[go: up one dir, main page]

WO2022052846A1 - Anticorps anti-cd47 et son utilisation - Google Patents

Anticorps anti-cd47 et son utilisation Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
seq
deletions
additions
substitutions
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/115946
Other languages
English (en)
Chinese (zh)
Inventor
李强
武翠
翁仕强
周利
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ampsource Biopharma Shanghai Inc
Original Assignee
Ampsource Biopharma Shanghai Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ampsource Biopharma Shanghai Inc filed Critical Ampsource Biopharma Shanghai Inc
Publication of WO2022052846A1 publication Critical patent/WO2022052846A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hospice & Palliative Care (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un anticorps anti-CD47 et son utilisation dans la préparation de médicaments pour le traitement de diverses maladies (y compris des tumeurs et des maladies infectieuses). L'anticorps peut reconnaître et se lier de manière spécifique à CD47 avec une affinité élevée, et ne provoque pas une réaction d'agglutination significative des globules rouges.
PCT/CN2021/115946 2020-09-09 2021-09-01 Anticorps anti-cd47 et son utilisation Ceased WO2022052846A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010941893.5 2020-09-09
CN202010941893.5A CN114230662A (zh) 2020-09-09 2020-09-09 抗cd47抗体及其用途

Publications (1)

Publication Number Publication Date
WO2022052846A1 true WO2022052846A1 (fr) 2022-03-17

Family

ID=80632619

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/115946 Ceased WO2022052846A1 (fr) 2020-09-09 2021-09-01 Anticorps anti-cd47 et son utilisation

Country Status (2)

Country Link
CN (1) CN114230662A (fr)
WO (1) WO2022052846A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024141063A1 (fr) * 2022-12-30 2024-07-04 甘李药业股份有限公司 Anticorps anti-vrs, composition, préparation et utilisation associées

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108738313A (zh) * 2016-10-20 2018-11-02 爱迈博 新的cd47单克隆抗体及其应用
CN109265547A (zh) * 2018-07-23 2019-01-25 中国科学院微生物研究所 一种抗cd47抗体及其应用
CN109422811A (zh) * 2017-08-29 2019-03-05 信达生物制药(苏州)有限公司 抗cd47抗体及其用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108738313A (zh) * 2016-10-20 2018-11-02 爱迈博 新的cd47单克隆抗体及其应用
CN109422811A (zh) * 2017-08-29 2019-03-05 信达生物制药(苏州)有限公司 抗cd47抗体及其用途
CN110214154A (zh) * 2017-08-29 2019-09-06 信达生物制药(苏州)有限公司 抗cd47抗体及其用途
CN109265547A (zh) * 2018-07-23 2019-01-25 中国科学院微生物研究所 一种抗cd47抗体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIE LIU, WANG LIJUAN, ZHAO FEIFEI, TSENG SERENA, NARAYANAN CYNDHAVI, SHURA LEI, WILLINGHAM STEPHEN, HOWARD MAUREEN, PROHASKA SUSAN: "Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, vol. 10, no. 9, 21 September 2015 (2015-09-21), pages 1 - 23, XP055223677, DOI: 10.1371/journal.pone.0137345 *
YANG QIN, LIU XIAO-BO: "Research Status of CD47-SIRPα Blockers in Solid and Blood Cancers", PROGRESS IN VETERINARY MEDICINE, vol. 41, no. 2, 20 February 2020 (2020-02-20), pages 104 - 108, XP055911137, ISSN: 1007-5038, DOI: 10.16437/j.cnki.1007-5038.2020.02.020 *

Also Published As

Publication number Publication date
CN114230662A (zh) 2022-03-25

Similar Documents

Publication Publication Date Title
JP6764474B2 (ja) 抗ｔｉｇｉｔ抗体及び使用方法
AU2013371957B2 (en) Antibodies that bind to TL1A and their uses
CA3170025A1 (fr) Proteine de liaison a pvgrig et ses utilisations medicales
KR20210013708A (ko) 항-ox40 항체 및 사용 방법
JP2024527606A (ja) 抗ｃ－ｃモチーフケモカイン受容体８（ｃｃｒ８）抗体及び使用方法
US10640564B2 (en) Antibodies that bind to CCR6 and their uses
CN115605507A (zh) 抗白介素-33抗体及其用途
CN114341181A (zh) 抗ms4a4a抗体及其使用方法
CN115151563A (zh) 使用抗ox40抗体与抗pd1或抗pdl1抗体组合治疗癌症的方法
KR20250044359A (ko) 항-ccr8 항체 및 사용 방법
WO2022166846A1 (fr) Anticorps anti-tnfr2 et son utilisation
CN114728063B (zh) 用抗ox40抗体和多重激酶抑制剂治疗癌症
WO2022052846A1 (fr) Anticorps anti-cd47 et son utilisation
CN115768800A (zh) 抗苗勒管激素受体2抗体和使用方法
CN114729050A (zh) 使用抗OX40抗体与PI3激酶δ抑制剂组合治疗癌症的方法
TW202442679A (zh) 抗－ｆｇｆｒ２ｂ抗體及其用途
TWI869582B (zh) 抗ｃｄ４７抗體及其用途
KR20220103105A (ko) 항-tim3 항체와 병용하여 항-ox40 항체를 사용한 암 치료 방법
JP2025524657A (ja) 抗ｔｉｇｉｔ抗体を使用したがんの治療方法
WO2025067469A1 (fr) Anticorps monoclonal anti-bdca2, protéine de fusion ainsi composée, taci, procédé et utilisation associés
HK40074321B (zh) 使用抗ox40抗体与抗tim3抗体的组合治疗癌症的方法
HK40076999A (en) Treatment of cancer with anti-ox40 antibodies and multi-kinase inhibitors
HK40076664A (en) Methods of cancer treatment using anti-ox40 antibodies in combination with anti-pd1 or anti-pdl1 antibodies
HK40087352A (zh) 抗白介素-33抗体及其用途
HK40063237A (en) Anti-ms4a4a antibodies and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21865901

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21865901

Country of ref document: EP

Kind code of ref document: A1