WO2017215585A1 - 抗cd47单克隆抗体及其应用 - Google Patents

抗cd47单克隆抗体及其应用 Download PDF

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WO2017215585A1
WO2017215585A1 PCT/CN2017/088013 CN2017088013W WO2017215585A1 WO 2017215585 A1 WO2017215585 A1 WO 2017215585A1 CN 2017088013 W CN2017088013 W CN 2017088013W WO 2017215585 A1 WO2017215585 A1 WO 2017215585A1
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seq
variable region
chain variable
set forth
sequence
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PCT/CN2017/088013
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French (fr)
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冯晓
金磊
肖亮
王涛
秦锁富
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长春金赛药业股份有限公司
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Priority to JP2018558409A priority Critical patent/JP6780021B2/ja
Priority to EP17812696.7A priority patent/EP3473649A4/en
Priority to CA3022484A priority patent/CA3022484A1/en
Priority to AU2017284157A priority patent/AU2017284157B2/en
Priority to US16/310,748 priority patent/US11155632B2/en
Priority to KR1020187037214A priority patent/KR102223187B1/ko
Publication of WO2017215585A1 publication Critical patent/WO2017215585A1/zh
Priority to US17/490,735 priority patent/US20220089727A1/en

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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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    • 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
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    • 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
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    • 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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • 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
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • G—PHYSICS
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    • 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
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the invention relates to the technical field of antibody drugs, in particular to anti-CD47 monoclonal antibodies and applications thereof.
  • CD47 also known as integrin-associated protein (IAP)
  • IAP integrin-associated protein
  • CD47 is a crucial marker on the cell surface, with a molecular weight between 47 and 55 kD. It structurally includes an amino-terminal extracellular variable region, a transmembrane region composed of 3-5 highly hydrophobic transmembrane segments and A hydrophilic carboxy-terminal cytoplasmic tail. It interacts with a variety of ligands such as integrins, SIRP ⁇ (signal regulatory protein alpha), SIRP ⁇ , and thrombospondin.
  • integrins such as integrins, SIRP ⁇ (signal regulatory protein alpha), SIRP ⁇ , and thrombospondin.
  • SIRP ⁇ Signaling protein alpha
  • ITIM immunoreceptor tyrosine inhibitory sequence
  • CD47/SIRP ⁇ is involved in the escape mechanism of tumor immunity
  • CD47 functions as a self-marker by transmitting an inhibitory "don't eat me” signal by binding to SIRP ⁇ expressed by myeloid cells such as macrophages, neutrophils, and dendritic cells.
  • SIRP ⁇ myeloid cells
  • myeloid cells such as macrophages, neutrophils, and dendritic cells.
  • the broad expression of CD47 under physiological conditions is to protect healthy cells from being eliminated by the innate immune system.
  • tumor cells effectively escape immune surveillance by overexpressing CD47.
  • CD47 and CD47-SIRP ⁇ signal systems have received extensive attention. Among them, the most remarkable is its potential drug target for cancer treatment.
  • Weissman of the University of Tennessee systematically studied the expression level of CD47 in various solid tumors. The results showed that all human solid tumor cells showed high expression of CD47, and the average expression level was about 3.3 times that of normal cells.
  • CD47 is highly expressed in many types of tumors and acts as a "don't eat me” signal to inhibit phagocytosis, which means that targeting the CD47-SIRP ⁇ pathway can be used as a treatment for many types of tumors.
  • RAUH et al. demonstrated in vitro and in vivo that blocking anti-CD47 monoclonal antibody can promote macrophage phagocytosis of tumor cells, inhibit the formation of acute myeloid leukemia (AML) in mice, and eliminate AML that has been successfully transplanted in vivo. It can also target the removal of leukemia stem cells (LSC).
  • AML acute myeloid leukemia
  • LSC leukemia stem cells
  • CHAO et al.'s study of acute lymphoblastic leukemia found that anti-CD47 monoclonal antibody combined with rituximab can not only eliminate tumors in the original transplant site, but also eliminate blood circulation and tumors that spread to the liver, spleen, lymph nodes, etc. Long-term survival and inhibition of tumor recurrence, while the use of anti-CD47 monoclonal antibody or anti-CD20 monoclonal antibody alone can only inhibit the growth rate of NHL but can not completely eliminate NHL.
  • anti-CD47 monoclonal antibodies used immunocompetent mice to establish a xenograft model.
  • Anti-mouse and anti-human CD47 monoclonal antibodies significantly inhibited tumor growth and confirmed anti-C D47 Antibodies can eliminate a variety of solid tumors and inhibit tumor metastasis and recurrence.
  • anti-C D47 monoclonal antibody also has anti-tumor effect on cancer stem cells (CSC) and its differentiated subtypes, and can transform tumorigenic TAM into Anti-tumor effector factors and enhance their phagocytosis.
  • Inhibition of mouse CD47 expression also enhances the sensitivity of tumor cells to radiotherapy, but has a protective effect on normal tissues, which may be related to the induction of protective autophagy in host immune cells.
  • anti-CD47 monoclonal antibodies phagocytose tumor cells by blocking the binding of CD47 on tumor cells to SIRP ⁇ on macrophages.
  • anti-CD47 antibodies can induce cytotoxicity of NK cells involved in anti-head and neck tumor cells. Again, tumor cells are cleared by direct induction of apoptosis.
  • studies on immunocompetent mice revealed that anti-CD47 monoclonal antibodies activate CD8 + T cells, causing acquired T cell immune responses that further kill tumor cells.
  • the technical problem to be solved by the present invention is to provide an anti-CD47 monoclonal antibody and an application thereof, and the anti-CD47 monoclonal antibody provided by the present invention can bind human CD47 and monkey CD47, and can block human SIRP and human in a dose-dependent manner. Binding of CD47; promoting the phagocytosis of tumor cells by macrophages. By blocking the binding signal of SIRP to human CD47, tumor cells are prevented from escaping the defense system of tumor immunity, and exert an anti-tumor effect.
  • the anti-CD47 monoclonal antibody provided by the present invention has a heavy chain variable region and a light chain variable region:
  • amino acid sequence of the heavy chain variable region is SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14;
  • the amino acid sequence of the anti-CD47 monoclonal antibody is substituted, deleted or added with one or more amino acids, wherein the plurality is 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.
  • substitution occurs in a hypervariable region
  • hypervariable regions of the heavy chain variable region are HVR-H1, HVR-H2, HVR-H3;
  • the hypervariable region HVR-H1 sequence of SEQ ID NO: 2 is set forth in SEQ ID NO: 45; the HVR-H2 sequence is set forth in SEQ ID NO: 46; and the HVR-H3 sequence is set forth in SEQ ID NO: 47;
  • the hypervariable region HVR-H1 sequence of SEQ ID NO: 5 is set forth in SEQ ID NO: 48; the HVR-H2 sequence is set forth in SEQ ID NO: 49; and the HVR-H3 sequence is set forth in SEQ ID NO: 50;
  • the hypervariable region HVR-H1 sequence of SEQ ID NO: 6 is set forth in SEQ ID NO: 51; the HVR-H2 sequence is set forth in SEQ ID NO: 52; and the HVR-H3 sequence is set forth in SEQ ID NO: 53;
  • hypervariable regions of the light chain variable region are HVR-L1, HVR-L2, HVR-L3;
  • the hypervariable region HVR-L1 sequence of SEQ ID NO: 9 is set forth in SEQ ID NO: 54; the HVR-L2 sequence is set forth in SEQ ID NO: 55; and the HVR-L3 sequence is set forth in SEQ ID NO: 56;
  • hypervariable region HVR-L1 sequence of SEQ ID NO: 12 is set forth in SEQ ID NO: 57; the HVR-L2 sequence is set forth in SEQ ID NO: 58; and the HVR-L3 sequence is set forth in SEQ ID NO: 59;
  • the hypervariable region HVR-L1 sequence of SEQ ID NO: 13 is set forth in SEQ ID NO: 60; the HVR-L2 sequence is set forth in SEQ ID NO: 61; and the HVR-L3 sequence is set forth in SEQ ID NO: 62.
  • amino acid sequence of the heavy chain variable region thereof is shown in any one of SEQ ID Nos: 1 to 7;
  • amino acid sequence of the light chain variable region thereof is shown in any one of SEQ ID NOS: 8 to 14.
  • the anti-CD47 monoclonal antibody has:
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the light chain variable region of amino acid sequence SEQ ID NO: 12;
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the light chain variable region of amino acid sequence SEQ ID NO: 13;
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the amino acid sequence is the light chain variable region of SEQ ID NO: 14.
  • the anti-CD47 monoclonal antibody provided by the present invention has a heavy chain type of IgG1, IgG3 or IgM; its light chain type is ⁇
  • the invention also provides nucleotides encoding the anti-CD47 monoclonal antibody.
  • the nucleotide has
  • (III) a sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;
  • the nucleotide has one or more nucleotides substituted, deleted or added to the nucleotide sequence shown in (I) or (II) or (III) or (IV) A nucleotide sequence obtained by a nucleotide and having the same or similar function as the nucleotide sequence shown in (I) or (II) or (III) or (IV).
  • the nucleotide has one or more nucleotides substituted, deleted or added to the nucleotide sequence shown in (I) or (II) or (III) or (IV) a nucleotide sequence obtained by the nucleotide, wherein the plurality is two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, and thirteen 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31 or 32.
  • the invention also provides an expression vector comprising a nucleotide encoding an anti-CD47 monoclonal antibody provided by the invention.
  • the invention also provides a host cell for transforming an expression vector of the invention.
  • the present invention also provides an antigen having the amino acid sequence shown in any one of SEQ ID NOs: 29 to 32.
  • the present invention also provides a hybridoma cell line which produces the anti-CD47 monoclonal antibody of the present invention.
  • the preparation method of the anti-CD47 monoclonal antibody provided by the invention comprises:
  • Step 1 After immunizing a mouse with the antigen provided by the present invention, obtaining spleen cells of the mouse;
  • Step 2 Fusion of the splenocytes and myeloma cells, screening for hybridoma cell lines capable of binding to CD47,
  • the anti-CD47 monoclonal antibody was prepared by in vitro culture.
  • a combination of the anti-CD47 monoclonal antibody of the present invention produced by chemical labeling or biomarking.
  • the chemical label is an isotope, an immunotoxin, and/or a chemical
  • the biomarker is biotin, avidin or an enzyme label.
  • the invention also provides a conjugate prepared by coupling the anti-CD47 monoclonal antibody or a combination thereof to a solid medium or a semi-solid medium.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a product for detecting expression of CD47.
  • the invention also provides a kit comprising the anti-CD47 monoclonal antibody, conjugate and/or conjugate.
  • the disease is leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and/or melanoma.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation that blocks the binding of CD47 to SIRP ⁇ .
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation for increasing the phagocytic index of tumor cells by macrophages.
  • the tumor cell is human peripheral blood leukemia T cell.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation for promoting apoptosis of tumor cells.
  • the tumor cells are human peripheral blood leukemia T cells.
  • the disease is leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and/or melanoma.
  • the invention also provides a medicament comprising the anti-CD47 monoclonal antibody of the invention, a combination thereof and/or a conjugate thereof.
  • a method for controlling a disease which comprises administering the medicament according to the invention;
  • the disease is leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreas Cancer, glioma and/or melanoma.
  • the anti-CD47 monoclonal antibody provided by the invention can effectively inhibit tumor growth; blocking human SIRP and human CD47 signaling can promote macrophage phagocytosis of tumor cells, prevent tumor cells from escaping tumor immune defense system, and exert anti-tumor effect. Blocking the binding of CD47 on the surface of tumor cells to SIRP on the surface of macrophages can block the "don't eat me" signal of tumor cells, promote the recognition and uptake of tumor cells by macrophages, and promote the phagocytosis of tumor cells. The binding of CD47 on the surface of tumor cells to SIRP on the surface of macrophages is a common "don't eat me" signal. Anti-CD47 antibody can be a very promising target in the tumor immune system and plays a powerful role in human tumor therapy. Effective role.
  • Figure 1 shows SDS-PAGE electrophoresis for detection of purified human and monkey CD47; Lane M: protein molecular weight marker; Figure 1-A: Lane 1: human CD47-linker peptide-hIgG1Fc; Lane 2: human CD47-linker peptide-His; Figure 1-B, lane 1: murine CD47-linker peptide-hIgG1Fc; lane 2: murine CD47-linker peptide-His;
  • FIG. 2 shows SDS-PAGE electrophoresis detection of positive antibody (PAB), lane M: protein molecular weight marker; lane 1: non-reducing PAB; lane 2: reducing PAB;
  • PAB positive antibody
  • Figure 3 shows the purified candidate antibody detected by non-reducing SDS-PAGE electrophoresis
  • lane M protein molecular weight marker
  • lane 1 059-1.11.1 purified antibody
  • lane 2 059-1.20.1 purified antibody
  • lane 3 059-1.30 .1 purified antibody
  • Lane 4 059-1.43.1 purified antibody
  • Lane 5 059-1.51.2 purified antibody
  • Lane 6 059-1.82.1 purified antibody
  • Lane 7 059-1.100.5 purified antibody
  • Figure 4 shows total RNA electrophoresis
  • M DL2000 molecular weight marker
  • lanes 1-7 059-1.11.1, 059-1.20.1, 059-1.30.1, 059-1.43.1, 059-1.51.2, respectively , 059-1.82.1, 059-1.100.5 total RNA electrophoresis bands;
  • Figure 5 shows the result of agarose electrophoresis detection of the PCR amplification candidate antibody heavy chain variable region and light chain variable region
  • Figure 6 is a graph showing the results of blocking human CD47 and SIRP ⁇ by antibodies 059-1.20.1, 059-1.30.1, 059-1.43.1, 059-1.82.1;
  • Figure 7 shows a FACS assay for binding of antibodies 059-1.20.1, 059-1.30.1, 059-1.43.1, 059-1.82.1 to jurkat cell surface CD47;
  • Figure 8 shows that antibodies 059-1.20.1, 059-1.30.1, 059-1.43.1, 059-1.82.1 promoted phagocytosis of Jurkat cells by mouse peritoneal primary macrophages.
