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WO2022052846A1 - Anti-cd47 antibody and use thereof - Google Patents

Anti-cd47 antibody and use thereof Download PDF

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Publication number
WO2022052846A1
WO2022052846A1 PCT/CN2021/115946 CN2021115946W WO2022052846A1 WO 2022052846 A1 WO2022052846 A1 WO 2022052846A1 CN 2021115946 W CN2021115946 W CN 2021115946W WO 2022052846 A1 WO2022052846 A1 WO 2022052846A1
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李强
武翠
翁仕强
周利
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Ampsource Biopharma Shanghai Inc
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Ampsource Biopharma Shanghai Inc
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/76—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Definitions

  • the present invention belongs to the field of therapeutic monoclonal antibodies, and more particularly, the present invention relates to an antibody against integrin-related protein (CD47); and also relates to the preparation of the antibody for the treatment of various diseases (including tumors and infectious diseases) Use in medicine.
  • CD47 integrin-related protein
  • CD47 also known as integrin-associated protein (IAP)
  • IAP integrin-associated protein
  • IAP integrin-associated protein
  • IgV-like domain at the extracellular amino-terminus
  • 5 transmembrane segments with highly hydrophobic extensions 1 short selection Sexually spliced carboxy-terminal cytoplasmic tail of which the IgV-like domain is its primary ligand binding site.
  • Its ligands include: Signal-regulatory protein ⁇ (SIRP ⁇ ), Thrombospondin (TSP) and Integrins.
  • CD47 is widely expressed on the cell membrane surface and is present on different cells in all tissues, especially leukocytes such as polymorphonuclear leukocytes (PMN), dendritic cells (DC), T cells, red blood cells, placenta, platelets and hematopoietic stem cells (hematopoietic stem cells). normal cells such as stem cells, HSCs).
  • leukocytes such as polymorphonuclear leukocytes (PMN), dendritic cells (DC), T cells, red blood cells, placenta, platelets and hematopoietic stem cells (hematopoietic stem cells).
  • PMN polymorphonuclear leukocytes
  • DC dendritic cells
  • T cells red blood cells
  • placenta placenta
  • platelets hematopoietic stem cells
  • normal cells such as stem cells, HSCs.
  • CD47 is highly expressed in most tumor cells in the human body, and it can be used as a standard for tumor diagnosis and progno
  • cancer stem cells such as leukemia stem cells, LSC
  • CD47 on the surface of tumor cells evades these cells by binding to SIRP ⁇ ligands on the surface of immune cells such as macrophages and DC cells, and delivering an inhibitory signal of "Don't eat me” to them.
  • phagocytosis Kershaw MH et al., Science, 2013, 341:41-42
  • phagocytosis evade the killing effect of T cells on tumor cells caused by the presentation of tumor antigens after phagocytosis (Vonderheide RH, Nature Medicine, 2015, 21: 1122–1123).
  • a few domestic and foreign antibody drugs with the same target are in the phase I-II clinical research stage, such as Forty Seven's Hu5F9-G4, which is in the clinical phase I/II stage, and is used alone to treat acute myeloid leukemia and lymphoma ; with rituximab for B-cell non-Hodgkin lymphoma; or with cetuximab for colorectal cancer.
  • Celgene's CC-90002 is in Phase I clinical trials, targeting solid tumors, multiple myeloma, and non-Hodgkin's lymphoma. Innovent's fully human monoclonal antibody IBI188 is undergoing clinical studies against advanced malignant tumors, including non-Hodgkin's lymphoma and ovarian cancer.
  • CD47 antibodies have been reported to induce hemagglutination of human erythrocytes. Hemagglutination is an example of a homotypic interaction, and treatment with a bivalent CD47 antibody can cause aggregation or agglutination of two CD47-expressing cells.
  • a fully IgG or F(ab') 2 configuration of the CD47 antibody MABL has been reported to induce hemagglutination of red blood cells, and this effect was only attenuated when MABL was changed to scFv or bivalent scFv (see e.g.
  • CD47 antibodies including B6H12, BRC126 and CC2C6, also cause hemagglutination of red blood cells. Therefore, causing hemagglutination is the main limitation of the current CD47-targeting full IgG or F(ab') 2 configuration antibodies in clinical application. Therefore, there is still an urgent need to develop CD47 antibodies that can not only effectively promote the phagocytosis of macrophages but also weakly bind to erythrocytes and do not cause erythrocyte agglutination.
  • the present invention provides an antibody that can specifically recognize and bind CD47 (especially human CD47) with high affinity.
  • the antibodies of the present invention are capable of modulating (eg, blocking, inhibiting, reducing, antagonizing, neutralizing or interfering with) the activity and/or signaling of CD47, and these antibodies do not result in significant hemagglutination of red blood cells.
  • the CD47 antibodies disclosed in the present invention can be used to treat, prevent and/or diagnose various diseases, such as tumors.
  • the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to CD47, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising at least one, two or Three complementarity determining regions (CDRs) selected from the group consisting of:
  • VH heavy chain variable region
  • CDRs Three complementarity determining regions
  • CDR-H1 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37
  • sequence of CDR-H1 contained in the VH or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or add) sequence;
  • CDR-H2 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37
  • CDR-H3 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37
  • sequence of CDR-H3 contained in the VH or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or add) sequence;
  • the light chain variable region (VL) it comprises comprises at least one, two or three complementarity determining regions (CDRs) selected from the group consisting of:
  • CDR-L1 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38
  • sequence of CDR-L1 contained in the VL or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or add) sequence;
  • CDR-L2 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38
  • CDR-L3 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH), and/or the light chain variable region (VL) CDR-L1, CDR-L2 and CDR-L3 are defined by the Kabat or IMGT numbering system.
  • the antibody or antigen-binding fragment thereof of the invention comprises: at least one of the heavy chain variable regions (VH) as set forth in SEQ ID NO: 5, 13, 15, 17 or 19 , two or three CDRs; and/or, at least one, two or three CDRs contained in the variable region (VL) of the light chain as set forth in SEQ ID NO: 6, 14, 16, 18 or 20;
  • VH heavy chain variable regions
  • VL variable region
  • the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and the CDR-L1, CDR-L2 and CDR-L1 contained in the light chain variable region (VL) L3 is determined by the IMGT or Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:79, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:79 substitutions, deletions or additions);
  • CDR-H2 consisting of the following sequence: SEQ ID NO:80, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:80) substitution, deletion or addition); and
  • CDR-H3 consisting of the following sequence: SEQ ID NO: 81, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 81) substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:82, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:82 substitutions, deletions or additions);
  • CDR-L2 consisting of the following sequence: SEQ ID NO:83, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:83 substitution, deletion or addition); and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the IMGT numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:79; CDR-H2 as set forth in SEQ ID NO:80; and, CDR-H3 as shown in SEQ ID NO: 81; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 82; CDR as shown in SEQ ID NO: 83 -L2; and, CDR-L3 as shown in SEQ ID NO:44.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions);
  • CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitution, deletion or addition); and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:42, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:42 substitutions, deletions or additions);
  • CDR-L2 consisting of the following sequence: SEQ ID NO:43, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:43) substitution, deletion or addition); and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 42; CDR as shown in SEQ ID NO: 43 -L2; and, CDR-L3 as shown in SEQ ID NO:44.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:45, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:45) substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:46, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:46) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 45; CDR as shown in SEQ ID NO: 46 -L2; and, CDR-L3 as shown in SEQ ID NO:44.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:47, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:47 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:48, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:48) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:49, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:49 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:50, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:50 substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:47; CDR-H2 as set forth in SEQ ID NO:48; and, CDR-H3 as shown in SEQ ID NO:41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:49; CDR as shown in SEQ ID NO:50 -L2; and, CDR-L3 as shown in SEQ ID NO:44.
  • the antibody or antigen-binding fragment thereof of the invention comprises: at least one of the heavy chain variable regions (VH) as set forth in SEQ ID NO: 7, 21, 23, 25 or 27 , two or three CDRs; and/or, at least one, two or three CDRs contained in the variable region (VL) of the light chain as set forth in SEQ ID NO: 8, 22, 24, 26 or 28;
  • VH heavy chain variable regions
  • VL variable region
  • the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and the CDR-L1, CDR-L2 and CDR-L1 contained in the light chain variable region (VL) L3 is determined by the IMGT or Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:84, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:84 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:85, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:85 substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:86, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:86) substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO: 87, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 87) substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:88, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:88) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the IMGT numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:84; CDR-H2 as set forth in SEQ ID NO:85; and, CDR-H3 as shown in SEQ ID NO: 86; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 87; CDR as shown in SEQ ID NO: 88 -L2; and, CDR-L3 as shown in SEQ ID NO:56.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:51, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:51 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:52, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:52 substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:54, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:54 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:55, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:55 substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:51; CDR-H2 as set forth in SEQ ID NO:52; and, CDR-H3 as shown in SEQ ID NO:53; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:54; CDR as shown in SEQ ID NO:55 -L2; and, CDR-L3 as shown in SEQ ID NO:56.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:51, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:51 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:57, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:57) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:54, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:54 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:55, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:55 substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:51; CDR-H2 as set forth in SEQ ID NO:57; and, CDR-H3 as shown in SEQ ID NO:53; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:54; CDR as shown in SEQ ID NO:55 -L2; and, CDR-L3 as shown in SEQ ID NO:56.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:58, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:58) substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:59, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:59) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:60, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:60) substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:61, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:61 substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:58; CDR-H2 as set forth in SEQ ID NO:59; and, CDR-H3 as shown in SEQ ID NO: 53; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 60; CDR as shown in SEQ ID NO: 61 -L2; and, CDR-L3 as shown in SEQ ID NO:56.
  • the antibody or antigen-binding fragment thereof of the invention comprises: at least one of the heavy chain variable regions (VH) as set forth in SEQ ID NO: 9, 29, 31, 33 or 35 , two or three CDRs; and/or, at least one, two or three CDRs contained in the variable region (VL) of the light chain as set forth in SEQ ID NO: 10, 30, 32, 34 or 36;
  • VH heavy chain variable regions
  • VL variable region
  • the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and the CDR-L1, CDR-L2 and CDR-L1 contained in the light chain variable region (VL) L3 is determined by the IMGT or Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:89, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:89) substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:90, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:90 substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:91, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:91) substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:92, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:92 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:93, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:93) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the IMGT numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:89; CDR-H2 as set forth in SEQ ID NO:90; and, CDR-H3 as shown in SEQ ID NO:91; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:92; CDR as shown in SEQ ID NO:93 -L2; and, CDR-L3 as shown in SEQ ID NO:67.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:62, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:62 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:63, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:63) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:65, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:65 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:66, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:66) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:62; CDR-H2 as set forth in SEQ ID NO:63; and, CDR-H3 as shown in SEQ ID NO:64; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:65; CDR as shown in SEQ ID NO:66 -L2; and, CDR-L3 as shown in SEQ ID NO:67.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:62, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:62 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO: 68, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 68) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:69, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:69 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:70, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:70 substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:62; CDR-H2 as set forth in SEQ ID NO:68; and, CDR-H3 as shown in SEQ ID NO:64; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:69; CDR as shown in SEQ ID NO:70 -L2; and, CDR-L3 as shown in SEQ ID NO:67.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:71, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:71 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:72, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:72) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:73, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:73 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:74, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:74) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:71; CDR-H2 as set forth in SEQ ID NO:72; and, CDR-H3 as shown in SEQ ID NO:64; and, the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:73; CDR as shown in SEQ ID NO:74 -L2; and, CDR-L3 as shown in SEQ ID NO:67.
  • the antibody or antigen-binding fragment thereof of the invention comprises: at least one, two or three contained in the variable region (VH) of the heavy chain as set forth in SEQ ID NO: 11 or 37 CDRs; and/or, at least one, two or three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 12 or 38; wherein the heavy chain variable region (VH)
  • the CDR-H1, CDR-H2 and CDR-H3 contained in the light chain variable region (VL) and the CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) are determined by the IMGT or Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:79, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:79 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:80, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:80) substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO: 81, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 81) substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:94, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:94) substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:83, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:83 substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the IMGT numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:79; CDR-H2 as set forth in SEQ ID NO:80; and, CDR-H3 as shown in SEQ ID NO: 81; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 94; CDR as shown in SEQ ID NO: 83 -L2; and, CDR-L3 as shown in SEQ ID NO:77.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:75, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:75 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:76, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:76) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 75; CDR as shown in SEQ ID NO: 76 -L2; and, CDR-L3 as shown in SEQ ID NO:77.
  • the antibody or antigen-binding fragment thereof comprises:
  • CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions),
  • CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), and
  • CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and
  • CDR-L1 consisting of the following sequence: SEQ ID NO:75, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:75 substitutions, deletions or additions),
  • CDR-L2 consisting of the following sequence: SEQ ID NO:78, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 compared to SEQ ID NO:78) substitutions, deletions or additions), and
  • CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions);
  • VH CDRs and VL CDRs are defined by the Kabat numbering system.
  • substitutions described in any of (i)-(vi) are conservative substitutions.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 75; CDR as shown in SEQ ID NO: 78 -L2; and, CDR-L3 as shown in SEQ ID NO:77.
  • the antibodies or antigen-binding fragments thereof of the invention further comprise framework regions (FRs) derived from mammalian (eg, murine or human) immunoglobulins.
  • FRs framework regions derived from mammalian (eg, murine or human) immunoglobulins.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) framework region (FR) derived from a murine immunoglobulin, and/or the antibody or its
  • the VL of the antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from a murine immunoglobulin.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) framework region (FR) derived from a human immunoglobulin, and/or the antibody or its
  • the VL of the antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from a human immunoglobulin.
  • the heavy chain variable region FR and/or light chain variable region FR of the antibody or antigen-binding fragment thereof of the invention may comprise one or more non-human (eg, murine) amino acid residues
  • the heavy chain framework region FR and/or the light chain framework region FR may comprise one or more amino acid back-mutations in which there are corresponding murine amino acid residues.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • the antibodies or antigen-binding fragments thereof of the invention are humanized.
  • the antibody or antigen-binding fragment thereof of the invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% humanized %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 5, 13, 15, 17 and 19; and/or , selected from the light chain variable regions shown in SEQ ID NOs: 6, 14, 16, 18 and 20.
  • VH heavy chain variable region
  • the antibodies or antigen-binding fragments thereof of the invention comprise:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibodies or antigen-binding fragments thereof of the invention comprise:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibodies or antigen-binding fragments thereof of the invention comprise:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibodies or antigen-binding fragments thereof of the invention comprise:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25 and 27; and/or , selected from the light chain variable regions shown in SEQ ID NOs: 8, 22, 24, 26 and 28.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9, 29, 31, 33 and 35; and/or , selected from the light chain variable regions shown in SEQ ID NOs: 10, 30, 32, 34 and 36.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 11 and 37; and/or, selected from the group consisting of SEQ ID NO: 11 and 37 : light chain variable regions shown at 12 and 38.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a mammalian (eg, murine or human) immunoglobulin-derived constant region sequence or variant thereof, which variant is derived from the same
  • the sequence comparison has one or more amino acid substitutions, deletions or additions.
  • the variant has one or more amino acid substitutions compared to the sequence from which it is derived.
  • the anti-CD47 antibody molecule has a heavy chain constant region (CH1 domain, hinge region, CH2 domain, and CH3 domain sequentially linked from N-terminus to C-terminus) selected from, eg, IgG1, IgG2, Heavy chain constant regions of IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular selected from heavy chain constant regions such as IgG1, IgG2, IgG3 and IgG4, more particularly selected from IgG1, IgG2 or IgG4 (e.g. heavy chain constant regions of human IgG1, IgG2 or IgG4).
  • a heavy chain constant region selected from, eg, IgG1, IgG2, Heavy chain constant regions of IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE
  • heavy chain constant regions such as IgG1, IgG2, IgG3 and IgG4, more particularly selected from IgG1, IgG2
  • the anti-CD47 antibody molecule has a light chain constant region selected from, eg, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (eg, a human kappa light chain).
  • the Fc fragment of the heavy chain constant region sequence is altered, eg, mutated, to modify properties of the antibody molecule of the invention (eg, to alter one or more of the following properties: eg, Fc receptor binding, effector cell function or complement function, Fab arm exchange, etc.).
  • the antibodies provided herein comprise Fc variants that have amino acid substitutions, deletions or additions with altered effector function (eg, reduction or elimination).
  • the Fc fragment of an antibody mediates several important effector functions, such as ADCC, ADCP, CDC, etc.
  • Methods of altering effector function by substituting amino acid residues in the Fc fragment of an antibody to alter the affinity of the antibody for effector ligands such as FcyR or complement C1q are known in the art (see, eg, EP 388,151A1 ; US 564,8260; US 562,4821; Natsume A et al, Cancer Res., 68:3863-3872, 2008; Idusogie EE et al, J.
  • amino acid L235 (EU numbering) on the heavy chain constant region is modified to alter the Fc receptor interaction, eg L235E or L235A.
  • amino acids 234 and 235 in the constant region of the antibody are modified simultaneously, such as L234A and L235A (L234A/L235A) (EU numbering).
  • amino acid D265 (EU numbering) on the heavy chain constant region of the antibody is modified to alter the Fc receptor interaction, eg D265A.
  • the interaction with C1q mainly affects the CDC activity of the antibody, and the corresponding amino acid positions are D270, K322, P329 and P331.
  • the amino acid of P331 in the constant region of the antibody is mutated to eliminate the CDC effect, such as P331S (EU numbering); in other preferred embodiments, the amino acid of P329 in the constant region of the antibody is substituted, such as P329A (EU number).
  • the antibodies provided herein can comprise amino acid substitutions, deletions or additions to Fc variants with extended circulating half-lives. Residues that affect the half-life mainly interact with FcRn, and the corresponding amino acid positions are L251-S254, L309-Q311 and N434-H435. For example, M252Y/S254T/T256E, M428L/N434S or T250Q/M428L were all able to prolong the half-life of antibodies in primates.
  • FcRn neonatal receptor
  • amino acid M428 (EU numbering) on the constant region of the antibody is modified to enhance the binding affinity of the FcRn receptor, eg M428L.
  • amino acids 250 and 428 (EU numbering) in the constant region of the antibody are modified simultaneously, eg, T250Q and M428L (T250Q/M428L).
  • the antibodies provided herein can also comprise Fc variants with amino acid substitutions, deletions or additions that reduce or eliminate Fc glycosylation.
  • IgG contains a conserved glycosylation site at amino acid N297 of the CH2 domain. Glycosylation at N297 greatly affects the activity of IgG. If the glycosylation at this site is removed, it will affect the IgG molecule. The conformation of the upper half of CH2, thereby losing the binding ability to Fc ⁇ Rs, affects antibody-related biological activities, such as ADCC effect.
  • amino acid N297 (EU numbering) on the human IgG constant region is modified to avoid glycosylation of the antibody, such as N297A.
  • the antibodies provided herein may also include Fc variants with Fab arm swapping that eliminates.
  • Fc variants with Fab arm swapping that eliminates.
  • replacing Ser at position 228 in the core hinge region of IgG4 with Pro(S228P) (EU numbering) can eliminate Fab exchange and enhance Fab stability.
  • the human IgG heavy chain constant region sequence is selected from the following variants:
  • the amino acid sequence of the heavy chain constant region of the antibody is preferably selected from the native or mutated human IgG heavy as set forth in SEQ ID NOs: 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105 chain constant region.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the anti-CD47 antibodies of the invention also encompass antibody fragments thereof, examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear Antibodies, single chain antibody molecules (eg, scFvs); and bispecific or multispecific antibodies formed from antibody fragments.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear Antibodies, single chain antibody molecules (eg, scFvs); and bispecific or multispecific antibodies formed from antibody fragments.
  • the present invention provides nucleic acids encoding any of the above anti-CD47 antibodies or antigen-binding fragments thereof.
  • the nucleotide sequence encoding the anti-CD47 antibody molecule is codon-optimized.
  • preferred embodiments of the present invention provide first and second nucleic acids encoding the heavy and light chains, respectively, of an anti-CD47 antibody molecule selected from any of the following: AB12W3, AB12W4, AB12W5, AB12W6, AB12W7, AB12W8, AB12W9, and AB12W10; or a sequence substantially identical thereto.
  • the first nucleic acid encoding the antibody heavy chain comprises the sequence set forth in SEQ ID NO: 106, 110, 114, 118, 122, 126, 130 or 134 or substantially identical thereto (eg, at least about 85 %, 90%, 95%, 99% or more highly similar sequences or sequences with one or more nucleotide substitutions (e.g.
  • the second nucleic acid encoding the antibody light chain comprises as shown in SEQ ID NO: 108, 112, 116, 120, 124, 128, 132 or 136 A sequence or a sequence that is substantially identical thereto (eg, a sequence that is at least about 85%, 90%, 95%, 99% or more similar or has one or more nucleotide substitutions (eg, conservative substitutions)) , or a sequence that differs by no more than 3, 5, 10, 15, 20, 25 or 30 nucleotides from the above sequence).
  • the present invention provides a vector comprising the above-mentioned nucleic acid.
  • the present invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention.
  • the vectors of the present invention are, for example, plasmids, cosmids, phages, and the like.
  • the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (eg, a mammal, eg, a human).
  • the present invention provides a host cell comprising the above-described isolated nucleic acid molecule or the above-described vector.
  • Host cells can be eukaryotic cells (eg, mammalian cells, insect cells, yeast cells) or prokaryotic cells (eg, E. coli).
  • Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • the host cells of the invention are mammalian cells, such as CHO (eg, CHO-K1, CHO-S, CHO DXB11, CHO DG44).
  • the present invention provides a method of making an anti-CD47 antibody or antigen-binding fragment thereof, wherein the method comprises culturing the host cell under conditions suitable for expression of nucleic acid encoding the antibody or antigen-binding fragment thereof, and isolating The antibody or antigen-binding fragment thereof. In certain embodiments, the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and/or excipient and/or stabilizer and at least one CD47 antibody or an antigen-binding fragment thereof of the present invention.
  • the present invention relates to a method of inhibiting CD47 binding to SIRP ⁇ in a subject, the method comprising administering to the subject an effective amount of the CD47 antibody or antigen-binding fragment thereof of the present invention.
  • the present invention also relates to the use of the CD47 antibody or antigen-binding fragment thereof in the preparation of a composition or medicament for inhibiting the binding of CD47 to SIRP ⁇ in a subject.
  • the present invention relates to a method of promoting phagocytosis of macrophages in a subject, the method comprising administering to the subject an effective amount of the CD47 antibody or antigen-binding fragment thereof of the present invention.
  • the present invention also relates to the use of any of the CD47 antibodies or antigen-binding fragments thereof in the manufacture of a composition or medicament for promoting phagocytosis by macrophages in a subject.
  • the present invention relates to a method of treating any CD47-related disease or disorder that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing CD47 activity, the method comprising administering to a subject or individual an effective amount of the CD47 antibody or antigen-binding fragment thereof of the present invention.
  • the present invention provides a method of treating (or preventing) a tumor in a subject, the method comprising: administering to the subject or individual the CD47 antibody or antigen-binding fragment thereof of the present invention.
  • the present invention also relates to the use of the CD47 antibody and antigen-binding fragment thereof in the preparation of a medicament for treating a CD47-mediated disorder or disease in a subject.
  • the antibody molecule can be used to treat hematological cancers, eg, hematological cancers selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (eg, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-lymphoblastic leukemia/lymphoma, and Burkitt's lymphoma), B-lymphoblastic leukemia/lymphoma; B-cell chronic Lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL) eg, transformed CLL, Richter syndrome, chronic myeloid leukemia (CML), follicular lymphoma, multiple myeloma, myelofibrosis, true Polycythemia, cutaneous T-cell lymphoma, monoclonal gam
  • the antibody molecules can be used to treat solid tumors.
  • the cancer is selected from the group consisting of: lung cancer (eg, non-small cell lung cancer, small cell lung cancer), pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, spine cancer, Brain Cancer, Cervical Cancer, Endometrial Cancer, Colon/Rectal Cancer, Anal Cancer, Endometrial Cancer, Esophagus Cancer, Gallbladder Cancer, Gastrointestinal Cancer, Skin Cancer, Prostate Cancer, Pituitary Cancer, Stomach Cancer, Uterine Cancer, Vaginal Cancer cancer and thyroid cancer.
  • the present invention relates to a method for detecting CD47 protein in a sample, the method comprising (a) contacting the sample with any anti-CD47 antibody or antigen-binding fragment thereof described herein; and (b) detecting the anti-CD47 antibody or Formation of a complex between its antigen-binding fragment and CD47 protein.
  • the CD47 is human CD47.
  • the detection method may be an in vitro or in vivo method.
  • an anti-CD47 antibody is used to select subjects suitable for treatment with an anti-CD47 antibody.
  • the anti-CD47 antibody is detectably labeled.
  • the present invention provides a kit for detecting the content or level of CD47 protein in a sample, which includes any of the anti-CD47 antibodies or antigen-binding fragments thereof described in the present invention and instructions for use.
  • the CD47 antibody or its antigen-binding fragment of the present invention binds to human CD47 with high affinity, blocks the interaction between CD47 and SIRP ⁇ , and promotes the phagocytic activity of macrophages on tumor cells expressing CD47, without causing significant erythrocyte hemorrhage coagulation reaction.
  • the CD47 antibody provided by the present invention shows extremely significant anti-tumor activity in the mouse xenograft model of human Burkkit's lymphoma.
  • the CD47 antibodies of the present invention overcome a key limitation for therapeutic targeting of CD47.
  • the CD47 antibody provided by the present invention can simultaneously bind to the CD47 natural antigen of human and cynomolgus monkey and/or rhesus monkey, so that the preclinical toxicology evaluation does not require the construction of a replacement molecule, and the obtained effective dose, toxic dose and toxicity
  • the side effects are more objective and accurate, and the clinical dose can be directly converted to reduce the risk of clinical research.
  • FR antibody framework regions immunoglobulin variable regions excluding CDR regions
  • V region A segment of an IgG chain whose sequence varies between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain.
  • agglutination refers to the agglutination of cells
  • hemagglutination refers to the agglutination of a specific subtype of cells, ie, red blood cells. Therefore, hemagglutination is a type of agglutination.
  • the antibodies of the invention do not cause significant levels of agglutination, such as red blood cell hemagglutination ("RBC hemagglutination").
  • the antibodies of the invention specifically bind to CD47 in a manner that does not promote agglutination of CD47 positive cells; and, the ability of the antibodies of the invention to bind CD47 on the cell surface without causing cell agglutination is not limited to red blood cells.
  • agglutination eg, the hemagglutination of RBCs
  • one skilled in the art can perform a hemagglutination assay in the presence of the CD47 antibodies of the invention, and thereafter measure the area of the RBC spots to determine the level of hemagglutination, as described in the Examples below.
  • the RBC spot area in the presence of the CD47 antibody of the invention versus the RBC spot area in the absence of the CD47 antibody of the invention (ie, under zero hemagglutination conditions), and the RBC spot area in the presence of other known CD47 antibodies comparisons were made.
  • hemagglutination response is quantified relative to a baseline control.
  • hemagglutination can also be quantified using densitometric analysis of RBC spots.
  • red blood cell and “erythrocyte” are synonymous and used interchangeably.
  • CDR region or “complementarity determining region” as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the CDR region sequences can be defined by the IMGT, Kabat, Chothia and AbM methods or the amino acid residues within the variable regions identified by any CDR region sequence determination method well known in the art.
  • Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al., eg, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and heavy Residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the chain variable domain, see Kabat et al., 1991, Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md.; CDR positions can also be identified as defined by the "hypervariable loop" (HVL) structure originally described by Chothia et al., e.g., light chains Residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the variable domain and 26-32 (H1), 52-56 (H2) and Residues at positions 95-102 (H3) (see, eg, Chothia et al,
  • IMGT immunoglobulin variable regions including CDRs
  • CDR regions are defined according to IMGT numbering, eg, 27-32 (L1), 50-52 (L2) and Residues 89-97 (L3) and residues 26-35 (H1), 51-57 (H2) and 93-102 (H3) of the heavy chain variable domain, see eg Dev Comp by Lefranc, MP et al. Immunol, 2003, 27:55-77, which is incorporated herein by reference.
  • CDR identification include "AbM definitions", which are a compromise between Kabat and Chothia and derived using Oxford Molecular's AbM antibody modeling software; or "contact definitions" of CDRs, which are based on observed antigen contacts and Described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745.
  • configuration definition approach of CDRs, the positions of CDRs can be identified as residues that make enthalpy contributions to antigen binding, see eg Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166.
  • the methods used in the present invention may utilize or define CDRs according to any of these methods, including but not limited to any of the Kabat definitions, IMGT definitions, Chothia definitions, AbM definitions, contact definitions and/or configuration definitions to define.
  • the terms “framework region”, “variable framework region”, “FR region” are variable domain residues other than the hypervariable region residues as defined herein.
  • the light chain variable framework region or the heavy chain variable framework region of the anti-CD47 antibody molecule may be selected from: (a) comprising at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 99% or preferably 100% of the amino acid residues from a human light or heavy chain variable framework region (e.g.
  • the light or heavy chain variable framework region comprises at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 99% identical or identical light or heavy chain variable framework region sequences.
  • the anti-CD47 antibody molecule comprises at least 1, 2, 3, 4, 5 amino acid sequences from, eg, FR regions in the entire variable region (eg, as shown in Table 1) 1, 6, 7, 10, 15, 20 or more altered heavy chain variable regions (eg, amino acid substitutions, insertions or deletions). In certain embodiments, the anti-CD47 antibody molecule comprises at least 1, 2, 3, 4, 5 amino acid sequences from, eg, FR regions in the entire variable region (eg, as shown in Table 1) 1, 6, 7, 10, 15, 20 or more altered (eg, amino acid substitutions, insertions or deletions) in the light chain variable region.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • Chimeric antibodies can be produced by any suitable recombinant DNA technique.
  • the antibody light chain variable region of the CD47 chimeric antibody further comprises a light chain FR region of a murine ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain variable region of the CD47 chimeric antibody further comprises the heavy chain FR region of murine IgG1, IgG2, IgG3 or a variant thereof.
  • the constant region of the human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising the heavy chain constant regions of human IgG1 or IgG4.
  • Human consensus framework refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. In general, selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, the subtypes of the sequences are as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR.
  • a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all HVRs (eg, CDRs) correspond to those of the non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source that utilizes a library of human antibodies or other human antibodies coding sequence. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • blocking refers to reduced CD47 signaling in the presence of an antibody of the invention.
  • the blocking of CD47-mediated signal transmission means that the CD47 signal transmission level in the presence of the CD47 antibody of the present invention is lower than the control level of CD47 (ie the CD47 signal transmission level in the absence of the antibody), and the reduction range is greater than or equal to 5%, 10% , 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100%.
  • Levels of CD47 signaling can be measured using a variety of standard techniques, such as, by way of non-limiting example, downstream gene activation and/or luciferase reporter assays in response to CD47 activation.
  • assays can be used to measure the level of CD47 signaling, including, for example, commercially available kits.
  • immunobinding and “immunobinding properties” as used herein refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength or affinity of an immune binding interaction can be expressed as the equilibrium dissociation constant (K D ) of the interaction, where a smaller K D value indicates a higher affinity.
  • K D equilibrium dissociation constant
  • the immunobinding properties of selected polypeptides can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both “association rate constants" (ka or kon) and “dissociation rate constants" (kd or koff) can be calculated from the concentrations and the actual rates of association and dissociation.
  • dissociation constant KD KD , ka and kd values can be measured by any effective method.
  • dissociation constants are measured using bioluminescence interferometry (eg, the ForteBio Octet method described in Example 2.5).
  • dissociation constants can be measured using surface plasmon resonance techniques (eg, Biacore) or Kinexa.
  • Antibodies of the invention are considered to specifically bind to the CD47 epitope when the equilibrium binding constant (K D ) is ⁇ 10 ⁇ M, preferably ⁇ 100 nM, more preferably ⁇ 10 nM, and most preferably ⁇ 100 pM to about 1 pM.
  • K D equilibrium binding constant
  • Anti-CD47 antibody of the present invention is an anti-CD47 antibody of the present invention.
  • CD47 integrated-associated protein (IAP)
  • ovarian cancer antigen OA3 ovarian cancer antigen OA3
  • Rh-associated antigen are synonymous and used interchangeably, and when used herein refer to origin from Any native CD47 of any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise stated.
  • the term encompasses "full-length” unprocessed CD47 as well as any form of CD47 or any fragment thereof produced by intracellular processing.
  • the term also includes naturally-occurring variants of CD47, eg, splice variants or allelic variants.
  • anti-CD47 antibody refers to an antibody that is capable of binding the CD47 protein or fragment thereof with sufficient affinity such that the antibody can be used with as diagnostic and/or therapeutic agents targeting CD47.
  • the anti-CD47 antibodies provided herein have a dissociation constant (Kd) ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, below 10 ⁇ 8 M , such as 10 -8 M to 10 -13 M, such as 10 -9 M to 10 -13 M).
  • the anti-CD47 antibodies or antigen-binding fragments thereof of the invention comprise substitutions, insertions or deletions. In preferred embodiments, substitutions, insertions or deletions occur in regions outside the CDRs (eg, in FRs).
  • the anti-CD47 antibodies of the invention include post-translational modifications to the light chain variable region, heavy chain variable region, light chain or heavy chain.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are substituted with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the present invention encompasses fragments of anti-CD47 antibodies.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear antibodies, single-chain antibody molecules (eg, scFv); and multispecifics formed from antibody fragments Sexual antibodies.
  • the anti-CD47 antibodies of the invention are humanized antibodies.
  • Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J, Frontiers in Bioscience, 2008, 13:1619-1633) .
  • Almagro & Fransson distinguish between rational and empirical approaches. A rational approach is characterized by generating a small number of engineered antibody variants and evaluating their binding or any other property of interest. If the designed variant does not produce the expected results, a new round of design and binding evaluation is initiated.
  • Rational methods include CDR grafting, surface reconstruction (Resurfacing), superhumanization (Superhumanization) and human string content optimization (Human StringContent Optimization).
  • empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening.
  • empirical approaches rely on reliable selection and/or screening systems capable of searching a large number of antibody variants.
  • In vitro display techniques such as phage display and ribosome display meet these requirements and are well known to the skilled person.
  • Empirical approaches include FR libraries, Guided selection, Framework-shuffling, and Humaneering.
  • the anti-CD47 antibodies of the invention are human antibodies.
  • Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol, 2001, 5:368-374 and Lonberg, Curr Opin Immunol, 2008, 20:450-459.
  • Antibody and antigen-binding fragments thereof suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric , humanized (especially CDR-grafted), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') 2 fragments, fragments generated from Fab expression libraries, Fd, Fv, Di Sulfide-linked Fv (dsFv), single chain antibody (eg scFv), diabody or tetrabody (Holliger P et al, Proc Natl Acad Sci USA, 1993, 90(14):6444-6448), nanobody (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against the antibodies of the invention), and epitope-binding fragments of any of the foregoing
  • the antibodies of the invention may be monospecific, bispecific or multispecific.
  • Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen binding domains specific for more than one target polypeptide. See, eg, Tutt et al., J Immunol, 1991, 147:60-69.
  • the anti-CD47 mAb can be linked or co-expressed with another functional molecule (eg, another peptide or protein).
  • another functional molecule eg, another peptide or protein
  • an antibody or fragment thereof can be functionally linked (eg, by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecules, such as another antibody or antibody fragment, to produce a second or Bispecific or multispecific antibodies with more binding specificities.
  • the antibodies of the invention bind to human CD47 protein, partially or fully modulate, block, inhibit, reduce, antagonize, neutralize or interfere with the binding of CD47 to SIRP ⁇ , thereby fully or partially inhibiting the functional activity of CD47.
  • Functional activities of CD47 include, for example, signaling through interaction with SIRP ⁇ , regulating (eg, increasing) intracellular calcium concentration after cell adhesion to the extracellular matrix, interacting with the C-terminal cell-binding domain of thrombospondin, and Fibrinogen interacts and interacts with various integrins.
  • CD47 antibodies reduce CD47-SIRP ⁇ -mediated signaling, promote phagocytosis, and inhibit tumor growth and/or migration by inhibiting SIRP- ⁇ binding to CD47.
  • the CD47 antibodies of the present invention exhibit various desirable properties, such as, by way of non-limiting example, effectively blocking the interaction of CD47 with its ligand, SIRP ⁇ , without causing significant erythrocyte hemagglutination, and being effective against tumors active.
  • the CD47 antibodies of the invention block at least 40%, at least 45%, at least 50%, at least 55% of the interaction between CD47 and SIRP ⁇ compared to the level of interaction between CD47 and SIRP ⁇ in the absence of the CD47 antibodies described herein %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 99% of interactions.
  • the CD47 antibody of the present invention does not cause significant cell agglutination, eg, the CD47 antibody of the present invention does not cause significant red blood cell hemagglutination.
  • significant cellular agglutination refers to the level of agglutination in the presence of existing CD47 antibodies.
  • the level of agglutination in the presence of a CD47 antibody of the invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 30%, compared to the level of agglutination in the presence of an existing CD47 antibody 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99%.
  • the level of agglutination in the presence of a CD47 antibody of the invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, if compared to the level of agglutination in the presence of an existing CD47 antibody %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, the CD47 antibody of the present invention does not cause a significant level of agglutination.
  • Antibodies of the invention are also significantly more effective in tumor models than antibodies known in the art.
  • the ability of macrophages to phagocytose tumor cells in the presence of CD47 antibodies of the invention is increased by at least 5%, at least 10%, at least 20%, compared to the ability of macrophages to phagocytose tumor cells in the presence of existing CD47 antibodies.
  • compositions and pharmaceutical preparations are provided.
  • the present invention also provides pharmaceutical compositions comprising one or more monoclonal antibodies that bind CD47 or an immunologically active fragment thereof. It should be understood that the anti-CD47 antibodies or pharmaceutical compositions provided by the present invention can be incorporated into suitable carriers, excipients and other agents in formulations for combined administration, thereby improving their transfer, delivery, tolerance and other properties.
  • composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered .
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.
  • treating means slowing, interrupting, retarding, relieving, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition or disease, and preventing recurrence of the associated disease.
  • the present invention also includes compositions (including pharmaceutical compositions or formulations) comprising anti-CD47 antibodies and compositions comprising polynucleotides encoding anti-CD47 antibodies.
  • the composition comprises one or more antibodies that bind CD47 or one or more polynucleotides encoding one or more antibodies that bind CD47.
  • suitable pharmaceutical carriers such as pharmaceutical excipients known in the art, including buffers.
  • the pharmaceutical composition of the present invention may include the antibody of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be included in kits, such as diagnostic kits.
  • Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , glycerol, propylene, glycol, water, ethanol, etc.
  • compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.
  • These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • Anti-CD47 antibodies of the invention of the desired purity can be prepared by mixing with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 1980, 16th Ed., Osol A. Ed.) Pharmaceutical formulations of anti-CD47 antibodies, preferably in the form of lyophilized formulations or aqueous solutions.
  • Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958.
  • Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.
  • compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other, the The active ingredients are suitably combined in amounts effective for the intended use.
  • the pharmaceutical composition of the present invention can be prepared as a sustained release formulation.
  • sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.
  • conservative modification is intended to mean that an amino acid modification does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitutions refer to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are well described in the art.
  • These families include those with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine) , proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g.
  • basic side chains eg lysine, arginine, histidine
  • acidic side chains eg aspartic acid, glutamic acid
  • uncharged polar side chains eg glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryp
  • one or more amino acid residues in the CDR regions of the antibodies of the invention can be replaced with other amino acid residues from the same family of side chains.
  • the present invention also provides a method of using the CD47 antibody for the treatment of cancer in a cell, tissue, organ, animal or patient.
  • cancers include, but are not limited to, solid tumors, soft tissue tumors, hematopoietic tumors, and metastatic lesions.
  • hematopoietic tumors include: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) such as transformed CLL, diffuse Large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt Lymphoma, multiple myeloma or Richter syndrome (Richter transformation).
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • DLBCL diffuse Large B-cell lymphoma
  • MDS myelodysplastic syndrome
  • lymphoma Hodgkin's disease
  • Additional blood cancers include: myelodysplastic syndromes (MDS) (eg, preleukemia, refractory anemia, Ph-negative chronic myeloid leukemia, chronic myelomonocytic leukemia, myeloid metaplasia), non-Hodgkin lymphoma neoplasms (eg, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-lymphoblastic leukemia/lymphoma, peripheral T-cell lymphoma, and Burkitt's lymphoma), B-lymphoblastic leukemia/ Lymphoma; B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma; B-cell-prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic marginal zone B-cell lymphoma ( ⁇ villous lymphocytes); hairy cell leukemia; Plasma cell myeloma/
  • solid tumors include malignancies of various organ systems, such as sarcomas, adenocarcinomas, and carcinomas, such as affecting the head and neck (including the pharynx), thyroid, lung (small cell or non-small cell lung cancer (NSCLC)), breast, lymphoid, Gastrointestinal (eg, oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genital and genitourinary tract (eg, kidney, urothelium, bladder, ovary, uterus, cervix, endometrium , prostate, testis), CNS (eg, nerve or glial cells, eg, neuroblastoma or glioma), or skin (eg, melanoma).
  • organ systems such as sarcomas, adenocarcinomas, and carcinomas, such as affecting the head and neck (including the pharynx), thyroid, lung (
  • the solid tumor is an NMDA receptor positive teratoma.
  • the cancer is selected from breast cancer, colon cancer, pancreatic cancer (eg, pancreatic neuroendocrine tumor (PNET) or pancreatic ductal adenocarcinoma (PDAC)), gastric cancer, uterine cancer, or ovarian cancer.
  • pancreatic cancer eg, pancreatic neuroendocrine tumor (PNET) or pancreatic ductal adenocarcinoma (PDAC)
  • gastric cancer uterine cancer, or ovarian cancer.
  • Anti-CD-47 antibodies can also be used to treat inflammatory disorders, autoimmune disorders, fibrotic disorders, fibroproliferative disorders, atopic disorders, or angiogenic disorders.
  • inflammatory disorders include, but are not limited to: chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), multiple sclerosis, psoriasis, Ischemia-reperfusion injury, septic shock, age-related macular degeneration (eg, wet age-related macular degeneration), atherosclerosis, Alzheimer's disease, Parkinson's disease, cardiovascular disease, vasculitis, I Type and type II diabetes, metabolic syndrome, diabetic retinopathy, restenosis.
  • chronic obstructive pulmonary disease asthma, rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), multiple sclerosis, psoriasis, Ischemia-reperfusion injury,
  • autoimmune diseases include, but are not limited to: asthma, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, type I diabetes, systemic lupus erythematosus (SLE), Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, Guillain-Barre syndrome, autoimmune hepatitis, and myasthenia gravis.
  • fibrotic diseases include, but are not limited to: scleroderma, liver fibrosis, pancreatic fibrosis, chronic obstructive pulmonary disease, diabetic nephropathy, sarcoidosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis, Neurofibromatosis, endometriosis, postoperative fibroids and restenosis.
  • atopic diseases include, but are not limited to, atopic dermatitis, atopic asthma, and allergic rhinitis.
  • the invention relates to a method of inhibiting, antagonizing the binding of CD47 to SIRP ⁇ in a subject, the method comprising administering to the subject an effective amount of any of the anti-CD47 antibodies or fragments thereof described herein.
  • the invention relates to a method of promoting phagocytosis by phagocytic cells of a subject, the method comprising administering to the subject an effective amount of any of the anti-CD47 antibodies or fragments thereof described herein.
  • the invention relates to a method of treating a related disease that targets CD47 for therapy, the method comprising administering to a subject an effective amount of any of the anti-CD47 antibodies or fragments thereof described herein.
  • the present invention relates to methods of ameliorating, slowing, inhibiting or preventing any disease or disorder that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing the binding of CD47 to SIRP ⁇ .
  • the present invention provides a method of treating cancer or tumor in a subject, a method of alleviating symptoms of cancer or tumor in a subject, and avoiding the method for tumor or cancer recurrence.
  • the anti-CD47 antibodies and antigen-binding fragments thereof provided by the present invention and pharmaceutical compositions comprising the same can be used as therapeutic agents for diagnosing, prognosing, monitoring, treating, alleviating and/or preventing abnormal CD47 in a subject Diseases and disorders related to expression, activity and/or signaling.
  • the anti-CD47 antibodies and antigen-binding fragments thereof disclosed herein and pharmaceutical compositions comprising the same can be administered by identifying the presence of diseases and disorders associated with aberrant CD47 expression, activity and/or signaling in a subject using standard methods .
  • the present invention provides the use of an anti-CD47 antibody in the manufacture or manufacture of a medicament for the treatment of the above-mentioned related diseases or disorders.
  • the antibodies of the invention can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • administration may be by any suitable route, eg, by injection, eg, intravenously or subcutaneously.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • an antibody of the invention will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment , the patient's clinical history and response to the antibodies, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in a single treatment or over a series of treatments.
  • the antibodies of the invention can be used to detect the progression of treatment of CD47-related diseases in vivo or in vitro, for example, by measuring an increase or decrease in the number of CD47-expressing cells (eg, cancer cells), it is possible to determine whether a certain Whether a specific therapy designed to treat the disease and relieve symptoms is working.
  • CD47-expressing cells eg, cancer cells
  • any of the anti-CD47 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of CD47 in a biological sample.
  • detection includes quantitative or qualitative detection.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.
  • Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H and 131I, fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbrella umbelliferone, luceriferase, eg, firefly luciferase and bacterial luciferase (US Patent No.
  • luciferin 2,3-dihydrophthalazine dione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase and xanthine oxidase, plus enzymes that utilize hydrogen peroxide dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/affinity Elements, spin tags, phage tags, stabilized free radicals, and more.
  • HR horseradish peroxidase
  • alkaline phosphatase beta-galactosidase
  • glucoamylase lysozyme
  • carbohydrate oxidase e.g., glucose
  • immunobinding and “immunobinding properties” refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength or affinity of an immunobinding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where the smaller the KD value, the higher the affinity.
  • KD equilibrium dissociation constant
  • the immunobinding properties of selected polypeptides can be determined using methods well known in the art. One assay involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • association rate constants Ka or Kon
  • dissociation rate constants Kd or Koff
  • immune cell includes cells of hematopoietic origin and that play a role in the immune response, including lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils cells, mast cells, basophils and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils cells, mast cells, basophils and granulocytes.
  • Immuno response refers to cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and the action of soluble macromolecules (including antibodies, cytokines and complement) produced by either of these cells or the liver that lead to selective targeting, binding, damage, destruction and/or clearance from vertebrates Invading pathogens, pathogen-infecting cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
  • An immune response includes, for example, activation or suppression of T cells (eg, effector T cells or Th cells, such as CD4+ or CD8+ T cells), or suppression of Treg cells.
  • immunogenicity refers to the ability of a particular substance to elicit an immune response.
  • effector cell refers to a cell of the immune system that expresses one or more FcRs and mediates one or more effector functions.
  • the cell expresses at least one type of activating Fc receptor, such as human Fc[gamma]RIII, and performs ADCC effector function.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, neutrophils, and eosinophils.
  • Effector cells also include, for example, T cells. They can be derived from any organism including, but not limited to, humans, mice, rats, rabbits and monkeys.
  • effector functions refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), and which vary among antibody isotypes.
  • antibody effector functions include, but are not limited to: Fc receptor binding affinity, ADCC, ADCP, CDC, downregulation of cell surface receptors (eg, B cell receptors), B cell activation, cytokine secretion, antibodies and antigens /half-life/clearance of antibody complexes, etc.
  • Methods of altering the effector function of antibodies are known in the art, eg, by introducing mutations in the Fc region.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as NK cells, neutrophils, or macrophages.
  • the FcR present on phagocytes binds these cytotoxic effector cells specifically to antibody-attached target cells, which then kill the target cells by secreting cytotoxins.
  • Methods for detecting ADCC activity of antibodies are known in the art and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an FcR (eg, CD16a).
  • ADCP antibody-dependent cell-mediated phagocytosis
  • complement-dependent cytotoxicity refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to an antibody Fc.
  • Methods for detecting the CDC activity of an antibody are known in the art, and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an Fc receptor (eg, C1q).
  • Monoclonal antibodies (mAbs) of the invention can be prepared by a variety of techniques, including conventional monoclonal antibody methodologies, such as standard somatic cell hybridization techniques as described in Kohler and Milstein, Nature, 1975; 256:495. Although somatic cell hybridization procedures are preferred, other methods of making monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can also be used in principle.
  • a preferred animal system for making hybridomas is the murine system.
  • the production of hybridomas in mice is a well established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion protocols are also known.
  • DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques such as PCR amplification or using cDNA cloning of hybridomas expressing the antibody of interest, and the DNA can be Inserted into an expression vector so that the gene of interest is operably linked to transcriptional and translational regulatory sequences, and transfected into a host cell for expression, preferably a eukaryotic expression vector, more preferably mammalian cells, such as CHO and its derived cell lines.
  • a host cell for expression preferably a eukaryotic expression vector, more preferably mammalian cells, such as CHO and its derived cell lines.
  • Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, specific antigens or epitopes thereof can be immobilized on columns to purify immunospecific antibodies by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • the chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared above.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • murine variable regions can be linked to human constant regions using methods known in the art (see, eg, US Patent No. 4,816,567 to Cabilly et al.).
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3).
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgGl or IgG4 constant region.
  • murine CDR regions can be inserted into human framework sequences using methods known in the art (see US Patent Nos. 5,225,539 to Winter and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.) .
  • Transgenic animals can also be used, eg, HuMAb mice (Medarex, Inc.) containing human immunoglobulin gene minilocus (miniloci) encoding unrearranged human heavy ( ⁇ and ⁇ ) and kappa light chain immunoglobulin sequences. ), plus targeted mutations inactivating the endogenous ⁇ and kappa chain loci (see, eg, Lonberg et al.
  • Figure 1 Determination of the binding ability of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to human CD47 antigen.
  • Figure 2-1 Determination of the cross-reactivity of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 with mouse CD47 antigen.
  • Figure 2-2 Determination of cross-reactivity of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 with cynomolgus monkey CD47 antigen.
  • Figure 3-1 The ability of mouse monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with the control antibody AB12W1 for binding to human CD47.
  • Figure 3-2 The ability of mouse monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with the control antibody AB12W2 for binding to human CD47.
  • Figure 3-3 The ability of murine mAbs Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with human SIRP ⁇ for binding to human CD47.
  • Figure 4-1 The binding ability of CD47 candidate mouse monoclonal antibodies Mo2A11971 and Mo2A21331 to CCRF-CEM cells was detected by flow cytometry.
  • Figure 5 Determination of the binding ability of humanized antibodies AB12W3 and AB12W6 to human CD47 antigen.
  • Figure 6 The ability of humanized antibodies AB12W3 and AB12W6 to block the binding of CD47 to the receptor SIRP ⁇ .
  • Figure 7-1 Determination of the binding ability of humanized antibodies AB12W3, AB12W4 and AB12W6 to lymphoma cell Jurkat.
  • Figure 7-2 Determination of the binding ability of humanized antibodies AB12W3, AB12W4 and AB12W6 to lymphoma cell Raji.
  • Figure 7-3 Determination of the binding ability of humanized antibody AB12W9 to human acute lymphoblastic leukemia cells CCRF-CEM.
  • Figure 8-1 Determination of the binding ability of humanized antibodies AB12W3 and AB12W6 to CHO/K1-hCD47 stable cell line.
  • Figure 8-2 Determination of the binding ability of humanized antibody AB12W9 to CHO/K1-hCD47 stable cell line.
  • Figure 9 CDC activity assay of humanized antibodies AB12W3, AB12W4, AB12W5 and AB12W6.
  • Figure 10 Humanized antibodies AB12W3, AB12W4 and AB12W6 mediate phagocytosis of CCRF-CEM of tumor cells by macrophages.
  • FIG. 11 Humanized antibody AB12W3 and AB12W4-mediated phagocytosis of erythrocyte RBCs by macrophages.
  • FIG. 14 Antitumor effects of CD47 humanized antibodies AB12W6, AB12W9 and AB12W10 in a mouse subcutaneous xenograft model of human Burkkit's lymphoma.
  • Figure 16 The tumor-suppressive effect of CD47 humanized antibody AB12W9 alone or in combination with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model.
  • mice After 50 ⁇ g of human CD47 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was fully emulsified with complete Freund's adjuvant, male Balb/C mice were immunized by multi-point immunization, and the immunization cycle was once every three weeks. On the 10th day after the third immunization, blood was collected from the eye socket, and plasma anti-human CD47 antibody titers were tested by ELISA to monitor the degree of immune response in mice. Then, 3 days before fusion, mice with the highest anti-human CD47 antibody titers were tested The mice were boosted once.
  • human CD47 antigen Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • mice After 3 days, the mice were sacrificed and the spleens of the mice were removed to fuse with the mouse myeloma Sp2/0 cell line. 2 ⁇ 10 8 Sp2/0 cells were mixed with 2 ⁇ 10 8 splenocytes in 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution.
  • polyethylene glycol molecular weight 1450
  • DMSO dimethyl sulfoxide
  • Iscove's medium containing 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM hypoxanthine, 0.4 ⁇ M aminopterin, and 16 ⁇ M thymidine
  • Iscove's medium containing 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM hypoxanthine, 0.4 ⁇ M aminopterin, and 16 ⁇ M thymidine
  • high-throughput ELISA was used to detect the ability of the antibody in the supernatant to compete with HRP-labeled human SIRP ⁇ for binding to CD47, so as to screen out the positive wells that compete with human SIRP ⁇ (see Example 2.3 for the method). Then, the above-mentioned fusion cells in the well containing the monoclonal antibody that can inhibit the binding of HRP-labeled SIRP ⁇ to human CD47 were subcloned, and 3 hybridoma cell lines expressing high-affinity mouse monoclonal antibody were obtained by competitive ELISA. #2A1621, #2A11971, #2A21331 and #2A2351.
  • Specific antibody-producing clones were grown in RPMI 1640 medium supplemented with 10% FCS. When the cell density reached approximately 5 x 105 cells/ml, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged and the culture supernatant was collected. Antibodies were purified on a protein G column and the monoclonal antibody eluate was dialyzed against 150 mM NaCl. The dialyzed solution was filter sterilized through a 0.2 ⁇ m filter to obtain the purified murine monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to be tested.
  • the ELISA plate was coated with human CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) overnight at room temperature. The coating solution was discarded, blocked with skim milk powder dissolved in PBS buffer for 0.5 h, and the plate was washed 3-4 times with PBST (pH 7.4, PBS containing 0.05% Tween 20). Then, 50 ⁇ l of purified anti-human CD47 murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 and humanized antibodies against CD47 AB12W1 and AB12W2 were added to each well as positive controls (AB12W1 was derived from the anti-human CD47 human antibody developed by Forty-seven Company).
  • Antibody Hu5F9-G4 whose VH and VL sequences are shown in U.S. Patent US9017675B2, respectively shown in SEQ ID NOs: 1 and 2 herein;
  • the variable region sequence of AB12W2 is derived from the anti-human CD47 humanized antibody CC- 90002, whose VH and VL sequences are shown in Chinese patent CN104271757B, respectively shown in SEQ ID NOs: 3 and 4), incubated at room temperature for 1 h, washed with PBS containing 0.05% Tween 20, and then added 50 ⁇ l to each well HRP-labeled goat anti-mouse or goat anti-human IgG polyclonal antibody (Jackson Laboratory) was used as the detection antibody, and then the plate was washed 3 to 4 times with PBST, the substrate TMB was added for 10 minutes, and then 0.2MH 2 SO 4 was added to stop the reaction. , and then read the absorbance value (OD value), and the results are shown in Figure 1.
  • Cynomolgus monkey CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) and mouse CD47 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) were diluted with PBS buffer to 0.1 ⁇ g/ml, and added in a volume of 100 ⁇ l/well. In a 96-well plate, place at 4°C for 16-20h. Aspirate the supernatant, wash the plate once with PBST buffer, add 200 ⁇ l PBST containing 1% nonfat dry milk (PBST/1% nonfat dry milk) to each well, and incubate at room temperature for 1 h to block.
  • PBST/1% nonfat dry milk 1% nonfat dry milk
  • Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 did not specifically bind to mouse CD47.
  • Figure 2-2 shows that Mo2A21331 has the strongest binding ability to cynomolgus monkey CD47, Mo2A1621 and Mo2A11971 have relatively weak binding ability to cynomolgus monkey CD47, and Mo2A2351 hardly binds to cynomolgus monkey CD47.
  • Human CD47 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was diluted to 0.1 ⁇ g/ml with PBS buffer, and 100 ⁇ l/well was added to a 96-well plate, overnight at room temperature. Discard the coating solution, add 200 ⁇ l PBST/1% nonfat dry milk to each well, and incubate for 1 h at room temperature for blocking.
  • the antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 can significantly and competitively block the binding of AB12W1 or AB12W2 to human CD47.
  • the antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A235 can compete with human SIRP ⁇ for binding to CD47, that is, they function by blocking the binding of CD47 and SIRP ⁇ .
  • Flow cytometry was used to detect the binding ability of CD47 candidate mouse antibody to two tumor cells with high CD47 expression.
  • Human Burkitt's lymphoma cells Raji-luc (Beijing Biositu Gene Biotechnology Co., Ltd.) and human acute lymphoblastic leukemia cells CCRF-CEM (Shanghai Institute of Cell, Chinese Academy of Sciences) were cultured respectively, and the cells were collected by centrifugation. The collected cells were resuspended in 1% PBSB, adjusted to a cell density of 2 ⁇ 10 6 /ml, plated in a 96-well plate, 100 ⁇ l per well (2 ⁇ 10 5 cells), and blocked at 4° C. for 0.5 h.
  • the CD47 mouse-derived antibodies Mo2A11971 and Mo2A21331 and the control antibody AB12W2 were diluted at the same time, starting from 10 ⁇ g/ml, and 5-fold serial dilution to obtain 7 concentration gradients for use.
  • the blocked cells were centrifuged to discard the supernatant, and the diluted CD47 humanized antibody was added, and incubated at 4°C for 1 h; the supernatant was removed by centrifugation, washed three times with 1% PBSB, and the diluted F4647-labeled goat anti-human IgG polyclonal antibody was added. (The Jackson Laboratory, Cat. No.
  • the binding affinity constants of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 and control antibodies AB12W1 and AB12W2 to antigen were determined by biofilm interferometry (BLI) (GE, Biacore T200).
  • BBI biofilm interferometry
  • the CD47 mouse antibody to be tested was immobilized on the surface of the Series S Protein A sensor chip (GE, Cat. No. 29-1275-56), and recombinant human CD47 protein (His-tag) (Acro Biosystems, Cat. No. CD-H5227) was used.
  • His-tag recombinant human CD47 protein
  • antigens are captured on the chip surface.
  • the data were processed and fitted with a 1:1 binding model of Biacore T200 analysis software.
  • the fitted data basically overlapped with the experimental data to obtain the association and dissociation rate constants ka and k d , and the equilibrium dissociation was obtained by dividing ka by k d Constant K D (see Table 1).
  • the results showed that the obtained humanized antibodies did not lose significant affinity, and the binding affinities of the mouse antibodies Mo2A1621, Mo2A11971 and Mo2A21331 to human CD47 were comparable to those of the control antibodies AB12W1 and AB12W2, and the K D values all reached the pM level.
  • the culture supernatant of hybridoma cells was taken, and IsoStrip TM mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology, product number sc-24958) was used to identify antibody subtypes.
  • the subtypes of the murine mAbs Mo2A21331, Mo2A1621, Mo2A11971 and Mo2A2351 were all identified as IgG1 (Kappa) type.
  • Antibody variable region amplification candidate hybridoma cells #2A21331, #2A1621, #2A11971 and #2A2351 were cultured to a total of about 10 7 cells, centrifuged at 1000 rpm for 10 min to collect cells, and extracted with Trizol kit (Invitrogen). RNA, the first-strand cDNA was synthesized by the reverse transcription kit SMARTer RACE, and the first-strand cDNA was used as the subsequent template to amplify the DNA sequence of the antibody variable region corresponding to the hybridoma cells. According to the subtype identification results, the heavy chain and light chain constant region sequences of the antibody subtype are obtained, and specific nested PCR primers are designed.
  • the primer sequences used in the amplification reaction are the same as the antibody variable region first framework region and Complementary constant regions.
  • the target gene was amplified by conventional PCR method, and the amplified products were sequenced to obtain the heavy chain variable regions of hybridoma clones #2A21331 secreting antibody Mo2A21331, #2A1621 secreting antibody Mo2A1621, #2A11971 secreting antibody Mo2A11971 and #2A2351 secreting antibody Mo2A2351, respectively. Sequences and light chain variable region sequences are shown in Table 2.
  • the antibodies were humanized by computer-aided three-dimensional modeling and structural analysis.
  • the inventors adopted two methods to humanize the murine antibody, one is CDR transplantation, and the other is express remodeling.
  • CDR-Grafting is a common antibody humanization method, which achieves the purpose of maintaining the activity and reducing the immunogenicity by replacing the FR of the human antibody with the FR of the mouse antibody.
  • the method for humanization transformation of CDR-grafted antibodies combined with the Discovery Studio analysis tool mainly includes the following steps: (1) modeling of the three-dimensional structure of the antibody; (2) analysis of key residues.
  • Residues that play a key role in the folding of the two domains There are three main categories: 1. Residues that play a key role in the folding of the two domains; 2. Residues close to the CDR region and embedded in the interior of the protein; 3. Residues that have direct interactions with the CDR region, including: hydrophobic interactions /Hydrogen bond/salt bridge; (3) Human template selection.
  • Resurfacing antibodies are humanized design of murine antibodies using computer-aided molecular design and surface residue replacement.
  • the principle of this method is to replace only the regions that are significantly different from the surface amino acids of human antibodies, and select amino acids similar to the surface residues of human antibodies on the basis of maintaining antibody activity and reducing heterology;
  • the humanization transformation process involves the following steps : 1. Compare the amino acid sequence of the antibody secreted by each hybridoma cell with the amino acid sequence of the human embryonic antibody to find the sequence with high homology; 2. For the side chain size, charge, hydrophobicity or possible formation of hydrogen The residues that affect the antibody CDR conformation are retained as much as possible; 3. Using computer simulation technology, when the solvent accessibility is greater than 30%, the exposed surface amino acids are replaced by adult embryonic antibody amino acids.
  • a total of 16 humanized antibodies were obtained, of which the humanized antibodies derived from the murine parental antibody Mo2A21331 were AB12W5, AB12W6, AB12W9, AB12Q1 and AB12Q2; the humanized antibodies derived from the murine parental antibody Mo2A1621 were AB12W3, AB12Q3, AB12Q4 and AB12Q5; the humanized antibodies derived from the murine parental antibody Mo2A11971 are AB12W4, AB12W7, AB12W8, AB12Q6, AB12Q7 and AB12Q8; the humanized antibodies derived from the murine parental antibody Mo2A2351 are AB12W10; the above human Among the humanized antibodies, except for AB12W5, AB12Q3, AB12Q6 and AB12W10, which were obtained by CDR transplantation, the rest of the antibodies were obtained by humanization by surface remodeling.
  • the amino acid sequences of the variable regions of the above-mentioned humanized antibodies are
  • the amino acid sequences of the CDR regions of the murine monoclonal antibodies Mo2A21331, Mo2A1621, Mo2A11971 and Mo2A2351 and their derived 16 humanized antibodies prepared by the present invention are shown in Table 4-1 to Table 4-4, wherein the variable regions of the antibodies comprise The amino acid sequences of the CDRs were defined using the Kabat and IMGT methods, respectively.
  • the VH and VL sequences shown in Table 3 were combined with the antibody heavy chain constant region (preferably from human IgG1, IgG2 or IgG4) and light chain constant
  • the sequences of the regions are spliced or assembled using conventional techniques. More preferably, the antibody heavy chain constant region sequence is modified and non-cleavable, and exhibits minimal Fc-mediated ADCC and CDC effects.
  • the anti-CD47 antibody molecule comprises the heavy chain constant region of wild-type human IgGl and the human kappa light chain constant region.
  • human IgG1 comprises a substitution at position 297 according to EU numbering (eg, Asn is substituted for Ala); in another embodiment , as shown in Table 5, the human IgG1 contains a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., substitution of Asp at position 265 to Ala and/or Pro at position 329 is substituted with Ala); in yet another embodiment, as shown in Table 5, the human IgG1 comprises a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or a combination of these Two substitutions (eg Leu to Ala at position 234 and/or Leu to Ala at position 235).
  • EU numbering e.g, Asn is substituted for Ala
  • the human IgG1 contains a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., substitution of Asp at position 265
  • the human IgGl comprises substitutions at positions 297, 250 and 428 according to EU numbering (eg, Asn is substituted by Ala, Thr is substituted by Gln, Met is substituted by Leu).
  • the anti-CD47 antibody molecule comprises the heavy chain constant region of wild-type human IgG2 and the human kappa light chain constant region shown in Table 5. Or use a modified human IgG2 constant region sequence; in one embodiment, as shown in Table 5, the anti-CD47 antibody molecule comprises human IgG2 mutated at position 331 according to EU numbering (eg, P to S).
  • the anti-CD47 antibody molecule comprises the heavy chain constant region of wild-type human IgG4 and the human kappa light chain constant region shown in Table 5. Or use a modified human IgG4 constant region sequence; in one embodiment, as shown in Table 5, the anti-CD47 antibody molecule comprises a human IgG4 mutated at position 228 according to EU numbering (eg, S to P); in another In one embodiment, as shown in Table 5, the anti-CD47 antibody molecule comprises human IgG4 with a substitution of Ser to Pro at position 228 and Leu to Glu at position 235 according to EU numbering.
  • the cDNA encoding the heavy chain and light chain of the CD47 humanized antibody obtained in the above method was inserted into PcDNA3.1 or its derivative plasmid, or other eukaryotic expression vector to construct a humanized antibody expression vector.
  • the vector plasmid used should contain the cytomegalovirus early gene promoter-enhancer required for high-level expression in mammalian cells.
  • the vector plasmid contains a selectable marker gene that confers ampicillin resistance in bacteria and G418 resistance in mammalian cells.
  • the vector plasmid contains the DHFR gene, and in a suitable host cell, the humanized antibody gene and the DHFR gene can be co-amplified with methotrexate (Methotrexate, MTX, Sigma) (for example, see patent CN103333917B).
  • methotrexate Metalhotrexate, MTX, Sigma
  • the recombinant expression vector plasmids constructed above are transfected into mammalian host cell lines to express humanized antibodies.
  • the preferred host cell line is dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells (see, eg, US Pat. No. 4,818,679 to Chasin, L. et al.).
  • DHFR dihydrofolate reductase
  • CHO Chinese hamster ovary
  • the secretion rate of each cell line was measured by the methods of limiting dilution subcloning transfectants and ELISA, and the cell line expressing the humanized antibody at a high level was selected. Conditioned media of humanized antibodies are collected for determination of their in vitro and in vivo biological activities.
  • nucleotide sequences encoding the heavy and light chains of humanized antibodies AB12W3, AB12W4, AB12W5, AB12W6, AB12W7, AB12W8, AB12W9 and AB12W10 shown in Table 6 were inserted into the expression vector constructed above, and subjected to pressure screening. , Subcloning cell lines that are stable and highly expressing the target antibody, and then culture and purify to obtain the target antibody.
  • Antibody number HC nucleotide sequence HC amino acid sequence LC nucleotide sequence LC amino acid sequence AB12W3 SEQ ID NO: 106 SEQ ID NO: 107 SEQ ID NO: 108 SEQ ID NO: 109 AB12W4 SEQ ID NO: 110 SEQ ID NO: 111 SEQ ID NO: 112 SEQ ID NO: 113 AB12W5 SEQ ID NO: 114 SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 117 AB12W6 SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 120 SEQ ID NO: 121 AB12W7 SEQ ID NO: 122 SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 AB12W8 SEQ ID NO: 126 SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO: 129 AB12W9 SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132 SEQ ID NO
  • the ELISA plate was coated with 0.2 ⁇ g/ml human CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Cat. No. 12283-HCCH) overnight at room temperature.
  • the coating solution was discarded, and 300 ⁇ l of PBST containing 2% nonfat dry milk (PBST/2% nonfat dry milk) was added to each well to block, and incubated at room temperature for 2 h. Then add 300 ⁇ l PBST to each well for washing, and repeat 3 to 4 times.
  • test antibodies AB12W3 and AB12W6 and the control antibodies AB12W1 and AB12W2 were diluted with PBST/2% nonfat dry milk, respectively, and added to the ELISA plate at 100 ⁇ l/well, incubated at room temperature for 1.5 hours, and then washed with PBST for 4 to 5 times. Then add HRP-goat anti-human IgG (L+H) enzyme-linked secondary antibody (Jackson Laboratory, Cat. No.
  • the humanized antibodies AB12W3 and AB12W6 can specifically bind to human CD47 antigen.
  • the EC50 values of AB12W3 and AB12W6 for binding to human CD47 were 11.18 ng/ml and 12.83 ng/ml, respectively, which were comparable to that of the control antibody AB12W2 (9.197 ng/ml), but lower than that of AB12W1 (26.66 ng/ml). ml).
  • Biotin-labeled recombinant human SIRP ⁇ (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number 11612-H08H-B) was used as a reagent.
  • Recombinant human CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number 12283-HCCH) was diluted to 2.0 ⁇ g/ml with PBS buffer, added to a 96-well plate in a volume of 100 ⁇ l/well, and incubated at 4°C overnight. Discard the coating solution, add 300 ⁇ l PBST/2% nonfat dry milk to each well, and incubate at room temperature for 2 h to block.
  • the binding ability of CD47 humanized antibody to CD47-expressing tumor cells was detected by FACS.
  • the lymphoma cell lines Raji-luc (Beijing Biocytogen Biotechnology Co., Ltd.) and Jurkat (Shanghai Cell Bank, Chinese Academy of Sciences) were cultured, and the cells were collected by centrifugation.
  • the collected cells were resuspended in 1% PBSB, adjusted to a cell density of 2 ⁇ 10 6 /ml, plated in a 96-well plate, 100 ⁇ l per well (2 ⁇ 10 5 cells), and blocked at 4° C. for 0.5 h.
  • the CD47 humanized antibodies AB12W3, AB12W4, AB12W6 and the control antibody AB12W2 were diluted at the same time, starting from 10 ⁇ g/ml, and 5-fold gradient dilution to obtain 7 concentration gradients for use.
  • the blocked cells were centrifuged to discard the supernatant, added with diluted CD47 humanized antibody, and incubated at 4°C for 1 h; centrifuged to remove the supernatant, washed three times with 1% PBSB, and added diluted F4647-labeled goat anti-human IgG polyclonal antibody ( Jackson, Cat. No.
  • the positive control group AB12W1 and the negative isotype control group (Isotype control) were set up, and 50 ⁇ l per well was added to the 96-well plate, fully mixed and left to stand. Incubate at 37°C for 1 h in a 5% CO2 incubator. The cells were collected by centrifugation, the supernatant was discarded, and the cells were washed three times with assay buffer, and the supernatant was discarded. Prepare an appropriate concentration of F4647-labeled goat anti-human IgG polyclonal antibody (Jackson, Cat. No. 109-605-088), resuspend cells in 100 ⁇ l per well, and incubate at room temperature for 1 h in the dark.
  • F4647-labeled goat anti-human IgG polyclonal antibody Jackson, Cat. No. 109-605-088
  • the cells were collected by centrifugation, the supernatant was discarded, and the cells were washed three times with assay buffer, and the supernatant was discarded.
  • the cells were resuspended in 100 ⁇ l of experimental buffer, and detected on the computer (BD Accuri C6), and the data were analyzed.
  • CD47 humanized antibodies AB12W6, AB12W3 and AB12W9 can specifically bind to CHO/K1-hCD47 cells overexpressing CD47, and the binding ability is stronger than that of the control antibody AB12W1.
  • Their EC50 values were 0.2167 ⁇ g/ml, 0.4608 ⁇ g/ml, 0.2515 ⁇ g/ml and 0.6926 ⁇ g/ml, respectively.
  • the constant regions are all of the human IgG4 subtype containing the S228P/L235E mutation.
  • Rituxan was used as a positive control antibody to detect its CDC killing effect on Raji cells (CD20 positive), and an isotype control group was set at the same time.
  • the binding affinity of the candidate antibodies AB12W3, AB12W4 and AB12W6 to human CD47 was comparable to that of the control antibodies AB12W1 and AB12W2.
  • the binding affinity of AB12W10 was one order of magnitude lower, and the humanized antibodies were better.
  • the affinity and specificity of the parental murine monoclonal antibody is preserved, and its immunogenicity is greatly reduced.
  • the extracted human peripheral blood, PBMC cells collected by gradient centrifugation, and human monocyte isolation kit (Miltenyi Biotechnology Co., Ltd.) were used to separate and collect monocytes.
  • the isolated monocytes were seeded into culture dishes at a cell density of 1 ⁇ 10 6 /ml, and the medium was changed on the 5th, 8th and 11th days after seeding, and the cells differentiated into macrophages after the 13th day. (M2 type), irregular adherent cell morphology can be observed with the naked eye.
  • Cell surface markers can also be detected by FACS, generally including CD11b, CD14, CD45, CD64, CD163, and CD206.
  • the CCRF-CEM (Shanghai Institute of Cells, Chinese Academy of Sciences) stained with PE fluorescence was seeded in a 96-well plate, 50 ⁇ l per well, about 2.5 ⁇ 10 5 cells.
  • the antibodies to be tested AB12W3, AB12W4 and AB12W6 were diluted with PBS to 200 ⁇ g/ml, 3-fold gradient diluted to 11 concentration points, and the AB12W1 positive control group and the isotype antibody negative control group were set at the same time. Then, different concentration gradients of the antibody to be tested were spread in the above-mentioned 96-well plate, 50 ⁇ l per well. Let stand for 1 h in a 37°C, 5% CO 2 incubator to fully bind the antibody to the target cells.
  • M2 macrophages prepared above, 100 ⁇ l per well, about 5 ⁇ 10 4 cells, and mix well. 37°C, 5% CO2 incubator for 1h. All cells were harvested and labeled with CD11b-APC fluorescent antibody (Invitrogen). Plates were washed according to conventional flow cytometry to remove unbound fluorescent antibodies. The total number of samples taken was 1 ⁇ 10 4 cells for on-machine detection, in which the target cells were PE fluorescence, the macrophages were APC fluorescence, and the phagocytosis was PE+APC dual fluorescence. read the fluorescence value, Data were analyzed and plotted with log concentration (lgC) as abscissa and percentage phagocytosis (%) as ordinate.
  • lgC log concentration
  • CD47 humanized antibodies AB12W3, AB12W4, and AB12W6 could mediate macrophage phagocytosis of tumor cells CCRF-CEM in a dose-dependent manner, which was comparable to the EC50 value of control antibody AB12W1.
  • CD47 is also highly expressed on the surface of red blood cells.
  • CD47 antibody binds to CD47 on the surface of red blood cells, it breaks the "don't eat me” signal on the surface of red blood cells, causing macrophages to clear red blood cells, which may induce symptoms of anemia.
  • red blood cell depletion occurred after CD47 antibody treatment, resulting in transient anemia as the main adverse reaction. Therefore, we measured the phagocytosis of erythrocytes by macrophages mediated by CD47 humanized antibodies AB12W3 and AB12W4 to evaluate the potential side effects of the CD47 humanized antibodies provided by the present invention in clinical application.
  • Erythrocytes were prepared by conventional methods and stained with PE fluorescence, and macrophages were labeled with CD11b-APC fluorescent antibody. Data were analyzed and plotted with log concentration (lgC) as abscissa and percentage phagocytosis (%) as ordinate.
  • control antibody AB12W1 was able to mediate stronger erythrocyte phagocytosis compared to the isotype control, while the humanized antibodies AB12W3 and AB12W4 hardly caused erythrocyte phagocytosis, and there was no significant difference compared with the isotype control antibody.
  • the detection method is as follows: collecting fresh human peripheral blood, centrifuging and washing the pellet, separating human red blood cells, and resuspending the cells to a density of 2 ⁇ 10 8 /ml. Add 50 ⁇ l of human erythrocyte suspension and 50 ⁇ l of the antibody to be tested (the highest concentration is 200 ⁇ g/ml, 3-fold gradient dilution, a total of 8 points) into a 96-well plate, and the negative control group (NC) is added with an equal volume of PBS buffer. Incubate at 37°C for 30 min. After the reaction, the 96-well plate was photographed with a scanner and the results were judged.
  • the criterion for determining the result is that if the red blood cells settle on the bottom of the well and are flattened into a mesh, then the red blood cell agglutination reaction has occurred (see the results of AB12W1 in Figure 12). (See the results of NC in Fig. 12 ), according to the area of the sedimented erythrocyte mass, the strength of the antibody on erythrocyte agglutination can be preliminarily determined.
  • CD47 humanized antibodies AB12W3, AB12W4, AB12W5, AB12W6, AB12W8, AB12W9 and AB12W10 did not cause obvious hemagglutination reaction like the negative control group, while the control antibody AB12W1 showed obvious cells at high concentrations. Agglomeration phenomenon. It is suggested that the CD47 antibody provided by the present invention has a significantly reduced effect of promoting hemagglutination, and it is expected that the related side reactions in its clinical application will also be greatly reduced or even eliminated.
  • the average tumor volume of the PBS control group was 1001.17 ⁇ 38.43 mm 3 ; the average tumor volume of the AB12W1 administration group was 554.07 ⁇ 122.11 mm 3 , and the TGI was 44.12%, relative to the control group.
  • the administration doses of AB12W6, AB12W9, AB12W10, control antibody AB12W2 and CD20 monoclonal antibody Rituxan were all 5 mg/kg, and the negative control group was given an equal volume of PBS accordingly.
  • the average tumor volume in the PBS control group was 1881.04 ⁇ 198.74; the average tumor volume in the Rituxan administration group was 806.79 ⁇ 206.63 mm 3 , and the TGI was 51.18%;
  • the mean tumor volume was 309.58 ⁇ 54.62 mm 3 , and the TGI was 84.21%; the mean tumor volume of the AB12W6-administered group was 370.54 ⁇ 116.72 mm 3 , and the TGI was 82.19%; the mean tumor volume of the AB12W9-administered group was 343.52 ⁇ 145.65 mm 3 , respectively , TGI was 83.94%; the mean tumor volume of AB12W10 administration group was 380.37 ⁇ 111.84mm 3 , TGI was 81.65%. Compared with the control group, all administration groups had very significant differences (P ⁇ 0.01).
  • the mean tumor volume of the PBS negative control group was 2139.04 ⁇ 309.53 mm 3 ; the mean tumor volume of the Rituxan administration group was 1303.69 ⁇ 481.03 mm 3 , and the TGI was 43.4%, which was 43.4% relative to There was no significant difference in the control group, but the tumor in 1 of the 6 mice completely regressed; the average tumor volume in the AB12W1-administered group was 859.40 ⁇ 241.19 mm 3 , and the TGI was 61.91%; the average tumor in the AB12W8-administered group The volume was 1838.71 ⁇ 263.19mm 3 , and there was no significant difference compared with the control group; the average tumor volume of the AB12W9 administration group was 555.13 ⁇ 189.79mm 3 , and the TGI was 76.44%; the Rituxan and AB12W1, AB12W8 and AB12W9 combined administration groups respectively The mean tumor volumes
  • the anti-CD47 humanized antibody AB12W9 has a good anti-tumor effect, and the anti-tumor effect of the anti-CD47 humanized antibody AB12W9 alone or in combination with Rituxan is better than that of the control antibody AB12W1 alone or in combination with Rituxan; AB12W8 alone has no significant effect It also significantly inhibited tumor growth when used in combination with Rituxan.
  • the combined effect of AB12W8 and AB12W9 and the control antibody AB12W1 and Rituxan was better than that of the single administration group.
  • the average tumor volume of the negative control group was 1711.49 ⁇ 540.38 mm 3 ; the average tumor volume of the Rituxan administration group was 1497.13 ⁇ 447.01 mm 3 , the TGI was 16.59%, relative to the negative
  • the average tumor volume of the AB12W2 administration group was 21.47 ⁇ 15.54mm 3
  • the TGI was 98.78%, which was significantly different from the negative control group (P ⁇ 0.0001); 1mg/kg, 3mg of AB12W9
  • the mean tumor volumes of the three doses/kg and 10 mg/kg were 770.48 ⁇ 218.83mm 3 , 179.76 ⁇ 186.32mm 3 and 78.13 ⁇ 63.00mm 3 , respectively, and the TGIs were 55.05%, 89.68% and 95.53%, respectively; There were very significant differences in the control group (P ⁇ 0.01); the mean tumor volumes of the three doses of Rituxan
  • the TGIs were 78.46%, 92.98% and 96.39%, respectively.
  • the animals were in good health and no animals died.
  • the body weight of the animals in each group increased. There was no significant difference in the body weight of the animals in the AB12W9 treatment group alone or in combination with the Rituxan treatment group and the solvent control group (p>0.05), indicating that the animals tolerated AB12W9 well. Animals have obvious toxic effects.
  • the anti-CD47 humanized antibody AB12W9 has a good anti-tumor effect alone, and the anti-tumor effect has a good dose-effect relationship; the anti-tumor effect of AB12W9 at a low dose of 1 mg/kg combined with Rituxan is better than that of AB12W9 alone Rituxan and Rituxan alone group; AB12W9 medium and high doses (3, 10 mg/kg) have achieved the best anti-tumor effect as a single drug, and there is no room for improvement, so the effect of combined use with Rituxan is comparable to that of AB12W9 alone.

