WO2020043188A1 - 抗cd47抗体及其应用 - Google Patents

抗cd47抗体及其应用 Download PDF

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WO2020043188A1
WO2020043188A1 PCT/CN2019/103673 CN2019103673W WO2020043188A1 WO 2020043188 A1 WO2020043188 A1 WO 2020043188A1 CN 2019103673 W CN2019103673 W CN 2019103673W WO 2020043188 A1 WO2020043188 A1 WO 2020043188A1
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seq
amino acid
acid sequence
antibody
variable region
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PCT/CN2019/103673
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English (en)
French (fr)
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王勇
赵立文
宋其峰
朱永强
张亚楠
韩璐薇
金亮
吴文明
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南京圣和药业股份有限公司
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Priority to CN201980056045.1A priority Critical patent/CN112601762B/zh
Priority to EP19853645.0A priority patent/EP3845561A4/en
Priority to JP2021510171A priority patent/JP2021535743A/ja
Priority to US17/270,947 priority patent/US20220119520A1/en
Priority to CA3110620A priority patent/CA3110620C/en
Priority to KR1020217008708A priority patent/KR102587442B1/ko
Publication of WO2020043188A1 publication Critical patent/WO2020043188A1/zh
Priority to JP2023041313A priority patent/JP2023075294A/ja

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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

Definitions

  • the invention relates to the technical field of antibody drugs, in particular to an anti-CD47 antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing the anti-CD47 antibody or an antigen-binding fragment thereof, and applications thereof
  • CD47 protein also known as integrin-related protein (IAP) is a five-transmembrane glycoprotein of the IgG superfamily and is widely expressed in different tissue cells. CD47 can bind to ligands TSP-1 or SIRP ⁇ and regulate different cell functions, including cell migration, adhesion, apoptosis, axon extension, cytokine production, and T cell activation.
  • SIRP ⁇ is a transmembrane protein containing a typical immune receptor tyrosine inhibitory motif (ITIM), which is mainly expressed on the surface of myeloid hematopoietic cells, such as macrophages and dendritic cells.
  • ITIM immune receptor tyrosine inhibitory motif
  • CD47 The binding of CD47 to SIRP ⁇ leads to phosphorylation of ITIMs, thereby recruiting SHP-1 / SHP-2, thereby inhibiting the accumulation of myosin IIA in phagocytic synapses, and ultimately inhibiting the phagocytic function of phagocytic cells.
  • Tumor cells express CD47 molecules and interact with SIRP ⁇ on the surface of macrophages, which can significantly inhibit the phagocytic activity of macrophages and avoid being swallowed by macrophages.
  • SIRP ⁇ Interleukin-1
  • CD47 blocking antibodies are considered as one of the most promising tumor treatment options.
  • the effectiveness of human CD47-blocking monoclonal antibodies has been demonstrated in various preclinical models, such as lymphoma, bladder cancer, colon cancer, glioblastoma, breast cancer, acute lymphocytic leukemia, and acute myeloid leukemia. .
  • red blood cells and platelets also express CD47 molecules
  • these cells may lose the protection of the "don't eat me" signal and be swallowed by macrophages. Therefore, to avoid the side effects of anti-CD47 antibodies, such as platelet degradation, red blood cell agglutination, red blood cell depletion, anemia, etc. are also an important point to consider when using anti-CD47 antibodies.
  • an anti-CD47 antibody or an antigen-binding fragment thereof which has excellent antitumor activity and does not have significant erythrocyte agglutination activity.
  • An aspect of the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a complementarity determining region of a heavy chain variable region 1 (HCDR1), complementarity determining region 2 (HCDR2) of the heavy chain variable region, and / or complementarity determining region 3 (HCDR3) of the heavy chain variable region, said light chain variable region comprising the complementarity of the light chain variable region Determining region 1 (LCDR1), complementarity determining region 2 (LCDR2) of the light chain variable region, and / or complementarity determining region 3 (LCDR3) of the light chain variable region.
  • HCDR1 complementarity determining region 1
  • HCDR2 complementarity determining region 2
  • HCDR3 complementarity determining region 3
  • the invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 selected from the group consisting of:
  • the light chain variable region comprises LCDR1, LCDR2, and LCDR3 selected from the group consisting of:
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 as SEQ ID NO: 1, 2 and 3, or SEQ ID NO: 1, respectively.
  • the light chain variable regions of the CDRs having an amino acid sequence of 13, 13 and 14 having at least 85% sequence identity.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 as SEQ ID NO: 10, 2 and 11 or SEQ ID NO: 10,
  • the light chain variable regions of the CDRs having an amino acid sequence of 13, 13 and 14 having at least 85% sequence identity.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 as SEQ ID NO: 4, 5 and 6, or SEQ ID NO: 4,
  • the heavy chain variable regions of the CDRs whose amino acid sequences shown in 5 and 6 have at least 85% sequence identity, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 15, 16 and 17 or SEQ ID NO: 15
  • the amino acid sequences shown in Figures 16, 16 and 17 have at least 85% of the light chain variable regions of the CDRs with sequence identity.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 as SEQ ID NO: 7, 8, and 9, or SEQ ID NO: 7,
  • the amino acid sequences shown in 8 and 9 have at least 85% of the heavy chain variable regions of the CDRs with sequence identity
  • the LCDR1, LCDR2, and LCDR3 are SEQ ID NO: 18, 19, and 20 or SEQ ID NO:
  • the light chain variable regions of the CDRs having an amino acid sequence of 18, 19, and 20 having at least 85% sequence identity.
  • the anti-CD47 antibody or antigen-binding fragment thereof according to the invention is a monoclonal antibody or an antigen-binding fragment thereof.
  • the anti-CD47 antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, or a humanized antibody or an antigen-binding fragment thereof.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the variable region of the heavy chain is selected from:
  • (b2) an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in (b1) and having the same or similar function to the amino acid sequence shown in (b1);
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 21, SEQ ID NO: 21 is substituted, deleted, or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 21 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 21, and the amino acid sequence of the light chain variable region is SEQ ID NO: 24, SEQ ID NO: 24 An amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 24 or having at least 85% sequence identity with SEQ ID NO: 24 Amino acid sequence.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 22, SEQ ID NO: 22 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 22 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 22, and the amino acid sequence of the light chain variable region is SEQ ID NO: 25, SEQ ID NO: 25 An amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 25 or having at least 85% sequence identity with SEQ ID NO: 25 Amino acid sequence.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 23, SEQ ID NO: 23 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 23 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is SEQ ID NO: 26, SEQ ID NO: 26 An amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 26 or having at least 85% sequence identity with SEQ ID NO: 26 Amino acid sequence.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 27, SEQ ID NO: 27 is substituted, deleted, or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 27 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is SEQ ID NO: 24, SEQ ID NO: 24 An amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 24 or having at least 85% sequence identity with SEQ ID NO: 24 Amino acid sequence.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 23, SEQ ID NO: 23 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 23 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is SEQ ID NO: 28, SEQ ID NO: 28 An amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 28 or having at least 85% sequence identity with SEQ ID NO: 28 Amino acid sequence.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 21, SEQ ID NO: 21 is substituted, deleted, or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 21 or having at least 85% sequence identity with SEQ ID NO: 21 and the HCDR1, HCDR2, and HCDR3 are as SEQ ID NO: 1, 2 And the amino acid sequence shown in Figure 3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 24, SEQ ID NO: 24 is obtained by substitution, deletion, or addition of one or more amino acids and is identical to SEQ ID NO: 24 is the functionally identical amino acid sequence or has at least 85% sequence identity with SEQ ID NO: 24 and the LCDR1, LCDR2, and LCDR3 are the amino acid sequences shown in SEQ ID NO: 12, 13, and 14.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 22, SEQ ID NO: 22 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 22 or having at least 85% sequence identity with SEQ ID NO: 22 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 4, 5 And 6 and the amino acid sequence of the light chain variable region is SEQ ID NO: 25, SEQ ID NO: 25 is obtained by substitution, deletion, or addition of one or more amino acids and is identical to SEQ ID NO: 25 The functionally identical amino acid sequence or at least 85% sequence identity with SEQ ID NO: 25 and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 15, 16 and 17.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 23, SEQ ID NO: 23 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 23 or having at least 85% sequence identity with SEQ ID NO: 23 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 7, 8 And 9 and the amino acid sequence of the light chain variable region is SEQ ID NO: 26, SEQ ID NO: 26 is obtained by substitution, deletion, or addition of one or more amino acids and is identical to SEQ ID NO: 26 is the functionally identical amino acid sequence or has at least 85% sequence identity with SEQ ID NO: 26 and the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO: 18, 19, and 20.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 27, SEQ ID NO: 27 is substituted, deleted, or The amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 27 or having at least 85% sequence identity with SEQ ID NO: 27 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 1, 2 And 11 and the amino acid sequence of the light chain variable region is SEQ ID NO: 24, SEQ ID NO: 24 is obtained by substitution, deletion, or addition of one or more amino acids and is identical to SEQ ID NO: 24 is the functionally identical amino acid sequence or has at least 85% sequence identity with SEQ ID NO: 24 and the LCDR1, LCDR2, and LCDR3 are the amino acid sequences shown in SEQ ID NO: 12, 13, and 14.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 23, SEQ ID NO: 23 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 23 or having at least 85% sequence identity with SEQ ID NO: 23 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 7, 8 And 9 and the amino acid sequence of the light chain variable region is SEQ ID NO: 28, SEQ ID NO: 28 is obtained by substitution, deletion, or addition of one or more amino acids and is identical to SEQ ID NO: 28 is a functionally identical amino acid sequence or has at least 85% sequence identity with SEQ ID NO: 28 and the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO: 18, 19, and 20.