  • the invention provides an anti-CD47 monoclonal antibody and an application thereof, and those skilled in the art can learn from the contents of the present article and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application of the present invention may be modified or combined and modified to achieve and apply the present invention without departing from the scope of the present invention. Invention technology.
  • Antibody refers to a protein composed of one or more polypeptides that specifically bind an antigen.
  • One form of antibody constitutes the basic building block of an antibody. This form is a tetramer consisting of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together to bind the antigen, while the constant region is responsible for the effector function of the antibody.
  • the "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain.
  • known types of antibodies include kappa and lambda light chains, as well as alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or other types of equivalents thereof.
  • the full length immunoglobulin "light chain” (approximately 25 kDa or approximately 214 amino acids) comprises a variable region formed by approximately 110 amino acids at the NH2-terminus, and a kappa or lambda constant region at the COOH-terminus.
  • a full length immunoglobulin "heavy chain” (approximately 50 kDa or approximately 446 amino acids) also contains a variable region (approximately 116 amino acids) and one of the heavy chain constant regions, such as gamma (approximately 330 amino acids).
  • Antibody includes antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and A fusion protein comprising an antigen binding portion of an antibody and a non-antibody protein.
  • the antibody can be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like, and detected.
  • the antibody may also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.
  • the present invention provides an anti-CD47 monoclonal antibody having a heavy chain variable region and a light chain variable region:
  • amino acid sequence of the heavy chain variable region is SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14;
  • the amino acid sequence of the anti-CD47 monoclonal antibody is substituted, deleted or added with one or more amino acids, wherein the plurality is 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.
  • substitution occurs in a hypervariable region
  • hypervariable regions of the heavy chain variable region are HVR-H1, HVR-H2, HVR-H3;
  • the hypervariable region HVR-H1 sequence of SEQ ID NO: 2 is set forth in SEQ ID NO: 45; the HVR-H2 sequence is set forth in SEQ ID NO: 46; and the HVR-H3 sequence is set forth in SEQ ID NO: 47;
  • the hypervariable region HVR-H1 sequence of SEQ ID NO: 5 is set forth in SEQ ID NO: 48; the HVR-H2 sequence is set forth in SEQ ID NO: 49; and the HVR-H3 sequence is set forth in SEQ ID NO: 50;
  • the hypervariable region HVR-H1 sequence of SEQ ID NO: 6 is set forth in SEQ ID NO: 51; the HVR-H2 sequence is set forth in SEQ ID NO: 52; and the HVR-H3 sequence is set forth in SEQ ID NO: 53;
  • hypervariable regions of the light chain variable region are HVR-L1, HVR-L2, HVR-L3;
  • the hypervariable region HVR-L1 sequence of SEQ ID NO: 9 is set forth in SEQ ID NO: 54; the HVR-L2 sequence is set forth in SEQ ID NO: 55; and the HVR-L3 sequence is set forth in SEQ ID NO: 56;
  • hypervariable region HVR-L1 sequence of SEQ ID NO: 12 is set forth in SEQ ID NO: 57; the HVR-L2 sequence is set forth in SEQ ID NO: 58; and the HVR-L3 sequence is set forth in SEQ ID NO: 59;
  • the hypervariable region HVR-L1 sequence of SEQ ID NO: 13 is set forth in SEQ ID NO: 60; the HVR-L2 sequence is set forth in SEQ ID NO: 61; and the HVR-L3 sequence is set forth in SEQ ID NO: 62.
  • the anti-CD47 monoclonal antibody has:
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the amino acid The sequence is the light chain variable region of SEQ ID NO:9;
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the light chain variable region of amino acid sequence SEQ ID NO: 12;
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the light chain variable region of amino acid sequence SEQ ID NO: 13;
  • amino acid sequence is the heavy chain variable region of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and the light chain variable region of amino acid sequence SEQ ID NO: 14;
  • the anti-CD47 monoclonal antibody provided by the present invention has a heavy chain type of IgG1, IgG3 or IgM; its light chain type is ⁇ .
  • amino acid sequence of the heavy chain variable region of the anti-CD47 monoclonal antibody is shown in SEQ ID NO: 1
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the anti-CD47 monoclonal antibody is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the anti-CD47 monoclonal antibody is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is as shown in SEQ ID NO: 3, light
  • amino acid sequence of the variable region of the chain is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • amino acid sequence of the heavy chain variable region of the anti-CD47 monoclonal antibody is set forth in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 9.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 3
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 11.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 5
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody against CD47 is set forth in SEQ ID NO: 7
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 14.
  • the heavy chain constant region of the antibody 059-1.82.1 of the present invention is mouse IgG3, the light chain constant region is the constant region of the mouse ⁇ chain, and the 059-1.30.1, 059-1.43.1, and 059-1.20.1 are all mice.
  • IgG1 the light chain constant region is the constant region of the mouse kappa chain
  • the heavy chain constant region of 059-1.11.1, 059-1.51.2, 059-1.100.5 is murine IgM
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1-7
  • the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 8-14.
  • the anti-CD47 mAb provided by the present invention is capable of binding to human CD47 and monkey CD47; in certain embodiments, the affinity between the antibody and its target is characterized by Ka, Kd (dissociation constant), KD (equilibrium dissociation constant)
  • Ka Ka
  • Kd dissociation constant
  • KD equilibrium dissociation constant
  • the present invention provides that the KD value of the antibody is not higher than 30 nM.
  • the anti-CD47 mAb provided by the present invention can block the binding of human SIRP to human CD47 in a dose-dependent manner; the blocking effect is represented by an EC50 value, and the present invention provides an antibody having an EC50 value of not less than 850 nM.
  • the anti-CD47 mAb provided by the present invention is capable of binding to cell surface CD47; the detection of the effect is carried out by the FACS method, and the result is expressed by MFI (fluorescence intensity), and the present invention provides that the MFI value of binding of the antibody to the cell surface CD47 is not less than 9547, Up to 18533.
  • the anti-CD47 monoclonal antibody provided by the present invention can promote the phagocytosis of tumor cells by macrophages, and the effect is measured by fluorescence imaging, and the results are expressed by the phagocytic index.
  • the present invention provides an antibody having a phagocytic index of up to 79 for jurkat cells.
  • the anti-CD47 monoclonal antibody provided by the invention can also induce apoptosis of tumor cells, and the effect is expressed by the cell apoptosis rate detected by the cytometry, and the results show that the antibody provides the apoptosis of the jurkat cells, and the apoptosis rate can reach 48%.
  • Jurkat cells belong to the acute T cell leukemia cell line and belong to one of a variety of malignant tumor cells. Like other malignant tumors, jurkat cells have high expression of CD47 on the surface.
  • the invention proves that the CD47 monoclonal antibody provided by the present invention can prevent tumor cells from escaping the defense system of tumor immunity by blocking the binding signal of SIRP and human CD47, and exerts an anti-tumor effect.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9
  • the amino acid sequence of the heavy chain variable region is As shown in SEQ ID NO: 3, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4, the amino acid of the light chain variable region The sequence is shown in SEQ ID NO: 11; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13,
  • Both CD47 and CD47 have good affinity, which can block the binding of CD47 to SIRP ⁇ , bind to CD47 on the cell surface, promote macrophage phagocytosis of tumor cells, and induce apoptosis of tumor cells.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region has the best effect of blocking the binding of CD47 to SIRP ⁇ by the monoclonal antibody shown in SEQ ID NO: It has the best effect on binding to CD47 on the surface of tumor cells, and its effect of inducing macrophage to phagocytose tumor cells is also optimal.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is superior to the monoclonal antibody shown in SEQ ID NO: 10. Effect.
  • the invention also provides nucleotides encoding the anti-CD47 monoclonal antibody.
  • the nucleotide has
  • (III) a sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;
  • the nucleotide has one or more nucleotides substituted, deleted or added to the nucleotide sequence shown in (I) or (II) or (III) or (IV) A nucleotide sequence obtained by a nucleotide and having the same or similar function as the nucleotide sequence shown in (I) or (II) or (III) or (IV).
  • the nucleotide has one or more nucleotides substituted, deleted or added to the nucleotide sequence shown in (I) or (II) or (III) or (IV) a nucleotide sequence obtained by the nucleotide, wherein the plurality is two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, and thirteen 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31 or 32.
  • the invention also provides an expression vector comprising the nucleotides of the invention providing a heavy chain variable region and/or a light chain variable region of an anti-CD47 monoclonal antibody.
  • the invention also provides a host cell for transforming an expression vector of the invention.
  • the present invention also provides an antigen having the amino acid sequence shown in any one of SEQ ID NOs: 29 to 32.
  • the present invention also provides a hybridoma cell line which produces the anti-CD47 monoclonal antibody of the present invention.
  • the preparation method of the anti-CD47 monoclonal antibody provided by the invention comprises:
  • Step 1 After immunizing a mouse with the antigen provided by the present invention, obtaining spleen cells of the mouse;
  • Step 2 The spleen cells and myeloma cells are fused, and a hybridoma cell strain capable of binding to CD47 is selected and cultured in vitro to obtain an anti-CD47 monoclonal antibody.
  • amino acid sequence of the antigen in the step 1 is as shown in SEQ ID NO: 29 or 31.
  • the antigen is mixed with an adjuvant to immunize the mouse.
  • the volume ratio of antigen to adjuvant was 1:1.
  • the immunization is specifically performed for the second immunization two weeks after the first immunization, and the mice having a serum titer greater than 1:200,000 after 3 days are boosted.
  • the doses of the first immunization, the second immunization, and the booster immunization were all 10 ⁇ g in terms of antigen mass.
  • the first immunization adjuvant is Freund's complete adjuvant
  • the second immunization and booster adjuvant is Freund's incomplete adjuvant.
  • mice used for immunization were BALB/C mice.
  • the myeloma cell is P3X63Ag8.653.
  • the fusion was carried out at a ratio of spleen cells to myeloma cells of 5:1.
  • the screening was screened using HAT medium.
  • the binding to CD47 specifically binds to the CD47 protein and is capable of binding to cells expressing the CD47 protein on the surface.
  • the CD47 mAb prepared by the method of the present invention was identified, the heavy chain type was IgG3, IgM or IgG1, and the light chain type was ⁇ .
  • a combination of the anti-CD47 monoclonal antibody of the present invention produced by chemical labeling or biomarking.
  • the chemical label is an isotope, an immunotoxin, and/or a chemical
  • the biomarker is biotin, avidin or an enzyme label.
  • the enzyme label is preferably horseradish peroxidase or alkaline phosphatase.
  • the immunotoxin is preferably aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, acacia protein, mistletoe lectin, lotus root toxin, PAP, herbicide, white toxin or loofah toxin.
  • the invention also provides a conjugate prepared by coupling the anti-CD47 monoclonal antibody or a combination thereof to a solid medium or a semi-solid medium.
  • the solid or non-solid medium is selected from the group consisting of colloidal gold, polystyrene plates or beads.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a product for detecting expression of CD47.
  • the CD47 monoclonal antibody provided by the present invention can bind to CD47 protein, and can also bind to cells expressing CD47 on the surface. Therefore, the CD47 monoclonal antibody provided by the present invention can be used for the detection of CD47 protein or cells expressing CD47 on the surface. Moreover, due to the high expression of the tumor cell surface marker CD47, the antibody provided by the present invention is capable of preparing a kit for detection of a tumor surface marker CD47. Among them, the detection of CD47 protein was carried out by ELISA, and the cells expressing CD47 on the surface were detected by FACS.
  • the invention also provides a kit comprising the anti-CD47 monoclonal antibody, conjugate and/or conjugate.
  • the kit for detecting CD47 protein further includes a coating buffer, a washing solution, a blocking solution, and/or a color developing solution.
  • the coating buffer is a carbonate buffer.
  • the washing solution includes PBS, Tween, sodium chloride, potassium chloride, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.
  • the blocking solution includes PBS and BSA.
  • the color developing solution includes a TMB solution, a substrate buffer, and a stop solution.
  • the substrate buffer includes citric acid and disodium hydrogen phosphate.
  • the stop solution is an aqueous hydrogen peroxide solution.
  • kits for detecting cells expressing CD47 on the surface PBS, goat anti-mouse IgG Fc and TITC secondary antibody are also included.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation that blocks the binding of CD47 to SIRP ⁇ .
  • the cells expressing CD47 on the surface are tumor cells.
  • the tumor cells are selected from the group consisting of leukemia cells, lymphoma cells, breast cancer cells, lung cancer cells, gastric cancer cells, intestinal cancer cells, esophageal cancer cells, ovarian cancer cells, cervical cancer cells, renal cancer cells, bladder cancer cells, pancreatic cancer cells. , glioma cells and / or melanoma cells.
  • the disease is leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and/or melanoma.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation that blocks the binding of CD47 to SIRP ⁇ .
  • the anti-CD47 mAb of the present invention blocks the binding of CD47 to SIRP ⁇ with an EC50 value of 850 nM to 2340 nM.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation for increasing the phagocytic index of tumor cells by macrophages.
