WO2021244627A1 - 分离的抗原结合蛋白及其用途 - Google Patents

分离的抗原结合蛋白及其用途 Download PDF

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WO2021244627A1
WO2021244627A1 PCT/CN2021/098261 CN2021098261W WO2021244627A1 WO 2021244627 A1 WO2021244627 A1 WO 2021244627A1 CN 2021098261 W CN2021098261 W CN 2021098261W WO 2021244627 A1 WO2021244627 A1 WO 2021244627A1
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amino acid
seq
acid sequence
antigen binding
binding protein
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PCT/CN2021/098261
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English (en)
French (fr)
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孙乃超
周若芸
高琪
马海立
刘恒
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天辰生物医药(苏州)有限公司
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Priority to US18/000,661 priority Critical patent/US20230212272A1/en
Publication of WO2021244627A1 publication Critical patent/WO2021244627A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • A—HUMAN NECESSITIES
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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
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    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • This application relates to the field of biomedicine, and specifically relates to an isolated antigen binding protein and its use.
  • the complement system plays a key role in the innate immune response.
  • the complement system is composed of more than 25 plasma proteins. These plasma proteins act through three currently known activation pathways: the classical pathway, the lectin pathway, and the alternative pathway. Although the initial steps of these pathways are different, they have the same late complement components (C5 to C9) that are responsible for activating and destroying target cells.
  • a normally functioning complement system can provide a strong defense against microbial infections, but the imbalance of complement can also lead to a variety of diseases.
  • paroxysmal nocturnal hemoglobinuria PNH
  • PIG-A paroxysmal nocturnal hemoglobinuria
  • GPI glycosylphosphatidylinositol
  • PNH paroxysmal nocturnal hemoglobinuria
  • GPI glycosylphosphatidylinositol
  • its clinical manifestations also include bone marrow failure and thrombosis.
  • the results of a large-scale clinical epidemiological survey showed that the mortality rate of PNH patients was 35% within 5 years after diagnosis and 50% within 10 years. Thrombotic events are the main cause of death of PNH patients in Europe and America, accounting for about 40-67%.
  • the application provides an isolated antigen binding protein, which comprises at least one CDR in a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 44-46 Or a variant thereof; and it comprises at least one CDR in the light chain variable region, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 70 or a variant thereof.
  • the amino acid sequence variant shown in SEQ ID NO: 70 comprises amino acid mutations selected from the group consisting of D32E, D32I, D32L, D32G, D32S, D32T, D32V, D32Y, G33A, G33S , G33I, G33Q, G33T and G33V.
  • the isolated antigen binding protein comprises HCDR1 in the heavy chain variable region shown in any one of SEQ ID NOs: 44-46 with an amino acid sequence.
  • the isolated antigen binding protein comprises HCDR2 in the heavy chain variable region shown in any one of SEQ ID NOs: 44-46 with an amino acid sequence.
  • the isolated antigen binding protein comprises HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs: 44-46 with an amino acid sequence.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 18.
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 19.
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein comprises LCDR1 in the light chain variable region shown in any one of SEQ ID NOs: 41-43 with an amino acid sequence.
  • the isolated antigen binding protein comprises LCDR2 in the light chain variable region shown in any one of SEQ ID NOs: 41-43 with an amino acid sequence.
  • the isolated antigen binding protein comprises LCDR3 in the light chain variable region shown in any one of SEQ ID NOs: 41-43.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 69.
  • the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NOs: 1 and 2.
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:17.
  • the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof.
  • the antigen binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the light chain variable region includes the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 21-23 .
  • the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 21-23.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 24-25.
  • the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 24-25.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 26-28.
  • the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 26-28.
  • the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 29-30.
  • the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 29-30.
  • the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID Nos: 41-43.
  • the isolated antigen binding protein includes an antibody light chain constant region, and the antibody light chain constant region includes a human Kappa light chain constant region.
  • the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:47.
  • the isolated antigen binding protein comprises an antibody light chain
  • the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 49, 51, and 53.
  • the heavy chain variable region includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 31-33 .
  • the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 31-33.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 34-35.
  • the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 34-35.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 36-38.
  • the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 36-38.
  • the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 39-40.
  • the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 39-40.
  • the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID Nos: 44-46.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.
  • the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48.
  • the isolated antigen binding protein comprises an antibody heavy chain
  • the antibody heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 50, 52, and 54.
  • the antigen is C5.
  • the C5 is human C5.
  • this application also provides an isolated nucleic acid molecule, which encodes the isolated antigen binding protein described in this application.
  • the present application also provides a vector, which contains the nucleic acid molecule described in the present application.
  • this application also provides a cell, which comprises the nucleic acid molecule described in this application or the vector described in this application.
  • this application also provides a method for preparing the isolated antigen binding protein described in this application, the method comprising culturing the isolated antigen binding protein described in this application under conditions cell.
  • this application also provides a pharmaceutical composition, which comprises the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and/or the cell described in this application, And optionally a pharmaceutically acceptable adjuvant.
  • the application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition described in the application in the preparation of medicines
  • the medicine is used to prevent, alleviate and/or treat C5-related diseases or disorders.
  • this application also provides a method for preventing, alleviating or treating C5-related diseases or disorders, the method comprising administering the isolated antigen binding protein described in this application, the nucleic acid to a subject in need
  • the molecule, the carrier, the cell and/or the pharmaceutical composition comprising administering the isolated antigen binding protein described in this application, the nucleic acid to a subject in need.
  • the present application also provides the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, which are used for prevention, alleviation or Methods of treating C5-related diseases or disorders.
  • this application also provides a method for detecting C5 in a sample, the method comprising administering the isolated antigen binding protein described in this application.
  • Figure 1 shows the results of the antigen binding capacity of the chimeric antibody described in this application and the positive control antibody.
  • Figure 2 shows the results of the hemolysis inhibition experiment of the chimeric antibody and the positive control antibody described in this application.
  • Figure 3 shows the experimental results of the changes in the yield of the chimeric antibody described in this application after site mutation.
  • Figure 4 shows the experimental results of changes in the antigen binding capacity of the chimeric antibody described in this application after site mutation.
  • Figure 5 shows the results of the classical pathway (CP) hemolysis inhibition experiment of the humanized antibody and the positive control antibody described in this application.
  • Figure 6 shows the results of the alternative pathway (AP) hemolysis inhibition experiment of the humanized antibody and the positive control antibody described in this application.
  • Figure 7 shows the results of the thermal stability experiment of the humanized antibody and the positive control antibody described in this application.
  • Figure 8 shows the results of the binding experiment between the humanized antibody described in this application and the human C5 antigen.
  • isolated generally refers to artificially obtained from the natural state or artificially synthesized. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide isolated from this natural state is called isolated.
  • isolated does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized.
  • the term "variant" generally refers to amino acid modifications (such as group substitutions, etc.) on the original protein sequence or one or more amino acid insertions, substitutions, and/or deletions, while retaining the original sequence function molecular.
  • the variant may have better biological activity (or function) than the original sequence.
  • the reservation need not be a complete reservation.
  • the variant may basically retain the original sequence function, for example, retain the original sequence at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%. %, 98%, or 99% functionality.
  • the amino acid sequence of the variant may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the original amino acid sequence.
  • the amino acid sequence variant shown in SEQ ID NO: 69 may include amino acid modifications (such as group substitutions, etc.) or one or more amino acids on the amino acid sequence shown in SEQ ID NO: 1. Insertion, substitution, and/or deletion, while retaining the original sequence function.
  • the function of the amino acid sequence shown in SEQ ID NO:1 may be basically retained, for example, the amino acid sequence shown in SEQ ID NO:1 may be retained at least 50%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, 96%, 97%, 98%, or 99% function. For example, it may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:1.
  • the identity can be achieved through the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277), version 3.0.0 or more
  • the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) executed in the higher version is determined.
  • the optional parameters used are gap penalty 10, gap extension penalty 0.5 and EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62).
  • isolated antigen binding protein generally refers to a protein with antigen binding ability obtained from a natural state by artificial means or artificially synthesized.
  • isolated antigen-binding protein may comprise an antigen-binding portion and optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that facilitates the antigen-binding portion to bind to the antigen.
  • the antigen binding protein may comprise, for example, an antibody-derived protein scaffold or alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).
  • peptide antibody mimics (“PAMs”) and scaffolds based on antibody mimics using fibronectin components can also be used as scaffolds.
  • antibody generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide that includes an antigen binding site, regardless of whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , Mutant and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAbs and other antibody fragments that retain the antigen-binding function (e.g., specifically bind to human C5). Generally, such fragments should include an antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • an IgM antibody is composed of 5 basic heterotetrameric units and another polypeptide called the J chain, while an IgA antibody includes 2-5 basic 4-chain units that can be combined with the J chain and polymerized to form a multivalent combination.
  • the 4-chain unit can generally be about 150,000 Daltons.
  • Each L chain can be connected to the H chain by a covalent disulfide bond, and two H chains can be connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • Each H and L chain may also have spaced intra-chain disulfide bridges.
  • Each H chain can have a variable domain/variable region (VH) at the N-terminus, followed by three constant domains/constant regions (CH) for the ⁇ and ⁇ chains, and for the ⁇ and ⁇ isotypes. It has four CH domains.
  • Each L chain may have a variable domain/variable region (VL) at the N-terminus, and may have a constant domain/constant region (CL) at the other end.
  • VL corresponds to VH
  • CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Certain amino acid residues are thought to form an interface between the light chain and heavy chain variable domains. VH and VL can pair together to form an antigen binding site.
  • L chains from any vertebrate species can be classified into one of two distinct types based on the amino acid sequence of their constant domains, called kappa and lambda.
  • immunoglobulins can also be classified into different classes or types.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes are further divided into subclasses. For example, humans include the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1. Antigen-binding fragments.
  • the term "antigen-binding fragment” generally refers to a part of a full-length antibody (for example, a target binding region or a variable region) or a functional fragment or analog thereof.
  • antibody fragments may include Fab, Fab', F(ab')2 and Fv fragments and the like.
  • a "functional fragment or analog” generally refers to a compound that has substantially the same qualitative biological activity as a full-length antibody.
  • a functional fragment or analog of an anti-C5 antibody can bind to the C5 protein so as to inhibit hemolysis of the classical pathway and/or the alternative pathway.
  • "Fv" fragments are usually the smallest antibody fragments that contain a complete target recognition and binding site. This region can be composed of a dimer (VH-VL dimer) in which a heavy chain variable domain and a light chain variable domain are non-covalently linked.
  • framework region generally refers to the part of the antibody variable region known in the art that exists between the more differentiated (ie, hypervariable) CDRs.
  • framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and are provided for the presentation of six CDRs (three from the heavy chain and three from the light chain) in a three-dimensional space
  • the backbone to form the antigen binding surface. That is, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).
  • the VL of the isolated antigen binding protein described in this application may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the VH of the isolated antigen binding protein described in this application may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • variable generally refers to the fact that certain segments of antibody variable regions (eg, heavy chain variable regions, light chain variable regions) differ in sequence between antibodies. Normally, the variable region mediates antigen binding and determines the specificity of a specific antibody to its specific antigen. Under normal circumstances, the variability is not evenly distributed throughout the variable domains, but concentrated in several regions called complementarity determining regions (CDR) or hypervariable regions (HVR) in the light chain and heavy chain variable domains. In the paragraph. The relatively conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • variable domains of the natural heavy and light chains can each contain four FR regions, most of which adopt a ⁇ -sheet configuration, are connected by three CDRs, and in some cases form part of a ⁇ -sheet structure.
  • the CDRs in each chain are held in close proximity by FR, and can work with the CDRs from the other chain to promote the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant region does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, for example, the antibody participates in antibody-dependent cytotoxicity.
  • light chain variable region generally refers to the amino terminal domain of an antibody light chain.
  • the light chain variable region may be referred to as "VL".
  • VL complementarity determining regions
  • HVR hypervariable regions
  • FR framework regions
  • variable region generally refers to the amino terminal domain of an antibody heavy chain.
  • the variable region of the heavy chain may be referred to as "VH".
  • VH variable region of the heavy chain
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • FR framework regions
  • the terms "subject” and “patient” are used interchangeably, and generally refer to mammals, such as human patients and non-human primates, and experimental animals, such as rabbits, rats, and mice. And other animals. Animals include all vertebrates, for example, mammals and non-mammals, such as dogs, cats, sheep, cows, pigs, rabbits, chickens and the like.
  • the subject used to implement Ben Shen Qi's treatment method is a human.
  • Subjects in need of treatment include patients who already have C5-related diseases or disorders, as well as people who tend to develop such disorders.
  • prevention generally refers to preventive administration of the combination to healthy patients to prevent the outbreak of the diseases and conditions described in this application.
  • prevention refers to the prophylactic administration of this combination to patients in the pre-stage of the allergic disease to be treated.
  • prevention does not require 100% elimination of the possibility of an event. More precisely, it means that the probability of an event occurring in the presence of the pharmaceutical composition or method is reduced.
  • treatment generally refers to the application or administration of a therapeutic agent to a patient, or the application or administration of a therapeutic agent to a tissue or cell line isolated from a patient who has a disease, has disease symptoms, or a disease tendency, which The purpose is to treat, cure, alleviate, alleviate, change, remedy, improve, improve or affect the disease, disease symptoms, or disease tendency. This can include improving the state of the disease, eliminating the focus of the disease, or improving the prognosis.
  • the term "cell” may generally be a single cell, cell line, or cell culture that is or has been the recipient of a nucleic acid molecule or vector.
  • the cell may include the nucleic acid molecule described in this application or the vector described in this application.
  • a cell can include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the offspring may not necessarily be exactly the same as the original parent cell (in terms of the morphology of the total DNA complement or in the genome).