  • the anti-CD47 antibody according to the present invention is a murine antibody, which further contains a murine heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and a murine ⁇ chain Or a variant light chain constant region.
  • the anti-CD47 murine antibody according to the present invention further contains a heavy chain constant region of murine IgG1 or IgG2 or a variant thereof, and a light chain constant region of murine kappa chain or a variant thereof.
  • the invention provides an anti-CD47 humanized antibody or antigen-binding fragment thereof, wherein:
  • amino acid sequence of the variable region of the heavy chain is selected from:
  • (c2) an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in (c1) and having the same or similar function as the amino acid sequence shown in (c1);
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 29, and SEQ ID NO: 29 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 29 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 29, and the amino acid sequence of the light chain variable region SEQ ID NO: 32, SEQ ID NO: 32 amino acid sequence obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 32 or having at least 85% sequence identity with SEQ ID NO: 32 Sexual amino acid sequence.
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acid sequence of the light chain variable region SEQ ID NO: 33, SEQ ID NO: 33 amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 33 or having at least 85% sequence identity with SEQ ID NO: 33 Sexual amino acid sequence.
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 31, and SEQ ID NO: 31 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 31 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 31, and the amino acid sequence of the light chain variable region SEQ ID NO: 34, SEQ ID NO: 34 amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO: 34 or having at least 85% sequence identity with SEQ ID NO: 34 Sexual amino acid sequence.
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 29, and SEQ ID NO: 29 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 29 or having at least 85% sequence identity with SEQ ID NO: 29 and the HCDR1, HCDR2, and HCDR3 are as SEQ ID NO: 10,
  • the amino acid sequences shown in 2 and 11 and the amino acid sequence of the light chain variable region is SEQ ID NO: 32
  • SEQ ID NO: 32 is obtained by substitution, deletion or addition of one or more amino acids, and is identical to SEQ ID ID NO : 32 has the same functional amino acid sequence or has at least 85% sequence identity with SEQ ID NO: 32 and the LCDR1, LCDR2, and LCDR3 are the amino acid sequences shown in SEQ ID NO: 12, 13, and 14.
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85% sequence identity with SEQ ID NO: 30 and the HCDR1, HCDR2, and HCDR3 are as SEQ ID NO: 4,
  • the amino acid sequences shown in 5 and 6, and the amino acid sequence of the variable region of the light chain is SEQ ID NO: 33, SEQ ID NO: 33 is obtained by substitution, deletion or addition of one or more amino acids, and is the same as SEQ ID NO : 33
  • the amino acid sequence that is functionally identical or has at least 85% sequence identity with SEQ ID NO: 33 and the LCDR1, LCDR2, and LCDR3 are the amino acid sequences shown in SEQ ID NO: 15, 16, and 17.
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 31, and SEQ ID NO: 31 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 31 or having at least 85% sequence identity with SEQ ID NO: 31 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 7,
  • the amino acid sequences shown in 8 and 9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 34, SEQ ID NO: 34 is obtained by substitution, deletion, or addition of one or more amino acids and is identical to SEQ ID ID NO : 34 has the same functional amino acid sequence or has at least 85% sequence identity with SEQ ID NO: 34 and the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO: 18, 19, and 20.
  • the present invention provides an anti-CD47 humanized antibody or an antigen-binding fragment thereof, wherein the heavy chain comprises a heavy chain constant region of human-derived IgG1, IgG2, IgG3, IgG4, or a variant thereof.
  • the light chain comprises a light chain constant region of a human-derived kappa, lambda chain, or a variant thereof.
  • the mouse-derived anti-CD47 antibody or antigen-binding fragment thereof may further include a light chain constant region of a mouse-derived ⁇ , ⁇ chain or a variant thereof, and / or further include a mouse-derived region.
  • the antibody light chain of the anti-CD47 chimeric antibody or antigen-binding fragment thereof further comprises a light chain constant region of a mouse-derived ⁇ , ⁇ chain, or a mutant sequence thereof.
  • the antibody heavy chain of the anti-CD47 chimeric antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of a mouse-derived IgG1, IgG2, IgG3, gG4 or a mutant sequence thereof, preferably a human IgG1 or IgG2 heavy chain constant region.
  • use of an IgG4 constant region that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutations.
  • the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and human ⁇ , ⁇ chains Or a variant light chain constant region.
  • the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a light chain of human ⁇ chain or a variant thereof. Chain constant region.
  • the present invention provides an anti-CD47 antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or F (ab) 2.
  • Another aspect of the present invention provides an isolated nucleic acid encoding an anti-CD47 antibody or an antigen-binding fragment thereof according to the present invention.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO: 35 or SEQ ID NO: 36 or SEQ ID NO: 37 ;
  • the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38 or SEQ ID NO: 39 or SEQ ID NO: 40.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 29 is shown in SEQ ID NO: 35; encoding the light chain variable region SEQ ID
  • the nucleotide sequence of NO: 32 is shown in SEQ ID NO: 38.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 30 is shown in SEQ ID NO: 36; encoding the light chain variable region SEQ ID
  • the nucleotide sequence of NO: 33 is shown in SEQ ID NO: 39.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID ID: 31 is shown in SEQ ID NO: 37; encoding the light chain variable region SEQ ID ID
  • the nucleotide sequence of NO: 34 is shown in SEQ ID NO: 40.
  • Another aspect of the present invention provides an expression vector expressing the anti-CD47 antibody or antigen-binding fragment thereof of the present invention.
  • An expression vector according to the invention comprises an isolated nucleic acid molecule of the invention.
  • Another aspect of the present invention provides a host cell transformed with an expression vector as described above.
  • the host cell according to the invention is selected from a prokaryotic cell and a eukaryotic cell.
  • the host cell is a bacterium, preferably E. coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method for preparing an anti-CD47 antibody or an antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention, and further comprises other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of a liposome.
  • the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent, or excipient to prepare a pharmaceutical preparation, which is suitable for oral or parenteral administration.
  • Methods of administration include, but are not limited to, the oral, intradermal, intramuscular, intraperitoneal, intravenous, brain, intraocular, intratracheal, subcutaneous, intranasal routes.
  • the formulations can be administered by any route, such as by infusion or bolus, by absorption through the epithelium or skin mucosa (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the formulations can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the field of pharmaceutical formulations.
  • Another aspect of the invention provides a method of inhibiting CD47 activity, the method comprising administering to an individual in need thereof an anti-CD47 antibody of the invention or an antigen-binding fragment thereof or a pharmaceutical composition of the invention.
  • Another aspect of the present invention provides the use of the anti-CD47 antibody or the antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for inhibiting the activity of CD47.
  • the drug that inhibits CD47 activity is used to treat leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, nerves Glioma and / or melanoma.
  • the present invention provides the use of the above-mentioned anti-CD47 antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of an antitumor medicament.
  • the tumor is selected from the group consisting of leukemia, lymphoma, and breast cancer. , Lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.
  • the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention has significant anti-tumor effects, can significantly inhibit tumor growth, and does not have significant erythrocyte agglutination activity.
  • the immunogenicity of humanized antibodies is greatly reduced, effectively eliminating human immunity.
  • the system's rejection response to exogenous monoclonal antibodies can be applied in the preparation of drugs for treating various tumor diseases, and has broad market prospects.
  • the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100 %. Sequence comparison and percent identity determination between two sequences can be performed by the BLASTN / BLASTP algorithm on the National Center for Biotechnology Institute website.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged at positions relative to each other in a three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs.”
  • CDRs complementarity determining regions
  • the "antigen-binding fragment” in the present invention refers to a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, and an scFv fragment that bind to human CD47, which have antigen-binding activity.
  • the Fv fragment contains the variable region of the heavy chain and light chain of the antibody, but has no constant region and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called single-chain antibodies or single-chain Fv (scFv).
  • the antibody in the present invention refers to an immunoglobulin molecule or an immunologically active portion thereof, that is, a molecule comprising an antigen binding site that specifically binds an antigen (immunoreactively reacts with it).
  • "Specific binding” refers to an antibody that reacts with one or more epitopes of an antigen without reacting with other polypeptides or binding other polypeptides with very low affinity (Kd> 10 -6 ).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab 'and F (ab') 2 fragments, Fv, scFv, and Fab expression libraries.
  • Monoclonal antibodies are antibodies obtained from a single cloned cell line, which is not limited to eukaryotic, prokaryotic, or phage cloned cell lines. Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as hybridoma technology, recombinant technology, phage display technology, and synthetic technologies such as CDR grafting or other existing technologies.
  • the "murine antibody” according to the present invention is a monoclonal antibody to human CD47 prepared according to the knowledge and skills in the art. Test subjects are injected with CD47 antigen during preparation, and hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.
  • the "chimeric antibody” is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody.
  • To establish a chimeric antibody first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody according to the need, The gene is linked to the human constant region gene to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the "humanized antibody” according to the present invention is also called a CDR-grafted antibody, and is an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework (FR).
