WO2021197401A1 - 结合cd47的抗原结合多肽及用途 - Google Patents

结合cd47的抗原结合多肽及用途 Download PDF

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WO2021197401A1
WO2021197401A1 PCT/CN2021/084802 CN2021084802W WO2021197401A1 WO 2021197401 A1 WO2021197401 A1 WO 2021197401A1 CN 2021084802 W CN2021084802 W CN 2021084802W WO 2021197401 A1 WO2021197401 A1 WO 2021197401A1
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seq
amino acid
acid sequence
identity
antigen
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PCT/CN2021/084802
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French (fr)
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WO2021197401A8 (zh
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桂秋
朱虹
王亮亮
张晓云
邹筱芳
申恒巧
徐宏江
杨玲
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正大天晴药业集团股份有限公司
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Priority to CN202180017777.7A priority Critical patent/CN115190887A/zh
Priority to AU2021250200A priority patent/AU2021250200A1/en
Priority to EP21780305.5A priority patent/EP4130042A1/en
Priority to KR1020227037553A priority patent/KR20220163991A/ko
Priority to CA3177519A priority patent/CA3177519A1/en
Priority to BR112022019795A priority patent/BR112022019795A2/pt
Priority to JP2022560185A priority patent/JP2023519620A/ja
Priority to IL296829A priority patent/IL296829A/en
Publication of WO2021197401A1 publication Critical patent/WO2021197401A1/zh
Publication of WO2021197401A8 publication Critical patent/WO2021197401A8/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to antigen-binding polypeptides, particularly to CD47-binding antigen-binding polypeptides and antigen-binding portions thereof, and to methods for using such antigen-binding polypeptides and antigen-binding portions thereof.
  • CD47 is a transmembrane glycoprotein widely expressed in multiple species and various tissues. It is also called integrin-related protein and is a member of the immunoglobulin superfamily. CD47 is about 50kD, which contains 1 extracellular Ig-like variable domain, 5 highly hydrophobic extended transmembrane fragments, and 1 short selectively spliced carboxy-terminal cytoplasmic tail.
  • SIRP ⁇ Inhibitory receptor signal regulator protein ⁇
  • CD47 binds to the IgV-like domain of the NH 2 terminal of SIRP ⁇ .
  • SIRP ⁇ is mainly expressed in cells of myeloid origin, including macrophages, granulocytes, DC cells, mast cells and their precursors, including hematopoietic stem cells.
  • CD47 can inhibit the phagocytic function of macrophages by binding to the protein SIRP ⁇ on the surface of macrophages.
  • PCD Programmed cell death
  • phagocytic cell removal are common ways for organisms to respond in order to remove damaged, precancerous or infected cells. Therefore, cells that survive in response to this organism (cancerous cells, long-term infected cells, etc.) have ways to evade PCD and phagocytic cell removal.
  • CD47 is constitutively upregulated in various types of diseased cells, cancer cells, and infected cells, allowing these cells to escape phagocytosis.
  • Anti-CD47 compounds that block the interaction between CD47 on one cell (cancerous cells, long-infected cells, etc.) and SIRP ⁇ on another cell (phagocytes) can counteract the increase in CD47 expression and promote cancer Phagocytosis of cells and/or infected cells.
  • CD47 expression and/or activity has been implicated in many diseases and disorders. Different studies have shown that almost all tumor cells and tumor tissues highly express CD47. CD47, which is highly expressed on the surface of tumor cells, binds to SIRP ⁇ on the surface of macrophages to release the "don't eat me" signal, which causes the macrophages in the tumor tissue infiltration area to not only get along with the tumor cells, but also It also promotes the proliferation of blood vessels in tumors, inhibits effector T cells, and promotes tumor cell expansion and growth. Therefore, there is a need for targeted CD47 therapy.
  • the present invention provides a new isolated CD47-binding antigen-binding polypeptide and antigen-binding portion thereof, and provides a solution for the treatment of targeting CD47.
  • the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, wherein the antigen-binding polypeptide comprises the following complementarity determining regions:
  • Heavy chain CDR1 which comprises an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5;
  • the heavy chain CDR2 which comprises an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10;
  • the heavy chain CDR3 which comprises an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15;
  • the light chain CDR1 which comprises an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20;
  • a light chain CDR2 which comprises an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25;
  • the light chain CDR3 includes an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30, or 32.
  • sequence shown in SEQ ID NO: 32 is QQFSX 2 STWT, and X 2 is D or E.
  • the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, wherein the antigen-binding polypeptide comprises the following complementarity determining regions:
  • Heavy chain CDR1 which comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5 or conservatively modified forms thereof;
  • Heavy chain CDR2 which comprises an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10 or a conservatively modified form thereof;
  • Heavy chain CDR3 which comprises an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15 or a conservatively modified form thereof;
  • Light chain CDR1 which comprises an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20 or a conservatively modified form thereof;
  • the light chain CDR2 which comprises an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25 or a conservatively modified form thereof;
  • the light chain CDR3 includes an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30, or 32 or a conservatively modified form thereof.
  • the present invention provides an isolated antigen-binding polypeptide that binds CD47 or an antigen-binding portion thereof, wherein the antigen-binding polypeptide comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises An amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, 41, 83, 85, or 87, and the light chain variable region includes an amino acid sequence selected from SEQ ID NO: : The amino acid sequence of 34, 36, 38, 40, 42, 84, 86 or 88 has at least 80% identity.