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Abstract

Provided are an antibody against CD47 and a use thereof in the preparation of drugs for the treatment of various diseases (including tumours and infectious diseases). The antibody can specifically recognise and bind to CD47 with high affinity, and will not cause a significant haemagglutination reaction of red blood cells.

Description

抗CD47抗体及其用途Anti-CD47 antibody and use thereof 技术领域technical field

本发明属于治疗性单克隆抗体领域，更具体地，本发明涉及一种针对整合素相关蛋白(CD47)的抗体；还涉及所述抗体用于制备治疗多种疾病(包括肿瘤和感染性疾病)药物中的用途。The present invention belongs to the field of therapeutic monoclonal antibodies, and more particularly, the present invention relates to an antibody against integrin-related protein (CD47); and also relates to the preparation of the antibody for the treatment of various diseases (including tumors and infectious diseases) Use in medicine.

背景技术Background technique

CD47又称整联蛋白(integrin-associated protein，IAP)，其全长含305个氨基酸，胞外氨基端为一个IgV样的结构域，高度疏水延伸的5个跨膜片段和1个短的选择性拼接的羧基端胞质尾区，其中IgV样结构域是其主要配体结合部位。其配体包括：信号调节蛋白α链(Signal-regulatory proteinα,SIRPα)、凝血酶敏感蛋白(Thrombospondin,TSP)和整合素(Integrins)。CD47广泛表达于细胞膜表面，存在于所有组织的不同细胞上，尤其是白细胞，如多形核白细胞(PMN)、树突状细胞(DC)、T细胞、红细胞、胎盘、血小板及造血干细胞(hematopoietic stem cells，HSCs)等正常细胞。同时，CD47在人体大部分肿瘤细胞中高表达，并且它可以作为肿瘤诊断及判断预后的一个标准。如它在急慢性髓系白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤以及膀胱癌、卵巢癌、乳腺癌、直肠癌、前列腺癌、肾癌、多发性骨髓瘤等多种实体肿瘤细胞或肿瘤干细胞上(如白血病干细胞leukemia stem cells,LSC)存在过表达，并且其高表达与临床预后差相关。CD47, also known as integrin-associated protein (IAP), contains 305 amino acids in its full length, an IgV-like domain at the extracellular amino-terminus, 5 transmembrane segments with highly hydrophobic extensions and 1 short selection Sexually spliced carboxy-terminal cytoplasmic tail of which the IgV-like domain is its primary ligand binding site. Its ligands include: Signal-regulatory proteinα (SIRPα), Thrombospondin (TSP) and Integrins. CD47 is widely expressed on the cell membrane surface and is present on different cells in all tissues, especially leukocytes such as polymorphonuclear leukocytes (PMN), dendritic cells (DC), T cells, red blood cells, placenta, platelets and hematopoietic stem cells (hematopoietic stem cells). normal cells such as stem cells, HSCs). At the same time, CD47 is highly expressed in most tumor cells in the human body, and it can be used as a standard for tumor diagnosis and prognosis. For example, it is used in acute and chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer, multiple myeloma and other solid tumor cells or There is overexpression on cancer stem cells (such as leukemia stem cells, LSC), and its high expression is associated with poor clinical prognosis.