  • FR human antibody variable region framework
  • Such variable region framework sequences can be obtained from public DNA databases or published references, such as from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN012441351.
  • FIG. 1 is a result of an anti-CD47 humanized antibody and monkey CD47 binding activity assay (ELISA).
  • Figure 2 is a result of an anti-CD47 humanized antibody and human CD47 binding activity assay (ELISA).
  • Figure 3 is a result of an anti-CD47 humanized antibody and cell surface CD47 binding activity assay (ELISA).
  • FIG. 4 is the result of the red blood cell agglutination experiment, in which RBC: a positive control; PBS: a blank control.
  • Figure 5 shows the results of FACS detection of anti-CD47 humanized antibody blocking activity.
  • Figure 6 is the anti-tumor test result of anti-CD47 humanized antibody Hu34-39-PE human gastric cancer NUGC-4 transplanted tumor model.
  • FIG. 7 shows the antitumor test results of the anti-CD47 humanized antibody Hu26T-31-PE human gastric cancer NUGC-4 xenograft model.
  • a gene fragment encoding the full length of the CD47 protein was synthesized, the amino acid sequence design was shown in SEQ ID NO: 41, and then cloned into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid pTargeT-CD47.
  • the extracellular region of human CD47 protein was fused with the hIgG1-Fc or his tag amino acid sequence.
  • the amino acid sequence design is shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.
  • the tagged CD47 protein extracellular domain gene fragments CD47-hFc and CD47-his were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmid pHR-CD47- hFc, pHR-CD47-his.
  • the amino acid sequence of the extracellular region of human CD47 protein and the amino acid sequence of mIgG1-Fc were fused.
  • the amino acid sequence design is shown in SEQ ID NO: 44. After codon optimization of this amino acid sequence, a complete expression plasmid pcDNA3.1 (+)-TPA-CD47-mIgG1-Fc was synthesized.
  • SIRP ⁇ is shown in SEQ ID NO: 45, and the complete expression plasmid pcDNA3.1 (+)-SIRP ⁇ -myc-His was synthesized after codon optimization of the sequence.
  • AB06.12-4P Using the antibody AB6.12-IgG4P (herein abbreviated as AB06.12-4P) disclosed in patent application WO2013 / 119714 as a positive control antibody, the amino acid sequence of AB06.12-4P is as follows:
  • Codon artificial optimization was performed on the amino acid sequence corresponding to the above antibody sequence to obtain the heavy and light chain expression plasmids pcDNA3.1 (+)-SHC025-hG4, pcDNA3.1 (+)-of the positive control antibody AB06.12-4P. SHC025-hk.
  • the heavy chain gene fragment was cloned into the eukaryotic expression plasmid pHR containing the constant region of the IgG4 light chain to obtain the heavy chain eukaryotic expression plasmid pHR-SHC025-hG4-4PE of AB06.12-4P.
  • the light chain expression plasmid was pcDNA3.1 (+)-SHC025-hk.
  • the eukaryotic expression plasmid pTargeT-CD47 was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) under a square pulse of 160V voltage and 15msec, and placed in an incubator at 37 ° C and 5% CO 2 ⁇ culturing. After 24 hours, the medium was cultured under pressure with 500 ug / ml G418. After 16 days, FACS was used to detect the pool positive rate. The cells after electroporation were plated (1x10 6 cells / ml cell density, 100ul / well), and the cells were incubated with PE mouse anti-human CD47 antibody (BD, 556046).
  • BD PE mouse anti-human CD47 antibody
  • the cytometer (BD, FACSJazz) reads the mean value at a wavelength of 585 nm, and uses GraphPad for data analysis.
  • the positive cell line was subcloned, and a cloned CHO-K1 cell line was selected.
  • the cell line expressed CD47 molecule at a high level, and was named CHO-K1-E5.
  • the PEI and a mixed solution of FreeStyle 293 Expression Medium was added to the plasmid, mixed, and then added to the cell culture, set 37 °C, 8% CO 2, humidified CO 2 incubator cultured.
  • Cells were fed on the first and third day after cell transfection, and 2.5 ml of glutamine (200 mM mother liquor concentration) and 5 ml of glucose (180 g / L mother liquor concentration) were added to each bottle.
  • glutamine 200 mM mother liquor concentration
  • glucose 180 g / L mother liquor concentration
  • AKTA (GE, AKTA Pure-150) was used for purification according to the properties of the protein using different affinity chromatography columns (see Table 1 for the affinity chromatography columns for different proteins).
  • the specific purification steps are as follows:
  • Loading Load the cell expression supernatant with a retention time of 5 min;
  • Rinse and store Rinse with purified water for 10 min, then 2 CV with 20% ethanol.
  • mice of the SJL strain were immunized with the anti-CD47 antigen protein with different tags and adjuvant co-immunization.
  • the first antigen was immunized with 50ug of antigen and the later immunized with 25ug of antigen.
  • the immune adjuvant can be Quick Antibody-Mouse5W (Beijing Boaolong Immunology Technology Co., Ltd.) or Titer Max (Sigma) and CpG (Jingsui Biotechnology Limited Synthesis) / Alum (thermo) adjuvant interval.
  • CD47 antigen protein samples of different labels are added dropwise to the adjuvant solution, and vortexed while adding dropwise to mix thoroughly.
  • the adjuvant dosage is performed according to the instructions. SJL mice were immunized after mixing to form a milky oil-in-water mixture.
  • CCRF-CEM human acute lymphoblastic leukemia cells
  • CHO-K1-E5 human acute lymphoblastic leukemia cells
  • Example 1 The human acute lymphoblastic leukemia cells (CCRF-CEM) and the CHO-K1-E5 positive single cells obtained in Example 1 were treated with trypsin digestion, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in PBS and sampled Count with a cell counter.
  • mice Immunized mice were immersed in 75% alcohol after sacrifice. The spleen was dissected out, ground with a grinding rod, and filtered through a cell sieve to prepare a single cell suspension. The spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Add 2 mL of erythrocyte lysate, lyse erythrocytes for 2 min at room temperature, add PBS to 20 mL, centrifuge at 1500 rpm for 7 min, discard the supernatant, resuspend and count for live cells.
  • the cell suspension was fused in a 9 mL electrofusion fusion tank. After fusion, the cell suspension was transferred to 15 mL of RPMI 1640 complete medium containing 20% FBS, and left at room temperature for 20 min. The fused cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, and 20% FBS. Add 100 ⁇ l / well of the cell suspension to several 96-well cell culture plates, ensure that the amount of cells per well is about 4 ⁇ 10 4 cells / well, and culture in a 37 ° C cell incubator. After 5 days, 100 ⁇ L / well of RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was added.
  • the cell supernatant was taken, and the hybridoma supernatant that could bind to the CD47-his protein or the cell surface CD47 was selected by ELISA, and the antibodies against CD47 other than hFc and mFc were selected using CD47-his.
  • the ability of the hybridoma supernatant to block CD47-SIRP ⁇ interaction was then analyzed by ELISA.
  • the positive cell lines were subcloned by limiting dilution. After one week of culture, the binding activity of the subclone supernatant to CD47 molecule and the activity of blocking the CD47-SIRP ⁇ interaction were detected by ELISA. Three types of double positive cell lines were obtained, which were labeled as SHC025-26, SHC025-34, SHC025-58.
  • the monoclonal antibody mother clones SHC025-26, SHC025-34, and SHC025-58 were determined, and they were expanded and cultured.
  • the culture conditions are 1640 medium containing 10% fetal bovine serum, 1x NAEE, 1x sodium pyruvate, and 1% penicillin double antibody.
  • the confluency of the cells is greater than> 80%, the cells are passaged and expanded and cultured.
  • the supernatant was collected to about 50 ml, and the antibody was purified. The obtained antibody was confirmed to be of good purity by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured. An appropriate amount of cells were extracted according to the instructions of RNeasy Plus Mini Kit (Qiagen, 74134) kit, and the reverse transcription was performed using PrimeScript 1st and cDNA Synthesis Kit (Takara, 6110A). The kit synthesizes the first strand of cDNA.
  • Design specific primers based on the mouse antibody subtype variable region (the 5 'end contains a homology arm sequence for homologous recombination with the eukaryotic expression vector), and use the cDNA as a template for PCR amplification of antibody variable region genes
  • design primers References: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester etal. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35–55; 2. Simon Koren Miha Colja Venturini etal.
  • Antibody variable-region sequencing as a method for hybridoma cell-line authentication, 2008, 78: 1071–1078). DNA sequencing was performed to obtain the sequence. The sequencing results are shown in Table 4.
  • the purified mouse antibody light chain and heavy chain variable region gene fragments were co-transformed with linearized eukaryotic expression plasmids containing human antibody light or heavy chain constant regions, respectively, after purification (see the purification procedure in Example 1).
  • For competent cells spread the mixture evenly on the surface of an agar plate containing the corresponding antibiotics, and then pick up several single colonies for DNA sequencing after overnight culture in a 37 ° C incubator; label the correctly sequenced chimeric antibodies as SHC025- 26CHI, SHC025-34CHI, SHC025-58CHI.
  • the chimeric antibody was transfected into HEK293E cells, and a large number of antibodies were obtained by expression and purification. Purity detection, activity analysis, and affinity detection were performed.