  • sequence shown in SEQ ID NO: 83 is:
  • SEQ ID NO: 84 The sequence shown in SEQ ID NO: 84 is:
  • sequence shown in SEQ ID NO: 85 is:
  • SEQ ID NO: 86 The sequence shown in SEQ ID NO: 86 is:
  • DX 14 QMTQSPSSLSASVGDRVTITCRSSQSLVHSNGYTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPPTFGQGTKLEIK, where X 14 is I or A;
  • SEQ ID NO: 87 The sequence shown in SEQ ID NO: 87 is:
  • QVQLVQSGAEVKKPGASVKVSCKX 15 SGFNIEDDYIEWVRQAPGQGLEWMGRIDPANDKTKYAQKFQGRVTMTX 16 DTSTX 17 TVYMELSSLRSEDTAVYYCX 18 RPGLRRYYSMDYWGQGTLVTVSS, wherein, X 15 is A or V, X 16 is R or G, X 17 is S or N, X 18 is A, or T;
  • SEQ ID NO: 88 The sequence shown in SEQ ID NO: 88 is:
  • the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47, wherein the antigen-binding polypeptide or antigen-binding portion thereof exhibits one or a combination of the following properties:
  • the melting temperature T of the antigen-binding polypeptide or its antigen-binding portion is greater than or equal to 62°C, and the polymerization temperature Tagg is greater than or equal to 61°C.
  • the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof, wherein the antigen-binding polypeptide or antigen-binding portion thereof binds the same on CD47 as any of the exemplary antigen-binding polypeptides or antigen-binding portions thereof provided herein gauge.
  • the present invention provides an isolated antigen-binding polypeptide or antigen-binding portion thereof, wherein the antigen-binding polypeptide or antigen-binding portion thereof competes with any of the exemplary antigen-binding polypeptides or antigen-binding portions thereof provided herein for binding to CD47, wherein The competition is measured by ELISA, flow cytometry or surface plasmon resonance assay.
  • the antigen binding portion may be selected from Fab fragment, Fab' fragment, F(ab') 2 fragment, Fd fragment, Fv fragment, dAb fragment, isolated complementarity determining region and Nanobody, but is not limited thereto.
  • the present invention provides an immunoconjugate comprising the antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 as described herein, and a therapeutic agent linked or conjugated to the antigen-binding polypeptide or antigen-binding portion thereof .
  • the antigen-binding polypeptide or antigen-binding portion thereof may be connected to the therapeutic agent through a linker.
  • the linker can be cleavable or non-cleavable.
  • the therapeutic agent may be selected from cytotoxic drugs, radioisotopes or immunomodulators, such as chemotherapeutics, immunosuppressants, immunostimulants, antimetabolites, alkylating agents, antibiotics, anti-angiogenic agents, Anti-mitotic agents, toxins, apoptosis agents, etc.
  • cytotoxic drugs such as chemotherapeutics, immunosuppressants, immunostimulants, antimetabolites, alkylating agents, antibiotics, anti-angiogenic agents, Anti-mitotic agents, toxins, apoptosis agents, etc.
  • the present invention provides a composition
  • a composition comprising component A and a pharmaceutically acceptable carrier, wherein component A is the CD47-binding antigen-binding polypeptide or antigen-binding portion thereof as described herein, or as described herein Of immunoconjugates.
  • the present invention provides an isolated hybridoma cell line selected from the group consisting of 2B2, 2H8, 3F10, 16E5, and 14A9.
  • the present invention provides an isolated nucleic acid that encodes the CD47-binding antigen-binding polypeptide or antigen-binding portion thereof described herein.
  • the invention provides a vector comprising the isolated nucleic acid described herein.
  • the present invention provides a host cell which contains the vector described herein or has integrated the isolated nucleic acid described herein in its genome.
  • the vector is an expression vector.
  • the present invention provides a method for preparing a CD47-binding antigen-binding polypeptide or antigen-binding portion thereof, comprising: under conditions suitable for expressing a nucleic acid encoding the CD47-binding antigen-binding polypeptide or antigen-binding portion thereof as described herein
  • the host cell described herein is cultivated, and the expressed antigen-binding polypeptide or antigen-binding portion thereof is isolated.
  • the present invention provides a method for reducing tumors or inhibiting the growth of tumor cells in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the CD47-binding antigen binding agent described herein.
  • the method comprises administering to the subject a therapeutically effective amount of the CD47-binding antigen binding agent described herein.
  • the present invention provides a method for treating cancer in a subject in need, wherein the method comprises administering to the subject a therapeutically effective amount of the CD47-binding antigen-binding polypeptide described herein or The antigen-binding portion thereof, the immunoconjugate described herein, or the composition described herein.
  • the present invention provides a method for promoting phagocytosis of macrophages in a subject, wherein the method comprises administering to the subject an effective amount of the CD47-binding antigen-binding polypeptide or antigen-binding polypeptide thereof described herein Part, the immunoconjugate described herein, or the composition described.
  • Figure 1 shows the effect of anti-CD47 chimeric antibody on erythrocyte agglutination; among them, the samples in the same row (antibody or control) have the same type but different concentrations, and the same column samples have the same concentration but different types; from left to right, the sample concentrations in different columns are in sequence Respectively 10000ng/ml, 3333.33ng/ml, 1111.11ng/ml, 370.37ng/ml, 123.46ng/ml, 41.15ng/ml, 13.72ng/ml, 4.57ng/ml; from top to bottom, different rows of samples They are Xi2B2, Xi2H8, Xi3F10, Xi16E5, Xi14A9, Xi16E5-2 (another repetition of Xi16E5), Hu5F9, IgG4, PBS, respectively.