  • the anti-CD47 mAb of the present invention increases the dose of macrophage to the tumor cell phagocytic index by an antibody concentration of 10 ⁇ g/mL.
  • the tumor cell is human peripheral blood leukemia T cell.
  • anti-CD47 monoclonal antibodies, conjugates and/or conjugates of the invention in the preparation of a formulation for promoting apoptosis of tumor cells.
  • the tumor cells are human peripheral blood leukemia T cells.
  • the anti-CD47 mAb of the present invention promotes tumor cell apoptosis at a dose of 10 ⁇ g/mL.
  • the disease is leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and/or melanoma.
  • the invention also provides a medicament comprising the anti-CD47 monoclonal antibody, conjugate and/or even of the invention Linkage.
  • a method for controlling a disease which comprises administering the medicament according to the invention;
  • the disease is leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreas Cancer, glioma and/or melanoma.
  • the medicine provided by the invention can promote the recognition and uptake of tumor cells by macrophages by blocking the "don't eat me” signal which can block tumor cells, and promote the phagocytosis of tumor cells.
  • the dosage form of the medicament provided by the present invention is an injection preparation or an injection injection.
  • the concentration of the antibody in the injection was 10 ⁇ g/mL.
  • the anti-CD47 monoclonal antibody provided by the invention can effectively inhibit tumor growth; blocking human SIRP and human CD47 signaling can promote macrophage phagocytosis of tumor cells, prevent tumor cells from escaping tumor immune defense system, and exert anti-tumor effect. Blocking the binding of CD47 on the surface of tumor cells to SIRP on the surface of macrophages can block the "don't eat me" signal of tumor cells, promote the recognition and uptake of tumor cells by macrophages, and promote the phagocytosis of tumor cells. The binding of CD47 on the surface of tumor cells to SIRP on the surface of macrophages is a common "don't eat me" signal. Anti-CD47 antibody can be a very promising target in the tumor immune system and plays a powerful role in human tumor therapy. Effective role.
  • the instruments used in the present invention are all commercially available and are commercially available.
  • amino acid sequence of the human and monkey-derived CD47 protein extracellular region is fused with the linker-hIgG1Fc or the linker-7His amino acid sequence, and the amino acid sequence is designed as shown in SEQ ID NO: 29, 30, 31, 32:
  • the amino acid sequences corresponding to the above designed human and monkey CD47 protein extracellular domain fusion protein (CD47ECD-linker peptide-hIgG1Fc or CD47ECD-linker peptide-7his) were codon-optimized, as SEQ ID NO: 33, 34, 35, At the 5' end, the HindIII restriction site and the Kozak sequence GCCGCCACC were added, and the 3' end was added with the stop codon TAG and EcoR I restriction sites, and the optimized DNA was synthesized by Kingsray and cloned into pUC57simple ( In the vector provided by Kingsray, human and monkey pUC57simple-CD47-linker peptide-hIgG1Fc and or CD47ECD-linker peptide-7his plasmid were obtained.
  • the pAb antibody sequences are as follows:
  • PABH as shown in SEQ ID NO: 37, 38:
  • PABL as shown in SEQ ID NO: 39, 40:
  • the amino acid sequence corresponding to the above antibody sequence was codon-optimized, and the HindIII restriction site and the Kozak sequence GCCGCCACC were added at the 5' end, and the 3' end plus the stop codon TAG and EcoR I restriction sites were entrusted.
  • the optimized DNA was synthesized by Kingsray and cloned into pUC57simple (provided by Kingsray) vector to obtain pUC57simple-PCABH, pUC57simple-PCABL plasmid, and plasmid pUC57simple-PCABH, pUC57simple-PCABL was digested (HindIII and EcoR). I), the gene fragment PCABH, PCABL recovered by electrophoresis was recombined with pcDNA3.1 vector to obtain pcDNA3.1-PCABH and pcDNA3.1-PCABL.
  • FreeStyle TM 293F cells using transient expression of transfected in Freestyle medium 24 hours before transfection, 0.6 ⁇ 10 6 cells/ml of 293F cells were inoculated in a 125 ml Erlenmeyer flask, and cultured in a 37° C. 5% CO 2 incubator at 130 rpm on a shaker.
  • 60 ⁇ l of 293fectin was added to 1 ml of OPtiMEM, mixed well, and incubated at room temperature for 5 minutes.
  • the recombinant vector was mixed at a ratio of 3:2:1, and the total amount of DNA was 30 ⁇ g. In 1 ml of OPtiMEM.
  • the DNA and 293fectin were then thoroughly mixed in a total volume of 2 ml, incubated for 15 minutes at room temperature, then the mixture was all added to the cell culture wells, mixed, and cultured in a 37 ° C 5% CO 2 incubator at 130 rpm on a shaker for 7 days.
  • the culture solution was subjected to high-speed centrifugation and vacuum filtration through a microporous membrane.
  • Purification was carried out according to the method provided by the manufacturer using a Protein A column (protein purification liquid chromatography system / AKTA Purifier 10, GE) to obtain a purified human PDL-1-mIgG2aFc fusion protein.
  • the AKTA was rinsed with ultrapure water and connected to a 1 ml rProtianA Fast Flow prepacked column to the AKTA; cleaning: 5 column volumes were washed with 1 M HAC. Equilibration: 20 mM PB 0.15 M NaCl (pH 7.0) equilibrated 5 column volumes.
  • Elution Elution with 20 mM sodium citrate buffer (pH 3.0) at a flow rate of 0.2 ml/min.
  • the collection started at UV 280 to 100 mAu and stopped when it was lowered to 100 mAu.
  • the pH of the sample was adjusted to pH 6-8 with 1 M Tris, as shown in Figures 1-2.
  • the immunogenic human CD47-hFc (prepared in Example 1) was emulsified at a ratio of 1:1 to the volume of the antigen and the adjuvant.
  • the first immunization was carried out using Freund's complete adjuvant, and after 2 weeks, the second immunization was started.
  • the antigen was emulsified using Freund's incomplete adjuvant, subcutaneous injection at 2 o'clock, and the amount of antigen injected per mouse was 10 ⁇ g, and the volume of injection per injection site was 25 ⁇ L.
  • mice were subjected to eyelid blood collection, and a small amount of blood samples were taken for serum titer detection. After indirect ELISA was used to detect serum titer titer of 1:200,000 or more, the mice were boosted. .
  • DMEM medium was aspirated by a syringe, injected into the peritoneal cavity of the mouse, and the peritoneal macrophages were washed and aspirated, collected in a centrifuge tube and centrifuged at 1500 rmp/min for 3 min, and the lower layer of the brown precipitate was resuspended and used. The above procedure was repeated to obtain peritoneal macrophages of 3 normal mice.
  • Myeloma cells were prepared, and P3X63Ag8.653 was resuscitated one week in advance, and cultured in complete medium containing 1X 8-azaguanine. The cells were replaced with 15% fetal bovine serum in DMEM two days before fusion to maintain the density of P3X63Ag8.653 at 70%. -80% to the day of integration.
  • splenocytes Two mice (labeled as L1 and L2) after booster immunization were collected, and the immune serum of L1 and L2 was collected, sacrificed and immersed in 75% alcohol. The skin and peritoneum of the abdomen of the immunized mice were cut open to expose the spleen. The spleen was removed by cutting the surrounding tissue with a cutting tip, ground with a grinding rod, and filtered through a cell sieve to prepare a single cell suspension.
  • Pre-fusion treatment P3X63Ag8.653 in the culture flask was collected, centrifuged at 1000 rpm/5 min, the supernatant was discarded, and resuspended, and viable cell count was performed.
  • the spleen cell suspension was centrifuged at 2000 rpm/5 min, the supernatant was discarded, resuspended, and viable cell counts were performed.
  • the number of viable cells of P3X63Ag8.653 and the number of viable cells of spleen cells were recorded.
  • HAT medium screening HAT screening medium containing 1 x HAT, 1 x cyan-streptomycin, 15% fetal bovine serum and 85% DMEM medium was prepared. The murine hybridoma cells and feeder cells were resuspended in the above HAT screening medium, and the two were mixed. The cell suspension was added to 20 96-well cell culture plates at 300 ⁇ l/well, and cultured in a 37 ° C cell culture incubator. After 1 week of culture, the first change was carried out with HT medium, and cultured in a 37 ° C cell culture incubator; after 3 days of culture, a second liquid exchange was performed with HT medium.
  • the cell supernatant was taken for ELISA, and the binding of the cell supernatant to human CD47-his protein was detected. After screening the cells with positive ELISA results, the second Elisa test was repeated; The result was a positive cell supernatant subjected to a FACS test to detect the binding of the cell supernatant to the surface CD47 protein of jurkat cells.
  • the double-positive cell line detected by ELISA and FACS was transferred from 96 wells to 24 wells, and after being overgrown, transferred to a 25 cm 2 culture flask for culture.
  • the positive cell strain was mixed by pipetting, and a small amount was taken for viable cell counting. Pipette about 100 cells into 40mL complete medium and mix them, and plate 2 pieces; add about 100 cells to 20mL complete medium and mix them, and plate 1 piece; add about 1000 cells and mix them into 20mL complete medium. Uniformly, 1 plate was plated; 4 plates were plated at 3 different cell densities, respectively 0.5 cells/well, 1 cell/well, 10 cells/well. The 96-well plate was cultured in a 37 ° C 5% CO 2 incubator.
  • the supernatant of the monoclonal cell well was taken for ELISA and FACS experiments, and the binding of the cell clone antibody to the human and monkey CD47-his tag and the binding of the CD47 protein on the surface of jurkat cells were detected.
  • ELISA and FACS were tested as double positive cell lines (7 in total, labeled 059-1.11.1, 059-1.20.1, 059-1.30.1, 059-1.43.1, 059-1.51.2, 059 -1.82.1, 059-1.100.5) Transfer from 96 wells into 24 wells, and transfer to a 25 cm 2 flask to grow.
  • the heavy chain constant region of antibody 059-1.82.1 is mouse IgG3
  • the light chain constant region is the constant region of mouse ⁇ chain
  • 059-1.30.1, 059-1.43.1 and 059-1.20.1 are all mouse IgG1.
  • the light chain constant region is the constant region of the mouse kappa chain
  • the heavy chain constant region of 059-1.11.1, 059-1.51.2, 059-1.100.5 is murine IgM
  • the amino acid sequence of the heavy chain variable region is SEQ ID One of NO: 1-7
  • the amino acid sequence of the light chain variable region is one of SEQ ID NOs: 8-14.
  • cryopreservation solution 90% fetal bovine serum, 10% DMSO.
  • the cells in the culture flask were resuspended, and after counting the cells, the cells were centrifuged at 1500 rpm/min for 3 min, and the supernatant was discarded.
  • the cells were boiled and suspended with fetal bovine serum containing 10% DMSO, and frozen at 5 ⁇ 10 6 cells per tube. In the box, overnight at -80 ° C, the next day was transferred to liquid nitrogen.
  • RNA was extracted from the positive monoclonal cell strain RNA extract and reverse transcribed into cDNA, which was permanently stored at -80 °C.
  • the hybridoma cell strain prepared in Example 2 was resuscitated by resuscitating DMEM medium containing 10% fetal bovine serum and 1% chelinomycin and using a vial culture, and after the cell confluence was about 90%, proceeding The cells were propagated by passage and expanded to a cell culture supernatant of about 200 mL.
  • 059-1.11.1, 059-1.20.1, 059-1.30.1, 059-1.43.1, 059-1.51.2, 059-1.82.1 were selected.
  • 059-1.100.5 monoclonal antibody cell line for total RNA extraction, and reverse transcription into cDNA, and then PCR amplification of the heavy chain variable region and light chain variable region of the antibody.
  • RNA was used as a template, and the random primers in the kit were reverse transcribed into the first strand cDNA, and then the heavy chain was designed with the constant region primer (mIgG R) and The linker primers in the kit were subjected to PCR amplification, and the light chain was subjected to PCR amplification using a constant region design primer (mIgK R) and a linker primer in the kit.
  • the sequences of mIgG R and mIgK R are as follows:
  • mIgG R CTCAGGGAARTARCCYTTGAC, (as shown in SEQ ID NO: 41);
  • mIgK R TCACTGCCATCAATCTTCCAC, (as shown in SEQ ID NO: 42);
  • the PCR fragment was recovered from the agarose gel recovery kit for TA cloning and then subjected to PCR for identification.
  • the primers were identified as M13-F and M13-R, and some samples selected from the correct hot strain were sent to Invitrogen for sequencing.
  • the heavy chain variable region protein sequence is finally determined to be SEQ ID NOS: 1 to 7; the light chain variable region protein sequence is SEQ ID NOS: 8 to 14; and the heavy chain nucleotide sequence is SEQ ID NO: 15 to 21;
  • the chain nucleotide sequence is SEQ ID NOs: 22-28:
  • hCD47-his was diluted to 1 ⁇ g/ml with PBS, added to 96 wells of the plate, 100 ⁇ L per well, and incubated overnight at 4 °C.
  • Blocking After washing the plate three times, it was blocked with 1% BSA + PBS, 300 ⁇ L per well, and incubated for 2 hours at room temperature.
  • Addition of candidate antibodies After washing the plate 3 times, add the cell culture supernatant of the candidate antibody or a positive control or a negative control. 100 ⁇ l per well, 2 hours at room temperature.
