制备用于预防或治疗受试者中血浆激肽释放酶或血浆前激肽释放酶相关的疾病的药物中的用途；所述与血浆激肽释放酶或血浆前激肽释放酶相关的疾病优选为水肿、类风湿性关节炎、痛风、肠道疾病、口腔粘膜炎、神经性疼痛、炎性疼痛、椎管狭窄-退行性脊柱疾病、糖尿病、动脉或静脉血栓形成、主动脉瘤、骨关节炎、脉管炎、肺栓塞、中风、败血病、系统性红斑狼疮性肾炎和烧伤、视网膜疾病；所述水肿优选自遗传性血管性水肿、脑水肿或头部外伤；所述视网膜疾病优选自糖尿病性黄斑水肿，视网膜静脉阻塞，年龄相关性黄斑变性，继发于视网膜静脉阻塞的黄斑水肿，葡萄膜炎、眼内炎或息肉状脉络膜血管病变。
本发明中提及的MHL(Lanadelumab)可参见现有技术(如专利WO2011085103A、WO2014113701A、WO2017100679A等)，并可根据本领域的常规技术手段进行构建及制备。 定义
本发明所述“结合”或“特异性结合”指在体外测定法中，抗体与抗原(如人PPK)的表位进行结合。结合的亲和力由KD(结合速率)或Kd(解离常数)进行表征。本发明所述的抗体结合或特异性结合人血浆激肽释放酶(PK)并不排除其可能对其他抗原或表位的结合，例如血浆前激肽释放酶(PPK)。
本发明所述“表位”意指抗原的与抗体特异性结合的部分。表位通常由部分(moiety)(诸如氨基酸或多糖侧链)的化学活性(诸如，极性、非极性或疏水性)表面基团构成，并且可以具有特定三维结构特征以及特定电荷特征。表位可由形成构象空间单元的连续和/或不连续氨基酸构成。对于不连续表位，来自抗原的线性序列的不同部分的氨基酸因蛋白质分子的折叠而在三维空间上靠近。
本发明所述“抗体”指包含抗原结合位点的结合蛋白。术语“结合位点”或“抗原结合位点”指配体实际上结合的抗体分子的区域。术语“抗原结合位点”包含抗体重链可变结构域(VH)和/或抗体轻链可变结构域(VL)或VH/VL对，并可源自完整的抗体或抗体片段如单链Fv、VH结构域和/或VL结构域、Fab或(Fab) 2。在本发明的一个实施方案中，每个抗原结合位点包含抗体重链可变结构域(VH)和/或抗体轻链可变结构域(VL)，和优选地由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)对组成。
本发明的抗体或可包含六个互补性决定区(CDRs)，其中三个重链可变结构域CDRs(CDRH1，CDRH2和CDRH3)和三个轻链可变结构域CDRs(CDRL1，CDRL2和CDRL3)。CDR和框架区(FRs)共同组成重链或轻链可变结构域。“互补性决定区”或“CDRs”中的氨基酸残基负责抗原结合。“构架”或“FR”区是互补性决定区之外的那些可变结构域区域。FR的可变性要低于CDR，FR分子共四个，分别为FR1、FR2、FR3和FR4。抗体的轻链和重链从N端到C端包括结构域FR1，CDR1、FR2、CDR2、FR3、CDR3和FR4。各条链上的CDR通过所述框架氨基酸分开。在识别抗体时，四个FR分子卷曲使CDR分子相互靠近。特别地，重链的CDR3是最有助于抗原结合的区域。
本发明的术语“Fab”指包含抗体重链可变结构域(VH)，抗体恒定结构域1(CH1)，抗体轻链可变结构域(VL)和抗体轻链恒定结构域(CL)的多肽，重链和轻链结构域之间通过二硫键稳定。一个Fab可形成一个抗原结合位点，两个“Fab”可通过二硫键形成“(Fab’)2”，因此具有两个抗原结合位点。“F(ab’)2”片段可以进一步被还原，形成两个Fab’片段。
本发明的术语“scFv”指包含抗体重链可变结构域(VH)和轻链可变结构域(VL)的多肽，重链和轻链结构域之间通过短肽稳定，scFv是抗原结合的关键区域。“Fv”指包含抗体重链 可变结构域(VH)和轻链可变结构域(VL)的多肽，重链和轻链结构域之间通过非共价键结合在一起。
本发明的结合分子或抗体还包含一个或多个免疫球蛋白种类的免疫球蛋白恒定区。免疫球蛋白种类包括IgG，IgM，IgA，IgD，和IgE同种型，并且在IgG和IgA的情形中，包括它们的亚型。在优选的实施方案中，本发明的抗体具有IgG型抗体的恒定结构域结构。
本发明的术语“恒定区”指除了可变区之外的抗体结构域的总合。恒定区不直接涉及抗原的结合，但是显示不同的效应子功能。根据重链的恒定区的氨基酸序列，抗体被分为下述类别：IgA，IgD，IgE，IgG和IgM，并且其中IgG和IgA被进一步细分为如下亚型：IgG1，IgG2，IgG3，和IgG4，IgA1和IgA2。对应于不同种类的抗体的重链恒定区分别被称为α、δ、ε、γ和μ。所有5种抗体中轻链恒定区被称为κ(kappa)和λ(lambda)。在本发明中来自人来源的恒定区指亚类IgG1，IgG2，IgG3，或IgG4的人抗体重链恒定区和/或κ或λ轻链恒定区。