  • Figure 2 shows the effect of partial anti-CD47 humanized antibodies on erythrocyte agglutination; among them, the same column has the same antibody type but different concentration, and the same row has the same antibody concentration but different type; from top to bottom, the antibody concentration of different rows is 10000ng respectively /ml, 3333.33ng/ml, 1111.11ng/ml, 370.37ng/ml, 123.46ng/ml, 41.15ng/ml, 13.72ng/ml, 4.57ng/ml; from left to right, the antibodies in columns 1-12 They are hz3F10-1.1, hz3F10-2.1, hz3F10-3.1, hz3F10-4.1, hz3F10-5.1, hz3F10-6.1, hz3F10-1.2, hz3F10-2.2, hz3F10-3.2, hz3F10-4.2, hz3F10-5.2, hz3F10-6.2
  • Figure 3 shows the effect of partial anti-CD47 humanized antibodies on erythrocyte agglutination; among them, the same column has the same antibody type but different concentration, and the same row has the same antibody concentration but different type; from top to bottom, the antibody concentration of different rows is 10000ng respectively /ml, 3333.33ng/ml, 1111.11ng/ml, 370.37ng/ml, 123.46ng/ml, 41.15ng/ml, 13.72ng/ml, 4.57ng/ml; from left to right, the antibodies in columns 1-8 They are hz16E5-1.1, hz16E5-3.1, hz16E5-1.2, hz16E5-3.2, hz16E5-1.3, hz16E5-3.3, hz14A9-2.3, hz14A9-2.4.
  • FIG. 4 shows the effect of anti-CD47 antibodies on the survival rate of mice.
  • Figure 5 shows the change of mouse body weight over time after a single administration of anti-CD47 antibody.
  • Figure 6 shows the weight change rate of mice at different times after a single administration of anti-CD47 antibody.
  • Figure 7 shows the change of mouse RBC over time after a single administration of anti-CD47 antibody.
  • Figure 8 shows the rate of change of mouse RBC at different times after a single administration of anti-CD47 antibody.
  • Figure 9 shows the changes in mouse HGB content over time after a single administration of anti-CD47 antibody.
  • Figure 10 shows the change rate of mouse HGB at different times after a single administration of anti-CD47 antibody.
  • Figure 11 shows the change of the average tumor volume with time after administration of anti-CD47 antibody in the MOLM-16 cell mouse model of human acute myeloid leukemia cells.
  • antigen-binding polypeptide refers to polypeptides and proteins having at least one antigen-binding domain.
  • antigen-binding polypeptides include, but are not limited to, monoclonal antibodies, multispecific antibodies, or fusion proteins.
  • antigen-binding portion of an antigen-binding polypeptide refers to one or more fragments of an antigen-binding polypeptide that retain the ability to specifically bind to an antigen (eg, CD47 protein). It has been shown that the antigen-binding function of an antigen-binding polypeptide can be performed by its fragments.
  • an antigen-binding portion of an antigen-binding polypeptide examples include: (i) Fab fragments, monovalent fragments composed of VL , VH , CL and CH1 domains; (ii) "Fab'fragments” and Fab The difference between the fragments is that several residues are added to the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody; (iii) F(ab') 2 fragments, including A bivalent fragment of two Fab fragments connected by a disulfide bridge in the hinge region; (iv) Fd fragment, composed of V H and CH1 domains; (v) Fv fragment, composed of VL and VH domains of one antibody arm ; (vi) dAb fragment (Ward et al (1989) Nature341: 544-546), the V H domains; (VII) an isolated complementarity determining region (CDRs of); and (viii) Nanobodies, comprising a single variable
  • the two domains of the Fv fragment, V L and V H encoded by separate genes may be used a synthetic linker linking them, so that they can be prepared as a single protein chain in which the V L and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. USA 85: 5879 -5883).
  • single chain antibodies are also intended to be encompassed by the term antigen binding polypeptide.
  • the antigen-binding portion herein can be obtained using conventional techniques well known to those skilled in the art, and can be used to screen fragments in the same manner as the complete antigen-binding polypeptide.
  • the term "isotype” refers to the antibody species encoded by the heavy chain constant region genes.
  • the CD47-binding antigen-binding polypeptide and its antigen-binding portion of the present invention can be derived from any species, including but not limited to mice, rats, rabbits, non-human primates (such as chimpanzees, cynomolgus monkeys, spider monkeys, Macaques), llamas and humans.
  • the CD47-binding antigen-binding polypeptide and its antigen-binding portion may be a chimeric antibody, a humanized antibody, or a complete human antibody.
  • the CD47-binding antigen-binding polypeptide is an antibody produced by a mouse-derived hybridoma cell line.
  • the CD47-binding antigen-binding polypeptide is a murine antibody. In other embodiments, the CD47-binding antigen-binding polypeptide is a chimeric antibody. In some embodiments, the chimeric antibody is a mouse-human chimeric antibody. In other embodiments, the CD47-binding antigen-binding polypeptide is a humanized antibody. In other embodiments, the CD47-binding antigen-binding polypeptide is derived from a murine antibody and is humanized.
  • chimeric antibody is an antibody that has at least a portion of a heavy chain variable region derived from one species and at least a portion of a light chain variable region; and at least a portion derived from a constant region of another species. Part.
  • chimeric antibodies may comprise murine variable regions and human constant regions.
  • a “humanized antibody” is an antibody that contains a complementarity determining region (CDR) derived from a non-human antibody; and a framework region and constant region derived from a human antibody.
  • the CD47-binding humanized antibodies provided herein can comprise CDRs derived from one or more murine antibodies as well as human framework and constant regions. Therefore, in some embodiments, the humanized antibody provided herein binds to the same epitope on CD47 as the murine antibody from which the CDR of the antibody is derived.
  • Exemplary humanized antibodies are provided herein. Additional CD47-binding humanized antibodies or variants thereof comprising the heavy chain CDRs and light chain CDRs provided herein can be produced using any human framework sequence and are also included in the present invention.
  • framework sequences suitable for use in the present invention include those framework sequences that are structurally similar to the framework sequences provided herein. Additional modifications can be made in the framework regions to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications, point mutations to reduce immunogenicity or remove T cell epitopes, or revert mutations to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including back mutations to the germline sequence. For example, in some embodiments, one or more amino acids in the human framework regions of the VH and/or VL of the humanized antibodies provided herein are backmutated to the corresponding amino acids in the parent murine antibody.