研究表明，肿瘤细胞表面的CD47通过与巨噬细胞、DC细胞等免疫细胞表面的SIRPα配体结合，向其传递―“别吃我”(Don't eat me)的抑制信号，从而逃避这些细胞的吞噬(Kershaw MH等,Science,2013,341:41-42)，并进而逃避吞噬后肿瘤抗原的递呈所导致的T细胞对肿瘤细胞的杀伤作用(Vonderheide RH,Nature Medicine,2015,21:1122–1123)。通过阻断肿瘤细胞表面的CD47与巨噬细胞或DC细胞表面的SIRPα的相互作用，可以促进巨噬细胞、DC细胞对肿瘤细胞的吞噬，并进一步增加肿瘤抗原的提呈，激活获得性免疫应答(Chao MP,Curr Opin Immunol,2012,24(2):225-32；Liu XJ,Nature medicine,2015,21(10):1209-1215)。因此，通过使用CD47抗体阻断CD47-SIRPα通路，介导巨噬细胞吞噬作用，能够靶向性杀伤肿瘤细胞。目前，国内外为数不多的同靶点抗体药物正处于I～II期临床研究阶段，例如Forty Seven公司的Hu5F9-G4，处于临床I/II期阶段，单用治疗急性骨髓性白血病及淋巴瘤；与利妥昔单抗联用治疗B细胞非霍奇金淋巴瘤；或与西妥昔单抗联用治疗结直肠癌。Celgene公司的CC-90002处于临床I期阶段，针对的适应症是实体瘤、多发性骨髓瘤，非霍奇金淋巴瘤。信达生物的全人单克隆抗体IBI188，正在开展针对晚期恶性肿瘤(包括非霍奇金淋巴瘤和卵巢癌)的临床研究。Studies have shown that CD47 on the surface of tumor cells evades these cells by binding to SIRPα ligands on the surface of immune cells such as macrophages and DC cells, and delivering an inhibitory signal of "Don't eat me" to them. phagocytosis (Kershaw MH et al., Science, 2013, 341:41-42), and then evade the killing effect of T cells on tumor cells caused by the presentation of tumor antigens after phagocytosis (Vonderheide RH, Nature Medicine, 2015, 21: 1122–1123). By blocking the interaction between CD47 on the surface of tumor cells and SIRPα on the surface of macrophages or DC cells, it can promote the phagocytosis of tumor cells by macrophages and DC cells, further increase the presentation of tumor antigens, and activate the adaptive immune response (Chao MP, Curr Opin Immunol, 2012, 24(2): 225-32; Liu XJ, Nature medicine, 2015, 21(10): 1209-1215). Therefore, by blocking the CD47-SIRPα pathway with CD47 antibody, the phagocytosis of macrophages can be mediated, and tumor cells can be targeted and killed. At present, a few domestic and foreign antibody drugs with the same target are in the phase I-II clinical research stage, such as Forty Seven's Hu5F9-G4, which is in the clinical phase I/II stage, and is used alone to treat acute myeloid leukemia and lymphoma ; with rituximab for B-cell non-Hodgkin lymphoma; or with cetuximab for colorectal cancer. Celgene's CC-90002 is in Phase I clinical trials, targeting solid tumors, multiple myeloma, and non-Hodgkin's lymphoma. Innovent's fully human monoclonal antibody IBI188 is undergoing clinical studies against advanced malignant tumors, including non-Hodgkin's lymphoma and ovarian cancer.

此外，多数CD47抗体被报道可引起人红细胞的血凝反应。血凝反应是同型相互作用的一个示例，使用二价CD47抗体处理后可引起两个表达CD47的细胞聚合或凝集。例如，一种全IgG或F(ab') 2构型的CD47抗体MABL被报道能够引起红细胞的血凝反应，且仅当MABL变为scFv或二价scFv时该作用减弱(参见例如UnoS等,Oncol Rep,2007,17:1189-1194；Kikuchi Y等,Biochem Biophys Res Commun,2004,315:912-918)。其它已知的CD47抗体(包括B6H12、BRC126和CC2C6)也会引起红细胞的血凝反应。因此，引起红细胞凝集是现有靶向CD47的全IgG或F(ab') 2构型抗体应用于临床的主要限制。因此，目前仍然迫切需要开发不仅能够有效促进巨噬细胞的吞噬作用而且与红细胞结合微弱，不导致红细胞凝集的CD47抗体。 In addition, most CD47 antibodies have been reported to induce hemagglutination of human erythrocytes. Hemagglutination is an example of a homotypic interaction, and treatment with a bivalent CD47 antibody can cause aggregation or agglutination of two CD47-expressing cells. For example, a fully IgG or F(ab') 2 configuration of the CD47 antibody MABL has been reported to induce hemagglutination of red blood cells, and this effect was only attenuated when MABL was changed to scFv or bivalent scFv (see e.g. UnoS et al., Oncol Rep, 2007, 17: 1189-1194; Kikuchi Y et al, Biochem Biophys Res Commun, 2004, 315: 912-918). Other known CD47 antibodies, including B6H12, BRC126 and CC2C6, also cause hemagglutination of red blood cells. Therefore, causing hemagglutination is the main limitation of the current CD47-targeting full IgG or F(ab') 2 configuration antibodies in clinical application. Therefore, there is still an urgent need to develop CD47 antibodies that can not only effectively promote the phagocytosis of macrophages but also weakly bind to erythrocytes and do not cause erythrocyte agglutination.

发明内容SUMMARY OF THE INVENTION

本发明提供了一种能够特异性识别并高亲和力结合CD47(尤其是人CD47)的抗体。本发明所述抗体能够调节(如阻断、抑制、减少、拮抗、中和或干扰)CD47的活性和/或信号传递，且这些抗体不会导致血红 细胞出现显著的血凝反应。本发明公开的CD47抗体可以用于治疗、预防和/或诊断多种疾病，例如肿瘤。The present invention provides an antibody that can specifically recognize and bind CD47 (especially human CD47) with high affinity. The antibodies of the present invention are capable of modulating (eg, blocking, inhibiting, reducing, antagonizing, neutralizing or interfering with) the activity and/or signaling of CD47, and these antibodies do not result in significant hemagglutination of red blood cells. The CD47 antibodies disclosed in the present invention can be used to treat, prevent and/or diagnose various diseases, such as tumors.

在第一个方面，本发明提供了一种能够特异性结合CD47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的重链可变区(VH)包含至少一个、两个或三个选自下组的互补决定区(CDR)：In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to CD47, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising at least one, two or Three complementarity determining regions (CDRs) selected from the group consisting of:

(i)CDR-H1，其具有如SEQ ID NO：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35或37所示的VH中含有的CDR-H1的序列，或者与所述VH中含有的CDR-H1的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(i) CDR-H1 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 The sequence of CDR-H1 contained in the VH, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or add) sequence;

(ii)CDR-H2，其具有如SEQ ID NO：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35或37所示的VH中含有的CDR-H2的序列，或者与所述VH中含有的CDR-H2的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(ii) CDR-H2 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 The sequence of CDR-H2 contained in the VH, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or added) sequence; and

(iii)CDR-H3，其具有如SEQ ID NO：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35或37所示的VH中含有的CDR-H3的序列，或者与所述VH中含有的CDR-H3的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 The sequence of CDR-H3 contained in the VH, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or add) sequence;

和/或，其包含的轻链可变区(VL)包含至少一个、两个或三个选自下组的互补决定区(CDR)：And/or, the light chain variable region (VL) it comprises comprises at least one, two or three complementarity determining regions (CDRs) selected from the group consisting of:

(iv)CDR-L1，其具有如SEQ ID NO：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36或38所示的VL中含有的CDR-L1的序列，或者与所述VL中含有的CDR-L1的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iv) CDR-L1 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 The sequence of CDR-L1 contained in the VL, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or add) sequence;

(v)CDR-L2，其具有如SEQ ID NO：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36或38所示的VL中含有的CDR-L2的序列，或者与所述VL中含有的CDR-L2的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(v) CDR-L2 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 The sequence of CDR-L2 contained in the VL, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions) compared to the sequence of the CDR-L2 contained in the VL added) sequence; and

(vi)CDR-L3，其具有如SEQ ID NO：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36或38所示的VL中含有的CDR-L3的序列，或者与所述VL中含有的CDR-L3的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 The sequence of CDR-L3 contained in the VL, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions) compared to the sequence of the CDR-L3 contained in the VL add) sequence;

或，or,

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，所述重链可变区(VH)中含有的CDR-H1、CDR-H2及CDR-H3，和/或所述轻链可变区(VL)中含有的CDR-L1、CDR-L2及CDR-L3由Kabat或IMGT编号系统定义。In certain preferred embodiments, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH), and/or the light chain variable region (VL) CDR-L1, CDR-L2 and CDR-L3 are defined by the Kabat or IMGT numbering system.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：如SEQ ID NO：5、13、15、17或19所示的重链可变区(VH)中含有的至少一个、两个或三个CDR；和/或，如SEQ ID NO：6、14、16、18或20所示的轻链可变区(VL)中含有的至少一个、两个或三个CDR；其中，所述重链可变区(VH)中含有的CDR-H1、CDR-H2及CDR-H3和所述轻链可变区(VL)中含有的CDR-L1、CDR-L2及CDR-L3通过IMGT或Kabat编号系统确定。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: at least one of the heavy chain variable regions (VH) as set forth in SEQ ID NO: 5, 13, 15, 17 or 19 , two or three CDRs; and/or, at least one, two or three CDRs contained in the variable region (VL) of the light chain as set forth in SEQ ID NO: 6, 14, 16, 18 or 20; Wherein, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and the CDR-L1, CDR-L2 and CDR-L1 contained in the light chain variable region (VL) L3 is determined by the IMGT or Kabat numbering system.

在某些更优选的实施方案中，所述抗体或其抗原结合片段包含：In certain more preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：79，或与SEQ ID NO：79相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(i) CDR-H1 consisting of the following sequence: SEQ ID NO:79, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:79 substitutions, deletions or additions);

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：80，或与SEQ ID NO：80相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:80, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:80) substitution, deletion or addition); and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：81，或与SEQ ID NO：81相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO: 81, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 81) substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：82，或与SEQ ID NO：82相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:82, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:82 substitutions, deletions or additions);

(v)CDR-L2，其由下述序列组成：SEQ ID NO：83，或与SEQ ID NO：83相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:83, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:83 substitution, deletion or addition); and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the IMGT numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：79所示的CDR-H1；如SEQ ID NO：80所示的CDR-H2；以及，如SEQ ID NO：81所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：82所示的CDR-L1；如SEQ ID NO：83所示的CDR-L2；以及，如SEQ ID NO：44所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:79; CDR-H2 as set forth in SEQ ID NO:80; and, CDR-H3 as shown in SEQ ID NO: 81; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 82; CDR as shown in SEQ ID NO: 83 -L2; and, CDR-L3 as shown in SEQ ID NO:44.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions);

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitution, deletion or addition); and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：42，或与SEQ ID NO：42相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:42, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:42 substitutions, deletions or additions);

(v)CDR-L2，其由下述序列组成：SEQ ID NO：43，或与SEQ ID NO：43相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:43, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:43) substitution, deletion or addition); and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：39所示的CDR-H1；如SEQ ID NO：40所示的CDR-H2；以及，如SEQ ID NO：41所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：42所示的CDR-L1；如SEQ ID NO：43所示的CDR-L2；以及，如SEQ ID NO：44所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 42; CDR as shown in SEQ ID NO: 43 -L2; and, CDR-L3 as shown in SEQ ID NO:44.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：45，或与SEQ ID NO：45相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:45, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:45) substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：46，或与SEQ ID NO：46相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:46, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:46) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：39所示的CDR-H1；如SEQ ID NO：40所示的CDR-H2；以及，如SEQ ID NO：41所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：45所示的CDR-L1；如SEQ ID NO：46所示的CDR-L2；以及，如SEQ ID NO：44所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 45; CDR as shown in SEQ ID NO: 46 -L2; and, CDR-L3 as shown in SEQ ID NO:44.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：47，或与SEQ ID NO：47相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:47, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:47 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：48，或与SEQ ID NO：48相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:48, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:48) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：49，或与SEQ ID NO：49相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:49, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:49 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：50，或与SEQ ID NO：50相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:50, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:50 substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：47所示的CDR-H1；如SEQ ID NO：48所示的CDR-H2；以及，如SEQ ID NO：41所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：49所示的CDR-L1；如SEQ ID NO：50所示的CDR-L2；以及，如SEQ ID NO：44所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:47; CDR-H2 as set forth in SEQ ID NO:48; and, CDR-H3 as shown in SEQ ID NO:41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:49; CDR as shown in SEQ ID NO:50 -L2; and, CDR-L3 as shown in SEQ ID NO:44.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：如SEQ ID NO：7、21、23、25或27所示的重链可变区(VH)中含有的至少一个、两个或三个CDR；和/或，如SEQ ID NO：8、22、24、26或28所示的轻链可变区(VL)中含有的至少一个、两个或三个CDR；其中，所述重链可变区(VH)中含有的CDR-H1、CDR-H2及CDR-H3和所述轻链可变区(VL)中含有的CDR-L1、CDR-L2及CDR-L3通过IMGT或Kabat编号系统确定。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: at least one of the heavy chain variable regions (VH) as set forth in SEQ ID NO: 7, 21, 23, 25 or 27 , two or three CDRs; and/or, at least one, two or three CDRs contained in the variable region (VL) of the light chain as set forth in SEQ ID NO: 8, 22, 24, 26 or 28; Wherein, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and the CDR-L1, CDR-L2 and CDR-L1 contained in the light chain variable region (VL) L3 is determined by the IMGT or Kabat numbering system.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：84，或与SEQ ID NO：84相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:84, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:84 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：85，或与SEQ ID NO：85相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:85, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:85 substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：86，或与SEQ ID NO：86相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:86, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:86) substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：87，或与SEQ ID NO：87相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO: 87, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 87) substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：88，或与SEQ ID NO：88相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:88, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:88) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the IMGT numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：84所示的CDR-H1；如SEQ ID NO：85所示的CDR-H2；以及，如SEQ ID NO：86所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：87所示的CDR-L1；如SEQ ID NO：88所示的CDR-L2；以及，如SEQ ID NO：56所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:84; CDR-H2 as set forth in SEQ ID NO:85; and, CDR-H3 as shown in SEQ ID NO: 86; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 87; CDR as shown in SEQ ID NO: 88 -L2; and, CDR-L3 as shown in SEQ ID NO:56.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：51，或与SEQ ID NO：51相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:51, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:51 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：52，或与SEQ ID NO：52相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:52, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:52 substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：53，或与SEQ ID NO：53相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：54，或与SEQ ID NO：54相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:54, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:54 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：55，或与SEQ ID NO：55相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:55, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:55 substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：51所示的CDR-H1；如SEQ ID NO：52所示的CDR-H2；以及，如SEQ ID NO：53所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：54所示的CDR-L1；如SEQ ID NO：55所示的CDR-L2；以及，如SEQ ID NO：56所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:51; CDR-H2 as set forth in SEQ ID NO:52; and, CDR-H3 as shown in SEQ ID NO:53; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:54; CDR as shown in SEQ ID NO:55 -L2; and, CDR-L3 as shown in SEQ ID NO:56.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：51，或与SEQ ID NO：51相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:51, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:51 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：57，或与SEQ ID NO：57相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:57, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:57) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：53，或与SEQ ID NO：53相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：54，或与SEQ ID NO：54相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:54, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:54 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：55，或与SEQ ID NO：55相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:55, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:55 substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：51所示的CDR-H1；如SEQ ID NO：57所示的CDR-H2；以及，如SEQ ID NO：53所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：54所示的CDR-L1；如SEQ ID NO：55所示的CDR-L2；以及，如SEQ ID NO：56所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:51; CDR-H2 as set forth in SEQ ID NO:57; and, CDR-H3 as shown in SEQ ID NO:53; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:54; CDR as shown in SEQ ID NO:55 -L2; and, CDR-L3 as shown in SEQ ID NO:56.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：58，或与SEQ ID NO：58相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:58, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:58) substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：59，或与SEQ ID NO：59相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:59, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:59) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：53，或与SEQ ID NO：53相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：60，或与SEQ ID NO：60相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:60, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:60) substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：61，或与SEQ ID NO：61相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:61, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:61 substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：58所示的CDR-H1；如SEQ ID NO：59所示的CDR-H2；以及，如SEQ ID NO：53所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：60所示的CDR-L1；如SEQ ID NO：61所示的CDR-L2；以及，如SEQ ID NO：56所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:58; CDR-H2 as set forth in SEQ ID NO:59; and, CDR-H3 as shown in SEQ ID NO: 53; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 60; CDR as shown in SEQ ID NO: 61 -L2; and, CDR-L3 as shown in SEQ ID NO:56.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：如SEQ ID NO：9、29、31、33或35所示的重链可变区(VH)中含有的至少一个、两个或三个CDR；和/或，如SEQ ID NO：10、30、32、34或36所示的轻链可变区(VL)中含有的至少一个、两个或三个CDR；其中，所述重链可变区(VH)中含有的CDR-H1、CDR-H2及CDR-H3和所述轻链可变区(VL)中含有的CDR-L1、CDR-L2及CDR-L3通过IMGT或Kabat编号系统确定。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: at least one of the heavy chain variable regions (VH) as set forth in SEQ ID NO: 9, 29, 31, 33 or 35 , two or three CDRs; and/or, at least one, two or three CDRs contained in the variable region (VL) of the light chain as set forth in SEQ ID NO: 10, 30, 32, 34 or 36; Wherein, the CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) and the CDR-L1, CDR-L2 and CDR-L1 contained in the light chain variable region (VL) L3 is determined by the IMGT or Kabat numbering system.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：89，或与SEQ ID NO：89相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:89, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:89) substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：90，或与SEQ ID NO：90相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:90, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:90 substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：91，或与SEQ ID NO：91相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:91, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:91) substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：92，或与SEQ ID NO：92相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:92, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:92 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：93，或与SEQ ID NO：93相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:93, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:93) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the IMGT numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：89所示的CDR-H1；如SEQ ID NO：90所示的CDR-H2；以及，如SEQ ID NO：91所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：92所示的CDR-L1；如SEQ ID NO：93所示的CDR-L2；以及，如SEQ ID NO：67所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:89; CDR-H2 as set forth in SEQ ID NO:90; and, CDR-H3 as shown in SEQ ID NO:91; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:92; CDR as shown in SEQ ID NO:93 -L2; and, CDR-L3 as shown in SEQ ID NO:67.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：62，或与SEQ ID NO：62相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:62, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:62 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：63，或与SEQ ID NO：63相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:63, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:63) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：64，或与SEQ ID NO：64相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：65，或与SEQ ID NO：65相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:65, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:65 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：66，或与SEQ ID NO：66相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:66, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:66) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：62所示的CDR-H1；如SEQ ID NO：63所示的CDR-H2；以及，如SEQ ID NO：64所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：65所示的CDR-L1；如SEQ ID NO：66所示的CDR-L2；以及，如SEQ ID NO：67所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:62; CDR-H2 as set forth in SEQ ID NO:63; and, CDR-H3 as shown in SEQ ID NO:64; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:65; CDR as shown in SEQ ID NO:66 -L2; and, CDR-L3 as shown in SEQ ID NO:67.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：62，或与SEQ ID NO：62相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:62, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:62 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：68，或与SEQ ID NO：68相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO: 68, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 68) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：64，或与SEQ ID NO：64相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：69，或与SEQ ID NO：69相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:69, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:69 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：70，或与SEQ ID NO：70相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:70, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:70 substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：62所示的CDR-H1；如SEQ ID NO：68所示的CDR-H2；以及，如SEQ ID NO：64所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：69所示的CDR-L1；如SEQ ID NO：70所示的CDR-L2；以及，如SEQ ID NO：67所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:62; CDR-H2 as set forth in SEQ ID NO:68; and, CDR-H3 as shown in SEQ ID NO:64; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:69; CDR as shown in SEQ ID NO:70 -L2; and, CDR-L3 as shown in SEQ ID NO:67.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：71，或与SEQ ID NO：71相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:71, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:71 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：72，或与SEQ ID NO：72相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:72, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:72) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：64，或与SEQ ID NO：64相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：73，或与SEQ ID NO：73相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:73, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:73 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：74，或与SEQ ID NO：74相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:74, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:74) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：71所示的CDR-H1；如SEQ ID NO：72所示的CDR-H2；以及，如SEQ ID NO：64所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：73所示的CDR-L1；如SEQ ID NO：74所示的CDR-L2；以及，如SEQ ID NO：67所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:71; CDR-H2 as set forth in SEQ ID NO:72; and, CDR-H3 as shown in SEQ ID NO:64; and, the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO:73; CDR as shown in SEQ ID NO:74 -L2; and, CDR-L3 as shown in SEQ ID NO:67.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：如SEQ ID NO：11或37所示的重链可变区(VH)中含有的至少一个、两个或三个CDR；和/或，如SEQ ID NO：12或38所示的轻链可变区(VL) 中含有的至少一个、两个或三个CDR；其中，所述重链可变区(VH)中含有的CDR-H1、CDR-H2及CDR-H3和所述轻链可变区(VL)中含有的CDR-L1、CDR-L2及CDR-L3通过IMGT或Kabat编号系统确定。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: at least one, two or three contained in the variable region (VH) of the heavy chain as set forth in SEQ ID NO: 11 or 37 CDRs; and/or, at least one, two or three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 12 or 38; wherein the heavy chain variable region (VH) The CDR-H1, CDR-H2 and CDR-H3 contained in the light chain variable region (VL) and the CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) are determined by the IMGT or Kabat numbering system.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：79，或与SEQ ID NO：79相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:79, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:79 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：80，或与SEQ ID NO：80相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:80, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:80) substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：81，或与SEQ ID NO：81相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO: 81, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 81) substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：94，或与SEQ ID NO：94相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:94, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:94) substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：83，或与SEQ ID NO：83相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:83, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:83 substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：77，或与SEQ ID NO：77相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the IMGT numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：79所示的CDR-H1；如SEQ ID NO：80所示的CDR-H2；以及，如SEQ ID NO：81所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：94所示的CDR-L1；如SEQ ID NO：83所示的CDR-L2；以及，如SEQ ID NO：77所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:79; CDR-H2 as set forth in SEQ ID NO:80; and, CDR-H3 as shown in SEQ ID NO: 81; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 94; CDR as shown in SEQ ID NO: 83 -L2; and, CDR-L3 as shown in SEQ ID NO:77.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)CDR：(b) the following three light chain variable region (VL) CDRs:

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：75，或与SEQ ID NO：75相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:75, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:75 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：76，或与SEQ ID NO：76相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:76, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:76) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：77，或与SEQ ID NO：77相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：39所示的CDR-H1；如SEQ ID NO：40所示的CDR-H2；以及，如SEQ ID NO：41所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：75所示的CDR-L1；如SEQ ID NO：76所示的CDR-L2；以及，如SEQ ID NO：77所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 75; CDR as shown in SEQ ID NO: 76 -L2; and, CDR-L3 as shown in SEQ ID NO:77.

在某些优选的实施方案中，所述抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)下述3个重链可变区(VH)CDR：(a) the following three heavy chain variable region (VH) CDRs:

(i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions),

(ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), and

(iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitution, deletion or addition); and

(b)下述3个轻链可变区(VL)的CDR：(b) CDRs of the following three light chain variable regions (VL):

(iv)CDR-L1，其由下述序列组成：SEQ ID NO：75，或与SEQ ID NO：75相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:75, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:75 substitutions, deletions or additions),

(v)CDR-L2，其由下述序列组成：SEQ ID NO：78，或与SEQ ID NO：78相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:78, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 compared to SEQ ID NO:78) substitutions, deletions or additions), and

(vi)CDR-L3，其由下述序列组成：SEQ ID NO：77，或与SEQ ID NO：77相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions);

其中，所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义。Wherein, the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs are defined by the Kabat numbering system.

在某些优选的实施方案中，(i)-(vi)任一项中所述的置换为保守置换。In certain preferred embodiments, the substitutions described in any of (i)-(vi) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：39所示的CDR-H1；如SEQ ID NO：40所示的CDR-H2；以及，如SEQ ID NO：41所示的CDR-H3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：75所示的CDR-L1；如SEQ ID NO：78所示的CDR-L2；以及，如SEQ ID NO：77所示的CDR-L3。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises: CDR-H1 as set forth in SEQ ID NO:39; CDR-H2 as set forth in SEQ ID NO:40; and, CDR-H3 as shown in SEQ ID NO: 41; and the VL of the antibody or antigen-binding fragment thereof comprises: CDR-L1 as shown in SEQ ID NO: 75; CDR as shown in SEQ ID NO: 78 -L2; and, CDR-L3 as shown in SEQ ID NO:77.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段进一步包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的构架区(FR)。In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention further comprise framework regions (FRs) derived from mammalian (eg, murine or human) immunoglobulins.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含来源于鼠免疫球蛋白的重链可变区(VH)构架区(FR)，和/或所述抗体或其抗原结合片段的VL包含来源于鼠免疫球蛋白的轻链可变区(VL)构架区(FR)。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) framework region (FR) derived from a murine immunoglobulin, and/or the antibody or its The VL of the antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from a murine immunoglobulin.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含来源于人免疫球蛋白的重链可变区(VH)构架区(FR)，和/或所述抗体或其抗原结合片段的VL包含来源于人免疫球蛋白的轻链可变区(VL)构架区(FR)。在此类实施方案中，本发明的抗体或其抗原结合片段的重链可变区FR和/或轻链可变区FR可以包含一个或多个非人源(例如，鼠源)氨基酸残基，例如所述重链构架区FR和/或轻链构架区FR可以 包含一或多个氨基酸回复突变，在这些回复突变中有相应的鼠源氨基酸残基。In certain preferred embodiments, the VH of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) framework region (FR) derived from a human immunoglobulin, and/or the antibody or its The VL of the antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from a human immunoglobulin. In such embodiments, the heavy chain variable region FR and/or light chain variable region FR of the antibody or antigen-binding fragment thereof of the invention may comprise one or more non-human (eg, murine) amino acid residues For example, the heavy chain framework region FR and/or the light chain framework region FR may comprise one or more amino acid back-mutations in which there are corresponding murine amino acid residues.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)人免疫球蛋白的重链构架区或其变体，所述变体与其所源自的序列相比具有至多20个氨基酸保守置换(例如至多15个、至多10个、或至多5个保守置换；例如1个，2个，3个，4个或5个保守置换)；和/或(a) a heavy chain framework region of a human immunoglobulin or a variant thereof having up to 20 amino acid conservative substitutions (eg, up to 15, up to 10, or up to 5) compared to the sequence from which it is derived conservative substitutions; e.g. 1, 2, 3, 4 or 5 conservative substitutions); and/or

(b)人免疫球蛋白的轻链构架区或其变体，所述变体与其所源自的序列相比具有至多20个氨基酸保守置换(例如至多15个、至多10个、或至多5个保守置换；例如1个，2个，3个，4个或5个保守置换)。(b) a light chain framework region of a human immunoglobulin or a variant thereof having up to 20 amino acid conservative substitutions (eg, up to 15, up to 10, or up to 5) compared to the sequence from which it is derived conservative substitutions; eg, 1, 2, 3, 4 or 5 conservative substitutions).

因此，在某些优选的实施方案中，本发明的抗体或其抗原结合片段是人源化的。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的人源化程度为至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。Accordingly, in certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention are humanized. In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% humanized %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NOs：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35和37任一项所示的序列；(i) the sequence shown in any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37;

(ii)与SEQ ID NOs：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35和37任一项所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) compared to the sequence set forth in any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37 A sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions); or

(iii)与SEQ ID NOs：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35和37任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least a 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequences of sequence identity;

和/或，and / or,

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NOs：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的序列；(iv) the sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38;

(v)与SEQ ID NOs：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) compared to the sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 A sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions); or

(vi)与SEQ ID NOs：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least a 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequences of sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：选自如SEQ ID NO：5、13、15、17和19所示的重链可变区(VH)；和/或，选自如SEQ ID NO：6、14、16、18和20所示的轻链可变区。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 5, 13, 15, 17 and 19; and/or , selected from the light chain variable regions shown in SEQ ID NOs: 6, 14, 16, 18 and 20.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：5所示的序列；(i) the sequence shown in SEQ ID NO: 5;

(ii)与SEQ ID NO：5所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 5; or

(iii)与SEQ ID NO：5所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：6所示的序列；(iv) the sequence shown in SEQ ID NO: 6;

(v)与SEQ ID NO：6所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 6 ;or

(vi)与SEQ ID NO：6所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：13所示的序列；(i) the sequence shown in SEQ ID NO: 13;

(ii)与SEQ ID NO：13所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 13 ;or

(iii)与SEQ ID NO：13所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：14所示的序列；(iv) the sequence shown in SEQ ID NO: 14;

(v)与SEQ ID NO：14所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 14 ;or

(vi)与SEQ ID NO：14所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：15所示的序列；(i) the sequence shown in SEQ ID NO: 15;

(ii)与SEQ ID NO：15所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 15 ;or

(iii)与SEQ ID NO：15所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：16所示的序列；(iv) the sequence shown in SEQ ID NO: 16;

(v)与SEQ ID NO：16所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 16 ;or

(vi)与SEQ ID NO：16所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：17所示的序列；(i) the sequence shown in SEQ ID NO: 17;

(ii)与SEQ ID NO：17所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 17 ;or

(iii)与SEQ ID NO：17所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：18所示的序列；(iv) the sequence shown in SEQ ID NO: 18;

(v)与SEQ ID NO：18所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 18 ;or

(vi)与SEQ ID NO：18所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：19所示的序列；(i) the sequence shown in SEQ ID NO: 19;

(ii)与SEQ ID NO：19所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 19 ;or

(iii)与SEQ ID NO：19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：20所示的序列；(iv) the sequence shown in SEQ ID NO: 20;

(v)与SEQ ID NO：20所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 20 ;or

(vi)与SEQ ID NO：20所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：选自如SEQ ID NO：7、21、23、25和27所示的重链可变区(VH)；和/或，选自如SEQ ID NO：8、22、24、26和28所示的轻链可变区。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 7, 21, 23, 25 and 27; and/or , selected from the light chain variable regions shown in SEQ ID NOs: 8, 22, 24, 26 and 28.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：7所示的序列；(i) the sequence shown in SEQ ID NO: 7;

(ii)与SEQ ID NO：7所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 7 ;or

(iii)与SEQ ID NO：7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：8所示的序列；(iv) the sequence shown in SEQ ID NO: 8;

(v)与SEQ ID NO：8所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 8 ;or

(vi)与SEQ ID NO：8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：21所示的序列；(i) the sequence shown in SEQ ID NO: 21;

(ii)与SEQ ID NO：21所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 21 ;or

(iii)与SEQ ID NO：21所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：22所示的序列；(iv) the sequence shown in SEQ ID NO: 22;

(v)与SEQ ID NO：22所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 22 ;or

(vi)与SEQ ID NO：22所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：23所示的序列；(i) the sequence shown in SEQ ID NO: 23;

(ii)与SEQ ID NO：23所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 23 ;or

(iii)与SEQ ID NO：23所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：24所示的序列；(iv) the sequence shown in SEQ ID NO: 24;

(v)与SEQ ID NO：24所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 24 ;or

(vi)与SEQ ID NO：24所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：25所示的序列；(i) the sequence shown in SEQ ID NO: 25;

(ii)与SEQ ID NO：25所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 25 ;or

(iii)与SEQ ID NO：25所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：26所示的序列；(iv) the sequence shown in SEQ ID NO: 26;

(v)与SEQ ID NO：26所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 26 ;or

(vi)与SEQ ID NO：26所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：27所示的序列；(i) the sequence shown in SEQ ID NO: 27;

(ii)与SEQ ID NO：27所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 27 ;or

(iii)与SEQ ID NO：27所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：28所示的序列；(iv) the sequence shown in SEQ ID NO: 28;

(v)与SEQ ID NO：28所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 28 ;or

(vi)与SEQ ID NO：28所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：选自如SEQ ID NO：9、29、31、33和35所示的重链可变区(VH)；和/或，选自如SEQ ID NO：10、30、32、34和36所示的轻链可变区。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9, 29, 31, 33 and 35; and/or , selected from the light chain variable regions shown in SEQ ID NOs: 10, 30, 32, 34 and 36.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：9所示的序列；(i) the sequence shown in SEQ ID NO: 9;

(ii)与SEQ ID NO：9所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 9; or

(iii)与SEQ ID NO：9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：10所示的序列；(iv) the sequence shown in SEQ ID NO: 10;

(v)与SEQ ID NO：10所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 10 ;or

(vi)与SEQ ID NO：10所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：29所示的序列；(i) the sequence shown in SEQ ID NO: 29;

(ii)与SEQ ID NO：29所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 29; or

(iii)与SEQ ID NO：29所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：30所示的序列；(iv) the sequence shown in SEQ ID NO: 30;

(v)与SEQ ID NO：30所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 30 ;or

(vi)与SEQ ID NO：30所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：31所示的序列；(i) the sequence shown in SEQ ID NO: 31;

(ii)与SEQ ID NO：31所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4 个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 31; or

(iii)与SEQ ID NO：31所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：32所示的序列；(iv) the sequence shown in SEQ ID NO: 32;

(v)与SEQ ID NO：32所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 32 ;or

(vi)与SEQ ID NO：32所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：33所示的序列；(i) the sequence shown in SEQ ID NO: 33;

(ii)与SEQ ID NO：33所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 33; or

(iii)与SEQ ID NO：33所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：34所示的序列；(iv) the sequence shown in SEQ ID NO: 34;

(v)与SEQ ID NO：34所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 34 ;or

(vi)与SEQ ID NO：34所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：35所示的序列；(i) the sequence shown in SEQ ID NO: 35;

(ii)与SEQ ID NO：35所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 35; or

(iii)与SEQ ID NO：35所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：36所示的序列；(iv) the sequence shown in SEQ ID NO: 36;

(v)与SEQ ID NO：36所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3 个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 36 ;or

(vi)与SEQ ID NO：36所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：选自如SEQ ID NO：11和37所示的重链可变区(VH)；和/或，选自如SEQ ID NO：12和38所示的轻链可变区。In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 11 and 37; and/or, selected from the group consisting of SEQ ID NO: 11 and 37 : light chain variable regions shown at 12 and 38.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：11所示的序列；(i) the sequence shown in SEQ ID NO: 11;

(ii)与SEQ ID NO：11所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 11; or

(iii)与SEQ ID NO：11所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：12所示的序列；(iv) the sequence shown in SEQ ID NO: 12;

(v)与SEQ ID NO：12所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 12 ;or

(vi)与SEQ ID NO：12所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：37所示的序列；(i) the sequence shown in SEQ ID NO: 37;

(ii)与SEQ ID NO：37所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 37; or

(iii)与SEQ ID NO：37所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：38所示的序列；(iv) the sequence shown in SEQ ID NO: 38;

(v)与SEQ ID NO：38所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 38 ;or

(vi)与SEQ ID NO：38所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些实施方案中，本发明的抗体或其抗原结合片段进一步包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的恒定区序列或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加。在某些优选的实施方案中，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换。在某些实施方案中，抗CD47抗体分子具有重链恒定区(从N端到C端顺序连接的CH1结构域、铰链区、CH2结构域和CH3结构域)，其选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；特别地选自例如IgG1、IgG2、IgG3和IgG4的重链恒定区，更特别地选自IgG1、IgG2或IgG4(例如是人IgG1、IgG2或IgG4)的重链恒定区。在一些实施方案中，抗CD47抗体分子具有轻链恒定区，其选自例如κ或λ的轻链恒定区，优选κ轻链恒定区(例如人κ轻链)。In certain embodiments, the antibody or antigen-binding fragment thereof of the invention further comprises a mammalian (eg, murine or human) immunoglobulin-derived constant region sequence or variant thereof, which variant is derived from the same The sequence comparison has one or more amino acid substitutions, deletions or additions. In certain preferred embodiments, the variant has one or more amino acid substitutions compared to the sequence from which it is derived. In certain embodiments, the anti-CD47 antibody molecule has a heavy chain constant region (CH1 domain, hinge region, CH2 domain, and CH3 domain sequentially linked from N-terminus to C-terminus) selected from, eg, IgG1, IgG2, Heavy chain constant regions of IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular selected from heavy chain constant regions such as IgG1, IgG2, IgG3 and IgG4, more particularly selected from IgG1, IgG2 or IgG4 (e.g. heavy chain constant regions of human IgG1, IgG2 or IgG4). In some embodiments, the anti-CD47 antibody molecule has a light chain constant region selected from, eg, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (eg, a human kappa light chain).

在一些优选实施方案中，所述重链恒定区序列的Fc片段被改变，例如被突变，以修饰本发明所述抗体分子的性质(例如改变下列中的一个或更多个特性：例如，Fc受体结合、效应细胞功能或补体功能、Fab臂交换等)。In some preferred embodiments, the Fc fragment of the heavy chain constant region sequence is altered, eg, mutated, to modify properties of the antibody molecule of the invention (eg, to alter one or more of the following properties: eg, Fc receptor binding, effector cell function or complement function, Fab arm exchange, etc.).

例如，本发明提供的抗体包含具有改变的效应子功能(例如降低或消除)的氨基酸置换、缺失或添加的Fc变体。抗体的Fc片段介导几种重要的效应子功能，例如ADCC、ADCP、CDC等。通过替换抗体的Fc片段中的氨基酸残基，以改变抗体对效应子配体(如FcγR或补体C1q)的亲和力，从而改变效应子功能的方法是本领域已知的(参见，例如EP 388,151A1；US 564,8260；US 562,4821；Natsume A等,Cancer Res.,68:3863-3872,2008；Idusogie EE等,J.Immunol.,166:2571-2575,2001；Lazar GA等,PNAS,103:4005-4010,2006；Shields RL等,JBC,276:6591-6604,2001；Stavenhagen JB等,Cancer Res.,67:8882-8890,2007；Stavenhagen JB等,Advan.Enzyme.Regul.,48:152-164,2008；Alegre ML等,J.Immunol.,148:3461-3468,1992；和Kaneko E等,Biodrugs,25:1-11,2011)。与FcγR相互作用主要影响抗体的ADCC活性，对应的氨基酸位点为P232-S239、D265-D270、Y296-T299和N325-I332。在本发明一些优选实施例中，对抗体重链恒定区上的氨基酸L235(EU编号)进行修饰以改变Fc受体相互作用，例如L235E或L235A。在本发明另一些优选实施例中，对抗体恒定区上的氨基酸234和235同时进行修饰，例如L234A和L235A(L234A/L235A)(EU编号)。在本发明另一些优选实施例中，对抗体重链恒定区上的氨基酸D265(EU编号)进行修饰以改变Fc受体相互作用，例如D265A。与C1q相互作用主要影响抗体的CDC活性，对应的氨基酸位点为D270、K322、P329和P331。在一些优选实施例中，对抗体恒定区上P331的氨基酸进行突变以消除CDC效应，例如P331S(EU编号)；在另一些优选实施例中，对抗体恒定区上P329的氨基酸进行置换，例如P329A(EU编号)。For example, the antibodies provided herein comprise Fc variants that have amino acid substitutions, deletions or additions with altered effector function (eg, reduction or elimination). The Fc fragment of an antibody mediates several important effector functions, such as ADCC, ADCP, CDC, etc. Methods of altering effector function by substituting amino acid residues in the Fc fragment of an antibody to alter the affinity of the antibody for effector ligands such as FcyR or complement C1q are known in the art (see, eg, EP 388,151A1 ; US 564,8260; US 562,4821; Natsume A et al, Cancer Res., 68:3863-3872, 2008; Idusogie EE et al, J. Immunol., 166:2571-2575, 2001; Lazar GA et al, PNAS, 103: 4005-4010, 2006; Shields RL et al, JBC, 276: 6591-6604, 2001; Stavenhagen JB et al, Cancer Res., 67: 8882-8890, 2007; Stavenhagen JB et al, Advan. Enzyme. Regul., 48 : 152-164, 2008; Alegre ML et al, J. Immunol., 148: 3461-3468, 1992; and Kaneko E et al, Biodrugs, 25: 1-11, 2011). The interaction with FcγR mainly affects the ADCC activity of the antibody, and the corresponding amino acid positions are P232-S239, D265-D270, Y296-T299 and N325-I332. In some preferred embodiments of the invention, amino acid L235 (EU numbering) on the heavy chain constant region is modified to alter the Fc receptor interaction, eg L235E or L235A. In other preferred embodiments of the present invention, amino acids 234 and 235 in the constant region of the antibody are modified simultaneously, such as L234A and L235A (L234A/L235A) (EU numbering). In other preferred embodiments of the invention, amino acid D265 (EU numbering) on the heavy chain constant region of the antibody is modified to alter the Fc receptor interaction, eg D265A. The interaction with C1q mainly affects the CDC activity of the antibody, and the corresponding amino acid positions are D270, K322, P329 and P331. In some preferred embodiments, the amino acid of P331 in the constant region of the antibody is mutated to eliminate the CDC effect, such as P331S (EU numbering); in other preferred embodiments, the amino acid of P329 in the constant region of the antibody is substituted, such as P329A (EU number).

例如，本发明提供的抗体可包含具有延长的循环半衰期的氨基酸置换、缺失或添加的Fc变体。影响半衰期的主要是与FcRn的相互作用的残基，对应的氨基酸位点为L251-S254、L309-Q311和N434-H435。例如，M252Y/S254T/T256E、M428L/N434S或T250Q/M428L都能够延长抗体在灵长类动物中的半衰期。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN 201280066663.2、US 2005/0014934A1、WO 97/43316、US 5,869,046、US 5,747,03、WO 96/32478。在本发明一些优选实施例中，对抗体恒定区上的氨基酸M428(EU编号)进行修饰以增强FcRn受体的结合亲和力，例如M428L。在另一些优选实施例中，对抗体恒定区上的氨基酸250和428(EU编号)同时进行修饰，例如T250Q和M428L(T250Q/M428L)。For example, the antibodies provided herein can comprise amino acid substitutions, deletions or additions to Fc variants with extended circulating half-lives. Residues that affect the half-life mainly interact with FcRn, and the corresponding amino acid positions are L251-S254, L309-Q311 and N434-H435. For example, M252Y/S254T/T256E, M428L/N434S or T250Q/M428L were all able to prolong the half-life of antibodies in primates. More mutation sites included in Fc variants with enhanced binding affinity to neonatal receptor (FcRn) can be found in Chinese Invention Patent CN 201280066663.2, US 2005/0014934A1, WO 97/43316, US 5,869,046, US 5,747,03, WO 96/32478. In some preferred embodiments of the invention, amino acid M428 (EU numbering) on the constant region of the antibody is modified to enhance the binding affinity of the FcRn receptor, eg M428L. In other preferred embodiments, amino acids 250 and 428 (EU numbering) in the constant region of the antibody are modified simultaneously, eg, T250Q and M428L (T250Q/M428L).

例如，本发明提供的抗体也可包含具有可以降低或消除Fc糖基化的氨基酸置换、缺失或添加的Fc变体。例如，IgG在CH2结构域的氨基酸N297处含有保守的糖基化位点，N297位糖基化对IgG的活性有很大影响，如果该位点糖基化被移除，则会影响IgG分子CH2上半部分的构象，从而丧失对FcγRs的结合能力，影响抗体相关的生物活性，如ADCC效应。在本发明的一些优选实施例中，对人IgG恒定区上的氨基 酸N297(EU编号)进行修饰以避免抗体的糖基化，例如N297A。For example, the antibodies provided herein can also comprise Fc variants with amino acid substitutions, deletions or additions that reduce or eliminate Fc glycosylation. For example, IgG contains a conserved glycosylation site at amino acid N297 of the CH2 domain. Glycosylation at N297 greatly affects the activity of IgG. If the glycosylation at this site is removed, it will affect the IgG molecule. The conformation of the upper half of CH2, thereby losing the binding ability to FcγRs, affects antibody-related biological activities, such as ADCC effect. In some preferred embodiments of the invention, amino acid N297 (EU numbering) on the human IgG constant region is modified to avoid glycosylation of the antibody, such as N297A.

例如，本发明提供的抗体也可包含具有可以消除Fab臂交换的Fc变体。例如，在本发明的一些优选实施例中，将IgG4的核心铰链区228位Ser替换为Pro(S228P)(EU编号)，可消除Fab交换作用，增强Fab稳定性。For example, the antibodies provided herein may also include Fc variants with Fab arm swapping that eliminates. For example, in some preferred embodiments of the present invention, replacing Ser at position 228 in the core hinge region of IgG4 with Pro(S228P) (EU numbering) can eliminate Fab exchange and enhance Fab stability.