  • the affinity constant of the human CD47 antibody was detected using a Biacore T200 instrument, and an anti-mouse Fc antibody (GE Healthcare, BR-1008-38) was coupled to a CM5 biosensor chip (GE Healthcare) by covalent attachment of the amino group.
  • Anti-mouse Fc antibody capture candidate monoclonal antibody or positive control B6H12 commercialized CD47 antibody, purchased from Abcam, article number: ab3283
  • different concentrations of human CD47 flowed through the candidate antibody on the chip at a flow rate of 30 ⁇ L/min
  • Human CD47 was bound to the candidate antibody with a binding time of 120 s and a dissociation time of 300 s.
  • the kinetic fit was performed using BIAevalution software (GE Healthcare) with an affinity of up to 059_1.82.1.
  • the affinity constants are obtained as shown in Table 4 below:
  • Antibody name EC50(nM) 059-1.11.1 2340 059-1.20.1 1190 059-1.30.1 1030 059-1.43.1 850 059-1.51.2 -- 059-1.82.1 1030 059-1.100.5 -- Positive control 1190 Negative control --
  • Jurkat cells were cultured, collected by centrifugation at 2000 rpm for 5 min, and washed once with PBS for viable cell counting; 2.0 ⁇ 10 5 cells were added to each tube, centrifuged at 2000 rpm for 5 min to remove supernatant, and 100 ⁇ L of candidate antibody cell supernatant was added to each tube.
  • Positive control Tubes were added with 1 ng/mL of B6H12 antibody (commercialized CD47 antibody, purchased from Abcam, article number: ab3283) 100 ⁇ L, negative control tubes were added to 100 ⁇ L of DMEM medium, and reacted for 1 hour at room temperature; centrifuged at 2000 rpm for 5 min, washed with PBS 3 times, each tube Add 4ng/mL goat anti-mouse IgG Fc, FITC secondary antibody 100 ⁇ L, react for 1 hour at room temperature; centrifuge for 5min at 2000rpm, wash PBS 3 times; add 300 ⁇ L PBS to each tube, transfer to flow-type tube, test on the machine . The results are shown in Table 6 and Figure 7.
  • the animal was killed by necking, and the whole mouse was immersed in 70% alcohol for 3 to 5 seconds. Place the animal on the dissection table, fix the limbs with a needle, pull the skin to both sides with both hands, expose the peritoneum, scrub the peritoneal wall with 70% alcohol, and inject 10ml of Eagle solution into the abdominal cavity with a syringe. Use your fingers to pry the peritoneal wall and allow the fluid to flow sufficiently in the abdominal cavity. Gently pick up the abdominal wall with a needle, so that the animal body slightly leans to one side, so that the liquid in the abdominal cavity is collected under the needle and sucked into the needle tube.
  • Serum medium in macrophages was changed to serum-free medium 2 hours before the Jurkat cells were plated, and various antibodies were added to the two suspension cells at a concentration of 10 ⁇ g/ml. After 2 hours of culture, Jurkat cells that were not phagocytized were washed away, and after cell imaging by fluorescence microscopy, the pros and cons of CD47 antibody phagocytosis were quantified by counting how many Jurkat cells, ie, phagocytic index, were phagocytized per 100 macrophages. . The results are shown in Figure 8 (phagocytic effect map) and Table 7 (phagocytic index).
  • B6H12 (commercialized CD47 antibody, purchased from Abcam, article number: ab3283), consistent with literature reports, has a weaker phagocytosis effect.
  • Example 5 Four selected antibody molecules have good phagocytosis, especially 059-1.30.1, 059-1.43.1, and 059-1.82.1.
  • B6H12 did not induce apoptosis in Jurkat cells, but four 059 antibodies were tested to induce apoptosis in Jurkat cells to varying degrees. 059-1.30.1 had the strongest effect. 059-1.30.1 performed best in promoting macrophage phagocytosis and inducing apoptosis in Jurkat cells.

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Abstract

抗CD47单克隆抗体及其应用。提供的抗CD47单抗能有效抑制肿瘤生长，阻断人SIRP与人CD47信号可以促进巨噬细胞对肿瘤细胞的吞噬作用，阻止肿瘤细胞逃避肿瘤免疫的防御系统，发挥抗肿瘤的作用。阻断肿瘤细胞表面CD47与巨噬细胞表面的SIRP的结合，可以阻断肿瘤细胞的"别吃我"信号，促进巨噬细胞对肿瘤细胞的识别摄取，促进肿瘤细胞被吞噬。肿瘤细胞表面的CD47与巨噬细胞表面SIRP的结合是一种普遍的"别吃我"信号，抗CD47抗体作为肿瘤免疫系统中的非常有前景的靶点，在人类肿瘤治疗方面发挥强大而有效的作用。

Description

抗CD47单克隆抗体及其应用
本申请要求于2016年06月17日提交中国专利局、申请号为201610436519.3、发明名称为“抗CD47单克隆抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及抗体药物技术领域，尤其涉及抗CD47单克隆抗体及其应用。
背景技术
CD47
CD47，也称作整联蛋白相关蛋白(IAP)，最初从人胎盘与整合素aVβ3共纯化及从血小板与β3整合素共免疫沉淀而为人所知，是一种广泛表达于细胞表面的跨膜糖蛋白，属于免疫球蛋白超家族。
CD47是细胞表面至关重要的标记物，分子量在47-55kD之间，在结构上包括一个氨基端细胞外可变区域，一个由3-5个高度疏水的跨膜片段构成的跨膜区域和一个亲水的羧基端胞质尾区。其与多种配体诸如整联蛋白、SIRPα(信号调节蛋白α)、SIRPγ和血小板反应蛋白相互作用。
SIRPα
信号调节蛋白α(SIRPα)也是一种跨膜蛋白，主要表达于巨噬细胞、树突状细胞和神经细胞表面。其胞外区含有3个免疫球蛋白超家族样区域，其中N末端的区域介导与CD47的结合，而其细胞内结构域具有典型的免疫受体酪氨酸抑制性序列(ITIM)。当与CD47结合后，SIRPα的ITIM被磷酸化，产生级联反应，抑制巨噬细胞的吞噬作用。
CD47/SIRPα参与肿瘤免疫的逃逸机制
在先天免疫系统中，CD47作为自身标志物、通过结合由髓样细胞诸如巨噬细胞、中性粒细胞和树突细胞表达的SIRPα而传递抑制性“别吃我”信号而发挥功能。因此，CD47在生理条件下广泛表达的作用是保护健康细胞免于被先天免疫系统消除。然而，肿瘤细胞则通过过表达CD47而有效逃脱免疫监视。
近年来，CD47和CD47-SIRPα信号系统受到广泛关注。其中，最令人瞩目的是其作为肿瘤治疗的潜在药靶。已有研究证实，CD47表达在大部分人类癌症(例如，NHL、AML、乳腺癌、结肠癌、成胶质细胞瘤、神经胶质瘤、卵巢癌、膀胱癌和前列腺癌)中上调，并且提高的CD47表达水平与侵袭性疾病和差的存活相关。坦福大学的Weissman系统地研究了多种实体瘤中CD47的表达水平，结果表明，所有人类实体瘤细胞中的CD47都呈高表达，其平均表达水平是对应正常细胞的3.3倍左右。而且，他们发现实体瘤病人CD47mRNA的水平与预后指数呈负相关。
进一步针对原位免疫缺陷性小鼠异种移植动物模型的实验发现，抗CD47单克隆抗体的施用能够抑制大型肿瘤的生长和转移，而对于小型肿瘤则可以治愈。
Willingham等人还在原位小鼠乳腺癌模型实验中证实了抗CD47单克隆抗体的有效性和安全性。该项研究不仅证实了高表达CD47是肿瘤细胞逃避免疫监视的普遍机制，也为通过阻断CD47-SIRPα信号通路来治疗肿瘤提供了有重要价值的借鉴。
治疗性抗CD47抗体
CD47在多类肿瘤中高表达并作为“别吃我”信号抑制吞噬作用，意味着靶向CD47-SIRPα通路可作为多类肿瘤的治疗方法。
RAUH等人通过体内外实验证实阻断性抗CD47单克隆抗体能促进巨噬细胞对肿瘤细胞进行吞噬，抑制急性粒细胞白血病(AML)在小鼠体内形成，并消除已在体内移植成功的AML，还可靶向清除白血病干细胞(LSC)。CHAO等人对急性淋巴细胞白血病的研究发现抗CD47单克隆抗体联合利妥昔单抗不仅可消除原始移植部位的肿瘤，还能消除血液循环及向肝、脾、淋巴结等部位扩散的肿瘤，达到长期生存且抑制肿瘤复发的效果，而单独使用抗CD47单克隆抗体或抗CD20单克隆抗体只能抑制NHL的生长速度却不能完全消除NHL。
为进一步证实抗CD47单克隆抗体对肿瘤的作用，WILLINGHAM等人用有免疫活性的小鼠建立移植瘤模型，抗鼠及抗人CD47单克隆抗体均可显著抑制肿瘤的生长，并证实抗C D47抗体可消除多种实体瘤肿并抑制肿瘤的转移及复发，此外抗C D47单克隆抗体对肿瘤干细胞(CSC)及其分化亚型也有抗瘤作用，并能将致瘤性的TAM、转变成抗瘤性的效应因子并增强其吞噬作用。抑制小鼠CD47的表达还能增强肿瘤细胞对放疗的敏感性，而对正常组织具有保护作用，这一作用可能与诱导宿主免疫细胞产生保护性自噬反应有关。
抗CD47单克隆抗体治疗肿瘤与多种机制有关。首先，抗CD47单克隆抗体通过阻断肿瘤细胞上的CD47与巨噬细胞上的SIRPα结合而使肿瘤细胞被吞噬。其次，与抗体依赖性的细胞介导的细胞毒作用和补体依赖性的细胞毒作有关，研究发现抗CD47抗体可诱导NK细胞参与的抗头颈肿瘤细胞的细胞毒作用。再次，通过直接诱导凋亡而清除肿瘤细胞。最后，对有免疫活性的小鼠进行研究发现抗CD47单克隆抗体可激活CD8+T细胞，引起获得性T细胞免疫反应，进一步杀伤肿瘤细胞。
随着人们对肿瘤分子发生机制研究的不断深入，分子免疫治疗逐渐成为除手术及化学药物治疗之外的另一有效的治疗手段。目前生物治疗在肿瘤治疗中的作用逐年提高，生物治疗在防止肿瘤复发、治疗晚期癌症及其并发症等诸多方面具有诸多优势。因此，存在对于能够靶向CD47的抗体和治疗的需要。
发明内容
有鉴于此，本发明要解决的技术问题在于提供抗CD47单克隆抗体及其应用，本发明提供的抗CD47单克隆抗体能够结合人CD47及猴CD47，可呈剂量依赖性阻断人SIRP与人CD47的结合；促进巨噬细胞对肿瘤细胞的吞噬作用。通过阻断SIRP与人CD47的结合信号，阻止肿瘤细胞逃避肿瘤免疫的防御系统，发挥抗肿瘤的作用。
