WO2021218953A1 - 针对人补体蛋白c5的抗体及其应用 - Google Patents

针对人补体蛋白c5的抗体及其应用 Download PDF

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WO2021218953A1
WO2021218953A1 PCT/CN2021/090185 CN2021090185W WO2021218953A1 WO 2021218953 A1 WO2021218953 A1 WO 2021218953A1 CN 2021090185 W CN2021090185 W CN 2021090185W WO 2021218953 A1 WO2021218953 A1 WO 2021218953A1
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amino acid
seq
acid sequence
variable region
chain variable
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PCT/CN2021/090185
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French (fr)
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王雪
徐晓红
高攀
陶春艳
邓小芳
吴建
李祥烽
毕建军
王骊淳
任红媛
林鉴
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上海普铭生物科技有限公司
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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • 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

Definitions

  • the present invention relates to the field of antibody medicines. Specifically, the present invention relates to antibodies against human complement protein C5 and their use for preparing medicines.
  • the complement system consists of dozens of proteins (C1 (C1q, C1r, C1s), C2, C3, C4, C5, C6, C7, C8, C9, etc.). It is a complex immune monitoring system that serves as a defense against other organisms.
  • the first host defense system of infection works, and it also distinguishes healthy host tissues from cell debris, apoptotic and necrotic cells; in addition, it participates in clearing immune complexes, regulating adaptive immune responses, and promoting tissue regeneration, Angiogenesis, stem cell mobilization and central nervous system development.
  • Complement protein 5 is the main component of the complement system and a key part of the innate immune system.
  • C5 is a 190kDa glycoprotein containing two polypeptide chains ⁇ and ⁇ connected by disulfide bonds, and its molecular masses are 115kDa and 75kDa.
  • the arginine residue at the 74th amino acid downstream of the N-terminus of the C5 ⁇ -chain is cleaved by C5 convertase to produce C5a and C5b complement cleavage products.
  • C5a is an anaphylactoxin that can stimulate vasodilation through histamine released by basophils and giant cells;
  • C5b can form a membrane attack complex (MAC) with complement proteins C6, C7, C8, and C9 on the cell surface.
  • MAC membrane attack complex
  • MAC MAC-binding protein
  • ischemia-reperfusion injury glomerulonephritis
  • glomerulonephritis systemic erythema Lupus
  • PNH paroxysmal nocturnal hemoglobinuria
  • aHUS atypical hemolytic uremic syndrome
  • myasthenia gravis and macular degeneration such as age-related macular degeneration.
  • complement protein C5 For complement protein C5, there have been different reports describing antibodies against C5, and some of the antibodies have been approved for the treatment of related indications or are in clinical development.
  • the monoclonal antibody Eculizumab developed by Alexion as a humanized IgG2/4 ⁇ monoclonal antibody targeting complement protein C5, has high affinity with complement protein C5.
  • the technical problem to be solved by the present invention is to obtain a high-affinity antibody that specifically binds to human C5 protein through hybridoma screening and humanization technology.
  • the crowd is effective.
  • the purpose of the present invention is to provide an antibody or functional fragment thereof that specifically binds to human C5 protein, and to provide its use.
  • the antibody fragments of the present invention cover various functional fragments of the antibody, for example, the antigen binding portion thereof, such as Fab, F(ab') 2 or scFv fragments.
  • the present invention provides an antibody or antigen-binding fragment thereof, which binds complement protein 5 (C5) with a higher affinity at neutral pH than at acidic pH.
  • the antibody or its antigen-binding fragment can effectively block the cleavage of C5 or its R885 mutant into C5a and C5b.
  • the antibody or antigen-binding fragment provided by the present invention comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and the heavy chain variable region and the light chain variable region comprise A combination of heavy chain CDRs and light chain CDRs selected from:
  • HCDR1 DYHLD
  • HCDR2 YIDPDNGGTFYNQKFKG
  • HCDR3 WHDYAPSFAY
  • LCDR1 HASQNINVWLS
  • LCDR2 KASNLHT
  • LCDR3 QQGQSYPLT
  • HCDR1 DYHLD
  • HCDR2 YIDPDTGGTFYNQKFKG
  • HCDR3 WHDYAPSFAY
  • LCDR1 HASQNINVWLS
  • LCDR2 KASNLHT
  • LCDR3 QQGQSYPLT
  • HCDR1 NFYLH
  • HCDR2 WIYPENLSTKYNDKFKD
  • HCDR3 SHYNDYLTGAMDH
  • LCDR1 RASKSISKYLA
  • LCDR2 SGSTLQF
  • LCDR3 QQHDQYPWT
  • HCDR1 NFYLH
  • HCDR2 WIYPENPSTKYNDKFKD
  • HCDR3 SHYNDYLTGAMDH
  • LCDR1 RASKSISKYLA
  • LCDR2 SGSTLQF
  • LCDR3 QQHDQYPWT
  • HCDR1 NFYLH
  • HCDR2 WIYPEQLSTKYNDKFKD
  • HCDR3 SHFTDYLTGAMDH
  • LCDR1 RASKSISKYLA
  • SGSTLQF LCDR2
  • LCDR3 QQHDQYPWT
  • HCDR1 NFYLH
  • HCDR2 WIYPEQLSTKYNDKFKD
  • HCDR3 SHFTDYLTGAMDH
  • LCDR1 RASKSISKYLA
  • SGSTLIF LCDR2
  • LCDR3 QQHDQYPWT
  • HCDR1 DIYIH
  • HCDR2 RIDPASGHTEYDPKFQA
  • HCDR3 EDYEGIGY
  • LCDR1 RASQNVDTHVA
  • LCDR2 LASYRYS
  • LCDR3 QQYNTYPLT
  • HCDR1 (DAWMD), HCDR2 (AIRNKTNNHATYYTESVKG), HCDR3 (QGDGYYVRFAY) shown in SEQ ID NO: 77, 78, 79; and LCDR1 (RASENTYSYLA) shown in SEQ ID NO: 80, 81, 82 , LCDR2 (DAKTLAE), LCDR3 (QHHYGTPYT).
  • the antibody or antigen-binding fragment thereof of the present invention further comprises a framework region (Framework Region; FR), the heavy chain variable region or
  • the light chain variable region contains the aforementioned domain components in the order of FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4 or in the order of FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4.
  • the heavy chain variable region comprises the same as shown in SEQ ID NO: 1, 9, 11, 13, 23, 25, 26, 27, 28, 30
  • the amino acid sequence of, 32, 33, 35, 37, 39, or 41 has an amino acid sequence that is at least 75% identical
  • the light chain variable region comprises the same as shown in SEQ ID NO: 2, 10, 12, 14,
  • the amino acid sequence of 24, 29, 31, 34, 36, 38, 40, or 42 has an amino acid sequence that is at least 75% identical.
  • the heavy chain variable region comprises at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1, 23, 25, 26 or 27
  • the light chain variable region comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 2 or 24; or,
  • the heavy chain variable region comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 9, 28, 30, 32 or 33; and, the light chain variable region comprises an amino acid sequence shown in The amino acid sequence of SEQ ID NO: 10, 29, 31 or 34 has an amino acid sequence with at least 75% identity; or
  • the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 11, 35, or 37; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence of 12, 36, or 38 has an amino acid sequence that is at least 75% identical; or,
  • the heavy chain variable region comprises an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 13, 39 or 41; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence of 14, 40, or 42 has an amino acid sequence that is at least 75% identical.
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 1; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 2 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 23; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 24 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 25; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 24 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 26; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 24 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 27; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 24 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 9; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 10 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 28; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 29 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 30; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 31 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 32; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 31 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 33; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 31 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 33; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 34 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 11; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 12 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 35; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 36 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 37; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 38 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 13; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 14 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 39; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 40 The sequence has an amino acid sequence with at least 75% identity;
  • Heavy chain variable region Contains an amino acid sequence that is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 41; and, Light chain variable region: Contains the amino acid sequence shown in SEQ ID NO: 42 The sequence has an amino acid sequence with at least 75% identity.
  • "at least 75% identity” is the identity of any percentage number between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • at most 25% difference in amino acid sequence caused by the "at least 75% identity” may exist in the heavy chain variable region or light chain variable region of the antibody or antigen-binding fragment thereof of the present invention other than the CDR In any framework region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region.
  • the difference can be caused by amino acid deletion, addition or substitution at any position, wherein the substitution can be a conservative substitution or a non-conservative substitution.
  • the heavy chain variable region and the light chain variable region in the antibody or antigen-binding fragment thereof of the present invention respectively comprise:
  • the CDR in the amino acid sequence of the divided variable region is defined according to Kabat.
  • the antibody or antigen-binding fragment thereof provided by the present invention binds to mammalian C5, preferably primate C5, more preferably human or cynomolgus monkey (cynomolgus monkey) C5, especially human C5.