本发明的一些优选实施例中，所述人IgG重链恒定区序列选自以下变体：In some preferred embodiments of the present invention, the human IgG heavy chain constant region sequence is selected from the following variants:

(i)含有Leu234Val和Leu235Ala突变的人IgG1的重链恒定区序列；(i) heavy chain constant region sequence of human IgG1 containing Leu234Val and Leu235Ala mutations;

(ii)含有Asn297Ala突变的人IgG1的重链恒定区序列；(ii) the heavy chain constant region sequence of human IgG1 containing the Asn297Ala mutation;

(iii)含有Thr250Gln、Asn297Ala和Met428Leu突变的人IgG1的重链恒定区序列；(iii) heavy chain constant region sequence of human IgG1 containing Thr250Gln, Asn297Ala and Met428Leu mutations;

(iv)含有Asp265Ala和Pro329Ala突变的人IgG1的重链恒定区序列；(iv) heavy chain constant region sequence of human IgG1 containing Asp265Ala and Pro329Ala mutations;

(v)含有Pro331Ser突变的人IgG2的重链恒定区序列；(v) the heavy chain constant region sequence of human IgG2 containing the Pro331Ser mutation;

(vi)含有Ser228Pro突变的人IgG4的重链恒定区序列；(vi) heavy chain constant region sequence of human IgG4 containing Ser228Pro mutation;

(vii)含有Ser228Pro和Leu235Glu突变的人IgG4的重链恒定区序列；(vii) heavy chain constant region sequence of human IgG4 containing Ser228Pro and Leu235Glu mutations;

更优选地，所述抗体重链恒定区的氨基酸序列优选自SEQ ID NOs：96、97、98、99、100、101、102、103、104或105所示的天然的或突变的人IgG重链恒定区。More preferably, the amino acid sequence of the heavy chain constant region of the antibody is preferably selected from the native or mutated human IgG heavy as set forth in SEQ ID NOs: 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105 chain constant region.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：107所示的序列；(i) the sequence shown in SEQ ID NO: 107;

(ii)与SEQ ID NO：107所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 107 ;or

(iii)与SEQ ID NO：107所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：109所示的序列；(iv) the sequence shown in SEQ ID NO: 109;

(v)与SEQ ID NO：109所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 109 ;or

(vi)与SEQ ID NO：109所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：111所示的序列；(i) the sequence shown in SEQ ID NO: 111;

(ii)与SEQ ID NO：111所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 111 ;or

(iii)与SEQ ID NO：111所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序 列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：113所示的序列；(iv) the sequence shown in SEQ ID NO: 113;

(v)与SEQ ID NO：113所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 113 ;or

(vi)与SEQ ID NO：113所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：115所示的序列；(i) the sequence shown in SEQ ID NO: 115;

(ii)与SEQ ID NO：115所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 115 ;or

(iii)与SEQ ID NO：115所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：117所示的序列；(iv) the sequence shown in SEQ ID NO: 117;

(v)与SEQ ID NO：117所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 117 ;or

(vi)与SEQ ID NO：117所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：119所示的序列；(i) the sequence shown in SEQ ID NO: 119;

(ii)与SEQ ID NO：119所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 119 ;or

(iii)与SEQ ID NO：119所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：121所示的序列；(iv) the sequence shown in SEQ ID NO: 121;

(v)与SEQ ID NO：121所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 121 ;or

(vi)与SEQ ID NO：121所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：123所示的序列；(i) the sequence shown in SEQ ID NO: 123;

(ii)与SEQ ID NO：123所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 123 ;or

(iii)与SEQ ID NO：123所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：125所示的序列；(iv) the sequence shown in SEQ ID NO: 125;

(v)与SEQ ID NO：125所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 125 ;or

(vi)与SEQ ID NO：125所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：127所示的序列；(i) the sequence shown in SEQ ID NO: 127;

(ii)与SEQ ID NO：127所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 127 ;or

(iii)与SEQ ID NO：127所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：129所示的序列；(iv) the sequence shown in SEQ ID NO: 129;

(v)与SEQ ID NO：129所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 129 ;or

(vi)与SEQ ID NO：129所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：131所示的序列；(i) the sequence shown in SEQ ID NO: 131;

(ii)与SEQ ID NO：131所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 131 ;or

(iii)与SEQ ID NO：131所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：133所示的序列；(iv) the sequence shown in SEQ ID NO: 133;

(v)与SEQ ID NO：133所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 133 ;or

(vi)与SEQ ID NO：133所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:

(a)重链，其包含选自下列的氨基酸序列：(a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：135所示的序列；(i) the sequence shown in SEQ ID NO: 135;

(ii)与SEQ ID NO：135所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 135 ;or

(iii)与SEQ ID NO：135所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；和(iii) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

(b)轻链，其包含选自下列的氨基酸序列：(b) a light chain comprising an amino acid sequence selected from the group consisting of:

(iv)SEQ ID NO：137所示的序列；(iv) the sequence shown in SEQ ID NO: 137;

(v)与SEQ ID NO：137所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 137 ;or

(vi)与SEQ ID NO：137所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity.

在某些优选的实施方案中，(ii)或(v)中所述的置换是保守置换。In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

在某些实施方案中，本发明的抗CD47抗体还涵盖其抗体片段，抗体片段的实例包括但不限于Fv、Fab、Fab′、Fab’-SH，F(ab’) 2、双抗体、线性抗体、单链抗体分子(例如scFv)；和由抗体片段形成的双特异性抗体或多特异性抗体。 In certain embodiments, the anti-CD47 antibodies of the invention also encompass antibody fragments thereof, examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear Antibodies, single chain antibody molecules (eg, scFvs); and bispecific or multispecific antibodies formed from antibody fragments.

再一方面，本发明提供了编码以上任何抗CD47抗体或其抗原结合片段的核酸。在某些实施方案中，编 码所述抗CD47抗体分子的核苷酸序列是密码子最优化的。例如，本发明优选实施例中提供分别地编码抗CD47的抗体分子的重链和轻链的第一和第二核酸，所述抗体分子选自下列中的任意一种：AB12W3、AB12W4、AB12W5、AB12W6、AB12W7、AB12W8、AB12W9和AB12W10；或与其基本上相同的序列。例如，编码所述抗体重链的第一核酸包含如SEQ ID NO：106、110、114、118、122、126、130或134所示的序列或与其基本上相同的序列(例如与其至少大约85％、90％、95％、99％或更高度相似的序列或具有一个或更多个核苷酸取代(例如保守性取代))的序列，或与上述序列相差不超过3、5、10、15、20、25或30个核苷酸的序列)；和编码所述抗体轻链的第二核酸包含如SEQ ID NO：108、112、116、120、124、128、132或136所示的序列或与其基本上相同的序列(例如与其至少大约85％、90％、95％、99％或更高度相似的序列或具有一个或更多个核苷酸取代(例如保守性取代))的序列，或与上述序列相差不超过3、5、10、15、20、25或30核苷酸的序列)。In yet another aspect, the present invention provides nucleic acids encoding any of the above anti-CD47 antibodies or antigen-binding fragments thereof. In certain embodiments, the nucleotide sequence encoding the anti-CD47 antibody molecule is codon-optimized. For example, preferred embodiments of the present invention provide first and second nucleic acids encoding the heavy and light chains, respectively, of an anti-CD47 antibody molecule selected from any of the following: AB12W3, AB12W4, AB12W5, AB12W6, AB12W7, AB12W8, AB12W9, and AB12W10; or a sequence substantially identical thereto. For example, the first nucleic acid encoding the antibody heavy chain comprises the sequence set forth in SEQ ID NO: 106, 110, 114, 118, 122, 126, 130 or 134 or substantially identical thereto (eg, at least about 85 %, 90%, 95%, 99% or more highly similar sequences or sequences with one or more nucleotide substitutions (e.g. conservative substitutions), or differing no more than 3, 5, 10, 15, 20, 25 or 30 nucleotide sequence); and the second nucleic acid encoding the antibody light chain comprises as shown in SEQ ID NO: 108, 112, 116, 120, 124, 128, 132 or 136 A sequence or a sequence that is substantially identical thereto (eg, a sequence that is at least about 85%, 90%, 95%, 99% or more similar or has one or more nucleotide substitutions (eg, conservative substitutions)) , or a sequence that differs by no more than 3, 5, 10, 15, 20, 25 or 30 nucleotides from the above sequence).

再一方面，本发明提供了包含上述核酸的载体。In yet another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid.

再一方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些优选的实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。In yet another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain preferred embodiments, the vectors of the present invention are, for example, plasmids, cosmids, phages, and the like. In certain preferred embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (eg, a mammal, eg, a human).

再一方面，本发明提供了一种宿主细胞，其包含上述分离的核酸分子或上述载体。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。适宜的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些优选的实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、CHO DG44)。In yet another aspect, the present invention provides a host cell comprising the above-described isolated nucleic acid molecule or the above-described vector. Host cells can be eukaryotic cells (eg, mammalian cells, insect cells, yeast cells) or prokaryotic cells (eg, E. coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain preferred embodiments, the host cells of the invention are mammalian cells, such as CHO (eg, CHO-K1, CHO-S, CHO DXB11, CHO DG44).

再一方面，本发明提供制备抗CD47抗体或其抗原结合片段的方法，其中所述方法包含在适于表达编码所述抗体或其抗原结合片段的核酸的条件下培养所述宿主细胞，以及分离所述抗体或其抗原结合片段。在某些实施方案中，所述方法还包括从宿主细胞回收所述抗体或其抗原结合片段。In yet another aspect, the present invention provides a method of making an anti-CD47 antibody or antigen-binding fragment thereof, wherein the method comprises culturing the host cell under conditions suitable for expression of nucleic acid encoding the antibody or antigen-binding fragment thereof, and isolating The antibody or antigen-binding fragment thereof. In certain embodiments, the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell.

再一方面，本发明提供了一种药物组合物，其包含药学上可接受的载体、和/或赋形剂和/或稳定剂和至少一种本发明所述CD47抗体或其抗原结合片段。In yet another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and/or excipient and/or stabilizer and at least one CD47 antibody or an antigen-binding fragment thereof of the present invention.

再一方面，本发明涉及在受试者中抑制CD47与SIRPα结合的方法，所述方法包括向受试者施用有效量的本发明所述CD47抗体或其抗原结合片段。In yet another aspect, the present invention relates to a method of inhibiting CD47 binding to SIRPα in a subject, the method comprising administering to the subject an effective amount of the CD47 antibody or antigen-binding fragment thereof of the present invention.

再一方面，本发明还涉及所述CD47抗体或其抗原结合片段制备用于在受试者中抑制CD47与SIRPα结合的组合物或药物中的用途。In yet another aspect, the present invention also relates to the use of the CD47 antibody or antigen-binding fragment thereof in the preparation of a composition or medicament for inhibiting the binding of CD47 to SIRPα in a subject.

再一方面，本发明涉及促进受试者的巨噬细胞发挥吞噬作用的方法，所述方法包括向受试者施用有效量的本发明所述CD47抗体或其抗原结合片段。In yet another aspect, the present invention relates to a method of promoting phagocytosis of macrophages in a subject, the method comprising administering to the subject an effective amount of the CD47 antibody or antigen-binding fragment thereof of the present invention.

再一方面，本发明还涉及任何所述CD47抗体或其抗原结合片段在制备用于促进受试者的巨噬细胞发挥吞噬作用的组合物或药物中的用途。In yet another aspect, the present invention also relates to the use of any of the CD47 antibodies or antigen-binding fragments thereof in the manufacture of a composition or medicament for promoting phagocytosis by macrophages in a subject.

再一方面，本发明涉及治疗可以通过消除、抑制或降低CD47活性而被改善、减缓、抑制或预防的任何与CD47相关的疾病或病症的方法，所述方法包括向受试者或个体施用有效量的本发明所述CD47抗体或其抗原结合片段。例如，本发明提供了治疗(或预防)对象中肿瘤的方法，所述方法包括：向所述受试者或个体施用本发明所述CD47抗体或其抗原结合片段。In yet another aspect, the present invention relates to a method of treating any CD47-related disease or disorder that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing CD47 activity, the method comprising administering to a subject or individual an effective amount of the CD47 antibody or antigen-binding fragment thereof of the present invention. For example, the present invention provides a method of treating (or preventing) a tumor in a subject, the method comprising: administering to the subject or individual the CD47 antibody or antigen-binding fragment thereof of the present invention.

再一方面，本发明还涉及所述CD47抗体及其抗原结合片段在制备针对治疗对象中CD47介导的病症或疾病的药物中的用途。In yet another aspect, the present invention also relates to the use of the CD47 antibody and antigen-binding fragment thereof in the preparation of a medicament for treating a CD47-mediated disorder or disease in a subject.

在某些实施方案中，所述抗体分子可以用于治疗血液癌症，例如，选自以下的血液癌症：急性成淋巴细胞白血病(ALL)、急性髓性白血病(AML)、非霍奇金淋巴瘤(例如，弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、套细胞淋巴瘤、B成淋巴细胞白血病/淋巴瘤和伯基特淋巴瘤)、B-成淋巴细胞白血病/淋巴瘤；B细胞慢性淋巴细胞白血病/小淋巴细胞淋巴瘤、慢性淋巴细胞白血病(CLL)例如，转化的CLL、Richter综合征、慢性髓细胞白血病(CML)、滤泡淋巴瘤、多发性骨髓瘤、骨髓纤维化、真性红细胞增多症、皮肤T细胞淋巴瘤、意义未明的单克隆丙种球蛋白病(MGUS)、骨髓增生异常综合征(MDS)、免疫母细胞性大细胞淋巴瘤、前体B-成淋巴细胞淋巴瘤和间变性大细胞淋巴瘤。在一些实施方案中，所述癌症优选自以下的血液癌症：多发性骨髓瘤、弥漫性大B细胞淋巴瘤、AML、CLL例如转化的CLL、Richter综合征或滤泡淋巴瘤。In certain embodiments, the antibody molecule can be used to treat hematological cancers, eg, hematological cancers selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (eg, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-lymphoblastic leukemia/lymphoma, and Burkitt's lymphoma), B-lymphoblastic leukemia/lymphoma; B-cell chronic Lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL) eg, transformed CLL, Richter syndrome, chronic myeloid leukemia (CML), follicular lymphoma, multiple myeloma, myelofibrosis, true Polycythemia, cutaneous T-cell lymphoma, monoclonal gammopathy of undetermined significance (MGUS), myelodysplastic syndrome (MDS), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and anaplastic large cell lymphoma. In some embodiments, the cancer is preferably selected from the following hematological cancers: multiple myeloma, diffuse large B-cell lymphoma, AML, CLL such as transformed CLL, Richter syndrome or follicular lymphoma.

在某些实施方案中，所述抗体分子可以用于治疗实体瘤。在某些实施方案中，所述癌症选自：肺癌(例如，非小细胞肺癌、小细胞肺癌)、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、膀胱癌、脊柱癌、脑癌、宫颈癌、子宫内膜癌、结肠/直肠癌、肛门癌、子宫内膜癌、食道癌、胆囊癌、胃肠道癌、皮肤癌、前列腺癌、垂体癌、胃癌、子宫癌、阴道癌和甲状腺癌。In certain embodiments, the antibody molecules can be used to treat solid tumors. In certain embodiments, the cancer is selected from the group consisting of: lung cancer (eg, non-small cell lung cancer, small cell lung cancer), pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, spine cancer, Brain Cancer, Cervical Cancer, Endometrial Cancer, Colon/Rectal Cancer, Anal Cancer, Endometrial Cancer, Esophagus Cancer, Gallbladder Cancer, Gastrointestinal Cancer, Skin Cancer, Prostate Cancer, Pituitary Cancer, Stomach Cancer, Uterine Cancer, Vaginal Cancer cancer and thyroid cancer.

再一方面，本发明涉及检测样品中CD47蛋白的方法，所述方法包括(a)将样品与本发明所述的任何抗CD47抗体或其抗原结合片段接触；和(b)检测抗CD47抗体或其抗原结合片段和CD47蛋白间的复合物的形成。在某些实施方案中，CD47是人CD47。在一个实施方案中，该检测方法可以是体外或体内方法。在一个实施方案中，抗CD47抗体被用于选择适合利用抗CD47抗体治疗的受试者。在一个实施方案中，抗CD47抗体是被可检测地标记的。In yet another aspect, the present invention relates to a method for detecting CD47 protein in a sample, the method comprising (a) contacting the sample with any anti-CD47 antibody or antigen-binding fragment thereof described herein; and (b) detecting the anti-CD47 antibody or Formation of a complex between its antigen-binding fragment and CD47 protein. In certain embodiments, the CD47 is human CD47. In one embodiment, the detection method may be an in vitro or in vivo method. In one embodiment, an anti-CD47 antibody is used to select subjects suitable for treatment with an anti-CD47 antibody. In one embodiment, the anti-CD47 antibody is detectably labeled.

再一方面，本发明提供了检测样品中CD47蛋白含量或水平的试剂盒，其包括任何本发明所述的抗CD47抗体或其抗原结合片段和使用说明书。In yet another aspect, the present invention provides a kit for detecting the content or level of CD47 protein in a sample, which includes any of the anti-CD47 antibodies or antigen-binding fragments thereof described in the present invention and instructions for use.

本发明所述CD47抗体或其抗原结合片段以高亲和力与人CD47结合，阻断CD47与SIRPα的相互作用，并促进巨噬细胞对表达CD47的肿瘤细胞的吞噬活性，同时不导致显著的红细胞血凝反应。此外，本发明提供的CD47抗体在人Burkkit’s淋巴瘤的小鼠移植瘤模型中显示出极其显著的抗肿瘤活性。因此，本发明的CD47抗体克服了用于治疗性靶向CD47的关键限制因素。The CD47 antibody or its antigen-binding fragment of the present invention binds to human CD47 with high affinity, blocks the interaction between CD47 and SIRPα, and promotes the phagocytic activity of macrophages on tumor cells expressing CD47, without causing significant erythrocyte hemorrhage coagulation reaction. In addition, the CD47 antibody provided by the present invention shows extremely significant anti-tumor activity in the mouse xenograft model of human Burkkit's lymphoma. Thus, the CD47 antibodies of the present invention overcome a key limitation for therapeutic targeting of CD47.

另外，本发明提供的CD47抗体可同时结合人和食蟹猴和/或恒河猴的CD47天然抗原，使其临床前毒理学评价不需要再构建替代分子，且获得的有效剂量、毒性剂量和毒副反应更客观、准确，可以直接进行临床剂量的转换，降低临床研究的风险。In addition, the CD47 antibody provided by the present invention can simultaneously bind to the CD47 natural antigen of human and cynomolgus monkey and/or rhesus monkey, so that the preclinical toxicology evaluation does not require the construction of a replacement molecule, and the obtained effective dose, toxic dose and toxicity The side effects are more objective and accurate, and the clinical dose can be directly converted to reduce the risk of clinical research.

发明详述Detailed description of the invention

在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。Before the present invention is described in detail below, it is to be understood that this invention is not limited to the specific methodology, protocols and reagents described herein. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which shall be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

缩写和定义Abbreviations and Definitions

CDR    用Kabat编号系统界定的免疫球蛋白可变区中的互补决定区CDRs Complementarity Determining Regions in Immunoglobulin Variable Regions Defined by the Kabat Numbering System

EC 50    产生50％功效或结合的浓度 EC 50 Concentration that produces 50% efficacy or binding

ELISA  酶联免疫吸附测定ELISA enzyme-linked immunosorbent assay

FR     抗体构架区：将CDR区排除在外的免疫球蛋白可变区FR antibody framework regions: immunoglobulin variable regions excluding CDR regions

HRP    辣根过氧化物酶HRP horseradish peroxidase

IC 50    产生50％抑制的浓度 IC 50 Concentration that produces 50% inhibition

IgG    免疫球蛋白GIgG Immunoglobulin G

Kabat  由Elvin A Kabat倡导的免疫球蛋白比对及编号系统Kabat Immunoglobulin alignment and numbering system initiated by Elvin A Kabat

mAb    单克隆抗体mAb Monoclonal Antibody

PCR    聚合酶链式反应PCR polymerase chain reaction

V区    在不同抗体之间序列可变的IgG链区段。其延伸到轻链的109位Kabat残基和重链的第113位残基。V region A segment of an IgG chain whose sequence varies between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain.

VH     免疫球蛋白重链可变区VH Immunoglobulin heavy chain variable region

VK     免疫球蛋白κ轻链可变区VK immunoglobulin kappa light chain variable region

K D     平衡解离常数 K D equilibrium dissociation constant

ka     结合速率常数ka association rate constant

Kd     解离速率常数Kd dissociation rate constant

术语“凝集”是指细胞凝集，而术语“血细胞凝集”是指特定细胞亚型即红细胞的凝集。因此，血细胞凝集是凝集的一种类型。本发明的所述抗体不导致显著的凝集水平，例如血红细胞血凝反应(“RBC血凝反应”)。我们观察到，本发明的抗体以不促进CD47阳性细胞凝集的方式特异性结合CD47；并且，本发明的抗体结合细胞表面的CD47且不产生细胞凝集现象的能力不限于红血球细胞。The term "agglutination" refers to the agglutination of cells, while the term "hemagglutination" refers to the agglutination of a specific subtype of cells, ie, red blood cells. Therefore, hemagglutination is a type of agglutination. The antibodies of the invention do not cause significant levels of agglutination, such as red blood cell hemagglutination ("RBC hemagglutination"). We observed that the antibodies of the invention specifically bind to CD47 in a manner that does not promote agglutination of CD47 positive cells; and, the ability of the antibodies of the invention to bind CD47 on the cell surface without causing cell agglutination is not limited to red blood cells.

本领域的技术人员可以通过常规实验对凝集水平进行定量，例如RBC的血凝反应。例如，本领域的技术人员可在本发明的CD47抗体存在的条件下进行血凝反应试验，其后测量RBC斑点的面积以确定血凝反应的水平，如下文实施例中所述。在一些情况下，对存在本发明CD47抗体时的RBC斑点面积与不存在本发明CD47抗体时(即零血凝反应条件下)的RBC斑点面积，以及存在其他已知CD47抗体时的RBC斑点面积进行了比较。在这种方式下，相对于基线对照对血凝反应进行定量。RBC斑点的面积越大表明血凝反应的水平越高。或者，也可使用RBC斑点的密度分析对血凝反应进行定量。Those skilled in the art can quantify the level of agglutination, eg, the hemagglutination of RBCs, by routine experimentation. For example, one skilled in the art can perform a hemagglutination assay in the presence of the CD47 antibodies of the invention, and thereafter measure the area of the RBC spots to determine the level of hemagglutination, as described in the Examples below. In some cases, the RBC spot area in the presence of the CD47 antibody of the invention versus the RBC spot area in the absence of the CD47 antibody of the invention (ie, under zero hemagglutination conditions), and the RBC spot area in the presence of other known CD47 antibodies comparisons were made. In this manner, the hemagglutination response is quantified relative to a baseline control. The larger the area of the RBC spot, the higher the level of blood coagulation. Alternatively, hemagglutination can also be quantified using densitometric analysis of RBC spots.

术语“血红细胞”和“红细胞”是同义词并可互换使用。The terms "red blood cell" and "erythrocyte" are synonymous and used interchangeably.

本文所用术语“超变区”或“CDR区"或“互补决定区”是指负责抗原结合的抗体氨基酸残基。CDR区序列可以由IMGT、Kabat、Chothia和AbM方法来定义或本领域熟知的任何CDR区序列确定方法而鉴定的可变区内的氨基酸残基。抗体CDR可鉴定为最初由Kabat等人定义的高变区，例如，轻链可变结构域的 24-34(L1)、50-56(L2)和89-97(L3)位残基和重链可变结构域的31-35(H1)、50-65(H2)和95-102(H3)位残基，参见Kabat等，1991，Sequences of Proteins of Immunological Interest(免疫目的物的蛋白质序列)，第5版，Public Health Service，National Institutes of Health，Bethesda，Md.；CDR的位置亦可鉴定为最初由Chothia等人描述的“超变环”(HVL)结构来界定的，例如，轻链可变结构域的26-32(L1)、50-52(L2)和91-96(L3)位残基和重链可变结构域的26-32(H1)、52-56(H2)和95-102(H3)位残基(参见例如Chothia等人,J Mol Biol,1992,227:799-817；Tomlinson等人,J Mol Biol,1992,227:776-798)。IMGT(ImMunoGeneTics)也提供了包括CDR的免疫球蛋白可变区的编号系统，根据IMGT编号定义CDR区，例如，轻链可变结构域的27-32(L1)、50-52(L2)和89-97(L3)位残基和重链可变结构域的26-35(H1)、51-57(H2)和93-102(H3)位残基，参见如Lefranc,M.P.等的Dev Comp Immunol,2003,27:55-77，其通过引用并入本文。用于CDR鉴定的其它方法包括“AbM定义”，其为Kabat与Chothia之间的折衷且使用Oxford Molecular′s AbM抗体模型软件得到；或CDR的“接触定义”，其基于所观察的抗原接触且阐述于MacCallum等人，1996，J.Mol.Biol.，262：732-745中。CDR的“构型定义”方法中，CDR的位置可鉴定为对抗原结合作出焓贡献的残基，参见例如Makabe等人，2008，Journal of Biological Chemistry，283：1156-1166。本发明所用的方法可利用或根据这些方法中的任一种所定义的CDR，包括但不限于Kabat定义、IMGT定义、Chothia定义、AbM定义、接触定义和/或构型定义中的任一者来定义。The term "hypervariable region" or "CDR region" or "complementarity determining region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The CDR region sequences can be defined by the IMGT, Kabat, Chothia and AbM methods or the amino acid residues within the variable regions identified by any CDR region sequence determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al., eg, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and heavy Residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the chain variable domain, see Kabat et al., 1991, Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md.; CDR positions can also be identified as defined by the "hypervariable loop" (HVL) structure originally described by Chothia et al., e.g., light chains Residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the variable domain and 26-32 (H1), 52-56 (H2) and Residues at positions 95-102 (H3) (see, eg, Chothia et al, J Mol Biol, 1992, 227:799-817; Tomlinson et al, J Mol Biol, 1992, 227:776-798). IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions including CDRs, and CDR regions are defined according to IMGT numbering, eg, 27-32 (L1), 50-52 (L2) and Residues 89-97 (L3) and residues 26-35 (H1), 51-57 (H2) and 93-102 (H3) of the heavy chain variable domain, see eg Dev Comp by Lefranc, MP et al. Immunol, 2003, 27:55-77, which is incorporated herein by reference. Other methods for CDR identification include "AbM definitions", which are a compromise between Kabat and Chothia and derived using Oxford Molecular's AbM antibody modeling software; or "contact definitions" of CDRs, which are based on observed antigen contacts and Described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In the "configuration definition" approach of CDRs, the positions of CDRs can be identified as residues that make enthalpy contributions to antigen binding, see eg Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166. The methods used in the present invention may utilize or define CDRs according to any of these methods, including but not limited to any of the Kabat definitions, IMGT definitions, Chothia definitions, AbM definitions, contact definitions and/or configuration definitions to define.

术语“构架区”“可变构架区”、“FR区”为除本发明定义的超变区残基之外的可变结构域残基。在某些实施方案中，抗CD47抗体分子的轻链可变构架区或重链可变构架区可以选自：(a)包含至少70、75、80、85、86、87、88、89、90、91、92、94、95、96、97、98、99％或优选地100％的来自人轻链或重链可变构架区的氨基酸残基(例如来自人成熟抗体、人种系序列或人共有序列的轻链或重链可变构架区残基)的轻链或重链可变构架区；(b)非人构架区(例如啮齿类动物构架)；或(c)已经被修饰的(例如被修饰以除去抗原决定簇或细胞毒性决定簇)的非人构架区，例如去免疫的或部分地人源化的构架。在一些实施方案中，轻链或重链可变构架区包含与人生殖细胞基因的V L或V H段的构架区至少70、75、80、85、86、87、88、89、90、91、92、94、95、96、97、98、99％相同或相同的轻或重链可变构架区序列。在某些实施方案中，抗CD47抗体分子包括来自例如完整可变区(例如在表1中所示的)中FR区的氨基酸序列的具有至少1个、2个、3个、4个、5个、6个、7个、10个、15个、20个或更多个改变(例如氨基酸取代、插入或缺失)的重链可变区。在某些实施方案中，抗CD47抗体分子包括来自例如完整可变区(例如在表1中所示的)中FR区的氨基酸序列的具有至少1个、2个、3个、4个、5个、6个、7个、10个、15个、20个或更多个改变(例如氨基酸取代、插入或缺失)的轻链可变区。 The terms "framework region", "variable framework region", "FR region" are variable domain residues other than the hypervariable region residues as defined herein. In certain embodiments, the light chain variable framework region or the heavy chain variable framework region of the anti-CD47 antibody molecule may be selected from: (a) comprising at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 99% or preferably 100% of the amino acid residues from a human light or heavy chain variable framework region (e.g. from a human mature antibody, human germline sequence or a light chain or heavy chain variable framework region residues of a human consensus sequence); (b) a non-human framework region (eg, a rodent framework); or (c) has been modified (eg, modified to remove antigenic or cytotoxic determinants) non-human framework regions, eg, deimmunized or partially humanized frameworks. In some embodiments, the light or heavy chain variable framework region comprises at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98, 99% identical or identical light or heavy chain variable framework region sequences. In certain embodiments, the anti-CD47 antibody molecule comprises at least 1, 2, 3, 4, 5 amino acid sequences from, eg, FR regions in the entire variable region (eg, as shown in Table 1) 1, 6, 7, 10, 15, 20 or more altered heavy chain variable regions (eg, amino acid substitutions, insertions or deletions). In certain embodiments, the anti-CD47 antibody molecule comprises at least 1, 2, 3, 4, 5 amino acid sequences from, eg, FR regions in the entire variable region (eg, as shown in Table 1) 1, 6, 7, 10, 15, 20 or more altered (eg, amino acid substitutions, insertions or deletions) in the light chain variable region.

术语“嵌合抗体(Chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。可以通过任何合适的重组DNA技术产生嵌合抗体。一些是本领域已知的(参见PCT/US86/02269；EP184,187；EP171,496；EP173,494；WO86/01533；US4,816,567；EP125,023；Better等人,Science,1988,240:1041-1043)；Liu等人,PNAS,1987,84:3439-3443；Liu等人,J Immunol,1987,139:3521-3526；Sun等人,PNAS,1987,84:214-218；Nishimura等人,Canc Res,1987,47:999-1005；Wood等人,Nature,1985,314:446-449；和Shaw等人,J Natl Cancer Inst,1988,80:1553-1559)。在本发明的一优选实施方案中，所述的CD47嵌合抗体的抗体轻链可变区进一步包含鼠源κ、λ链或其变体的轻链FR区。所述CD47嵌合抗体的抗体重链可变区进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链FR区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区。The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody. Chimeric antibodies can be produced by any suitable recombinant DNA technique. Some are known in the art (see PCT/US86/02269; EP184,187; EP171,496; EP173,494; WO86/01533; US4,816,567; EP125,023; -1043); Liu et al., PNAS, 1987, 84:3439-3443; Liu et al., J Immunol, 1987, 139:3521-3526; Sun et al., PNAS, 1987, 84:214-218; Nishimura et al. , Canc Res, 1987, 47:999-1005; Wood et al, Nature, 1985, 314:446-449; and Shaw et al, J Natl Cancer Inst, 1988, 80:1553-1559). In a preferred embodiment of the present invention, the antibody light chain variable region of the CD47 chimeric antibody further comprises a light chain FR region of a murine κ, λ chain or a variant thereof. The antibody heavy chain variable region of the CD47 chimeric antibody further comprises the heavy chain FR region of murine IgG1, IgG2, IgG3 or a variant thereof. The constant region of the human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising the heavy chain constant regions of human IgG1 or IgG4.

“人共有框架”是指这样的框架，即在选择人免疫球蛋白VL或VH框架序列中，其代表最常出现的氨基酸残基。一般而言，对人免疫球蛋白VL或VH序列的选择是从可变结构域序列的亚型中选择。一般而言， 该序列的亚型是如Kabat等，Sequences of Proteins ofImmunological Interest，第五版，NIH Publication 91-3242，Bethesda MD(1991)，1-3卷中的亚型。"Human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. In general, selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, the subtypes of the sequences are as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

“人源化”抗体是指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的HVR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all HVRs (eg, CDRs) correspond to those of the non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

“人抗体”指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于这样抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。"Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source that utilizes a library of human antibodies or other human antibodies coding sequence. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

本文所用术语“阻断”表示存在本发明的抗体时减少的CD47信号传递。CD47介导的信号传递阻断指本发明的CD47抗体存在时CD47信号传递水平低于CD47的对照水平(即不存在抗体时的CD47信号传递水平)，降低的幅度大于或等于5％、10％、20％、25％、30％、40％、50％、60％、70％、75％、80％、90％、95％、99％或100％。可使用多种标准技术测量CD47信号传递水平，如作为非限制性实施例，测量下游基因激活和/或响应CD47激活的荧光素酶报告试验。本领域技术人员应理解可使用多种试验测量CD47信号传递水平，包括例如市售可得的试剂盒。The term "blocking" as used herein refers to reduced CD47 signaling in the presence of an antibody of the invention. The blocking of CD47-mediated signal transmission means that the CD47 signal transmission level in the presence of the CD47 antibody of the present invention is lower than the control level of CD47 (ie the CD47 signal transmission level in the absence of the antibody), and the reduction range is greater than or equal to 5%, 10% , 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100%. Levels of CD47 signaling can be measured using a variety of standard techniques, such as, by way of non-limiting example, downstream gene activation and/or luciferase reporter assays in response to CD47 activation. One of skill in the art will appreciate that a variety of assays can be used to measure the level of CD47 signaling, including, for example, commercially available kits.

本文使用的术语“免疫结合”和“免疫结合性质”是指一种非共价相互作用，其发生在免疫球蛋白分子和抗原(对于该抗原而言免疫球蛋白为特异性的)之间。免疫结合相互作用的强度或亲和力可以相互作用的平衡解离常数(K D)表示，其中K D值越小，表示亲和力越高。所选多肽的免疫结合性质可使用本领域中公知的方法定量。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kd或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出。(参见Malmqvist M,Nature,1993,361:186-187)。kd/ka的比率等于解离常数K D(通常参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量K D、ka和kd值。在优选的实施方案中，用生物发光干涉测量法(例如，实施例2.5中所述的ForteBio Octet法)来测量解离常数。在其它优选的实施方案中，可用表面等离子共振技术(例如Biacore)或Kinexa来测量解离常数。当平衡结合常数(K D)为≤10μM，优选为≤100nM，更优选为≤10nM，和最优选为≤100pM～约1pM时，本发明的抗体被认为特异性地结合至CD47表位。 The terms "immunobinding" and "immunobinding properties" as used herein refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immune binding interaction can be expressed as the equilibrium dissociation constant (K D ) of the interaction, where a smaller K D value indicates a higher affinity. The immunobinding properties of selected polypeptides can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both "association rate constants" (ka or kon) and "dissociation rate constants" (kd or koff) can be calculated from the concentrations and the actual rates of association and dissociation. (See Malmqvist M, Nature, 1993, 361:186-187). The ratio of kd/ka is equal to the dissociation constant KD (see generally Davies et al., Annual Rev Biochem, 1990;59:439-473). KD , ka and kd values can be measured by any effective method. In a preferred embodiment, dissociation constants are measured using bioluminescence interferometry (eg, the ForteBio Octet method described in Example 2.5). In other preferred embodiments, dissociation constants can be measured using surface plasmon resonance techniques (eg, Biacore) or Kinexa. Antibodies of the invention are considered to specifically bind to the CD47 epitope when the equilibrium binding constant (K D ) is ≤ 10 μM, preferably ≤ 100 nM, more preferably ≤ 10 nM, and most preferably ≤ 100 pM to about 1 pM.

本发明的抗CD47抗体Anti-CD47 antibody of the present invention

除非另外说明，术语“CD47”、“整合素相关蛋白(IAP)”、“卵巢癌抗原OA3”和“Rh-相关的抗原”同义并且可互换使用，当用于本文中时是指来自任何脊椎动物来源(包括哺乳动物如灵长类动物(例如人)和啮齿类动物(例如，小鼠和大鼠))的任何天然CD47，除非另有说明。该术语涵盖“全长”未加工的CD47以及由细胞内加工产生的任何形式的CD47或其任何片段。该术语还包括天然存在的CD47的变体，例如，剪接变体或等位变体。Unless otherwise specified, the terms "CD47", "integrin-associated protein (IAP)", "ovarian cancer antigen OA3" and "Rh-associated antigen" are synonymous and used interchangeably, and when used herein refer to origin from Any native CD47 of any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise stated. The term encompasses "full-length" unprocessed CD47 as well as any form of CD47 or any fragment thereof produced by intracellular processing. The term also includes naturally-occurring variants of CD47, eg, splice variants or allelic variants.

术语“抗CD47抗体”、“抗CD47”、“CD47抗体”或“结合CD47的抗体”是指这样的抗体，所述抗体能够以足够的亲合力结合CD47蛋白或其片段以致所述抗体可以用作靶向CD47中的诊断剂和/或治疗剂。在一些实施方案中，本文提供的抗CD47的抗体的解离常数(Kd)≤1μM、≤100nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10 -8M以下，例如10 -8M至10 -13M，例如10 -9M至10 -13M)。 The terms "anti-CD47 antibody", "anti-CD47", "CD47 antibody" or "CD47-binding antibody" refer to an antibody that is capable of binding the CD47 protein or fragment thereof with sufficient affinity such that the antibody can be used with as diagnostic and/or therapeutic agents targeting CD47. In some embodiments, the anti-CD47 antibodies provided herein have a dissociation constant (Kd) ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (eg, below 10 −8 M , such as 10 -8 M to 10 -13 M, such as 10 -9 M to 10 -13 M).

在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段包含取代、插入或缺失。在优选的实施方案中，取代、插入或缺失发生在CDR外的区域(例如在FR中)。任选地，本发明的抗CD47抗体包括对轻 链可变区、重链可变区、轻链或重链的翻译后修饰。In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof of the invention comprise substitutions, insertions or deletions. In preferred embodiments, substitutions, insertions or deletions occur in regions outside the CDRs (eg, in FRs). Optionally, the anti-CD47 antibodies of the invention include post-translational modifications to the light chain variable region, heavy chain variable region, light chain or heavy chain.

在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如取代)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.

在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基取代。In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are substituted with cysteine residues.

在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof.

在一些实施方案中，本发明涵盖抗CD47抗体的片段。抗体片段的实例包括但不限于Fv、Fab、Fab′、Fab’-SH，F(ab’) 2、双抗体、线性抗体、单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。 In some embodiments, the present invention encompasses fragments of anti-CD47 antibodies. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear antibodies, single-chain antibody molecules (eg, scFv); and multispecifics formed from antibody fragments Sexual antibodies.

在一些实施方案中，本发明的抗CD47抗体是人源化抗体。用于使抗体人源化的不同方法是技术人员已知的，如由Almagro&Fransson综述的，其内容通过引用完整并入本文(Almagro JC和Fransson J,Frontiers in Bioscience,2008,13:1619-1633)。Almagro&Fransson区分理性办法和经验办法。理性办法的特征在于生成少数工程化抗体变体并评估其结合或任何其它感兴趣的特性。如果设计的变体不产生预期的结果，那么启动新一轮的设计和结合评估。理性办法包括CDR嫁接、表面重建(Resurfacing)、超人源化(Superhumanization)和人字符串内容优化(Human StringContent Optimization)。相比之下，经验办法基于生成大的人源化变体库并使用富集技术或高通量筛选选出最佳克隆。因而，经验办法依赖于能够对大量抗体变体进行搜索的可靠的选择和/或筛选系统。体外展示技术，如噬菌体展示和核糖体展示满足这些要求并且是技术人员公知的。经验办法包括FR库、导向选择(Guided selection)、框架改组(Framework-shuffling)和Humaneering。In some embodiments, the anti-CD47 antibodies of the invention are humanized antibodies. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J, Frontiers in Bioscience, 2008, 13:1619-1633) . Almagro & Fransson distinguish between rational and empirical approaches. A rational approach is characterized by generating a small number of engineered antibody variants and evaluating their binding or any other property of interest. If the designed variant does not produce the expected results, a new round of design and binding evaluation is initiated. Rational methods include CDR grafting, surface reconstruction (Resurfacing), superhumanization (Superhumanization) and human string content optimization (Human StringContent Optimization). In contrast, empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening. Thus, empirical approaches rely on reliable selection and/or screening systems capable of searching a large number of antibody variants. In vitro display techniques such as phage display and ribosome display meet these requirements and are well known to the skilled person. Empirical approaches include FR libraries, Guided selection, Framework-shuffling, and Humaneering.

在一些实施方案中，本发明的抗CD47抗体是人抗体。可使用本领域中已知的各种技术来制备人抗体。人抗体一般描述于van Dijk和van de Winkel,Curr Opin Pharmacol,2001,5:368-374以及Lonberg,Curr Opin Immunol,2008,20:450-459。In some embodiments, the anti-CD47 antibodies of the invention are human antibodies. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol, 2001, 5:368-374 and Lonberg, Curr Opin Immunol, 2008, 20:450-459.

适用于本发明的“抗体及其抗原结合片段”包括但不限于多克隆、单克隆、单价、双特异性、异缀合物、多特异性、重组、异源、异源杂合、嵌合、人源化(特别是嫁接有CDR的)、去免疫的、或人的抗体、Fab片段、Fab′片段、F(ab′) 2片段、由Fab表达库产生的片段、Fd、Fv、二硫化物连接的Fv(dsFv)、单链抗体(例如scFv)、双抗体或四抗体(Holliger P等,Proc Natl Acad Sci USA,1993,90(14):6444-6448)、纳米抗体(nanobody)(也称为单域抗体)、抗独特型(抗Id)抗体(包括例如针对本发明抗体的抗Id抗体)和上述任一种的表位结合片段。 "Antibody and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric , humanized (especially CDR-grafted), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') 2 fragments, fragments generated from Fab expression libraries, Fd, Fv, Di Sulfide-linked Fv (dsFv), single chain antibody (eg scFv), diabody or tetrabody (Holliger P et al, Proc Natl Acad Sci USA, 1993, 90(14):6444-6448), nanobody (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against the antibodies of the invention), and epitope-binding fragments of any of the foregoing.

在一些实施方案中，本发明的抗体可以是单特异性的、双特异性或多特异性的。多特异性单抗可以对一种靶多肽的不同表位是特异性的或可以含有对超过一种靶多肽特异性的抗原结合域。参见例如，Tutt等,J Immunol,1991,147:60-69。抗CD47单抗可以与另一种功能性分子(例如另一种肽或蛋白质)连接或共表达。例如抗体或其片段可以功能性与一或多种其它分子，如另一种抗体或抗体片段连接(例如通过化学偶联、遗传融合、非共价联合或以其它方式)以产生具有第二或更多结合特异性的双特异性或多特异性抗体。In some embodiments, the antibodies of the invention may be monospecific, bispecific or multispecific. Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen binding domains specific for more than one target polypeptide. See, eg, Tutt et al., J Immunol, 1991, 147:60-69. The anti-CD47 mAb can be linked or co-expressed with another functional molecule (eg, another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (eg, by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecules, such as another antibody or antibody fragment, to produce a second or Bispecific or multispecific antibodies with more binding specificities.