本发明提供的抗CD47单克隆抗体具有重链可变区和轻链可变区：
(Ⅰ)所述重链可变区的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4或SEQ ID NO:5或SEQ ID NO:6或SEQ ID NO:7所示；
(Ⅱ)所述轻链可变区的氨基酸序列如SEQ ID NO:8或SEQ ID NO:9或SEQ ID NO:10或SEQ ID NO:11或SEQ ID NO:12或SEQ ID NO:13或SEQ ID NO:14所示；
(Ⅲ)(Ⅰ)或(Ⅱ)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(Ⅰ)或(Ⅱ)所示的氨基酸序列功能相同或相似的氨基酸序列；或
(Ⅳ)与(Ⅰ)或(Ⅱ)所述序列至少有80％同源性的氨基酸序列。本发明中，抗CD47的单克隆抗体中，
在本发明的一些具体实施方案中，所述抗CD47单克隆抗体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氨基酸序列中，所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个或32个。
所述取代发生于高变区；
所述重链可变区的高变区为HVR-H1，HVR-H2，HVR-H3；
SEQ ID NO:2的高变区HVR-H1序列如SEQ ID NO:45所示；HVR-H2序列如SEQ ID NO:46所示；HVR-H3序列如SEQ ID NO:47所示；
SEQ ID NO:5的高变区HVR-H1序列如SEQ ID NO:48所示；HVR-H2序列如SEQ ID NO:49所示；HVR-H3序列如SEQ ID NO:50所示；
SEQ ID NO:6的高变区HVR-H1序列如SEQ ID NO:51所示；HVR-H2序列如SEQ ID NO:52所示；HVR-H3序列如SEQ ID NO:53所示；
所述轻链可变区的高变区为HVR-L1，HVR-L2，HVR-L3；
SEQ ID NO:9的高变区HVR-L1序列如SEQ ID NO:54所示；HVR-L2序列如SEQ ID NO:55所示；HVR-L3序列如SEQ ID NO:56所示；
SEQ ID NO:12的高变区HVR-L1序列如SEQ ID NO:57所示；HVR-L2序列如SEQ ID NO:58所示；HVR-L3序列如SEQ ID NO:59所示；
SEQ ID NO:13的高变区HVR-L1序列如SEQ ID NO:60所示；HVR-L2序列如SEQ ID NO:61所示；HVR-L3序列如SEQ ID NO:62所示。
其重链可变区的氨基酸序列如SEQ ID NO:1～7任一项所示；
其轻链可变区的氨基酸序列如SEQ ID NO:8～14任一项所示。
在本发明的一些具体实施方案中，所述抗CD47单克隆抗体具有：
(ⅰ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:8的轻链可变区；
(ⅱ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:9的轻链可变区；
(ⅲ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸 序列为SEQ ID NO:10的轻链可变区；
(ⅳ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:11的轻链可变区；
(Ⅴ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:12的轻链可变区；
(Ⅵ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:13的轻链可变区；
(Ⅶ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:14的轻链可变区。
本发明提供的抗CD47单克隆抗体的重链类型为IgG1，IgG3或IgM；其轻链类型为κ
本发明还提供了编码所述抗CD47单克隆抗体的核苷酸。
在本发明的一些具体实施方案中，所述核苷酸具有
(Ⅰ)如SEQ ID NO:15-21所示的重链可变区的核苷酸序列；如SEQ ID NO:22-28所示的轻链可变区的核苷酸序列；或
(Ⅱ)如SEQ ID NO:15-21所示的重链可变区的核苷酸序列的互补序列；如SEQ ID NO:22-28所示的轻链可变区的核苷酸序列的互补序列；或
(Ⅲ)与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的序列；或
(Ⅳ)与(Ⅰ)或(Ⅱ)或(Ⅲ)所述序列至少有80％同源性的序列。
在本发明的一些具体实施方案中，所述核苷酸具有与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸获得的、且与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列功能相同或相似的核苷酸序列。
在本发明的一些具体实施方案中，所述核苷酸具有与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸获得的核苷酸序列，所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个或32个。
本发明还提供了一种表达载体，包括本发明提供的编码抗CD47单克隆抗体的核苷酸。
本发明还提供了一种宿主细胞，转化本发明所述的表达载体。
本发明还提供了一种抗原，其具有如SEQ ID NO:29～32任一项所示的氨基酸序列。
本发明还提供了产生本发明所述抗CD47单克隆抗体的杂交瘤细胞株。
本发明提供的抗CD47单克隆抗体的制备方法包括：
步骤1：以本发明提供的抗原免疫小鼠后，获得小鼠的脾细胞；
步骤2：融合所述脾细胞和骨髓瘤细胞，筛选能够与CD47结合的杂交瘤细胞株， 经体外培养，制得抗CD47单克隆抗体。
本发明所述抗CD47单克隆抗体经化学标记或生物标记制得的结合物。
所述化学标记为同位素、免疫毒素和/或化学药物；
所述生物标记为生物素、亲和素或酶标记。
本发明还提供了所述抗CD47单克隆抗体或的结合物与固体介质或半固体介质偶联制得的偶联物。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备检测CD47表达的产品中的应用。
本发明还提供了一种试剂盒，包括所述抗CD47单克隆抗体、结合物和/或偶联物。
一种疾病的诊断方法，以本发明提供的试剂盒检测CD47表达，根据CD47的表达量判断是否患有疾病；
所述疾病为白血病、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。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备阻断CD47与SIRPα结合的制剂中的应用。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备提高巨噬细胞对肿瘤细胞吞噬指数的制剂中的应用。
本发明实施例中，肿瘤细胞为人外周血白血病T细胞。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备促进肿瘤细胞凋亡的制剂中的应用。
本发明的实施例中，肿瘤细胞为人外周血白血病T细胞。
所述抗CD47单克隆抗体、结合物和/或偶联物在制备防治疾病的药物中的应用；
所述疾病为白血病、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。
本发明还提供了一种药物，包括本发明所述抗CD47单克隆抗体、其结合物和/或偶联物。
一种疾病的防治方法，给予本发明所述的药物；所述疾病为白血病、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。
本发明提供的抗CD47单抗能有效抑制肿瘤生长；阻断人SIRP与人CD47信号可以促进巨噬细胞对肿瘤细胞的吞噬作用，阻止肿瘤细胞逃避肿瘤免疫的防御系统，发挥抗肿瘤的作用。阻断肿瘤细胞表面CD47与巨噬细胞表面的SIRP的结合，可以阻断肿瘤细胞的“别吃我”信号，促进巨噬细胞对肿瘤细胞的识别摄取，促进肿瘤细胞被吞噬。肿瘤细胞表面的CD47与巨噬细胞表面SIRP的结合是一种普遍的“别吃我”信号，抗CD47抗体可以作为肿瘤免疫系统中的非常有前景的靶点，在人类肿瘤治疗方面发挥强大而有效的作用。
附图说明
图1示SDS-PAGE电泳检测纯化的人和猴的CD47；泳道M：蛋白质分子量标记；图1-A：泳道1：人CD47-连接肽-hIgG1Fc；泳道2:人CD47-连接肽-His；图1-B,泳道1：鼠CD47-连接肽-hIgG1Fc；泳道2:鼠CD47-连接肽-His；
图2示SDS-PAGE电泳检测阳性抗体(PAB)，泳道M：蛋白质分子量标记；泳道1：非还原PAB；泳道2：还原PAB；
图3示非还原SDS-PAGE电泳检测纯化的候选抗体；泳道M:蛋白质分子量标记；泳道1:059-1.11.1纯化抗体；泳道2:059-1.20.1纯化抗体；泳道3:059-1.30.1纯化抗体；泳道4:059-1.43.1纯化抗体；泳道5:059-1.51.2纯化抗体；泳道6：059-1.82.1纯化抗体；泳道7：059-1.100.5纯化抗体；
图4示总RNA电泳检测；M：DL2000分子量标记，泳道1-7：分别为059-1.11.1、059-1.20.1、059-1.30.1、059-1.43.1、059-1.51.2、059-1.82.1、059-1.100.5总RNA电泳条带；
图5示PCR扩增候选抗体重链可变区和轻链可变区琼脂糖电泳检测结果；
图6示抗体059-1.20.1、059-1.30.1、059-1.43.1、059-1.82.1阻断人CD47与SIRPα的结果图；
图7示抗体059-1.20.1、059-1.30.1、059-1.43.1、059-1.82.1与jurkat细胞表面CD47结合的FACS测定实验；
图8示抗体059-1.20.1、059-1.30.1、059-1.43.1、059-1.82.1促进小鼠腹腔原代巨噬细胞对Jurkat细胞的吞噬。
具体实施方式
本发明提供了抗CD47单克隆抗体及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
“抗体”是指由能特异结合抗原的一种或多种多肽构成的蛋白质。抗体的一种形式构成了抗体的基本结构单元。这种形式是四聚物，它由两对完全相同的抗体链构成，每一对都有一个轻链和一个重链。在每对抗体链中，轻链和重链的可变区联合在一起共同负责结合抗原，而恒定区则负责抗体的效应器功能。
抗体重链或轻链的“可变区”是该链的N端成熟区域。目前已知的抗体类型包括κ和λ轻链，以及α，γ(IgG1，IgG2，IgG3，IgG4)，δ，ε和μ重链或它们的其它类型等价物。全长的免疫球蛋白“轻链”(大约25kDa或大约214个氨基酸)包含一个由NH2-末端上大约110个氨基酸形成的可变区，以及一个COOH-末端上的κ或λ恒定区。全长的免疫球蛋白“重链”(大约50kDa或大约446个氨基酸)，同样包含一个可变区(大约116个氨基酸)，以及重链恒定区之一，例如γ(大约330个氨基酸)。
“抗体”包括任何同型体的抗体或免疫球蛋白，或保持与抗原特异结合的抗体片段，包括但不限于Fab，Fv，scFv和Fd片段、嵌合抗体、人源化抗体、单链抗体以及包含抗体的抗原结合部分和非抗体蛋白质的融合蛋白质。抗体可以被标记和检测，例如，可以通过放射性同位素、能产生可检测物的酶、荧光蛋白质、生物素等等进行标记并被检测。抗体还可以结合于固相载体，包括但不限于聚苯乙烯平板或珠粒等等。
本发明提供了一种抗CD47单克隆抗体具有重链可变区和轻链可变区：
(Ⅰ)所述重链可变区的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4或SEQ ID NO:5或SEQ ID NO:6或SEQ ID NO:7所示；
(Ⅱ)所述轻链可变区的氨基酸序列如SEQ ID NO:8或SEQ ID NO:9或SEQ ID NO:10或SEQ ID NO:11或SEQ ID NO:12或SEQ ID NO:13或SEQ ID NO:14所示；
(Ⅲ)(Ⅰ)或(Ⅱ)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(Ⅰ)或(Ⅱ)所示的氨基酸序列功能相同或相似的氨基酸序列；或
(Ⅳ)与(Ⅰ)或(Ⅱ)所述序列至少有80％同源性的氨基酸序列。
在本发明的一些具体实施方案中，所述抗CD47单克隆抗体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氨基酸序列中，所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个或32个。
所述取代发生于高变区；
所述重链可变区的高变区为HVR-H1，HVR-H2，HVR-H3；
SEQ ID NO:2的高变区HVR-H1序列如SEQ ID NO:45所示；HVR-H2序列如SEQ ID NO:46所示；HVR-H3序列如SEQ ID NO:47所示；
SEQ ID NO:5的高变区HVR-H1序列如SEQ ID NO:48所示；HVR-H2序列如SEQ ID NO:49所示；HVR-H3序列如SEQ ID NO:50所示；
SEQ ID NO:6的高变区HVR-H1序列如SEQ ID NO:51所示；HVR-H2序列如SEQ ID NO:52所示；HVR-H3序列如SEQ ID NO:53所示；
所述轻链可变区的高变区为HVR-L1，HVR-L2，HVR-L3；
SEQ ID NO:9的高变区HVR-L1序列如SEQ ID NO:54所示；HVR-L2序列如SEQ ID NO:55所示；HVR-L3序列如SEQ ID NO:56所示；
SEQ ID NO:12的高变区HVR-L1序列如SEQ ID NO:57所示；HVR-L2序列如SEQ ID NO:58所示；HVR-L3序列如SEQ ID NO:59所示；
SEQ ID NO:13的高变区HVR-L1序列如SEQ ID NO:60所示；HVR-L2序列如SEQ ID NO:61所示；HVR-L3序列如SEQ ID NO:62所示。
在本发明的一些具体实施方案中，所述抗CD47单克隆抗体具有：
(ⅰ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:8的轻链可变区；
(ⅱ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸 序列为SEQ ID NO:9的轻链可变区；