  • the antibody or antigen-binding fragment thereof of the present invention can bind to human C5 with high affinity, has a significant inhibitory effect on the cleavage of human C5 into C5a and C5b, significantly inhibits complement-dependent hemolysis, and blocks complement Dependent cytotoxicity, and has significant binding affinity to cynomolgus monkey C5; and also has high affinity to human C5 (R885) mutant, inhibiting the cleavage of C5 variant R885 into C5a and C5b.
  • the antibody or antigen-binding fragment thereof of the present invention has the characteristics of pH-dependent binding to C5 antigen, and the affinity is lower in acidic conditions than in neutral conditions.
  • the antibody provided by the present invention may be a murine antibody, a chimeric antibody, or a fully or partially humanized antibody; the antigen-binding fragment provided may be the antibody capable of specifically binding antigen, such as human C5 or its R885H mutant Any fragment of, such as scFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv fragment.
  • the antibody provided by the present invention is a monoclonal antibody or a single chain antibody.
  • the antibody or antigen-binding fragment thereof provided by the present invention further comprises a human or murine constant region, preferably a murine or human heavy chain constant region (CH) and/or light chain constant region (CL); preferably, the The antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, for example, two heavy chains and a light chain. More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.
  • the antibody provided by the present invention is a monoclonal antibody, preferably a humanized monoclonal antibody.
  • the heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region of the monoclonal antibody is of ⁇ type.
  • the heavy chain constant region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 45; the light chain constant region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 46.
  • the present invention provides a nucleic acid molecule that includes a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, and a heavy chain CDR contained in the antibody or antigen-binding fragment thereof of the present invention.
  • the nucleotide sequence of the chain or light chain is not limited to the sequence of the chain or light chain.
  • the nucleic acid molecule of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Therefore, in yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • the vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
  • the vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preservation or expression of antibodies. Therefore, in yet another aspect, the present invention provides a host cell that contains the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • the antibodies provided by the present invention can be obtained by any method known in the art.
  • the host cell provided by the present invention is allowed to express the heavy chain and the light chain of the antibody to assemble into the antibody
  • the host cell is cultured.
  • the method further includes the step of recovering the produced antibody.
  • the means for obtaining the antibody or its antigen-binding fragment are well known in the art.
  • the present invention also provides a composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector and/or host cell provided by the present invention.
  • the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.
  • the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell and/or composition in the preparation of a medicament for the prevention or treatment of complement (e.g., complement protein).
  • complement e.g., complement protein
  • C5 Mediated diseases.
  • the disease is rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis, optic neuromyelitis or macular degeneration such as age-related Sexual macular degeneration.
  • the present invention also provides a method for preventing or treating diseases mediated by complement (for example, complement protein C5), the method comprising administering the antibody or antigen-binding fragment thereof of the present invention to a subject in need thereof , Nucleic acid molecules, vectors, host cells and/or compositions.
  • complement for example, complement protein C5
  • the disease is rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis, optic neuromyelitis or macular degeneration such as age-related Sexual macular degeneration.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell and/or composition in the preparation of reagents for diagnosing diseases, which are caused by complement (e.g., complement protein).
  • complement e.g., complement protein
  • C5 Mediated.
  • the disease is rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis, optic neuromyelitis or macular degeneration such as age-related Sexual macular degeneration.
  • the present invention also provides a method for diagnosing diseases, which comprises combining the antibody or antigen-binding fragment, nucleic acid molecule, vector, host cell and/or composition of the present invention with a sample from a subject In contact, the disease is mediated by complement (for example, complement protein C5).
  • complement for example, complement protein C5
  • the disease is rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis, optic neuromyelitis or macular degeneration such as age-related Sexual macular degeneration.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and/or composition of the present invention.
  • the kit can be used for therapeutic or diagnostic purposes.
  • antibodies or antigen-binding fragments, nucleic acid molecules, vectors, host cells and/or compositions provided by the present invention can be used for the prevention, treatment or diagnosis of diseases related to the C5Arg885 mutant.
  • an antibody library is established using hybridoma technology, and anti-C5 monoclonal antibodies with high affinity to C5 and biological activity are obtained by screening.
  • Obtain multiple lead antibody molecules which not only have high affinity with C5, but also have hemolysis inhibitory activity.
  • Recombinantly express human-mouse chimeric antibody and humanized antibody based on the mouse-derived lead antibody molecule, and finally obtain a humanized antibody molecule with similar biological activity as the mouse-derived antibody.
  • In vitro pharmacodynamic studies show that the antibody of the present invention can effectively block the hemolytic activity mediated by human serum and inhibit the complement-dependent cytotoxic activity.
  • the antibody of the present invention also has high affinity to human C5 (R885) mutant, and inhibits the cleavage of C5 variant R885 into C5a and C5b.
  • the antibody of the present invention has the characteristics of pH-dependent binding to C5 antigen, and its affinity under acidic conditions is lower than that under neutral conditions. Experiments have shown that its dissociation rate constant Koff and affinity constant KD are better at pH 5.8 and pH 7.4. The ratios are all greater than the control antibody eculizumab, indicating that the antibody of the present invention is easier to dissociate from the antigen C5 under acidic conditions than eculizumab.
  • epitope competition experiments show that the antibody of the present invention and eculizumab have different antigen binding epitopes, and are new candidate antibody drugs with different binding characteristics.
  • Figure 1 shows the ELISA identification results of the supernatant of the hybridoma cell line.
  • Figure 2 shows the inhibitory effect of hybridoma cell line supernatant on complement-dependent hemolysis.
  • Figure 3 shows the binding activity of the chimeric antibody to human C5.
  • Figure 4 shows the binding activity of the chimeric antibody to cynomolgus monkey C5.
  • Figure 5 shows the binding activity of the chimeric antibody to the human C5 (R885H) mutant.
  • Figure 6 shows the binding activity of chimeric antibodies and humanized antibodies to human C5.
  • Human C5 protein purchased from Yiqiao Shenzhou, Catalog No. 13416-H18H, NP_001726.2 (Gln19-Cys1676);
  • Heavy chain constant region of hIgG1 subtype SEQ ID NO: 45;
  • the light chain constant region of hIgG-Kappa subtype SEQ ID NO: 46.
  • Antigen recombinant human complement protein C5 (Gln19-Cys1676) was purchased from Yiqiao Shenzhou (Cat. No. 13416-H18H). The protein was recombinantly expressed by human 293 cells (HEK293), with a histidine tag at the C-terminus and a Flag tag at the N-terminus.
  • the method for screening human C5 binding agents by ELISA is as follows: Dilute human C5 protein to 1 ⁇ g/ml with PBS buffer, and coat 100 ⁇ l per well in a 96-well plate (Microwell 96F 167008, Thermo) and incubate overnight at 4°C; take it out the next day For 96-well plates, wash the plates with PBST (containing 0.5% Tween 20), and thoroughly dry the residual water after each infiltration for 1 min. Add 200 ⁇ l of PBST containing 2% BSA to the sample wells and place them at 37° C. for blocking for 1 h; then wash the plate with PBST, and shake off the water in the wells.
  • PBST containing 0.5% Tween 20
  • the ELISA-positive clones were transferred from the 384-well plate to the 96-well plate for expansion, and then 30 ⁇ l of hybridoma cell culture supernatant was taken for hemolysis test to evaluate the inhibitory effect of complement-dependent hemolysis.
  • the method is as follows: Dilute Human Serum Complement (Normal Human Serum Complement, Quidel Corporation, A112) 4 times with PBS to prepare human complement stock solution, add 30 ⁇ l/well to a 96-well U-shaped plate, and add 30 ⁇ l hybridoma cell culture supernatant ; 10% sheep red blood cells were washed with PBS, centrifuged at 3000 rpm for 3 min, washed 3 times, 30 ⁇ l/well was added to the 96-well U-shaped plate with human complement and hybridoma cell culture supernatant before, and gently mixed; Place the plate at 37°C and incubate for 1 hour; after incubation, place the 96-well plate in a 96-well plate centrifuge and centrifuge at 3000 rpm for 3 minutes; take 60 ⁇ l of centrifuge supernatant and add it to a new 96-well plate, and measure the OD405 absorbance with a microplate reader value. Part of the results is shown in Figure 2.
  • the hybridoma parent clone that secretes anti-human C5 antibody and inhibits complement-dependent hemolysis activity is added to a 96-well plate by the limiting dilution method, and the monoclonal cells are observed and labeled under a microscope after 2-3 days. After the 7th day, the ELISA test is carried out.
  • the hemolysis test screens monoclonal hybridoma cells that can secrete anti-human C5 monoclonal antibodies.
  • RNAfast200 kit Shanghai Feijie Biotechnology Co., Ltd.