在一些实施方案中，本发明的抗体结合人CD47蛋白，部分或完全地调节、阻断、抑制、减少、拮抗、中和或干扰CD47与SIRPα的结合从而完全或部分地抑制CD47的功能活性。CD47的功能活性包括例如通 过与SIRPα的相互作用传导信号、在细胞粘附至细胞外基质后调节(如增加)细胞内钙离子浓度、与血小板反应蛋白的C端细胞结合结构域相互作用、与血纤蛋白原相互作用以及与多种整联蛋白相互作用。例如，CD47抗体通过抑制SIRP-α与CD47结合，减少CD47-SIRPα介导的信号传导，促进吞噬作用，并且抑制肿瘤生长和/或迁移。In some embodiments, the antibodies of the invention bind to human CD47 protein, partially or fully modulate, block, inhibit, reduce, antagonize, neutralize or interfere with the binding of CD47 to SIRPα, thereby fully or partially inhibiting the functional activity of CD47. Functional activities of CD47 include, for example, signaling through interaction with SIRPα, regulating (eg, increasing) intracellular calcium concentration after cell adhesion to the extracellular matrix, interacting with the C-terminal cell-binding domain of thrombospondin, and Fibrinogen interacts and interacts with various integrins. For example, CD47 antibodies reduce CD47-SIRPα-mediated signaling, promote phagocytosis, and inhibit tumor growth and/or migration by inhibiting SIRP-α binding to CD47.

本发明的CD47抗体表现出多种需要的特性，如通过非限制性实施例的方式有效地阻断CD47与其配体SIRPα的相互作用，同时不导致显著的红细胞血凝反应，以及有效的抗肿瘤活性。例如，与不存在本文所述CD47抗体时CD47与SIRPα之间的相互作用水平相比，本发明的CD47抗体阻断了CD47与SIRPα之间至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少95％或至少99％的相互作用。The CD47 antibodies of the present invention exhibit various desirable properties, such as, by way of non-limiting example, effectively blocking the interaction of CD47 with its ligand, SIRPα, without causing significant erythrocyte hemagglutination, and being effective against tumors active. For example, the CD47 antibodies of the invention block at least 40%, at least 45%, at least 50%, at least 55% of the interaction between CD47 and SIRPα compared to the level of interaction between CD47 and SIRPα in the absence of the CD47 antibodies described herein %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 99% of interactions.

本发明的CD47抗体不会导致显著的细胞凝集，例如本发明的CD47抗体不会导致显著的血红细胞血凝反应。在一些情况下，显著的细胞凝集指现有CD47抗体存在时的凝集水平。在一个方面，与现有CD47抗体存在时的凝集水平相比，本发明的CD47抗体存在时的凝集水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％。在一些实施方式中，如果与现有CD47抗体存在时的凝集水平相比，本发明的CD47抗体存在时的凝集水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％，则说明本发明的CD47抗体未导致显著的凝集水平。The CD47 antibody of the present invention does not cause significant cell agglutination, eg, the CD47 antibody of the present invention does not cause significant red blood cell hemagglutination. In some instances, significant cellular agglutination refers to the level of agglutination in the presence of existing CD47 antibodies. In one aspect, the level of agglutination in the presence of a CD47 antibody of the invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 30%, compared to the level of agglutination in the presence of an existing CD47 antibody 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99%. In some embodiments, the level of agglutination in the presence of a CD47 antibody of the invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, if compared to the level of agglutination in the presence of an existing CD47 antibody %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, the CD47 antibody of the present invention does not cause a significant level of agglutination.

与本领域已知的抗体相比，在肿瘤模型中本发明的抗体也显著有效。例如，与现有CD47抗体存在时巨噬细胞吞噬肿瘤细胞的能力相比，本发明的CD47抗体存在时巨噬细胞吞噬肿瘤细胞的能力升高了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％。Antibodies of the invention are also significantly more effective in tumor models than antibodies known in the art. For example, the ability of macrophages to phagocytose tumor cells in the presence of CD47 antibodies of the invention is increased by at least 5%, at least 10%, at least 20%, compared to the ability of macrophages to phagocytose tumor cells in the presence of existing CD47 antibodies. At least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

药物组合物和药物制剂Pharmaceutical compositions and pharmaceutical preparations

本发明还提供了包括一种或多种结合CD47或其免疫活性片段的单克隆抗体的药物组合物。应理解，本发明提供的抗CD47抗体或药物组合物可以整合至制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而改善其转移、递送、耐受等性质。The present invention also provides pharmaceutical compositions comprising one or more monoclonal antibodies that bind CD47 or an immunologically active fragment thereof. It should be understood that the anti-CD47 antibodies or pharmaceutical compositions provided by the present invention can be incorporated into suitable carriers, excipients and other agents in formulations for combined administration, thereby improving their transfer, delivery, tolerance and other properties.

术语“药物组合物”指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical composition" refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered .

术语“药用载体”指与治疗剂一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂或媒介物。The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性，以及避免相关疾病的复发。As used herein, "treating" means slowing, interrupting, retarding, relieving, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition or disease, and preventing recurrence of the associated disease.

本发明还包括包含抗CD47抗体的组合物(包括药物组合物或药物制剂)和包含编码抗CD47抗体的多核苷酸的组合物。在某些实施方案中，组合物包含一种或多种结合CD47的抗体或一种或多种编码一种或多种结合CD47的抗体的多核苷酸。这些组合物还可以包含合适的药用载体，如本领域中已知的药用赋形剂，包括缓冲剂。The present invention also includes compositions (including pharmaceutical compositions or formulations) comprising anti-CD47 antibodies and compositions comprising polynucleotides encoding anti-CD47 antibodies. In certain embodiments, the composition comprises one or more antibodies that bind CD47 or one or more polynucleotides encoding one or more antibodies that bind CD47. These compositions may also contain suitable pharmaceutical carriers, such as pharmaceutical excipients known in the art, including buffers.

本发明的药物组合物可包括本发明的抗体和药用载体。这些药物组合物可包括于试剂盒中，如诊断试剂盒。The pharmaceutical composition of the present invention may include the antibody of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be included in kits, such as diagnostic kits.

适用于本发明的药用载体可以是无菌液体，如水和油，包括那些具有石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶 液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Hand book of Pharmaceutical Excipients”,第五版,R.C.Rowe等,PharmaceuticalPress,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准载体，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Hand book of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe et al., Pharmaceutical Press, London, Chicago. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

可以通过将具有所需纯度的本发明的抗CD47抗体与一种或多种任选的药用载体(Remington’s Pharmaceutical Sciences,1980,第16版,Osol A.编)混合来制备包含本文所述的抗CD47抗体的药物制剂，优选地以冻干制剂或水溶液的形式。Anti-CD47 antibodies of the invention of the desired purity can be prepared by mixing with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 1980, 16th Ed., Osol A. Ed.) Pharmaceutical formulations of anti-CD47 antibodies, preferably in the form of lyophilized formulations or aqueous solutions.

示例性的冻干抗体制剂描述于美国专利号6,267,958。水性抗体制剂包括美国专利号6,171,586和WO2006/044908中所述的那些，后一种制剂包括组氨酸-乙酸盐缓冲剂。Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分，所述活性成分以对于目的用途有效的量合适地组合存在。The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other, the The active ingredients are suitably combined in amounts effective for the intended use.

本发明的药物组合物可制备成持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。The pharmaceutical composition of the present invention can be prepared as a sustained release formulation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.

具有保守修饰的抗体Antibodies with conservative modifications

术语“保守修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。此类保守修饰包括氨基酸的取代、添加和缺失。修饰可以通过本领域已知的标准技术，例如定点诱变和PCR介导的优点引入到本发明的抗体中。保守氨基酸取代指氨基酸残基用具有类似侧链的氨基酸残基替换。本领域中对具有类似侧链的氨基酸残基家族已有详细说明。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区中的一个或多个氨基酸残基。The term "conservative modification" is intended to mean that an amino acid modification does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitutions refer to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are well described in the art. These families include those with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine) , proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine) , tryptophan, histidine) amino acids. Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention can be replaced with other amino acid residues from the same family of side chains.

CD47抗体的治疗用途Therapeutic uses of CD47 antibodies

本发明还提供了所述CD47抗体用于治疗细胞、组织、器官、动物或患者中癌症的方法。The present invention also provides a method of using the CD47 antibody for the treatment of cancer in a cell, tissue, organ, animal or patient.

癌症的实例包括但不限于：实体瘤、软组织肿瘤、造血系统肿瘤和转移性病变。Examples of cancers include, but are not limited to, solid tumors, soft tissue tumors, hematopoietic tumors, and metastatic lesions.

造血系统肿瘤的实例包括：白血病、急性白血病、急性成淋巴细胞白血病(ALL)、急性髓性白血病(AML)、慢性髓细胞白血病(CML)、慢性淋巴细胞白血病(CLL)例如转化的CLL、弥漫性大B细胞淋巴瘤(DLBCL)、滤泡淋巴瘤、毛细胞白血病、骨髓增生异常综合征(MDS)、淋巴瘤、霍奇金病、恶性淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、多发性骨髓瘤或Richter综合征(Richter转化)。Examples of hematopoietic tumors include: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) such as transformed CLL, diffuse Large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt Lymphoma, multiple myeloma or Richter syndrome (Richter transformation).

另外的血液癌症包括：骨髓增生异常综合征(MDS)(例如，白血病前期、难治性贫血、Ph阴性慢性髓细胞白血病、慢性髓单核细胞白血病、髓样化生)、非霍奇金淋巴瘤(例如，弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、套细胞淋巴瘤、B成淋巴细胞白血病/淋巴瘤、外周T细胞淋巴瘤和伯基特淋巴瘤)、B成淋巴 细胞白血病/淋巴瘤；B细胞慢性淋巴细胞白血病/小淋巴细胞淋巴瘤；B细胞-幼淋巴细胞白血病；淋巴浆细胞性淋巴瘤；脾边缘区B细胞淋巴瘤(±绒毛状淋巴细胞)；毛细胞白血病；浆细胞骨髓瘤/浆细胞瘤；MALT类型的结外边缘区B细胞淋巴瘤；结节性边缘区B细胞淋巴瘤(±单核细胞样B细胞)；滤泡淋巴瘤；套细胞淋巴瘤；弥漫性大B细胞淋巴瘤；伯基特淋巴瘤；前体T成淋巴细胞淋巴瘤/白血病；T细胞幼淋巴细胞白血病；T细胞颗粒淋巴细胞白血病；侵袭性NK细胞白血病；成人T细胞淋巴瘤/白血病(HTLV 1阳性)；结外NK/T细胞淋巴瘤，鼻型；肠病型T细胞淋巴瘤；肝脾型γ-δT细胞淋巴瘤；皮下脂膜炎样T细胞淋巴瘤；蕈样霉菌病/塞扎里综合征；间变性大细胞淋巴瘤，T/裸细胞(null cell)，原发皮肤型；间变性大细胞淋巴瘤，T/裸细胞，原发系统型；外周T细胞淋巴瘤(没有对其它方面进行表征)；血管免疫母细胞性T细胞淋巴瘤、慢性淋巴细胞白血病(CLL)、慢性髓细胞白血病(CML)、多发性骨髓瘤、真性红细胞增多症或骨髓纤维化、皮肤T细胞淋巴瘤、小淋巴细胞淋巴瘤(SLL)、边缘区淋巴瘤、CNS淋巴瘤、免疫母细胞性大细胞淋巴瘤和前体B成淋巴细胞淋巴瘤。Additional blood cancers include: myelodysplastic syndromes (MDS) (eg, preleukemia, refractory anemia, Ph-negative chronic myeloid leukemia, chronic myelomonocytic leukemia, myeloid metaplasia), non-Hodgkin lymphoma neoplasms (eg, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-lymphoblastic leukemia/lymphoma, peripheral T-cell lymphoma, and Burkitt's lymphoma), B-lymphoblastic leukemia/ Lymphoma; B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma; B-cell-prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic marginal zone B-cell lymphoma (± villous lymphocytes); hairy cell leukemia; Plasma cell myeloma/plasmacytoma; extranodal marginal zone B-cell lymphoma of MALT type; nodular marginal zone B-cell lymphoma (±monocyte-like B-cell); follicular lymphoma; mantle cell lymphoma; Diffuse large B-cell lymphoma; Burkitt's lymphoma; precursor T-lymphoblastic lymphoma/leukemia; T-cell prolymphocytic leukemia; T-cell granular lymphocytic leukemia; aggressive NK-cell leukemia; adult T-cell lymphoma / Leukemia (HTLV 1 positive); Extranodal NK/T-cell lymphoma, nasal type; Enteropathic T-cell lymphoma; Hepatosplenic gamma-delta T-cell lymphoma; Subcutaneous panniculitis-like T-cell lymphoma; Mycosis fungoides Mycosis/Sezari syndrome; anaplastic large cell lymphoma, T/null cell, primary cutaneous; anaplastic large cell lymphoma, T/null cell, primary systemic; peripheral T cells Lymphoma (not otherwise characterized); angioimmunoblastic T-cell lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, polycythemia vera, or myelofibrosis , cutaneous T-cell lymphoma, small lymphocytic lymphoma (SLL), marginal zone lymphoma, CNS lymphoma, immunoblastic large cell lymphoma and precursor B lymphoblastic lymphoma.

实体瘤的实例包括各种器官系统的恶性肿瘤，例如肉瘤、腺癌和癌，如影响头颈(包括咽)、甲状腺、肺(小细胞或非小细胞肺癌(NSCLC))、乳腺、淋巴样、胃肠(例如，口腔、食道、胃、肝脏、胰腺、小肠、结肠和直肠、肛管)、生殖器和泌尿生殖道(例如，肾、泌尿上皮、膀胱、卵巢、子宫、子宫颈、子宫内膜、前列腺、睾丸)、CNS(例如，神经或胶质细胞，例如，神经母细胞瘤或胶质瘤)或皮肤(例如，黑色素瘤)的那些。在某些实施方案中，所述实体瘤为NMDA受体阳性畸胎瘤。在某些实施方案中，所述癌症选自：乳腺癌、结肠癌、胰腺癌(例如，胰腺神经内分泌肿瘤(PNET)或胰腺导管腺癌(PDAC))、胃癌、子宫癌或卵巢癌。Examples of solid tumors include malignancies of various organ systems, such as sarcomas, adenocarcinomas, and carcinomas, such as affecting the head and neck (including the pharynx), thyroid, lung (small cell or non-small cell lung cancer (NSCLC)), breast, lymphoid, Gastrointestinal (eg, oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genital and genitourinary tract (eg, kidney, urothelium, bladder, ovary, uterus, cervix, endometrium , prostate, testis), CNS (eg, nerve or glial cells, eg, neuroblastoma or glioma), or skin (eg, melanoma). In certain embodiments, the solid tumor is an NMDA receptor positive teratoma. In certain embodiments, the cancer is selected from breast cancer, colon cancer, pancreatic cancer (eg, pancreatic neuroendocrine tumor (PNET) or pancreatic ductal adenocarcinoma (PDAC)), gastric cancer, uterine cancer, or ovarian cancer.

抗CD-47抗体，包括例如本文所述的抗体分子，也可以用于治疗炎性病症、自身免疫性病症、纤维化病症、纤维增生性病症、特应性病症或血管生成病症。炎性病症的实例包括但不限于：慢性阻塞性肺病、哮喘、类风湿性关节炎、炎症性肠病(包括克罗恩氏病和溃疡性结肠炎)、多发性硬化症、银屑病、缺血再灌注损伤、败血性休克、年龄相关性黄斑变性(例如，湿性年龄相关性黄斑变性)、动脉粥样硬化、阿尔茨海默氏病、帕金森病、心血管疾病、血管炎、I型和II型糖尿病、代谢综合征、糖尿病视网膜病变、再狭窄。自身免疫性疾病的实例包括但不限于：哮喘、类风湿性关节炎、炎症性肠病、多发性硬化症、银屑病、I型糖尿病、系统性红斑狼疮(SLE)、斯耶格伦氏综合征、桥本氏甲状腺炎、格雷夫斯氏病、格林巴利综合征、自身免疫性肝炎和重症肌无力。纤维化疾病的实例包括但不限于：硬皮病、肝纤维化、胰腺纤维化、慢性阻塞性肺病、糖尿病性肾病、结节病、特发性肺纤维化、肝硬化、囊性纤维化、神经纤维瘤病、子宫内膜异位、术后肌瘤和再狭窄。特应性疾病的实例包括但不限于：特应性皮炎、特应性哮喘和过敏性鼻炎。Anti-CD-47 antibodies, including, for example, the antibody molecules described herein, can also be used to treat inflammatory disorders, autoimmune disorders, fibrotic disorders, fibroproliferative disorders, atopic disorders, or angiogenic disorders. Examples of inflammatory disorders include, but are not limited to: chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), multiple sclerosis, psoriasis, Ischemia-reperfusion injury, septic shock, age-related macular degeneration (eg, wet age-related macular degeneration), atherosclerosis, Alzheimer's disease, Parkinson's disease, cardiovascular disease, vasculitis, I Type and type II diabetes, metabolic syndrome, diabetic retinopathy, restenosis. Examples of autoimmune diseases include, but are not limited to: asthma, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, type I diabetes, systemic lupus erythematosus (SLE), Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, Guillain-Barre syndrome, autoimmune hepatitis, and myasthenia gravis. Examples of fibrotic diseases include, but are not limited to: scleroderma, liver fibrosis, pancreatic fibrosis, chronic obstructive pulmonary disease, diabetic nephropathy, sarcoidosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis, Neurofibromatosis, endometriosis, postoperative fibroids and restenosis. Examples of atopic diseases include, but are not limited to, atopic dermatitis, atopic asthma, and allergic rhinitis.

CD47抗体的治疗方法和用途Therapeutic methods and uses of CD47 antibodies

在一方面中，本发明涉及在受试者中抑制、拮抗CD47与SIRPα结合的方法，所述方法包括向受试者施用有效量的本文所述的任何抗CD47抗体或其片段。在另一方面中，本发明涉及促进受试者的吞噬细胞的吞噬作用的方法，所述方法包括向受试者施用有效量的本文所述的任何抗CD47抗体或其片段。在一方面中，本发明涉及治疗以CD47为治疗靶点的相关疾病的方法，所述方法包括向受试者施用有效量的本文所述的任何抗CD47抗体或其片段。在一方面中，本发明涉及可以通过消除、抑制或降低CD47与SIRPα的结合而改善、减缓、抑制或预防的任何疾病或病症的方法。在另一方面，本发明通过向有需要的受试者施用本发明的抗CD47抗体或其片段而提供治疗受试者癌症或者肿瘤的方法、缓解受试者癌症或者肿瘤症状的方法、避免受试者肿瘤或者癌症复发的方法。In one aspect, the invention relates to a method of inhibiting, antagonizing the binding of CD47 to SIRPα in a subject, the method comprising administering to the subject an effective amount of any of the anti-CD47 antibodies or fragments thereof described herein. In another aspect, the invention relates to a method of promoting phagocytosis by phagocytic cells of a subject, the method comprising administering to the subject an effective amount of any of the anti-CD47 antibodies or fragments thereof described herein. In one aspect, the invention relates to a method of treating a related disease that targets CD47 for therapy, the method comprising administering to a subject an effective amount of any of the anti-CD47 antibodies or fragments thereof described herein. In one aspect, the present invention relates to methods of ameliorating, slowing, inhibiting or preventing any disease or disorder that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing the binding of CD47 to SIRPα. In another aspect, the present invention provides a method of treating cancer or tumor in a subject, a method of alleviating symptoms of cancer or tumor in a subject, and avoiding the method for tumor or cancer recurrence.

在一方面，本发明提供的抗CD47抗体及其抗原结合片段和包含其的药物组合物可以用作治疗剂，用于诊断、预后、监控、治疗、缓和和/或预防受试者中异常CD47的表达、活性和/或信号传递相关的疾病和病 症。通过使用标准方法鉴定受试者中存在异常CD47的表达、活性和/或信号传递相关的疾病和病症时，可以给药本发明公开的抗CD47抗体及其抗原结合片段和包含其的药物组合物。In one aspect, the anti-CD47 antibodies and antigen-binding fragments thereof provided by the present invention and pharmaceutical compositions comprising the same can be used as therapeutic agents for diagnosing, prognosing, monitoring, treating, alleviating and/or preventing abnormal CD47 in a subject Diseases and disorders related to expression, activity and/or signaling. The anti-CD47 antibodies and antigen-binding fragments thereof disclosed herein and pharmaceutical compositions comprising the same can be administered by identifying the presence of diseases and disorders associated with aberrant CD47 expression, activity and/or signaling in a subject using standard methods .

在其他方面，本发明提供抗CD47抗体在生产或制备药物中的用途，所述药物用于治疗上文提及的相关疾病或病症。In other aspects, the present invention provides the use of an anti-CD47 antibody in the manufacture or manufacture of a medicament for the treatment of the above-mentioned related diseases or disorders.

本发明的抗体(以及任何另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending in part on whether the administration is short-term or long-term, administration may be by any suitable route, eg, by injection, eg, intravenously or subcutaneously. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

为了预防或治疗疾病，本发明的抗体的合适剂量将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment , the patient's clinical history and response to the antibodies, and the judgment of the attending physician. The antibody is suitably administered to the patient in a single treatment or over a series of treatments.

在另一方面，本发明的抗体可以用于体内或者体外检测与CD47相关的疾病的治疗的进程，例如，通过测量表达CD47的细胞(例如癌症细胞)的数目的增加或者减少，可以确定是否某种特定的旨在治疗疾病、缓解症状的疗法是否有效。In another aspect, the antibodies of the invention can be used to detect the progression of treatment of CD47-related diseases in vivo or in vitro, for example, by measuring an increase or decrease in the number of CD47-expressing cells (eg, cancer cells), it is possible to determine whether a certain Whether a specific therapy designed to treat the disease and relieve symptoms is working.

用于诊断和检测的方法和组合物Methods and compositions for diagnosis and detection

在某些实施方案中，本文中提供的任何抗CD47抗体或其抗原结合片段可以用于检测CD47在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。In certain embodiments, any of the anti-CD47 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of CD47 in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample is blood, serum, or other fluid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues.

在某些实施方案中，提供标记的抗CD47抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。示例性标记包括但不限于，放射性同位素32P、14C、125I、3H和131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，荧光素酶(luceriferase)，例如，萤火虫荧光素酶和细菌荧光素酶(美国专利号4,737,456)，荧光素，2，3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，加上利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。In certain embodiments, labeled anti-CD47 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H and 131I, fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbrella umbelliferone, luceriferase, eg, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazine dione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase and xanthine oxidase, plus enzymes that utilize hydrogen peroxide dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/affinity Elements, spin tags, phage tags, stabilized free radicals, and more.

免疫结合及免疫应答Immune binding and immune response

术语“免疫结合”和“免疫结合性质”是指一种非共价相互作用，其发生在免疫球蛋白分子和抗原(对于该抗原而言免疫球蛋白为特异性的)之间。免疫结合相互作用的强度或亲和力可以用相互作用的平衡解离常数(KD)表示，其中KD值越小，表示亲和力越高。所选多肽的免疫结合性质可使用本领域中公知的方法测定。一种测定方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(Ka或Kon)和“解离速率常数”(Kd或Koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,1993,Nature,361:186-187)。kd/ka的比率等于解离常数KD(通常参见Davies DR et al,1990,Annual Rev Biochem,59:439-473)。可用任何有效的方法测量KD、ka和kd值。The terms "immunobinding" and "immunobinding properties" refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunobinding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where the smaller the KD value, the higher the affinity. The immunobinding properties of selected polypeptides can be determined using methods well known in the art. One assay involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both "association rate constants" (Ka or Kon) and "dissociation rate constants" (Kd or Koff) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, 1993, Nature, 361 :186-187). The ratio of kd/ka is equal to the dissociation constant KD (see generally Davies DR et al, 1990, Annual Rev Biochem, 59:439-473). KD, ka and kd values can be measured by any valid method.

术语“免疫细胞”包括具有造血起源并在免疫应答中起作用的细胞，包括淋巴细胞，例如B细胞和T细 胞；天然杀伤细胞；髓样细胞，例如单核细胞，巨噬细胞，嗜酸性粒细胞，肥大细胞，嗜碱性粒细胞和粒细胞。The term "immune cell" includes cells of hematopoietic origin and that play a role in the immune response, including lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils cells, mast cells, basophils and granulocytes.

“免疫应答”是指免疫系统的细胞(例如T淋巴细胞，B淋巴细胞，自然杀伤(NK)细胞，巨噬细胞，嗜酸性粒细胞，肥大细胞，树突状细胞和嗜中性粒细胞)和由这些细胞或肝脏中的任何一种产生的可溶性大分子(包括抗体，细胞因子和补体)的作用，该作用导致选择性地靶向，结合，损伤，破坏和/或从脊椎动物体内清除入侵的病原体，感染病原体的细胞或组织，癌细胞或其他异常细胞，或者在自身免疫或病理性炎症的情形下，是正常的人类细胞或组织。免疫反应包括例如T细胞(例如效应T细胞或Th细胞，如CD4+或CD8+T细胞)的活化或抑制，或Treg细胞的抑制。"Immune response" refers to cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and the action of soluble macromolecules (including antibodies, cytokines and complement) produced by either of these cells or the liver that lead to selective targeting, binding, damage, destruction and/or clearance from vertebrates Invading pathogens, pathogen-infecting cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An immune response includes, for example, activation or suppression of T cells (eg, effector T cells or Th cells, such as CD4+ or CD8+ T cells), or suppression of Treg cells.

术语“免疫原性”指特定物质引发免疫应答的能力。The term "immunogenicity" refers to the ability of a particular substance to elicit an immune response.

效应子功能effector function

术语“效应细胞”是指免疫系统的一种细胞，其表达一种或多种FcR并介导一种或多种效应器功能。优选地，该细胞表达至少一种类型的激活性Fc受体，例如人FcγRIII，并执行ADCC效应器功能。介导ADCC的人白细胞的实例包括外周血单个核细胞(PBMC)、NK细胞、单核细胞、巨噬细胞、中性粒细胞和嗜酸性粒细胞。效应细胞也包括例如T细胞。他们可以来源于包括但不限于人、小白鼠、大鼠、兔子和猴的任何生物体。The term "effector cell" refers to a cell of the immune system that expresses one or more FcRs and mediates one or more effector functions. Preferably, the cell expresses at least one type of activating Fc receptor, such as human Fc[gamma]RIII, and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, neutrophils, and eosinophils. Effector cells also include, for example, T cells. They can be derived from any organism including, but not limited to, humans, mice, rats, rabbits and monkeys.

术语“效应子功能”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体不同种型而变化。抗体效应子功能的例子包括但不限于：Fc受体结合亲和性、ADCC、ADCP、CDC、细胞表面受体(例如B细胞受体)的下调、B细胞活化、细胞因子分泌、抗体和抗原/抗体复合物的半衰期/清除率等。改变抗体的效应子功能的方法是本领域已知的，例如通过在Fc区引入突变来完成。The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), and which vary among antibody isotypes. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, ADCC, ADCP, CDC, downregulation of cell surface receptors (eg, B cell receptors), B cell activation, cytokine secretion, antibodies and antigens /half-life/clearance of antibody complexes, etc. Methods of altering the effector function of antibodies are known in the art, eg, by introducing mutations in the Fc region.

术语“抗体依赖性细胞介导的细胞毒性(ADCC)”是指一种细胞毒性形式，抗体作为一种桥联形式，通过与免疫细胞或细胞毒性细胞(例如NK细胞、中性粒细胞或巨噬细胞)上存在的FcR结合，使这些细胞毒性效应细胞特异性结合到抗体附着的靶细胞上，然后通过分泌细胞毒素杀死靶细胞。检测抗体的ADCC活性的方法是本领域已知的，例如可通过测定待测抗体与FcR(例如CD16a)之间的结合活性来评价。The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which antibodies act as a bridging form by interacting with immune cells or cytotoxic cells such as NK cells, neutrophils, or macrophages. The FcR present on phagocytes) binds these cytotoxic effector cells specifically to antibody-attached target cells, which then kill the target cells by secreting cytotoxins. Methods for detecting ADCC activity of antibodies are known in the art and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an FcR (eg, CD16a).

术语“抗体依赖细胞介导的吞噬作用(ADCP)”指一种细胞介导的反应，在该反应中，表达FcγR的非特异性细胞毒活性细胞识别靶细胞上结合的抗体并随后引起该靶细胞的吞噬。The term "antibody-dependent cell-mediated phagocytosis (ADCP)" refers to a cell-mediated reaction in which non-specific cytotoxicly active cells expressing FcγRs recognize bound antibodies on a target cell and subsequently elicit that target cell devoured.

术语“补体依赖的细胞毒性(CDC)”是指通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的CDC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价。The term "complement-dependent cytotoxicity (CDC)" refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to an antibody Fc. Methods for detecting the CDC activity of an antibody are known in the art, and can be assessed, for example, by measuring the binding activity between the antibody to be tested and an Fc receptor (eg, C1q).

单克隆抗体制备Monoclonal Antibody Preparation

本发明的单克隆抗体(mAb)可以通过多种技术进行制备，包括常规单克隆抗体方法学，例如Kohler和Milstein,Nature,1975；256:495中所述的标准体细胞杂交技术。虽然优选体细胞杂交规程，但是原则上也可以使用制备单克隆抗体的其它方法，例如B淋巴细胞的病毒或致癌转化。Monoclonal antibodies (mAbs) of the invention can be prepared by a variety of techniques, including conventional monoclonal antibody methodologies, such as standard somatic cell hybridization techniques as described in Kohler and Milstein, Nature, 1975; 256:495. Although somatic cell hybridization procedures are preferred, other methods of making monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can also be used in principle.

用于制备杂交瘤的优选动物系统为鼠科动物系统。在小鼠中制备杂交瘤是非常完善的规程。分离用于融合的经免疫的脾细胞的免疫方案和技术是本领域已知的。融合配偶体(例如鼠骨髓瘤细胞)和融合规程也是已知的。A preferred animal system for making hybridomas is the murine system. The production of hybridomas in mice is a well established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion protocols are also known.

为表达抗体或其抗体片段，可以通过标准分子生物学技术(例如PCR扩增或使用表达目标抗体的杂交 瘤的cDNA克隆)获得编码部分或全长轻链和重链的DNA，并且可以将DNA插入到表达载体中，从而使得目的基因与转录和翻译调控序列可操作的连接，转染宿主细胞进行表达，表达宿主优选真核表达载体，更优选哺乳动物细胞，例如CHO及其衍生细胞系。To express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques such as PCR amplification or using cDNA cloning of hybridomas expressing the antibody of interest, and the DNA can be Inserted into an expression vector so that the gene of interest is operably linked to transcriptional and translational regulatory sequences, and transfected into a host cell for expression, preferably a eukaryotic expression vector, more preferably mammalian cells, such as CHO and its derived cell lines.

抗体可通过公知的技术，例如使用蛋白A或蛋白G的亲和层析纯化。随后或备选地，可将特异性抗原或其表位固定在柱上以通过免疫亲和层析而纯化免疫特异性抗体。免疫球蛋白的纯化例如由D.Wilkinson论述(The Scientist,由The Scientist,Inc.,Philadelphia PA,Vol.14,No.8(2000年4月17日),25-28页公布)。Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, specific antigens or epitopes thereof can be immobilized on columns to purify immunospecific antibodies by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

本发明的嵌合或人源化抗体可以根据上述制备的鼠单克隆抗体的序列进行制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。例如，为创造嵌合抗体，可使用本领域已知的方法将鼠可变区连接至人恒定区(参见例如Cabilly等人的美国专利No.4,816,567)。通过将编码VH的DNA可操作的连接至编码重链恒定区(CH1、CH2和CH3)的另一DNA分子可以将编码VH区的分离的DNA转变为全长重链基因。人重链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人,1991,Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是最优选为IgG1或IgG4恒定区。The chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared above. DNA encoding heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques. For example, to create chimeric antibodies, murine variable regions can be linked to human constant regions using methods known in the art (see, eg, US Patent No. 4,816,567 to Cabilly et al.). The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgGl or IgG4 constant region.

为创造人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539及Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370)。还可以利用转基因动物，例如，HuMAb小鼠(Medarex,Inc.)含有编码未重排的人重链(μ和γ)和κ轻链免疫球蛋白序列的人免疫球蛋白基因微型基因座(miniloci)，加之使内源μ和κ链基因座失活的靶向突变(参见例如Lonberg等人(1994)Nature 368(6474):856-859)；或携带人重链转基因和人轻链转染色体的“KM小鼠 TM”(参见专利WO02/43478)进行抗体人源化改造。其他抗体人源化改造的方法包括噬菌体展示技术。 To create humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see US Patent Nos. 5,225,539 to Winter and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.) . Transgenic animals can also be used, eg, HuMAb mice (Medarex, Inc.) containing human immunoglobulin gene minilocus (miniloci) encoding unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences. ), plus targeted mutations inactivating the endogenous μ and kappa chain loci (see, eg, Lonberg et al. (1994) Nature 368(6474):856-859); or carrying a human heavy chain transgene and a human light chain transchromosome The "KM mouse TM " (see patent WO02/43478) was used for antibody humanization. Other methods of humanizing antibodies include phage display technology.

通过下列实施例进一步说明本发明，所述实施例不应解释为进一步限制。在此将整篇申请中引用的所有附图和所有参考文献、专利和已公开专利申请的内容明确收入本文作为参考。The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all drawings and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

附图说明Description of drawings

图1、纯化的鼠源抗体Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与人CD47抗原的结合能力测定。Figure 1. Determination of the binding ability of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to human CD47 antigen.

图2-1、纯化的鼠源抗体Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与小鼠CD47抗原的交叉反应性测定。Figure 2-1. Determination of the cross-reactivity of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 with mouse CD47 antigen.

图2-2、纯化的鼠源抗体Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与食蟹猴CD47抗原的交叉反应性测定。Figure 2-2. Determination of cross-reactivity of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 with cynomolgus monkey CD47 antigen.

图3-1、鼠源单抗Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与对照抗体AB12W1竞争结合人CD47的能力。Figure 3-1. The ability of mouse monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with the control antibody AB12W1 for binding to human CD47.

图3-2、鼠源单抗Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与对照抗体AB12W2竞争结合人CD47的能力。Figure 3-2. The ability of mouse monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with the control antibody AB12W2 for binding to human CD47.

图3-3、鼠源单抗Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与人SIRPα竞争结合人CD47的能力。Figure 3-3. The ability of murine mAbs Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with human SIRPα for binding to human CD47.

图4-1、流式细胞术检测CD47候选鼠源单抗Mo2A11971和Mo2A21331与CCRF-CEM细胞的结合能力。Figure 4-1. The binding ability of CD47 candidate mouse monoclonal antibodies Mo2A11971 and Mo2A21331 to CCRF-CEM cells was detected by flow cytometry.

图4-2、流式细胞术检测CD47候选鼠源单抗Mo2A11971和Mo2A21331与Raji细胞的结合能力。Figure 4-2. The binding ability of CD47 candidate mouse monoclonal antibodies Mo2A11971 and Mo2A21331 to Raji cells was detected by flow cytometry.

图5、人源化抗体AB12W3和AB12W6与人CD47抗原的结合能力测定。Figure 5. Determination of the binding ability of humanized antibodies AB12W3 and AB12W6 to human CD47 antigen.

图6、人源化抗体AB12W3和AB12W6阻断CD47与受体SIRPα结合的能力。Figure 6. The ability of humanized antibodies AB12W3 and AB12W6 to block the binding of CD47 to the receptor SIRPα.

图7-1、人源化抗体AB12W3、AB12W4和AB12W6与淋巴瘤细胞Jurkat的结合能力测定。Figure 7-1. Determination of the binding ability of humanized antibodies AB12W3, AB12W4 and AB12W6 to lymphoma cell Jurkat.

图7-2、人源化抗体AB12W3、AB12W4和AB12W6与淋巴瘤细胞Raji的结合能力测定。Figure 7-2. Determination of the binding ability of humanized antibodies AB12W3, AB12W4 and AB12W6 to lymphoma cell Raji.

图7-3、人源化抗体AB12W9与人急性淋巴白血病细胞CCRF-CEM的结合能力测定。Figure 7-3. Determination of the binding ability of humanized antibody AB12W9 to human acute lymphoblastic leukemia cells CCRF-CEM.

图8-1、人源化抗体AB12W3和AB12W6与CHO/K1-hCD47稳定细胞株的结合能力测定。Figure 8-1. Determination of the binding ability of humanized antibodies AB12W3 and AB12W6 to CHO/K1-hCD47 stable cell line.

图8-2、人源化抗体AB12W9与CHO/K1-hCD47稳定细胞株的结合能力测定。Figure 8-2. Determination of the binding ability of humanized antibody AB12W9 to CHO/K1-hCD47 stable cell line.

图9、人源化抗体AB12W3、AB12W4、AB12W5和AB12W6的CDC活性测定。Figure 9. CDC activity assay of humanized antibodies AB12W3, AB12W4, AB12W5 and AB12W6.

图10、人源化抗体AB12W3、AB12W4和AB12W6介导巨噬细胞对肿瘤细胞的CCRF-CEM的吞噬作用。Figure 10. Humanized antibodies AB12W3, AB12W4 and AB12W6 mediate phagocytosis of CCRF-CEM of tumor cells by macrophages.

图11、人源化抗体AB12W3和AB12W4介导的巨噬细胞对红细胞RBC的吞噬能力测定。Figure 11. Humanized antibody AB12W3 and AB12W4-mediated phagocytosis of erythrocyte RBCs by macrophages.

图12、人源化抗体AB12W3、AB12W4、AB12W5、AB12W6、AB12W8、AB12W9和AB12W10促进红细胞凝集活性测定。Figure 12. Humanized antibodies AB12W3, AB12W4, AB12W5, AB12W6, AB12W8, AB12W9 and AB12W10 promoting hemagglutination activity assay.

图13、CD47人源化抗体AB12W3、AB12W4、AB12W5和AB12W6在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的抑瘤作用。Figure 13. Antitumor effects of CD47 humanized antibodies AB12W3, AB12W4, AB12W5 and AB12W6 in human Burkkit's lymphoma mouse subcutaneous xenograft model.

图14、CD47人源化抗体AB12W6、AB12W9和AB12W10在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的抑瘤作用。Figure 14. Antitumor effects of CD47 humanized antibodies AB12W6, AB12W9 and AB12W10 in a mouse subcutaneous xenograft model of human Burkkit's lymphoma.

图15、CD47人源化抗体AB12W8和AB12W9单用或与Rituxan联用在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的抑瘤作用。Figure 15. Antitumor effects of CD47 humanized antibodies AB12W8 and AB12W9 alone or in combination with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model.

图16、CD47人源化抗体AB12W9单用或与Rituxan联用在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的抑瘤作用。Figure 16. The tumor-suppressive effect of CD47 humanized antibody AB12W9 alone or in combination with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model.

具体实施方式detailed description

实施例1：抗人CD47鼠源单克隆抗体的制备Example 1: Preparation of anti-human CD47 mouse monoclonal antibody

将人CD47抗原50μg(北京义翘神州生物技术有限公司)以完全弗氏佐剂充分乳化后，采用多点免疫方式免疫雄性Balb/C小鼠，免疫周期为三周一次。在第3次免疫后第10天，通过眼窝取血，ELISA测试血浆抗人CD47抗体滴度以监测小鼠免疫应答程度，然后在融合前3天，对产生抗人CD47抗体滴度最高的小鼠加强免疫一次。3天后，处死小鼠并取出该小鼠脾脏与小鼠骨髓瘤Sp2/0细胞株融合。混合2×10 8Sp2/0细胞与2×10 8脾细胞在50％聚乙二醇(分子量为1450)和5％二甲基亚砜(DMSO)溶液中融合。用Iscove培养基(含有10％胎牛血清，100单位/mL青霉素，100μg/mL链霉素，0.1mM次黄嘌呤，0.4μM氨基蝶呤和16μM胸苷)来调整脾脏细胞数至5×10 5/mL，以0.3ml加入96孔培养板孔内，并置于37℃，5％CO 2培养箱内。培养10天后，采用高通量ELISA法分别检测上清中抗体与HRP标记的人SIRPα竞争结合CD47的能力，以此筛选出与人SIRPα竞争的阳性孔(方法参见实施例2.3)。再将上述含有能够抑制HRP标记SIRPα与人CD47结合的单克隆抗体的孔内融合细胞进行亚克隆，同样以竞争ELISA方法筛选得到3个表达高亲和力鼠单克隆抗体的杂交瘤细胞株，分别是#2A1621、#2A11971、#2A21331和#2A2351。 After 50 μg of human CD47 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was fully emulsified with complete Freund's adjuvant, male Balb/C mice were immunized by multi-point immunization, and the immunization cycle was once every three weeks. On the 10th day after the third immunization, blood was collected from the eye socket, and plasma anti-human CD47 antibody titers were tested by ELISA to monitor the degree of immune response in mice. Then, 3 days before fusion, mice with the highest anti-human CD47 antibody titers were tested The mice were boosted once. After 3 days, the mice were sacrificed and the spleens of the mice were removed to fuse with the mouse myeloma Sp2/0 cell line. 2×10 8 Sp2/0 cells were mixed with 2×10 8 splenocytes in 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution. Use Iscove's medium (containing 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) to adjust the spleen cell count to 5 x 10 5 /mL, add 0.3 ml to the well of a 96-well culture plate, and place in a 37°C, 5% CO 2 incubator. After 10 days of culture, high-throughput ELISA was used to detect the ability of the antibody in the supernatant to compete with HRP-labeled human SIRPα for binding to CD47, so as to screen out the positive wells that compete with human SIRPα (see Example 2.3 for the method). Then, the above-mentioned fusion cells in the well containing the monoclonal antibody that can inhibit the binding of HRP-labeled SIRPα to human CD47 were subcloned, and 3 hybridoma cell lines expressing high-affinity mouse monoclonal antibody were obtained by competitive ELISA. #2A1621, #2A11971, #2A21331 and #2A2351.

在补充10％FCS的RPMI 1640培养基中培养产生特异性抗体的克隆。当细胞密度达到大约5×10 5个细胞/ml时，用无血清培养基替换该培养基。2至4天后，将培养过的培养基离心，收集培养上清液。用蛋白G柱纯化抗体，然后用150mM NaCl透析单克隆抗体洗脱液。通过0.2μm滤器将透析的溶液过滤除菌，以获得待测试的纯化的鼠单克隆抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351。 Specific antibody-producing clones were grown in RPMI 1640 medium supplemented with 10% FCS. When the cell density reached approximately 5 x 105 cells/ml, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged and the culture supernatant was collected. Antibodies were purified on a protein G column and the monoclonal antibody eluate was dialyzed against 150 mM NaCl. The dialyzed solution was filter sterilized through a 0.2 μm filter to obtain the purified murine monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to be tested.