根据本发明所述的抗体或抗原结合片段还包括具有“保守序列修饰”的结合分子或抗体(“变体”)。这意味着不影响或改变上述特征的核苷酸和氨基酸序列修饰。可以通过本领域已知的技术对核苷酸或氨基酸进行修饰，如位点定向诱变和PCR介导的诱变。在抗体或抗原结合片段中的非必需氨基酸残基可以优选地被来自相同侧链家族的另一种氨基酸残基置换。因此，“变体”抗体或抗原结合片段是指其氨基酸序列与“母体”氨基酸序列相比，具有一个或多个氨基酸的添加、缺失和/或置换。因此本发明所述抗体或抗原结合片段的核苷酸或氨基酸序列均包括具有“保守序列修饰”与母体核苷酸或氨基酸具有至少80％、85％、90％、95％、98％、99％或以上同源性的序列。
本发明的术语“嵌合抗体”指抗体包括来自一种来源或物种的抗体可变结构域，以及源自另外来源或物种的恒定区的至少一部分，通常通过重组DNA技术进行制备。例如，在一个实施方案中，包括鼠可变区和人恒定区的嵌合抗体。
本发明的术语“人源化抗体”指抗体的恒定区部分(即CH区和CL区)或抗体全部由人类抗体基因编码。人源化抗体可以大大降低异源抗体对人类机体造成的免疫反应。
本发明的术语“宿主细胞”指可以被改造从而产生根据本发明所述的抗体的任何细胞系。在一个实施方案中，将HEK293细胞和CHO细胞用作宿主细胞。在本申请中，表述“细胞”、“细胞系”和“细胞培养物”可交替使用，且包括他们的后代。
本发明的术语“药学上可接受的赋形剂”是指能够递送本发明有效量活性分子、不干扰活性分子生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质，包括任何本领域普通技术人员已知的溶剂、分散介质、表面活性剂、抗氧剂、防腐剂(例如抗菌剂、抗真菌剂)、 等张剂、吸收延迟剂、盐、防腐剂、药物、药物稳定剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料等及其组合。优选地，所述赋形剂适合用于眼内、静脉内、肌内、皮下、肠胃外、关节内等方式给药。
说明书附图
图1为实施例6中恒河猴眼底荧光素钠血管造影图片
图2为实施例6中视网膜微血管渗漏情况结果
图3为恒河猴眼底荧光素钠血管造影评分标准图(以视网膜微血管渗漏严重情况从0-10分进行评分，生理盐水组FFA图片为0分，以只给予CA-I组为8分，透光不完全，眼底模糊不清为9分，无法透光(动物无光感)为10分)
图4为实施例七中恒河猴眼底荧光素钠血管造影图
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
本发明描述的抗体重轻链的CDR移植，PCR引入位点突变及突变文库的筛选是通过常规基因重组技术及基于抗原抗体相互作用的免疫学技术所完成，具体实验方法步骤如<<分子克隆>>第三版(Joseph Sambrook，科学出版社，2002年8月1日)及类似实验手册所记载。
实施例一 小鼠免疫及杂交瘤筛选：采用标准腹腔免疫或足底免疫两种方式对BALB/C雌性小鼠(鼠龄6-8周，购于达硕实验动物有限公司)进行免疫，10ug抗原Human kallikrein(Enzyme research，货号：3792A)/只小鼠，每2至3周进行一次注射，所有小鼠初次免疫使用等体积CFA(SIGMA，货号：SLBW7430)与抗原Humankallikrein(Enzyme research，货号：3792A)，混合乳化，冲刺免疫前的加强免疫(约第二次免疫-第四次免疫)使用IFA(SIGMA，货号：F5506)免疫；在融合前三天对小鼠进行冲刺免疫，融合前冲刺免疫所用抗原Human kallikrein为PBS配制(20ug/只小鼠)。拧脖处死小鼠，用70％乙醇浸泡小鼠5min进行消毒。摘取脾脏及淋巴结，适当研磨脾脏和淋巴结，分别将获得的淋巴B细胞或脾脏B细胞同P3X63Ag8.653骨髓瘤细胞(货号：
Figure PCTCN2022073460-appb-000001