  • the light chain variable region has positions 2 and/or 33 and/or 48 and/or 63 and/or 68 and/or 71 and/or 72 and/or 94
  • the amino acid of is backmutated to the corresponding amino acid found at the position in the mouse 3F10, 14A9 or 16E5 light chain variable region.
  • the heavy chain variable region is at positions 24 and/or 46 and/or 63 and/or 72 and/or 73 and/or 74 and/or 77 and/or 97
  • the amino acid of was backmutated to the corresponding amino acid found at the position in the variable region of the mouse 3F10, 14A9 or 16E5 heavy chain.
  • the humanized 3F10 antibody comprises a heavy chain variable region whose mutation type is selected from one or more of the following positions: the amino acid at position 73 is mutated from Asp(D) to Glu(E) ; The amino acid at position 74 is mutated from Glu(E) to Ile(I); and the amino acid at position 77 is mutated from Ser(S) to Asn(N), and the humanized 3F10 antibody contains light chain variable Region, the mutation type is selected from one or more of the following positions: the amino acid at position 48 is mutated from Leu(L) to Trp(W); the amino acid at position 71 is mutated from Asp(D) to Ser( S); the amino acid at position 72 was mutated from Phe (F) to Tyr (Y); and the amino acid at position 94 was mutated from Asp (D) to Glu (E).
  • the humanized 14A9 antibody comprises a heavy chain variable region whose mutation type is selected from one or more of the following positions: the amino acid at position 24 is mutated from Ala(A) to Val(V) ; The amino acid at position 72 was mutated from Arg(R) to Gly(G); the amino acid at position 77 was mutated from Ser(S) to Asn(N); and the amino acid at position 97 was mutated from Ala(A) It is Thr(T), and the humanized 14A9 contains the light chain variable region, and its mutation type is selected from one or more of the following positions: the amino acid at position 63 is mutated from Ser(S) to Ile(I) ; The amino acid at position 68 is mutated from Gly(G) to Ser(S).
  • the humanized 16E5 antibody comprises a heavy chain variable region whose mutation type is selected from one or more of the following positions: the amino acid at position 46 is mutated from Glu(E) to Lys(K) ; The amino acid at position 63 is mutated from Lys(K) to Glu(E); the amino acid at position 72 is mutated from Thr(T) to Leu(L); the amino acid at position 77 is mutated from Ser(S) to Arg(R); and the amino acid at position 97 is mutated from Ala(A) to Thr(T), and the humanized 16E5 antibody contains a light chain variable region, and the mutation type is selected from one or more of the following positions One: the amino acid at position 2 is mutated from Ile (I) to Ala (A); and the amino acid at position 33 is mutated from Asn (N) to Gln (Q).
  • the description of the sequence number of the mutation site in this article starts counting sequentially from the first amino acid of the variable region. Additional or alternative back mutations can be made in the framework regions of the humanized antibodies provided herein to improve the properties of the antibodies.
  • the present invention also includes humanized antibodies that bind to CD47 and contain framework modifications corresponding to the exemplary modifications described herein relative to any suitable framework sequence, as well as those that improve the properties of the antibody in other ways Other frame decorations.
  • the term "derived" when used to refer to a molecule or polypeptide relative to a reference antibody or other binding protein means a molecule or polypeptide capable of specifically binding to the same epitope as the reference antibody or other binding protein.
  • isolated refers to a target compound (e.g., antibody or nucleic acid) that has been separated from its natural environment.
  • EC50 refers to the effective concentration, 50% of the maximum response of the antibody.
  • IC50 refers to the inhibitory concentration, 50% of the maximum response of the antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • K D when used herein refers to the dissociation constant, expressed in molar concentration (M).
  • M molar concentration
  • the K D value of the antibody can be measured using methods known in the art.
  • a preferred method for determining the K D of an antibody is to use surface plasmon resonance, and more preferably to use a biosensor system, such as a Biacore system.
  • treatment means to treat, cure, alleviate, alleviate, change, remedy, ameliorate, ameliorate, or affect a disease (such as a disease), symptoms of a disease, or prevent or delay symptoms, complications, or biochemistry in a statistically significant manner
  • a disease such as a disease
  • symptoms of a disease or prevent or delay symptoms, complications, or biochemistry in a statistically significant manner
  • terapéuticaally effective amount refers to the amount of a compound or composition necessary to provide a therapeutic and/or prophylactic benefit to a subject.
  • the term “subject” includes any human or non-human animal.
  • the term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, anchors, horses, cows, chickens, amphibians, reptiles, and the like.
  • the subject according to the invention is a human.
  • the terms “patient” or “subject” can be used interchangeably.
  • “about” means within the acceptable error range for a specific value determined by a person of ordinary skill in the art, and it depends in part on how the value is measured or determined, that is, the limitation of the measurement system. For example, “about” can mean within 1 time or more than 1 standard deviation according to practice in the field. Alternatively, “about” may mean a range of up to ⁇ 5%, such as fluctuating within ⁇ 2%, ⁇ 1%, or ⁇ 0.5% of a given specific numerical range. When a specific value is given in the application or the claims, unless otherwise stated, the meaning of "about” should be considered to be within an acceptable error range of the specific value. In this article, unless otherwise specified, the values of step parameters or conditions are modified by "about” by default.
  • identity also known as identity.
  • a mathematical algorithm can be used to compare sequences and determine the percent identity between two sequences. The algorithm of E. Meyers and W. Miller (Comput. Appl.
  • Biosci., 4:11-17 (1988)) can be used to determine the percent identity between two amino acid sequences, which has been included in the ALIGN program (version 2.0) , It uses the PAM120 residue weight table, the gap length penalty is 12, and the gap penalty is 4.