  • the cells may include cells transfected in vitro with the vectors described in this application.
  • the cells may be bacterial cells (e.g., E.
  • the cell is a mammalian cell.
  • the mammalian cell is a HEK293T cell.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, such as a human patient.
  • the pharmaceutical composition described in this application may comprise the isolated antigen binding protein described in this application, the carrier described in this application and/or the cell described in this application, and optionally a pharmaceutically acceptable Adjuvant.
  • the pharmaceutical composition may also contain one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. Preparations.
  • the acceptable ingredients of the composition are non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • pharmaceutically acceptable adjuvant generally refers to any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration. Generally safe, non-toxic, and neither biologically nor otherwise undesirable.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed.
  • the vector can be transformed, transduced, or transfected into the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage And animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papilloma viruses such as papilloma viruses
  • papillary polyoma vacuoles Virus such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the carrier may
  • directly or indirectly connected generally refers to the relative “directly connected” or “indirectly connected”.
  • Directly connected usually refers to a direct connection.
  • the direct connection may be a situation where the connected substances (for example, amino acid sequence segments) are directly connected without spacer components (for example, amino acid residues or derivatives thereof); for example, the amino acid sequence segment X is directly connected to another amino acid.
  • the sequence segment Y is directly connected by an amide bond formed between the C-terminal amino acid of the amino acid sequence segment X and the N-terminal amino acid of the amino acid sequence segment Y.
  • Indirect connection generally refers to the case where there are spacers (such as amino acid residues or derivatives thereof) between connected substances (such as amino acid sequence segments) and indirectly connected.
  • spacers such as amino acid residues or derivatives thereof
  • connected substances such as amino acid sequence segments
  • the term "complementarity determining region” or the term “CDR” generally refers to the variable region that collectively defines the binding site of an antigen binding protein (for example, a natural immunoglobulin, a chimeric antibody or a humanized antibody) Amino acid sequences with binding affinity and specificity (see, for example, Chothia et al., J.Mol. Biol. 196:901-917 (1987); Kabat et al., US Department of Health and Human Services NIH publication (USDept of Health and Human Services NIH) Publication) No. 913242 (1991)).
  • an antigen binding protein for example, a natural immunoglobulin, a chimeric antibody or a humanized antibody
  • Amino acid sequences with binding affinity and specificity see, for example, Chothia et al., J.Mol. Biol. 196:901-917 (1987); Kabat et al., US Department of Health and Human Services NIH publication (USDept of Health and
  • an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3).
  • Naturally occurring camel antibodies consisting only of heavy chains can maintain normal and stable functions even in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • C5 also known as “complement component 5" or “complement factor 5" generally refers to the serum protein of the complement cascade or its variants.
  • the C5 protein can contain two chains ⁇ and ⁇ . This protein represents the focal point involved in the three complement activation pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin pathway.
  • the amino acid sequence of the full-length C5 protein can be exemplified by the amino acid sequence provided in GenBank as the accession number NP_001726.2.
  • the term “C5" may include recombinant C5 protein or fragments thereof.
  • the term can also include C5 proteins or fragments thereof that bind, for example, a histidine tag, murine or human Fc, or a signal sequence (such as ROR1).
  • the term may include the sequence exemplified by the sequence shown in SEQ ID NO:60.
  • C5-related disease or disorder generally refers to any disease or disorder that can be treated or alleviated by interfering with C5 protein.
  • complement-related diseases include inflammatory diseases and autoimmune diseases, distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis, kidney Glomerulonephritis, renal vasculitis, cardiopulmonary bypass, coronary endothelial dysfunction induced by heart failure, type II membrane proliferative glomerulonephritis, acute renal failure, antiphospholipid syndrome, macular degeneration, endophthalmitis, new vascular diseases , Allograft transplantation, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disease (COPD) respiratory distress syndrome, asthma, paroxysmal nocturnal hemoglobinuria (PNH) and aspiration pneumonia.
  • COPD chronic obstructive pulmonary disease
  • KD also known as "K D ", "affinity constant” or “equilibrium dissociation constant” usually refers to the titration measurement at equilibrium, or by the dissociation rate constant ( kd) divided by the binding rate constant (K a ).
  • K a association rate constant
  • K d dissociation rate constant
  • K D equilibrium dissociation constant
  • the K D value can be measured by Octet, and other experimental methods and instruments such as BIAcore (Biomolecular Interaction Analysis) can also be used.
  • electrochemiluminescence analysis-solution equilibrium titration measures the K D value. The measurement method is as described in Estep P. et al., MAbs, 2013.5(2): p.270-8.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • this application provides an antigen binding protein.
  • the antigen binding protein includes at least one CDR in the light chain variable region VL.
  • the antigen binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 69:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR1 may include at least amino acid substitutions selected from the following positions: amino acid substitutions at X 9 and/or X 10 .
  • the LCDR1 may include at least the amino acid substitution at X 9 and/or X 10 , wherein the amino acid at X 9 may be substituted Is D, E, I, L, G, S, T, V or Y; the amino acid at X 10 can be substituted with G, A, S, I, Q, T or V.
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 1-15.
  • the antigen binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 17.
  • the antigen binding protein may include L-FR1, the C-terminus of L-FR1 is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 may include SEQ ID NO: 21- The amino acid sequence shown in any one of 23.
  • the antigen binding protein may include L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include any of SEQ ID NOs: 24-25 The amino acid sequence shown in the item.
  • the antigen binding protein may include L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may include any of SEQ ID NOs: 26-28 The amino acid sequence shown in the item.
  • the antigen binding protein may include L-FR4, the N-terminus of L-FR4 is directly or indirectly connected to the C-terminus of LCDR3, and the L-FR4 may include SEQ ID NO: 29-30 The amino acid sequence shown in any one of them.
  • the antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in any one of SEQ ID NOs: 41-43.
  • the antigen binding protein may include a light chain constant region CL, and the CL may include a human Kappa light chain constant region.
  • the CL may include the amino acid sequence shown in SEQ ID NO:47.
  • the antigen binding protein may include a light chain, and the light chain may include the amino acid sequence shown in any one of SEQ ID NOs: 49, 51, and 53.
  • the antigen binding protein comprises at least one CDR in the VH of the heavy chain variable region.
  • the antigen binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18.
  • the antigen binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 19.
  • the antigen binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the antigen binding protein may include H-FR1, the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 may include SEQ ID NO: 31- The amino acid sequence shown in any one of 33.
  • the antigen binding protein may include H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include any of SEQ ID NOs: 34-35 The amino acid sequence shown in the item.
  • the antigen binding protein may include H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include any of SEQ ID NOs: 36-38 The amino acid sequence shown in the item.
  • the antigen binding protein may include H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 may include SEQ ID NO: 39-40 The amino acid sequence shown in any one of them.
  • the antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in any one of SEQ ID NO: 44-46.
  • the antigen binding protein may include a heavy chain constant region CH, and the CH may include a human IgG heavy chain constant region.
  • the IgG heavy chain constant region may comprise a human IgG1 heavy chain constant region.
  • the CH may include the amino acid sequence shown in SEQ ID NO:48.
  • the antigen binding protein may include a heavy chain, and the heavy chain may include the amino acid sequence shown in any one of SEQ ID NOs: 50, 52, and 54.
  • the antigen binding protein may include LCDR1-3
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 69
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.
  • the antigen binding protein may include LCDR1-3
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 1-15
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as CHI-16, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.
  • the antigen-binding protein described in the present application may include the same LCDR1-3 as 16H1L2m and 16H46L39am
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 2
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.
  • the antigen binding protein may include L-FR1-4
  • the L-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 21-23
  • the L-FR2 may include SEQ The amino acid sequence shown in any one of ID NO: 24-25
  • the L-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 26-28
  • the L-FR4 may include SEQ ID The amino acid sequence shown in any one of NO: 29-30.
  • the antigen binding protein described in the present application may include the same L-FR1-4 as CHI-16, the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 21, and the L-FR2 may include SEQ The amino acid sequence shown in ID NO: 24, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 26, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may include the same L-FR1-4 as 16H1L2m
  • the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 22
  • the L-FR2 may include SEQ ID NO
  • the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 27
  • the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in the present application may include the same L-FR1-4 as 16H46L39am
  • the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 23
  • the L-FR2 may include SEQ ID NO
  • the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 28
  • the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein may include HCDR1-3
  • the HCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 18, and the HCDR2 may include any one of SEQ ID NO: 19
  • the amino acid sequence shown and the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 20.
  • the antigen binding protein described in the present application may include the same HCDR1-3 as CHI-16, 16H1L2m and 16H46L39am
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18
  • the HCDR2 may include SEQ ID NO The amino acid sequence shown in: 19
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 20.
  • the antigen binding protein may include H-FR1-4
  • the H-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 31-33
  • the H-FR2 may include SEQ The amino acid sequence shown in any one of ID NO: 34-35
  • the H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 36-38
  • the H-FR4 may include SEQ ID NO: the amino acid sequence shown in any one of 39-40.
  • the antigen binding protein described in the present application may include the same H-FR1-4 as CHI-16
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 31
  • the H-FR2 may include SEQ
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 36
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 39.
  • the antigen binding protein described in the present application may include the same H-FR1-4 as 16H1L2m
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 32
  • the H-FR2 may include SEQ ID NO
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 37
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 40.
  • the antigen binding protein described in the present application may include the same H-FR1-4 as 16H46L39am
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 33
  • the H-FR2 may include SEQ ID NO
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 38
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 40.
  • the antigen binding protein may include LCDR1-3 and HCDR1-3
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 69
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18,
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 19,
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the antigen binding protein may include LCDR1-3 and HCDR1-3
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 1-15
  • the LCDR2 may include SEQ ID NO
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17
  • the amino acid sequence shown, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 20.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 that are the same as CHI-16
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO:1
  • the LCDR2 may include SEQ ID NO
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the HCDR2 may include SEQ ID NO: 19
  • the amino acid sequence shown, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 20.
  • the antigen binding protein described in the present application may include the same LCDR1-3 and HCDR1-3 as 16H1L2m and 16H46L39am
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 2
  • the LCDR2 may include SEQ ID NO
  • the amino acid sequence shown in SEQ ID NO: 16 and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 19, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 20.
  • the antigen binding protein may include a light chain variable region VL and a heavy chain variable region VH, and the VL may include the amino acid sequence shown in any one of SEQ ID NO: 41-43, and The VH may include the amino acid sequence shown in any one of SEQ ID NOs: 44-46.
  • the antigen binding protein may include the same light chain variable region VL and heavy chain variable region VH as CHI-16, the VL may include the amino acid sequence shown in SEQ ID NO: 41, and the VH may Contains the amino acid sequence shown in SEQ ID NO: 44.
  • the antigen binding protein may include the same light chain variable region VL and heavy chain variable region VH as 16H1L2m, the VL may include the amino acid sequence shown in SEQ ID NO: 42, and the VH may include SEQ ID NO: 45 shows the amino acid sequence.
  • the antigen binding protein may include the same light chain variable region VL and heavy chain variable region VH as 16H46L39am, the VL may include the amino acid sequence shown in SEQ ID NO: 43, and the VH may include SEQ ID NO: 46 amino acid sequence.
  • the antigen binding protein may include a light chain constant region CL and a heavy chain constant region CH
  • the CL may include the amino acid sequence shown in SEQ ID NO: 47
  • the CH may include SEQ ID NO: 48 shown in the amino acid sequence.
  • the antigen binding protein may include the same light chain constant region CL and heavy chain constant region CH as CHI-16, 16H1L2m and 16H46L39am
  • the CL may include the amino acid sequence shown in SEQ ID NO: 47
  • the CH may include the amino acid sequence shown in SEQ ID NO: 48.
  • the antigen binding protein may include a light chain and a heavy chain
  • the light chain may include the amino acid sequence shown in any one of 49, 51, and 53
  • the heavy chain may include SEQ ID NO: The amino acid sequence shown in any one of 50, 52, and 54.
  • the antigen binding protein may include the same light chain and heavy chain as CHI-16, the light chain may include the amino acid sequence shown in SEQ ID NO: 49, and the heavy chain may include SEQ ID NO: 50 The amino acid sequence shown.
  • the antigen binding protein may include the same light chain and heavy chain as 16H1L2m, the light chain may include the amino acid sequence shown in SEQ ID NO: 51, and the heavy chain may include the amino acid sequence shown in SEQ ID NO: 52 The amino acid sequence.
  • the antigen binding protein may include the same light chain and heavy chain as 16H46L39am, the light chain may include the amino acid sequence shown in SEQ ID NO: 53, and the heavy chain may include the amino acid sequence shown in SEQ ID NO: 54 The amino acid sequence.
  • the antigen binding protein may include LCDR1-3 and L-FR1-4, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17, the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 21, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 24, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 26, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 29;
  • the antigen binding protein It may also include VL and CL, the VL may include the amino acid sequence shown in SEQ ID NO: 41, and the CL may include the amino acid sequence shown in SEQ ID NO: 47;
  • the antigen binding protein may also include HCDR1 -3 and H-FR1-4, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, the
  • the antigen binding protein may include LCDR1-3 and L-FR1-4, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17, the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 22, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 24, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 27, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 30;
  • the antigen binding protein It may also include VL and CL, the VL may include the amino acid sequence shown in SEQ ID NO: 42, and the CL may include the amino acid sequence shown in SEQ ID NO: 47;
  • the antigen binding protein may also include HCDR1 -3 and H-FR1-4, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, the
  • the antigen binding protein may include LCDR1-3 and L-FR1-4, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17, the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 23, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 25, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 28, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 30;
  • the antigen binding protein It may also include VL and CL, the VL may include the amino acid sequence shown in SEQ ID NO: 43, and the CL may include the amino acid sequence shown in SEQ ID NO: 47;
  • the antigen binding protein may also include HCDR1 -3 and H-FR1-4, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, the HC
  • the antigen binding protein provided in this application can compete with the reference antibody for binding to C5.