  • the total RNA of the hybridoma cells was reversed using 5 ⁇ PrimeScript RT Master Mix (Takara) Record into cDNA; use degenerate primers and Extaq PCR reagents (Takara) to amplify antibody light chain variable region IgVL ( ⁇ ) and heavy chain variable region VH sequences; use PCR clean-up Gel extraction kit (Macherey-Nagel) Purify the PCR amplification product; follow the pmd19T Simple Vector Kit (Takara) instructions to connect the amplified PCR product to the T vector and transform E. coli competent cells. After the strain is amplified and the plasmid is extracted, DNA sequencing is performed to obtain monoclonal antibodies. Variable region sequence.
  • variable region sequence of the murine antibody (named with the clone number), see Table 1.
  • the complete light and heavy chain variable regions of the murine antibody were cloned into a recombinant expression vector.
  • the constant regions of the heavy chain were all selected from hIgG1 subtype, and the light chain was selected from hIgG-Kappa subtype.
  • the resulting recombinant chimeric antibody was named "Mouse Abbreviated as -xi", used for antibody production and purification.
  • the control antibody Eculizumab, heavy and light chain variable region sequence (SEQ ID NO: 43; SEQ ID NO: 44) is also constructed into a chimeric expression vector of hIgG1 and hIgG-Kappa subtypes after the full gene synthesis, here Name it "Ref1".
  • the recombinant chimeric antibody protein was expressed and purified by the following method: HEK293-6E cells were diluted and passaged with F17 medium at a density of 1.0 ⁇ 10 6 cells/ml the day before transfection. After 24 hours, the cell density reaches 1.8-2.0x10 6 cells/ml and can be used for transfection.
  • Each 1.0x10 6 cells contains 1 ⁇ g DNA (heavy chain and light chain plasmid ratio is 1:1.5), 4 ⁇ g PEI (polyethyleneimine-linear, Polysciences, 24885-2), 0.4 ⁇ l 293Expression MAX-1 (Acro, EXP-711) transfection complex, diluted with Optimal MEM medium (Gibco, 31985-070) and incubated at room temperature for 15 minutes, then added to the cells.
  • the transfected cells were cultured in a culture flask under the conditions of 5% CO 2, 37° C. and 125 rpm shaking speed. 1% peptone medium was added 22-26h after transfection.
  • the cultured cells were collected and centrifuged at 3000 rpm for 30 min to retain the supernatant.
  • the culture supernatant was purified with a Mabselect Sure column, and the eluted antibody protein was dialyzed into PBS and stored at -80°C for a long time. Purified antibodies with a purity of more than 95% detected by SDS-PAGE can be used in subsequent experiments.
  • a complement-dependent hemolysis experiment was performed. Dilute the human serum complement 4 times with PBS to prepare the human complement mother solution, add 30 ⁇ l/well to the 96-well U-shaped plate for later use; dilute the anti-human C5 chimeric antibody to 100 ⁇ g/mL with PBS to start, then 2-fold dilution, set 8 gradients, two replicate wells, 30 ⁇ l/well were added to a 96-well U-shaped plate with human complement, placed at room temperature for 1 hour; 10% sheep red blood cells were washed with PBS, then centrifuged at 3000 rpm for 3 minutes, and the washing was repeated 3 times.
  • CDC complement-dependent cytotoxicity
  • a 4-parameter fitting curve was performed using the softmax pro software by absorbance value, and the EC50 value of the sample was calculated.
  • Each plate can measure the EC50 of 3 samples, one of which is the control antibody Ref1 as the internal reference sample.
  • the ratio of the EC50 of each chimeric antibody to be tested to the control antibody is calculated as the titer. The results are shown in Table 3.
  • the Octet RED 96 instrument was used to analyze the cross-reactivity of the chimeric antibody with cynomolgus monkey C5.
  • the difference is only that the antigen human C5 protein is replaced with the antigen cynomolgus C5 protein (Acro, article number CO5-C52Hx).
  • the results are shown in Figure 4 in Figure 4A, Figure 4B, Figure 4C, Figure 4D, and Figure 4E.
  • the results show that the control antibody Ref1 has a very low affinity with the cynomolgus C5 protein, while other molecules to be tested have a high affinity with the cynomolgus C5 protein.
  • the Octet RED 96 instrument was used to analyze the binding activity of the chimeric antibody to the human C5 (R885H) mutant.
  • the same was carried out with reference to Example 4, except that the antigen human C5 protein was replaced with the antigen human C5 protein (R885H) mutant (Acro, catalog number CO5-H52Hx).
  • the results are shown in Figure 5 in Figure 5A, Figure 5B, Figure 5C, Figure 5D, and Figure 5E.
  • the results show that the control antibody Ref1 has a low affinity with human C5 (R885H) protein, while other test molecules have high affinity with human C5 (R885H) protein.
  • the mouse antibodies 13F21, 11D5, 8K13, 4E5 were humanized. After comprehensively analyzing the sequence of the murine antibody heavy chain, determining the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the antibody’s conserved three-dimensional conformation, search for known human antibody sequences through analysis and select The human antibody heavy chain sequence that is most similar to the murine antibody, such as IGHV1-3*01, selects its antibody framework region sequence as a template. The murine antibody heavy chain CDR is inserted into the human antibody framework region to generate a humanized antibody heavy chain sequence (heavy chain version 0).
  • CDR antigen complementarity determinant
  • humanized antibody heavy chain sequences versions 1, 2, 3
  • a humanized antibody light chain sequence version 0, 1, 2
  • the designed and synthesized humanized antibody light and heavy chains are co-transfected into 293 cells, and the humanized antibody is expressed recombinantly (for example, the version naming method: heavy chain version 0 + light chain version 0 co-expression, that is, H0L0).
  • the sequences of the heavy and light chain variable regions and CDR regions of each humanized antibody are shown in Table 5.
  • Example 8 Biacore system detects the binding kinetics (K on , K off ) and affinity constant K D of monoclonal antibodies and human C5 under different pH conditions
  • the sensor chip CM4 analysis channel and the control sample channel are saturated and coupled with the maximum amount of anti-human Fc antibody, and then 7.5 ⁇ g/ml antibody is flowed through the analysis channel to make the purified monoclonal antibody evenly distributed, and finally in the analysis channel and sample
  • the channel flows through the gradient dilution antigen sample (the initial concentration is 50 nM, 1:2 dilution 6-8 concentration points, and the 0.741 nm concentration point is set to repeat), and the light response value after the binding of the antibody antigen is measured.
  • the binding constant Kon and the dissociation rate constant Koff of the antibody, and the affinity constant KD are finally obtained. The results are shown in Table 8.