  • the algorithm of Needleman and Wunsch J.Mol.Biol.484-453 (1970) can be used to determine the percent identity of two amino acid sequences, which has been incorporated into the GCG software package (available at www.gcg.com) In the GAP program in ), it uses the Blossum 62 matrix or the PAM250 matrix, the gap weight is 16, 14, 12, 10, 8, 6, or 4, and the length weight is 1, 2, 3, 4, 5, or 6.
  • Xn and Xaa are equivalent and refer to the unspecified amino acid (Unspecified Amino Acid), and the scope of its coverage is specified by the subsequent definition in the relevant expression.
  • CD47 Integrin-associated protein
  • IAP Integrin-associated protein
  • OA3 Integrin-associated protein
  • MER6 can be used interchangeably with each other.
  • human CD47 and hCD47 etc. are used interchangeably herein, and refer to human CD47 and variants or isotypes of human CD47.
  • antibody that binds to CD47 and “anti-CD47 antibody” are used interchangeably with each other.
  • the present invention provides an antigen-binding polypeptide that binds to CD47 or an antigen-binding portion thereof, and provides a new solution for the treatment of targeting CD47.
  • the antigen-binding polypeptides provided herein also show many other desirable characteristics for targeted therapy. Specifically, these desired characteristics can be, for example, the K D value of 1.53E-08 or less binds to CD47, blocks the binding of CD47 to SIRP ⁇ , promotes macrophage-mediated phagocytosis of cells expressing CD47, and has no significant effect.
  • CD47-binding antigen-binding polypeptides exhibit better therapeutic effects; in some embodiments, CD47-binding antigen-binding polypeptides exhibit reduced side effects.
  • the present invention provides CD47-binding antigen-binding polypeptides and antigen-binding portions thereof comprising specific sequences.
  • an isolated CD47-binding antigen-binding polypeptide or an antigen-binding portion thereof comprising one or more CDRs selected from the group consisting of SEQ ID NOs: 1 to 32.
  • the isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 comprises a heavy chain CDR1 sequence comprising an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5.
  • a heavy chain CDR1 sequence comprising an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5.
  • identity at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • An amino acid sequence that is identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical.
  • the isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 comprises a heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10. At least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88 % Identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity Identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.
  • the isolated antigen-binding polypeptide that binds CD47 or an antigen-binding portion thereof comprises a heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15. At least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88 % Identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity Identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.
  • the isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 comprises a light chain CDR1 that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19, or 20. At least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88 % Identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity Identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.
  • the isolated antigen-binding polypeptide that binds CD47 or an antigen-binding portion thereof comprises a light chain CDR2 comprising an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25 At least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88 % Identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity Identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.
  • the isolated antigen-binding polypeptide that binds CD47 or an antigen-binding portion thereof comprises a light chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30, or 32 At least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88 % Identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity Amino acid sequence with identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.
  • the heavy chain CDRs and light chain CDRs of the antigen-binding polypeptides provided herein can be independently selected or mixed and matched to form any heavy chain CDR1, CDR2, and CDR3 from the antigen-binding polypeptides provided herein. ; And any light chain CDR1, CDR2, and CDR3 antigen-binding polypeptide or antigen-binding portion thereof. Therefore, in some embodiments, the present invention provides an isolated antigen-binding polypeptide that binds CD47, comprising three heavy chain CDRs (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) and three light chain CDRs (light chain CDR1). , Light chain CDR2, light chain CDR3), where:
  • the heavy chain CDR1 which comprises at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5. At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity Amino acid sequence of
  • the heavy chain CDR2 which comprises at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10, At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity Amino acid sequence of
  • the heavy chain CDR3 which comprises at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15, At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity Amino acid sequence of
  • a light chain CDR1 comprising at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity with an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19 or 20, At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity Amino acid sequence of
  • a light chain CDR2 comprising at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity with an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25, At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity
  • a light chain CDR3 comprising at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity with an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32, At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity
  • the amino acid sequence comprising at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity with an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32, At least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92 % Identity
  • the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining region: heavy chain CDR1, which comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5; heavy chain CDR2, It comprises an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10; a heavy chain CDR3, which comprises an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15; a light chain CDR1, It includes an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19, or 20; a light chain CDR2, which includes an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25; and a light chain CDR3 , which comprises an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3.
  • the chain CDR3 respectively contains at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and at least 84% identity with the amino acid sequences shown in SEQ ID NOs: 1, 6 and 11 , At least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least Amino acid sequence of 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity, so
  • the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise at least 80% identity, at least 81% identity, at least 82% identity, and at least the amino acid sequence shown in SEQ ID NOs: 16, 21 and 26.
  • identity at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% Identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity Or 100% identical amino acid sequences.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining regions: heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6; comprising SEQ The heavy chain CDR3 of the amino acid sequence of ID NO: 11; the light chain CDR1 of the amino acid sequence of SEQ ID NO: 16; the light chain CDR2 of the amino acid sequence of SEQ ID NO: 21; and the amino acid sequence of SEQ ID NO: 26 The light chain CDR3.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3.
  • the chain CDR3 respectively contains at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and at least 84% identity with the amino acid sequences shown in SEQ ID NOs: 2, 7 and 12 , At least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least Amino acid sequence of 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity, so
  • the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise at least 80% identity, at least 81% identity, at least 82% identity, and at least the amino acid sequence shown in SEQ ID NOs: 17, 22 and 27.