  • the reference antibody may include LCDR1-3
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 69
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17; in some cases, the antigen binding protein of the present application may include LCDR1-3, and the LCDR1 may include any one of SEQ ID NO: 1-15.
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.
  • the reference antibody may include HCDR1-3
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 19
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the reference antibody may include LCDR1-3 and HCDR1-3
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 69
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17,
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 18,
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 19,
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 20;
  • the antigen binding protein of the present application may include LCDR1-3 and HCDR1-3
  • the LCDR1 may include SEQ ID NO: 1 -15
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 17,
  • the reference antibody may include a light chain variable region VL and a heavy chain variable region VH
  • the VL may include the amino acid sequence shown in any one of SEQ ID NO: 41-43
  • the VH may include the amino acid sequence shown in any one of SEQ ID NOs: 44-46.
  • this application also provides immunoconjugates.
  • the isolated antigen binding protein can be cross-linked with other compounds (such as therapeutic agents) to form the immunoconjugate.
  • therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerases Inhibitors of I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents, such as SN-38.
  • the antibody and the therapeutic agent can be cross-linked by a linker, which can be cleaved, such as a peptide linker, a disulfide linker, or a hydrazone linker.
  • the linker may be a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.
  • ADC can be like US Patent 7,087,600; 6,989,452; and 7,129,261; PCT Publication WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; US Patent Publication 20060024317; Prepare as described in 20060004081; and 20060247295.
  • this application also provides an isolated nucleic acid molecule, which can encode the isolated antigen binding protein described in this application.
  • the isolated nucleic acid molecule described in this application can be an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or artificially synthesized, which can encode the present The isolated antigen binding protein described in the application.
  • this application also provides a vector, which may contain the nucleic acid molecule described in this application.
  • the vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.
  • the vector may include: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage and Animal viruses and so on.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • the vector may contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication site.
  • the carrier may also include components that assist it to enter cells, such as viral particles, liposomes or protein coats, but not only these substances.
  • this application also provides a cell, which may contain the nucleic acid molecule described in this application or the vector described in this application.
  • the cell may include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the offspring may not necessarily be exactly the same as the original parent cell (in terms of the morphology of the total DNA complement or in the genome).
  • the cells may include prokaryotic cells, yeast cells, or higher eukaryotic cells.
  • Prokaryotes suitable for this purpose include Gram-negative bacteria and Gram-positive bacteria, for example, enteric bacteria such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus (Proteus), Salmonella, Serratia, and Shigella, as well as Bacillus, Pseudomonas and Streptomyces.
  • eukaryotic microorganisms such as filamentous fungi or yeasts can also be suitable cloning or expression hosts for the vectors shown in this application.
  • Saccharomyces cerevisiae is most commonly used in lower eukaryotic host microorganisms.
  • a variety of other genera, species and strains are also commonly available and can be used in this application, for example, Schizosaccharomyces pombe, Kluyveromyces, Candida, Trichoderma, Streptomyces crassa ( Neurospora crassa), and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergi 11us host cells, such as A. nidulans And Aspergillus niger (A. niger).
  • vertebrate cells and vertebrate cells proliferated in culture are suitable cloning or expression hosts for the vectors shown in this application.
  • the cell can include a mammalian host cell line.
  • a mammalian host cell line for example, monkey kidney cells, human embryonic kidney cell lines, young hamster kidney cells, Chinese hamster ovary cells, mouse Sertoli cells, human cervical cancer cells (HELA), canine kidney cells, human lung cells, humans Liver cells, mouse breast cancer cells or NSO cells.
  • HELA human cervical cancer cells
  • the cells may also include cells transfected in vitro with the vectors described in this application.
  • the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or myeloma cells.
  • the cell may be a mammalian cell.
  • the mammalian cell may be a HEK293 cell.
  • this application also provides a method for preparing the isolated antigen binding protein described in this application, and the method may include culturing the isolated antigen binding protein described in this application under conditions Cell.
  • the application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition in the preparation of medicines.
  • Drugs are used to prevent, alleviate and/or treat C5-related diseases or disorders.
  • the present application also provides methods for preventing, alleviating or treating C5-related diseases or disorders.
  • the method may include administering the isolated antigen binding protein and the nucleic acid described in this application to a subject in need.
  • the molecule, the carrier, the cell and/or the pharmaceutical composition are administered to a subject in need.
  • the administration can be performed in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the application also provides the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, which can be used for prevention, alleviation or Treatment of C5-related diseases or conditions.
  • the subject can include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • this application also provides a pharmaceutical composition, which comprises the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and/or the cell described in this application, And optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may additionally contain one or more other therapeutic agents suitable for the treatment or prevention of C5-related diseases or disorders.
  • the pharmaceutically acceptable adjuvant enhances or stabilizes the composition, or facilitates the preparation of the composition.
  • the pharmaceutically acceptable adjuvant may include physiologically compatible solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the pharmaceutical composition can be administered by various methods known in the art, depending on the desired result, the route of administration, and/or the mode of administration.
  • administration can be intravenous, intramuscular, intraperitoneal or subcutaneous, or adjacent to the target site.
  • the pharmaceutical compositions are formulated so that they can be administered intravitreally into the eye.
  • the isolated antigen binding protein e.g., antibody, bispecific and multispecific molecule
  • the isolated antigen binding protein can be coated with a material to protect the compound from the action of acid or other natural conditions. Can inactivate the compound.
  • the pharmaceutical composition can be sterile and fluid.
  • a coating material such as lecithin
  • a surfactant proper fluidity can be maintained.
  • isotonic agents are included in the composition, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride.
  • the long-term absorption of the injectable pharmaceutical composition can be achieved by including an agent that delays absorption (for example, aluminum monostearate or gelatin) in the pharmaceutical composition.
  • the isolated antigen binding protein, the nucleic acid molecule, the carrier and/or the cell are mixed with optional pharmaceutically acceptable carriers, excipients or stabilizers to prepare for storage.
  • the pharmaceutical composition may be in the form of a lyophilized preparation or an aqueous solution.
  • the pharmaceutically acceptable adjuvant may include a pharmaceutically acceptable carrier, excipient or stabilizer.
  • the acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration employed, and includes buffers such as phosphate citrate, acetate, and other organic acids.
  • the pharmaceutical composition comprising the isolated antigen binding protein described in this application may be in a water-soluble form.
  • the pharmaceutical composition for in vivo administration may be sterile. This can be easily achieved by sterile filter membrane filtration or other methods.
  • administration of the pharmaceutical composition containing the isolated antigen binding protein described in this application in the form of a sterile aqueous solution can be carried out in a variety of ways, including but not limited to oral, subcutaneous, intravenous, intranasal, intra-aural, transdermal, topical (E.g., gel, ointment, lotion, cream, etc.), intraperitoneal, intramuscular, intrapulmonary, parenteral, rectal, or intraocular.
  • the isolated antigen binding protein can be directly applied as a solution or spray.
  • the pharmaceutical composition can be prepared according to methods well known and routinely practiced in the art.
  • the pharmaceutical composition can be prepared under GMP conditions.
  • a therapeutically effective dose or effective dose of C5 binding protein is used in the pharmaceutical composition of the present application.
  • the C5 binding protein can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.
  • the dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response).
  • the actual dosage level of the active ingredient in the pharmaceutical composition of the present application can be changed in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response, composition, and mode of administration for a particular patient without being toxic to the patient.
  • the dosage level selected depends on a variety of pharmacokinetic factors, for example, the activity of the specific composition or its ester, salt or amide used in this application, the route of administration, the time of administration, the excretion rate of the specific compound being used, and the therapeutic effect Duration, other drugs, compounds and/or substances used in combination with the particular composition used, age, sex, weight, status, general health and previous medical history of the subject being treated, and other factors.
  • subcutaneous administration can be used in situations where the patient can self-administer the pharmaceutical composition.
  • Many protein therapeutics are not effective enough to allow the formulation of a therapeutically effective dose of the maximum acceptable volume for subcutaneous administration.
  • the antigen binding protein disclosed in the present application may be suitable for subcutaneous administration, for example, increased potency, increased plasma half-life, and increased solubility.
  • protein therapeutics can be delivered by intravenous infusion or bolus injection.
  • the antigen binding proteins disclosed in this application can also be delivered using such methods.
  • this application also provides a method for detecting C5 in a sample, the method comprising administering the isolated antigen binding protein described in this application.
  • a sample obtained from a subject is contacted with the isolated antigen binding protein (e.g., C5 binding protein) described in this application.
  • C5 binding protein e.g., C5 binding protein
  • C5 binding protein is labeled with a detectable label or reporter molecule or anti-C5 binding protein is used as a capture ligand to selectively isolate C5 from patient samples.
  • the unlabeled anti-C5 binding protein can be used in detection applications in conjunction with a second antibody that itself has a detectable label.
  • the detectable label or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; it can also be a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate, rhodamine; or an enzyme, such as alkaline Phosphatase, ⁇ -galactosidase, horseradish peroxidase, or luciferase; specific exemplary analyses that can be used to detect or measure C5 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • the sample that can be used in the C5 detection of the present application may include any tissue or fluid sample obtained from a subject under normal or pathological conditions, which contains a detectable content of C5 protein or a fragment thereof.
  • the level of C5 protein in a specific sample obtained from a healthy subject e.g., a subject not suffering from a C5-related disease
  • a healthy subject e.g., a subject not suffering from a C5-related disease
  • This baseline level of C5 is then compared to the C5 level measured in a sample obtained from an individual suspected of having a C5-related disease or disorder or symptoms related to the disorder.
  • the C5 binding protein may not contain additional labels, and may also contain N-terminal or C-terminal labels.
  • the label is biotin.
  • the position of the label can determine the orientation of the peptide relative to the surface on which the peptide is bound. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will keep the C-terminal part of the peptide away from the surface.
  • Antigen binding protein 16H1L2m 16H46L39am Heavy chain variable region SEQ ID NO: 61 SEQ ID NO: 62 Light chain variable region SEQ ID NO: 63 SEQ ID NO: 64 Light chain SEQ ID NO: 65 SEQ ID NO: 67 Heavy chain SEQ ID NO: 66 SEQ ID NO: 68
  • the ELISA method was used to determine the binding of the culture supernatants of 9 hybridoma cell lines expressing high-affinity murine monoclonal antibodies to the human C5 antigen. Coat the ELISA plate with human C5 (shown in SEQ ID NO: 60), overnight at 4 degrees Celsius. The coating solution was discarded, each well was blocked with bovine serum albumin (BSA) dissolved in phosphate buffered saline (PBS) for 1 hour, and the wells were washed with PBST solution (20% Tween in PBS).
  • BSA bovine serum albumin
  • PBS phosphate buffered saline
  • mice monoclonal antibody hybridoma cell line culture supernatant and eculizumab ECU
  • ECU mouse monoclonal antibody hybridoma cell line culture supernatant and eculizumab
  • the m16 murine antibody was selected for subsequent antibody preparation.
  • the method for obtaining the DNA fragment containing the coding sequence of the murine antibody m16 heavy chain and light chain variable region is: isolating mRNA from m16 mouse hybridoma cells to prepare cDNA.
  • the DNA fragments of the variable region of the heavy chain and the variable region of the light chain are separated from the cDNA by polymerase chain reaction (PCR).
  • the DNA fragment obtained after gel recovery was cloned into the TOPO-TA vector (Yeasen) and sequenced, and the amino acid sequence (SEQ ID NO: 44) of the variable region (VH) encoding the m16 heavy chain (SEQ ID NO: 44) was obtained by using the software Discovery Studio to predict
  • the amino acid sequence of the complementarity determining region is HCDR1 (SEQ ID NO: 18), HCDR2 (SEQ ID NO: 19) and HCDR3 (SEQ ID NO: 20).
  • the murine heavy chain variable region and light chain variable region gene sequences were cloned from mouse hybridoma cells, and the human IgG1 constant region without ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation was selected. Sequence (amino acid sequence shown in SEQ ID NO: 48), linking murine antibody m16 heavy chain variable region gene and human IgG1 constant region gene to form a chimeric gene, light chain variable region gene and human Kappa light chain constant
  • the region sequence (amino acid sequence shown in SEQ ID NO: 47) is connected to form a chimeric gene, and then inserted into the vector pcDNA3.1 (Biofeng Lab), and finally in human kidney epithelial cells (293) (purchased from Thermofisher, catalog number: XP293) ) Express the chimeric antibody molecule in the expression system to obtain the chimeric antibody CHI-16.
  • Eculizumab (ECU, its light chain amino acid sequence SEQ ID NO: 55, heavy chain amino acid sequence SEQ ID NO: 56) produced under the same conditions, Ravulizumab (RAV, its light chain amino acid sequence SEQ ID NO: 55 , The heavy chain amino acid sequence of SEQ ID NO: 57), SKY59 (the light chain amino acid sequence of SEQ ID NO: 58, and the heavy chain amino acid sequence of SEQ ID NO: 59) are positive control antibodies.
  • Enzyme-labeled plates purchased from Costar, catalog number: 42592 were coated with antibodies Eculizumab (ECU), Ravulizumab (RAV), SKY59, and CHI-16, respectively, overnight at 4°C. The coating solution was discarded, and each well was blocked with 2.5% skimmed milk dissolved in phosphate buffered saline (PBS) for 1 hour, and the wells were washed with PBST solution (20% Tween in PBS).