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Abstract

本发明公开了一种抗人C5蛋白的抗体或其抗原结合片段，该抗体或其抗原结合片段以高亲和力结合人C5蛋白，阻断C5蛋白或其R885突变体裂解为C5a和C5b，可以用于治疗类风湿性关节炎、阵发性睡眠性血红蛋白尿、非典型溶血性尿毒症综合征、重症肌无力、视神经脊髓炎或黄斑变性等疾病从而使患者受益。

Description

针对人补体蛋白C5的抗体及其应用
相关申请的交叉引用
本专利申请要求于2020年4月28日提交的申请号为CN202010347154.3的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明涉及抗体药物领域，具体而言，本发明涉及针对人补体蛋白C5的抗体及其用于制备药物的用途。
背景技术
已知补体系统由数十种蛋白(C1(C1q、C1r、C1s)、C2、C3、C4、C5、C6、C7、C8、C9等)组成，是一个复杂的免疫监测系统，作为抵御其他生物感染的第一道宿主防御系统起作用，并且也将健康的宿主组织与细胞碎片、凋亡的和坏死的细胞区别开；此外，参与清除免疫复合体、调节适应性免疫反应、促进组织再生、血管生成、干细胞动员和中枢神经系统的发育。
补体蛋白5(C5)是补体系统的主要成分，是先天免疫系统的关键部分。C5是190kDa的糖蛋白，包含两个由二硫键连接的多肽链α和β，其分子质量是115kDa和75kDa。在C5α-链N末端下游第74个氨基酸的精氨酸残基处被C5转化酶切割，会产生C5a和C5b补体裂解产物。其中，C5a是一种过敏毒素，可通过嗜碱性细胞和巨细胞释放的组胺刺激血管舒张；C5b可在细胞表面依次与补体蛋白C6、C7、C8、C9一同形成膜攻击复合体(MAC)的初始组分，MAC在靶细胞表面的积累最终导致细胞胶体渗透性裂解，促成促炎环境和细胞损失。
正常条件下，补体激活及其末端效应均处于严密调控之下，从而有效地维持机体的自稳功能，MAC的形成也会由于存在细胞表面组分和可溶性调节组分而不影响外围组织。但是，不受控制或不适当的补体激活或调节也会对自身组织细胞造成损伤，诱导急性和慢性疾病状态，例如类风湿性关节炎、缺血再灌注损伤、肾小球肾炎、系统性红斑狼疮、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力以及黄斑变性例如年龄相关性黄斑变性等。
针对补体蛋白C5，已有不同报道描述了抗C5的抗体，部分抗体已经被 批准上市用于相关适应症的治疗，或处于临床开发中。例如，由亚力兄制药(Alexion)研发的单克隆抗体依库珠单抗(Eculizumab)，作为人源化的靶向补体蛋白C5的IgG2/4κ型单克隆抗体，能与补体蛋白C5高亲和性结合且有效抑制其裂解为C5a和C5b，阻止终端补体复合物C5b-9的生成，从而有效抑制补体末端介导的血管内溶血，已被批准用于治疗成人和儿童阵发性睡眠性血红蛋白尿症(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力和视神经脊髓炎谱系疾病。依库珠单抗的第二代Ravulizumab，则通过抗体工程实现了长效化：与C5结合为pH敏感性的，在细胞外中性条件下与C5结合，被内皮细胞内吞后，在酸性环境中与C5解离，避免了抗体被降解，从而再循环到细胞外继续结合C5，达到长效目的。
但是，虽然以上两种药物可以有效治疗大部分PNH、aHUS患者，但由于部分患者C5的多态性(Arg885突变型)，导致患者对二者不响应。因此，目前仍需要开发新型的针对补体蛋白C5的结合剂，例如抗体，特别是适用于在具有补体蛋白C5多态性的患者中作为替代或更佳的相关适应症治疗药物。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合人C5蛋白的高亲和力抗体，其不但能够有效阻断C5裂解为C5a和C5b，而且对R885多态性人群有效。
针对上述技术问题，本发明的目的是提供一种特异性结合人C5蛋白的抗体或其功能片段，并提供其用途。其中，本发明所述的抗体的片段涵盖抗体的各种功能性片段，例如其抗原结合部分，如Fab、F(ab’) 2或scFv片段。
本发明的技术方案如下：
一方面，本发明提供一种抗体或其抗原结合片段，所述抗体或其抗原结合片段在中性pH下结合补体蛋白5(C5)的亲和力比在酸性pH下更高。并且，所述抗体或其抗原结合片段能够有效阻断C5或其R885突变体裂解为C5a和C5b。
根据序列结构域的组成，本发明提供的抗体或其抗原结合片段包含重链可变区(HCVR)和轻链可变区(LCVR)，所述重链可变区和轻链可变区包含选自以下的重链CDR和轻链CDR的组合：
(1)依次示于SEQ ID NO:47、48、49的HCDR1(DYHLD)、HCDR2 (YIDPDNGGTFYNQKFKG)、HCDR3(WHDYAPSFAY)；和，依次示于SEQ ID NO:50、51、52的LCDR1(HASQNINVWLS)、LCDR2(KASNLHT)、LCDR3(QQGQSYPLT)；
(2)依次示于SEQ ID NO:47、97、49的HCDR1(DYHLD)、HCDR2(YIDPDTGGTFYNQKFKG)、HCDR3(WHDYAPSFAY)；和，依次示于SEQ ID NO:50、51、52的LCDR1(HASQNINVWLS)、LCDR2(KASNLHT)、LCDR3(QQGQSYPLT)；
(3)依次示于SEQ ID NO:65、66、67的HCDR1(NFYLH)、HCDR2(WIYPENLSTKYNDKFKD)、HCDR3(SHYNDYLTGAMDH)；和，依次示于SEQ ID NO:68、69、70的LCDR1(RASKSISKYLA)、LCDR2(SGSTLQF)、LCDR3(QQHDQYPWT)；
(4)依次示于SEQ ID NO:65、98、67的HCDR1(NFYLH)、HCDR2(WIYPENPSTKYNDKFKD)、HCDR3(SHYNDYLTGAMDH)；和，依次示于SEQ ID NO:68、69、70的LCDR1(RASKSISKYLA)、LCDR2(SGSTLQF)、LCDR3(QQHDQYPWT)；
(5)依次示于SEQ ID NO:65、99、100的HCDR1(NFYLH)、HCDR2(WIYPEQLSTKYNDKFKD)、HCDR3(SHFTDYLTGAMDH)；和，依次示于SEQ ID NO:68、69、70的LCDR1(RASKSISKYLA)、LCDR2(SGSTLQF)、LCDR3(QQHDQYPWT)；
(6)依次示于SEQ ID NO:65、99、100的HCDR1(NFYLH)、HCDR2(WIYPEQLSTKYNDKFKD)、HCDR3(SHFTDYLTGAMDH)；和，依次示于SEQ ID NO:68、101、70的LCDR1(RASKSISKYLA)、LCDR2(SGSTLIF)、LCDR3(QQHDQYPWT)；
(7)依次示于SEQ ID NO:71、72、73的HCDR1(DIYIH)、HCDR2(RIDPASGHTEYDPKFQA)、HCDR3(EDYEGIGY)；和，依次示于SEQ ID NO:74、75、76的LCDR1(RASQNVDTHVA)、LCDR2(LASYRYS)、LCDR3(QQYNTYPLT)；
(8)依次示于SEQ ID NO:77、78、79的HCDR1(DAWMD)、HCDR2(AIRNKTNNHATYYTESVKG)、HCDR3(QGDGYYVRFAY)；和，依次示于SEQ ID NO:80、81、82的LCDR1(RASENTYSYLA)、LCDR2(DAKTLAE)、LCDR3(QHHYGTPYT)。
按照本领域公知的抗体中重链可变区、轻链可变区的结构域组成，本发 明的抗体或其抗原结合片段还包含框架区(Framework Region；FR)，该重链可变区或轻链可变区以FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4的顺序或以FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4的顺序包含上述结构域组分。
优选地，在本发明提供的抗体或其抗原结合片段中，所述重链可变区包含与示于SEQ ID NO:1、9、11、13、23、25、26、27、28、30、32、33、35、37、39或41的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:2、10、12、14、24、29、31、34、36、38、40或42的氨基酸序列具有至少75％同一性的氨基酸序列。
更优选地，在本发明提供的抗体或其抗原结合片段中，所述重链可变区包含与示于SEQ ID NO:1、23、25、26或27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:2或24的氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含与示于SEQ ID NO:9、28、30、32或33的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:10、29、31或34的氨基酸序列具有至少75％同一性的氨基酸序列；或者
所述重链可变区包含与示于SEQ ID NO:11、35或37的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:12、36或38的氨基酸序列具有至少75％同一性的氨基酸序列；或者，
所述重链可变区包含与示于SEQ ID NO:13、39或41的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:14、40或42的氨基酸序列具有至少75％同一性的氨基酸序列。
根据本发明的具体实施方式，所述抗体或其抗原结合片段中：
(1)重链可变区：包含与示于SEQ ID NO:1的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:2的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)重链可变区：包含与示于SEQ ID NO:23的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)重链可变区：包含与示于SEQ ID NO:25的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的 氨基酸序列具有至少75％同一性的氨基酸序列；