CRL-1580，购于ATCC)或FO骨髓瘤细胞(货号：TCM31，购于中科院细胞库)按一定比例进行混合，使用电融合仪进行融合。在杂交瘤细胞恢复到最佳状态时，进行ELISA检测。根据ELISA检测检测结果，挑选阳性杂交瘤细胞扩大培养。2-3天后进行第二次检测(结合ELISA与功能活性)，将有功能活性的杂交瘤细胞株扩大培养。使用Trizol法提取杂交瘤细胞的总RNA，取5μg总RNA进行反转录，得 到cDNA；然后以cDNA为模板，使用5’RACE技术进行PCR扩增，将PCR产物连接进酶切载体pcDNA3.4中，连接产物通过热激转化进感受态Stellar Competent Cells中，进行菌落PCR鉴定，将阳性克隆提取少量质粒送测序，获得杂交瘤抗体基因轻重链可变区序列。
实施例二、嵌合抗体构建：根据小鼠杂交瘤抗体可变区基因序列与人轻重链恒定区设计引物，对小鼠杂交瘤抗体轻重链可变区基因序列进行PCR扩增，得到嵌合抗体Q片段；对人轻链恒定区和人重链恒定区(hIgG4或者hIgG1)PCR扩增，得到嵌合抗体H片段；利用同源重组的方法将Q片段和H片段同时连接进酶切载体pcDNA3.4中。将测序正确的质粒转染进expiCHO细胞或者HEK293细胞进行蛋白表达，细胞培养约7-10天左右，离心收集细胞上清；细胞上清利用AKTA蛋白纯化仪进行亲和纯化，得到嵌合抗体蛋白。
实施例三、抗体人源化：由于鼠源抗体容易引起人的HAMA反应，为了减少鼠源抗体在人体内的的免疫原性，需要对鼠源抗体进行人源化抗体改造。本实施例采用传统的CDR移植，框架改组和关键氨基酸回复突变设计等方法，对多个候选分子进行人源化。具体方法为首先通过NCBI数据库比对，将鼠源抗体轻重链CDR区分别移植进与之序列高度相似的人胚系基因FR区中，得到人源化后的抗体可变区；然后利用同源重组方法分别将轻重链的恒定区同相应的抗体可变区进行拼接并连接进表达载体中，通过菌落PCR筛选，测序鉴定获得正确的抗体轻重链表达质粒。然后将测序正确的轻重链质粒转染expiCHO或HEK293细胞中进行表达；7-10天后，细胞上清利用AKTA蛋白纯化仪进行亲和纯化，得到人源化抗体蛋白。随后将人源化抗体进行活性测定。对优选的抗体分子同母本抗体分子进行序列比较分析，根据氨基酸位置、极性、分子量、空间构象等多方面确定对活性影响较大的氨基酸分子，将这些氨基酸进行回复突变，以进一步增加抗体分子的活性，通过组合筛选最终获得人源化抗体分子。
实施例四 与人PK结合亲和力测试
用OctetQKe refurb检测仪检测候选抗体与抗原Human kallikrein(Enzyme research，货号:HPK1302)的亲和力，将候选抗体loading于传感器上，与不同浓度的抗原进行结合解离反应，条件为baseline时间：120s，loading时间：300s，association时间：600s，dissociation时间：2000s，反应完成后，用Octet分析软件进行结果分析，结果如表2。
表2
Figure PCTCN2022073460-appb-000002
其中MHL(Lanadelumab)可参见现有技术(如专利WO2011085103A、WO2014113701A、 WO2017100679A等)，并可根据本领域的常规技术手段进行构建及制备。
实施例五 PK活性检测方法
PK可以特异性的从精氨酸的C端切割H-Pro-Phe-Arg-AMC底物，从而释放出AMC(7-氨基-4-甲基香豆素)，AMC可以发出荧光(激发光360nm、发射光480nm)，检测AMC的荧光值的变化可以反映PK的活性高低，以此来达到检测PK抑制剂的生物学活性的目的。首先是抗体样品准备，用检测缓冲液(20mM Tris-HCl，PH＝7.50、50mM NaCl、1mM EDTA、0.1％PEG-8000和0.1％Triton X-100)将抗体蛋白稀释至200μg/ml作为起始浓度，进行4倍梯度稀释，共11个浓度梯度，稀释好后待用。然后用检测缓冲液将PK稀释至250ng/ml的溶液备用，将H-Pro-Phe-Arg-AMC底物用检测缓冲液稀释至1000μM备用。分别吸取50μl检测缓冲液(阴性对照)及稀释后的各个浓度梯度蛋白溶液至96孔不透光酶标板中；另取40μl 250ng/ml PK稀释液(终浓度为100ng/ml)至上述96孔板中；最后加入10μl 1000μM H-Pro-Phe-Arg-AMC底物(终浓度为100μM)至上述96孔板中。将准备好的96孔板放入多功能酶标仪(Spectra Max i3X)进行检测，检测参数：激发光360nm、发射光480nm，每60s读一次，共读10min。将酶反应速率(斜率)进行归一化处理后与浓度(nM)进行四参数曲线拟合，计算出IC50(nM)。检测结果如表2。