  • ECU Eculizumab
  • RAV Ravulizumab
  • SKY59 SKY59
  • CHI-16 CHI-16
  • sheep red blood cells purchased from Bio-channel, item number: BC-RBC-S003
  • GVB++ buffer including Mg++ and Ca++, purchased from Tianenze, item number: 25-02080
  • Hemolysin (1:2000, rabbit anti-sheep red blood cell antibody) purchased from Beijing Boerxi Technology, Item: BM351Y
  • the activated sheep red blood cells were washed twice with GVB++ buffer, and diluted to 30 ⁇ l (5x106 cells) with GVB++ buffer after centrifugation.
  • inhibition rate (%) 100 ⁇ [ (OD without antibody sample reading-OD serum background value)-(OD with antibody sample reading-OD serum background value)]/(OD without antibody sample reading-OD serum background value).
  • the 32D and 32D of the light chain CDR1 region of the CHI-16 humanized antibody 16HDa 33G carries out multiple single-point mutations of amino acids, and screens out the mutant amino acids that have the least impact on antigen binding and yield.
  • the D32E, D32I, D32L, D32G, D32S, D32T, D32V, D32Y single-point mutation vectors were constructed for the 32D site in the 16HDa light chain variable region, and the G33A, D32I, D32L, D32G, D32S, D32T, D32V, and D32Y sites were respectively constructed for the 33G site in the 16HDa light chain variable region.
  • G33S, G33I, G33Q, G33T, G33V single-point mutation vectors using 293 cells to produce D32 single-point mutation humanized antibodies in a 24-well plate, the unmutated humanized antibody 16HDa expressed under the same conditions as a control, take The supernatant of the culture medium was preliminarily quantified by ELISA competition method to detect the yield of mutant antibody and control antibody (Titer). The method is as follows. The diluted supernatant to be tested and the HRP-labeled anti-human IgG as the detection antibody are mixed in equal volume and then added to an ELISA plate coated with 1ug/ml CHI-16, and incubated at room temperature for 1 hour.
  • the chimeric antibody CHI-16 was humanized, and finally two different humanized antibodies were obtained: 16H1L2m and 16H46L39am.
  • the 16H1L2m heavy chain nucleotide sequence is shown in SEQ ID NO: 66
  • the 16H1L2m light chain nucleotide sequence is shown in SEQ ID NO: 65
  • the 16H1L2m heavy chain variable region nucleotide sequence is shown in SEQ ID NO: 61
  • the nucleotide sequence of the 16H1L2m light chain variable region is shown in SEQ ID NO: 63.
  • the nucleotide sequence of the heavy chain of 16H46L39am is shown in SEQ ID NO: 68
  • the nucleotide sequence of the light chain of 16H46L39am is shown in SEQ ID NO: 67
  • the nucleotide sequence of the heavy chain variable region of 16H46L39am is shown in SEQ ID NO: 62
  • the nucleotide sequence of the light chain variable region of 16H46L39am is shown in SEQ ID NO: 64.
  • the murine heavy chain variable region and light chain variable region gene sequences were cloned from mouse hybridoma cells, and the human IgG1 constant region without ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation was selected.
  • Gene sequence (SEQ ID NO: 48) the humanized antibody 16H1L2m and 16H46L39am heavy chain variable region gene and human IgG1 constant region gene are connected to form a chimeric gene, and the light chain variable region gene is connected to the human Kappa light chain constant region (As shown in SEQ ID NO: 47) ligated to form a chimeric gene, and then inserted into the transient expression vector pcDNA3.1, and 100 mL of 293 cells were used to transiently produce humanized antibody proteins to obtain antibody proteins 16H1L2m and 16H46L39am.
  • Eculizumab (ECU), Ravulizumab (RAV), SKY59 and chimeric antibody CHI-16 produced under the same conditions were used
  • the in vitro hemolysis inhibition test was used to detect the inhibitory activity of anti-human C5 humanized antibodies in the classical pathway (CP) and the alternative pathway (AP) on complement activation.
  • step 4 The method of the classical pathway (CP) in vitro hemolysis inhibition experiment is shown in step 4) of Example 2.
  • the antibody 16H1L2m and 16H46L39am prepared by GVB++ is a gradient concentration dilution solution (antibody dilution concentration initial concentration 2ug/ml (Final concentration), 2-fold dilution, 6 dilution gradients).
  • the method of the alternative pathway (AP) in vitro hemolysis inhibition experiment is as follows. Normal rabbit red blood cells (purchased from Bio-channel, catalog number: BC-RBC-RAB003) are mixed with EGTA to inhibit the classical pathway of complement activation. GVB Mg+EGTA ( Tianenze/25-02090) normal human serum (NHS) with a final concentration of 5% and gradient dilutions of each antibody (the initial concentration (final concentration) of the antibody is 200ug/ml, 4-fold dilution, 6 gradients, rabbit red blood cells The quantity is 5 ⁇ 10 6 , wash three times with GVB Mg+EGTA before adding) in the well plate that has been incubated, and incubate at room temperature for 1 hour.
  • Inhibition rate (%) 100X[(OD without antibody sample reading -OD serum background value)-(OD with antibody sample reading-OD serum background value)]/(OD without antibody sample reading-OD serum background value).
  • the ELISA plate (purchased from Corning, catalog number: 42592) was coated with the chimeric antibody CHI-16, overnight at 4°C. The coating solution was discarded, the wells were blocked with 2.5% skimmed milk dissolved in phosphate buffered saline (PBS) for 1 hour, and the wells were washed with PBST solution (20% Tween in PBS).
  • PBS phosphate buffered saline
  • the antibodies to be tested were divided into three groups, and each group was treated at room temperature (37°C), 66°C, and 67.5°C for 1 hour. The antibodies treated at different temperatures were diluted to 2000ng/ml, 500ng/ml, and 500ng/ml respectively.
  • the ELISA plate (purchased from Corning, catalog number: 42592) was coated with each antibody (16H1L2m, 16H46L39am, ECU and RAV), overnight at 4°C. The coating solution was discarded, and each well was blocked with 2.5% skimmed milk dissolved in phosphate buffered saline (PBS) for 1 hour, and the wells were washed with PBST solution (20% Tween in PBS).
  • PBS phosphate buffered saline
  • the results are shown in Figure 8.
  • the EC 50 can evaluate the concentration of the antigen used when each antibody binds to the antigen at 50% .
  • the lower the EC 50 value the stronger the binding capacity of the antibody to the human C5 antigen.
  • the results are shown in Figure 8.
  • the binding of 16H1L2m and 16H46L39am to the antigen Power is stronger than ECU and RAV.
  • the EC 50 value is shown in Table 5.
  • the molecular interaction analyzer (ForteBio Octet) was used to determine the binding affinity constants of the control antibody ECU and the humanized antibodies 16H1L2m and 16H46L39am with the antigen using the biofilm interference technology (BLI). Analyze the affinity analysis of the kinetic fitting curve and the measurement results of the kinetic parameters of each channel, and calculate the affinity constants of each candidate-derived antibody and ECU (see Table 6).
  • K a association rate constant
  • K d dissociation rate constant
  • K D affinity constant equal to K d / K a.

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Abstract

提供了一种分离的抗原结合蛋白，其包含重链可变区中的至少一个CDR，所述重链可变区包含如SEQ ID NO:44-46所示的氨基酸序列或其变体；且其包含轻链可变区中的至少一个CDR，所述轻链可变区包含如SEQ ID NO:41-43所示的氨基酸序列或其变体。

Description

分离的抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种分离的抗原结合蛋白及其用途。
背景技术
补体系统在先天免疫应答中起到关键作用，补体系统由超过25种血浆蛋白组成，这些血浆蛋白通过三种目前已知的激活途径起作用：经典途径、凝集素途径和旁路途径。虽然这些途径的初始步骤有所不同，但具有相同的负责激活和摧毁靶细胞的晚期补体组分(C5至C9)。
功能正常的补体系统能够对微生物感染提供强有力的防御，但补体的失调也会导致多种病症的出现。例如，阵发性夜间血红蛋白尿(PNH)，该病源于造血干细胞PIG-A基因突变引起一组通过糖肌醇磷脂(glycosylphosphatidylinositol，GPI)锚连在细胞表面的膜蛋白的缺失，从而导致细胞性能发生变化。其临床表现除了补体介导的慢性血管内溶血外，还包括骨髓衰竭和血栓。大规模的临床流行病学调查结果显示，PNH患者在诊断后5年内死亡率35％，10年内死亡率为50％。血栓事件是导致欧美PNH患者死亡的主要原因，约占40-67％。
PNH的治疗主要有两种方式，基于症状的对症治疗和造血干细胞移植。后者仅用于伴有再生障碍性贫血的严重PNH患者或者已经进展为白血病的患者。目前经FDA批准用于治疗PNH的药物有Eculizumab(依库珠单抗，商品名
Figure PCTCN2021098261-appb-000001
Soliris)和Ravulizumab-cwvz(商品名Ultomiris)，然而其治疗效果有待于进一步完善改进。因此，研究开发其他的治疗药物仍然十分必要。
发明内容
本申请提供了一种分离的抗原结合蛋白，其包含重链可变区中的至少一个CDR，所述重链可变区包含如SEQ ID NO:44-46中任一项所示的氨基酸序列或其变体；且其包含轻链可变区中的至少一个CDR，所述轻链可变区包含如SEQ ID NO:70所示的氨基酸序列或其变体。
在某些实施方式中，所述如SEQ ID NO:70所示的氨基酸序列变体包含选自如下组的氨基酸突变：D32E、D32I、D32L、D32G、D32S、D32T、D32V、D32Y、G33A、G33S、G33I、G33Q、G33T和G33V。
在某些实施方式中，所述分离的抗原结合蛋白包含氨基酸序列如SEQ ID NO:44-46中任一项所示的重链可变区中的HCDR1。
在某些实施方式中，所述分离的抗原结合蛋白包含氨基酸序列如SEQ ID NO:44-46中任一项所示的重链可变区中的HCDR2。
在某些实施方式中，所述分离的抗原结合蛋白包含氨基酸序列如SEQ ID NO:44-46中任一项所示的重链可变区中的HCDR3。