  • identity at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% Identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity Or 100% identical amino acid sequences.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining regions: heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2; heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7; comprising SEQ The heavy chain CDR3 of the amino acid sequence of ID NO: 12; the light chain CDR1 of the amino acid sequence of SEQ ID NO: 17; the light chain CDR2 of the amino acid sequence of SEQ ID NO: 22; and the amino acid sequence of SEQ ID NO: 27 The light chain CDR3.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3.
  • the chain CDR3 contains at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and at least 84% identity with the amino acid sequences shown in SEQ ID NOs: 3, 8 and 13, respectively.
  • the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise at least 80% identity, at least 81% identity, at least 82% identity, and at least the amino acid sequence shown in SEQ ID NOs: 18, 23 and 28.
  • identity at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% Identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity Or 100% identical amino acid sequences.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining regions: heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 3; heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8; comprising SEQ The heavy chain CDR3 of the amino acid sequence of ID NO: 13; the light chain CDR1 of the amino acid sequence of SEQ ID NO: 18; the light chain CDR2 of the amino acid sequence of SEQ ID NO: 23; and the amino acid sequence of SEQ ID NO: 28 The light chain CDR3.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3.
  • the chain CDR3 contains at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and at least 84% identity with the amino acid sequences shown in SEQ ID NOs: 4, 9 and 14, respectively.
  • the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise at least 80% identity, at least 81% identity, at least 82% identity, and at least the amino acid sequence shown in SEQ ID NOs: 19, 24 and 32.
  • identity at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% Identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity Or 100% identical amino acid sequences.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining regions: heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9; comprising SEQ The heavy chain CDR3 of the amino acid sequence of ID NO: 14; the light chain CDR1 of the amino acid sequence of SEQ ID NO: 19; the light chain CDR2 of the amino acid sequence of SEQ ID NO: 24; and the amino acid sequence of SEQ ID NO: 32 The light chain CDR3.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3.
  • the chain CDR3 contains at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, and at least 84% identity with the amino acid sequences shown in SEQ ID NOs: 5, 10, and 15, respectively.
  • the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise at least 80% identity, at least 81% identity, at least 82% identity, and at least the amino acid sequence shown in SEQ ID NOs: 20, 25 and 30.
  • identity at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% Identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity Or 100% identical amino acid sequences.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises the following complementarity determining regions: heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5; heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; comprising SEQ The heavy chain CDR3 of the amino acid sequence of ID NO: 15; the light chain CDR1 of the amino acid sequence of SEQ ID NO: 20; the light chain CDR2 of the amino acid sequence of SEQ ID NO: 25; and the amino acid sequence of SEQ ID NO: 30 The light chain CDR3.
  • the present invention also provides conservatively modified forms of antigen-binding polypeptides and antigen-binding parts thereof.
  • a conservative amino acid substitution is the replacement of an amino acid with another amino acid having a similar structure or chemical properties (e.g., like a similar side chain).
  • Exemplary conservative substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Bengamin/Cummings Publication Company, 4th edition (1987).
  • the isolated antigen-binding polypeptide that binds to CD47 comprises: heavy chain CDR1, which comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, or 5 or conservatively modified forms thereof; heavy chain CDR2, It includes an amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9 or 10 or its conservatively modified form; heavy chain CDR3, which includes an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14 or 15 or its conservative A modified form of amino acid sequence; light chain CDR1, which comprises an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 19, or 20 or a conservatively modified form thereof; light chain CDR2, which comprises an amino acid sequence selected from SEQ ID NO: 21, The amino acid sequence of 22, 23, 24 or 25 or its conservatively modified form; and the light chain CDR3, which comprises an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 30 or 32 or its conservatively modified form. Conservative modifications are present in any one or more light chain CDRs or heavy chain CDRs.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises: a heavy chain CDR1, which comprises an amino acid sequence selected from SEQ ID NO: 3, 4, or 5; and a heavy chain CDR2, which comprises a heavy chain CDR2, which comprises a sequence selected from SEQ ID NO: 8. , 9 or 10; heavy chain CDR3, which includes an amino acid sequence selected from SEQ ID NO: 13, 14 or 15; light chain CDR1, which includes an amino acid sequence selected from SEQ ID NO: 18, 19 or 20;
  • the light chain CDR2 includes an amino acid sequence selected from SEQ ID NO: 23, 24, or 25; and the light chain CDR3 includes an amino acid sequence selected from SEQ ID NO: 28, 30, or 32.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the heavy chain CDR1 comprises SEQ ID NO:1 Or the amino acid sequence of its conservatively modified form, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 6 or its conservatively modified form, and the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 11 or its conservatively modified form, so
  • the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 16 or a conservatively modified form thereof, the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a conservatively modified form thereof, and the light chain CDR3 comprises SEQ ID NO: 26 Or its conservatively modified form of amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the heavy chain CDR1 comprises SEQ ID NO: 2
  • the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 7 or its conservatively modified form
  • the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 12 or its conservatively modified form
  • the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 17 or a conservatively modified form thereof
  • the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 22 or a conservatively modified form thereof
  • the light chain CDR3 comprises SEQ ID NO: 27 Or its conservatively modified form of amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the heavy chain CDR1 comprises SEQ ID NO: 3
  • the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 8 or its conservatively modified form
  • the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 13 or its conservatively modified form
  • the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 18 or its conservatively modified form
  • the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 23 or its conservatively modified form
  • the light chain CDR3 comprises SEQ ID NO: 28 Or its conservatively modified form of amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the heavy chain CDR1 comprises SEQ ID NO: 4
  • the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or its conservatively modified form
  • the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 14 or its conservatively modified form
  • the light chain CDR1 includes the amino acid sequence of SEQ ID NO: 19 or a conservatively modified form thereof
  • the light chain CDR2 includes the amino acid sequence of SEQ ID NO: 24 or a conservatively modified form thereof
  • the light chain CDR3 includes SEQ ID NO: 32 Or its conservatively modified form of amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the heavy chain CDR1 comprises SEQ ID NO: 5
  • the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 10 or its conservatively modified form
  • the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or its conservatively modified form
  • the light chain CDR1 includes the amino acid sequence of SEQ ID NO: 20 or a conservatively modified form thereof
  • the light chain CDR2 includes the amino acid sequence of SEQ ID NO: 25 or a conservatively modified form thereof
  • the light chain CDR3 includes SEQ ID NO: 30 Or its conservatively modified form of amino acid sequence.