  • amino acid sequences of SHC025-26CHI and SHC025-58CHI were modified as follows: the C118 of the SHC025-26CHI heavy chain was mutated to T and the label was SHC025-26CHI-T; the C56 of the SHC025-58CHI light chain was mutated to A, labeled as SHC025-58CHI-A, and the mutant gene was constructed using the method of site-directed mutagenesis. See Table 5 for chimeric antibody sequencing results.
  • SHC025-34CHI, SHC025-58CHI-A, SHC025-26CHI-T were selected for humanized antibody transformation.
  • the humanization of antibodies is first performed by comparing the mouse antibody sequences in the Immunogene Database (IMGT) with the mouse source to confirm the murine origin of the SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T antibody variable regions.
  • IMGT Immunogene Database
  • Germline after homology alignment, the FR regions of the heavy chain variable region sequences of the SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T antibodies were respectively compared with the mouse antibody germline genes IGHV1-8 * 01, IGHV3 -21 * 04 and IGHV1-2 * 02 are most similar; the FR sequences of the antibody light chain variable regions are most similar to the mouse antibodies IGKV3-11 * 01, IGKV1-5 * 01, and IGKV4-1 * 01, respectively.
  • SHC025-34CHI / SHC025-58CHI-A antibody framework region sequence FR1-FR3 as a template, a full human framework with similar 3D structure but low immunogenicity was replaced in the human framework region library to replace SHC025-34CHI / SHC025-58CHI-A FR1-FR3 sequence, heavy chain / light chain full-length sequence for 3D modeling and structural comparison analysis with the original antibody heavy / light chain sequence, considering the antigenicity and 3D structural similarity, finally choose SHC025-34CHI 6 humanized heavy chain variable regions (see SEQ ID NO: 48, 49, 50, 51, 52, 53) and 4 humanized light chain variable regions (see SEQ ID NO: 54, 55, 56 , 57) and 6 humanized heavy chain variable regions of SHC025-58CHI-A (see SEQ ID NO: 58, 59, 60, 61, 62, 63) and 5 humanized light chain variable regions ( (See SEQ ID NO: 64, 65, 66, 67
  • sequences of non-CDR regions of SHC025-34CHI / SHC025-58CHI-A humanized antibodies have reached more than 95% humanization.
  • Four humanized heavy chain variable regions see SEQ ID NO: 69, 70, 71, 72
  • two humanized light chain variable regions were finally obtained from SHC025-26CHI-T. , 74).
  • the above designed humanized antibody light chain and heavy chain variable region amino acid sequences were reverse transcribed into corresponding nucleotide sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments were generated. Oligonucleotide fragments are ligated by PCR after annealing, and specific primers (containing the homology arm sequence at the 5 'end for homologous recombination with the eukaryotic expression vector) are used to amplify the complete light and heavy chains.
  • Variable region nucleotide fragments co-transformed purified light chain variable region nucleotide fragments with linearized IgG4 light chain constant region-containing eukaryotic expression plasmids into E.
  • variable region nucleotide fragment and the eukaryotic expression plasmid of the IgG4 heavy chain constant region containing the S228P / L235E mutation were co-transformed into E. coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were evenly coated on agar containing the corresponding antibiotics On the surface of the plate, several single colonies were picked for DNA sequencing after overnight culture in a 37 ° C incubator.
  • Positively sequenced positive clones were inoculated into 2 ⁇ YT liquid medium containing the corresponding antibiotics, cultured with shaking at 37 ° C for more than 12 hours, and then the cells were collected for plasmid extraction to obtain humanized antibody light and heavy chain expression plasmids. Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the plasmid was transfected into HEK293E cells, and a large number of antibodies were obtained by expression and purification. Purity detection, activity analysis, and affinity detection were performed.
  • Humanized antibodies with good purity, activity, and affinity were selected and labeled Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE. The sequences are shown in Table 6. See Table 7 for sources of humanized antibodies.
  • Protein-based Elisa was used to analyze antibody binding activity.
  • Cynomolgus CD47-His (0.1 ⁇ g / well, ACRO Biosystems, Cat. No. CD7-C52H1-50ug) coated a 96-well microtiter plate.
  • the anti-CD47 antibody provided by the present invention starts with 2 ⁇ g / mL as a primary antibody, and is added to the enzyme-labeled plate by a 5-fold gradient dilution.
  • concentrations There are 8 concentrations, the concentrations are 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64ng / mL, 0.128ng / mL, 0ng / mL, and incubated at 37 ° C for 1.5h.
  • the positive control antibody was AB06.12-4P.
  • Anti-Human IgG HRP Jackson, 109-035-003, 1: 10000 was used as the secondary antibody, and TMB (3,3 ', 5,5'-tetramethylbenzidine) was added as the coloring solution.
  • the instrument thermo, Multiskan FC
  • Graph50 was used to generate EC50, and the results are shown in Figure 1.
  • the binding activity of the antibodies was analyzed by ELISA.
  • the human CD47-His protein (0.1 ug / well, prepared in Examples 1 and 2) was coated onto a 96-well microtiter plate and incubated at 37 ° C for 2 h. After washing 3 times with 1xPBST, blocking with 5% skim milk at 4 ° C overnight.
  • the anti-CD47 antibody provided by the present invention was used as a primary antibody starting from 2 ⁇ g / mL, 5 times gradient dilution was added to the enzyme plate, a total of 8 concentrations, the concentrations were 2000ng / mL, 400ng / mL, 80ng / mL, 16ng / mL, 3.2ng / mL, 0.64ng / mL, 0.128ng / mL, 0ng / mL, and incubate at 37 ° C for 1.5h.
  • the positive control antibody was AB06.12-4P.
  • the experimental results show that the humanized anti-CD47 antibodies Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE provided by the present invention have the ability to bind to human CD47, and the binding ability is similar to that of the positive control antibody AB06.12- 4P is equivalent.
  • CHO-K1-E5 cells were plated at 1 ⁇ 10 5 cells per well and cultured overnight at 37 ° C and 5% CO 2 ; fixed with 4% paraformaldehyde and blocked with skim milk the next day 1h; gently wash the cell plate with 1xPBS; the anti-CD47 antibody provided by the present invention is used as a primary antibody starting from 2 ⁇ g / mL, 5 times gradient dilution is added to the cell plate, a total of 8 concentrations, the concentrations are 2000ng / mL, 400ng / mL, 80ng / mL, 16ng / mL, 3.2ng / mL, 0.64ng / mL, 0.128ng / mL, 0ng / mL, and incubated at 37 ° C for 1.5h.
  • the positive control antibody was AB06.12-4P; the secondary antibody used Anti-Human IgG HRP (Jackson, 109-035-003, 1: 10000), the color development solution TMB was added, and after termination, the OD450 value was read using a microplate reader (thermo, Multiskan FC). EC50 was generated using GraphPad, and the results are shown in Figure 3.
  • the experimental results show that the humanized anti-CD47 antibodies Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE provided by the present invention can all bind to CD47 on the cell surface, and the binding ability is similar to that of the positive control antibody AB06.12- 4P is equivalent.
  • the affinity of the humanized anti-CD47 antibody prepared in Examples 1 and 2 to the antigen CD47 (19-136) -hFC was measured using Fortebio Octet. Firstly, the antigen CD47 (19-136) -hFc was biotinylated, and then desalted 3-4 times with a 10kD ultrafiltration tube and PBS centrifugal ultrafiltration. Nanodrop was used to determine the actual concentration of the antigen CD47-hFc-Biotin after biotin labeling.
  • CD47-hFc-Biotin was diluted with SD buffer (0.02% Tween20 + 0.1% BSA solution) to a concentration of 5ug / ml, and the humanized anti-CD47 antibody was diluted with a 4-fold concentration gradient in SD buffer to a concentration of 10ug / ml, 2.5ug / ml, 0.625ug / ml, 0ug / ml, the SA sensor was used to cure the antigen, and the affinity was measured according to the operating procedures of fortebioOctetRED96. The specific parameters and experimental results are shown in Table 8.
  • Hu26T-31-PE has a higher affinity for binding to human CD47 protein.
  • the initial concentration of the antibody to be analyzed is between 1-20 uM, and a 2-fold gradient dilution is performed, for a total of 24 concentration gradients.
  • a round bottom 96-well plate was added with the above-mentioned 50 ⁇ L of antibodies at different concentrations, and then 50 ul of the above 2% red blood cell suspension was added, mixed, and left at room temperature. Observe whether there is agglutination after 2h.
  • the rabbit polyclonal antibody RBC antibody (Rockland, 109-4139) was used as a positive control for occurrence of agglutination of red blood cells, and the results are shown in FIG. 4.
  • the concentration of the antibodies (rabbit polyclonal antibody RBC antibody, AB06.12-4P antibody and the antibody to be tested in the present invention) added from left to right in a 96-well plate was sequentially diluted from 20uM to a 2-fold gradient, where RBC Denotes a positive control group (using rabbit polyclonal antibody RBC antibody, which significantly causes agglutination of red blood cells), and PBS represents a blank control group.
  • the erythrocytes in the well are small dots, and the edges are neat to indicate that they do not cause cell agglutination; the edges that are slightly irregular are to indicate a small amount of red blood cell agglutination;
  • the anti-CD47 antibody Hu5F9-G4 disclosed in patent application WO2011 / 143624 can cause significant agglutination of most red blood cells in the same concentration range, which is a common adverse phenomenon of anti-CD47 antibodies.
  • the Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE of the present invention do not cause the phenomenon of agglutination of red blood cells, and the antibody of the present invention is significantly superior to this aspect.