在某些实施方式中，所述HCDR1包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:20所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含氨基酸序列如SEQ ID NO:41-43中任一项所示的轻链可变区中的LCDR1。
在某些实施方式中，所述分离的抗原结合蛋白包含氨基酸序列如SEQ ID NO:41-43中任一项所示的轻链可变区中的LCDR2。
在某些实施方式中，所述分离的抗原结合蛋白包含氨基酸序列如SEQ ID NO:41-43中任一项所示的轻链可变区中的LCDR3。
在某些实施方式中，所述LCDR1包含SEQ ID NO:69所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NO:1和2中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述轻链可变区包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
在某些实施方式中，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:21-23中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR1包含SEQ ID NO:21-23中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:24-25中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR2包含SEQ ID NO:24-25中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包 含SEQ ID NO:26-28中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR3包含SEQ ID NO:26-28中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR4包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
在某些实施方式中，所述轻链可变区包含SEQ ID NO:41-43中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Kappa轻链恒定区。
在某些实施方式中，所述抗体轻链恒定区包含SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链，且所述抗体轻链包含SEQ ID NO:49、51和53中任一项所示的氨基酸序列。
在某些实施方式中，所述重链可变区包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在某些实施方式中，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:31-33中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR1包含SEQ ID NO:31-33中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:34-35中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR2包含SEQ ID NO:34-35中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:36-38中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR3包含SEQ ID NO:36-38中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:39-40中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR4包含SEQ ID NO:39-40中任一项所示的氨基酸序列。
在某些实施方式中，所述重链可变区包含SEQ ID NO:44-46中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区源自人IgG重链恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区源自人IgG1重链恒定区。
在某些实施方式中，所述抗体重链恒定区包含SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链，且所述抗体重链包含SEQ ID NO:50、52和54中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原为C5。
在某些实施方式中，所述C5为人C5。
另一方面，本申请还提供了分离的核酸分子，其编码本申请所述的分离的抗原结合蛋白。
另一方面，本申请还提供了载体，其包含本申请所述的核酸分子。
另一方面，本申请还提供了细胞，其包含本申请所述的核酸分子或本申请所述的载体。
另一方面，本申请还提供了制备本申请所述的分离的抗原结合蛋白的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。
另一方面，本申请还提供了药物组合物，其包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。
另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗C5相关的疾病或病症。
另一方面，本申请还提供了预防、缓解或治疗C5相关的疾病或病症的方法，所述方法包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请还提供了所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其用于预防、缓解或治疗C5相关的疾病或病症的方法。
另一方面，本申请还提供了检测样品中C5的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的 示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述的嵌合抗体与阳性对照抗体的抗原结合力实验结果图。
图2显示的是本申请所述的嵌合抗体和阳性对照抗体生溶血抑制实验结果图。
图3显示的是本申请所述的嵌合抗体位点突变后的产量变化的实验结果图。
图4显示的是本申请所述的嵌合抗体位点突变后的抗原结合力变化实验结果图。
图5显示的是本申请所述的人源化抗体和阳性对照抗体经典途径(CP)溶血抑制实验结果图。
图6显示的是本申请所述的人源化抗体和阳性对照抗体旁路途径(AP)溶血抑制实验结果图。
图7显示的是本申请所述的人源化抗体和阳性对照抗体热稳定性实验结果图。
图8显示的是本申请所述的人源化抗体与人C5抗原的结合实验结果图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的或者人工合成的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的核酸分子”通常是指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“变体”通常是指在原蛋白序列上进行氨基酸修饰(例如基团取代等)或者进行一个或多个氨基酸的插入、取代、和/或缺失，而保留原序列功能的分子。例如，所述变体可以比原序列具有更好的生物活性(或功能)。例如，所述保留不必是完全保留。例如，所述变体可以基本保留原序列功能，例如，保留原序列至少50％、60％、65％、70％、75％、 80％、85％、90％、95％、96％、97％、98％，或99％的功能。例如，所述变体的氨基酸序列可以与原氨基酸序列至少70％，75％，80％，85％，90％，95％，96％，97％，98％，或99％相同。例如，所述如SEQ ID NO:69所示的氨基酸序列变体可以包括在如SEQ ID NO:1所示的氨基酸序列上进行氨基酸修饰(例如基团取代等)或者进行一个或多个氨基酸的插入、取代、和/或缺失，而保留原序列功能的分子。例如，可以基本保留如SEQ ID NO:1所示的氨基酸序列功能，例如，保留如SEQ ID NO:1所示的氨基酸序列至少50％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％，或99％的功能。例如，可以与如SEQ ID NO:1所示的氨基酸序列至少70％，75％，80％，85％，90％，95％，96％，97％，98％，或99％相同。在本申请中，同一性，例如可以通过EMBOSS软件包(EMBOSS：欧洲分子生物学开放软件套组，Rice等，2000，Trends in Genetics 16：276-277)的Needle程序，3.0.0版或更高版本中执行的Needleman-Wunsch算法(Needleman和Wunsch，1970，J.Mol.Biol.48：443-453)来确定。使用的任选参数为缺口罚分(gap penalty)10，缺口延伸罚分(gap extension penalty)0.5和EBLOSUM62取代矩阵(BLOSUM62的EMBOSS版)。使用Needle标记为“最长同一性(longest identity)”(使用nobrief选项获得)的输出结果作为百分比同一性，并计算如下：(相同的残基×100)/(比对的长度-在比对中的空位总数)。
在本申请中，术语“分离的抗原结合蛋白”通常指从天然状态下经人工手段获得或者人工合成的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的支架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白支架或具有移植的CDR或CDR衍生物的备选蛋白支架或人工支架。此类支架包括但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的支架，以及包含例如生物相容性聚合物的完全合成的支架。参见例如Korndorfer等,2003,Proteins:Structure,Function,andBioinformatics,53(1):121-129(2003)；Roque等,Biotechnol.Prog.20:639-654(2004)。此外，还可以包括肽抗体模拟物("PAMs")以及利用纤连蛋白组分基于抗体模拟物的支架可以用作支架。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本申请的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合人C5)的其它抗体片段。通常，这样的片段应当包括抗原结合结 构域。例如，基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。例如，IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般可以为约150,000道尔顿。每个L链可以通过共价二硫键与H链连接，而两个H链可以通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还可以具有间隔的链内二硫化桥键。每个H链在N末端可以具有可变结构域/可变区(VH)，对于α和γ链各自继之以三个恒定结构域/恒定区(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端可以具有可变结构域/可变区(VL)，在其另一端可以具有恒定结构域/恒定区(CL)。在某些实施方式中，VL与VH对应，CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为可以在轻链和重链可变结构域之间形成界面。VH和VL可以配对一起形成抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton & Lange,Norwalk,Conn.,1994，第71页和第6章。在某些实施方式中，来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。取决于其重链恒定结构域/恒定区(CH)的氨基酸序列，还可以将免疫球蛋白分为不同的类别或种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人包含有下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。抗原结合片段。
在本申请中，术语“抗原结合片段”通常是指全长抗体的一部分(例如靶结合区或可变区)或其功能片段或类似物。抗体片段的实例可以包括Fab，Fab'，F(ab')2和Fv片段等。“功能片段或类似物”通常是指与全长抗体具有基本一样的定性生物活性的化合物。例如，抗C5抗体的功能片段或类似物是可以与C5蛋白结合以便可抑制经典途径和/或旁路途径的溶血现象。“Fv”片段通常是最小的抗体片段，其包含一完整的靶识别和结合位点。该区域可以由一个重链可变结构域与一个轻链可变结构域呈非共价联接的二聚体(VH—VL二聚体)组成。
在本申请中，术语“框架区”通常是指本领域所知的抗体可变区中存在于差异性更高的(即高变)CDR之间的部分。通常情况下，此类框架区典型地称为框架1至4(FR1、FR2、FR3和FR4)且提供用于在三维空间中呈现六个CDR(三个来自重链且三个来自轻链)的骨架，以形成抗原结合表面。即在VH中四个(H-FR1，H-FR2，H-FR3，和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3，和L-FR4)。例如，本申请所述的分离的抗原结合蛋白的VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。本申请所述的分离的抗原结合蛋白的VH可以 包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在本申请中，术语“可变”通常指抗体可变区(例如，重链可变区，轻链可变区)的某些区段在抗体之间序列上存在差异的事实。通常情况下，可变区介导抗原结合并决定特定抗体对其特定抗原的特异性。通常情况下，可变性并非在整个可变结构域范围内均匀分布，而是集中在轻链和重链可变结构域中称为互补决定区(CDR)或高变区(HVR)的若干区段中。可变区相对保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自可以包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR紧密靠近地保持在一起，可以与来自于另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。通常恒定区不直接参与抗体与抗原的结合，但显示出各种效应子功能，例如抗体参与抗体依赖性细胞毒性。
在本申请中，术语“轻链可变区”通常指抗体轻链的氨基末端结构域。轻链可变区可以称为“VL”。这些结构域通常是抗体轻链中变化最大的部分(相对于相同类型的其它抗体)，可以包括互补决定区(CDR)或高变区(HVR)和框架区(FR)。
在本申请中，术语“重链可变区”通常指抗体重链的氨基末端结构域。重链可变区可以称为“VH”。这些结构域通常是抗体重链中变化最大的部分(相对于相同类型的其它抗体)，可以包括互补决定区(CDR)或高变区(HVR)和框架区(FR)。
在本申请中，术语“受试者”和“患者”可互换使用，通常是指哺乳动物，如人患者和非人灵长类，以及实验用动物，如兔、大鼠和小鼠，以及其他动物。动物包括所有的脊椎动物，例如，哺乳动物和非哺乳动物，如狗、猫、绵羊、牛、猪、兔、鸡等。例如，用于实施本沈琪的治疗方法的受试者是人。需要治疗的受试者包括已经患有与C5相关的疾病或病症的患者以及倾向于发展此类病症的人。
本申请中，术语“预防”通常是指对健康患者预防性地施用组合，以预防本申请所述疾病和病症的爆发。此外，术语“预防”指对处于待治疗变应性疾病前期的患者预防性地施用这种组合。术语“预防”不需要100％消除事件的可能性。更准确地说，它表示在所述药物组合物或方法存在下事件发生的可能性降低了。
在本申请中，术语“治疗”通常是指将治疗剂施加或给予患者，或将治疗剂施加或给予由患者分离的组织或细胞系，该患者患有疾病、具有疾病症状或疾病倾向，其目的是治疗、治愈、减轻、缓解、改变、补救、改善、提高或影响该疾病、疾病症状或疾病倾向。可以包括改善病状态、消除病灶或改善的预后。
在本申请中，术语“细胞”通常可以是或已经是核酸分子或载体的接受者的单个细胞、细胞系或细胞培养物。细胞可以包括本申请所述的核酸分子或本申请所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或骨髓瘤细胞。在某些实施方案中，细胞为哺乳动物细胞。在某些实施方案中，哺乳动物细胞为HEK293T细胞。
在本申请中，术语“药物组合物”通常指涉及适合施用于患者、例如人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。例如，组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的佐剂”通常指与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂等。通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“直接或间接相连”通常是指相对的“直接相连”或“间接相连”。“直接相连”通常是指直接连接。例如，所述直接相连可以为相连的物质(例如氨基酸序列区段)之间没有间隔成分(例如氨基酸残基或其衍生物)而直接相连接的情况；例如氨基酸序列区段X 与另一氨基酸序列区段Y通过氨基酸序列区段X的C端氨基酸与氨基酸序列区段Y的N端氨基酸形成的酰胺键直接连接。“间接相连”通常是指相连的物质(例如氨基酸序列区段)之间有间隔成分(例如氨基酸残基或其衍生物)而间接相连接的情况例如，在本申请所述的分离的抗原结合蛋白中，所述L-FR1的C末端与所述LCDR1的N末端可以直接或间接相连。
在本申请中，术语“互补决定区”或术语“CDR”通常是指共同界定了抗原结合蛋白(例如，天然免疫球蛋白，嵌合抗体或人源化抗体)结合位点的可变区域的结合亲和力和特异性的氨基酸序列(参见例如，Chothia等，J.Mol.Biol.196：901-917(1987)；Kabat等，美国健康和人类服务部NIH出版物(U.S.Deptof Health and Human Services NIH Publication)No.913242(1991))。通常，抗体包括六个CDR；VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下也能够保持功能正常且稳定。参见，例如,Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。
在本申请中，术语“C5”，也称为“补体组分5”或“补体因子5”通常是指补体级联的血清蛋白或其变体。C5蛋白可以包含两条链α和β。该蛋白表示参与三条补体激活途径的集中点：经典途径、旁路途径和甘露糖结合凝集素途径。全长C5蛋白的氨基酸序列可通过GenBank中作为登录号NP_001726.2中提供的氨基酸序列来举例说明。术语“C5”可以包括重组C5蛋白或其片段。该术语还可以包括结合例如组氨酸标签、鼠或人Fc或信号序列(如ROR1)的C5蛋白或其片段。例如，该术语可以包括通过SEQ ID NO:60中所示的序列举例说明的序列。