(ⅲ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:10的轻链可变区；
(ⅳ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:11的轻链可变区；
(Ⅴ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:12的轻链可变区；
(Ⅵ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:13的轻链可变区；
(Ⅶ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:14的轻链可变区；
本发明提供的抗CD47单克隆抗体其重链类型为IgG1，IgG3或IgM；其轻链类型为κ。
具体的，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻 链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
在本发明的实施例中，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示。
或，抗CD47的单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:14所示。
本发明的抗体059-1.82.1的重链恒定区为鼠IgG3，轻链恒定区为鼠κ链的恒定区，059-1.30.1、059-1.43.1和059-1.20.1都为鼠IgG1，轻链恒定区为鼠κ链的恒定区，059-1.11.1、059-1.51.2、059-1.100.5的重链恒定区为鼠IgM,重链可变区的氨基酸序列为SEQ ID NO:1-7中的一个，轻链可变区的氨基酸序列为SEQ ID NO:8-14中的一个。
本发明提供的抗CD47单抗能够结合人CD47及猴CD47；在某些实施方案中，抗体与其靶点之间的亲和力用Ka、Kd(解离常数)、KD(平衡解离常数)来表征，本发明提供抗体的KD值不高于30nM。
本发明提供的抗CD47单抗可呈剂量依赖性阻断人SIRP与人CD47的结合；其阻断效果通过EC50值表示，本发明提供抗体的EC50值不低于850nM。
本发明提供的抗CD47单抗能够结合细胞表面CD47；该效果的检测通过FACS法进行，其结果通过MFI(荧光强度)表示，本发明提供抗体与细胞表面CD47结合的MFI值不低于9547，最高可达18533。
本发明提供的抗CD47单抗能够促进巨噬细胞对肿瘤细胞的吞噬作用，该效果通过荧光成像法测定，结果以吞噬指数表示。本发明提供抗体对jurkat细胞的吞噬指数可达79。
本发明提供的抗CD47单抗还能够诱导肿瘤细胞的凋亡，该效果通过流失细胞仪检测细胞凋亡率表示，结果表明，本发明提供抗体能够诱导jurkat细胞的凋亡，凋亡率可达48％。
Jurkat细胞属急性T细胞白血病细胞系，属多种恶性肿瘤细胞中的一种，与其他恶性肿瘤相同，jurkat细胞表面CD47呈高表达。本发明通过对jurkat的实验证明，本发明提供的CD47单抗能够通过阻断SIRP与人CD47的结合信号，阻止肿瘤细胞逃避肿瘤免疫的防御系统，发挥抗肿瘤的作用。
本发明提供的单抗中，重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:9所示；重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示；重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示；重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:13所示，的单克隆抗体与人CD47、猴CD47皆具有良好的亲和力，其能够阻断CD47与SIRPα的结合，能够与细胞表面CD47结合，促进巨噬细胞吞噬肿瘤细胞，并诱导肿瘤细胞的凋亡。
其中，重链可变区的氨基酸序列如SEQ ID NO:4所示，轻链可变区的氨基酸序列如SEQ ID NO:11所示的单克隆抗体阻断CD47与SIRPα的结合的效果最佳；其与肿瘤细胞表面CD47结合的效果也最佳，其诱导巨噬细胞吞噬肿瘤细胞的效果也最佳。但在诱导肿瘤细胞凋亡方面，重链可变区的氨基酸序列如SEQ ID NO:3所示，轻链可变区的氨基酸序列如SEQ ID NO:10所示的单克隆抗体则具有更优的效果。
本发明还提供了编码所述抗CD47单克隆抗体的核苷酸。
在本发明的一些具体实施方案中，所述核苷酸具有
(Ⅰ)如SEQ ID NO:15-21所示的重链可变区的核苷酸序列；如SEQ ID NO:22-28所示的轻链可变区的核苷酸序列；或
(Ⅱ)如SEQ ID NO:15-21所示的重链可变区的核苷酸序列的互补序列；如SEQ ID NO:22-28所示的轻链可变区的核苷酸序列的互补序列；或
(Ⅲ)与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的序列；或
(Ⅳ)与(Ⅰ)或(Ⅱ)或(Ⅲ)所述序列至少有80％同源性的序列。
在本发明的一些具体实施方案中，所述核苷酸具有与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸获得的、且与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列功能相同或相似的核苷酸序列。
在本发明的一些具体实施方案中，所述核苷酸具有与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸获得的核苷酸序列，所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个或32个。
本发明还提供了一种表达载体，包括本发明提供抗CD47单克隆抗体重链可变区和/或轻链可变区的核苷酸。
本发明还提供了一种宿主细胞，转化本发明所述的表达载体。
本发明还提供了一种抗原，其具有如SEQ ID NO:29～32任一项所示的氨基酸序列。
本发明还提供了产生本发明所述抗CD47单克隆抗体的杂交瘤细胞株。
本发明提供的抗CD47单克隆抗体的制备方法包括：
步骤1：以本发明提供的抗原免疫小鼠后，获得小鼠的脾细胞；
步骤2：融合所述脾细胞和骨髓瘤细胞，筛选能够与CD47结合的杂交瘤细胞株，经体外培养，制得抗CD47单克隆抗体。
本发明中，步骤1中所述抗原的氨基酸序列如SEQ ID NO:29或31所示。
本发明中，抗原与佐剂混合后对小鼠进行免疫。
抗原与佐剂的体积比为1:1。
所述免疫具体为首次免疫后二周进行第二次免疫，3天后区血清效价大于1:200000的小鼠进行加强免疫。
首次免疫、第二次免疫和加强免疫的剂量以抗原质量计皆为10μg。
免疫采用两点注射。
首次免疫的佐剂为弗氏完全佐剂，第二次免疫和加强免疫的佐剂为弗氏不完全佐剂。
免疫采用的小鼠为BALB/C小鼠。
骨髓瘤细胞为P3X63Ag8.653。
以脾细胞与骨髓瘤细胞的个数比为5:1进行融合。
所述筛选采用HAT培养基筛选。
所述与CD47结合具体为能与CD47蛋白结合且能够与表面表达CD47蛋白的细胞结合。
经鉴定，采用本发明提供方法制备的CD47单抗，重链的类型为IgG3、IgM或IgG1类，轻链的类型为κ。
本发明所述抗CD47单克隆抗体经化学标记或生物标记制得的结合物。
所述化学标记为同位素、免疫毒素和/或化学药物；
所述生物标记为生物素、亲和素或酶标记。
所述酶标记优选为辣根过氧化物酶或碱性磷酸酶。
所述免疫毒素优选为黄曲霉毒素、白喉毒素、绿脓杆菌外毒素、蓖麻毒蛋白、相思子毒蛋白、槲寄生凝集素、蒴莲根毒素、PAP、造草素、白树毒素或丝瓜毒素。
本发明还提供了所述抗CD47单克隆抗体或的结合物与固体介质或半固体介质偶联制得的偶联物。
所述固体介质或非固体介质选自胶体金、聚苯乙烯平板或珠粒。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备检测CD47表达的产品中的应用。
实验表明，本发明提供的CD47单抗能够与CD47蛋白相结合，也可与表面表达CD47的细胞相结合。故而，本发明提供的CD47单抗能够用于CD47蛋白或表面表达CD47的细胞的检测。并且，由于肿瘤细胞表面标志物CD47的高表达，本发明提供的抗体能够制备用于肿瘤表面标志物CD47检测的试剂盒。其中，CD47蛋白的检测采用ELISA法，表面表达CD47的细胞的检测采用FACS法。
本发明还提供了一种试剂盒，包括所述抗CD47单克隆抗体、结合物和/或偶联物。
检测CD47蛋白的试剂盒中，还包括包被缓冲液、洗涤液、封闭液和/或显色液。
所述包被缓冲液为碳酸盐缓冲液。
所述洗涤液中包括PBS、Tween、氯化钠、氯化钾、磷酸氢二钠、磷酸氢二钾。
所述封闭液中包括PBS和BSA。
所述显色液包括TMB溶液、底物缓冲液和终止液。
所述底物缓冲液包括柠檬酸和磷酸氢二钠。
所述终止液为过氧化氢水溶液。
检测表面表达CD47的细胞的试剂盒中，还包括PBS、羊抗鼠IgG Fc和TITC二抗。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备阻断CD47与SIRPα结合的制剂中的应用。
所述表面表达CD47的细胞为肿瘤细胞。
所述肿瘤细胞选自白血病细胞、淋巴瘤细胞、乳腺癌细胞、肺癌细胞、胃癌细胞、肠癌细胞、食管癌细胞、卵巢癌细胞、宫颈癌细胞、肾癌细胞、膀胱癌细胞、胰腺癌细胞、神经胶质瘤细胞和/或黑素瘤细胞。
一种疾病的诊断方法，以本发明提供的试剂盒检测CD47表达，根据CD47的表达量判断是否患有疾病；
所述疾病为白血病、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。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备阻断CD47与SIRPα结合的制剂中的应用。
本发明所述的抗CD47单抗阻断CD47与SIRPα结合的EC50值为850nM～2340nM。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备提高巨噬细胞对肿瘤细胞吞噬指数的制剂中的应用。
本发明所述的抗CD47单抗提高巨噬细胞对肿瘤细胞吞噬指数的剂量以抗体浓度计为10μg/mL。
本发明实施例中，肿瘤细胞为人外周血白血病T细胞。
本发明所述抗CD47单克隆抗体、结合物和/或偶联物在制备促进肿瘤细胞凋亡的制剂中的应用。
本发明的实施例中，肿瘤细胞为人外周血白血病T细胞。
本发明所述的抗CD47单抗促进肿瘤细胞凋亡的剂量为10μg/mL。
所述抗CD47单克隆抗体、其结合物和/或偶联物在制备防治肿瘤的药物中的应用；
所述疾病为白血病、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。
本发明还提供了一种药物，包括本发明所述抗CD47单克隆抗体、结合物和/或偶 联物。
一种疾病的防治方法，给予本发明所述的药物；所述疾病为白血病、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。
本发明提供的药物能够通过阻断可以阻断肿瘤细胞的“别吃我”信号，促进巨噬细胞对肿瘤细胞的识别摄取，促进肿瘤细胞被吞噬。
本发明提供的药物的剂型为注射液剂或注射粉针剂。
所述注射液中，抗体的浓度为10μg/mL。
本发明提供的抗CD47单抗能有效抑制肿瘤生长；阻断人SIRP与人CD47信号可以促进巨噬细胞对肿瘤细胞的吞噬作用，阻止肿瘤细胞逃避肿瘤免疫的防御系统，发挥抗肿瘤的作用。阻断肿瘤细胞表面CD47与巨噬细胞表面的SIRP的结合，可以阻断肿瘤细胞的“别吃我”信号，促进巨噬细胞对肿瘤细胞的识别摄取，促进肿瘤细胞被吞噬。肿瘤细胞表面的CD47与巨噬细胞表面SIRP的结合是一种普遍的“别吃我”信号，抗CD47抗体可以作为肿瘤免疫系统中的非常有前景的靶点，在人类肿瘤治疗方面发挥强大而有效的作用。
本发明采用的仪器皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1抗原蛋白及阳性对照抗体的制备
1.1抗原基因合成及表达载体构建：
融合人源和猴源CD47蛋白胞外区氨基酸序列与连接肽-hIgG1Fc或连接肽-7his氨基酸序列，氨基酸序列设计如SEQ ID NO:29,30,31,32所示：
上述设计的人和猴CD47蛋白胞外区融合蛋白(CD47ECD-连接肽-hIgG1Fc或CD47ECD-连接肽-7his)所对应的氨基酸序列进行密码子人工优化，如SEQ ID NO:33，34,35,36在5’端加上HindⅢ酶切位点和Kozak序列GCCGCCACC，3’端加上终止密码子TAG和EcoR I酶切位点，并委托金斯瑞公司合成优化后的DNA，克隆到pUC57simple(金斯瑞公司提供)载体中，获得人和猴pUC57simple-CD47-连接肽-hIgG1Fc和或CD47ECD-连接肽-7his质粒。
酶切(HindⅢ和EcoR I)人和猴的质粒pUC57simple-CD47-连接肽-hIgG1Fc、pUC57simple-CD47ECD-连接肽-7his和载体pcDNA3.1，电泳回收得到的融合基因片段CD47-连接肽-hIgG1Fc和CD47ECD-连接肽-7his，并与pcDNA3.1载体进行连接反应重组构建获得表达质粒：
pcDNA3.1-人CD47-连接肽-hIgG1Fc；
pcDNA3.1-人CD47ECD-连接肽-7his；
pcDNA3.1-猴CD47-连接肽-hIgG1Fc；
pcDNA3.1-猴CD47ECD-连接肽-7his；
阳性抗体基因合成及表达载体构建
pAb抗体序列如下：
PABH，如SEQ ID NO:37,38所示：
PABL，如SEQ ID NO:39,40所示：
上述的抗体序列所对应的氨基酸序列进行密码子人工优化，在5’端加上HindⅢ酶切位点和Kozak序列GCCGCCACC，3’端加上终止密码子TAG和EcoR I酶切位点，并委托金斯瑞公司合成优化后的DNA，克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-PCABH，pUC57simple-PCABL质粒，将质粒pUC57simple-PCABH，pUC57simple-PCABL进行酶切后(HindⅢ和EcoR I)，电泳回收得到的基因片段PCABH，PCABL，并与pcDNA3.1载体进行连接反应重组构建获得pcDNA3.1-PCABH、pcDNA3.1-PCABL。
1.2瞬转表达
对pcDNA3.1-PCABH、
pcDNA3.1-PCABL.