  • the isolated antigen-binding polypeptides that bind CD47 provided herein comprise a heavy chain variable region and a light chain variable region, and the variable regions of the heavy chain and light chain comprise CDR regions and framework regions FR (including FR1, FR2, FR3 and FR4), the order of formation is the variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the present invention provides an isolated antigen-binding polypeptide that binds CD47 or an antigen-binding portion thereof, the antigen-binding polypeptide comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region Contains at least 80%, at least 85% identity, at least 86% identity, at least 87% identity, at least an amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 34, 36, 38 , 40, 42, 84, 86 or 88 amino acid sequence has at least 80% identity, at least 85% identity, at least 86%
  • sequence shown in SEQ ID NO: 83 is:
  • SEQ ID NO: 84 The sequence shown in SEQ ID NO: 84 is:
  • sequence shown in SEQ ID NO: 85 is:
  • SEQ ID NO: 86 The sequence shown in SEQ ID NO: 86 is:
  • SEQ ID NO: 87 The sequence shown in SEQ ID NO: 87 is:
  • QVQLVQSGAEVKKPGASVKVSCKX 15 S GFNIEDDYIE WVRQAPGQGLEWMG RIDPANDKTK YAQKFQGRVTMTX 16 DTSTX 17 TVYMELSSLRSEDTAVYYCX 18 R PGLRRYYSMDY WGQGTLVTVSS, wherein, X 15 is A or V, X 16 is R or G, X 17 is S or N, X 18 is A, or T;
  • SEQ ID NO: 88 The sequence shown in SEQ ID NO: 88 is:
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the following sequence or consists of the following sequence: selected from SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87 amino acid sequence
  • the light chain variable region comprises the following sequence or consists of the following sequence: selected from SEQ ID NO: 34, 36, 38, 40, 42, 84, 86 or 88 amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the following sequence or consists of the following sequence: selected from SEQ ID NO: 35, 39, 41, 83, 85 or 87 amino acid sequence
  • the light chain variable region comprises the following sequence or consists of the following sequence: selected from SEQ ID NO: 36, 40, 42, 84, 86 or 88 Amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 79 or is represented by SEQ ID NO:
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80 or is composed of the amino acid sequence of SEQ ID NO: 80.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 or is represented by SEQ ID NO:
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82 or is composed of the amino acid sequence of SEQ ID NO: 82.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 33 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 34 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 33, so The variable region of the light chain includes the amino acid sequence of SEQ ID NO: 34.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 35 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 36 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the isolated antigen-binding polypeptide that binds to anti-CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, The light chain variable region includes the amino acid sequence of SEQ ID NO: 36.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 37 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 38 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, so The variable region of the light chain includes the amino acid sequence of SEQ ID NO: 38.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 39 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 40 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the isolated antigen-binding polypeptide that binds to CD47 provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, so The variable region of the light chain includes the amino acid sequence of SEQ ID NO: 40.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 41 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 42 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 83 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 84 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 85%
  • the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83, and the light chain variable region
  • the chain variable region includes the amino acid sequence of SEQ ID NO: 84.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 85 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 86 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 9 least 9
  • the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 85, and the light chain variable region
  • the chain variable region includes the amino acid sequence of SEQ ID NO: 86.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising at least 80% identity to the amino acid sequence of SEQ ID NO: 87 Identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, An amino acid sequence of at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity,
  • the variable region of the light chain comprises at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least the amino acid sequence of SEQ ID NO: 88 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 90% identity
  • the isolated antigen-binding polypeptide that binds CD47 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87, and the light chain variable region
  • the chain variable region includes the amino acid sequence of SEQ ID NO: 88.
  • the heavy chain variable region and the light chain variable region described herein contain the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 described herein. Sequence characteristics.
  • the isolated antigen-binding polypeptide that binds to CD47 provided herein comprises a heavy chain and a light chain.
  • the heavy chain and the light chain also include Constant region.
  • the constant region is humanized.
  • the framework region FRs of the constant and variable regions are both humanized. The immunogenicity is reduced by constructing chimeric antibodies (e.g., humanized constant regions and non-humanized variable regions) or humanized antibodies (e.g., humanized constant regions and FR regions).
  • the light chain constant region of the antigen binding polypeptide is a human kappa chain constant region. In some embodiments, the light chain constant region of the antigen binding polypeptide is a human lambda chain constant region.
  • the heavy chain constant region of an antigen-binding polypeptide can be derived from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, such as IgG1, IgG2, IgG3, and IgG4.
  • the isolated antigen-binding polypeptide that binds CD47 is of the IgG1 isotype.
  • the isolated antigen-binding polypeptide that binds CD47 is of the IgG4 isotype.
  • the antigen binding polypeptide comprises a modified constant region.
  • the hinge region in the constant region of human IgG4 is modified to avoid or reduce chain exchange.
  • an IgG4 type antibody has a Ser228Pro (S228P) mutation according to the EU numbering index.
  • the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising or consisting of the following sequence: An amino acid sequence selected from SEQ ID NO: 33, 35, 37, 39, or 41, and the light chain variable region comprises or consists of the following sequence: selected from SEQ ID NO: 34, 36, 38, 40 or 42 amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody, which comprises a heavy chain and a light chain, the heavy chain having a compound selected from SEQ ID NO: 89, 93, 97, 101, or 105.