(4)重链可变区：包含与示于SEQ ID NO:26的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)重链可变区：包含与示于SEQ ID NO:27的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)重链可变区：包含与示于SEQ ID NO:9的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:10的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)重链可变区：包含与示于SEQ ID NO:28的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:29的氨基酸序列具有至少75％同一性的氨基酸序列；
(8)重链可变区：包含与示于SEQ ID NO:30的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:31的氨基酸序列具有至少75％同一性的氨基酸序列；
(9)重链可变区：包含与示于SEQ ID NO:32的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:31的氨基酸序列具有至少75％同一性的氨基酸序列；
(10)重链可变区：包含与示于SEQ ID NO:33的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:31的氨基酸序列具有至少75％同一性的氨基酸序列；
(11)重链可变区：包含与示于SEQ ID NO:33的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:34的氨基酸序列具有至少75％同一性的氨基酸序列；
(12)重链可变区：包含与示于SEQ ID NO:11的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:12的氨基酸序列具有至少75％同一性的氨基酸序列；
(13)重链可变区：包含与示于SEQ ID NO:35的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:36的氨基酸序列具有至少75％同一性的氨基酸序列；
(14)重链可变区：包含与示于SEQ ID NO:37的氨基酸序列具有至少 75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:38的氨基酸序列具有至少75％同一性的氨基酸序列；
(15)重链可变区：包含与示于SEQ ID NO:13的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:14的氨基酸序列具有至少75％同一性的氨基酸序列；
(16)重链可变区：包含与示于SEQ ID NO:39的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:40的氨基酸序列具有至少75％同一性的氨基酸序列；
(17)重链可变区：包含与示于SEQ ID NO:41的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:42的氨基酸序列具有至少75％同一性的氨基酸序列。
在本发明的上下文中，“至少75％同一性”为75％至100％之间的任何百分比数字的同一性，例如75％、80％、85％、90％，甚至91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。可选地，所述“至少75％同一性”导致的氨基酸序列的至多25％差异可存在于本发明的抗体或其抗原结合片段的重链可变区或轻链可变区中CDR以外的任意框架区中，或者存在于重链可变区和轻链可变区以外的任意结构域或序列中。所述差异可以由任何位置的氨基酸缺失、添加或置换产生，其中置换可以是保守置换或非保守置换。
进一步地，本发明的抗体或其抗原结合片段中的重链可变区和轻链可变区分别包含：
(1)示于SEQ ID NO:1和SEQ ID NO:2的氨基酸序列；
(2)示于SEQ ID NO:23和SEQ ID NO:24的氨基酸序列；
(3)示于SEQ ID NO:25和SEQ ID NO:24的氨基酸序列；
(4)示于SEQ ID NO:26和SEQ ID NO:24的氨基酸序列；
(5)示于SEQ ID NO:27和SEQ ID NO:24的氨基酸序列；
(6)示于SEQ ID NO:9和SEQ ID NO:10的氨基酸序列；
(7)示于SEQ ID NO:28和SEQ ID NO:29的氨基酸序列；
(8)示于SEQ ID NO:30和SEQ ID NO:31的氨基酸序列；
(9)示于SEQ ID NO:32和SEQ ID NO:31的氨基酸序列；
(10)示于SEQ ID NO:33和SEQ ID NO:31的氨基酸序列；
(11)示于SEQ ID NO:33和SEQ ID NO:34的氨基酸序列；
(12)示于SEQ ID NO:11和SEQ ID NO:12的氨基酸序列；
(13)示于SEQ ID NO:35和SEQ ID NO:36的氨基酸序列；
(14)示于SEQ ID NO:37和SEQ ID NO:38的氨基酸序列；
(15)示于SEQ ID NO:13和SEQ ID NO:14的氨基酸序列；
(16)示于SEQ ID NO:39和SEQ ID NO:40的氨基酸序列；或
(17)示于SEQ ID NO:41和SEQ ID NO:42的氨基酸序列。
此外，基于本发明提供的上述重链可变区和轻链可变区的具体氨基酸序列的组合，本领域技术人员可以常规地确定其中包含的重链CDR和轻链CDR的氨基酸序列，以本领域其他已知方法确定得到的重轻链CDR及其组合也被涵盖在本发明的范围内。例如，在本发明的实施例中，划分的可变区氨基酸序列中的CDR按照Kabat定义。
本发明提供的所述抗体或其抗原结合片段结合哺乳动物C5，优选灵长类动物C5，进一步优选人或食蟹猴(cynomolgus monkey)C5，特别是人C5。
根据本发明的具体实施方式，本发明的抗体或其抗原结合片段能够以高亲和力结合人C5，对人C5的裂解为C5a和C5b具有明显的抑制作用，显著抑制补体依赖性溶血，阻断补体依赖性细胞毒性，且对食蟹猴C5具有显著的结合亲和力；并且，对人C5(R885)突变体也具有高亲和力，抑制C5变体R885裂解为C5a和C5b。此外，本发明的抗体或其抗原结合片段具有pH依赖性结合C5抗原的特点，亲和力在酸性条件低于中性条件。
优选地，本发明提供的抗体可以为鼠源抗体、嵌合抗体或者完全或部分人源化抗体；提供的抗原结合片段可以为所述抗体的能够特异性结合抗原例如人C5或其R885H突变体的任何片段，例如scFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv片段。
优选地，本发明提供的抗体为单克隆抗体或单链抗体。
优选地，本发明提供的抗体或其抗原结合片段还包含人或鼠的恒定区，优选包含鼠或人的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体或其抗原结合片段包含重链和轻链，例如两条重链和轻链。更优选地，所述抗体或其抗原结合片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
根据本发明的具体实施方式，本发明提供的抗体为单克隆抗体，优选为人源化的单克隆抗体。优选地，所述单克隆抗体的重链恒定区为IgG1型， 轻链恒定区为κ型。例如，所述单克隆抗体的重链恒定区包含示于SEQ ID NO:45的氨基酸序列；所述单克隆抗体的轻链恒定区包含示于SEQ ID NO:46的氨基酸序列。
另一方面，本发明提供一种核酸分子，其包含编码本发明所述的抗体或其抗原结合片段中包含的重链CDR、轻链CDR、重链可变区、轻链可变区、重链或轻链的核苷酸序列。
本发明的核酸分子可以被克隆到载体中，进而转化或转染宿主细胞。因此，还一方面，本发明提供一种载体，其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或者以任何方式进入宿主细胞内，用于保存或表达抗体等目的。因此，又一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
本发明提供的抗体可以利用本领域已知的任何方法获得。例如，在允许本发明提供的宿主细胞表达所述抗体的重链和轻链以组装成所述抗体的情况下，培养所述宿主细胞。任选地，所述方法还包括回收产生的抗体的步骤。在本发明提供的抗体或其抗原结合片段的如上结构域氨基酸序列的情况下，获得抗体或其抗原结合片段的手段是本领域公知的。
本发明提供的抗体或其抗原结合片段、核酸分子、载体或宿主细胞可以被包含在组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种组合物，所述组合物包含本发明提供的抗体或其抗原结合片段、核酸分子、载体和/或宿主细胞。优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
再一方面，本发明还提供所述抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物在制备药物中的用途，所述药物用于预防或治疗由补体(例如补体蛋白C5)介导的疾病。优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性。
另一方面，本发明还提供一种预防或治疗由补体(例如补体蛋白C5)介导的疾病的方法，所述方法包括给有此需要的受试者施用本发明的抗体或 其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物。优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性。其中，所述受试者为哺乳动物，优选灵长类动物，更优选人。
再一方面，本发明还提供所述抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物在制备用于诊断疾病的试剂中的用途，所述疾病由补体(例如补体蛋白C5)介导。优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性。
由此相应地，本发明还提供一种诊断疾病的方法，所述方法包括使本发明的抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物与来自受试者的样品相接触，所述疾病由补体(例如补体蛋白C5)介导。优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性。其中，所述受试者为哺乳动物，优选灵长类动物，更优选人。
由此相应地，又一方面，本发明提供一种试剂盒，所述试剂盒包括本发明的抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物。所述试剂盒可以用于治疗或诊断用途。
特别是，本发明提供的抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物可用于与C5Arg885突变型相关的疾病的预防、治疗或诊断。