  • the ability of the anti-CD47 antibody provided by the present invention to block the binding of SIRPa to CD47 on the cell surface was measured using FACS.
  • CHO-K1-E5 positive cell line was used to observe the binding capacity of CD47 to SIRPa in the presence of a gradient dilution of anti-CD47 antibody.
  • the secondary antibody used PE Streptavidin (Biolegend, 405203, 1: 200) to monitor changes in SIRPa-Biotin.
  • AB06.12-4P served as a positive control that blocked SIRPa binding to cell surface CD47.
  • a flow cytometer (BD, FACSJazz) reads the mean value at a wavelength of 585 nm, and generates a IC50 using a GraphPad. The results are shown in FIG. 5.
  • NUGC-4 human gastric cancer cells were purchased from the American Type Culture Collection (ATCC);
  • NOD-Scid mice female, 5-8 weeks old, weighing 18-20 grams, purchased from Shanghai Lingchang Biotechnology Co., Ltd .;
  • the reference substance Isotype IgG4 (article number AB170091) was purchased from Sino-American Crown Scientific Biotechnology Co., Ltd. and used as a negative control substance;
  • the humanized anti-CD47 antibody of the present invention was formulated with PBS at two concentrations of 0.6 mg / mL and 0.3 mg / mL, and Isotype IgG4 and AB 06.12-4P were formulated at 0.6 mg / mL.
  • NUGC-4 human gastric cancer cells were cultured in a RMPI1640 medium containing 10% fetal calf serum, 100 U / mL penicillin, and 100 ⁇ g / mL streptomycin in a 37 ° C, 5% CO 2 incubator. The passages were digested once a week with 2 mL of 1 ⁇ EDTA solution. When the cell saturation is 80% -90%, the cells are collected, counted, and seeded. PBS containing 5 ⁇ 10 6 cells was mixed with 100 uL of Matrigel (final volume 200 ⁇ L) to inoculate the right rear side of the mice, and the number of the inoculated cells was 5 ⁇ 10 6 / head.
  • RTV V21 / V0, where V0 is the tumor volume measured during group administration (ie, d0), and V21 is the tumor volume measured during 21 days of administration.
  • the tumor volume on the last day (day21) of the administration group and the vehicle group was analyzed by T-test and GraphPad Prism. The results are shown in Table 9.
  • the anti-CD47 antibody provided by the present invention has a significant anti-tumor effect and can significantly suppress tumor growth, suggesting that the antibody can be used in the preparation of anti-tumor drugs and has a predictable market prospect.

Abstract

涉及抗体药物技术领域，尤其涉及一种抗CD47抗体或其抗原结合片段，包含抗CD47抗体或其抗原结合片段的药物组合物以及它们的应用。抗CD47抗体或其抗原结合片段具有显著的抗肿瘤活性，与人CD47蛋白的亲和力较高，可阻断SIRPa结合到细胞表面CD47的能力，且不具有显著的红细胞凝集活性，可在制备抗肿瘤的药物中应用。

Description

抗CD47抗体及其应用 技术领域
本发明涉及抗体药物技术领域，尤其涉及一种抗CD47抗体或其抗原结合片段，包含抗CD47抗体或其抗原结合片段的药物组合物以及它们的应用
背景技术
CD47蛋白又称整合素相关蛋白(IAP)，属于IgG超家族的一个五次跨膜糖蛋白，广泛表达于不同组织细胞。CD47可与配体TSP-1或SIRPα结合，调控不同的细胞功能，包括细胞迁移、粘附、凋亡，轴突延伸，细胞因子产生及T细胞激活。SIRPα是一个含有典型免疫受体酪氨酸抑制基序(ITIM)跨膜蛋白，主要表达于髓系造血细胞膜表面，如巨噬细胞，树突状细胞等。CD47与SIRPα结合后，导致ITIMs的磷酸化，从而招募SHP-1/SHP-2，进而抑制肌球蛋白IIA在吞噬突触的积累，最终抑制吞噬细胞的吞噬功能。
肿瘤细胞的“免疫逃逸”被认为是肿瘤发生、发展和抗药的主要机制。肿瘤细胞通过高表达CD47分子，与巨噬细胞表面的SIRPα相互作用，可显著抑制巨噬细胞的吞噬活性，避免被巨噬细胞吞噬掉。当阻断CD47与SIRPα的结合时，可消除因肿瘤导致的免疫抑制或免疫耐受，有效杀伤肿瘤细胞。这为CD47的肿瘤免疫靶向治疗提供了极为有力的理论依据。
近年来，国内外针对CD47/SIRPα信号通路的各种治疗方式进行了大量研究，例如抗CD47抗体、抗SIRPα抗体、重组SIRPα蛋白、双特异性抗体。其中，CD47阻断性抗体被认为是其中最有希望的肿瘤治疗方案。人CD47阻断型单抗的有效性已经在多种临床前模型中证实，比如淋巴瘤、膀胱癌、结肠癌、成胶质细胞瘤、乳腺癌、急性淋巴细胞性白血病和急性骨髓性白血病等。
此外，由于红细胞、血小板也表达CD47分子，当抗体阻断CD47与SIRPα的相互作用时，可能导致这些细胞失去“别吃我”信号的保护，进而被巨噬细胞吞噬。因此要避免抗CD47抗体的副作用，比如血小板降解，红细胞凝集，红细胞耗竭，贫血等也是应用抗CD47抗体需要考虑的一个重点。
因此，需要提供一种抗CD47抗体或其抗原结合片段，其具有优异的抗肿瘤活性且不具有显著的红细胞凝集活性。
发明内容
本发明一方面提供一种抗CD47抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(HCDR1)、重链可变区的互补决定区2(HCDR2)和/或重链可变区的互补决定区3(HCDR3)，所述轻链可变区包含轻链可变区的互补决定区1(LCDR1)、轻链可变区的互补决定区2(LCDR2)和/或轻链可变区的互补决定区3(LCDR3)。
在一些实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的HCDR1、HCDR2和HCDR3：
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(a2)如SEQ ID NO:10、2和11所示的氨基酸序列；
(a3)如SEQ ID NO:4、5和6所示的氨基酸序列；和
(a4)如SEQ ID NO:7、8和9所示的氨基酸序列；
(a5)与(a1)、(a2)、(a3)或(a4)所示的氨基酸序列具有至少85％序列同一性的CDR；和
(2)所述轻链可变区包含选自如下组的LCDR1、LCDR2和LCDR3：
(a6)如SEQ ID NO:12、13和14所示的氨基酸序列；
(a7)如SEQ ID NO:15、16和17所示的氨基酸序列；和
(a8)如SEQ ID NO:18、19和20所示的氨基酸序列；
(a9)与(a6)、(a7)或(a8)所示的氨基酸序列具有至少85％序列同一性的CDR。
在一个具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14或与SEQ ID NO:12、13和14所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:10、2和11或与SEQ ID NO:10、2和11所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14或与SEQ ID NO:12、13和14所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％，序列同一性的CDR的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85％，序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:18、19和20或与SEQ ID NO:18、19和20所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
在一些具体的实施方案中，根据本发明的抗CD47抗体或其抗原结合片段是单克隆抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的抗CD47抗体或其抗原结合片段是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段或人源化抗体或其抗原结合片段。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:27所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:28所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:21，SEQ ID NO:21经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:21功能相同的氨基酸序列或与SEQ ID NO:21具有至少85％序列同一性的 氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:21，SEQ ID NO:21经取代、缺失或添加一个或多个氨基酸获 得的且与SEQ ID NO:21功能相同的氨基酸序列或与SEQ ID NO:21具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:12、13和14所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:15、16和17所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:7、8和9所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:18、19和20所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和11所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:12、13和14所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性且 所述HCDR1、HCDR2和HCDR3如SEQ ID NO:7、8和9所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:18、19和20所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗CD47抗体为鼠源抗体，其还含有鼠源的IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区，和鼠源的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗CD47鼠源抗体还含有鼠源的IgG1或IgG2或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失 或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:34，SEQ ID NO:34经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:34功能相同的氨基酸序列或与SEQ ID NO:34具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:10、2和11所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:12、13和14所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:15、16和17所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:7、8和9所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:34，SEQ ID NO:34经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:34功能相同的氨基酸序列或与SEQ ID NO:34具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:18、19和20所示的氨基酸序列。