实施例2、抗人CD47鼠源单克隆抗体的功能鉴定Example 2. Functional identification of anti-human CD47 mouse monoclonal antibody

2.1 ELISA法测定鼠源抗体与人CD47抗原的结合能力2.1 ELISA method to determine the binding ability of mouse antibody to human CD47 antigen

以人CD47(北京义翘神州生物技术有限公司)包被酶标板，室温过夜。弃去包被溶液，用溶解在PBS缓冲液的脱脂奶粉封闭0.5h，用PBST(pH7.4，PBS含0.05％吐温20)洗板3～4次。然后每孔分别加入50μl纯化的抗人CD47鼠源抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351及针对CD47的人源化抗体AB12W1和AB12W2作为阳性对照(AB12W1来源于Forty-seven公司开发的抗人CD47人源化抗体Hu5F9-G4，其VH和VL序列参见美国专利US9017675B2，分别如本文SEQ ID NO：1和2所示；AB12W2的可变区序列来源于celgene公司开发的抗人CD47人源化抗体CC-90002，其VH和VL序列参见中国专利CN104271757B，分别如本文SEQ ID NO：3和4所示)，室温孵育1h，用含有0.05％吐温(Tween)20的PBS洗孔，然后每孔加入50μl HRP标记的羊抗鼠或羊抗人IgG多克隆抗体(Jackson Laboratory)作为检测抗体，再以PBST洗板3～4次、加入底物TMB显色10分钟、然后加入0.2M H 2SO 4终止反应，后读取吸光度值(OD值)，其结果示于图1。 The ELISA plate was coated with human CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) overnight at room temperature. The coating solution was discarded, blocked with skim milk powder dissolved in PBS buffer for 0.5 h, and the plate was washed 3-4 times with PBST (pH 7.4, PBS containing 0.05% Tween 20). Then, 50 μl of purified anti-human CD47 murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 and humanized antibodies against CD47 AB12W1 and AB12W2 were added to each well as positive controls (AB12W1 was derived from the anti-human CD47 human antibody developed by Forty-seven Company). Antibody Hu5F9-G4, whose VH and VL sequences are shown in U.S. Patent US9017675B2, respectively shown in SEQ ID NOs: 1 and 2 herein; the variable region sequence of AB12W2 is derived from the anti-human CD47 humanized antibody CC- 90002, whose VH and VL sequences are shown in Chinese patent CN104271757B, respectively shown in SEQ ID NOs: 3 and 4), incubated at room temperature for 1 h, washed with PBS containing 0.05% Tween 20, and then added 50 μl to each well HRP-labeled goat anti-mouse or goat anti-human IgG polyclonal antibody (Jackson Laboratory) was used as the detection antibody, and then the plate was washed 3 to 4 times with PBST, the substrate TMB was added for 10 minutes, and then 0.2MH 2 SO 4 was added to stop the reaction. , and then read the absorbance value (OD value), and the results are shown in Figure 1.

2.2抗CD47鼠源抗体种属交叉反应测定2.2 Species cross-reactivity assay of anti-CD47 murine antibody

对获得的鼠源单抗Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351与小鼠和食蟹猴的CD47的结合能力进行了测定。The binding ability of the obtained murine monoclonal antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to CD47 of mouse and cynomolgus monkey was determined.

用PBS缓冲液将食蟹猴CD47(北京义翘神州生物技术有限公司)及小鼠CD47抗原(北京义翘神州生物技术有限公司)，稀释至0.1μg/ml，以100μl/孔的体积加于96孔板中，4℃放置16～20h。吸掉上清，用PBST缓冲液洗板1次后，每孔加入200μl含1％脱脂奶粉的PBST(PBST/1％脱脂奶粉)，室温孵育1h封闭。移去封闭液，用PBST缓冲液洗板3次后，加入待测抗CD47抗体，100μl/孔，室温孵育1.5h。移去反应体系，PBST洗板3次后，50μl/孔加入1：4000稀释的HRP标记的羊抗鼠IgG二抗(The Jackson Laboratory)，室温孵育1h。再以PBST洗板3次后，每孔加入100μl TMB，室温孵育5-10min。最后，每孔加入50μl 0.2M H 2SO 4终止反应，并用酶标仪在双波长450/620nm处读取OD值。 Cynomolgus monkey CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) and mouse CD47 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) were diluted with PBS buffer to 0.1 μg/ml, and added in a volume of 100 μl/well. In a 96-well plate, place at 4°C for 16-20h. Aspirate the supernatant, wash the plate once with PBST buffer, add 200 μl PBST containing 1% nonfat dry milk (PBST/1% nonfat dry milk) to each well, and incubate at room temperature for 1 h to block. Remove the blocking solution, wash the plate with PBST buffer three times, add the anti-CD47 antibody to be tested, 100 μl/well, and incubate at room temperature for 1.5 h. The reaction system was removed, and after washing the plate three times with PBST, 50 μl/well of HRP-labeled goat anti-mouse IgG secondary antibody (The Jackson Laboratory) diluted 1:4000 was added, and incubated at room temperature for 1 h. After washing the plate three times with PBST, 100 μl of TMB was added to each well and incubated at room temperature for 5-10 min. Finally, 50 μl of 0.2M H 2 SO 4 was added to each well to stop the reaction, and the OD value was read at dual wavelengths 450/620 nm with a microplate reader.

如图2-1所示，Mo2A1621、Mo2A11971、Mo2A21331以及Mo2A2351与鼠CD47均不能特异结合。图2-2中显示Mo2A21331与食蟹猴CD47的结合能力最强，Mo2A1621和Mo2A11971与食蟹猴CD47的结合能力相对较弱，而Mo2A2351几乎不与食蟹猴CD47结合。As shown in Figure 2-1, Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 did not specifically bind to mouse CD47. Figure 2-2 shows that Mo2A21331 has the strongest binding ability to cynomolgus monkey CD47, Mo2A1621 and Mo2A11971 have relatively weak binding ability to cynomolgus monkey CD47, and Mo2A2351 hardly binds to cynomolgus monkey CD47.

2.3 ELISA法测定鼠源抗体的相对亲和力2.3 Determination of relative affinity of murine antibodies by ELISA

用PBS缓冲液将人CD47抗原(北京义翘神州生物技术有限公司)稀释至0.1μg/ml，以100μl/孔加于96孔板中，室温过夜。弃去包被溶液，每孔加入200μl PBST/1％脱脂奶粉，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板3次后，然后每孔加入50μL生长培养基(DMEM+5％FBS)与50μl以辣根过氧化物酶(HRP)标记的对照抗体AB12W1或AB12W2，分别以PBS洗去未结合的HRP标记的AB12W1或AB12W2。然后分别加入抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351；以培养基作为空白对照。充分孵育后以PBS洗去未结合的HRP标记的AB12W1或AB12W2，然后用酶标仪在双波长450/620nm处读取OD值。Human CD47 antigen (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was diluted to 0.1 μg/ml with PBS buffer, and 100 μl/well was added to a 96-well plate, overnight at room temperature. Discard the coating solution, add 200 μl PBST/1% nonfat dry milk to each well, and incubate for 1 h at room temperature for blocking. Remove the blocking solution, wash the plate 3 times with PBST buffer, then add 50 μL growth medium (DMEM+5% FBS) and 50 μL horseradish peroxidase (HRP)-labeled control antibody AB12W1 or AB12W2 to each well, Unbound HRP-labeled AB12W1 or AB12W2 were washed away with PBS, respectively. Then antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 were added respectively; the medium was used as a blank control. After sufficient incubation, the unbound HRP-labeled AB12W1 or AB12W2 was washed away with PBS, and then the OD value was read at dual wavelengths of 450/620 nm with a microplate reader.

另外，还测定了抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351与HRP标记的人SIRPα(北京义翘神州生物技术有限公司，货号11612-H08H)竞争结合人CD47的能力，同样以抗体AB12W1和AB12W2作为阳性对照，方法同上。In addition, the ability of antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 to compete with HRP-labeled human SIRPα (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Cat. No. 11612-H08H) for binding to human CD47 was also determined. Antibodies AB12W1 and AB12W2 were also used as positive controls. , the method is the same as above.

结果如图3-1和图3-2所示，抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351都能显著地竞 争性阻断AB12W1或AB12W2与人CD47的结合。The results are shown in Fig. 3-1 and Fig. 3-2, the antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 can significantly and competitively block the binding of AB12W1 or AB12W2 to human CD47.

图3-3中显示，抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351同对照抗体AB12W1和AB12W2一样，都能与人SIRPα竞争结合CD47，即通过阻断CD47和SIRPα的结合发挥功能。As shown in Figure 3-3, the antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351, like the control antibodies AB12W1 and AB12W2, can compete with human SIRPα for binding to CD47, that is, they function by blocking the binding of CD47 and SIRPα.

2.4流式细胞术测定鼠源抗体与CD47表达阳性细胞的结合能力2.4 Determination of the binding ability of mouse-derived antibodies to CD47-positive cells by flow cytometry

用流式细胞术(FACS)检测CD47候选鼠抗与两株CD47高表达肿瘤细胞的结合能力。分别培养人Burkitt's淋巴瘤细胞Raji-luc(北京百奥赛图基因生物技术有限公司)和人急性淋巴白血病细胞CCRF-CEM(中科院上海细胞所)，离心收集细胞。将收集的细胞用1％PBSB重悬，调整细胞密度为2×10 6/ml，铺于96孔板中，每孔100μl(2×10 5个细胞)，4℃封闭0.5h。同时稀释CD47鼠源抗体Mo2A11971和Mo2A21331以及对照抗体AB12W2，从10μg/ml开始，5倍梯度稀释，得到7个浓度梯度，备用。封闭后的细胞离心弃上清，加入稀释好的CD47人源化抗体，4℃孵育1h；离心去上清，用1％PBSB洗3遍，加入稀释好的F4647标记的山羊抗人IgG多抗(The Jackson Laboratory，货号109-605-088)，4℃避光孵育1h；离心去上清，1％PBSB洗2遍，每孔再用100μl 1％PF重悬，上机检测(BD Accuri C6)。再以平均荧光强度，通过软件OriginPro8.0进行分析，计算CD47人源化抗体与这2株肿瘤细胞结合的EC 50值，并比较CD47鼠源抗体Mo2A11971和Mo2A33和对照抗体AB12W2的结合活性。 Flow cytometry (FACS) was used to detect the binding ability of CD47 candidate mouse antibody to two tumor cells with high CD47 expression. Human Burkitt's lymphoma cells Raji-luc (Beijing Biositu Gene Biotechnology Co., Ltd.) and human acute lymphoblastic leukemia cells CCRF-CEM (Shanghai Institute of Cell, Chinese Academy of Sciences) were cultured respectively, and the cells were collected by centrifugation. The collected cells were resuspended in 1% PBSB, adjusted to a cell density of 2×10 6 /ml, plated in a 96-well plate, 100 μl per well (2×10 5 cells), and blocked at 4° C. for 0.5 h. At the same time, the CD47 mouse-derived antibodies Mo2A11971 and Mo2A21331 and the control antibody AB12W2 were diluted at the same time, starting from 10 μg/ml, and 5-fold serial dilution to obtain 7 concentration gradients for use. The blocked cells were centrifuged to discard the supernatant, and the diluted CD47 humanized antibody was added, and incubated at 4°C for 1 h; the supernatant was removed by centrifugation, washed three times with 1% PBSB, and the diluted F4647-labeled goat anti-human IgG polyclonal antibody was added. (The Jackson Laboratory, Cat. No. 109-605-088), incubated at 4°C for 1 h in the dark; centrifuged to remove the supernatant, washed twice with 1% PBSB, resuspended in 100 μl of 1% PF per well, and detected on the machine (BD Accuri C6 ). The average fluorescence intensity was then analyzed by the software OriginPro 8.0 to calculate the EC 50 value of CD47 humanized antibody binding to these two tumor cells, and compare the binding activities of CD47 mouse antibodies Mo2A11971 and Mo2A33 with the control antibody AB12W2.

图4-1和图4-2结果显示，候选鼠抗Mo2A11971和对照抗体AB12W2与这两株细胞表面的CD47结合能力相当，Mo2A21331结合能力相对较弱。The results of Figure 4-1 and Figure 4-2 show that the candidate mouse anti-Mo2A11971 and the control antibody AB12W2 have comparable binding abilities to CD47 on the surface of these two cell lines, while the binding ability of Mo2A21331 is relatively weak.

2.5生物薄膜干涉技术测定抗CD47鼠源抗体的动力学常数和亲和力2.5 Determination of kinetic constants and affinity of anti-CD47 murine antibody by biofilm interferometry

采用生物薄膜干涉技术(BLI)对纯化的鼠源抗体Mo2A1621、Mo2A11971、Mo2A21331和Mo2A2351以及对照抗体AB12W1和AB12W2与抗原的结合亲和力常数进行测定(GE，Biacore T200)。测定时，将待测CD47鼠源抗体固定在Series S Protein A传感芯片(GE，货号29-1275-56)表面，用重组人CD47蛋白(His-tag)(Acro Biosystems，货号CD-H5227)作为流动相，抗原被捕获到芯片表面。处理数据，并用Biacore T200的分析软件1：1结合的模型进行拟合，拟合数据与实验数据基本重叠，得到结合和解离速率常数k a和k d，用k d除k a得到平衡解离常数K D(见表1)。结果显示，获得的人源化抗体亲和力没有明显损失，鼠源抗体Mo2A1621、Mo2A11971和Mo2A21331与人CD47的结合亲和力与对照抗体AB12W1和AB12W2相当，K D值均达到pM级。 The binding affinity constants of purified murine antibodies Mo2A1621, Mo2A11971, Mo2A21331 and Mo2A2351 and control antibodies AB12W1 and AB12W2 to antigen were determined by biofilm interferometry (BLI) (GE, Biacore T200). During the measurement, the CD47 mouse antibody to be tested was immobilized on the surface of the Series S Protein A sensor chip (GE, Cat. No. 29-1275-56), and recombinant human CD47 protein (His-tag) (Acro Biosystems, Cat. No. CD-H5227) was used. As the mobile phase, antigens are captured on the chip surface. The data were processed and fitted with a 1:1 binding model of Biacore T200 analysis software. The fitted data basically overlapped with the experimental data to obtain the association and dissociation rate constants ka and k d , and the equilibrium dissociation was obtained by dividing ka by k d Constant K D (see Table 1). The results showed that the obtained humanized antibodies did not lose significant affinity, and the binding affinities of the mouse antibodies Mo2A1621, Mo2A11971 and Mo2A21331 to human CD47 were comparable to those of the control antibodies AB12W1 and AB12W2, and the K D values all reached the pM level.

表1、抗体的亲和力测定结果Table 1. Affinity determination results of antibodies

Figure PCTCN2021115946-appb-000001
Figure PCTCN2021115946-appb-000001

实施例3、抗CD47鼠源单抗的亚型鉴定及可变区扩增Example 3. Subtype identification and variable region amplification of anti-CD47 mouse monoclonal antibody

抗体亚型鉴定：取杂交瘤细胞培养上清液，采用IsoStrip TM小鼠单克隆抗体亚型鉴定试剂盒(Santa Cruz  Biotechnology，货号sc-24958)鉴定抗体亚型。鼠单抗Mo2A21331、Mo2A1621、Mo2A11971和Mo2A2351的亚型经鉴定都为IgG1(Kappa)型。 Identification of antibody subtypes: The culture supernatant of hybridoma cells was taken, and IsoStrip TM mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology, product number sc-24958) was used to identify antibody subtypes. The subtypes of the murine mAbs Mo2A21331, Mo2A1621, Mo2A11971 and Mo2A2351 were all identified as IgG1 (Kappa) type.

抗体可变区扩增：将候选杂交瘤细胞#2A21331、#2A1621、#2A11971和#2A2351分别培养至总量约10 7个细胞，1000rpm离心10min收集细胞，并以Trizol试剂盒(Invitrogen)提取总RNA，用反转录试剂盒SMARTer RACE合成第一链cDNA，以第一链cDNA为后续模板扩增杂交瘤细胞所对应的抗体可变区DNA序列。根据亚型鉴定结果，获取该抗体亚型的重链和轻链恒定区序列，设计特异性的巢式PCR引物，该扩增反应中所使用的引物序列与抗体可变区第一框架区和恒定区互补。采用常规PCR方法扩增目的基因，将扩增产物测序后，分别得到杂交瘤克隆#2A21331分泌抗体Mo2A21331、#2A1621分泌抗体Mo2A1621、#2A11971分泌抗体Mo2A11971以及#2A2351分泌抗体Mo2A2351的重链可变区序列和轻链可变区序列，如表2所示。 Antibody variable region amplification: candidate hybridoma cells #2A21331, #2A1621, #2A11971 and #2A2351 were cultured to a total of about 10 7 cells, centrifuged at 1000 rpm for 10 min to collect cells, and extracted with Trizol kit (Invitrogen). RNA, the first-strand cDNA was synthesized by the reverse transcription kit SMARTer RACE, and the first-strand cDNA was used as the subsequent template to amplify the DNA sequence of the antibody variable region corresponding to the hybridoma cells. According to the subtype identification results, the heavy chain and light chain constant region sequences of the antibody subtype are obtained, and specific nested PCR primers are designed. The primer sequences used in the amplification reaction are the same as the antibody variable region first framework region and Complementary constant regions. The target gene was amplified by conventional PCR method, and the amplified products were sequenced to obtain the heavy chain variable regions of hybridoma clones #2A21331 secreting antibody Mo2A21331, #2A1621 secreting antibody Mo2A1621, #2A11971 secreting antibody Mo2A11971 and #2A2351 secreting antibody Mo2A2351, respectively. Sequences and light chain variable region sequences are shown in Table 2.

表2、鼠源抗体可变区氨基酸序列Table 2. Amino acid sequence of murine antibody variable region

Figure PCTCN2021115946-appb-000002
Figure PCTCN2021115946-appb-000002

实施例4、抗CD47鼠单克隆抗体的人源化Example 4. Humanization of anti-CD47 mouse monoclonal antibody

根据上述获得的Mo2A21331、Mo2A1621、Mo2A11971和Mo2A2351鼠源抗体可变区序列，利用计算机辅助抗体三维建模以及结构分析进行抗体人源化改造。本发明人采用两种方法进行鼠源抗体的人源化改造，一种是CDR移植，另一种是表明重塑。其中，CDR移植(CDR-Grafting)是一种常见的抗体人源化方法，是通过将人源抗体的FR替换鼠源抗体的FR来达到保持活性、降低免疫原性的目的。结合Discovery Studio分析工具进行CDR移植抗体人源化改造的方法主要包括以下步骤：(1)抗体三维结构建模；(2)关键残基分析。利用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式以确定对于保持CDR区构象至关重要的关键残基，主要有三类：1、位于VL和VH结合界面上的残基，对于两个结构域的折叠起到关键作用；2、靠近CDR区并且包埋于蛋白内部的残基；3、与CDR区有直接相互作用的残基，相互作用包括：疏水相互作用/氢键/盐桥；(3)人源模板选择。须同时满足下面两个条件：首先，把各杂交瘤细胞分泌的抗体氨基酸序列与人胚胎系抗体氨基酸序列进行比对，找出同源性高的序列；其次，选择其中与MHC II(HLA-DR)亲和力低的人胚胎系(germline)抗体框架序列，以降低其免疫原性；(4)基于关键残基分析的反向嫁接，获得人源化抗体序列。According to the variable region sequences of Mo2A21331, Mo2A1621, Mo2A11971 and Mo2A2351 murine antibodies obtained above, the antibodies were humanized by computer-aided three-dimensional modeling and structural analysis. The inventors adopted two methods to humanize the murine antibody, one is CDR transplantation, and the other is express remodeling. Among them, CDR-Grafting is a common antibody humanization method, which achieves the purpose of maintaining the activity and reducing the immunogenicity by replacing the FR of the human antibody with the FR of the mouse antibody. The method for humanization transformation of CDR-grafted antibodies combined with the Discovery Studio analysis tool mainly includes the following steps: (1) modeling of the three-dimensional structure of the antibody; (2) analysis of key residues. Molecular docking was used to analyze the framework amino acid sequences of the variable region and its surrounding areas, and the spatial binding mode was investigated to determine the key residues that are crucial for maintaining the conformation of the CDR region. There are three main categories: 1. Residues that play a key role in the folding of the two domains; 2. Residues close to the CDR region and embedded in the interior of the protein; 3. Residues that have direct interactions with the CDR region, including: hydrophobic interactions /Hydrogen bond/salt bridge; (3) Human template selection. The following two conditions must be met at the same time: first, compare the amino acid sequence of the antibody secreted by each hybridoma cell with the amino acid sequence of the human embryonic antibody to find out the sequence with high homology; DR) low-affinity human germline antibody framework sequence to reduce its immunogenicity; (4) reverse grafting based on key residue analysis to obtain humanized antibody sequences.

表面重塑抗体是利用计算机辅助的分子设计和表面残基替换法进行鼠源抗体的人源化设计。该方法的原则是仅替换与人源抗体表面氨基酸差别明显的区域，在维持抗体活性并兼顾减少异源性基础上选用与人抗体表面残基相似的氨基酸替换；人源化改造过程涉及以下步骤：1、把各杂交瘤细胞分泌的抗体的氨基酸序列与人胚胎系抗体氨基酸序列进行比对，找出同源性高的序列；2、对于影响侧链大小、电荷、疏水性或可能形成氢键从而影响到抗体CDR构象的残基尽量保留；3、利用计算机模拟技术，选择溶剂可及性(solvent accessibility)大于30％时，暴露的表面氨基酸替换成人胚胎系抗体氨基酸。Resurfacing antibodies are humanized design of murine antibodies using computer-aided molecular design and surface residue replacement. The principle of this method is to replace only the regions that are significantly different from the surface amino acids of human antibodies, and select amino acids similar to the surface residues of human antibodies on the basis of maintaining antibody activity and reducing heterology; the humanization transformation process involves the following steps : 1. Compare the amino acid sequence of the antibody secreted by each hybridoma cell with the amino acid sequence of the human embryonic antibody to find the sequence with high homology; 2. For the side chain size, charge, hydrophobicity or possible formation of hydrogen The residues that affect the antibody CDR conformation are retained as much as possible; 3. Using computer simulation technology, when the solvent accessibility is greater than 30%, the exposed surface amino acids are replaced by adult embryonic antibody amino acids.

共获得16株人源化抗体，其中由鼠源亲本抗体Mo2A21331衍生获得的人源化抗体为AB12W5、 AB12W6、AB12W9、AB12Q1和AB12Q2；由鼠源亲本抗体Mo2A1621衍生获得的人源化抗体为AB12W3、AB12Q3、AB12Q4和AB12Q5；由鼠源亲本抗体Mo2A11971衍生获得的人源化抗体为AB12W4、AB12W7、AB12W8、AB12Q6、AB12Q7和AB12Q8；由鼠源亲本抗体Mo2A2351衍生获得的人源化抗体为AB12W10；上述人源化抗体中，除AB12W5、AB12Q3、AB12Q6和AB12W10是采用CDR移植法获得的，其余抗体均由表面重塑法进行人源化改造而获得。上述人源化抗体的可变区氨基酸序列如表3所示。A total of 16 humanized antibodies were obtained, of which the humanized antibodies derived from the murine parental antibody Mo2A21331 were AB12W5, AB12W6, AB12W9, AB12Q1 and AB12Q2; the humanized antibodies derived from the murine parental antibody Mo2A1621 were AB12W3, AB12Q3, AB12Q4 and AB12Q5; the humanized antibodies derived from the murine parental antibody Mo2A11971 are AB12W4, AB12W7, AB12W8, AB12Q6, AB12Q7 and AB12Q8; the humanized antibodies derived from the murine parental antibody Mo2A2351 are AB12W10; the above human Among the humanized antibodies, except for AB12W5, AB12Q3, AB12Q6 and AB12W10, which were obtained by CDR transplantation, the rest of the antibodies were obtained by humanization by surface remodeling. The amino acid sequences of the variable regions of the above-mentioned humanized antibodies are shown in Table 3.

表3、人源化抗体可变区氨基酸序列Table 3. Amino acid sequence of humanized antibody variable region

Figure PCTCN2021115946-appb-000003
Figure PCTCN2021115946-appb-000003

本发明制备的鼠源单抗Mo2A21331、Mo2A1621、Mo2A11971和Mo2A2351及其衍生的16个人源化抗体的CDR区的氨基酸序列如表4-1至表4-4所示，其中抗体可变区所包含的CDR的氨基酸序列分别采用Kabat和IMGT方法来定义。The amino acid sequences of the CDR regions of the murine monoclonal antibodies Mo2A21331, Mo2A1621, Mo2A11971 and Mo2A2351 and their derived 16 humanized antibodies prepared by the present invention are shown in Table 4-1 to Table 4-4, wherein the variable regions of the antibodies comprise The amino acid sequences of the CDRs were defined using the Kabat and IMGT methods, respectively.

表4-1、关于示例性的抗CD47抗体Mo2A21331及其衍生的人源化抗体的CDR区序列Table 4-1. CDR region sequences for exemplary anti-CD47 antibody Mo2A21331 and its derived humanized antibodies

Figure PCTCN2021115946-appb-000004
Figure PCTCN2021115946-appb-000004

Figure PCTCN2021115946-appb-000005
Figure PCTCN2021115946-appb-000005

Figure PCTCN2021115946-appb-000006
Figure PCTCN2021115946-appb-000006

表4-2、关于示例性的抗CD47抗体Mo2A1621及其衍生的人源化抗体的CDR区序列Table 4-2. CDR region sequences for exemplary anti-CD47 antibody Mo2A1621 and its derived humanized antibodies

Figure PCTCN2021115946-appb-000007
Figure PCTCN2021115946-appb-000007

Figure PCTCN2021115946-appb-000008
Figure PCTCN2021115946-appb-000008

Figure PCTCN2021115946-appb-000009
Figure PCTCN2021115946-appb-000009

表4-3、关于示例性的抗CD47抗体Mo2A11971及其衍生的人源化抗体的CDR区序列Table 4-3. CDR region sequences for exemplary anti-CD47 antibody Mo2A11971 and its derived humanized antibodies

Figure PCTCN2021115946-appb-000010
Figure PCTCN2021115946-appb-000010

Figure PCTCN2021115946-appb-000011
Figure PCTCN2021115946-appb-000011

Figure PCTCN2021115946-appb-000012
Figure PCTCN2021115946-appb-000012

表4-4、关于示例性的抗CD47抗体Mo2A2351及其衍生的人源化抗体的CDR区序列Table 4-4. CDR region sequences for exemplary anti-CD47 antibody Mo2A2351 and derived humanized antibodies

Figure PCTCN2021115946-appb-000013
Figure PCTCN2021115946-appb-000013

Figure PCTCN2021115946-appb-000014
Figure PCTCN2021115946-appb-000014

为了获得由两条重链和两条轻链组成的全长抗体序列，将表3中所示VH和VL序列的与抗体重链恒定区(优选自人IgG1、IgG2或IgG4)和轻链恒定区(优选自人κ轻链)序列采用常规技术进行拼接或组装。更优选地，抗体重链恒定区序列是修饰的且非裂解性的，并显示出极小的Fc-介导ADCC和CDC效应。例如，在一种实施方案中，抗CD47抗体分子包含野生型人IgG1的重链恒定区和人κ轻链恒定区。或采用修饰的人IgG1恒定区序列，例如在一种实施方案中，如表5所示，人IgG1在根据EU编号的第297位包含取代(例如Asn取代为Ala)；在另一种实施方案中，如表5所示，人IgG1在根据EU编号的第265位包含取代、在根据EU编号的第329位包含取代或包含这两种取代(例如在第265位Asp取代为Ala和/或在第329位Pro取代为Ala)；在又一种实施方案中，如表5所示，人IgG1在根据EU编号的第234位包含取代、在根据EU编号的第235位包含取代或包含这两种取代(例如在第234位Leu取代为Ala和/或在第235位Leu取代为Ala)。在又一种实施方案中，如表5所示，人IgG1在根据EU编号的第297、250和428位包含取代(例如，Asn被Ala取代，Thr被Gln取代、Met被Leu取代)。在另一种实施方案中，抗CD47抗体分子包含表5中所示的野生型人IgG2的重链恒定区和人κ轻链恒定区。或采用修饰的人IgG2恒定区序列；在一种实施方案中，如表5所示，抗CD47抗体分子包括在根据EU编号的第331位突变(例如P变为S)的人IgG2。在另一种实施方案中，抗CD47抗体分子包含表5中所示的野生型人IgG4的重链恒定区和人κ轻链恒定区。或采用修饰的人IgG4恒定区序列；在一种实施方案中，如表5所示，抗CD47抗体分子包括在根据EU编号的第228位突变(例如S变为P)的人IgG4；在另一种实施方案中，如表5所示，抗CD47抗体分子包括在根据EU编号的第228位Ser取代为Pro和第235位Leu取代为Glu的人IgG4。In order to obtain a full-length antibody sequence consisting of two heavy chains and two light chains, the VH and VL sequences shown in Table 3 were combined with the antibody heavy chain constant region (preferably from human IgG1, IgG2 or IgG4) and light chain constant The sequences of the regions (preferably from human kappa light chains) are spliced or assembled using conventional techniques. More preferably, the antibody heavy chain constant region sequence is modified and non-cleavable, and exhibits minimal Fc-mediated ADCC and CDC effects. For example, in one embodiment, the anti-CD47 antibody molecule comprises the heavy chain constant region of wild-type human IgGl and the human kappa light chain constant region. Or use a modified human IgG1 constant region sequence, eg, in one embodiment, as shown in Table 5, human IgG1 comprises a substitution at position 297 according to EU numbering (eg, Asn is substituted for Ala); in another embodiment , as shown in Table 5, the human IgG1 contains a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., substitution of Asp at position 265 to Ala and/or Pro at position 329 is substituted with Ala); in yet another embodiment, as shown in Table 5, the human IgG1 comprises a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or a combination of these Two substitutions (eg Leu to Ala at position 234 and/or Leu to Ala at position 235). In yet another embodiment, as shown in Table 5, the human IgGl comprises substitutions at positions 297, 250 and 428 according to EU numbering (eg, Asn is substituted by Ala, Thr is substituted by Gln, Met is substituted by Leu). In another embodiment, the anti-CD47 antibody molecule comprises the heavy chain constant region of wild-type human IgG2 and the human kappa light chain constant region shown in Table 5. Or use a modified human IgG2 constant region sequence; in one embodiment, as shown in Table 5, the anti-CD47 antibody molecule comprises human IgG2 mutated at position 331 according to EU numbering (eg, P to S). In another embodiment, the anti-CD47 antibody molecule comprises the heavy chain constant region of wild-type human IgG4 and the human kappa light chain constant region shown in Table 5. Or use a modified human IgG4 constant region sequence; in one embodiment, as shown in Table 5, the anti-CD47 antibody molecule comprises a human IgG4 mutated at position 228 according to EU numbering (eg, S to P); in another In one embodiment, as shown in Table 5, the anti-CD47 antibody molecule comprises human IgG4 with a substitution of Ser to Pro at position 228 and Leu to Glu at position 235 according to EU numbering.

表5、人IgG重链和人κ轻链的恒定区氨基酸序列Table 5. The constant region amino acid sequences of human IgG heavy chain and human kappa light chain

Figure PCTCN2021115946-appb-000015
Figure PCTCN2021115946-appb-000015

Figure PCTCN2021115946-appb-000016
Figure PCTCN2021115946-appb-000016

Figure PCTCN2021115946-appb-000017
Figure PCTCN2021115946-appb-000017

实施例5、人源化抗体表达载体的构建和蛋白表达Example 5. Construction and protein expression of humanized antibody expression vector

用上述方法中获得的CD47人源化抗体的重链和轻链的编码cDNA插入到PcDNA3.1或其衍生质粒，或其它真核表达载体中，构建人源化抗体表达载体。优选地，使用的载体质粒应含有在哺乳动物细胞中高水平表达所需的巨细胞病毒早期基因启动因子-增强子。同时，载体质粒中含有可选择标记基因，从而在细菌中赋予氨苄青霉素抗性，而在哺乳动物细胞中赋予G418抗性。另外，载体质粒中含有DHFR基因，在合适的宿主细胞中，能以氨甲喋呤(Methotrexate,MTX,Sigma)共扩增人源化抗体基因和DHFR基因(例如，参见专利CN103333917B)。The cDNA encoding the heavy chain and light chain of the CD47 humanized antibody obtained in the above method was inserted into PcDNA3.1 or its derivative plasmid, or other eukaryotic expression vector to construct a humanized antibody expression vector. Preferably, the vector plasmid used should contain the cytomegalovirus early gene promoter-enhancer required for high-level expression in mammalian cells. At the same time, the vector plasmid contains a selectable marker gene that confers ampicillin resistance in bacteria and G418 resistance in mammalian cells. In addition, the vector plasmid contains the DHFR gene, and in a suitable host cell, the humanized antibody gene and the DHFR gene can be co-amplified with methotrexate (Methotrexate, MTX, Sigma) (for example, see patent CN103333917B).

将上述已构建的重组表达载体质粒转染入哺乳动物宿主细胞系，以表达人源化抗体。为了稳定高水平的表达，优选的宿主细胞系是二氢叶酸还原酶(DHFR)缺陷型的中国仓鼠卵巢(CHO)细胞(参见，例如Chasin,L.等人的美国专利4818679号)。优选的转染方法是电穿孔，也可以使用其他方法，包括磷酸钙共沉降，脂转染和原生质融合等。在电穿孔中，用设为250V电场和960μFd电容的Gene Pulser(Bio-Rad Laboratories)，在比色杯内加入2×10 7个细胞悬浮在0.8ml的PBS中，并含有10μg用PvuI(Takara)线性化的表达载体质粒DNA。转染2天后，加入含有0.2mg/mL G418以及200nM氨甲喋呤(methotrexate或MTX)。为了实现较高水平的表达，用受MTX药物抑制的DHFR基因共扩增转染的人源化抗体基因。用极限稀释亚克隆转染子及ELISA的方法测定各细胞系的分泌率，选出高水平表达人源化抗体的细胞株。收集人源化抗体的条件培养基，用于测定其体外和体内生物学活性。 The recombinant expression vector plasmids constructed above are transfected into mammalian host cell lines to express humanized antibodies. For stable high-level expression, the preferred host cell line is dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells (see, eg, US Pat. No. 4,818,679 to Chasin, L. et al.). The preferred method of transfection is electroporation, but other methods can also be used, including calcium phosphate co-precipitation, lipofection, and protoplast fusion, among others. In electroporation, using a Gene Pulser (Bio-Rad Laboratories) set to an electric field of 250 V and a capacitance of 960 μFd, 2×10 7 cells were added to a cuvette suspended in 0.8 ml of PBS containing 10 μg of PvuI (Takara ) linearized expression vector plasmid DNA. Two days after transfection, G418 containing 0.2 mg/mL and 200 nM methotrexate (methotrexate or MTX) was added. To achieve higher levels of expression, the transfected humanized antibody gene was co-amplified with the DHFR gene inhibited by the MTX drug. The secretion rate of each cell line was measured by the methods of limiting dilution subcloning transfectants and ELISA, and the cell line expressing the humanized antibody at a high level was selected. Conditioned media of humanized antibodies are collected for determination of their in vitro and in vivo biological activities.

例如，将表6所示编码人源化抗体AB12W3、AB12W4、AB12W5、AB12W6、AB12W7、AB12W8、AB12W9和AB12W10的重链和轻链的核苷酸序列插入上述构建的表达载体中，经过加压筛选、亚克隆稳定、高表达目的抗体的细胞株，再经培养、纯化得到各目的抗体。For example, the nucleotide sequences encoding the heavy and light chains of humanized antibodies AB12W3, AB12W4, AB12W5, AB12W6, AB12W7, AB12W8, AB12W9 and AB12W10 shown in Table 6 were inserted into the expression vector constructed above, and subjected to pressure screening. , Subcloning cell lines that are stable and highly expressing the target antibody, and then culture and purify to obtain the target antibody.

表6、人源化抗体重链和轻链的氨基酸序列及其编码核苷酸序列Table 6. Amino acid sequences of humanized antibody heavy and light chains and their encoding nucleotide sequences

抗体编号Antibody number HC核苷酸序列HC nucleotide sequence HC氨基酸序列HC amino acid sequence LC核苷酸序列LC nucleotide sequence LC氨基酸序列LC amino acid sequence AB12W3AB12W3 SEQ ID NO：106SEQ ID NO: 106 SEQ ID NO：107SEQ ID NO: 107 SEQ ID NO：108SEQ ID NO: 108 SEQ ID NO：109SEQ ID NO: 109 AB12W4AB12W4 SEQ ID NO：110SEQ ID NO: 110 SEQ ID NO：111SEQ ID NO: 111 SEQ ID NO：112SEQ ID NO: 112 SEQ ID NO：113SEQ ID NO: 113 AB12W5AB12W5 SEQ ID NO：114SEQ ID NO: 114 SEQ ID NO：115SEQ ID NO: 115 SEQ ID NO：116SEQ ID NO: 116 SEQ ID NO：117SEQ ID NO: 117 AB12W6AB12W6 SEQ ID NO：118SEQ ID NO: 118 SEQ ID NO：119SEQ ID NO: 119 SEQ ID NO：120SEQ ID NO: 120 SEQ ID NO：121SEQ ID NO: 121 AB12W7AB12W7 SEQ ID NO：122SEQ ID NO: 122 SEQ ID NO：123SEQ ID NO: 123 SEQ ID NO：124SEQ ID NO: 124 SEQ ID NO：125SEQ ID NO: 125 AB12W8AB12W8 SEQ ID NO：126SEQ ID NO: 126 SEQ ID NO：127SEQ ID NO: 127 SEQ ID NO：128SEQ ID NO: 128 SEQ ID NO：129SEQ ID NO: 129 AB12W9AB12W9 SEQ ID NO：130SEQ ID NO: 130 SEQ ID NO：131SEQ ID NO: 131 SEQ ID NO：132SEQ ID NO: 132 SEQ ID NO：133SEQ ID NO: 133 AB12W10AB12W10 SEQ ID NO：134SEQ ID NO: 134 SEQ ID NO：135SEQ ID NO: 135 SEQ ID NO：136SEQ ID NO: 136 SEQ ID NO：137SEQ ID NO: 137

实施例6、CD47人源化抗体的定性Example 6. Characterization of CD47 humanized antibody

6.1 CD47人源化抗体与人CD47的结合能力的测定6.1 Determination of the binding ability of CD47 humanized antibody to human CD47

用0.2μg/ml的人CD47(北京义翘神州生物技术有限公司，货号12283-HCCH)包被酶标板，室温过夜。弃去包被溶液，每孔加入300μl含2％脱脂奶粉的PBST(PBST/2％脱脂奶粉)封闭，置于室温孵育2h。然后每孔加入300μl PBST洗涤，重复3～4次。待测抗体AB12W3和AB12W6及对照抗体AB12W1和AB12W2分别用PBST/2％脱脂奶粉进行梯度稀释，以100μl/孔加入酶标板中，室温孵育1.5h，再用PBST洗板4～5次。然后加入以PBST/2％脱脂奶粉稀释的HRP-羊抗人IgG(L+H)酶联二抗(Jackson Laboratory，货号109-035-003)，100μl/孔，置室温孵育1.5h，再以PBST洗板4～5次，每孔加入100μl的TMB底物室温显色 10min，用1M HCl终止显色，最后在350nm处读取OD值。数据用GraphPadPrism5软件分析，以浓度对数(lgC)为横坐标，OD值为纵坐标作图。The ELISA plate was coated with 0.2 μg/ml human CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Cat. No. 12283-HCCH) overnight at room temperature. The coating solution was discarded, and 300 μl of PBST containing 2% nonfat dry milk (PBST/2% nonfat dry milk) was added to each well to block, and incubated at room temperature for 2 h. Then add 300 μl PBST to each well for washing, and repeat 3 to 4 times. The test antibodies AB12W3 and AB12W6 and the control antibodies AB12W1 and AB12W2 were diluted with PBST/2% nonfat dry milk, respectively, and added to the ELISA plate at 100 μl/well, incubated at room temperature for 1.5 hours, and then washed with PBST for 4 to 5 times. Then add HRP-goat anti-human IgG (L+H) enzyme-linked secondary antibody (Jackson Laboratory, Cat. No. 109-035-003) diluted with PBST/2% nonfat dry milk, 100 μl/well, incubate at room temperature for 1.5 hours, and then add The plate was washed 4-5 times with PBST, and 100 μl of TMB substrate was added to each well for 10 min at room temperature for color development. The color development was stopped with 1 M HCl, and the OD value was finally read at 350 nm. The data were analyzed with GraphPad Prism5 software, and the logarithm of concentration (lgC) was used as the abscissa and the OD value was plotted as the ordinate.

结果见图5，人源化抗体AB12W3和AB12W6同对照抗体AB12W1和AB12W2一样，都能特异地结合人CD47抗原。AB12W3和AB12W6结合人CD47的EC 50值分别为11.18ng/ml和12.83ng/ml，与对照抗体AB12W2的EC 50值(9.197ng/ml)相当，但低于AB12W1的EC 50值(26.66ng/ml)。 The results are shown in Figure 5. The humanized antibodies AB12W3 and AB12W6, like the control antibodies AB12W1 and AB12W2, can specifically bind to human CD47 antigen. The EC50 values of AB12W3 and AB12W6 for binding to human CD47 were 11.18 ng/ml and 12.83 ng/ml, respectively, which were comparable to that of the control antibody AB12W2 (9.197 ng/ml), but lower than that of AB12W1 (26.66 ng/ml). ml).

6.2 CD47人源化抗体阻断人CD47与受体SIRPα结合实验6.2 Experiment of CD47 humanized antibody blocking the binding of human CD47 to receptor SIRPα

以生物素标记的重组人SIRPα(北京义翘神州生物技术有限公司，货号11612-H08H-B)作为试剂。用PBS缓冲液将重组人CD47(北京义翘神州生物技术有限公司，货号12283-HCCH)稀释至2.0μg/ml，以100μl/孔的体积加于96孔板中，于4℃孵育过夜。弃去包被溶液，每孔加入300μl PBST/2％脱脂奶粉，室温孵育2h封闭。移去封闭液，用PBST缓冲液洗板3次后，然后每孔加入50μl以PBST/2％脱脂奶粉梯度稀释的抗CD47人源化抗体AB12W3和AB12W6以及对照抗体AB12W1和AB12W2，然后每孔再加入50μl以PBST/2％脱脂奶粉稀释至0.2μg/ml的生物素标记的重组人SIRPα，充分孵育后以PBST洗去未结合的抗体及生物素标记的SIRPα。然后加入1：1000倍稀释的Streptavidin-HRP(Pierce，货号21132)，100μL/孔，充分孵育后以PBST洗去未结合的Streptavidin-HRP，每孔加入100μl的TMB显色液，室温显色10分钟。用1M的HCL终止显色反应，以酶标仪在波长350nm处读取OD值。在GraphPadPrism5软件中，以浓度对数(lgC)为横坐标，OD值为纵坐标做非线性回归可变斜率方程。Biotin-labeled recombinant human SIRPα (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number 11612-H08H-B) was used as a reagent. Recombinant human CD47 (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number 12283-HCCH) was diluted to 2.0 μg/ml with PBS buffer, added to a 96-well plate in a volume of 100 μl/well, and incubated at 4°C overnight. Discard the coating solution, add 300 μl PBST/2% nonfat dry milk to each well, and incubate at room temperature for 2 h to block. Remove the blocking solution, wash the plate three times with PBST buffer, and then add 50 μl of anti-CD47 humanized antibodies AB12W3 and AB12W6 and control antibodies AB12W1 and AB12W2 diluted in PBST/2% nonfat dry milk to each well. 50 μl of biotin-labeled recombinant human SIRPα diluted with PBST/2% nonfat dry milk to 0.2 μg/ml was added, and after sufficient incubation, unbound antibody and biotin-labeled SIRPα were washed away with PBST. Then add 1:1000-fold diluted Streptavidin-HRP (Pierce, Cat. No. 21132), 100 μL/well, after sufficient incubation, wash off the unbound Streptavidin-HRP with PBST, add 100 μl of TMB chromogenic solution to each well, and develop color at room temperature for 10 minute. The color reaction was terminated with 1 M HCL, and the OD value was read at a wavelength of 350 nm with a microplate reader. In GraphPad Prism5 software, take the logarithm of concentration (lgC) as abscissa and OD value as ordinate to do nonlinear regression variable slope equation.