本发明中，利用重组补体蛋白C5成功免疫小鼠后取脾细胞，利用杂交瘤技术建立抗体库，筛选获得与C5具有高亲和力并且具有生物学活性的抗C5单克隆抗体。获得多个先导抗体分子，不仅和C5具有高亲和力，而且具有溶血抑制活性。基于鼠源先导抗体分子进行重组表达人鼠嵌合抗体及人源化抗体，最终获得与鼠源抗体具有相似生物学活性的人源化抗体分子。体外药效学研究表明，本发明的抗体能够有效阻断人血清介导的溶血活性、抑制补体依赖性细胞毒活性。本发明的抗体对人C5(R885)突变体也具有高亲和力，抑制C5变体R885裂解为C5a和C5b。此外，本发明的抗体具有pH依赖性结合C5抗原的特点，亲和力在酸性条件低于中性条 件，实验证明，其解离速率常数Koff和亲和力常数KD在pH5.8和pH7.4条件下的比值均大于对照抗体依库珠单抗，表明本发明的抗体比依库珠单抗更容易在酸性条件下与抗原C5解离。并且，表位竞争实验表明本发明的抗体与依库珠单抗具有不同的抗原结合表位，为具有不同结合特征的新型候选抗体药物。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了杂交瘤细胞株上清的ELISA鉴定结果。
图2显示了杂交瘤细胞株上清对补体依赖型溶血的抑制作用。
图3显示了嵌合抗体与人C5的结合活性。
图4显示了嵌合抗体与食蟹猴C5的结合活性。
图5显示了嵌合抗体与人C5(R885H)突变体的结合活性。
图6显示了嵌合抗体、人源化抗体与人C5的结合活性。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
人C5蛋白：购自义翘神州，货号13416-H18H，NP_001726.2(Gln19-Cys1676)；
hIgG1亚型的重链恒定区：SEQ ID NO:45；
hIgG-Kappa亚型的轻链恒定区：SEQ ID NO:46。
实施例1 人补体蛋白C5对Balb/c小鼠的免疫
抗原重组人补体蛋白C5(Gln19-Cys1676)购自义翘神州(货号13416-H18H)，该蛋白由人293细胞(HEK293)重组表达，C端含有组氨酸标签，N端含有Flag标签。
参考Antibodies:A Laboratory Manual,Second Edition(Edward A.Greenfield 2012)，以14天为间隔共计42天的过程免疫8周龄Balb/c小鼠。将人C5蛋白在完全或不完全弗氏佐剂中乳化，将其以单侧的方式注射于小 鼠颈背部、尾根部、腹股沟3处皮下组织和腹膜腔内。在免疫第35天尾静脉采血，用ELISA方法检测抗体滴度后，取免疫小鼠脾细胞与骨髓瘤细胞融合。
实施例2 抗人C5单克隆抗体杂交瘤细胞株的筛选、鉴定
取人C5蛋白免疫Balb/c小鼠脾脏细胞，与骨髓瘤细胞使用PEG或者电融合方法进行融合，将融合后的杂交瘤细胞以1x10 7个/孔密度接种于含HAT培养基中铺于384孔板中，培养5天后换含HT培养基继续培养2-3天筛选杂交瘤细胞。于384孔板中培养7-10天后，取细胞上清进行ELISA实验，筛选能够分泌抗人C5抗体的杂交瘤母克隆。
用ELISA法筛选人C5结合剂的方法如下：将人C5蛋白用PBS缓冲液稀释至1μg/ml，每孔100μl包被于96孔板(Microwell 96F 167008，Thermo)4℃孵育过夜；次日取出96孔板，用PBST(含0.5％吐温20)洗板，每次浸润1min后彻底甩干残余水分。样品孔中加入200μl的含2％BSA的PBST，置于37℃封闭1h；然后用PBST洗板，并甩干孔中水分。向96孔板中加入待测样品100μl 4℃孵育过夜。取出96孔板后用PBST洗板后每孔加入1:20000稀释的二抗羊抗鼠偶联辣根过氧化物酶100μl，并置于37℃孵育1h。再用PBST洗5次，每孔加入100μl Substrate Solution(Invitrogen)，于37℃孵育10min；每孔加入2N硫酸50μl终止反应后于酶标仪(M5e，Molecular Device)450nm波长处检测吸光度。部分结果见图1中的图1A、图1B。
随后将ELISA阳性的克隆从384孔板转到96孔板扩大培养，然后取30μl杂交瘤细胞培养上清进行溶血实验检测，评估补体依赖性溶血的抑制作用。方法如下：将人血清补体(Normal Human Serum Complement,Quidel Corporator,A112)使用PBS稀释4倍，制备人补体母液，30μl/孔加于96孔U型板中，并加入30μl杂交瘤细胞培养上清；10％绵羊血红细胞使用PBS洗涤，3000rpm离心3min，重复洗涤3次，30μl/孔加入前面加有人补体和杂交瘤细胞培养上清的96孔U型板中，轻轻混匀；将96孔板放置于37℃孵育1h；孵育完成后将96孔板置于96孔板离心机以3000rpm转速离心3min；取60μl离心上清液加入到新的96孔板中，用酶标仪测OD405吸光度值。部分结果见图2，发现克隆6A2和1P5的上清无溶血抑制活性，5N19等克隆的上清有溶血抑制活性。
实施例3 抗人C5单克隆抗体序列测定
将分泌抗人C5抗体且有抑制补体依赖性溶血活性的杂交瘤母克隆采用有限稀释法加入96孔板中，第2-3天后显微镜下观察并标记单克隆细胞，第7天后通过ELISA实验和溶血实验筛选能够分泌抗人C5单克隆抗体的单克隆杂交瘤细胞。
将筛选的单克隆杂交瘤细胞扩大培养后，按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用5×PrimeScript RT Master Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列；利用PCR clean-up Gel extraction试剂盒(Macherey-Nagel)纯化PCR扩增产物；按照pmd19T Simple Vector Kit试剂盒(Takara)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。
获取鼠源抗体(以克隆编号命名)的可变区序列并分析，见表1。
表1 鼠源抗体可变区及其CDR(按Kabat定义)的序列
Figure PCTCN2021090185-appb-000001
实施例4 重组嵌合抗体表征
将鼠源抗体的完整轻、重链可变区克隆进入重组表达载体，重链恒定区 均选用hIgG1亚型，轻链均选用hIgG-Kappa亚型，得到的重组嵌合抗体命名为“鼠源抗体简称-xi”，用于抗体生产和纯化。对照抗体依库珠单抗，重轻链可变区序列(SEQ ID NO:43；SEQ ID NO:44)全基因合成后也构建入hIgG1和hIgG-Kappa亚型的嵌合表达载体，本文中命名为“Ref1”。
通过下述方法对上述重组嵌合抗体蛋白进行表达和纯化：转染前一天HEK293-6E细胞以1.0x10 6个细胞/ml密度用F17培养基稀释传代。24小时后，细胞密度达到1.8-2.0x10 6个细胞/ml可用于转染。以每1.0x10 6个细胞含有1μg DNA(重链和轻链质粒比例为1:1.5)、4μg PEI(聚乙烯亚胺-线形，Polysciences,24885-2)、0.4μl 293Expression MAX-1(Acro,EXP-711)的转染复合物，用Optimal MEM培养基(Gibco,31985-070)稀释并在室温下孵育15min后加入细胞中。在5％CO 2、37℃和125rpm振荡速度的条件下，在培养瓶中培养转染细胞。转染22-26h后加入1％蛋白胨培养基。转染后第6天收集培养的细胞并以3000rpm离心30min保留上清液。将培养上清用Mabselect Sure柱纯化，洗脱的抗体蛋白透析至PBS并在-80℃下长期保存。用SDS-PAGE检测抗体纯度为95％以上的纯化抗体可用于后续实验。
采用Fortebio公司Octet RED 96仪器分析嵌合抗体的亲和力。配制缓冲液1(磷酸盐缓冲液(PBS)+0.1％BSA+0.02％吐温20(Tween 20)+0.05％Priclin300，pH7.4)，用于稀释抗原和各抗体。在pH7.4条件下，使用抗人IgG Fc捕获(AHC)传感器，先结合浓度稀释为100nM的嵌合抗体，再在pH 7.4条件下结合缓冲液1稀释的浓度为50nM抗原人C5蛋白，随后在pH 7.4的缓冲液1中解离。AHC传感器通过甘氨酸脉冲再生可多次使用。用Fortebio的Data analysis 10.0处理数据，结果见图3中图3A、图3B、图3C。
为评估嵌合抗体对人C5蛋白裂解的抑制作用，进行了补体依赖性溶血实验。将人血清补体使用PBS稀释4倍，制备人补体母液，30μl/孔加于96孔U型板中备用；抗人C5嵌合抗体使用PBS稀释至100μg/mL起始，然后2倍稀释，设置8个梯度，两个复孔，30μl/孔加于带有人补体的96孔U型板中，室温放置1h；10％绵羊血红细胞使用PBS洗涤，再以3000rpm转速离心3min，重复洗涤3次，30μl/孔加入前面加有人补体和抗C5抗体的96孔U型板中，轻轻混匀；将96孔板放置于37℃孵育1h；孵育完成后将96孔板置于96孔板离心机3000rpm离心3min；取60μL离心上清液加入到新的96孔板中，用酶标仪测OD405吸光度值。最后使用酶标仪自带softMax软件处理数据，使用四参数拟合法计算R2和IC50值。结果见表2。
表2 溶血实验检测嵌合抗体对人C5蛋白裂解的抑制作用
样品名称 IC50(μg/ml) R2
Ref1 10.62 1.000
10L15-xiIgG 7.605 1.000
11E14-xiIgG 7.676 1.000
13F21-xiIgG 7.408 1.000
7H3-xiIgG 8.346 0.999
10M9-xiIgG 8.006 0.999
6K23-xiIgG 8.54 0.999
11D5-xiIgG 6.934 0.997
8K13-xiIgG 7.871 0.999
7B5-xiIgG 7.516 0.998
4E5-xiIgG 9.323 1.000
9B3-xiIgG 9.271 1.000
为进一步验证嵌合抗体对补体活性的抑制作用，进行了补体依赖性细胞毒性(CDC)阻断分析实验。利用抗CD20单克隆抗体利妥昔单抗(Rituximab)在一定条件下致Raji细胞发生CDC反应为基础，观察使用抗C5抗体对此反应是否有阻断作用。首先，将利妥昔单抗使用稀释液(RPMI1640培养基)稀释至10μg/mL，50μL/孔加入96孔平底板中；将待测抗C5抗体使用稀释液稀释至10μg/mL，然后2倍稀释，设置9个梯度，2个复孔，50μL/孔加入96孔平底板中；Raji细胞计数调整密度至2x10 6个/mL，50μL/孔加入96孔平底板中；人血清补体稀释5倍，20μL/孔加入96孔平底板中；37℃孵育2.5h；30μL/孔CCK-8试剂加入96孔平底板中，37℃继续孵育3h；通过酶标仪检测450nm吸光度。通过吸光值采用softmax pro软件进行4参数拟合曲线，计算样品的EC50值。每块板可以测得3个样品的EC50，其中1个为对照抗体Ref1作为内参样品，计算每种待测嵌合抗体与对照抗体EC50的比值即为效价，结果见表3。