在一些实施方案中，本发明提供一种抗CD47人源化抗体或其抗原结合片段，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的鼠源抗CD47抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区。
在本发明一个优选的实施方案中，所述的抗CD47嵌合抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗CD47嵌合抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2、IgG3、gG4或其突变序列的重链恒定区，优选包含人源IgG1或IgG2重链恒定区，或者使用氨基酸突变后显著降低ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。
在一些具体的实施方案中，本发明的抗CD47人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗CD47人源化抗体或其抗原结合片段还包含人源IgG1或IgG2或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗CD47抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv或F(ab)2。
本发明的另一方面提供一种分离的核酸，其编码根据本发明的抗CD47抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:35或SEQ ID NO:36或SEQ ID NO:37所示；编码轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:38或SEQ ID NO:39或SEQ ID NO:40所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:29的核苷酸序列如SEQ ID NO:35所示；编码轻链可变区SEQ ID NO:32的核苷酸序列如SEQ ID NO:38所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:30的核苷酸序列如SEQ ID NO:36所示；编码轻链可变区SEQ ID NO:33的核苷酸序列如SEQ ID NO:39所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:31 的核苷酸序列如SEQ ID NO:37所示；编码轻链可变区SEQ ID NO:34的核苷酸序列如SEQ ID NO:40所示。
本发明的另一方面提供一种表达载体，其表达本发明的抗CD47抗体或其抗原结合片段。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗CD47抗体或其抗原结合片段的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗CD47人源化抗体或其抗原结合片段和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗CD47人源化抗体或其抗原结合片段，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
可以将本发明的抗CD47人源化抗体或其抗原结合片段与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供抑制CD47活性的方法，所述方法包括向有此需要的个体施用本发明的抗CD47抗体或其抗原结合片段或本发明的药物组合物。
本发明的另一方面提供本发明的抗CD47抗体或其抗原结合片段或本发明的药物组合物在制备抑制CD47活性的药物中的应用。在一些实施方案中，所述抑制CD47活性的药物用于治疗白血病、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。在一些实施方案中，本发明提供上述抗CD47抗体或其抗原结合片段或本发明的药物组合物在制备抗肿瘤的药物中的应用，优选地，所述肿瘤选自白血病、淋巴 瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和黑素瘤。
本发明提供的抗CD47抗体或其抗原结合片段具有显著的抗肿瘤作用，可明显抑制肿瘤增长，且不具有显著的红细胞凝集活性，人源化后的抗体免疫原性大大降低，有效消除人体免疫系统对外源性单抗的排异反应，可在制备用于治疗各类肿瘤疾病的药物中应用，具有广阔的市场前景。
定义
除非另有定义，本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual)(第2版，Cold Spring Harbor Laboratory Press，纽约冷泉港(1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中使用的命名以及实验室方法和技术是本领域公知且普遍使用的。
在本发明中，术语“至少80％序列同一性”是指至少80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在本发明中，术语“至少85％序列同一性”是指至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在一些优选的实施方案中，本发明所述的序列同一性可以至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。
在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补，且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院，马里兰州贝塞斯达(1987和1991))或Chothia和Lesk，J.Mol.Biol.196:901-917(1987)，Chothia等，Nature 342:878-883(1989)定义。
本发明所述的“抗原结合片段”是指具有抗原结合活性的Fab片段、Fab’片段、F(ab’) 2片段及 与人CD47结合的Fv片段、scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体或单链Fv(scFv)。
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10 -6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。
本发明所述的“鼠源抗体”为根据本领域知识和技能制备的对人CD47的单克隆抗体。制备时用CD47抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
本发明所述的“人源化抗体”也称为CDR移植抗体，是将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。此类可变区框架序列可以从公共的DNA数据库或公开的参考文献获得，例如从ImMunoGeneTics(IMGT)网站http://imgt.cines.fr得到或从免疫球蛋白杂志，2001ISBN012441351上获得。
附图说明
图1是抗CD47人源化抗体与猴CD47结合活性测定(ELISA)结果。
图2是抗CD47人源化抗体与人CD47结合活性测定(ELISA)结果。
图3是抗CD47人源化抗体与细胞表面CD47结合活性测定(ELISA)结果。
图4是红细胞凝集实验结果，其中RBC：阳性对照；PBS：空白对照。
图5是FACS检测抗CD47人源化抗体阻断活性实验结果。
图6是抗CD47人源化抗体Hu34-39-PE人胃癌NUGC-4移植瘤模型抗肿瘤试验结果。
图7是抗CD47人源化抗体Hu26T-31-PE人胃癌NUGC-4移植瘤模型抗肿瘤试验结果。
具体实施方式
下面代表性的实施例是为了更好地说明本发明，而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件，如冷泉港的抗体技术实验手册、分子克隆手册等，或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。
实施例1 抗原蛋白及阳性对照抗体的制备
1、抗原蛋白及阳性对照抗体的表达载体构建
(1)抗原蛋白的表达载体构建
合成编码CD47蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:41所示，然后克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-CD47。
融合人源CD47蛋白胞外区氨基酸序列与hIgG1-Fc或his标签氨基酸序列，氨基酸序列设计分别如SEQ ID NO:42和SEQ ID NO:43所示。对上述氨基酸序列进行密码子优化后合成带有标签的CD47蛋白胞外区基因片段CD47-hFc、CD47-his，并将其分别克隆至真核表达质粒pHR中，获得其表达质粒pHR-CD47-hFc、pHR-CD47-his。
融合人源CD47蛋白胞外区氨基酸序列与mIgG1-Fc氨基酸序列，氨基酸序列设计如SEQ ID NO:44所示。对该氨基酸序列进行密码子优化后合成完整的表达质粒pcDNA3.1(+)-TPA-CD47-mIgG1-Fc。
SIRPα的序列如SEQ ID NO:45所示，对所述序列进行密码子优化后合成完整的表达质粒pcDNA3.1(+)-SIRPα-myc-His。
(2)阳性对照抗体的表达载体构建
使用专利申请WO2013/119714中公开的抗体AB6.12-IgG4P(本文简称AB06.12-4P)作为阳性对照抗体，AB06.12-4P的氨基酸序列如下所示：
AB06.12-4P重链氨基酸序列：SEQ ID NO:46；
AB06.12-4P轻链氨基酸序列：SEQ ID NO:47。
对上述抗体序列所对应的氨基酸序列进行密码子人工优化，获得阳性对照抗体AB06.12-4P的重链及轻链表达质粒pcDNA3.1(+)-SHC025-hG4、pcDNA3.1(+)-SHC025-hk。并将其重链基因 片段克隆到含有IgG4轻链恒定区的真核表达质粒pHR上，获得AB06.12-4P的重链真核表达质粒pHR-SHC025-hG4-4PE，其轻链表达质粒为pcDNA3.1(+)-SHC025-hk。
2、抗原蛋白及阳性对照抗体的表达与纯化
(1)抗原蛋白的稳转细胞株的构建
将真核表达质粒pTargeT-CD47在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置37℃，5％CO 2的培养箱中培养。24h后采用含500ug/ml G418的培养基加压培养。16天后采用FACS检测pool阳性率，将电转质粒后的细胞铺板(1x10 6个/ml的细胞密度，100ul/孔)，采用PE mouse anti-human CD47抗体(BD，556046)与细胞孵育，以流式细胞仪(BD，FACSJazz)读取585nm波长下mean值，使用GraphPad进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达CD47分子，命名为CHO-K1-E5。
(2)标签抗原蛋白及阳性对照抗体的表达
在1L细胞培养瓶中接种密度为0.5x 10 6个细胞/ml的293F细胞，加入新鲜的预热的FreeStyle293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8.5mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500ul，混合均匀，取250ug待转染质粒加入8.5ml FreeStyle 293表达培养基中，混合均匀，其中标签抗原蛋白质粒pHR-CD47-hFc、pHR-CD47-his、pcDNA3.1(+)-TPA-CD47-mIgG1-Fc、pcDNA3.1(+)-SIRPα-myc-His分别转染；阳性对照抗体AB06.12-4P重链质粒pHR-SHC025-hG4-4PE和轻链质粒pcDNA3.1(+)-SHC025-hk共同转染。将PEI与FreeStyle 293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)和5ml的葡萄糖(母液浓度为180g/L)。当细胞细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
(3)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用不同的亲和层析柱进行纯化(不同蛋白适配的亲和层析柱见表1)，具体纯化步骤如下：
表1不同蛋白适配的亲和层析柱
Figure PCTCN2019103673-appb-000001
Figure PCTCN2019103673-appb-000002
清洗：超纯水清洗设备及管路2min，流速10mL/min，后用0.1M NaOH清洗层析系统；
接柱：将层析柱接入层析设备，并用超纯水冲洗5min；后0.1M NaOH冲洗30min，保留时间5min；
平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV(柱体积)；
上样：将细胞表达上清上样，保留时间5min；
后平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV；
洗脱：50mM醋酸，pH＝3.4洗脱，保留时间5min。UV280至50mAu左右时开始收集，降至50mAu左右时停止收集。用1M Tris-HCl，pH 9.0将样品pH调节至7.0；
再平衡：20mM PB+0.15M NaCl，pH 7.2平衡3个CV，保留时间5min；
在线清洗：0.1M NaOH清洗30min，保留时间5min；
清洗保存：纯化水清洗10min，后20％乙醇2个CV。
实施例2 单克隆抗体的制备
1、杂交瘤单克隆的制备
(1)动物免疫
采用不同标签的抗CD47抗原蛋白与佐剂共同免疫的方法免疫SJL品系的实验小鼠，首次抗原使用50ug抗原，后期使用25ug抗原免疫。
免疫佐剂可以是Quick Antibody-Mouse5W(北京博奥龙免疫技术有限公司)或Titer Max(Sigma)与CpG(金斯瑞生物科技有限合成)/Alum(thermo)佐剂间隔。将不同标签的CD47抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫SJL小鼠。
高水平表达CD47分析的细胞系如CCRF-CEM和CHO-K1-E5也用来免疫小鼠，使之产生抗体。用胰蛋白酶消化处理正在培养的人急性淋巴白血病细胞(CCRF-CEM)和实施例1中获得的CHO-K1-E5阳性单细胞，1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，取样用细胞计数仪计数，剩余样品1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，计入适量的PBS以获得1x10 8个细胞/ml的细胞悬液。实验组小鼠每只免疫1x10 7个细胞。