表2部分抗体PKa的活性结果
  KH01 KH02 KH10 KH11 KH12 KH13 KH14 KH15 KH16 KH19 KH20 KH21 MHL
IC50(nM) 0.297 0.235 0.227 0.188 0.174 0.221 0.398 0.224 0.230 0.870 1.1 0.82 0.990
实施例六 人血浆活性检测方法
人血浆中存在PPK、PK等蛋白，但通过检测发现人血浆原液或稀释液中PK并不显现出活性，可能是一个酶活抑制的平衡状态。如果额外加入Factor XIIa因子将打破平衡，产生具有酶活性的PK蛋白，以此原理检测PK抑制剂的活性。首先是抗体样品准备，用检测缓冲液(20mM Tris-HCl，PH＝7.50、150mM NaCl、1mM EDTA、0.1％PEG-8000和0.1％Triton X-100)将抗体蛋白进行稀释至200μg/ml作为起始浓度，进行4倍梯度稀释，共11个浓度梯度，稀释好后待用。用检测缓冲液将人血浆稀释40倍，Factor XIIa因子稀释至100ng/ml，底物肽(H-Pro-Phe-Arg-AMC)稀释至1000μM备用。分别吸取50μl检测缓冲液及稀释后的各个浓度梯度抗体蛋白溶液加至96孔不透光酶标板；另取20μl人血浆40倍稀释样至上述96孔板中，37℃孵育30min；再加入浓度为100ng/ml Factor XIIa 20μl，37℃孵育45min；最后向上述96孔不透光酶标板各孔中加入浓度1000μM底物肽(H-Pro-Phe-Arg-AMC)10μl。将准备好的96孔板放入多功能酶标仪(Spectra Max i3X)进行检测，检测参数：激发光360nm、发射光480nm，每60s读一次，共读10min。将酶反应速率(斜率)进行归一化处理后与浓度(nM) 进行四参数曲线拟合，计算出IC50(nM)，结果见表3。
表3部分抗体人血浆中活性结果
样品 KH01 KH02 KH03 KH04 KH05 KH06 KH07 KH08
IC50(nM) 0.753 0.52 0.949 0.987 0.921 0.918 0.863 0.578
样品 KH09 KH11 KH18 KH19 KH20 KH21 MHL  
IC50(nM) 0.764 0.1032 0.419 0.93 1.09 1.29 1.17  
实施例七 动物药效研究
1、灵长类动物疾病模型构建
在糖尿病视网膜病变病人的玻璃体内，有显著的碳酸酐酶I(CA-I)过量表达。CA-I的表达量增加会引起玻璃体内微环境的改变，从而激发KKS信号通路的激活，引起视网膜血管通透性增加，导致病人的视网膜血管渗漏和黄斑水肿(DME)。向恒河猴(4岁，3.2-4.9Kg)玻璃体内注射碳酸酐酶量为0.4mg—0.55mg/眼，30min荧光素眼底血管造影(FFA)观察微血管渗漏越严重。
2、含有受试药物的辅料配方见表4：
表4受试药物的辅料配方
Acetic Acid/Sodium Acetate 20mM，PH 5-5.2
sodium chloride 40mM
sucrose 8.8％
吐温80 0.01％
3、给药方式
通过将受试药物组(KH02)(500μg/眼)玻璃体注射给药后30min，碳酸酐酶CA-I玻璃体注射造模，造模后30min通过眼底荧光素钠血管造影(FFA)进行检测观测药物分子对CA-I诱导视网膜血管渗漏的抑制作用。将三只恒河猴分为药物组、MHL组和阴性组(阴性组为不含药物的制剂缓冲液)，每组一只恒河猴(2只眼睛)。
4、实验结果
实验眼底荧光素钠血管造影图片见图1，眼底渗漏情况见图2。本发明药物能有效抑制渗漏。
实施例八 动物药效研究
动物模型构建等见实施例七。
眼底荧光素钠血管造影评分标准：以视网膜微血管渗漏严重情况从0-10分进行评分，生理盐水组FFA图片为0分，以只给予CA-I组为8分，透光不完全，眼底模糊不清为9分，无法透光(动物无光感)为10分，具体评分标准见图3。
通过将受试药物(KH02)100μg/眼玻璃体注射给药后30min，碳酸酐酶CA-I玻璃体注射造 模，造模后30min通过眼底荧光素钠血管造影(FFA)进行第一次检测观测药物分子对CA-I诱导视网膜血管渗漏的抑制作用；14天后再次用CA-I造模，造模后30min进行第二次FFA检测。将分为药物组(2只恒河猴、4只眼睛)、对照组为生理盐水组和CA-I组。眼底荧光素钠血管造影图片见图4。本发明药物能显著抑制渗漏。