如图6中所示，人源化抗体AB12W3和AB12W6以及对照抗体AB12W1和AB12W2都能特异地阻断CD47与SIRPα的结合，其中AB12W3和AB12W6与对照抗体AB12W2的EC 50值相当，分别为1173ng/ml、1288ng/ml和1023ng/ml，阻断能力都显著高于对照抗体AB12W1(EC 50＝3022ng/ml)。 As shown in Figure 6, the humanized antibodies AB12W3 and AB12W6 and the control antibodies AB12W1 and AB12W2 can specifically block the binding of CD47 to SIRPα, wherein the EC50 values of AB12W3 and AB12W6 and the control antibody AB12W2 are comparable, which are 1173ng/ ml, 1288ng/ml and 1023ng/ml, the blocking ability was significantly higher than that of the control antibody AB12W1 (EC 50 =3022ng/ml).

6.3利用FACS检测CD47人源化抗体与淋巴瘤细胞的结合活性6.3 Detection of the binding activity of CD47 humanized antibody to lymphoma cells by FACS

用FACS检测CD47人源化抗体与CD47高表达肿瘤细胞的结合能力。培养淋巴瘤细胞系Raji-luc(北京百奥赛图基因生物技术有限公司)和Jurkat(中科院上海细胞库)细胞，离心收集细胞。将收集的细胞用1％PBSB重悬，调整细胞密度为2×10 6/ml，铺于96孔板中，每孔100μl(2×10 5个细胞)，4℃封闭0.5h。同时稀释CD47人源化抗体AB12W3、AB12W4、AB12W6以及对照抗体AB12W2，从10μg/ml开始，5倍梯度稀释，得到7个浓度梯度，备用。封闭后的细胞离心弃上清，加入稀释好的CD47人源化抗体，4℃孵育1h；离心去上清，用1％PBSB洗3次，加入稀释的F4647标记的山羊抗人IgG多抗(Jackson，货号109-605-088)，4℃避光孵育1h；离心去上清，再以1％PBSB洗两次，用100μl 1％PF重悬，上机检测(BD Accuri C6)。再以平均荧光强度(MFI)，通过软件OriginPro 8.0进行分析，计算CD47人源化抗体与淋巴瘤细胞结合的EC 50值，并比较CD47人源化抗体AB12W3、AB12W4、AB12W6和对照抗体AB12W2的结合活性。 The binding ability of CD47 humanized antibody to CD47-expressing tumor cells was detected by FACS. The lymphoma cell lines Raji-luc (Beijing Biocytogen Biotechnology Co., Ltd.) and Jurkat (Shanghai Cell Bank, Chinese Academy of Sciences) were cultured, and the cells were collected by centrifugation. The collected cells were resuspended in 1% PBSB, adjusted to a cell density of 2×10 6 /ml, plated in a 96-well plate, 100 μl per well (2×10 5 cells), and blocked at 4° C. for 0.5 h. At the same time, the CD47 humanized antibodies AB12W3, AB12W4, AB12W6 and the control antibody AB12W2 were diluted at the same time, starting from 10 μg/ml, and 5-fold gradient dilution to obtain 7 concentration gradients for use. The blocked cells were centrifuged to discard the supernatant, added with diluted CD47 humanized antibody, and incubated at 4°C for 1 h; centrifuged to remove the supernatant, washed three times with 1% PBSB, and added diluted F4647-labeled goat anti-human IgG polyclonal antibody ( Jackson, Cat. No. 109-605-088), incubated at 4°C for 1 h in the dark; centrifuged to remove the supernatant, washed twice with 1% PBSB, resuspended with 100 μl of 1% PF, and detected on the machine (BD Accuri C6). Then, the mean fluorescence intensity (MFI) was analyzed by the software OriginPro 8.0, the EC 50 value of CD47 humanized antibody binding to lymphoma cells was calculated, and the binding of CD47 humanized antibody AB12W3, AB12W4, AB12W6 and control antibody AB12W2 was compared. active.

结果显示，CD47人源化抗体AB12W3、AB12W4、AB12W6与两株淋巴瘤细胞均具有良好的结合活性(图7-1和图7-2)。在同种细胞系中横向比较各人源化抗体与淋巴瘤细胞的结合能力发现，AB12W6与两株细胞的结合力最强，EC 50值比对照抗体低了50％，AB12W4与对照抗体相当，AB12W3与两株淋巴瘤细胞的结合能力最弱(表7)。 The results showed that the CD47 humanized antibodies AB12W3, AB12W4, and AB12W6 had good binding activity to the two lymphoma cells (Fig. 7-1 and Fig. 7-2). In the horizontal comparison of the binding ability of each humanized antibody to lymphoma cells in the same cell line, it was found that AB12W6 had the strongest binding ability to the two cell lines, and the EC 50 value was 50% lower than that of the control antibody, and AB12W4 was comparable to the control antibody. AB12W3 had the weakest binding ability to the two lymphoma cell lines (Table 7).

表7、CD47人源化抗体与淋巴瘤细胞系结合的EC50值Table 7. EC50 values of CD47 humanized antibody binding to lymphoma cell lines

Figure PCTCN2021115946-appb-000018
Figure PCTCN2021115946-appb-000018

Figure PCTCN2021115946-appb-000019
Figure PCTCN2021115946-appb-000019

同时还测定了人源化抗体AB12W9以及对照抗体AB12W1、AB12W2和人急性淋巴白血病细胞CCRF-CEM(中科院上海细胞所)的结合能力，待测样品准备及测试方法同上。结果如图7-3所示，AB12W9与CCRF-CEM的结合能力与对照抗体AB12W2相当(EC 50值分别为0.1615μg/ml和0.1568μg/ml)，显著强于另一个对照抗体AB12W1(EC 50值为1.078μg/ml)。 At the same time, the binding ability of humanized antibody AB12W9 and control antibodies AB12W1, AB12W2 and human acute lymphoblastic leukemia cells CCRF-CEM (Shanghai Institute of Cell, Chinese Academy of Sciences) was also determined. The results are shown in Figure 7-3, the binding ability of AB12W9 to CCRF-CEM was comparable to that of the control antibody AB12W2 (EC 50 values were 0.1615 μg/ml and 0.1568 μg/ml, respectively), and significantly stronger than another control antibody AB12W1 (EC 50 value was 1.078 μg/ml).

6.4 FACS测定人源化抗体与CD47过表达细胞的结合能力6.4 Determination of the binding ability of humanized antibody to CD47 overexpressing cells by FACS

消化并收集稳定过表达CD47的细胞株CHO/K1-hCD47(Genscript，货号：M00581)，于800RPM离心3min，弃上清，用实验缓冲液(含1％灭活胎牛血清的DPBS培养基)重悬细胞，以台盼蓝染色法对细胞进行计数，用实验缓冲液调整细胞密度为1×10 6/ml，按每孔50μl接种于“U”型底的96孔板中。用上述实验缓冲液配制不同浓度的待测抗体AB12W3、AB12W6和AB12W9，同时设置阳性对照组AB12W1以及阴性同型对照组(Isotype control)，按每孔50μl加入96孔板中，充分混匀后静置于5％CO2的培养箱中，37℃孵育1h。离心收集细胞，吸弃上清，用实验缓冲液洗涤细胞3次，弃上清。配制适当浓度的F4647标记的山羊抗人IgG多抗(Jackson，货号109-605-088)，按照每孔100μl重悬细胞，室温避光孵育1h。离心收集细胞，吸弃上清，用实验缓冲液洗涤细胞3次，弃上清。用100μl实验缓冲液重悬细胞，上机检测(BD Accuri C6)，分析数据，以浓度对数(lgC)为横坐标，以MFI为纵坐标作图。 Digest and collect the cell line CHO/K1-hCD47 (Genscript, catalog number: M00581) stably overexpressing CD47, centrifuge at 800 RPM for 3 min, discard the supernatant, and use experimental buffer (DPBS medium containing 1% inactivated fetal bovine serum) Resuspend the cells, count the cells by trypan blue staining, adjust the cell density to 1×10 6 /ml with experimental buffer, and inoculate 50 μl per well in a “U” bottom 96-well plate. The test antibodies AB12W3, AB12W6 and AB12W9 of different concentrations were prepared with the above experimental buffer. At the same time, the positive control group AB12W1 and the negative isotype control group (Isotype control) were set up, and 50 μl per well was added to the 96-well plate, fully mixed and left to stand. Incubate at 37°C for 1 h in a 5% CO2 incubator. The cells were collected by centrifugation, the supernatant was discarded, and the cells were washed three times with assay buffer, and the supernatant was discarded. Prepare an appropriate concentration of F4647-labeled goat anti-human IgG polyclonal antibody (Jackson, Cat. No. 109-605-088), resuspend cells in 100 μl per well, and incubate at room temperature for 1 h in the dark. The cells were collected by centrifugation, the supernatant was discarded, and the cells were washed three times with assay buffer, and the supernatant was discarded. The cells were resuspended in 100 μl of experimental buffer, and detected on the computer (BD Accuri C6), and the data were analyzed.

如图8-1和图8-2所示，CD47人源化抗体AB12W6、AB12W3和AB12W9都能与过表达CD47的CHO/K1-hCD47细胞特异性结合，并且结合能力都强于对照抗体AB12W1，它们的EC 50值分别为0.2167μg/ml、0.4608μg/ml、0.2515μg/ml和0.6926μg/ml。 As shown in Figure 8-1 and Figure 8-2, CD47 humanized antibodies AB12W6, AB12W3 and AB12W9 can specifically bind to CHO/K1-hCD47 cells overexpressing CD47, and the binding ability is stronger than that of the control antibody AB12W1. Their EC50 values were 0.2167 μg/ml, 0.4608 μg/ml, 0.2515 μg/ml and 0.6926 μg/ml, respectively.

6.5 CD47人源化抗体的CDC活性测定6.5 Determination of CDC activity of CD47 humanized antibody

使用CD47表达阳性的源于Burkitt淋巴瘤的细胞系Raji细胞(ATCC：CCL-86)，检测CD47人源化抗体AB12W3、AB12W4、AB12W5和AB12W6的CDC活性测定，四种人源化抗体的重链恒定区都是含有S228P/L235E突变的人IgG4亚型。为做方法验证，以Rituxan作为阳性对照抗体，检测其对Raji细胞(CD20阳性)的CDC杀伤作用，同时设置同型对照组。The CDC activity assay of CD47 humanized antibodies AB12W3, AB12W4, AB12W5 and AB12W6 using the CD47 expression-positive Burkitt lymphoma-derived cell line Raji cells (ATCC: CCL-86), the heavy chains of the four humanized antibodies The constant regions are all of the human IgG4 subtype containing the S228P/L235E mutation. For method validation, Rituxan was used as a positive control antibody to detect its CDC killing effect on Raji cells (CD20 positive), and an isotype control group was set at the same time.

消化并收集Raji细胞，于800RPM离心3min，弃上清，用实验缓冲液(含1％灭活胎牛血清的无酚红MEM培养基)重悬细胞，以台盼蓝染色法对细胞进行计数，并用含15％正常人血清(NHS)的无酚红MEM培养基调整细胞密度为1×10 6/ml，按每孔50μl接种于96孔板中。用上述实验缓冲液配制不同浓度的待测抗体，按每孔50μl加入96孔板中，充分混匀后静置于5％CO2的培养箱中，37℃孵育4h。孵育结束后，每孔加入100μl CellTiter-Glo TM细胞活性检测试剂(Promega)，室温孵育10min，用多功能酶标仪(Biotek NEO2)检测并分析数据，以浓度对数(lgC)为横坐标，以细胞裂解百分比(％)为纵坐标作图。 Digest and collect Raji cells, centrifuge at 800 RPM for 3 min, discard the supernatant, resuspend the cells in experimental buffer (phenol red-free MEM medium containing 1% inactivated fetal bovine serum), and count the cells by trypan blue staining , and adjusted the cell density to 1×10 6 /ml with phenol red-free MEM medium containing 15% normal human serum (NHS), and inoculated 50 μl per well in a 96-well plate. Different concentrations of the antibody to be tested were prepared with the above experimental buffer, 50 μl per well was added to a 96-well plate, mixed well and then placed in a 5% CO2 incubator, and incubated at 37°C for 4h. After the incubation, 100 μl of CellTiter-Glo TM cell viability detection reagent (Promega) was added to each well, incubated at room temperature for 10 min, and the data was detected and analyzed with a multifunctional microplate reader (Biotek NEO2). The ordinate is plotted with the percentage of cell lysis (%).

如图9所示，CD47人源化抗体AB12W3、AB12W4、AB12W5和AB12W6对靶细胞Raji都不具有CDC细胞毒作用。含野生型人IgG4Fc片段的抗体本身介导较弱的ADCC和CDC效应，而本发明人源化抗体所含的IgG4Fc片段将L235突变为E，使其ADCC效应进一步去除，试验证实它们对靶细胞同样不具有显著的CDC杀伤作用。提示本发明提供的针对CD47的人源化抗体具有良好的安全性，不会介导其基于 阻断作用杀伤肿瘤细胞之外所不必要的ADCC和CDC等细胞毒作用，从而对表达CD47的正常细胞起到保护作用，减少不必要的临床副反应。As shown in Figure 9, none of the CD47 humanized antibodies AB12W3, AB12W4, AB12W5 and AB12W6 had CDC cytotoxicity on the target cell Raji. The antibody containing the wild-type human IgG4 Fc fragment itself mediates weak ADCC and CDC effects, while the IgG4 Fc fragment contained in the humanized antibody of the present invention mutates L235 to E, which further removes the ADCC effect. It also does not have significant CDC killing effect. It is suggested that the humanized antibody against CD47 provided by the present invention has good safety, and will not mediate its unnecessary cytotoxic effects such as ADCC and CDC in addition to killing tumor cells based on the blocking effect, thereby preventing normal CD47-expressing cells. Cells play a protective role, reducing unnecessary clinical side effects.

6.6抗CD47人源化抗体动力学参数和亲和力测定6.6 Anti-CD47 Humanized Antibody Kinetic Parameters and Affinity Determination

对CD47人源化抗体AB12W3、AB12W4、AB12W6和AB12W10的亲和力测定也是利用BLI技术，根据仪器Biacore T200的说明书进行操作，方法参照实施例2.5。The affinity determination of CD47 humanized antibodies AB12W3, AB12W4, AB12W6 and AB12W10 also used BLI technology, and the operation was performed according to the instructions of the instrument Biacore T200, and the method was referred to in Example 2.5.

结果显示，获得的人源化抗体亲和力没有明显损失，候选抗体AB12W3、AB12W4和AB12W6与人CD47的结合亲和力与对照抗体AB12W1和AB12W2相当，AB12W10的结合亲和力低一个数量级，人源化抗体较好地保留了亲本鼠单克隆抗体的亲和力和特异性，大大降低了其免疫原性。The results showed that the obtained humanized antibodies did not lose significant affinity. The binding affinity of the candidate antibodies AB12W3, AB12W4 and AB12W6 to human CD47 was comparable to that of the control antibodies AB12W1 and AB12W2. The binding affinity of AB12W10 was one order of magnitude lower, and the humanized antibodies were better The affinity and specificity of the parental murine monoclonal antibody is preserved, and its immunogenicity is greatly reduced.

6.7抗CD47人源化抗体介导巨噬细胞的吞噬作用6.7 Anti-CD47 Humanized Antibody Mediates Phagocytosis of Macrophages

(1)对肿瘤细胞CCRF-CEM吞噬作用(1) Phagocytosis of tumor cells CCRF-CEM

抽取的人外周血，利用梯度离心法收集的PBMC细胞，利用人单核细胞分离试剂盒(美天旎生物科技公司)，分离并收集单核细胞。将分离的单核细胞按照1×10 6/ml的细胞密度接种到培养皿中，分别在接种后第5天、第8天和第11天换液，第13天后细胞即分化成为巨噬细胞(M2型)，可肉眼观察到细胞形态为不规则贴壁。也可用FACS检测细胞表面的标志物，一般包括CD11b、CD14、CD45、CD64、CD163和CD206。 The extracted human peripheral blood, PBMC cells collected by gradient centrifugation, and human monocyte isolation kit (Miltenyi Biotechnology Co., Ltd.) were used to separate and collect monocytes. The isolated monocytes were seeded into culture dishes at a cell density of 1×10 6 /ml, and the medium was changed on the 5th, 8th and 11th days after seeding, and the cells differentiated into macrophages after the 13th day. (M2 type), irregular adherent cell morphology can be observed with the naked eye. Cell surface markers can also be detected by FACS, generally including CD11b, CD14, CD45, CD64, CD163, and CD206.

将用PE荧光染色的CCRF-CEM(中科院上海细胞所)接种于96孔板，每孔50μl，约2.5×10 5个细胞。将待测抗体AB12W3、AB12W4和AB12W6用PBS稀释至200μg/ml，3倍梯度稀释至11个浓度点，同时设置AB12W1阳性对照组和同型抗体阴性对照组。然后将不同浓度梯度的待测抗体，铺于上述96孔板中，每孔50μl。37℃，5％CO 2培养箱中静置1h，使抗体与靶细胞充分结合。然后加入上述制备的M2型巨噬细胞，每孔100μl，约5×10 4个细胞，混匀。37℃，5％CO 2培养箱放置1h。收集所有细胞，以CD11b-APC荧光抗体(Invitrogen)标记。按照常规流式方法洗板，洗掉未结合的荧光抗体。上机检测，取样本总数为1×10 4个细胞，其中靶细胞为PE荧光，巨噬细胞为APC荧光，发生吞噬作用的为PE+APC双荧光。读取荧光值，

Figure PCTCN2021115946-appb-000020
分析数据，以浓度对数(lgC)为横坐标，以吞噬百分比(％)为纵坐标作图。 The CCRF-CEM (Shanghai Institute of Cells, Chinese Academy of Sciences) stained with PE fluorescence was seeded in a 96-well plate, 50 μl per well, about 2.5×10 5 cells. The antibodies to be tested AB12W3, AB12W4 and AB12W6 were diluted with PBS to 200 μg/ml, 3-fold gradient diluted to 11 concentration points, and the AB12W1 positive control group and the isotype antibody negative control group were set at the same time. Then, different concentration gradients of the antibody to be tested were spread in the above-mentioned 96-well plate, 50 μl per well. Let stand for 1 h in a 37°C, 5% CO 2 incubator to fully bind the antibody to the target cells. Then add the M2 macrophages prepared above, 100 μl per well, about 5×10 4 cells, and mix well. 37°C, 5% CO2 incubator for 1h. All cells were harvested and labeled with CD11b-APC fluorescent antibody (Invitrogen). Plates were washed according to conventional flow cytometry to remove unbound fluorescent antibodies. The total number of samples taken was 1×10 4 cells for on-machine detection, in which the target cells were PE fluorescence, the macrophages were APC fluorescence, and the phagocytosis was PE+APC dual fluorescence. read the fluorescence value,
Figure PCTCN2021115946-appb-000020
Data were analyzed and plotted with log concentration (lgC) as abscissa and percentage phagocytosis (%) as ordinate.

如图10所示，CD47人源化抗体AB12W3、AB12W4和AB12W6都能介导巨噬细胞吞噬肿瘤细胞CCRF-CEM，并且呈剂量依赖性，与对照抗体AB12W1的EC 50值相当。 As shown in Figure 10, CD47 humanized antibodies AB12W3, AB12W4, and AB12W6 could mediate macrophage phagocytosis of tumor cells CCRF-CEM in a dose-dependent manner, which was comparable to the EC50 value of control antibody AB12W1.

(2)对红细胞RBC的吞噬作用(2) Phagocytosis of erythrocyte RBC

CD47也高表达于红细胞表面，当CD47抗体结合红细胞表面的CD47后，打破红细胞表面的“别吃我”信号，导致巨噬细胞清除红细胞，可能诱发贫血症状。临床I期试验中，CD47抗体治疗后，发生红细胞枯竭，造成短暂性的贫血是其主要的不良反应。因此，我们测定CD47人源化抗体AB12W3、和AB12W4介导的巨噬细胞对红细胞的吞噬作用大小，来评价本发明提供的CD47人源化抗体在临床应用中的潜在副作用。红细胞采用常规方法制备，并以PE荧光染色，巨噬细胞以CD11b-APC荧光抗体标记，待测抗体样品稀释方法及测定步骤同上。分析数据，以浓度对数(lgC)为横坐标，以吞噬百分比(％)为纵坐标作图。CD47 is also highly expressed on the surface of red blood cells. When CD47 antibody binds to CD47 on the surface of red blood cells, it breaks the "don't eat me" signal on the surface of red blood cells, causing macrophages to clear red blood cells, which may induce symptoms of anemia. In phase I clinical trials, red blood cell depletion occurred after CD47 antibody treatment, resulting in transient anemia as the main adverse reaction. Therefore, we measured the phagocytosis of erythrocytes by macrophages mediated by CD47 humanized antibodies AB12W3 and AB12W4 to evaluate the potential side effects of the CD47 humanized antibodies provided by the present invention in clinical application. Erythrocytes were prepared by conventional methods and stained with PE fluorescence, and macrophages were labeled with CD11b-APC fluorescent antibody. Data were analyzed and plotted with log concentration (lgC) as abscissa and percentage phagocytosis (%) as ordinate.

如图11所示，与同型对照相比，对照抗体AB12W1能够介导较强的红细胞吞噬能力，而人源化抗体AB12W3和AB12W4几乎不引起红细胞吞噬，与同型对照抗体相比无显著性差异。As shown in Figure 11, the control antibody AB12W1 was able to mediate stronger erythrocyte phagocytosis compared to the isotype control, while the humanized antibodies AB12W3 and AB12W4 hardly caused erythrocyte phagocytosis, and there was no significant difference compared with the isotype control antibody.

6.8抗CD47人源化抗体促进红细胞凝集活性测定6.8 Determination of anti-CD47 humanized antibody promoting hemagglutination activity

利用红细胞凝集反应对候选CD47人源化抗体是否引发血凝反应进行评估。通过对存在CD47抗体时的RBC斑点面积与不存在CD47抗体时(即零血凝反应条件下)的RBC斑点面积进行比较，RBC斑点面积越大表明血凝反应的水平越高，也可使用RBC斑点的密度分析对血凝反应进行定量。Whether candidate CD47 humanized antibodies elicit hemagglutination was assessed using hemagglutination. By comparing the RBC spot area in the presence of CD47 antibody with the RBC spot area in the absence of CD47 antibody (ie under zero hemagglutination conditions), a larger RBC spot area indicates a higher level of hemagglutination, and RBC can also be used Densitometric analysis of the spots quantified hemagglutination.

检测方法如下：采集新鲜人外周血，离心并洗涤沉淀，分离人红细胞，将细胞重悬至密度为2×10 8/ml。将50μl人红细胞悬液与50μl待测抗体(最高浓度为200μg/ml，3倍梯度稀释，总共8个点)加入96孔板中，阴性对照组(NC)加入等体积的PBS缓冲液，在37℃孵育30min，反应结束后用扫描仪对96孔板进行拍照并判断结果。结果判定标准为如果红细胞沉于孔底，平铺呈网状，则发生了红细胞凝集反应(参见图12中AB12W1的结果)，如果红细胞未发生凝集反应，则红细胞将呈点状沉于孔底(参见图12中NC的结果)，根据沉降的红细胞团面积，可以初步判定抗体对红细胞凝集作用的强弱。 The detection method is as follows: collecting fresh human peripheral blood, centrifuging and washing the pellet, separating human red blood cells, and resuspending the cells to a density of 2×10 8 /ml. Add 50 μl of human erythrocyte suspension and 50 μl of the antibody to be tested (the highest concentration is 200 μg/ml, 3-fold gradient dilution, a total of 8 points) into a 96-well plate, and the negative control group (NC) is added with an equal volume of PBS buffer. Incubate at 37°C for 30 min. After the reaction, the 96-well plate was photographed with a scanner and the results were judged. The criterion for determining the result is that if the red blood cells settle on the bottom of the well and are flattened into a mesh, then the red blood cell agglutination reaction has occurred (see the results of AB12W1 in Figure 12). (See the results of NC in Fig. 12 ), according to the area of the sedimented erythrocyte mass, the strength of the antibody on erythrocyte agglutination can be preliminarily determined.

由图12可见，CD47人源化抗体AB12W3、AB12W4、AB12W5、AB12W6、AB12W8、AB12W9和AB12W10同阴性对照组一样都不会引起明显的红细胞凝集反应，而对照抗体AB12W1在高浓度下呈现明显的细胞凝集现象。提示本发明提供的CD47抗体具有显著降低的促血细胞凝集作用，预期其临床应用中的相关副反应也会极大程度的降低甚至消除。It can be seen from Figure 12 that CD47 humanized antibodies AB12W3, AB12W4, AB12W5, AB12W6, AB12W8, AB12W9 and AB12W10 did not cause obvious hemagglutination reaction like the negative control group, while the control antibody AB12W1 showed obvious cells at high concentrations. Agglomeration phenomenon. It is suggested that the CD47 antibody provided by the present invention has a significantly reduced effect of promoting hemagglutination, and it is expected that the related side reactions in its clinical application will also be greatly reduced or even eliminated.

实施例7、CD47人源化抗体的体内药效学研究Example 7. In vivo pharmacodynamics study of CD47 humanized antibody

7.1AB12W3、AB12W4、AB12W5和AB12W6在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的药效学研究7.1 Pharmacodynamic study of AB12W3, AB12W4, AB12W5 and AB12W6 in human Burkkit's lymphoma mouse subcutaneous xenograft model

选取7～8周龄的雌性NOD/SCID小鼠(北京维通利华实验动物技术有限公司)，收集处于对数生长期的Raji细胞(中科院细胞库)，将4×10 6个Raji细胞和等体积Matrigel混合，接种于NOD/SCID小鼠右侧背部皮下。待小鼠肿瘤体积生长到平均87mm 3左右，小鼠按肿瘤体积和体重随机分为7组，每组6只，给药组分别腹腔给予AB12W3、AB12W4、AB12W5和AB12W6以及对照抗体AB12W1和AB12W2，阴性对照组相应给予等体积的PBS。所有给药组均为每周给药两次，给药组的剂量均为5mg/kg。给药当天记为第0天，每周用电子游标卡尺测量肿瘤的最大直径(D)和最小直径(d)，使用以下公式计算肿瘤体积(mm 3)＝[D×d 2]/2，并根据公式计算各给药组的肿瘤生长抑制率TGI(％)＝(1-给药组RTV/对照组RTV)×100％；其中，相对肿瘤体积(RTV)为每一次测量(即D n)所得肿瘤体积Vn与分组给药时(即D 0)测量所得肿瘤体积V 0的比值。 Select 7-8-week-old female NOD/SCID mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), collect Raji cells in logarithmic growth phase (Cell Bank of Chinese Academy of Sciences), and 4×10 6 Raji cells and An equal volume of Matrigel was mixed and inoculated subcutaneously on the right back of NOD/SCID mice. When the tumor volume of the mice grew to an average of about 87 mm 3 , the mice were randomly divided into 7 groups according to the tumor volume and body weight, with 6 mice in each group. The negative control group was given an equal volume of PBS accordingly. All administration groups were administered twice a week, and the doses of administration groups were all 5 mg/kg. The day of administration was recorded as day 0, the maximum diameter (D) and the minimum diameter (d) of the tumor were measured with an electronic vernier caliper every week, and the tumor volume (mm 3 )=[D×d 2 ]/2 was calculated using the following formula, and The tumor growth inhibition rate of each administration group was calculated according to the formula TGI (%)=(1-RTV of administration group/RTV of control group)×100%; wherein, the relative tumor volume (RTV) was the value of each measurement (ie D n ) The ratio of the obtained tumor volume Vn to the obtained tumor volume V 0 was measured at the time of group administration (ie, D 0 ).

如图13所示，在给药后第21天，PBS对照组平均肿瘤体积为1001.17±38.43mm 3；AB12W1给药组的平均肿瘤体积为554.07±122.11mm 3，TGI为44.12％，相对于对照组具有非常显著性差异(P<0.01)；AB12W2给药组的平均肿瘤体积为181.39±54.07mm 3，TGI为82.74％，相对于对照组具有非常显著性差异(P<0.0001)；AB12W3给药组的平均肿瘤体积为544.04±61.44mm 3，TGI为47.56％，相对于对照组具有非常显著性差异(P<0.01)；AB12W4给药组的平均肿瘤体积为429.30±66.15mm 3，TGI为59.35％，相对于对照组具有非常显著性差异(P<0.0001)；AB12W5给药组的平均肿瘤体积为194.89±33.71mm 3，TGI为80.76％，相对于对照组具有非常显著性差异(P<0.0001)；AB12W6给药组的平均肿瘤体积为180.74±77.36mm 3，TGI为83.13％，相对于对照组具有非常显著性差异(P<0.0001)。 As shown in Figure 13, on the 21st day after administration, the average tumor volume of the PBS control group was 1001.17±38.43 mm 3 ; the average tumor volume of the AB12W1 administration group was 554.07 ± 122.11 mm 3 , and the TGI was 44.12%, relative to the control group. There was a very significant difference between the two groups (P<0.01); the average tumor volume of the AB12W2 administration group was 181.39±54.07mm 3 , and the TGI was 82.74%, which was significantly different from the control group (P<0.0001); AB12W3 administration The average tumor volume of the group was 544.04±61.44mm 3 and the TGI was 47.56%, which was significantly different from the control group (P<0.01); the average tumor volume of the AB12W4 administration group was 429.30±66.15mm 3 and the TGI was 59.35 %, there was a very significant difference compared with the control group (P<0.0001); the average tumor volume of the AB12W5 administration group was 194.89±33.71mm 3 , and the TGI was 80.76%, which had a very significant difference compared with the control group (P<0.0001). ); the mean tumor volume of the AB12W6 administration group was 180.74±77.36 mm 3 , and the TGI was 83.13%, which was significantly different from the control group (P<0.0001).

以上结果表明，抗CD47人源化抗体AB12W3、AB12W4、AB12W5和AB12W6都显示出良好的抗肿瘤效果，抑瘤效果均优于对照抗体AB12W1，其中AB12W5和AB12W6的TGI同对照抗体AB12W2相当，均高于80％。The above results show that the anti-CD47 humanized antibodies AB12W3, AB12W4, AB12W5 and AB12W6 all showed good anti-tumor effects, and the tumor-inhibitory effects were all better than those of the control antibody AB12W1. The TGI of AB12W5 and AB12W6 were comparable to those of the control antibody AB12W2, and both were higher than those of the control antibody AB12W2. at 80%.

7.2 AB12W6、AB12W9和AB12W10在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的药效学研究7.2 Pharmacodynamics study of AB12W6, AB12W9 and AB12W10 in human Burkkit's lymphoma mouse subcutaneous xenograft model

选取7～8周龄的雌性NOD/SCID小鼠(北京维通利华实验动物技术有限公司)，收集处于对数生长期的Raji细胞(中科院细胞库)，将5×10 6个Raji细胞和等体积Matrigel混合，接种于NOD/SCID小鼠右侧背部皮下。待小鼠肿瘤体积生长到平均85mm 3左右，小鼠按肿瘤体积和体重随机分为8组，每组6只，分别腹腔给予相应药物。所有给药组均为每周给药两次，AB12W6、AB12W9、AB12W10、对照抗体AB12W2以及CD20单抗Rituxan的给药剂量均为5mg/kg，阴性对照组相应给予等体积的PBS。给药当天记为第0天，每周用电子游标卡尺测量肿瘤的最大直径(D)和最小直径(d)，使用以下公式计算肿瘤体积(mm3)＝[D×d 2]/2，并根据公式计算各给药组的肿瘤生长抑制率TGI(％)＝(1-给药组RTV/对照组RTV)×100％；其中，相对肿瘤体积(RTV)为每一次测量(即D n)所得肿瘤体积Vn与分组给药时(即D 0)测量所得肿瘤体积V 0的比值。 Select 7-8 week old female NOD/SCID mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), collect Raji cells in logarithmic growth phase (Cell Bank of Chinese Academy of Sciences), and transfer 5×10 6 Raji cells and An equal volume of Matrigel was mixed and inoculated subcutaneously on the right back of NOD/SCID mice. When the tumor volume of the mice grew to an average of about 85 mm 3 , the mice were randomly divided into 8 groups according to the tumor volume and body weight, with 6 mice in each group, and the corresponding drugs were administered intraperitoneally. All administration groups were administered twice a week. The administration doses of AB12W6, AB12W9, AB12W10, control antibody AB12W2 and CD20 monoclonal antibody Rituxan were all 5 mg/kg, and the negative control group was given an equal volume of PBS accordingly. The day of administration was recorded as day 0, the maximum diameter (D) and the minimum diameter (d) of the tumor were measured with an electronic vernier caliper every week, and the tumor volume (mm3)=[D×d 2 ]/2 was calculated using the following formula, and calculated according to The formula calculates the tumor growth inhibition rate of each administration group TGI (%)=(1-RTV of administration group/RTV of control group)×100%; wherein, the relative tumor volume (RTV) is obtained by each measurement (ie D n ) The ratio of tumor volume Vn to the tumor volume V0 measured at the time of group administration (ie, D0 ).

如图14所示，在给药后第15天，PBS对照组平均肿瘤体积为1881.04±198.74；Rituxan给药组的平均肿瘤体积为806.79±206.63mm 3，TGI为51.18％；AB12W2给药组的平均肿瘤体积为309.58±54.62mm 3，TGI为84.21％；AB12W6给药组的平均肿瘤体积为370.54±116.72mm 3，TGI为82.19％；AB12W9给药组的平均肿瘤体积分别为343.52±145.65mm 3，TGI为83.94％；AB12W10给药组的平均肿瘤体积为380.37±111.84mm 3，TGI为81.65％。所有给药组相对于对照组均有非常显著性差异(P<0.01)。 As shown in Figure 14, on the 15th day after administration, the average tumor volume in the PBS control group was 1881.04±198.74; the average tumor volume in the Rituxan administration group was 806.79±206.63 mm 3 , and the TGI was 51.18%; The mean tumor volume was 309.58±54.62 mm 3 , and the TGI was 84.21%; the mean tumor volume of the AB12W6-administered group was 370.54±116.72 mm 3 , and the TGI was 82.19%; the mean tumor volume of the AB12W9-administered group was 343.52±145.65 mm 3 , respectively , TGI was 83.94%; the mean tumor volume of AB12W10 administration group was 380.37±111.84mm 3 , TGI was 81.65%. Compared with the control group, all administration groups had very significant differences (P<0.01).

以上结果表明，抗CD47人源化抗体AB12W6、AB12W9和AB12W10同对照抗体AB12W2都显示出良好的抗肿瘤效果，抑瘤率均高于80％，显著优于Rituxan给药组。The above results showed that the anti-CD47 humanized antibodies AB12W6, AB12W9 and AB12W10 showed good anti-tumor effects with the control antibody AB12W2, and the tumor inhibition rates were all higher than 80%, which was significantly better than the Rituxan administration group.

7.3 AB12W8或AB12W9与Rituxan联用在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的药效学研究7.3 Pharmacodynamic study of AB12W8 or AB12W9 combined with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model

选取7～8周龄的雌性NOD/SCID小鼠(购自北京维通利华实验动物技术有限公司)，收集处于对数生长期的Raji细胞(购自中科院上海细胞库)，将5×10 6个Raji细胞和等体积Matrigel混合，接种于NOD/SCID小鼠右侧背部皮下。待小鼠肿瘤体积生长到平均112mm 3左右，小鼠按肿瘤体积和体重随机分为8组，每组6只，分别腹腔给予相应药物。所有给药组均为每周给药两次，给药组的剂量均为5mg/kg，分组情况及给药方案见表8。给药当天记为第0天，每周用电子游标卡尺测量肿瘤的最大直径(D)和最小直径(d)，使用以下公式计算肿瘤体积(mm3)＝[D×d 2]/2，并根据公式计算各给药组的肿瘤生长抑制率TGI(％)＝(1-给药组RTV/对照组RTV)×100％；其中，相对肿瘤体积(RTV)为每一次测量(即D n)所得肿瘤体积Vn与分组给药时(即D 0)测量所得肿瘤体积V 0的比值。 Select 7-8-week-old female NOD/SCID mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), collect Raji cells in logarithmic growth phase (purchased from Shanghai Cell Bank of Chinese Academy of Sciences), and transfer 5 × 10 Six Raji cells were mixed with an equal volume of Matrigel and inoculated subcutaneously on the right back of NOD/SCID mice. When the tumor volume of the mice grew to an average of about 112 mm 3 , the mice were randomly divided into 8 groups according to the tumor volume and body weight, with 6 mice in each group, and the corresponding drugs were administered intraperitoneally. All the administration groups were administered twice a week, and the doses of the administration groups were all 5 mg/kg. The day of administration was recorded as day 0, the maximum diameter (D) and the minimum diameter (d) of the tumor were measured with an electronic vernier caliper every week, and the tumor volume (mm3)=[D×d 2 ]/2 was calculated using the following formula, and calculated according to The formula calculates the tumor growth inhibition rate of each administration group TGI (%)=(1-RTV of administration group/RTV of control group)×100%; wherein, the relative tumor volume (RTV) is obtained by each measurement (ie D n ) The ratio of tumor volume Vn to the tumor volume V0 measured at the time of group administration (ie, D0 ).

如图15所示，在给药后第21天，PBS阴性对照组平均肿瘤体积为2139.04±309.53mm 3；Rituxan给药组的平均肿瘤体积为1303.69±481.03mm 3，TGI为43.4％，相对于对照组无显著性差异，但6只小鼠里有1只小鼠的肿瘤完全消退；AB12W1给药组的平均肿瘤体积为859.40±241.19mm 3，TGI为61.91％；AB12W8给药组的平均肿瘤体积为1838.71±263.19mm 3，相对于对照组无显著性差异；AB12W9给药组的平均肿瘤体积为555.13±189.79mm 3，TGI为76.44％；Rituxan分别和AB12W1、AB12W8以及AB12W9联合给药组的平均肿瘤体积分别为701.89±301.99mm 3、915.32±409.09mm 3和397.32±171.98mm 3，TGI分别为67.71％、62.18％和82.83％。Rituxan和AB12W1或AB12W9联合给药组各有一只小鼠的肿瘤完全消退。另外，整个实验过程中，动物健康状态良好，无动物死亡。实验结束时，各组动物体重均出现增长，AB12M8和AB12W9单用或联用Rituxan处理组动物与溶剂对照组动物体重比较无显著性差异(p>0.05)，表明动物对AB12M8和AB12W9耐受良好，未对实验动物产生明显毒性作用。 As shown in Figure 15, on the 21st day after administration, the mean tumor volume of the PBS negative control group was 2139.04±309.53 mm 3 ; the mean tumor volume of the Rituxan administration group was 1303.69±481.03 mm 3 , and the TGI was 43.4%, which was 43.4% relative to There was no significant difference in the control group, but the tumor in 1 of the 6 mice completely regressed; the average tumor volume in the AB12W1-administered group was 859.40±241.19 mm 3 , and the TGI was 61.91%; the average tumor in the AB12W8-administered group The volume was 1838.71±263.19mm 3 , and there was no significant difference compared with the control group; the average tumor volume of the AB12W9 administration group was 555.13±189.79mm 3 , and the TGI was 76.44%; the Rituxan and AB12W1, AB12W8 and AB12W9 combined administration groups respectively The mean tumor volumes were 701.89±301.99mm 3 , 915.32±409.09mm 3 and 397.32±171.98mm 3 , respectively, and the TGIs were 67.71%, 62.18%, and 82.83%, respectively. One mouse in each of the Rituxan and AB12W1 or AB12W9 co-administered groups had complete tumor regression. In addition, during the whole experiment, the animals were in good health and no animals died. At the end of the experiment, the body weight of the animals in each group increased. There was no significant difference in the body weight of the animals in the AB12M8 and AB12W9 treatment groups alone or in combination with the solvent control group (p>0.05), indicating that the animals tolerated AB12M8 and AB12W9 well. , did not produce obvious toxic effects on experimental animals.

以上结果表明，抗CD47人源化抗体AB12W9具有良好的抗肿瘤作用，单用或与Rituxan联用的抑瘤效果均优于对照抗体AB12W1单用或与Rituxan联用组；AB12W8单用不具有显著的抑瘤作用，当与Rituxan联用时也能显著地抑制肿瘤生长。AB12W8和AB12W9及对照抗体AB12W1与Rituxan联用的效果均好于单独给药组。The above results show that the anti-CD47 humanized antibody AB12W9 has a good anti-tumor effect, and the anti-tumor effect of the anti-CD47 humanized antibody AB12W9 alone or in combination with Rituxan is better than that of the control antibody AB12W1 alone or in combination with Rituxan; AB12W8 alone has no significant effect It also significantly inhibited tumor growth when used in combination with Rituxan. The combined effect of AB12W8 and AB12W9 and the control antibody AB12W1 and Rituxan was better than that of the single administration group.

表8、CD47人源化抗体与Rituxan联用在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的抑瘤作用Table 8. Antitumor effect of CD47 humanized antibody combined with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model

Figure PCTCN2021115946-appb-000021
Figure PCTCN2021115946-appb-000021

7.4 AB12W9单用或与Rituxan联用在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的药效学研究7.4 Pharmacodynamic study of AB12W9 alone or in combination with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model

选取7～8周龄的雌性NOD/SCID小鼠(北京维通利华实验动物技术有限公司)，收集处于对数生长期的Raji细胞(中科院上海细胞库)，将5×10 6个Raji细胞和等体积Matrigel混合，接种于NOD/SCID小鼠右侧背部皮下。待小鼠肿瘤体积生长到平均110mm 3左右，小鼠按肿瘤体积和体重随机分为9组，每组6只，分别腹腔给予相应药物。所有给药组均为每周给药两次，具体分组情况、给药方案及主要实验结果(包括各给药组肿瘤消退情况)见表9。给药当天记为第0天，每周用电子游标卡尺测量肿瘤的最大直径(D)和最小直径(d)，使用以下公式计算肿瘤体积(mm3)＝[D×d 2]/2，并根据公式计算各给药组的肿瘤生长抑制率TGI(％)＝(1-给药组RTV/对照组RTV)×100％；其中，相对肿瘤体积(RTV)为每一次测量(即D n)所得肿瘤体积Vn与分组给药时(即D 0)测量所得肿瘤体积V 0的比值。 Select 7-8 week old female NOD/SCID mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), collect Raji cells in logarithmic growth phase (Shanghai Cell Bank of Chinese Academy of Sciences), and transfer 5 × 10 6 Raji cells It was mixed with an equal volume of Matrigel and inoculated subcutaneously on the right back of NOD/SCID mice. When the tumor volume of the mice grew to an average of about 110 mm 3 , the mice were randomly divided into 9 groups according to the tumor volume and body weight, with 6 mice in each group, and the corresponding drugs were administered intraperitoneally. All administration groups were administered twice a week. The specific grouping, administration schedule and main experimental results (including tumor regression in each administration group) are shown in Table 9. The day of administration was recorded as day 0, the maximum diameter (D) and the minimum diameter (d) of the tumor were measured with an electronic vernier caliper every week, and the tumor volume (mm3)=[D×d 2 ]/2 was calculated using the following formula, and calculated according to The formula calculates the tumor growth inhibition rate of each administration group TGI (%)=(1-RTV of administration group/RTV of control group)×100%; wherein, the relative tumor volume (RTV) is obtained by each measurement (ie D n ) The ratio of tumor volume Vn to the tumor volume V0 measured at the time of group administration (ie, D0 ).