免疫方案如表2所示：
表2小鼠免疫方案
组别 抗原 佐剂 免疫途径*
1 PBS 无  
2 CD47-his/CD47-mFc Quick Antibody-Mouse5W i.m.
3 CD47-his/CD47-mFc Titer Max/CpG/Alum s.c./i.m.
4 CD47-mFc Quick Antibody-Mouse5W i.m.
5 CD47-mFc Titer Max/CpG/Alum s.c./i.m.
6 CCRF-CEM/CHO-K1-E5 无 i.p.
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。
(2)杂交瘤融合
脾细胞的获取和制备：将加强免疫后的小鼠处死后浸泡75％的酒精中。解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1:1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
(3)杂交瘤及亚克隆上清的筛选
融合一周后，取细胞上清，通过ELISA筛选出能结合CD47-his蛋白或细胞表面CD47的杂交瘤上清，利用CD47-his筛选针对CD47而非hFc、mFc的抗体。然后利用ELISA分析杂交瘤上清阻断CD47-SIRPα相互作用的能力。包被SIRPα-myc-his于酶标板上，加入重组人源蛋白CD47-hFc与杂交瘤上清的混合物孵育2h，加入HRP标记的anti human IgG Fc特异性抗体(Jackson Immuno Research)孵育1h，利用酶标仪检测450nm处的吸光值。将筛选获得的具有结合能力及阻断能力的杂交瘤母克隆扩大培养，进行结合活性及阻断活性的复测，再次筛选获得 具有结合及阻断能力的杂交瘤阳性克隆。
利用有限稀释法将阳性细胞株进行亚克隆，培养一周后利用ELISA检测亚克隆上清与CD47分子的结合活性以及阻断CD47-SIRPα相互作用的活性，获得3种双阳性细胞株，分别标记为SHC025-26、SHC025-34、SHC025-58。
2、亚型的鉴定
参照鼠抗体亚型鉴定试剂盒SBA Clonotyping Systerm-C57BL/6-HRP(SouthernBiotech，货号：5300-05B)说明书对抗体进行亚型鉴定，结果如表3所示：
表3抗体亚型鉴定结果
命名 抗体亚型
SHC025-26 IgG1/k
SHC025-34 IgG2c/k
SHC025-58 IgG2b/k
3、单克隆抗体的制备
根据亚克隆上清活性分析结果确定单克隆抗体母克隆株SHC025-26、SHC025-34、SHC025-58，将其扩大培养。培养条件是含有10％胎牛血清、1x NAEE、1x丙酮酸钠、1％青链霉素双抗的1640培养基，待细胞汇合度大于>80％时，进将细胞传代扩培，待培养至约50ml时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。
4、单克隆抗体测序
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据小鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得小鼠抗体轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester etal.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.Simon KorenMiha
Figure PCTCN2019103673-appb-000003
Colja Venturini etal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列，测序结果见表4。
表4抗CD47鼠源单克隆抗体序列表
抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHC025-26 SEQ ID NO:21 SEQ ID NO:24
SHC025-34 SEQ ID NO:22 SEQ ID NO:25
SHC025-58 SEQ ID NO:23 SEQ ID NO:26
实施例4 嵌合抗体的构建
将纯化后(纯化步骤见实施例1)的小鼠抗体轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将混合液均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序；将测序正确的嵌合抗体分别标记为SHC025-26CHI、SHC025-34CHI、SHC025-58CHI。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得嵌合抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将嵌合抗体转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
测序发现，在SHC025-26的重链CDR第118位，以及SHC025-58的轻链CDR第56位各自含有一个半胱氨酸，CDR区域的半胱氨酸在表达时会与抗体分子上的其他半胱氨酸随机配对氧化形成二硫桥，从而极大的影响抗体纯度。为解决之一问题，对SHC025-26CHI、SHC025-58CHI氨基酸序列进行如下改造：将SHC025-26CHI重链之C118突变为T，标记为SHC025-26CHI-T；将SHC025-58CHI轻链之C56突变为A，标记为SHC025-58CHI-A，利用定点突变的方法构建突变基因。嵌合抗体测序结果见表5。
表5抗CD47嵌合抗体序列表
嵌合抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHC025-26CHI SEQ ID NO:21 SEQ ID NO:24
SHC025-26CHI-T SEQ ID NO:27 SEQ ID NO:24
SHC025-34CHI SEQ ID NO:22 SEQ ID NO:25
SHC025-58CHI SEQ ID NO:23 SEQ ID NO:26
SHC025-58CHI-A SEQ ID NO:23 SEQ ID NO:28
实施例5 人源化抗体的构建及生产
根据嵌合抗体的活性分析、亲和力KD值等结果选择SHC025-34CHI、SHC025-58CHI-A、SHC025-26CHI-T进行人源化抗体改造。
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对，确认SHC025-34CHI、SHC025-58CHI-A、SHC025-26CHI-T抗体可变区的鼠源种系，经过同源比对，SHC025-34CHI、SHC025-58CHI-A、SHC025-26CHI-T抗体的重链可变区序列的FR区分别与小鼠抗体种系基因IGHV1-8*01、IGHV3-21*04以及IGHV1-2*02最为相似；抗体轻链可变区的FR序列则分别与小鼠抗体IGKV3-11*01、IGKV1-5*01以及IGKV4-1*01最为相似。以SHC025-34CHI/SHC025-58CHI-A抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代SHC025-34CHI/SHC025-58CHI-A的FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，最终选择SHC025-34CHI的6条人源化重链可变区(参见SEQ ID NO:48、49、50、51、52、53)和4条人源化轻链可变区(参见SEQ ID NO:54、55、56、57)及SHC025-58CHI-A的6条人源化重链可变区(参见SEQ ID NO:58、59、60、61、62、63)和5条人源化轻链可变区(参见SEQ ID NO:64、65、66、67、68)进行下一步优化。SHC025-34CHI/SHC025-58CHI-A人源化抗体非CDR区序列均达到95％以上人源化。用SHC025-26CHI-T重轻链的可变区序列，在Protein Data Bank中进行结构比对分析，选择最相近的FR1-FR3序列替换掉鼠源序列，并将在结构模拟中显示对抗体结构稳定起到关键作用的的氨基酸位点回突变为鼠源性氨基酸残基。最终获得SHC025-26CHI-T的4条人源化重链可变区(参见SEQ ID NO:69、70、71、72)和2条人源化轻链可变区(参见SEQ ID NO:73、74)。
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有IgG4轻链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与含S228P/L235E突变的IgG4重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
挑选纯度、活性和亲和力均较好的人源化抗体，标记为Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE，序列见表6。人源化抗体来源见表7。
表6抗CD47人源化抗体序列表
Figure PCTCN2019103673-appb-000004
表7人源化序列设计信息
Figure PCTCN2019103673-appb-000005
实施例6 与猴CD47结合活性测定(ELISA)
采用protein based Elisa分析抗体的结合活性。食蟹猴CD47-His(0.1μg/孔，ACRO Biosystems，Cat.No.CD7-C52H1-50ug)包被96孔酶标板。本发明提供的抗CD47抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、 80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h，阳性对照抗体为AB06.12-4P。二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB(3,3',5,5'-四甲基联苯胺)，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如图1所示。
实验结果显示本发明提供的人源化抗CD47抗体Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均具有与食蟹猴CD47结合的能力，且结合能力与阳性对照抗体AB06.12-4P相当。
实施例7 与人CD47结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人CD47-His蛋白(0.1ug/孔，实施例1、2中制得)包被到96孔酶标板，37℃孵育2h。用1xPBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1xPBST清洗3次后，本发明提供的抗CD47抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h，阳性对照抗体为AB06.12-4P；用1xPBST清洗5次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，37℃孵育40min。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如图2所示。
实验结果显示本发明提供的人源化抗CD47抗体Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均具有与人CD47结合的能力，且结合能力与阳性对照抗体AB06.12-4P相当。
实施例8 与细胞表面CD47结合的测定(ELISA)
采用Cell based Elisa分析抗体的结合活性。CHO-K1-E5细胞以每孔1x10 5个细胞的方式铺细胞板，置于37℃，5％CO 2的条件下过夜培养；第二天用4％的多聚甲醛固定后用脱脂牛奶封闭1h；用1xPBS轻柔清洗细胞板；本发明提供的抗CD47抗体作为一抗从2μg/mL开始，5倍梯度稀释加入细胞板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h，阳性对照抗体为AB06.12-4P；二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如图3所示。
实验结果显示，本发明提供的人源化抗CD47抗体Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均可与细胞表面CD47结合，且结合能力与阳性对照抗体AB06.12-4P相当。
实施例9 与人CD47蛋白的亲和力测定