pcDNA3.1-人CD47-连接肽-hIgG1Fc；
pcDNA3.1-人CD47ECD-连接肽-7his；
pcDNA3.1-猴CD47-连接肽-hIgG1Fc；
pcDNA3.1-猴CD47ECD-连接肽-7his；进行瞬时表达。
使用FreeStyleTM293F细胞在Freestyle培养基中进行瞬转表达。转染前24小时，在125ml锥形瓶中接种0.6×106细胞/ml的293F细胞，37℃5％CO2温箱中130rpm摇床培养。转染时先取60微升的293fectin加入到1ml的OPtiMEM中，充分混匀后，室温孵育5分钟；同时将重组载体和按3:2:1的比例进行混合，总DNA的量为30μg，溶于1ml的OPtiMEM中。然后将DNA和293fectin充分混合，总体积为2ml，室温孵育15分钟，然后将混合物全部加入细胞培养孔中，混匀，37℃5％CO2温箱中130rpm摇床培养7天。将培养液进行高速离心、微孔滤膜抽真空过滤。
1.3蛋白纯化
根据厂家提供的操作方法采用Protein A柱(蛋白纯化液相色谱系统/AKTA Purifier 10,GE)进行纯化，得到纯化的人PDL-1-mIgG2aFc融合蛋白。AKTA用超纯水冲洗干净连接1ml rProtianA Fast Flow预装柱到AKTA上；清洗：用1M HAC清洗5个柱体积。平衡：20mM PB 0.15M NaCl(pH 7.0)平衡5个柱体积。
上样：将细胞表达上清样品1000rpm×5min离心，取上清，8000rpm×30min离心，取20ml离心后上清上样，流速0.2ml/min。平衡：20mM PB 0.15M NaCl(pH 7.0)平衡5个柱体积，0.2ml/min。清洗：20mM PB 1M NaCl(pH 7.0)清洗5个柱体积。平衡：20mM PB 0.15M NaCl(pH 7.0)平衡5个柱体积。
洗脱：20mM柠檬酸钠缓冲液(pH 3.0)洗脱，流速0.2ml/min。UV280至100mAu时开始收集，降至100mAu时停止收集。用1M Tris将样品pH调节至pH6～8，如图1～2。
实施例2单克隆杂交瘤的制备
1、小鼠的免疫：
以抗原与佐剂的体积为1:1的比例对免疫原人CD47-hFc(实施例1制得)进行乳化，首次免疫使用弗氏完全佐剂乳化抗原，2周后，开始第二次免疫，使用弗氏不完全佐剂乳化抗原，皮下2点注射，每只小鼠注射的抗原的量为10μg，每个注射点注射的体积为25μL。
在第二次免疫后3天，对小鼠进行眼眶采血，取少量血液样本进行血清效价检测，经间接ELISA方法检测血清效价滴度达到1:200000或者以上后，对小鼠进行加强免疫。
共进行3组免疫，每组为5只小鼠。
2、饲养细胞和骨髓瘤细胞的准备
饲养细胞的准备，剪开正常BALB/C小鼠(拉颈处死)的腹部皮肤。暴露腹膜。用注射器吸取DMEM培养基后，注入小鼠腹腔中，洗涤吸取出腹腔巨噬细胞，收集于离心管中1500rmp/min离心3min，取下层褐色沉淀重悬后备用。重复上述步骤获取3只正常小鼠的腹腔巨噬细胞。
骨髓瘤细胞准备，提前一周复苏P3X63Ag8.653，并用含1X 8-氮鸟嘌呤的完全培养基培养，融合前两天换用15％胎牛血清的DMEM培养，维持P3X63Ag8.653的密度为70％-80％至融合当天。
3、细胞融合及HAT筛选：
脾细胞的获取和制备：取加强免疫后的小鼠2只(标记为L1、L2)，采集L1、L2的免疫血清后处死并浸泡于75％酒精中。剪开免疫小鼠的腹部侧面的皮肤和腹膜，暴露脾脏。用剪尖剔除周围组织获取脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。
细胞融合前处理：收集培养瓶内的P3X63Ag8.653，1000rpm/5min离心后弃上清，重悬后进行活细胞计数。2000rpm/5min离心脾细胞悬液，弃上清后重悬并进行活细胞计数。记录P3X63Ag8.653活细胞数，脾细胞活细胞数。
细胞融合：按脾细胞：P3X63Ag8.653＝5:1的比例混合细胞，2000rpm/5min离心后倒净上清，震散细胞沉淀，在37℃水浴中缓慢滴加1mL预热的50％PEG1500溶液，并使管底在37℃水中划圈摇动，上述操作时间控制在1min左右，静置反应30s，于管内由慢到快地加入37℃预热的DMEM培养基，终止反应。将终止反应后的细胞悬液800rpm离心3min后弃去上清，轻轻震散细胞沉淀。
HAT培养基筛选：配制含1×HAT、1×青-链霉素、15％胎牛血清和85％DMEM培养基的HAT筛选培养基。用上述的HAT筛选培养基重悬鼠杂交瘤细胞和饲养细胞，并混合两者。按300μl/孔将细胞悬液加到20块96孔细胞培养板中，置于37℃细胞培养箱中培养。培养1周后，用HT培养基进行第一次换液，并置于37℃细胞培养箱中培养；培养3天后，用HT培养基进行第二次换液。
4、阳性细胞株的筛选
融合后2周，取细胞上清进行ELISA实验，检测细胞上清与人CD47-his蛋白的结合情况，筛选出ELISA结果为阳性的细胞后，进行第二次Elisa实验的复测；取复测结果为阳性的细胞上清进行FACS实验，检测细胞上清与jurkat细胞表面CD47蛋白的结合情况。
5、扩大培养
将ELISA和FACS检测为双阳性的细胞株从96孔中转入24孔中培养，待长满后转入25cm2培养瓶中培养。
6、有限稀释法亚克隆
吹打混匀阳性细胞株，并吸取少量进行活细胞计数。吸取约100个细胞加入40mL完全培养基中混匀，铺板2块；另吸取约100个细胞加入20mL完全培养基中混匀，铺板1块；另吸取约1000个细胞加入20mL完全培养基中混匀，铺板1块；共以3个不同的细胞密度铺板4块，分别为0.5细胞/孔,1细胞/孔,10细胞/孔。将96孔板置37℃5％CO2培养箱中培养。
7、克隆检测和扩大培养
取单克隆细胞孔上清进行ELISA和FACS实验，分别检测细胞克隆抗体与人、猴CD47-his标签的结合及与jurkat细胞表面CD47蛋白的结合情况。将ELISA和FACS检测为双阳性的细胞株(共7株，分别标记为059-1.11.1、059-1.20.1、059-1.30.1、059-1.43.1、059-1.51.2、059-1.82.1、059-1.100.5)从96孔中转入24孔中培养，待长满后转入25cm2培养瓶中培养。
8、亚类的鉴定
包被羊抗鼠IgG(Fc)，2μg/ml，每孔50μL，4℃过夜，BSA封闭，加入待测细胞上清，室温，2小时，加入酶标亚类二抗IgG1，IgG2a，IgG2b，IgG2c，IgG3，κ，λ(abcam)，显色，450nm读数，判断所测细胞株的亚类为IgG3或IgM或IgG1，κ。
其中，抗体059-1.82.1的重链恒定区为鼠IgG3，轻链恒定区为鼠κ链的恒定区，059-1.30.1、059-1.43.1和059-1.20.1都为鼠IgG1，轻链恒定区为鼠κ链的恒定区，059-1.11.1、059-1.51.2、059-1.100.5的重链恒定区为鼠IgM,重链可变区的氨基酸序列为SEQ ID NO:1-7中的一个，轻链可变区的氨基酸序列为SEQ ID NO:8-14中的一个。
在加液前每步用PBST洗涤3次。
9、细胞冻存
冻存液配制：90％胎牛血清，10％DMSO。
重悬培养瓶内细胞，细胞计数后，以1500rpm/min离心3min后弃去上清，用含10％DMSO的胎牛血清进行吹打悬浮，以每管5×106细胞数冻存于冻存盒内，-80℃过夜，次日转入液氮中。
10、单克隆杂交瘤基因保存
取阳性单克隆细胞株RNA抽提液提取RNA,并反转录成cDNA,于-80℃永久保存。
实施例3单克隆抗体制备及鉴定
1.采用体外培养法制备抗体
对实施例2制得的杂交瘤细胞株进行复苏，方法为将含有10％胎牛血清、1％青链霉素的DMEM培养基复苏并使用小瓶培养，待细胞汇合度约90％后，进行传代扩培，扩培至细胞培养上清共约200mL。
2.抗体纯化
收集培养约7天左右的细胞上清，测量体积(约200mL)，加入NaCl使上清中NaCl为2.5M，经0.22μm混合纤维素微孔滤膜真空过滤后，4℃保存，Protein A亲和层析进行抗体纯化。上样：含2.5M NaCl的细胞培养上清经0.22μm虑膜过滤后浓缩至30ml后直接上样；流洗：pH 7.4 2.5M PBS流洗，冲至UV280基线为0；洗脱：pH3.50.1M柠檬酸溶液，每段2ml收集洗脱液，每管加入100μl 1M Tris溶液；浓缩收集液，使用PBS洗脱至初始成分比例小于0.1％。跑SDS-PAGE胶核实纯化抗体纯度。(图3)
实施例4单克隆抗体基因测序和重组抗体的制备
1.单克隆抗体基因测序
经免疫、融合以及单克隆化后，基于亲和力实验结果选取059-1.11.1、059-1.20.1、059-1.30.1、059-1.43.1、059-1.51.2、059-1.82.1、059-1.100.5、单克隆抗体细胞株进行总RNA提取，并反转录成cDNA，然后以cDNA为模板PCR扩增抗体的重链可变区和轻链可变区。
抗体基因重链和轻链的序列分析。7株单克隆抗体总RNA采用Invitrogen公司的
Figure PCTCN2017088013-appb-000001
reagent试剂盒(15596-026)试剂盒按照其说明书进行提取，提取结果见图4。
接着采用Takara公司的5’RACE FULL试剂盒(D315)，以总RNA为模板，试剂盒中的随机引物进行反转录为第一链cDNA，然后重链以恒定区设计引物(mIgG R)和试剂盒中的接头引物进行PCR扩增，轻链以恒定区设计引物(mIgK R)和试剂盒中的接头引物进行PCR扩增。mIgG R和mIgK R的序列如下:
mIgG R：CTCAGGGAARTARCCYTTGAC，(如SEQ ID NO:41所示)；
mIgK R：TCACTGCCATCAATCTTCCAC，(如SEQ ID NO:42所示)；
7个单克隆抗体细胞株的重链可变区和轻链可变区PCR扩增后电泳检测如图5，
琼脂糖凝胶回收试剂盒回收PCR片段进行TA克隆后调单克隆进行PCR鉴定，鉴定引物为M13-F和M13-R，鉴定正确热菌株中选取部分样品送至Invitrogen测序。最终确定重链可变区蛋白序列为SEQ ID NO:1～7；轻链可变区蛋白序列为SEQ ID NO:8～14；重链核苷酸序列为SEQ ID NO:15～21；轻链核苷酸序列为SEQ ID NO:22～28：
鉴定引物M13-F，如下：
5'TGTAAAACGACGGCCAGT3'，(如SEQ ID NO:43所示)；
鉴定引物M13-R，如下：
5'CAGGAAACAGCTATGACC3'，(如SEQ ID NO:44所示)。
表1序列
Figure PCTCN2017088013-appb-000002
实施例5亲和力测定及竞争性ELISA检测
1.与人CD47结合测定(ELISA)
包被：hCD47-his用PBS稀释成1μg/ml，加至酶标板96孔中，每孔100μL，4℃下孵育过夜。封闭：洗板三次后用1％BSA+PBS封闭，每孔300μL，室温条件下孵育2小时。加候选抗体：洗板3次后，加入候选抗体的细胞培养上清或阳性对照或阴性对照。每孔100μl，室温条件下2小时。加二抗：洗板3次后，加入山羊抗小鼠IgG Fc,HRP(1:10000)，每孔100μL，室温条件下反应1小时。显色：洗板4次后，加TMB显色液，每孔100μL，室温下避光显色10分钟。终止：直接加入100μL每孔的终止液终止反应，。检测：终止反应后立即把酶标板放入酶标仪中，在450nm处测其OD值，保存原始数据整理如下表2：
表2候选克隆与人CD47的ELISA检测OD450值
抗体名称 OD450
059-1.11.1 3.6718
059-1.20.1 4.0
059-1.30.1 4.0
059-1.43.1 4.0
059-1.51.2 0.7709
059-1.82.1 4.0
059-1.100.5 0.81
阴性对照 0.086
2.与猴CD47结合测定(ELISA)
包被：猴CD47-his用PBS稀释成1μg/ml，加至酶标板96孔中，每孔100μL，4℃下孵育过夜。封闭：洗板三次后用1％BSA+PBS封闭，每孔300μL，室温条件下孵育2小时。加候选抗体：洗板3次后，加入候选抗体的细胞培养上清或阴性对照。每孔100μl，室温条件下2小时。加二抗：洗板3次后，加入山羊抗小鼠IgG Fc,HRP (1:10000)，每孔100μL，室温条件下反应1小时。显色：洗板4次后，加TMB显色液，每孔100μL，室温下避光显色10分钟。终止：直接加入100μL每孔的终止液终止反应，。检测：终止反应后立即把酶标板放入酶标仪中，在450nm处测其OD值，保存原始数据整理如下表3：
表3候选克隆与猴CD47的ELISA检测OD值
抗体名称 OD450
059-1.11.1 4
059-1.20.1 4
059-1.30.1 4
059-1.43.1 4
059-1.51.2 0.6336
059-1.82.1 3.896
059-1.100.5 0.6166
阴性对照 0.0883
3.与人CD47蛋白的亲和力测定
使用Biacore T200仪器检测人CD47抗体的亲和力常数,通过氨基共价结合将抗小鼠Fc抗体(GE Healthcare公司,BR-1008-38)偶联在CM5生物传感芯片(GE Healthcare公司)上，芯片上的抗小鼠Fc抗体捕获候选单克隆抗体或阳性对照B6H12(商业化CD47抗体，购自Abcam，货号：ab3283)，不同浓度的人CD47以30μL/min流速流过过芯片上的候选抗体，人CD47与候选抗体进行结合，结合时间为120s，解离时间为300s.用BIAevalution软件(GE Healthcare公司)进行动力学拟合，亲和力最高为059_1.82.1获得亲和力常数结果如下表4：
表4候选克隆与人CD47的亲和力测定结果
抗体名称 Ka(1/Ms) Kd(1/s) KD(nM)
059-1.11.1 2.60E5 0.004015 15.5
059-1.20.1 2.17E5 0.00548 25.3
059-1.30.1 4.24E5 0.01276 30.1
059-1.43.1 2.36E5 0.006657 28.3
059-1.51.2 3.70E5 0.01009 27.3
059-1.82.1 2.56E5 0.003643 14.2
059-1.100.5 1.57E5 0.0069 44.0
阳性对照 1.32E5 0.003636 27.5
实施例6剂量依赖性阻断人CD47与人SIRP的结合(ELISA法)
包被：人CD47-hFc用PBS稀释成2μg/ml，加至酶标板96孔中，每孔100μL，4℃下孵育过夜。封闭：洗板3次后用1％BSA+PBS封闭，每孔300μL，室温下孵育1小时。候选抗体与SIRP-his的混合：纯化的候选抗体分别用PBST稀释成20μg/ml， 以PBST溶液进行3倍梯度稀释，共稀释7个梯度；人SIRP-his蛋白用PBST稀释成500ng/mL,1:1混合不同稀释梯度的候选抗体和人SIRP-his蛋白，室温下孵育30min。加候选抗体与人SIRP-his蛋白的混合物：每孔100μL,室温下反应1h，对照孔加入IgG同种型对照与人SIRP-his蛋白的混合物。加二抗：洗板3次后，加入抗His标签抗体,HRP(1:3000)，每孔100μL，室温条件下反应1小时。显色：洗板4次后，加TMB显色液，每孔100μL，室温下避光显色10分钟。终止：直接加终止液终止反应，每孔100μL。检测：终止反应后立即把酶标板放入酶标仪中，在450nm处测其OD值，保存原始数据。数据处理：将原始数据输入到软件SoftMax Pro 6.2.1中进行数据处理。结果如表5、图6所示(图中的数据为最终计算所得数据)。
表5候选抗体阻断CD47与SIRP结合的结果
抗体名称 EC50(nM)
059-1.11.1 2340
059-1.20.1 1190
059-1.30.1 1030
059-1.43.1 850
059-1.51.2 --
059-1.82.1 1030
059-1.100.5 --
阳性对照 1190
阴性对照 --
结果表明候选抗体059-1.11.1、059-1.20.1、059-1.30.1、059-1.43.1和059-1.82.1均有较强的阻断效应，059-1.51.2和059-1.100.5没有阻断人CD47和人SIRP结合的效应。
实施例7与细胞表面CD47结合的测定(FACS法)
培养Jurkat细胞后收集，2000rpm离心5min，PBS洗涤1次后进行活细胞计数；每管加入2.0×105的细胞，2000rpm离心5min去上清，每管加入100μL候选抗体的细胞上清，阳性对照管加入1ng/mL的B6H12抗体(商业化CD47抗体，购自Abcam，货号：ab3283)100μL,阴性对照管加入100μL DMEM培养基，室温下反应1小时；2000rpm离心5min，PBS洗涤3次，每管加入4ng/mL山羊抗小鼠IgG Fc，FITC二抗100μL,室温条件下反应1小时；2000rpm离心5min，PBS洗3次；每管加入300μL PBS，转移至流式上样管中，上机测试。结果见表6、图7所示。
表6候选抗体与Jurkat细胞表面CD47结合的结果
Figure PCTCN2017088013-appb-000003
Figure PCTCN2017088013-appb-000004
结果显示，候选抗体与细胞表面CD47有明显的结合活性。
实施例8CD47抗体介导小鼠腹腔原代巨噬细胞对Jurkat细胞的吞噬实验
1.C57鼠腹腔原代巨噬细胞制备
引颈杀死动物，手提鼠尾将全鼠浸入70％酒精中3～5秒。置动物于解剖台上，用针头固定四肢，双手持镊撕开皮肤拉向两侧，暴露出腹膜，用70％酒精擦洗腹膜壁后，用注射器注10ml Eagle液入腹腔中，同时从两侧用手指揉压腹膜壁，令液体在腹腔内充分流动。用针头轻轻挑起腹壁，使动物体微倾向一侧，使腹腔中液体集于针头下吸取入针管内。小心拔出针头，把液体注入离心管中，250g 4℃离心10分钟，去上清，加10ml Eagle培养基，计数细胞。为获取3×105贴附细胞/平方厘米，需接种2.5×106/ml。为纯化培养细胞，去除其它白细胞，接种数小时后，去除培养液，用Eagle液冲洗1～2次后，再加新Eagle培养液置37℃，5％CO2温箱中培养。
2.巨噬细胞吞噬实验
通过荧光成像法考察实施例5制备的CD47抗体对C57BL小鼠腹腔原代巨噬细胞对Jurkat细胞吞噬作用的影响。提前一天用PKH26染料对小鼠巨噬细胞进行染色(4微摩、5min)，以2万细胞/孔接种于96孔板；次日用CFSE染料染色Jurkat细胞(2微摩、10min)，清洗后Jurkat细胞重悬于无血清培养基中，以8万细胞/孔接种于巨噬细胞上。接板Jurkat细胞前2小时将巨噬细胞中有血清培养基换成无血清培养基，将各种抗体以10微克/ml的浓度加入到两种混悬细胞中。培养2小时后，洗去未被吞噬的Jurkat细胞，荧光显微镜进行细胞成像后，通过计算每100个巨噬细胞中吞噬多少Jurkat细胞，即吞噬指数，来定量分析CD47抗体促吞噬作用的优劣。结果见图8(吞噬效果图)和表7(吞噬指数)。B6H12(商业化CD47抗体，购自Abcam，货号：ab3283)，与文献报道一致，促吞噬作用较弱。实施例5四个筛选出的抗体分子，都有良好促吞噬作用，尤其是059-1.30.1，059-1.43.1,和059-1.82.1.