表3 嵌合抗体的补体依赖性细胞毒性阻断分析
样品名称 R2 EC50(μg/ml)
Ref1 1 2.598
10L15-xiIgG 0.989 2.42
11E14-xiIgG 0.999 2.715
13F21-xiIgG 0.983 2.284
7H3-xiIgG 0.988 3.379
10M9-xiIgG 0.991 2.442
6K23-xiIgG 0.99 3.31
11D5-xiIgG 0.999 1.628
8K13-xiIgG 0.995 2.228
7B5-xiIgG 0.997 2.153
4E5-xiIgG 0.99 3.825
9B3-xiIgG 0.997 4.422
实施例5 抗体的种属交叉性和R885H突变体结合活性
用Octet RED 96仪器分析嵌合抗体与食蟹猴(cynomolgus monkey)C5的交叉反应性。参考实施例4进行，区别仅在于将抗原人C5蛋白替换为抗原食蟹猴C5蛋白(Acro，货号CO5-C52Hx)。结果见图4中图4A、图4B、图4C、图4D、图4E。结果表明，对照抗体Ref1与食蟹猴C5蛋白亲和力很低，而其它待测分子与食蟹猴C5蛋白均有高亲和力。
用Octet RED 96仪器分析嵌合抗体与人C5(R885H)突变体的结合活性。同样参考实施例4进行，区别仅在于将抗原人C5蛋白替换为抗原人C5蛋白(R885H)突变体(Acro，货号CO5-H52Hx)。结果见图5中图5A、图5B、图5C、图5D、图5E。结果表明，对照抗体Ref1与人C5(R885H)蛋白亲和力很低，而其它待测分子与人C5(R885H)蛋白均有高亲和力。
实施例6 抗体的表位分析
采用Fortebio公司Octet RED 96仪器分析嵌合抗体与对照抗体Ref1的表位竞争关系。配制缓冲液1(磷酸盐缓冲液(PBS)+0.1％BSA+0.02％吐温20(Tween 20)+0.05％Priclin300，pH7.4)，用于稀释抗原和各抗体。在pH 7.4条件下，使用NTA芯片，先结合浓度为50nM抗原人C5蛋白，再结合浓度为100nM的对照抗体Ref1，随后结合浓度为100nM的嵌合抗体。用Fortebio的Data analysis 10.0处理数据，结果见表4，13F21、8K13、4E5与对照抗体Ref1不竞争表位，而11D5与Ref1表位有竞争关系，结合种属交 叉和R885H突变体的结合特点，表明11D5和Ref1有相近表位。
表4 嵌合抗体与Ref1的抗原表位竞争关系
克隆号 竞争比例 竞争关系
11D5-xiIgG 27.31％ 竞争
8K13-xiIgG 106.15％ 不竞争
13F21-xiIgG 92.85％ 不竞争
4E5-xiIgG 93.56％ 不竞争
实施例7 抗体人源化改造及人源化抗体的表征
对鼠源抗体13F21、11D5、8K13、4E5进行人源化改造。在对鼠源抗体重链序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)后，通过分析搜索已知人源抗体序列，选择与鼠源抗体最为相似接近的人源抗体重链序列，如IGHV1-3*01，选择其抗体框架区序列作为模板。将鼠源抗体重链CDR嵌入到人源抗体框架区，生成人源化抗体重链序列(重链版本0)。随后，对可能参与抗原抗体结合的鼠源框架区个别氨基酸位点进行回复，生成人源化抗体重链序列(版本1，2，3)。同样过程，生成人源化抗体轻链序列(版本0，1，2……)。将设计合成的人源化抗体轻重链共转染293细胞，重组表达人源化抗体(版本命名方式例如：重链版本0+轻链版本0共表达，即为H0L0)。各人源化抗体的重链和轻链可变区及CDR区序列见表5。
表5 人源化抗体可变区和CDR(按Kabat定义)序列
Figure PCTCN2021090185-appb-000002
Figure PCTCN2021090185-appb-000003
采用Fortebio公司Octet RED 96仪器分析比较嵌合抗体和人源化抗体的亲和力。同样参照实施例4进行，人源化抗体也为100nM的检测浓度。结果见图6中图6A、图6B、图6C、图6D、图6E和图6F。
为评估人源化抗体对人C5裂解的抑制作用，部分抗体进行了补体依赖性溶血实验。参考实施例4进行，结果见表6。
表6 溶血实验检测人源化抗体对人C5裂解的抑制作用
样品名称 IC50(ug/ml) R2
Ref1 8.728 0.999
13F21-H0L0 6.471 0.994
13F21-H1L0 6.858 0.993
13F21-H3L1 7.962 0.999
13F21-H4L1 6.702 0.990
13F21-H10L1 8.846 0.999
13F21-H11L1 8.69 0.999
为进一步验证人源化抗体对补体活性的抑制作用，进行了补体依赖性细胞毒性(CDC)阻断分析实验。参考实施例4进行，结果见表7。
表7 人源化抗体的补体依赖性细胞毒性阻断分析
样品名称 EC50(ug/ml) R2
Ref1 2.627 0.995
13F21-H0L0 2.422 0.987
13F21-H1L0 2.632 0.993
13F21-H3L1 28.34 0.998
13F21-H4L1 4.273 0.997
13F21-H10L1 2.269 0.999
13F21-H11L1 2.427 0.997
实施例8 Biacore系统检测单克隆抗体与人C5在不同pH条件下的结合动力学(K on，K off)和亲和力常数K D
采用GE公司BIAcore仪器S200测定抗体抗原相互作用力。参考GE公司Human antibody capture kit商品试剂盒(货号BR-1008-39，Lot 10261753)操作说明，分别在pH7.4和pH5.8条件下测定抗体与人C5在不同pH条件下的结合动力学(K on，K off)和亲和力常数K D。首先在传感芯片CM4分析通道和对照样品通道都饱和偶联最大量抗人Fc抗体，然后在分析通道流过7.5μg/ml抗体，使纯化的单克隆抗体均匀分布，最后在分析通道和样品通道一起流过梯度稀释的抗原样品(起始浓度50nM，1:2稀释6-8个浓度点，并且设定0.741nm浓度点重复)，测定抗体抗原结合后发生的光反应值。随后，经仪器软件拟合分析，最终得到抗体的结合常数Kon和解离速率常数Koff，以及亲和力常数KD。结果见表8，中性条件下，13F21-H4L1、13F21-H10L1、13F21-H11L1亲和力优于对照抗体Ref1；13F21-H3L1、13F21-H4L1、13F21-H10L1、13F21-H11L1、4E5-H9L1和4E5-H9L3解离速率常数Koff和亲和力常数KD在pH5.8和pH7.4条件下的比值均大于对照抗体Ref1，表明这些抗体比Ref1更容易在酸性条件下与抗原C5蛋白解离。
表8 人源化抗体与抗原在中性和酸性条件下的结合动力学和亲和力常数
Figure PCTCN2021090185-appb-000004
Figure PCTCN2021090185-appb-000005
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (16)

  1. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段在中性pH下结合补体蛋白5(C5)的亲和力比在酸性pH下更高。
  2. 根据权利要求1所述的抗体，其中所述抗体或其抗原结合片段能够有效阻断C5或其R885突变体裂解为C5a和C5b。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区(HCVR)和轻链可变区(LCVR)，所述重链可变区和轻链可变区包含选自以下的重链CDR和轻链CDR的组合：
    (1)依次示于SEQ ID NO:47、48、49的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:50、51、52的LCDR1、LCDR2、LCDR3；
    (2)依次示于SEQ ID NO:47、97、49的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:50、51、52的LCDR1、LCDR2、LCDR3；
    (3)依次示于SEQ ID NO:65、66、67的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:68、69、70的LCDR1、LCDR2、LCDR3；
    (4)依次示于SEQ ID NO:65、98、67的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:68、69、70的LCDR1、LCDR2、LCDR3；
    (5)依次示于SEQ ID NO:65、99、100的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:68、69、70的LCDR1、LCDR2、LCDR3；
    (6)依次示于SEQ ID NO:65、99、100的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:68、101、70的LCDR1、LCDR2、LCDR3；
    (7)依次示于SEQ ID NO:71、72、73的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:74、75、76的LCDR1、LCDR2、LCDR3；
    (8)依次示于SEQ ID NO:77、78、79的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO:80、81、82的LCDR1、LCDR2、LCDR3。
  4. 根据权利要求1至3中任一项所述的抗体或其抗原结合片段，其特征在于，所述重链可变区包含与示于SEQ ID NO:1、9、11、13、23、25、26、27、28、30、32、33、35、37、39或41的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:2、10、12、14、24、29、31、34、36、38、40或42的氨基酸序列具有至少75％同一性的氨基酸序列；
    优选地，所述抗体或其抗原结合片段中，所述重链可变区包含与示于 SEQ ID NO:1、23、25、26或27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:2或24的氨基酸序列具有至少75％同一性的氨基酸序列；或者，
    所述重链可变区包含与示于SEQ ID NO:9、28、30、32或33的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:10、29、31或34的氨基酸序列具有至少75％同一性的氨基酸序列；或者