在本申请中，术语“C5相关的疾病或病症”通常是指可以通过干预C5蛋白而实现治疗或缓解目的任何疾病或病症。例如，补体-相关的疾病包括炎症性疾病和自身免疫疾病，局部缺血和再灌注之后的远端组织损伤，心肺旁路手术期间的补体激活，皮肌炎，天疱疮，狼疮肾炎，肾小球肾炎，肾血管炎，心肺旁路、心力衰竭诱导的冠状内皮功能障碍，II型膜增生性肾小球肾炎，急性肾衰竭，抗磷脂综合征，黄斑变性，眼内炎，新型血管疾病，同种异体移植物移植，超急性排斥，血液透析，慢性阻塞性肺病(COPD)呼吸窘迫综合征，哮喘，阵发性睡眠性血红蛋白尿(PNH)和吸入性肺炎等。
在本申请中，术语“KD”，也称作“K D”、“亲和常数”或“平衡解离常数”，通常是指在滴定测量中在平衡时、或者通过将解离速率常数(kd)除以结合速率常数(K a)所获得的值。使用结合速率常数(K a)、解离速率常数(K d)和平衡解离常数(K D)表示结合蛋白(例如本申请所述的分离的抗原结合蛋白)对抗原(例如人C5)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术提供了高灵敏度以及在生理缓冲液中在平衡时 检查样品的能力。例如，可以通过Octet测定所述K D值，也可以使用其他实验途径和仪器例如BIAcore(生物分子相互作用分析)测定。另外，电化学发光分析-溶液平衡滴定法(MSD-SET)测定所述K D值。测量方法如Estep P.等人,MAbs,2013.5(2):p.270-8所述。
在本申请中，术语“包含”或“包括”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供一种抗原结合蛋白。所述抗原结合蛋白包含轻链可变区VL中的至少一个CDR。
在本申请中，所述抗原结合蛋白可包含LCDR1，且所述LCDR1可包含SEQ ID NO:69所示的氨基酸序列：
KASQSVDYX 9X 10DSYLN(SEQ ID NO:69)：其中，X 9＝D，E，I，L，G，S，T，V或Y；X 10＝G，A，S，I，Q，T或V。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:1所示抗原结合蛋白的LCDR1相比，所述LCDR1可至少包含在选自以下位置处的氨基酸取代：在X 9和/或X 10处的氨基酸取代。
在某些情形中，与SEQ ID NO:1所示抗原结合蛋白的LCDR1相比，所述LCDR1可至少包含在X 9和/或X 10处的氨基酸取代，其中X 9处的氨基酸可被取代为D，E，I，L，G，S，T，V或Y；X 10处的氨基酸可被取代为G，A，S，I，Q，T或V。
例如，所述LCDR1可包含SEQ ID NO:1-15中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR2，且所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR3，且所述LCDR3可包含SEQ ID NO:17中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1可包含SEQ ID NO:21-23中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2可包含SEQ ID NO:24-25中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3可包含SEQ ID NO:26-28中任一项所示的氨基酸序列。
本申请中，所述抗原结合蛋白可包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4可包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链可变区VL，且所述VL可包含SEQ ID NO:41-43中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链恒定区CL，且所述CL可包含人Kappa轻链恒定区。例如，所述CL可包含SEQ ID NO:47所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链，且所述轻链可包含SEQ ID NO:49、51和53中任一项所示的氨基酸序列。
所述抗原结合蛋白包含重链可变区VH中的至少一个CDR。
在本申请中，所述抗原结合蛋白可包含HCDR1，且所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR2，且所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR3，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可包含SEQ ID NO:31-33中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可包含SEQ ID NO:34-35中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可包含SEQ ID NO:36-38中任一项所示的氨基酸序列。
本申请中，所述抗原结合蛋白可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4可包含SEQ ID NO:39-40中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含重链可变区VH，且所述VH可包含SEQ ID NO:44-46中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含重链恒定区CH，且所述CH可包含人IgG重链恒定区。在某些情形中，所述IgG重链恒定区可包含人IgG1重链恒定区。例如，所述CH可包 含SEQ ID NO:48所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含重链，且所述重链可包含SEQ ID NO:50、52和54中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1-3，所述LCDR1可包含SEQ ID NO:69所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1-3，所述LCDR1可包含SEQ ID NO:1-15中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与CHI-16相同的LCDR1-3，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与16H1L2m和16H46L39am相同的LCDR1-3，所述LCDR1可包含SEQ ID NO:2所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR1-4，所述L-FR1可包含SEQ ID NO:21-23中任一项所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:24-25中任一项所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:26-28中任一项所示的氨基酸序列，且所述L-FR4可包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与CHI-16相同的L-FR1-4，所述L-FR1可包含SEQ ID NO:21所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:24所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:26所示的氨基酸序列，且所述L-FR4可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与16H1L2m相同的L-FR1-4，所述L-FR1可包含SEQ ID NO:22所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:24所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:27所示的氨基酸序列，且所述L-FR4可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与16H46L39am相同的L-FR1-4，所述L-FR1可包含SEQ ID NO:23所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:25所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:28所示的氨基酸序列，且所述L-FR4可包含SEQ ID  NO:30所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1-3，所述HCDR1可包含SEQ ID NO:18中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20中任一项所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与CHI-16、16H1L2m和16H46L39am相同的HCDR1-3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR1-4，所述H-FR1可包含SEQ ID NO:31-33中任一项所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:34-35中任一项所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:36-38中任一项所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:39-40中任一项所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与CHI-16相同的H-FR1-4，所述H-FR1可包含SEQ ID NO:31所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:34所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:36所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:39所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与16H1L2m相同的H-FR1-4，所述H-FR1可包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:37所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:40所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与16H46L39am相同的H-FR1-4，所述H-FR1可包含SEQ ID NO:33所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:38所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:40所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1-3和HCDR1-3，所述LCDR1可包含SEQ ID NO:69所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1-3和HCDR1-3，所述LCDR1可包含SEQ ID NO:1-15中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸 序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与CHI-16相同的LCDR1-3和HCDR1-3，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与16H1L2m和16H46L39am相同的LCDR1-3和HCDR1-3，所述LCDR1可包含SEQ ID NO:2所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链可变区VL和重链可变区VH，所述VL可包含SEQ ID NO:41-43中任一项所示的氨基酸序列，且所述VH可包含SEQ ID NO:44-46中任一项所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与CHI-16相同的轻链可变区VL和重链可变区VH，所述VL可包含SEQ ID NO:41所示的氨基酸序列，且所述VH可包含SEQ ID NO:44所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与16H1L2m相同的轻链可变区VL和重链可变区VH，所述VL可包含SEQ ID NO:42所示的氨基酸序列，且所述VH可包含SEQ ID NO:45所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与16H46L39am相同的轻链可变区VL和重链可变区VH，所述VL可包含SEQ ID NO:43所示的氨基酸序列，且所述VH可包含SEQ ID NO:46所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链恒定区CL和重链恒定区CH，所述CL可包含SEQ ID NO:47所示的氨基酸序列，且所述CH可包含SEQ ID NO:48所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与CHI-16、16H1L2m和16H46L39am相同的轻链恒定区CL和重链恒定区CH，所述CL可包含SEQ ID NO:47所示的氨基酸序列，且所述CH可包含SEQ ID NO:48所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链和重链，所述轻链可包含49、51和53中任一项所示的氨基酸序列，且所述重链可包含SEQ ID NO:50、52和54中任一项所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与CHI-16相同的轻链和重链，所述轻链可包含SEQ ID NO:49所示的氨基酸序列，且所述重链可包含SEQ ID NO:50所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与16H1L2m相同的轻链和重链，所述轻链可包含SEQ ID NO:51所示的氨基酸序列，且所述重链可包含SEQ ID NO:52所示的氨基酸序列。
例如，所述抗原结合蛋白可包含与16H46L39am相同的轻链和重链，所述轻链可包含SEQ ID NO:53所示的氨基酸序列，且所述重链可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述的抗原结合蛋白可包含LCDR1-3和L-FR1-4，所述LCDR1可包含SEQ ID NO:1所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:21所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:24所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:26所示的氨基酸序列，且所述L-FR4可包含SEQ ID NO:29所示的氨基酸序列；所述的抗原结合蛋白还可包含VL和CL，所述VL可包含SEQ ID NO:41所示的氨基酸序列，且所述CL可包含SEQ ID NO:47所示的氨基酸序列；所述的抗原结合蛋白还可包含HCDR1-3和H-FR1-4，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:31所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:34所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:36所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:39所示的氨基酸序列；所述的抗原结合蛋白还可包含VH和CH，所述VH可包含SEQ ID NO:44所示的氨基酸序列，且所述CH可包含SEQ ID NO:48所示的氨基酸序列；且所述抗原结合蛋白还可包含轻链和重链，所述轻链可包含SEQ ID NO:49所示的氨基酸序列，且所述重链可包含SEQ ID NO:50所示的氨基酸序列。例如，所述的抗原结合蛋白可包含与CHI-16相同的抗体轻链和重链。
在本申请中，所述的抗原结合蛋白可包含LCDR1-3和L-FR1-4，所述LCDR1可包含SEQ ID NO:2所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:22所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:24所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:27所示的氨基酸序列，且所述L-FR4可包含SEQ ID NO:30所示的氨基酸序列；所述 的抗原结合蛋白还可包含VL和CL，所述VL可包含SEQ ID NO:42所示的氨基酸序列，且所述CL可包含SEQ ID NO:47所示的氨基酸序列；所述的抗原结合蛋白还可包含HCDR1-3和H-FR1-4，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:32所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:37所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:40所示的氨基酸序列；所述的抗原结合蛋白还可包含VH和CH，所述VH可包含SEQ ID NO:45所示的氨基酸序列，且所述CH可包含SEQ ID NO:48所示的氨基酸序列；且所述抗原结合蛋白还可包含轻链和重链，所述轻链可包含SEQ ID NO:51所示的氨基酸序列，且所述重链可包含SEQ ID NO:52所示的氨基酸序列。例如，所述的抗原结合蛋白可包含与16H1L2m相同的抗体轻链和重链。