表7CD47抗体促进巨噬细胞对Jurkat细胞的吞噬
Figure PCTCN2017088013-appb-000005
Figure PCTCN2017088013-appb-000006
实施例9CD47抗体体外诱导Jurkat细胞凋亡实验
收集对数生长的Jurkat细胞，无血清培养基清洗后，用5％FBS-1640制备单细胞悬液，将细胞重悬至10×105/ml，按照5×105/孔，即500μl/孔加入24孔板培养。实验设计加入CD47抗体的(终浓度为10μg/ml)，50μl/孔，设置anti-Fas阳性对照孔，无需加入抗体的孔则用相同体积的培养基补入。5h后收集细胞悬液到1.5ml EP管中，离心500g×5min。弃上清，各管加入100μl PBS重悬混匀细胞，膜联蛋白-V(Roche Diagnostics)4℃避光染色30分钟。PBS清洗3次，各管加入500μl PBS重悬混匀细胞，流式上机前10-15min加入PI(终浓度为1μg/ml)流式上机检测膜联蛋白-V阳性和膜联蛋白-V、PI双阳性细胞占总细胞比例即为Jurkat细胞的凋亡率。结果见表8：
表8CD47抗体诱导Jurkat细胞凋亡
抗体名称 凋亡率(％)
059-1.20.1 33
059-1.30.1 48
059-1.43.1 37
059-1.82.1 24
对照抗体(B6H12) 8
阴性对照 5
B6H12不诱导Jurkat细胞凋亡，而测定四个059抗体都不同程度诱导Jurkat细胞的凋亡，059-1.30.1作用最强。059-1.30.1在促进巨噬细胞吞噬和诱导Jurkat细胞凋亡均表现最佳。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2017088013-appb-000007
Figure PCTCN2017088013-appb-000008
Figure PCTCN2017088013-appb-000009
Figure PCTCN2017088013-appb-000010
Figure PCTCN2017088013-appb-000011
Figure PCTCN2017088013-appb-000012
Figure PCTCN2017088013-appb-000013
Figure PCTCN2017088013-appb-000014
Figure PCTCN2017088013-appb-000015
Figure PCTCN2017088013-appb-000016
Figure PCTCN2017088013-appb-000017
Figure PCTCN2017088013-appb-000018
Figure PCTCN2017088013-appb-000019
Figure PCTCN2017088013-appb-000020
Figure PCTCN2017088013-appb-000021
Figure PCTCN2017088013-appb-000022
Figure PCTCN2017088013-appb-000023
Figure PCTCN2017088013-appb-000024
Figure PCTCN2017088013-appb-000025
Figure PCTCN2017088013-appb-000026
Figure PCTCN2017088013-appb-000027
Figure PCTCN2017088013-appb-000028
Figure PCTCN2017088013-appb-000029
Figure PCTCN2017088013-appb-000030
Figure PCTCN2017088013-appb-000031
Figure PCTCN2017088013-appb-000032
Figure PCTCN2017088013-appb-000033
Figure PCTCN2017088013-appb-000034
Figure PCTCN2017088013-appb-000035
Figure PCTCN2017088013-appb-000036
Figure PCTCN2017088013-appb-000037
Figure PCTCN2017088013-appb-000038
Figure PCTCN2017088013-appb-000039
Figure PCTCN2017088013-appb-000040

Claims (28)

  1. 一种抗CD47单克隆抗体，其特征在于，其具有重链可变区和轻链可变区：
    (Ⅰ)所述重链可变区的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4或SEQ ID NO:5或SEQ ID NO:6或SEQ ID NO:7所示；
    (Ⅱ)所述轻链可变区的氨基酸序列如SEQ ID NO:8或SEQ ID NO:9或SEQ ID NO:10或SEQ ID NO:11或SEQ ID NO:12或SEQ ID NO:13或SEQ ID NO:14所示；
    (Ⅲ)(Ⅰ)或(Ⅱ)所述氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(Ⅰ)或(Ⅱ)所示的氨基酸序列功能相同或相似的氨基酸序列；或
    (Ⅳ)与(Ⅰ)或(Ⅱ)所述序列至少有80％同源性的氨基酸序列。
  2. 根据权利要求1所述的抗CD47单克隆抗体，其特征在于，所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个或32个。
  3. 根据权利要求1所述的抗CD47单克隆抗体，其特征在于，所述取代发生于高变区；
    所述重链可变区的高变区为HVR-H1，HVR-H2，HVR-H3；
    SEQ ID NO:2的高变区HVR-H1序列如SEQ ID NO:45所示；HVR-H2序列如SEQ ID NO:46所示；HVR-H3序列如SEQ ID NO:47所示；
    SEQ ID NO:5的高变区HVR-H1序列如SEQ ID NO:48所示；HVR-H2序列如SEQ ID NO:49所示；HVR-H3序列如SEQ ID NO:50所示；
    SEQ ID NO:6的高变区HVR-H1序列如SEQ ID NO:51所示；HVR-H2序列如SEQ ID NO:52所示；HVR-H3序列如SEQ ID NO:53所示；
    所述轻链可变区的高变区为HVR-L1，HVR-L2，HVR-L3；
    SEQ ID NO:9的高变区HVR-L1序列如SEQ ID NO:54所示；HVR-L2序列如SEQ ID NO:55所示；HVR-L3序列如SEQ ID NO:56所示；
    SEQ ID NO:12的高变区HVR-L1序列如SEQ ID NO:57所示；HVR-L2序列如SEQ ID NO:58所示；HVR-L3序列如SEQ ID NO:59所示；
    SEQ ID NO:13的高变区HVR-L1序列如SEQ ID NO:60所示；HVR-L2序列如SEQ ID NO:61所示；HVR-L3序列如SEQ ID NO:62所示。
  4. 根据权利要求1所述的抗CD47单克隆抗体，其特征在于，其具有：
    (ⅰ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:8的轻链可变区；
    (ⅱ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:9的轻链可变区；
    (ⅲ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:10的轻链可变区；
    (ⅳ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:11的轻链可变区；
    (Ⅴ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:12的轻链可变区；
    (Ⅵ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:13的轻链可变区；
    (Ⅶ)氨基酸序列为SEQ ID NO:1,2,3,4,5,6,7的重链可变区，和氨基酸序列为SEQ ID NO:14的轻链可变区。
  5. 根据权利要求1～4任一项所述的抗CD47单克隆抗体，其特征在于，其重链类型为IgG1，IgG3或IgM；其轻链类型为κ。
  6. 编码如权利要求1～5任一项所述的抗CD47单克隆抗体的核苷酸。
  7. 根据权利要求6所述的核苷酸，其特征在于，具有
    (Ⅰ)如SEQ ID NO:15-21所示的重链可变区的核苷酸序列；如SEQ ID NO:22-28所示的轻链可变区的核苷酸序列；或
    (Ⅱ)如SEQ ID NO:15-21所示的重链可变区的核苷酸序列的互补序列；如SEQ ID NO:22-28所示的轻链可变区的核苷酸序列的互补序列；或
    (Ⅲ)与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的序列；或
    (Ⅳ)与(Ⅰ)或(Ⅱ)或(Ⅲ)所述序列至少有80％同源性的序列。
  8. 根据权利要求6或7所述的核苷酸，其特征在于，具有与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸而获得的、且与(Ⅰ)或(Ⅱ)或(Ⅲ)或(Ⅳ)所示的核苷酸序列功能相同或相似的核苷酸序列。
  9. 根据权利要求6至8任一项所述的核苷酸，其特征在于，所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个或32个。
  10. 一种表达载体，其特征在于，包括权利要求6～9任一项所述的核苷酸。
  11. 一种宿主细胞，其特征在于，转化权利要求10所述的表达载体。
  12. 一种抗原，其特征在于，具有如SEQ ID NO:29～32任一项所示的氨基酸序列。
  13. 产生权利要求1～5任一项所述抗CD47单克隆抗体的杂交瘤细胞株。
  14. 权利要求1～5任一项所述抗CD47单克隆抗体的制备方法，其特征在于，包括：
    步骤1：以权利要求12所述的抗原免疫小鼠后，获得小鼠的脾细胞；
    步骤2：融合所述脾细胞和骨髓瘤细胞，筛选能够与CD47结合的杂交瘤细胞株，经体外培养，制得抗CD47单克隆抗体。
  15. 权利要求1～5任一项所述抗CD47单克隆抗体经化学标记或生物标记制得的 结合物。
  16. 根据权利要求15所述的结合物，其特征在于，所述化学标记为同位素、免疫毒素和/或化学药物；所述生物标记为生物素、亲和素或酶标记。
  17. 权利要求1～5任一项所述抗CD47单克隆抗体或权利要求15～16任一项所述的结合物与固体介质或半固体介质偶联制得的偶联物。
  18. 权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物在制备检测CD47表达的产品中的应用。
  19. 一种试剂盒，其特征在于，权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物。
  20. 权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物在制备阻断CD47与SIRPα结合的制剂中的应用。
  21. 权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物在制备提高巨噬细胞对肿瘤细胞吞噬指数的制剂中的应用。
  22. 根据权利要求21所述的应用，其特征在于，所述肿瘤细胞为人外周血白血病T细胞。
  23. 权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物在制备促进肿瘤细胞凋亡的制剂中的应用。
  24. 根据权利要求23所述的应用，其特征在于，所述肿瘤细胞为人外周血白血病T细胞。
  25. 权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物在制备防治疾病的药物中的应用；
    所述疾病为白血病、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。
  26. 一种药物，其特征在于，包括权利要求1～5任一项所述抗CD47单克隆抗体、权利要求15～16任一项所述的结合物和/或权利要求17所述的偶联物。
  27. 一种疾病的诊断方法，其特征在于，以权利要求19所述的试剂盒检测CD47表达，根据CD47的表达量判断是否患有疾病；
    所述疾病为白血病、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。
  28. 一种疾病的防治方法，其特征在于，给予权利要求26所述的药物；所述疾病为白血病、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。
PCT/CN2017/088013 2016-06-17 2017-06-13 抗cd47单克隆抗体及其应用 WO2017215585A1 (zh)

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