利用Fortebio Octet对实施例1、2中制得的人源化抗CD47抗体结合抗原CD47(19-136)-hFC的亲和力进行测定。先将抗原CD47(19-136)-hFc生物素化标记，然后用10kD的超滤管，PBS离心超滤脱盐3-4次，Nanodrop测定生物素标记后抗原CD47-hFc-Biotin的实际浓度。将CD47-hFc-Biotin用SD缓冲液(0.02％Tween20+0.1％BSA溶液)稀释到浓度5ug/ml，所述人源化抗CD47抗体用SD缓冲液4倍浓度梯度稀释，使其浓度为10ug/ml、2.5ug/ml、0.625ug/ml、0ug/ml，选用SA传感器固化该抗原，按fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表8所示。
表8与人CD47蛋白的亲和力测定
Figure PCTCN2019103673-appb-000006
实验结果显示，与阳性对照抗体相比，Hu26T-31-PE与人CD47蛋白结合具有更高的亲和力。
实施例10 红细胞凝集实验
取5mL血液加入40mL PBS，2000rpm轻柔离心5min，弃上清，用PBS洗涤三次，然后用PBS重悬红细胞，按红细胞积压，配制2％的红细胞悬液。待分析抗体起始浓度为1-20uM之间，2倍梯度稀释，共24个浓度梯度。圆底96孔板中加入上述50μL不同浓度抗体，然后加入50ul上述2％的红细胞悬液，混匀，室温放置。2h后观察是否有凝集现象。兔多克隆抗体RBC抗体(Rockland，109-4139)作为红细胞凝集发生的阳性对照，结果如图4所示。如图4所示，96孔板从左至右加入的抗体(兔多克隆抗体RBC抗体、AB06.12-4P抗体及本发明待测抗体)浓度从20uM起始2倍梯度依次稀释，其中RBC表示阳性对照组(使用兔多克隆抗体RBC抗体，显著引起红细胞凝集)，PBS表示空白对照组。孔中红细胞小圆点状，边缘整齐表示不引起细胞凝集；边缘略不整齐表示少量红细胞凝集；片状，布满孔底表示大部分红细胞凝集。
经测定，专利申请WO2011/143624中公开的抗CD47抗体Hu5F9-G4在相同浓度范围内能引起显著的大部分红细胞凝集，此为抗CD47抗体的普遍不良现象。而在相同的条件下，如实验结果显示，本发明的Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均不会引起红细胞凝集现象，本发明的抗体在此方面明显优于抗体Hu5F9-G4。
实施例11 FACS检测CD47阻断活性实验
采用FACS检测本发明提供的抗CD47抗体阻断SIRPa结合到细胞表面CD47的能力。
CHO-K1-E5阳性细胞株作为CD47提供者，在梯度稀释的抗CD47抗体存在的情况下，观察CD47与SIRPa的结合能力。二抗使用PE Streptavidin(Biolegend，405203，1:200)来监测SIRPa-Biotin的变化。AB06.12-4P作为阻断SIRPa结合到细胞表面CD47的阳性对照。流式细胞仪(BD，FACSJazz)读取585nm波长下mean值，使用GraphPad生成IC50，结果如图5所示。
实验结果显示，阻断活性依次排序为Hu26T-31-PE≧Hu34-39-PE≧AB06.12-P﹥Hu58A-14-PE。
实施例12 人胃癌NUGC-4移植瘤模型抗肿瘤试验
1、实验材料
(1)实验细胞及动物
NUGC-4人胃癌细胞购自美国典型培养物保藏中心(ATCC)；
NOD-Scid小鼠，雌性，5-8周龄，体重18-20克，购自上海灵畅生物科技有限公司；
(2)供试品及对照品
对照品Isotype IgG4(货号AB170091)购自中美冠科生物技术有限公司，用作阴性对照品；
试验前，将本发明的人源化抗CD47抗体用PBS配制为0.6mg/mL和0.3mg/mL两个浓度，Isotype IgG4和AB 06.12-4P配制为0.6mg/mL。
(3)实验方法
用含有10％胎牛血清，100U/mL的青霉素和100μg/mL的链霉素的RMPI1640培养基在37℃、5％CO 2的培养箱中培养NUGC-4人胃癌细胞。一周一次用2mL 1×EDTA溶液消化处理传代。当细胞饱和度为80％-90％时，收取细胞，计数，接种。将含有5×10 6细胞PBS同100uL的Matrigel混合(终体积200μL)接种于小鼠的右后边，接种细胞数目为5×10 6/只。待肿瘤生长至体积达150-200mm 3时开始分组，腹腔给药每周3次，每周三次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。抗体的抑瘤疗效用相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。RTV＝V21/V0，其中V0是分组给药时(即d0)测量所得肿瘤体积，V21为给药21天测量时的肿瘤体积。将给药组与溶媒组最后一天(day21)瘤体积用T-test进行分析，用GraphPad Prism进行。结果如表9所示。
表9人胃癌NUGC-4移植瘤模型抗肿瘤试验结果
Figure PCTCN2019103673-appb-000007
***p＜0.001vs.Isotype IgG4；**p＜0.005vs.Isotype IgG4
实验结果显示，本发明提供的抗体在人胃癌NUGC-4细胞接种NOD-SCID小鼠移植瘤模型中具有较显著的抗肿瘤作用。3mg/kg剂量的Hu26T-31-PE和Hu34-39-PE与6mg/kg的参照抗体AB06.12-4P抑瘤作用相当，而6mg/kg剂量Hu26T-31-PE和Hu34-39-PE抑瘤作用优于6mg/kg参照抗体AB06.12-4P。停药一周后，6mg/kg的参照抗体组出现肿瘤复发现象，而Hu26T-31-PE和Hu34-39-PE组无复发现象(图6、图7)。提示本发明提供的抗CD47抗体出乎意料的具有更加显著抑制肿瘤生长的作用。
以上实施例证明本发明提供的抗CD47抗体具有显著的抗肿瘤作用，可明显抑制肿瘤增长，提示该抗体可在制备抗肿瘤药物中的应用，具有可预期的市场前景。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。

Claims (16)

  1. 一种抗CD47抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含选自如下组的HCDR1、HCDR2和HCDR3：
    (a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
    (a2)如SEQ ID NO:10、2和11所示的氨基酸序列；
    (a3)如SEQ ID NO:4、5和6所示的氨基酸序列；和
    (a4)如SEQ ID NO:7、8和9所示的氨基酸序列；
    (a5)与(a1)、(a2)、(a3)或(a4)所示的氨基酸序列具有至少85％序列同一性的CDR；和
    (2)所述轻链可变区包含选自如下组的LCDR1、LCDR2和LCDR3：
    (a6)如SEQ ID NO:12、13和14所示的氨基酸序列；
    (a7)如SEQ ID NO:15、16和17所示的氨基酸序列；和
    (a8)如SEQ ID NO:18、19和20所示的氨基酸序列；
    (a9)与(a6)、(a7)或(a8)所示的氨基酸序列具有至少85％序列同一性的CDR。
  2. 如权利要求1所述的抗CD47抗体或其抗原结合片段，其具有：
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14或与SEQ ID NO:12、13和14所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:10、2和11或与SEQ ID NO:10、2和11所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14或与SEQ ID NO:12、13和14所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；或
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:18、19和20或与SEQ ID NO:18、19和20所示的氨基酸序列具有至 少85％序列同一性的CDR的轻链可变区。
  3. 如权利要求1或2所述的抗CD47抗体或其抗原结合片段，其中所述抗体是鼠源单克隆抗体、人鼠嵌合抗体或人源化抗体。
  4. 如权利要求1-3之任一项所述的抗CD47抗体或其抗原结合片段，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:27所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:28所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  5. 如权利要求4所述的CD47抗体或其抗原结合片段，其中所述抗体还含有鼠源的IgG1、IgG2或其变体的重链恒定区，鼠源的k链或其变体的轻链恒定区。
  6. 如权利要求1-3之任一项所述的抗CD47抗体或其抗原结合片段，其中
    所述重链可变区的氨基酸序列为SEQ ID NO:21，SEQ ID NO:21经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:21功能相同的氨基酸序列或与SEQ ID NO:21具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ  ID NO:26具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性的氨基酸序列；或
    所述重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列。
  7. 如权利要求1-3之任一项所述的抗CD47抗体或其抗原结合片段，其中所述抗CD47抗体为人源化抗体，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (c1)如SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31所示的氨基酸序列；
    (c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (c4)如SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34所示的氨基酸序列；
    (c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  8. 如权利要求7所述的抗CD47抗体或其抗原结合片段，其中
    所述重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％ 序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性的氨基酸序列；或
    所述重链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:34，SEQ ID NO:34经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:34功能相同的氨基酸序列或与SEQ ID NO:34具有至少85％序列同一性的氨基酸序列。
  9. 一种分离的核酸，其编码权利要求1-8之任一项所述的抗CD47抗体或其抗原结合片段。
  10. 如权利要求9所述的核酸，其中：
    (1)编码所述重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:35、SEQ ID NO:36或SEQ ID NO:37所示；
    (2)编码所述轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:38、SEQ ID NO:39或SEQ ID NO:40所示。
  11. 一种表达载体，其包含如权利要求9或10所述的核酸。
  12. 一种宿主细胞，其转化如权利要求11所述的表达载体，所述宿主细胞选自原核细胞和真核细胞，优先为哺乳动物细胞。
  13. 制备权利要求1-8任一项所述的抗CD47抗体或其抗原结合片段的方法，包括在如权利要求12所述的宿主细胞中表达抗体，以及从宿主细胞中分离所述抗体的步骤。
  14. 一种药物组合物，其包含权利要求1-8之任一项所述的抗CD47抗体或其抗原结合片段和药学可接受的载体。
  15. 如权利要求1-8之任一项所述的抗CD47抗体或其抗原结合片段或如权利要求14的药物组合物在制备用于抑制CD47活性的药物中的应用。
  16. 如权利要求15所述的应用，所述抑制CD47活性的药物用于治疗白血病、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。
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