如图16所示，在给药后第27天，阴性对照组平均肿瘤体积为1711.49±540.38mm 3；Rituxan给药组的平均肿瘤体积为1497.13±447.01mm 3，TGI为16.59％，相对于阴性对照组无显著性差异；AB12W2给药组的平均肿瘤体积为21.47±15.54mm 3，TGI为98.78％，相对于阴性对照组具有非常显著性差异(P<0.0001)；AB12W9的1mg/kg、3mg/kg和10mg/kg三个剂量的平均肿瘤体积分别为770.48±218.83mm 3、179.76±186.32mm 3和78.13±63.00mm 3，TGI分别为55.05％、89.68％和95.53％；所有给药组相对于对照组均有非常显著性差异(P<0.01)；Rituxan分别和AB12W9三个剂量联合给药组的平均肿瘤体积分别为389.48±360.90mm 3、126.00±112.53mm 3和63.10±59.45mm 3，TGI分别为78.46％、92.98％和96.39％。另外，整个实验过程中，动物健康状态良好，无动物死亡。实验结束时，各组动物体重均出现增长，AB12W9单用或联用Rituxan处理组动物与溶剂对照组动物体重比较无显著性差异(p>0.05)，表明动物对AB12W9耐受良好，未对实验动物产生明显毒性作用。 As shown in Figure 16, on the 27th day after administration, the average tumor volume of the negative control group was 1711.49 ± 540.38 mm 3 ; the average tumor volume of the Rituxan administration group was 1497.13 ± 447.01 mm 3 , the TGI was 16.59%, relative to the negative There was no significant difference in the control group; the average tumor volume of the AB12W2 administration group was 21.47±15.54mm 3 , and the TGI was 98.78%, which was significantly different from the negative control group (P<0.0001); 1mg/kg, 3mg of AB12W9 The mean tumor volumes of the three doses/kg and 10 mg/kg were 770.48±218.83mm 3 , 179.76±186.32mm 3 and 78.13±63.00mm 3 , respectively, and the TGIs were 55.05%, 89.68% and 95.53%, respectively; There were very significant differences in the control group (P<0.01); the mean tumor volumes of the three doses of Rituxan and AB12W9 were 389.48±360.90mm 3 , 126.00±112.53mm 3 and 63.10±59.45mm 3 , respectively. The TGIs were 78.46%, 92.98% and 96.39%, respectively. In addition, during the whole experiment, the animals were in good health and no animals died. At the end of the experiment, the body weight of the animals in each group increased. There was no significant difference in the body weight of the animals in the AB12W9 treatment group alone or in combination with the Rituxan treatment group and the solvent control group (p>0.05), indicating that the animals tolerated AB12W9 well. Animals have obvious toxic effects.

以上结果表明，抗CD47人源化抗体AB12W9单用就具有良好的抗肿瘤作用，且抗肿瘤作用具有良好 的量效关系；AB12W9低剂量1mg/kg与Rituxan联用的抑瘤效果优于AB12W9单用和Rituxan单用组；AB12W9中、高剂量(3、10mg/kg)因单用药效已经达到最佳抗肿瘤效果，没有提高的余地，故与Rituxan联用的效果与单用AB12W9相当。The above results show that the anti-CD47 humanized antibody AB12W9 has a good anti-tumor effect alone, and the anti-tumor effect has a good dose-effect relationship; the anti-tumor effect of AB12W9 at a low dose of 1 mg/kg combined with Rituxan is better than that of AB12W9 alone Rituxan and Rituxan alone group; AB12W9 medium and high doses (3, 10 mg/kg) have achieved the best anti-tumor effect as a single drug, and there is no room for improvement, so the effect of combined use with Rituxan is comparable to that of AB12W9 alone.

表9、CD47人源化抗体与Rituxan联用在人Burkkit’s淋巴瘤小鼠皮下移植瘤模型中的抑瘤作用Table 9. Antitumor effect of CD47 humanized antibody combined with Rituxan in human Burkkit's lymphoma mouse subcutaneous xenograft model

Figure PCTCN2021115946-appb-000022
Figure PCTCN2021115946-appb-000022

在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (31)

一种特异性结合CD47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(VH)和轻链可变区(VL)，且其VH和VL分别包含选自下组的互补决定区(CDR)：An antibody or antigen-binding fragment thereof that specifically binds to CD47, the antibody or antigen-binding fragment thereof comprises a variable region of a heavy chain (VH) and a variable region of a light chain (VL), and its VH and VL respectively comprise a variable region selected from the group consisting of Complementarity determining regions (CDRs) of the following groups: 其重链可变区(VH)包含的CDR选自：Its heavy chain variable region (VH) comprises CDRs selected from: (i)CDR-H1，其具有如SEQ ID NO：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35或37所示的VH中含有的CDR-H1的序列，或者与所述VH中含有的CDR-H1的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(i) CDR-H1 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 The sequence of CDR-H1 contained in the VH, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or add) sequence; (ii)CDR-H2，其具有如SEQ ID NO：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35或37所示的VH中含有的CDR-H2的序列，或者与所述VH中含有的CDR-H2的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(ii) CDR-H2 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 The sequence of CDR-H2 contained in the VH, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or added) sequence; and (iii)CDR-H3，其具有如SEQ ID NO：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35或37所示的VH中含有的CDR-H3的序列，或者与所述VH中含有的CDR-H3的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 having as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 The sequence of CDR-H3 contained in the VH, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or add) sequence; 和/或，and / or, 其轻链可变区(VL)的CDR选自：The CDRs of its light chain variable region (VL) are selected from: (iv)CDR-L1，其具有如SEQ ID NO：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36或38所示的VL中含有的CDR-L1的序列，或者与所述VL中含有的CDR-L1的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iv) CDR-L1 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 The sequence of CDR-L1 contained in the VL, or with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 substitutions, deletions or add) sequence; (v)CDR-L2，其具有如SEQ ID NO：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36或38所示的VL中含有的CDR-L2的序列，或者与所述VL中含有的CDR-L2的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；和(v) CDR-L2 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 The sequence of CDR-L2 contained in the VL, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions) compared to the sequence of the CDR-L2 contained in the VL added) sequence; and (vi)CDR-L3，其具有如SEQ ID NO：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36或38所示的VL中含有的CDR-L3的序列，或者与所述VL中含有的CDR-L3的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 having as SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 The sequence of CDR-L3 contained in the VL, or with one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 substitutions, deletions or additions) compared to the sequence of the CDR-L3 contained in the VL add) sequence; 优选地，(i)-(vi)任一项中所述的置换为保守置换。Preferably, the substitutions described in any of (i)-(vi) are conservative substitutions. 如权利要求1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包含：如SEQ ID NO：5、13、15、17或19所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO：6、14、16、18或20所示的轻链可变区(VL)中含有的3个CDR；其中，所述重链可变区(VH)中含有的3个CDR和所述轻链可变区(VL)中含有的3个CDR通过IMGT或Kabat编号系统确定。The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region shown in SEQ ID NO: 5, 13, 15, 17 or 19 3 CDRs contained in (VH); and/or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 6, 14, 16, 18 or 20; wherein, the The three CDRs contained in the variable region of the heavy chain (VH) and the three CDRs contained in the variable region of the light chain (VL) are identified by the IMGT or Kabat numbering system. 如权利要求1或2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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(a) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：79，或与SEQ ID NO：79相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:79, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:79 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：80，或与SEQ ID NO：80相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:80, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:80) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：81，或与SEQ ID NO：81相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO: 81, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 81) substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：82，或与SEQ ID NO：82相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:82, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:82 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：83，或与SEQ ID NO：83相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:83, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:83 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions); 其中，组(a)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义；wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs in group (a) are defined by the IMGT numbering system; (b)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(b) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：42，或与SEQ ID NO：42相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:42, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:42 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：43，或与SEQ ID NO：43相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:43, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:43) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions); (c)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(c) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：45，或与SEQ ID NO：45相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:45, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:45) substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：46，或与SEQ ID NO：46相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:46, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:46) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions); (d)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(d) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：47，或与SEQ ID NO：47相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:47, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:47 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：48，或与SEQ ID NO：48相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:48, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:48) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：49，或与SEQ ID NO：49相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:49, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:49 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：50，或与SEQ ID NO：50相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:50, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:50 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：44，或与SEQ ID NO：44相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:44, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:44 substitutions, deletions or additions); 其中，组(b)、(c)和(d)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义；wherein said heavy chain variable region (VH) CDRs and said light chain variable region (VL) CDRs in groups (b), (c) and (d) are defined by the Kabat numbering system; 优选地，组(a)、(b)、(c)和(d)中(i)-(vi)任一项中所述的置换为保守置换。Preferably, the substitutions described in any of (i)-(vi) in groups (a), (b), (c) and (d) are conservative substitutions. 如权利要求1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包含：如SEQ ID NO：7、21、23、25或27所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO：8、22、24、26或28所示的轻链可变区(VL)中含有的3个CDR；其中，所述重链可变区(VH)中含有的3个CDR和所述轻链可变区(VL)中含有的3个CDR通过IMGT或Kabat编号系统确定。The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region shown in SEQ ID NO: 7, 21, 23, 25 or 27 3 CDRs contained in (VH); and/or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 8, 22, 24, 26 or 28; wherein, the The three CDRs contained in the variable region of the heavy chain (VH) and the three CDRs contained in the variable region of the light chain (VL) are identified by the IMGT or Kabat numbering system. 如权利要求1或4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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 4, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(a) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：84，或与SEQ ID NO：84相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:84, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:84 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：85，或与SEQ ID NO：85相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:85, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:85 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：86，或与SEQ ID NO：86相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:86, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:86) substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：87，或与SEQ ID NO：87相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO: 87, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 87) substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：88，或与SEQ ID NO：88相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:88, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:88) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions); 其中，组(a)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义；wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs in group (a) are defined by the IMGT numbering system; (b)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(b) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：51，或与SEQ ID NO：51相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:51, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:51 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：52，或与SEQ ID NO：52相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:52, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:52 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：53，或与SEQ ID NO：53相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：54，或与SEQ ID NO：54相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:54, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:54 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：55，或与SEQ ID NO：55相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:55, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:55 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions); (c)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(c) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：51，或与SEQ ID NO：51相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:51, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:51 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：57，或与SEQ ID NO：57相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:57, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:57) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：53，或与SEQ ID NO：53相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：54，或与SEQ ID NO：54相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:54, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:54 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：55，或与SEQ ID NO：55相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:55, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:55 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions); (d)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(d) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：58，或与SEQ ID NO：58相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:58, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:58) substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：59，或与SEQ ID NO：59相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:59, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:59) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：53，或与SEQ ID NO：53相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:53, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:53 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：60，或与SEQ ID NO：60相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:60, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:60) substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：61，或与SEQ ID NO：61相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:61, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:61 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：56，或与SEQ ID NO：56相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:56, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:56) substitutions, deletions or additions); 其中，组(b)、(c)和(d)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义；wherein said heavy chain variable region (VH) CDRs and said light chain variable region (VL) CDRs in groups (b), (c) and (d) are defined by the Kabat numbering system; 优选地，组(a)、(b)、(c)和(d)中的(i)-(vi)任一项中所述的置换为保守置换。Preferably, the substitutions described in any of (i)-(vi) in groups (a), (b), (c) and (d) are conservative substitutions. 如权利要求1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包含：如SEQ ID NO：9、29、31、33或35所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO：10、30、32、34或36所示的轻链可变区(VL)中含有的3个CDR；其中，所述重链可变区(VH)中含有的3个CDR和所述轻链可变区(VL)中含有的3个CDR通过IMGT或Kabat编号系统确定。The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region shown in SEQ ID NO: 9, 29, 31, 33 or 35 3 CDRs contained in (VH); and/or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 10, 30, 32, 34 or 36; wherein, the The three CDRs contained in the variable region of the heavy chain (VH) and the three CDRs contained in the variable region of the light chain (VL) are identified by the IMGT or Kabat numbering system. 如权利要求1或6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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 6, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(a) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：89，或与SEQ ID NO：89相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:89, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:89) substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：90，或与SEQ ID NO：90相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:90, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:90 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：91，或与SEQ ID NO：91相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:91, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:91) substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：92，或与SEQ ID NO：92相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:92, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:92 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：93，或与SEQ ID NO：93相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:93, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:93) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions); 其中，组(a)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义；wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs in group (a) are defined by the IMGT numbering system; (b)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(b) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：62，或与SEQ ID NO：62相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:62, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:62 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：63，或与SEQ ID NO：63相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:63, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:63) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：64，或与SEQ ID NO：64相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：65，或与SEQ ID NO：65相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:65, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:65 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：66，或与SEQ ID NO：66相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:66, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:66) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions); (c)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(c) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：62，或与SEQ ID NO：62相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:62, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:62 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：68，或与SEQ ID NO：68相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO: 68, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 68) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：64，或与SEQ ID NO：64相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：69，或与SEQ ID NO：69相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:69, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:69 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：70，或与SEQ ID NO：70相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:70, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:70 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions); (d)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(d) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：71，或与SEQ ID NO：71相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:71, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:71 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：72，或与SEQ ID NO：72相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:72, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:72) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：64，或与SEQ ID NO：64相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:64, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:64 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：73，或与SEQ ID NO：73相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:73, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:73 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：74，或与SEQ ID NO：74相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:74, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:74) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：67，或与SEQ ID NO：67相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:67, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:67) substitutions, deletions or additions); 其中，组(b)、(c)和(d)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义；wherein said heavy chain variable region (VH) CDRs and said light chain variable region (VL) CDRs in groups (b), (c) and (d) are defined by the Kabat numbering system; 优选地，组(a)、(b)、(c)和(d)中(i)-(vi)任一项中所述的置换为保守置换。Preferably, the substitutions described in any of (i)-(vi) in groups (a), (b), (c) and (d) are conservative substitutions. 如权利要求1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包含：如SEQ ID NO：11或37所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO：12或38所示的轻链可变区(VL)中含有的3个CDR；其中，所述重链可变区(VH)中含有的3个CDR和所述轻链可变区(VL)中含有的3个CDR通过IMGT或Kabat编号系统确定。The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a variable region (VH) of a heavy chain represented by SEQ ID NO: 11 or 37 3 CDRs; and/or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 12 or 38; wherein, the 3 CDRs contained in the heavy chain variable region (VH) The three CDRs and the three CDRs contained in the light chain variable region (VL) are identified by the IMGT or Kabat numbering system. 如权利要求1或8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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 8, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(a) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：79，或与SEQ ID NO：79相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:79, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:79 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：80，或与SEQ ID NO：80相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:80, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:80) substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：81，或与SEQ ID NO：81相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO: 81, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO: 81) substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：94，或与SEQ ID NO：94相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:94, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:94) substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：83，或与SEQ ID NO：83相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:83, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:83 substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：77，或与SEQ ID NO：77相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions); 其中，组(a)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由IMGT编号系统定义；wherein the heavy chain variable region (VH) CDRs and the light chain variable region (VL) CDRs in group (a) are defined by the IMGT numbering system; (b)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(b) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸 置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：75，或与SEQ ID NO：75相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:75, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:75 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：76，或与SEQ ID NO：76相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:76, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:76) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：77，或与SEQ ID NO：77相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions); (c)包含下述3个重链可变区(VH)CDR和3个轻链可变区(VL)CDR：(c) comprises the following 3 heavy chain variable region (VH) CDRs and 3 light chain variable region (VL) CDRs: (i)CDR-H1，其由下述序列组成：SEQ ID NO：39，或与SEQ ID NO：39相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(i) CDR-H1 consisting of the following sequence: SEQ ID NO:39, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:39 substitutions, deletions or additions), (ii)CDR-H2，其由下述序列组成：SEQ ID NO：40，或与SEQ ID NO：40相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(ii) CDR-H2 consisting of the following sequence: SEQ ID NO:40, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:40 substitutions, deletions or additions), (iii)CDR-H3，其由下述序列组成：SEQ ID NO：41，或与SEQ ID NO：41相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(iii) CDR-H3 consisting of the following sequence: SEQ ID NO:41, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared to SEQ ID NO:41 substitutions, deletions or additions); (iv)CDR-L1，其由下述序列组成：SEQ ID NO：75，或与SEQ ID NO：75相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，(iv) CDR-L1 consisting of the following sequence: SEQ ID NO:75, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3) compared to SEQ ID NO:75 substitutions, deletions or additions), (v)CDR-L2，其由下述序列组成：SEQ ID NO：78，或与SEQ ID NO：78相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列，和(v) CDR-L2 consisting of the following sequence: SEQ ID NO:78, or having one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 compared to SEQ ID NO:78) substitutions, deletions or additions), and (vi)CDR-L3，其由下述序列组成：SEQ ID NO：77，或与SEQ ID NO：77相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个或3个置换、缺失或添加)的序列；(vi) CDR-L3 consisting of the following sequence: SEQ ID NO:77, or having one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 compared to SEQ ID NO:77) substitutions, deletions or additions); 其中，组(b)和(c)中所述重链可变区(VH)CDR和所述轻链可变区(VL)CDR由Kabat编号系统定义；wherein said heavy chain variable region (VH) CDRs and said light chain variable region (VL) CDRs in groups (b) and (c) are defined by the Kabat numbering system; 优选地，组(a)、(b)和(c)中(i)-(vi)任一项中所述的置换为保守置换。Preferably, the substitutions described in any of (i)-(vi) in groups (a), (b) and (c) are conservative substitutions. 如权利要求1-9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包含来源于哺乳动物免疫球蛋白的构架区(FR)；优选地，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的构架区或其变体；其中，所述变体与其所源自的序列相比具有至多20个氨基酸保守置换(例如至多15个、至多10个、或至多5个保守置换；例如1个，2个，3个，4个或5个保守置换)。The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from mammalian immunoglobulin; preferably, the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from a mammalian immunoglobulin; The antibody or antigen-binding fragment thereof comprises a framework region derived from a human immunoglobulin or a variant thereof; wherein the variant has up to 20 amino acid conservative substitutions (e.g. up to 15, up to 10, or up to 5 conservative substitutions; eg, 1, 2, 3, 4 or 5 conservative substitutions). 如权利要求1或10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包含：The antibody or antigen-binding fragment thereof of claim 1 or 10, wherein the antibody or antigen-binding fragment thereof comprises: (a)重链可变区(VH)，其包含选自下列的氨基酸序列：(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i)SEQ ID NOs：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35和37任一项所示的序列；(i) the sequence set forth in any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37; (ii)与SEQ ID NOs：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35和37任一项所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) compared to the sequence set forth in any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37 A sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions); or (iii)与SEQ ID NOs：5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35和37任一项 所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least a 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequences of sequence identity; 和/或，and / or, (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv)SEQ ID NOs：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的序列；(iv) the sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38; (v)与SEQ ID NOs：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) compared to the sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 A sequence having one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions); or (vi)与SEQ ID NOs：6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least a 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequences of sequence identity; 优选地，(ii)或(v)中所述的置换是保守置换。Preferably, the substitutions described in (ii) or (v) are conservative substitutions. 如权利要求1或11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包含选自如SEQ ID NO：5、13、15、17和19所示的重链可变区(VH)；和/或，选自如SEQ ID NO：6、14、16、18和20所示的轻链可变区。The antibody or antigen-binding fragment thereof of claim 1 or 11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain selectable from the group consisting of SEQ ID NOs: 5, 13, 15, 17 and 19. variable region (VH); and/or, selected from the light chain variable regions as set forth in SEQ ID NOs: 6, 14, 16, 18 and 20. 如权利要求1或12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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 12, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)其重链可变区(VH)包含选自下列的氨基酸序列：(a) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：5所示的序列；(i) the sequence shown in SEQ ID NO: 5; (ii)与SEQ ID NO：5所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 5; or (iii)与SEQ ID NO：5所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：6所示的序列；(iv) the sequence shown in SEQ ID NO: 6; (v)与SEQ ID NO：6所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 6 ;or (vi)与SEQ ID NO：6所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (b)其重链可变区(VH)包含选自下列的氨基酸序列：(b) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：13所示的序列；(i) the sequence shown in SEQ ID NO: 13; (ii)与SEQ ID NO：13所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 13 ;or (iii)与SEQ ID NO：13所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：14所示的序列；(iv) the sequence shown in SEQ ID NO: 14; (v)与SEQ ID NO：14所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 14 ;or (vi)与SEQ ID NO：14所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (c)其重链可变区(VH)包含选自下列的氨基酸序列：(c) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：15所示的序列；(i) the sequence shown in SEQ ID NO: 15; (ii)与SEQ ID NO：15所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 15 ;or (iii)与SEQ ID NO：15所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：16所示的序列；(iv) the sequence shown in SEQ ID NO: 16; (v)与SEQ ID NO：16所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 16 ;or (vi)与SEQ ID NO：16所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (d)其重链可变区(VH)包含选自下列的氨基酸序列：(d) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：17所示的序列；(i) the sequence shown in SEQ ID NO: 17; (ii)与SEQ ID NO：17所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 17 ;or (iii)与SEQ ID NO：17所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：18所示的序列；(iv) the sequence shown in SEQ ID NO: 18; (v)与SEQ ID NO：18所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 18 ;or (vi)与SEQ ID NO：18所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (e)其重链可变区(VH)包含选自下列的氨基酸序列：(e) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：19所示的序列；(i) the sequence shown in SEQ ID NO: 19; (ii)与SEQ ID NO：19所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 19 ;or (iii)与SEQ ID NO：19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)，包含选自下列的氨基酸序列：and, its light chain variable region (VL), comprising an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：20所示的序列；(iv) the sequence shown in SEQ ID NO: 20; (v)与SEQ ID NO：20所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 20 ;or (vi)与SEQ ID NO：20所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 优选地，组(a)-(e)中(ii)或(v)中所述的置换是保守置换。Preferably, the substitutions described in (ii) or (v) in groups (a)-(e) are conservative substitutions. 如权利要求1或11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包含选自如SEQ ID NO：7、21、23、25和27所示的重链可变区(VH)；和/或，选自如SEQ ID NO：8、22、24、26和28所示的轻链可变区。The antibody or antigen-binding fragment thereof of claim 1 or 11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain selectable from the group consisting of SEQ ID NOs: 7, 21, 23, 25 and 27. The variable region (VH); and/or, selected from the light chain variable regions as set forth in SEQ ID NOs: 8, 22, 24, 26 and 28. 如权利要求1或14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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 14, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)其重链可变区(VH)包含选自下列的氨基酸序列：(a) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：7所示的序列；(i) the sequence shown in SEQ ID NO: 7; (ii)与SEQ ID NO：7所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 7 ;or (iii)与SEQ ID NO：7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：8所示的序列；(iv) the sequence shown in SEQ ID NO: 8; (v)与SEQ ID NO：8所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 8 ;or (vi)与SEQ ID NO：8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (b)其重链可变区(VH)包含选自下列的氨基酸序列：(b) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：21所示的序列；(i) the sequence shown in SEQ ID NO: 21; (ii)与SEQ ID NO：21所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 21 ;or (iii)与SEQ ID NO：21所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：22所示的序列；(iv) the sequence shown in SEQ ID NO: 22; (v)与SEQ ID NO：22所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 22 ;or (vi)与SEQ ID NO：22所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (c)其重链可变区(VH)包含选自下列的氨基酸序列：(c) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：23所示的序列；(i) the sequence shown in SEQ ID NO: 23; (ii)与SEQ ID NO：23所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 23 ;or (iii)与SEQ ID NO：23所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：24所示的序列；(iv) the sequence shown in SEQ ID NO: 24; (v)与SEQ ID NO：24所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 24 ;or (vi)与SEQ ID NO：24所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (d)其重链可变区(VH)包含选自下列的氨基酸序列：(d) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：25所示的序列；(i) the sequence shown in SEQ ID NO: 25; (ii)与SEQ ID NO：25所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 25 ;or (iii)与SEQ ID NO：25所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：26所示的序列；(iv) the sequence shown in SEQ ID NO: 26; (v)与SEQ ID NO：26所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 26 ;or (vi)与SEQ ID NO：26所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (e)其重链可变区(VH)包含选自下列的氨基酸序列：(e) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：27所示的序列；(i) the sequence shown in SEQ ID NO: 27; (ii)与SEQ ID NO：27所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 27 ;or (iii)与SEQ ID NO：27所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：28所示的序列；(iv) the sequence shown in SEQ ID NO: 28; (v)与SEQ ID NO：28所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 28 ;or (vi)与SEQ ID NO：28所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 优选地，组(a)-(e)中(ii)或(v)中所述的置换是保守置换。Preferably, the substitutions described in (ii) or (v) in groups (a)-(e) are conservative substitutions. 如权利要求1或11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包含选自如SEQ ID NO：9、29、31、33和35所示的重链可变区(VH)；和或，选自如SEQ ID NO：10、30、32、34和36所示的轻链可变区。The antibody or antigen-binding fragment thereof of claim 1 or 11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain selectable from the group consisting of SEQ ID NOs: 9, 29, 31, 33 and 35. variable region (VH); and or, selected from the light chain variable regions as set forth in SEQ ID NOs: 10, 30, 32, 34 and 36. 如权利要求1或16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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 16, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)其重链可变区(VH)包含选自下列的氨基酸序列：(a) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：9所示的序列；(i) the sequence shown in SEQ ID NO: 9; (ii)与SEQ ID NO：9所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 9; or (iii)与SEQ ID NO：9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：10所示的序列；(iv) the sequence shown in SEQ ID NO: 10; (v)与SEQ ID NO：10所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 10 ;or (vi)与SEQ ID NO：10所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (b)其重链可变区(VH)包含选自下列的氨基酸序列：(b) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：29所示的序列；(i) the sequence shown in SEQ ID NO: 29; (ii)与SEQ ID NO：29所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 29; or (iii)与SEQ ID NO：29所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：30所示的序列；(iv) the sequence shown in SEQ ID NO: 30; (v)与SEQ ID NO：30所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 30 ;or (vi)与SEQ ID NO：30所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (c)其重链可变区(VH)包含选自下列的氨基酸序列：(c) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：31所示的序列；(i) the sequence shown in SEQ ID NO: 31; (ii)与SEQ ID NO：31所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 31; or (iii)与SEQ ID NO：31所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：32所示的序列；(iv) the sequence shown in SEQ ID NO: 32; (v)与SEQ ID NO：32所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 32 ;or (vi)与SEQ ID NO：32所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (d)其重链可变区(VH)包含选自下列的氨基酸序列：(d) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：33所示的序列；(i) the sequence shown in SEQ ID NO: 33; (ii)与SEQ ID NO：33所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 33; or (iii)与SEQ ID NO：33所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：34所示的序列；(iv) the sequence shown in SEQ ID NO: 34; (v)与SEQ ID NO：34所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 34 ;or (vi)与SEQ ID NO：34所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (e)其重链可变区(VH)包含选自下列的氨基酸序列：(e) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：35所示的序列；(i) the sequence shown in SEQ ID NO: 35; (ii)与SEQ ID NO：35所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 35; or (iii)与SEQ ID NO：35所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：36所示的序列；(iv) the sequence shown in SEQ ID NO: 36; (v)与SEQ ID NO：36所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 36 ;or (vi)与SEQ ID NO：36所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 优选地，组(a)-(e)中(ii)或(v)中所述的置换是保守置换。Preferably, the substitutions described in (ii) or (v) in groups (a)-(e) are conservative substitutions. 如权利要求1或11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包含选自如SEQ ID NO：11和37所示的重链可变区(VH)；和/或，选自如SEQ ID NO：12和38所示的轻链可变区。The antibody or antigen-binding fragment thereof of claim 1 or 11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 11 and 37; and/or, selected from the light chain variable regions as set forth in SEQ ID NOs: 12 and 38. 如权利要求1或18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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 18, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)其重链可变区(VH)包含选自下列的氨基酸序列：(a) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：11所示的序列；(i) the sequence shown in SEQ ID NO: 11; (ii)与SEQ ID NO：11所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 11; or (iii)与SEQ ID NO：11所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：12所示的序列；(iv) the sequence shown in SEQ ID NO: 12; (v)与SEQ ID NO：12所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 12 ;or (vi)与SEQ ID NO：12所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (b)其重链可变区(VH)包含选自下列的氨基酸序列：(b) its heavy chain variable region (VH) comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：37所示的序列；(i) the sequence shown in SEQ ID NO: 37; (ii)与SEQ ID NO：37所示的序列相比具有一个或几个置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 37; or (iii)与SEQ ID NO：37所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链可变区(VL)包含选自下列的氨基酸序列：and, its light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：38所示的序列；(iv) the sequence shown in SEQ ID NO: 38; (v)与SEQ ID NO：38所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 38 ;or (vi)与SEQ ID NO：38所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 优选地，组(a)和(b)中(ii)或(v)中所述的置换是保守置换。Preferably, the substitutions described in (ii) or (v) in groups (a) and (b) are conservative substitutions. 如权利要求1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包含来源于哺乳动物免疫球蛋白的恒定区序列或其变体；优选地，所述抗体或其抗原结合片段来源于人免疫球蛋白的恒定区序列或其变体；更优选地，所述抗体或其抗原结合片段包含选自例如IgG1、IgG2、IgG3和IgG4的重链恒定区；更特别地选自人IgG1、IgG2或IgG4的重链恒定区。The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a constant region sequence derived from mammalian immunoglobulin or a variant thereof; preferably, the antibody or The antigen-binding fragment thereof is derived from a constant region sequence of a human immunoglobulin or a variant thereof; more preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from, for example, IgGl, IgG2, IgG3 and IgG4; more particularly is selected from the heavy chain constant regions of human IgG1, IgG2 or IgG4. 如权利要求20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包含人免疫球蛋白的重链恒定区变体；所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加；优选地，所述变体具有改变的一个或更多个特性：例如，Fc受体结合、效应细胞功能；更优选地，所述人IgG重链恒定区变体选自：The antibody or antigen-binding fragment thereof of claim 20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region variant of a human immunoglobulin; the variant is identical to the sequence from which it is derived than having one or more amino acid substitutions, deletions or additions; preferably, the variant has altered one or more properties: eg, Fc receptor binding, effector cell function; more preferably, the human IgG Heavy chain constant region variants are selected from: (i)含有Leu234Val和Leu235Ala突变的人IgG1的重链恒定区；(i) the heavy chain constant region of human IgG1 containing Leu234Val and Leu235Ala mutations; (ii)含有Asn297Ala突变的人IgG1的重链恒定区；(ii) the heavy chain constant region of human IgG1 containing the Asn297Ala mutation; (iii)含有Thr250Gln、Asn297Ala和Met428Leu突变的人IgG1的重链恒定区；(iii) the heavy chain constant region of human IgG1 containing Thr250Gln, Asn297Ala and Met428Leu mutations; (iv)含有Asp265Ala和Pro329Ala突变的人IgG1的重链恒定区；(iv) the heavy chain constant region of human IgG1 containing Asp265Ala and Pro329Ala mutations; (v)含有Pro331Ser突变的人IgG2的重链恒定区；(v) the heavy chain constant region of human IgG2 containing the Pro331Ser mutation; (vi)含有Ser228Pro突变的人IgG4的重链恒定区；(vi) the heavy chain constant region of human IgG4 containing the Ser228Pro mutation; (vii)含有Ser228Pro和Leu235Glu突变的人IgG4的重链恒定区。(vii) Heavy chain constant region of human IgG4 containing Ser228Pro and Leu235Glu mutations. 最优选地，所述抗体重链恒定区的氨基酸序列选自SEQ ID NOs：96、97、98、99、100、101、102、103、104和105。Most preferably, the amino acid sequence of the heavy chain constant region of the antibody is selected from the group consisting of SEQ ID NOs: 96, 97, 98, 99, 100, 101, 102, 103, 104 and 105. 如权利要求1或20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选自下组：The antibody or antigen-binding fragment thereof of claim 1 or 20, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: (a)其重链包含选自下列的氨基酸序列：(a) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：107所示的序列；(i) the sequence shown in SEQ ID NO: 107; (ii)与SEQ ID NO：107所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 107 ;or (iii)与SEQ ID NO：107所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：109所示的序列；(iv) the sequence shown in SEQ ID NO: 109; (v)与SEQ ID NO：109所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 109 ;or (vi)与SEQ ID NO：109所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (b)其重链包含选自下列的氨基酸序列：(b) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：111所示的序列；(i) the sequence shown in SEQ ID NO: 111; (ii)与SEQ ID NO：111所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 111 ;or (iii)与SEQ ID NO：111所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：113所示的序列；(iv) the sequence shown in SEQ ID NO: 113; (v)与SEQ ID NO：113所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 113 ;or (vi)与SEQ ID NO：113所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (c)其重链包含选自下列的氨基酸序列：(c) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：115所示的序列；(i) the sequence shown in SEQ ID NO: 115; (ii)与SEQ ID NO：115所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 115 ;or (iii)与SEQ ID NO：115所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：117所示的序列；(iv) the sequence shown in SEQ ID NO: 117; (v)与SEQ ID NO：117所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 117 ;or (vi)与SEQ ID NO：117所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (d)其重链包含选自下列的氨基酸序列：(d) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：119所示的序列；(i) the sequence shown in SEQ ID NO: 119; (ii)与SEQ ID NO：119所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 119 ;or (iii)与SEQ ID NO：119所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：121所示的序列；(iv) the sequence shown in SEQ ID NO: 121; (v)与SEQ ID NO：121所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 121 ;or (vi)与SEQ ID NO：121所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (e)其重链包含选自下列的氨基酸序列：(e) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：123所示的序列；(i) the sequence shown in SEQ ID NO: 123; (ii)与SEQ ID NO：123所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 123 ;or (iii)与SEQ ID NO：123所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：125所示的序列；(iv) the sequence shown in SEQ ID NO: 125; (v)与SEQ ID NO：125所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 125 ;or (vi)与SEQ ID NO：125所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (f)其重链包含选自下列的氨基酸序列：(f) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：127所示的序列；(i) the sequence shown in SEQ ID NO: 127; (ii)与SEQ ID NO：127所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 127 ;or (iii)与SEQ ID NO：127所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：129所示的序列；(iv) the sequence shown in SEQ ID NO: 129; (v)与SEQ ID NO：129所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 129 ;or (vi)与SEQ ID NO：129所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (g)其重链包含选自下列的氨基酸序列：(g) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：131所示的序列；(i) the sequence shown in SEQ ID NO: 131; (ii)与SEQ ID NO：131所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 131 ;or (iii)与SEQ ID NO：131所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：133所示的序列；(iv) the sequence shown in SEQ ID NO: 133; (v)与SEQ ID NO：133所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 133 ;or (vi)与SEQ ID NO：133所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; (h)其重链包含选自下列的氨基酸序列：(h) its heavy chain comprises an amino acid sequence selected from the group consisting of: (i)SEQ ID NO：135所示的序列；(i) the sequence shown in SEQ ID NO: 135; (ii)与SEQ ID NO：135所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(ii) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 135 ;or (iii)与SEQ ID NO：135所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(iii) at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 和，其轻链包含选自下列的氨基酸序列：and, its light chain comprises an amino acid sequence selected from the group consisting of: (iv)SEQ ID NO：137所示的序列；(iv) the sequence shown in SEQ ID NO: 137; (v)与SEQ ID NO：137所示的序列相比具有一个或几个氨基酸置换、缺失或添加(例如1个，2个，3个，4个或5个置换、缺失或添加)的序列；或(v) a sequence having one or several amino acid substitutions, deletions or additions (eg 1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 137 ;or (vi)与SEQ ID NO：137所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；(vi) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 94%, at least 95%, at least 96%, Sequences of at least 97%, at least 98%, at least 99%, or 100% sequence identity; 优选地，组(a)-(h)中(ii)或(v)中所述的置换是保守置换。Preferably, the substitutions described in (ii) or (v) in groups (a)-(h) are conservative substitutions. 如权利要求1-22任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体片段包括但不限于Fv、Fab、Fab′、Fab’-SH，F(ab’)2、双抗体、线性抗体、单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody fragment includes but is not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, Diabodies, linear antibodies, single chain antibody molecules (eg, scFvs); and multispecific antibodies formed from antibody fragments. 编码如权利要求1-23任一项所述抗体或其抗原结合片段的核酸；优选地，编码所述抗体重链的第一核酸包含如SEQ ID NO：106、110、114、118、122、126、130或134所示的序列或与其基本上相同的序列(例如与其至少大约85％、90％、95％、99％或更高度相似的序列或具有一个或更多个核苷酸取代(例如保守性取代))的序列，或与上述序列相差不超过3、6、15、30或45个核苷酸的序列)；和编码所述抗体轻链的第二核酸包含如SEQ ID NO：108、112、116、120、124、128、132或136所示的序列或与其基本上相同的序列(例如与其至少大约85％、90％、95％、99％或更高度相似的序列或具有一个或更多个核苷酸取代(例如保守性取代))的序列，或与上述序列相差不超过3、6、15、30或45个核苷酸的序列)。Nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-23; preferably, the first nucleic acid encoding the antibody heavy chain comprises as SEQ ID NO: 106, 110, 114, 118, 122, 126, 130 or 134, or a sequence substantially identical thereto (e.g. a sequence that is at least about 85%, 90%, 95%, 99% or more similar thereto or has one or more nucleotide substitutions ( For example, conservative substitutions))), or sequences that differ from the above sequences by no more than 3, 6, 15, 30, or 45 nucleotides); and the second nucleic acid encoding the antibody light chain comprises as SEQ ID NO: 108, 112, 116, 120, 124, 128, 132, or 136, or a sequence substantially identical thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more similar thereto or has A sequence of one or more nucleotide substitutions (eg, conservative substitutions), or a sequence that differs from the above sequence by no more than 3, 6, 15, 30, or 45 nucleotides). 包含如权利要求24所述核酸的载体。A vector comprising the nucleic acid of claim 24. 包含如权利要求25所述载体的宿主细胞；优选地，所述宿主细胞是真核细胞；更优选地，所述细胞是哺乳动物细胞；最优选地，所述宿主细胞为CHO细胞。A host cell comprising the vector of claim 25; preferably, the host cell is a eukaryotic cell; more preferably, the cell is a mammalian cell; most preferably, the host cell is a CHO cell. 制备如权利要求1-23任一项所述的抗体或其抗原结合片段的方法，其特征在于，所述方法包含在适于表达编码所述抗体或其抗原结合片段的核酸的条件下培养所述宿主细胞，以及分离所述抗体或其抗原结合片段。A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-23, characterized in that the method comprises culturing the plant under conditions suitable for expression of nucleic acid encoding the antibody or antigen-binding fragment thereof. the host cell, and the isolation of the antibody or antigen-binding fragment thereof. 一种药物组合物，其包含药学上可接受的载体、和/或赋形剂和/或稳定剂和至少一种如权利要求1-23任一项所述的抗体或其抗原结合片段。A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and/or excipient and/or stabilizer and at least one antibody or antigen-binding fragment thereof according to any one of claims 1-23. 如权利要求1-23任一项所述的抗体或其抗原结合片段在制备针对治疗对象中CD47介导的病症或疾病的药物中的用途；优选地，所述病症或疾病是血液癌症和实体肿瘤；其中，血液癌症选自：急性成淋巴细胞白血病(ALL)、急性髓性白血病(AML)、非霍奇金淋巴瘤(例如，弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、套细胞淋巴瘤、B成淋巴细胞白血病/淋巴瘤和伯基特淋巴瘤)、B-成淋巴细胞白血病/淋巴瘤；B细胞慢性淋巴细胞白血病/小淋巴细胞淋巴瘤、慢性淋巴细胞白血病(CLL)例如，转化的CLL、Richter综 合征、慢性髓细胞白血病(CML)、滤泡淋巴瘤、多发性骨髓瘤、骨髓纤维化、真性红细胞增多症、皮肤T细胞淋巴瘤、意义未明的单克隆丙种球蛋白病(MGUS)、骨髓增生异常综合征(MDS)、免疫母细胞性大细胞淋巴瘤、前体B-成淋巴细胞淋巴瘤和间变性大细胞淋巴瘤。在一些实施方案中，所述癌症优选自以下的血液癌症：多发性骨髓瘤、弥漫性大B细胞淋巴瘤、AML、CLL例如转化的CLL、Richter综合征或滤泡淋巴瘤；其中，实体瘤选自：肺癌(例如，非小细胞肺癌、小细胞肺癌)、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、膀胱癌、脊柱癌、脑癌、宫颈癌、子宫内膜癌、结肠/直肠癌、肛门癌、子宫内膜癌、食道癌、胆囊癌、胃肠道癌、皮肤癌、前列腺癌、垂体癌、胃癌、子宫癌、阴道癌和甲状腺癌。Use of the antibody or antigen-binding fragment thereof of any one of claims 1-23 in the preparation of a medicament for a CD47-mediated disorder or disease in a subject; preferably, the disorder or disease is hematological cancer and solid Tumor; wherein the blood cancer is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (eg, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, mantle cell Lymphoma, B-lymphoblastic leukemia/lymphoma and Burkitt's lymphoma), B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL) such as , transformed CLL, Richter syndrome, chronic myeloid leukemia (CML), follicular lymphoma, multiple myeloma, myelofibrosis, polycythemia vera, cutaneous T-cell lymphoma, monoclonal gamma globulin of undetermined significance disease (MGUS), myelodysplastic syndrome (MDS), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and anaplastic large cell lymphoma. In some embodiments, the cancer is preferably selected from the following hematological cancers: multiple myeloma, diffuse large B-cell lymphoma, AML, CLL such as transformed CLL, Richter syndrome or follicular lymphoma; wherein the solid tumor Selected from: lung cancer (eg, non-small cell lung cancer, small cell lung cancer), pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, spine cancer, brain cancer, cervical cancer, endometrial cancer , colon/rectal, anal, endometrial, esophageal, gallbladder, gastrointestinal, skin, prostate, pituitary, gastric, uterine, vaginal and thyroid cancers. 检测样品中CD47蛋白的方法，所述方法包括(a)将样品与如权利要求1-23任一项所述的抗体或其抗原结合片段接触；和(b)检测所述抗体或其片段和CD47蛋白间的复合物的形成。A method for detecting CD47 protein in a sample, the method comprising (a) contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-23; and (b) detecting the antibody or fragment thereof and Formation of complexes between CD47 proteins. 检测样品中CD47蛋白含量或水平的试剂盒，其包括如权利要求1-23任一项所述的抗体或其抗原结合片段和使用说明书。A kit for detecting the content or level of CD47 protein in a sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-23 and an instruction manual.
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