在本申请中，所述的抗原结合蛋白可包含LCDR1-3和L-FR1-4，所述LCDR1可包含SEQ ID NO:2所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:23所示的氨基酸序列，所述L-FR2可包含SEQ ID NO:25所示的氨基酸序列，所述L-FR3可包含SEQ ID NO:28所示的氨基酸序列，且所述L-FR4可包含SEQ ID NO:30所示的氨基酸序列；所述的抗原结合蛋白还可包含VL和CL，所述VL可包含SEQ ID NO:43所示的氨基酸序列，且所述CL可包含SEQ ID NO:47所示的氨基酸序列；所述的抗原结合蛋白还可包含HCDR1-3和H-FR1-4，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:33所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:38所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:40所示的氨基酸序列；所述的抗原结合蛋白还可包含VH和CH，所述VH可包含SEQ ID NO:46所示的氨基酸序列，且所述CH可包含SEQ ID NO:48所示的氨基酸序列；且所述抗原结合蛋白还可包含轻链和重链，所述轻链可包含SEQ ID NO:53所示的氨基酸序列，且所述重链可包含SEQ ID NO:54所示的氨基酸序列。例如，所述的抗原结合蛋白可包含与16H46L39am相同的抗体轻链和重链。
参比抗体
本申请提供的抗原结合蛋白，其可与参比抗体竞争结合C5。
在本申请中，所述参比抗体可包含LCDR1-3，所述LCDR1可包含SEQ ID NO:69所示 的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列；某些情形中，本申请所述抗原结合蛋白可包含LCDR1-3，所述LCDR1可包含SEQ ID NO:1-15中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述参比抗体可包含HCDR1-3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述参比抗体可包含LCDR1-3和HCDR1-3，所述LCDR1可包含SEQ ID NO:69所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列；在某些情形中，本申请所述抗原结合蛋白可包含LCDR1-3和HCDR1-3，所述LCDR1可包含SEQ ID NO:1-15中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述参比抗体可包含轻链可变区VL和重链可变区VH，所述VL可包含SEQ ID NO:41-43中任一项所示的氨基酸序列，且所述VH可包含SEQ ID NO:44-46中任一项所示的氨基酸序列。
免疫偶联物
另一方面，本申请还提供了免疫偶联物。在本申请中，所述分离的抗原结合蛋白可以与其他化合物(例如治疗剂)交联，形成所述免疫偶联物。例如抗体-药物偶联物(ADC)。合适的治疗剂包括细胞毒素、烷化剂、DNA小沟结合分子、DNA嵌入剂、DNA交联剂、组蛋白去乙酰化酶抑制剂、核输出抑制剂、蛋白酶体抑制剂、拓扑异构酶I或II的抑制剂、热激蛋白抑制剂、酪氨酸激酶抑制剂、抗生素和抗有丝分裂剂，例如SN-38。在ADC中，抗体和治疗剂可以通过接头交联，该接头可切割，例如肽类接头、二硫类接头或腙类接头。例如，接头可以是肽类接头，例如Val-Cit、Ala-Val、Val-Ala-Val、Lys-Lys、Pro-Val-Gly-Val-Val、Ala-Asn-Val、Val-Leu-Lys、Ala-Ala-Asn、Cit-Cit、Val-Lys、Lys、Cit、Ser或Glu。ADC可以如美 国专利7,087,600；6,989,452；和7,129,261；PCT公开WO 02/096910；WO 07/038,658；WO 07/051,081；WO 07/059,404；WO 08/083,312；和WO 08/103,693；美国专利公开20060024317；20060004081；和20060247295中描述般进行制备。
核酸
另一方面，本申请还提供了分离的核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。本申请所述的分离的核酸分子可以为任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物，其可以编码本申请所述的分离的抗原结合蛋白。
载体
另一方面，本申请还提供了载体，其可以包含本申请所述的核酸分子。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
细胞
另一方面，本申请还提供了细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。
例如，所述细胞可以包括原核细胞、酵母菌细胞或高等真核细胞。适用于此目的的原核生物包括格兰氏阴性菌和格兰氏阳性菌，例如，肠细菌，例如大肠杆菌、肠杆菌、欧文氏菌(Erwinia)、克雷臼氏杆菌(Klebsiella)、变形菌(Proteus)、沙门氏菌(Salmonella)、粘质沙雷氏菌(Serratia)、和志贺氏杆菌(Shigella),以及杆菌、假单胞菌和链霉菌。
例如，丝状真菌或酵母菌等真核微生物也可以作为本申请所示载体的适宜克隆或表达宿主。在低等真核宿主微生物中最常使用的是酿酒酵母。多种其他属、种和菌株也是通常可得到并可用于本申请中，例如，稷酒裂殖酵母菌(Schizosaccharomycespombe)，克鲁维酵母属， 念珠菌属，木霉属，粗糙链抱霉(Neurospora crassa)，及丝状真菌，例如，脉抱菌(Neurospora)、青霉菌(Penicillium)、弯颈霉(To1ypocladium)和曲霉(aspergi 11us)宿主细胞，例如，构巢曲菌(A.nidulans)和黑曲霉菌(A.niger)。
例如，脊椎动物细胞及在培养物(组织培养物)中增殖的脊椎动物细胞本申请所示载体的适宜克隆或表达宿主。
例如，所述细胞可以包括哺乳动物宿主细胞系。例如，猴肾脏细胞，人类胚胎肾脏细胞系，幼小仓鼠肾脏细胞，中国仓鼠卵巢细胞小鼠塞尔托利(sertoli)细胞，人类子宫颈癌细胞(HELA)，犬肾脏细胞，人类肺细胞，人类肝脏细胞，小鼠乳腺癌细胞或NSO细胞。
例如，所述细胞还可以包括用本申请所述的载体在体外转染的细胞。例如，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或骨髓瘤细胞。在某些实施方案中，所述细胞可以为哺乳动物细胞。例如，所述哺乳动物细胞可以为HEK293细胞。
制备
另一方面，本申请还提供了制备本申请所述的分离的抗原结合蛋白的方法，所述方法可以包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。
预防、缓解和/或治疗
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗C5相关的疾病或病症。
另一方面，本申请还提供了预防、缓解或治疗C5相关的疾病或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
例如，所述施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其可以用于预防、缓解或治疗C5相关的疾病或病症。
例如，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
药物组合物
另一方面，本申请还提供了药物组合物，其包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。
例如，药物组合物可以另外地含有适合治疗或预防C5相关的疾病或病症的一种或多种其它治疗剂。
例如，所述药学上可接受的佐剂增强或稳定组合物，或促进组合物的制备。例如，所述药学上可接受的佐剂可以包括生理上相容的溶剂、分散介质、包衣材料、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。
例如，所述药物组合物可以通过本领域已知的多种方法施用，取决于期望结果、施用途径和/或施用模式改变。例如，施用可以为静脉内、肌内、腹膜内或皮下、或邻近靶位点施用。例如，配制所述的药物组合物使得可将它们玻璃体内施用入眼。例如，取决于施用途径，可以将所述分离的抗原结合蛋白(例如，抗体、双特异性和多特异性分子)用材料包覆以保护化合物免于酸或其它天然条件的作用，所述作用可以使化合物失活。
例如，所述药物组合物可以为无菌的且为流体。例如，通过使用包衣材料(诸如卵磷脂)、或在分散液的情况下通过保持所要求的颗粒大小、以及通过使用表面活性剂可以保持适当的流动性。
例如，在组合物中包括等渗剂，例如，糖、多元醇，诸如甘露糖醇、山梨糖醇或氯化钠。例如，可注射的所述药物组合物的长期吸收可以通过在所述药物组合物中包括延迟吸收的试剂(例如，单硬脂酸铝或明胶)来实现。
例如，所述分离的抗原结合蛋白、所述核酸分子、所述载体和/或所述细胞与任选的药学可接受载体、赋形剂或稳定剂混合而制备以贮存。例如，所述药物组合物的形式可以为冻干制剂或水溶液。
例如，所述药学上可接受的佐剂可以包括药学可接受载体、赋形剂或稳定剂。
例如，所述可接受的载体、赋形剂或稳定剂以采用的剂量和浓度对接受者是无毒的，并且包括缓冲剂例如磷酸盐柠檬酸盐、乙酸盐和其他有机酸。
例如，包含本申请所述的分离的抗原结合蛋白的药物组合物可以是水溶性形式。
例如，用于体内施用的药物组合物可以是无菌的。这可以通过无菌过滤膜过滤或其他方法而容易地实现。例如，以无菌水溶液形式施用包含本申请所述的分离的抗原结合蛋白的药物组合物可以多种方式进行，包括但不限于口服、皮下、静脉内、鼻内、耳内、透皮、局部(例如，凝胶、油膏、洗剂、乳霜等)、腹膜内、肌肉内、肺内、胃肠外、直肠或眼内。在一些 情况下，例如治疗伤口、炎症等，所述的分离的抗原结合蛋白可以作为溶液或喷雾剂直接应用。
例如，所述药物组合物可以根据本领域熟知且常规实践的方法制备。例如，所述药物组合物可以在GMP条件下制备。通常，在本申请的药物组合物中使用C5结合蛋白的治疗有效剂量或有效剂量。例如，通过本领域技术人员已知的常规方法可将C5结合蛋白配制成药学上可接受的剂量形式。调节剂量方案以提供最佳的期望响应(例如，治疗响应)。本申请的药物组合物中活性成分的实际剂量水平可发生改变以便获得有效实现特定患者所期望的治疗响应、组合物和施用模式而对患者不具有毒性的活性成分的量。选择的剂量水平取决于多种药代动力学因素，例如，本申请使用的特定组合物或其酯、盐或酰胺的活性，施用途径，施用时间，正使用的特定化合物的排泄速率，治疗的持续时间，与使用的特定组合物组合使用的其它药物、化合物和/或物质，治疗的受试者的年龄、性别、体重、状态、一般健康和先前病史以及其它因素。
例如，皮下施用可以用于其中患者可以自己施用药物组合物的情况。许多蛋白治疗剂不足够有效以允许配制皮下施用最大可接受体积的治疗有效剂量。本申请公开的抗原结合蛋白可适用于皮下施用，例如，可以增加的效价、提高的血浆半衰期和提高的溶解度。
本领域已知，蛋白治疗剂可以通过经静脉输注或快速浓注来递送。本申请公开的抗原结合蛋白也可以使用此类方法递送。
检测方法
另一方面本申请还提供了检测样品中C5的方法，所述方法包括施用本申请中所述的分离的抗原结合蛋白。
例如，将获取自受试者的样品接触本申请所述的分离的抗原结合蛋白(例如，C5结合蛋白)。例如，其中用可检测标记或报告分子标记C5结合蛋白或将抗C5结合蛋白用作捕获配体以选择性地从患者样品分离C5。或者，未标记的抗C5结合蛋白可以结合第二抗体用于检测应用中，所述第二抗体自身是具有可检测标记的。可检测标记或报告分子可以是放射性同位素，例如如3H、14C、32P、35S或125I；还可以是荧光或化学发光部分，例如异硫氰酸荧光素、若丹明；或酶，例如碱性磷酸酶、β-半乳糖苷酶、辣根过氧化物酶或荧光素酶；可以用于检测或测量样品中的C5的特定示例性分析包括酶联免疫吸附分析(ELISA)、放射性免疫分析(RIA)和荧光激活的细胞分选(FACS)。
可以用于本申请的C5检测中的样品可以包括获取自正常或病理状况下的受试者的任何组织或流体样品，其含有可检测含量的C5蛋白或其片段。例如，可以将测量获自健康受试者 (例如，未患C5相关疾病的受试者)的特定样品的C5蛋白的水平，以最初建立C5的基线或标准水平。随后将这个C5的基线水平相对于获取自疑似患有C5相关疾病或病症或与该病症相关症状的个体的样品中测量的C5水平进行比较。
C5结合蛋白可以不含另外的标记，也可以含有N-末端或C-末端标记。例如，所述标记是生物素。在结合分析中，标记(如果存在的话)的位置可以确定肽相对于其上结合肽的表面的方向。例如，如果表面包被了抗生物素蛋白，则含有N-末端生物素的肽将使得肽的C-末端部分远离表面。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的分离的抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
表1 抗原结合蛋白氨基酸序列表
Figure PCTCN2021098261-appb-000002
Figure PCTCN2021098261-appb-000003
表2 抗原结合蛋白核苷酸序列表
抗原结合蛋白 16H1L2m 16H46L39am
重链可变区 SEQ ID NO:61 SEQ ID NO:62
轻链可变区 SEQ ID NO:63 SEQ ID NO:64
轻链 SEQ ID NO:65 SEQ ID NO:67
重链 SEQ ID NO:66 SEQ ID NO:68
实施例1抗人C5鼠源单克隆抗体
1)ELISA法测定与人C5抗原的结合
使用ELISA法测定9个表达高亲和力鼠单克隆抗体的杂交瘤细胞株培养上清液与人C5抗原的结合。以人C5(SEQ ID NO:60所示)包被酶标板，4摄氏度过夜。弃去包被液，用溶解在磷酸盐缓冲盐水(PBS)的牛血清白蛋白(BSA)封闭各孔1小时，用PBST溶液(20％吐温的PBS溶液)洗孔。然后分别加入100ng/mL和20ng/mL的鼠单克隆抗体的杂交瘤细胞株培养上清液和依库珠单抗(ECU)作为阳性对照，室温孵育1小时，然后使用PBST溶液室温孵育1小时，用PBST溶液洗孔后加入HRP标记的检测抗体，检测各抗体的结合能力。
选择其中的m16鼠源抗体用于后续抗体制备。
实施例2抗人C5嵌合抗体
1)抗人C5鼠源单克隆抗体m16重链和轻链可变区基因的克隆
获得含鼠源抗体m16重链和轻链可变区编码序列的DNA片段的方法为：从m16小鼠杂交瘤细胞中分离出mRNA，以此制备cDNA。通过聚合酶链式反应(PCR)从cDNA中分离重链可变区和轻链可变区DNA片段。将胶回收后得到的DNA片段克隆入TOPO-TA载体(Yeasen)并测序，通过使用软件Discovery Studio进行预测得到编码m16重链的可变区(VH)氨基酸序列(SEQ ID NO:44)及其互补决定区的氨基酸序列HCDR1(SEQ ID NO:18)，HCDR2(SEQ ID NO:19)和HCDR3(SEQ ID NO:20)。轻链的可变区(VL)氨基酸序列(SEQ ID NO:41)及其互补决定区的氨基酸序列LCDR1(SEQ ID NO:1)，LCDR2(SEQ ID NO:16)和LCDR3(SEQ ID NO:17)。
2)抗人C5嵌合抗体的制备
从小鼠杂交瘤细胞中克隆出鼠源重链可变区和轻链可变区基因序列，选择使用氨基酸突变后无ADCC(抗体依赖的细胞介导的细胞毒性作用)毒性的人源IgG1恒定区序列(氨基酸序列如SEQ ID NO:48所示)，将鼠源抗体m16重链可变区基因与人源IgG1恒定区基因连接成嵌合基因，轻链可变区基因与人Kappa轻链恒定区序列(氨基酸序列如SEQ ID NO:47所示)连接成嵌合基因，之后插入载体pcDNA3.1(Biofeng Lab)中，最后在人肾上皮细胞(293)(购自Thermo fisher，货号：XP293)表达系统中表达嵌合抗体分子，获得嵌合抗体CHI-16。以相同条件下生产的依库珠单抗(ECU，其轻链氨基酸序列SEQ ID NO:55，重链氨基酸序列SEQ ID NO:56)、Ravulizumab(RAV，其轻链氨基酸序列SEQ ID NO:55，重链氨基酸序列SEQ ID NO:57)、SKY59(其轻链氨基酸序列SEQ ID NO:58，重链氨基酸序列SEQ ID NO: 59)为阳性对照抗体。
3)ELISA法测定嵌合抗体与人C5抗原的结合
以抗体依库珠单抗(ECU)、Ravulizumab(RAV)、SKY59、CHI-16分别包被酶标板(购自Costar，货号：42592)，4℃过夜。弃去包被液，用溶解在磷酸盐缓冲盐水(PBS)的2.5％的脱脂奶封闭各孔1小时，用PBST溶液(20％吐温的PBS溶液)洗孔。然后每孔分别加入100μl人C5梯度稀释液，37℃孵育1小时，用PBST洗孔，最后每孔加入100μl HRP标记的抗组氨酸抗体(购自Sino Biological，货号：105327-MM02T-H)作为检测抗体，其结果如图1所示，抗体与人C5的结合力越强，相同人C5抗原浓度下颜色越深，OD450值越高。由图1中显示可知，嵌合抗体CHI-16与人C5的结合活性比ECU和Ravulizumab(RAV)更好。
4)嵌合抗体对补体介导的体外溶血的抑制作用
体外溶血抑制实验的方法如下：绵羊红细胞(购自Bio-channel，货号：BC-RBC-S003)用GVB++缓冲液(包含Mg++和Ca++，购自天恩泽，货号：25-02080)洗涤三次，加入溶血素(1:2000，兔抗羊红细胞抗体)(购自北京博尔西科技，货号：BM351Y)室温放置10分钟以激活绵羊红细胞。将激活后的绵羊红细胞用GVB++缓冲液洗涤两次，离心后用GVB++缓冲液稀释至30μl(5x106个细胞)。使用50μl、2.6％的普通人血清(NHS，用GVB++稀释)(购自Stem Express，货号：PBSR015C)与50μl用GVB++配制的抗体梯度稀释液(抗体稀释浓度初始浓度2ug/ml(终浓度)，2倍稀释，6个稀释梯度)混合，室温孵育孔板(购自Costar，货号：3799)约30分钟，同时设置无抗体样品对照组，即只加入与上述抗体混合液同体积的1％的NHS(完全溶血对照组)。将30μl激活后的绵羊红细胞加入NHS和抗体已经孵育好的孔板中，置于室温孵育1小时。离心后收集上清在405nm条件下用读板机读取吸光值，抗C5抗体对溶血的抑制作用通过由如下公式得出的抑制率(％)来体现：抑制率(％)＝100×[(OD无抗体样品读数-OD血清背景值)-(OD有抗体样品读数-OD血清背景值)]/(OD无抗体样品读数-OD血清背景值)。
其结果示于图2，终浓度为1％的NHS可以使激活后的绵羊红细胞发生溶血，加入抗体后能够抑制溶血，并且体CHI-16的抑制效果好于阳性对照抗体依库珠单抗(ECU)、Ravulizumab(RAV)、SKY59的抑制效果。IC 50可以评估各抗体抑制50％溶血时的使用浓度，各组IC 50如下表3所示，嵌合抗体CHI-16的IC 50比阳性对照抗体依库珠单抗(ECU)、Ravulizumab(RAV)、SKY59的IC 50更低，表明体外溶血抑制效果更好。
表3 嵌合抗体的溶血抑制效果
  ECU RAV SKY59 CHI-16
IC 50(ng/ml) 401.3 1186 542.5 204.5
实施例3去除抗体异构化的点突变抗体
1)轻链CDR1区单点突变抗体的产量变化
由于CDR区中的天冬氨酸(D)和甘氨酸(G)相连时容易脱氨，导致抗原与抗体的结合不稳定，因此对CHI-16人源化抗体16HDa的轻链CDR1区的32D和33G进行多个氨基酸的单点突变，筛选出对抗原结合力和产量影响最小的突变氨基酸。
针对16HDa轻链可变区中的32D位点分别构建D32E、D32I、D32L、D32G、D32S、D32T、D32V、D32Y单点突变载体，针对16HDa轻链可变区中的33G位点分别构建G33A、G33S、G33I、G33Q、G33T、G33V单点突变载体，使用293细胞在24孔板中生产D32单点突变的人源化抗体，相同条件下表达的未突变的人源化抗体16HDa作为对照，取培养液上清用ELISA竞争法进行初步定量，检测突变抗体和对照抗体的产量(Titer)。方法如下，将稀释后的待测上清与作为检测抗体的HRP标记的抗人IgG等体积混合后加入包被1ug/ml的CHI-16的ELISA板中，室温孵育1小时。同时以CHI-16为标准品按照同样的方法制作标准曲线。显色后读值，根据标准曲线可以计算得到上清中的Titer。结果如图3所示，在这些突变抗体中，与突变前的对照抗体相比，C32L、D32G和D32T产量无明显变化。