    所述重链可变区包含与示于SEQ ID NO:11、35或37的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:12、36或38的氨基酸序列具有至少75％同一性的氨基酸序列；或者，
    所述重链可变区包含与示于SEQ ID NO:13、39或41的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含与示于SEQ ID NO:14、40或42的氨基酸序列具有至少75％同一性的氨基酸序列。
  5. 根据权利要求1至4中任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段中：
    (1)重链可变区：包含与示于SEQ ID NO:1的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:2的氨基酸序列具有至少75％同一性的氨基酸序列；
    (2)重链可变区：包含与示于SEQ ID NO:23的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
    (3)重链可变区：包含与示于SEQ ID NO:25的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
    (4)重链可变区：包含与示于SEQ ID NO:26的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
    (5)重链可变区：包含与示于SEQ ID NO:27的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列；
    (6)重链可变区：包含与示于SEQ ID NO:9的氨基酸序列具有至少75％ 同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:10的氨基酸序列具有至少75％同一性的氨基酸序列；
    (7)重链可变区：包含与示于SEQ ID NO:28的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:29的氨基酸序列具有至少75％同一性的氨基酸序列；
    (8)重链可变区：包含与示于SEQ ID NO:30的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:31的氨基酸序列具有至少75％同一性的氨基酸序列；
    (9)重链可变区：包含与示于SEQ ID NO:32的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:31的氨基酸序列具有至少75％同一性的氨基酸序列；
    (10)重链可变区：包含与示于SEQ ID NO:33的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:31的氨基酸序列具有至少75％同一性的氨基酸序列；
    (11)重链可变区：包含与示于SEQ ID NO:33的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:34的氨基酸序列具有至少75％同一性的氨基酸序列；
    (12)重链可变区：包含与示于SEQ ID NO:11的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:12的氨基酸序列具有至少75％同一性的氨基酸序列；
    (13)重链可变区：包含与示于SEQ ID NO:35的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:36的氨基酸序列具有至少75％同一性的氨基酸序列；
    (14)重链可变区：包含与示于SEQ ID NO:37的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:38的氨基酸序列具有至少75％同一性的氨基酸序列；
    (15)重链可变区：包含与示于SEQ ID NO:13的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:14的氨基酸序列具有至少75％同一性的氨基酸序列；
    (16)重链可变区：包含与示于SEQ ID NO:39的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:40的 氨基酸序列具有至少75％同一性的氨基酸序列；或
    (17)重链可变区：包含与示于SEQ ID NO:41的氨基酸序列具有至少75％同一性的氨基酸序列；和，轻链可变区：包含与示于SEQ ID NO:42的氨基酸序列具有至少75％同一性的氨基酸序列。
  6. 根据权利要求1至5中任一项所述的抗体或其抗原结合片段，其特征在于，所述重链可变区和轻链可变区分别包含：
    (1)示于SEQ ID NO:1和SEQ ID NO:2的氨基酸序列；
    (2)示于SEQ ID NO:23和SEQ ID NO:24的氨基酸序列；
    (3)示于SEQ ID NO:25和SEQ ID NO:24的氨基酸序列；
    (4)示于SEQ ID NO:26和SEQ ID NO:24的氨基酸序列；
    (5)示于SEQ ID NO:27和SEQ ID NO:24的氨基酸序列；
    (6)示于SEQ ID NO:9和SEQ ID NO:10的氨基酸序列；
    (7)示于SEQ ID NO:28和SEQ ID NO:29的氨基酸序列；
    (8)示于SEQ ID NO:30和SEQ ID NO:31的氨基酸序列；
    (9)示于SEQ ID NO:32和SEQ ID NO:31的氨基酸序列；
    (10)示于SEQ ID NO:33和SEQ ID NO:31的氨基酸序列；
    (11)示于SEQ ID NO:33和SEQ ID NO:34的氨基酸序列；
    (12)示于SEQ ID NO:11和SEQ ID NO:12的氨基酸序列；
    (13)示于SEQ ID NO:35和SEQ ID NO:36的氨基酸序列；
    (14)示于SEQ ID NO:37和SEQ ID NO:38的氨基酸序列；
    (15)示于SEQ ID NO:13和SEQ ID NO:14的氨基酸序列；
    (16)示于SEQ ID NO:39和SEQ ID NO:40的氨基酸序列；或
    (17)示于SEQ ID NO:41和SEQ ID NO:42的氨基酸序列。
  7. 根据权利要求1至6中任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段结合哺乳动物C5，优选灵长类动物C5，进一步优选人或食蟹猴C5，特别是人C5；
    优选地，所述抗体为鼠源抗体、嵌合抗体或者完全或部分人源化抗体；所述抗原结合片段为所述抗体的scFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv片段；
    优选地，所述抗体为单克隆抗体或单链抗体；优选地，所述抗体或其抗原结合片段还包含恒定区，优选包含鼠或人的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体或其抗原结合片段包含重链和轻链；
    更优选地，所述抗体或其抗原结合片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区；
    进一步优选地，所述抗体为单克隆抗体，优选为人源化的单克隆抗体；
    优选地，所述单克隆抗体的重链恒定区为IgG1型，轻链恒定区为κ型。
  8. 一种核酸分子，其包含编码权利要求1至7中任一项所述的抗体或其抗原结合片段中包含的重链CDR、轻链CDR、重链可变区、轻链可变区、重链或轻链的核苷酸序列。
  9. 一种载体，其包含权利要求8所述的核酸分子。
  10. 一种宿主细胞，其包含权利要求8所述的核酸分子和/或权利要求9所述的载体，或者所述宿主细胞被权利要求8所述的核酸分子和/或权利要求9所述的载体转化或转染。
  11. 一种组合物，其包含权利要求1至7中任一项所述的抗体或其抗原结合片段、权利要求8所述的核酸分子、权利要求9所述的载体和/或权利要求10所述的宿主细胞；
    优选地，所述组合物为药物组合物，其可选地还包含药学上可接受的载体、辅料或赋形剂。
  12. 权利要求1至7中任一项所述的抗体或其抗原结合片段、权利要求8所述的核酸分子、权利要求9所述的载体、权利要求10所述的宿主细胞和/或权利要求11所述的组合物在制备药物中的用途，所述药物用于预防或治疗由补体(例如补体蛋白C5)介导的疾病；
    优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性。
  13. 一种预防或治疗由补体(例如补体蛋白C5)介导的疾病的方法，所述方法包括给有此需要的受试者施用权利要求1至7中任一项所述的抗体或其抗原结合片段、权利要求8所述的核酸分子、权利要求9所述的载体、权利要求10所述的宿主细胞和/或权利要求11所述的组合物；
    优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性；
    其中，所述受试者为哺乳动物，优选灵长类动物，更优选人。
  14. 权利要求1至7中任一项所述的抗体或其抗原结合片段、权利要求8 所述的核酸分子、权利要求9所述的载体、权利要求10所述的宿主细胞和/或权利要求11所述的组合物在制备用于诊断疾病的试剂中的用途，所述疾病由补体(例如补体蛋白C5)介导；
    优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性。
  15. 一种诊断疾病的方法，所述方法包括使权利要求1至7中任一项所述的抗体或其抗原结合片段、权利要求8所述的核酸分子、权利要求9所述的载体、权利要求10所述的宿主细胞和/或权利要求11所述的组合物与来自受试者的样品相接触，所述疾病由补体(例如补体蛋白C5)介导；
    优选地，所述疾病为类风湿性关节炎、阵发性睡眠性血红蛋白尿(PNH)、非典型溶血性尿毒症综合征(aHUS)、重症肌无力、视神经性脊髓炎或黄斑变性例如年龄相关性黄斑变性；
    其中，所述受试者为哺乳动物，优选灵长类动物，更优选人。
  16. 一种试剂盒，所述试剂盒包括权利要求1至7中任一项所述的抗体或其抗原结合片段、权利要求8所述的核酸分子、权利要求9所述的载体、权利要求10所述的宿主细胞和/或权利要求11所述的组合物。
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