Claims (10)

  1. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列X 1X 2X 3X 4X 5，其中，X 1为D或N，X 2为T、I或Y，X 3为Y或W，X 4为I或M，X 5为F或H，或者HCDR1为TSGMGVS；CDR2含有序列Y 1IY 2PY 3Y 4Y 5Y 6Y 7Y 8Y 9Y 10Y 11Y 12Y 13Y 14Y 15，其中Y 1为R或A，Y 2为D或Y，Y 3为E或G，Y 4为N或D，Y 5为D、G或S，Y 6为N或D，Y 7为T或I，Y 8为K、V或S，Y 9为Y或F，Y 10为D或N，Y 11为P或Q，Y 12为K或R，Y 13为I或F，Y 14为Q或K，Y 15为G或D，或者CDR2为HIYWDDDKRYNPSLKS；CDR3含有序列Z 1GZ 2Z 3Z 4Z 5Y，其中Z 1为G或A，Z 2为G或S，Z 3为L或I，Z 4为F或P，Z 5为A或S，或者CDR3为PHYYAFDGFGY或ERAYYRYDEDFDY；
    其中轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列L 1ASL 2DINL 3YL 4L 5，其中L 1为E或K，L 2为H或Q，L 3为N、K或S，L 4为I或L，L 5为A或S，或者CDR1为SATSIINSNYFH；CDR2含有序列M 1M 2M 3M 4LM 5M 6，其中M 1为Y、F或R，M 2为T或A，M 3为S或N，M 4为T、S、R或N，M 5为Q、H、V或A，M 6为S、Q、D或P；CDR3含有序列LQYDN 1LN 2T，其中N 1为N或D，N 2为F或W，或者CDR3为VQYDEFPLT或QQGSSLPRT。
  2. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段重链可变区：CDR1含有序列SEQ ID NO:1-5中任一个，CDR2含有序列SEQ ID NO:6-11中任一个，CDR3含有序列SEQ ID NO:12-15中任一个；轻链可变区：CDR1含有序列SEQ ID NO:16-21中任一个，CDR2含有序列SEQ ID NO:22-27中任一个，CDR3含有序列SEQ ID NO:28-31中任一个；优选的，所述重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:1；CDR2含有序列SEQ ID NO:6；CDR3含有序列SEQ ID NO:12；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:16；CDR2含有序列SEQ ID NO:22；CDR3含有序列SEQ ID NO:28；
    或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:1；CDR2含有序列SEQ ID NO:7；CDR3含有序列SEQ ID NO:12；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:17；CDR2含有序列SEQ ID NO:23；CDR3含有序列SEQ ID NO:28；
    或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:2；CDR2含有序列SEQ ID NO:8；CDR3含有序列SEQ ID NO:13；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:18；CDR2含有序列SEQ ID NO:24；CDR3含有序列SEQ ID NO:29；
    或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:3；CDR2含有序列SEQ ID NO:9；CDR3含有序列SEQ ID NO:14；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:19；CDR2含有序列SEQ ID NO:25；CDR3含有序列SEQ ID NO:30；