2)ELISA检测轻链CDR1区单点突变抗体的抗原结合力变化
使用293细胞在24孔板中生产D32和G33单点突变的人源化抗体，相同条件下表达的未突变的人源化抗体16HDa作为对照，取培养液上清用竞争ELISA法检测突变抗体和对照抗体对抗原的结合力。方法如下：以对照抗体包被酶标板，4度过夜。弃去包被液，用溶解在磷酸盐缓冲盐水(PBS)的脱脂奶封闭各孔1小时，用PBST溶液(20％吐温的PBS溶液)洗孔。然后每孔分别加入突变抗体与C5抗原的混合液，室温孵育1小时，用PBST洗孔，然后每孔加入HRP标记的抗组氨酸抗体作为检测抗体。其结果示于图4，若抗体突变后与抗原的结合力降低，则OD450值变大。因此在所有的突变抗体中，D32E突变后可以保持与之前一样的抗原结合能力，其次与抗原结合能力相近的突变是G33A。综合之前的产量变化结果，因此选择将抗体轻链可变区D32E突变引入后续人源化的抗体中以避免与抗原结合中可能的脱氨反应。
实施例4抗人C5人源化抗体
1)抗人C5人源化抗体的制备
将嵌合抗体CHI-16进行人源化，最终得到两种不同的人源化抗体：16H1L2m和 16H46L39am。16H1L2m重链核苷酸序列如SEQ ID NO:66所示，16H1L2m轻链核苷酸序列如SEQ ID NO:65所示，16H1L2m重链可变区核苷酸序列如SEQ ID NO:61所示，16H1L2m轻链可变区核苷酸序列如SEQ ID NO:63所示。16H46L39am重链核苷酸序列如SEQ ID NO:68所示，16H46L39am轻链核苷酸序列如SEQ ID NO:67所示，16H46L39am重链可变区核苷酸序列如SEQ ID NO:62所示，16H46L39am轻链可变区核苷酸序列如SEQ ID NO:64所示。
从小鼠杂交瘤细胞中克隆出鼠源重链可变区和轻链可变区基因序列，选择使用氨基酸突变后无ADCC(抗体依赖的细胞介导的细胞毒性作用)毒性的人源IgG1恒定区基因序列(SEQ ID NO:48)，将人源化抗体16H1L2m和16H46L39am重链可变区基因与人源IgG1恒定区基因连接成嵌合基因，轻链可变区基因与人Kappa轻链恒定区(如SEQ ID NO:47所示)连接成嵌合基因，之后插入瞬转表达载体pcDNA3.1中，使用100mL的293细胞瞬转生产人源化抗体蛋白，得到抗体蛋白16H1L2m和16H46L39am。以相同条件下生产的依库珠单抗(ECU)、Ravulizumab(RAV)、SKY59以及嵌合抗体CHI-16为阳性对照抗体。
2)人源化抗体对补体介导的体外溶血的抑制作用
通过使用体外溶血抑制试验分别检测经典途径(CP)和旁路途径(AP)中抗人C5人源化抗体对补体激活的抑制活性。
经典途径(CP)体外溶血抑制实验的方法如实施例2步骤4)所示。按照实施例2步骤4的方法检测人源化抗体16H1L2m和16H46L39am对补体介导的体外溶血的抑制作用，其中，GVB++配制的抗体16H1L2m和16H46L39am的梯度浓度稀释液(抗体稀释浓度初始浓度2ug/ml(终浓度)，2倍稀释，6个稀释梯度)。
实验结果如图5所示，16H1L2m和16H46L39am与嵌合抗体CHI-16有相近的抑制效果，均好于依库珠单抗(ECU)、Ravulizumab(RAV)、SKY59对照组的效果。IC 50和IC 90分别表示各抗体溶血抑制率为50％和90％的使用浓度。
各组的IC 50和IC 90值如表4所示：
表4 抗人C5候选人源化抗体的溶血抑制效果
  ECU RAV SKY59 CHI-16 16H1L2m 16H46L39am
IC 50(ng/ml) 558.80 2681.00 918.70 301.60 346.70 329.70
IC 90(ng/ml) 1342.42 9062.67 3036.32 499.80 510.59 507.17
旁路途径(AP)体外溶血抑制实验的方法如下，正常的兔红细胞(购自Bio-channel，货号：BC-RBC-RAB003)与EGTA混合以抑制补体激活的经典途径，加入GVB Mg+EGTA(天 恩泽/25-02090)处理的终浓度为5％的普通人血清(NHS)与各抗体梯度稀释液(抗体初始浓度(终浓度)为200ug/ml，4倍稀释，6个梯度，兔红细胞的数量为5×10 6，加入前用GVB Mg+EGTA清洗三次)孵育好的孔板中，室温孵育1小时。离心后收集上清在405nm条件下读取吸光值，抗C5抗体对溶血的抑制作用通过下列公式得出的抑制率(％)来体现：抑制率(％)＝100X[(OD无抗体样品读数-OD血清背景值)-(OD有抗体样品读数-OD血清背景值)]/(OD无抗体样品读数-OD血清背景值)。
结果如图6所示，293细胞表达的16H1L2m和16H46L39am对于旁路途径溶血抑制作用均比依库珠单抗(ECU)、Ravulizumab(RAV)、SKY59三个阳性对照抗体生物学活性更好。
3)ELISA测定人源化抗体的热稳定性
用嵌合抗体CHI-16包被酶标板(购自Corning，货号：42592)，4℃过夜。弃去包被液，用溶解在磷酸盐缓冲盐水(PBS)的2.5％的脱脂奶封闭各孔1小时，用PBST溶液(20％吐温的PBS溶液)洗孔。将待测抗体分三组，每组分别放到室温(37℃)、66℃、67.5℃温度下处理1h，将不同温度处理过的抗体稀释到2000ng/ml、500ng/ml，分别与500ng/ml的人C5抗原等体积混合作为一抗加入各孔中，室温孵育1小时，用PBST洗孔，然后每孔加入100μl HRP标记的抗组氨酸抗体(购自Sino Biological，货号：105327-MM02T-H)作为检测抗体。抗体在高温下失活，不能与人C5抗原结合，多余的人C5抗原就会与包被的CHI-16结合显色，因此可以估计抗体的热稳定性，抗体的热稳定性越好，OD450值越低，抑制人C5抗原结合的能力越强。结果如图7所示，16H1L2m和16H46L39am的热稳定性较对照抗体更好。
4)ELISA测定人源化抗体与人C5抗原的结合
用各抗体(16H1L2m、16H46L39am、ECU和RAV)包被酶标板(购自Corning，货号：42592)，4℃过夜。弃去包被液，用溶解在磷酸盐缓冲盐水(PBS)的2.5％的脱脂奶封闭各孔1小时，用PBST溶液(20％吐温的PBS溶液)洗孔。然后每孔分别加入100μl人C5梯度稀释液(C5稀释到1ug/ml，然后3倍稀释，依次稀释7个梯度，100ul/孔)，室温孵育1小时，用PBST洗孔，然后每孔以1:8000比例加入100μl HRP标记的抗组氨酸抗体(购自Sino Biological，货号：105327-MM02T-H)作为检测抗体。
其结果如图8所示，抗体与人C5的结合力越强，相同的人C5浓度下颜色越深，OD450值越高。EC 50可以评估各抗体与抗原50％结合力时的抗原的使用浓度，EC 50值越低说明抗体与人C5抗原的结合力越强，结果如图8所示，16H1L2m和16H46L39am与抗原的结合力都比ECU和RAV更强。EC 50值如表5所示。
表5 抗人C5候选人源化抗体与抗原的结合力
  ECU RAV 16H1L2m 16H46L39am
EC 50(ng/ml) 180.9 941.9 117.5 168.8
5)人源化抗体的亲和力分析试验
采用分子互作分析仪(ForteBio Octet)利用生物薄膜干涉技术(BLI)测定对照抗体ECU和人源化抗体16H1L2m和16H46L39am与抗原的结合亲和力常数。分析亲和力分析动力学拟合曲线及各通道动力学参数测定结果，计算得出各个候选人源化抗体和ECU的亲和力常数(见表6)。
表6.抗人C5候选人源化抗体和ECU结合人C5的亲和力(Octet)
  K D(M) K a(1/Ms) K d(1/s)
ECU 3.02×10 -10 2.16×10 5 6.52×10 -5
16H1L2m 3.59×10 -10 2.17×10 5 7.81×10 -5
16H46L39am 3.69×10 -10 2.13×10 5 7.86×10 -5
表6中，K a：结合速率常数；K d：解离速率常数；K D：亲和常数，等于K d/K a。
其结果显示16H1L2m和16H46L39am与对照抗体ECU结合人C5的亲和力(Octet)相近。

Claims (56)

  1. 分离的抗原结合蛋白，其包含重链可变区中的至少一个CDR，所述重链可变区包含如SEQ ID NO:44-46中任一项所示的氨基酸序列或其变体；且其包含轻链可变区中的至少一个CDR，所述轻链可变区包含如SEQ ID NO:70所示的氨基酸序列或其变体。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其中所述如SEQ ID NO:70所示的氨基酸序列变体包含选自如下组的氨基酸突变：D32E、D32I、D32L、D32G、D32S、D32T、D32V、D32Y、G33A、G33S、G33I、G33Q、G33T和G33V。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含氨基酸序列如SEQ ID NO:
    44-46中任一项所示的重链可变区中的HCDR1。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包含氨基酸序列如SEQ ID NO:44-46中任一项所示的重链可变区中的HCDR2。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含氨基酸序列如SEQ ID NO:44-46中任一项所示的重链可变区中的HCDR3。
  6. 根据权利要求3-5中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:18所示的氨基酸序列。
  7. 根据权利要求4-6中任一项所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:19所示的氨基酸序列。
  8. 根据权利要求5-7中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:20所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其包含氨基酸序列如SEQ ID NO:41-43中任一项所示的轻链可变区中的LCDR1。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含氨基酸序列如SEQ ID NO:41-43中任一项所示的轻链可变区中的LCDR2。
  11. 根据权利要求1-10中任一项所述的分离的抗原结合蛋白，其包含氨基酸序列如SEQ ID NO:41-43中任一项所示的轻链可变区中的LCDR3。
  12. 根据权利要求9-11中任一项所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:69所示的氨基酸序列。
  13. 根据权利要求9-11中任一项所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:1和2中任一项所示的氨基酸序列。
  14. 根据权利要求10-13中任一项所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:16所示的氨基酸序列。
  15. 根据权利要求11-14中任一项所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:17所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  17. 根据权利要求16所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其中所述轻链可变区包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
  19. 根据权利要求18所述的分离的抗原结合蛋白，其中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:21-23中任一项所示的氨基酸序列。
  20. 根据权利要求18-19中任一项所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:21-23中任一项所示的氨基酸序列。
  21. 根据权利要求18-20中任一项所述的分离的抗原结合蛋白，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:24-25中任一项所示的氨基酸序列。
  22. 根据权利要求18-21中任一项所述的分离的抗原结合蛋白，其中所述L-FR2包含SEQ ID NO:24-25中任一项所示的氨基酸序列。
  23. 根据权利要求18-22中任一项所述的分离的抗原结合蛋白，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:26-28中任一项所示的氨基酸序列。
  24. 根据权利要求18-23中任一项所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:26-28中任一项所示的氨基酸序列。
  25. 根据权利要求18-24中任一项所述的分离的抗原结合蛋白，其中所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
  26. 根据权利要求18-25中任一项所述的分离的抗原结合蛋白，其中所述L-FR4包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的分离的抗原结合蛋白，其中所述轻链可变区包含SEQ ID NO:41-43中任一项所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所 述抗体轻链恒定区包括人Kappa轻链恒定区。
  29. 根据权利要求28所述的分离的抗原结合蛋白，其中所述抗体轻链恒定区包含SEQ ID NO:47所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的分离的抗原结合蛋白，其包含抗体轻链，且所述抗体轻链包含SEQ ID NO:49、51和53中任一项所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其中所述重链可变区包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
  32. 根据权利要求31所述的分离的抗原结合蛋白，其中所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:31-33中任一项所示的氨基酸序列。
  33. 根据权利要求31-32中任一项所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:31-33中任一项所示的氨基酸序列。
  34. 根据权利要求31-33中任一项所述的分离的抗原结合蛋白，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:34-35中任一项所示的氨基酸序列。
  35. 根据权利要求31-34中任一项所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:34-35中任一项所示的氨基酸序列。
  36. 根据权利要求31-35中任一项所述的分离的抗原结合蛋白，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:36-38中任一项所示的氨基酸序列。
  37. 根据权利要求31-36中任一项所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:36-38中任一项所示的氨基酸序列。
  38. 根据权利要求31-37中任一项所述的分离的抗原结合蛋白，其中所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:39-40中任一项所示的氨基酸序列。
  39. 根据权利要求31-38中任一项所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:39-40中任一项所示的氨基酸序列。
  40. 根据权利要求1-39中任一项所述的分离的抗原结合蛋白，其中所述重链可变区包含SEQ ID NO:44-46中任一项所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所 述抗体重链恒定区源自人IgG重链恒定区。
  42. 根据权利要求1-41中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区源自人IgG1重链恒定区。
  43. 根据权利要求41-42中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区包含SEQ ID NO:48所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的分离的抗原结合蛋白，其包含抗体重链，且所述抗体重链包含SEQ ID NO:50、52和54中任一项所示的氨基酸序列。
  45. 根据权利要求1-44中任一项所述的分离的抗原结合蛋白，所述抗原为补体因子5(C5)蛋白。
  46. 根据权利要求45所述的分离的抗原结合蛋白，所述C5为人C5。
  47. 分离的核酸分子，其编码权利要求1-46中任一项所述的分离的抗原结合蛋白。
  48. 载体，其包含根据权利要求47所述的核酸分子。
  49. 细胞，其包含根据权利要求47所述的核酸分子或根据权利要求48所述的载体。
  50. 制备权利要求1-46中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-46中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求49所述的细胞。
  51. 免疫偶联物，其包含权利要求1-46中任一项所述的分离的抗原结合蛋白。
  52. 药物组合物，其包含权利要求1-46中任一项所述的分离的抗原结合蛋白、权利要求47所述的核酸分子、权利要求48所述的载体、权利要求49所述的细胞和/或权利要求51所述的免疫偶联物，以及任选地药学上可接受的佐剂。
  53. 权利要求1-46中任一项所述的分离的抗原结合蛋白、权利要求47所述的核酸分子、权利要求48所述的载体、权利要求49所述的细胞、权利要求51所述的免疫偶联物和/或权利要求52所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗C5相关的疾病或病症。
  54. 预防、缓解或治疗C5相关的疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-46中任一项所述的分离的抗原结合蛋白、权利要求51所述的免疫偶联物和/或权利要求52所述的药物组合物。
  55. 权利要求1-46中任一项所述的分离的抗原结合蛋白、权利要求47所述的核酸分子、权利要求48所述的载体、权利要求49所述的细胞、权利要求51所述的免疫偶联物和/或权利要求52所述的药物组合物，其用于预防、缓解或治疗C5相关的疾病或病症。
  56. 检测样品中C5的方法，所述方法包括施用权利要求1-46中任一项所述的分离的抗原结合蛋白。
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