    或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:4；CDR2含有序列SEQ ID NO:10；CDR3含有序列SEQ ID NO:12；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:20；CDR2含有序列SEQ ID NO:26；CDR3含有序列SEQ ID NO:29；
    或重链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:5；CDR2含有序列SEQ ID NO:11；CDR3含有序列SEQ ID NO:15；轻链可变区包括以下三个互补决定区(CDR)：CDR1含有序列SEQ ID NO:21；CDR2含有序列SEQ ID NO:27；CDR3含有序列SEQ ID NO:31。
  3. 根据前述任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或全人源化抗体或其抗原结合片段。
  4. 根据前述任一项所述的抗体或抗原结合片段，其重链可变区序列选自SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：46、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：54或SEQ ID NO：56中的任一种，轻链可变区序列选自SEQ ID NO：34、SEQ ID NO：36-42、SEQ ID NO：45、SEQ ID NO：47-49、SEQ ID NO：53、SEQ ID NO：55或SEQ ID NO：57的任一种；优选的，重链可变区序列选自SEQ ID NO：35，轻链可变区序列选自SEQ ID NO：36-42中的任一种；或其重链可变区序列选自SEQ ID NO：44或46，轻链可变区序列为SEQ ID NO：45；或其重链可变区序列选自SEQ ID NO：50或SEQ ID NO：32，轻链可变区序列选自SEQ ID NO：47-49中的任一种；或其重链可变区序列为SEQ ID NO：33，轻链可变区序列为SEQ ID NO：34；或其重链可变区序列为SEQ ID NO：43，轻链可变区序列为SEQ ID NO：42；或其重链可变区序列为SEQ ID NO：51，轻链可变区序列为SEQ ID NO：48；或其重链可变区序列为SEQ ID NO：52，轻链可变区序列为SEQ ID NO：53；或其重链可变区序列为SEQ ID NO：54，轻链可变区序列为SEQ ID NO：55；或其重链可变区序列为SEQ ID NO：56，轻链可变区序列为SEQ ID NO：57。
  5. 根据前述任一项所述的抗体或抗原结合片段，其特征在于，所述抗原结合片段选自Fab、Fab’、Fv、scFv或(Fab’)2片段。
  6. 分离的核酸，其编码权利要求1～5任一项所述的抗体或抗原结合片段。
  7. 包含权利要求6的核酸的载体，优选地所述载体是表达载体；以及所述载体的宿主细胞，优选地，所述宿主细胞是原核细胞或真核细胞，更优选的选自酵母细胞、哺乳动物细胞(例如293细胞或CHO细胞)。
  8. 一种药物组合物，其特征在于包含权利要求1-5中任一项所述的抗体或抗原结合片段，和药学上可接受的赋形剂；所述药物组合物优选为玻璃体内注射剂、视网膜下注射制剂、脉络膜内注射制剂、静脉注射制剂、肿瘤内注射制剂或肌肉注射制剂。
  9. 权利要求1-5中任一项所述的体或抗原结合片段在制备用于预防或治疗受试者中血浆激肽释放酶或血浆前激肽释放酶相关的疾病的药物中的用途；其中所述与血浆激肽释放酶或血浆前激肽释放酶相关的疾病优选为水肿、类风湿性关节炎、痛风、肠道疾病、口腔粘膜炎、神经性疼痛、炎性疼痛、椎管狭窄-退行性脊柱疾病、糖尿病、动脉或静脉血栓形成、主动脉瘤、骨关节炎、脉管炎、肺栓塞、中风、败血病、系统性红斑狼疮性肾炎和烧伤、视网膜疾病。
  10. 根据权利要求9中所述的用途，其中所述水肿选自遗传性血管性水肿、脑水肿或头部外伤；所述视网膜疾病优选自糖尿病性黄斑水肿，视网膜静脉阻塞，年龄相关性黄斑变性，继发于视网膜静脉阻塞的黄斑水肿，葡萄膜炎、眼内炎或息肉状脉络膜血管病变。
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