  • the amino acid sequence of has at least 80% identity, at least 85% identity, at least 90% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity Identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity
  • the light chain has an amino acid sequence selected from SEQ ID NO: 91, 95, 99, 103 or 107
  • the amino acid sequence shown has at least 80% identity, at least 85% identity, at least 90% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96%
  • the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 89 and a light chain according to SEQ ID NO: 91. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody, comprising a heavy chain according to SEQ ID NO: 93 and a light chain according to SEQ ID NO: 95. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 97 and a light chain according to SEQ ID NO: 99.
  • the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 101 and a light chain according to SEQ ID NO: 103. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a chimeric antibody comprising a heavy chain according to SEQ ID NO: 105 and a light chain according to SEQ ID NO: 107.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising or consisting of the following sequence : An amino acid sequence selected from SEQ ID NO: 83, 85, or 87, and the light chain variable region comprises or consists of the following sequence: an amino acid sequence selected from SEQ ID NO: 84, 86 or 88.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody
  • the heavy chain has a heavy chain selected from SEQ ID NO: 109, 113, 117, 121, 125, 129, 133, 137, 141
  • the amino acid sequence represented by 145, 149, 153, 157, 161, 165, 169, 173, 177, 181 or 185 has at least 80% identity, at least 85% identity, at least 90% identity, at least 92% identity , At least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or 100% identity amino acid sequence
  • the light chain has an ID number selected from SEQ ID NO: 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 109 and a light chain according to SEQ ID NO: 111. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 113 and a light chain according to SEQ ID NO: 115. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 117 and a light chain according to SEQ ID NO: 119.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 121 and a light chain according to SEQ ID NO: 123. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 125 and a light chain according to SEQ ID NO: 127. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 129 and a light chain according to SEQ ID NO: 131.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 133 and a light chain according to SEQ ID NO: 135. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 137 and a light chain according to SEQ ID NO: 139. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 141 and a light chain according to SEQ ID NO: 143.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 145 and a light chain according to SEQ ID NO: 147. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 149 and a light chain according to SEQ ID NO: 151. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 153 and a light chain according to SEQ ID NO: 155.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 157 and a light chain according to SEQ ID NO: 159. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 161 and a light chain according to SEQ ID NO: 163. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody comprising a heavy chain according to SEQ ID NO: 165 and a light chain according to SEQ ID NO: 167.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 169 and a light chain according to SEQ ID NO: 171. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 173 and a light chain according to SEQ ID NO: 175. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 177 and a light chain according to SEQ ID NO: 179.
  • the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 181 and a light chain according to SEQ ID NO: 183. In one embodiment, the isolated antigen-binding polypeptide that binds to CD47 is a humanized antibody, comprising a heavy chain according to SEQ ID NO: 185 and a light chain according to SEQ ID NO: 187.
  • the isolated antigen-binding polypeptide that binds to CD47 comprises a heavy chain and a light chain
  • the heavy chain comprises or consists of the following sequence: selected from SEQ ID NO: 93, 101, 105, 109, 113, The amino acid sequence of 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181 or 185
  • the light chain comprises or consists of the following sequence Composition: selected from SEQ ID NO: 95, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183 or 187 amino acid sequence.
  • the isolated antigen-binding polypeptide that binds to CD47 or/and its antigen-binding portion binds to CD47 with a K D value of 1.53E-08M or less.
  • the antigen-binding polypeptide or/and its antigen-binding portion bind to human CD47.
  • the antigen-binding polypeptide or/and its antigen-binding portion binds to cynomolgus CD47.
  • the antigen-binding polypeptide or/and its antigen-binding portion bind to human CD47 and cynomolgus CD47.
  • the antigen-binding polypeptide or/and its antigen-binding portion has the following binding affinity (K D ) for human CD47: from about 1E-12M to about 1E-05M, from about 1E-12M to about 1.89E -06M, about 1E-12M to about 1.89E-07M, about 1E-11M to about 1.89E-07M, about 9.46E-10M to about 1.89E-07M, about 9.46E-10M to about 1.89E-08M, about In the range of 2.04E-10M to about 1.53E-07M or about 2.04E-10M to about 1.89E-08M.
  • K D binding affinity for human CD47: from about 1E-12M to about 1E-05M, from about 1E-12M to about 1.89E -06M, about 1E-12M to about 1.89E-07M, about 1E-11M to about 1.89E-07M, about 9.46E-10M to about 1.89E-07M, about 9.46E-10M to about 1.89E-08M
  • the antigen-binding polypeptide or/and its antigen-binding portion has the following binding affinity (K D ) for human CD47: about 1E-05M or less, about 1.89E-06M or less, about 1.89 E-07M or less, about 1.89E-08M or less, about 1.53E-08M or less, about 9.31E-08M or less, about 9.31E-09M or less, about 8.55E-09M or more Small, about 2.35E-09M or less, or about 2.04E-010M or less.
  • K D binding affinity for human CD47: about 1E-05M or less, about 1.89E-06M or less, about 1.89 E-07M or less, about 1.89E-08M or less, about 1.53E-08M or less, about 9.31E-08M or less, about 9.31E-09M or less, about 8.55E-09M or more Small, about 2.35E-09M or less, or about 2.04E-010M or less.
  • the antigen-binding polypeptide or/and its antigen-binding portion has the following binding affinity (K D ) to human CD47: about 7.48E-09M, about 3.50E-10M, about 7.67E-10M, about 9.06E-09M, about 2.35E-09M, about 9.46E-10M, about 8.36E-10M, about 1.53E-09M, about 1.42E-09M, about 9.60E-10M, about 5.92E-10M, about 9.04E -10M, about 7.79E-10M, about 1.15E-09M, about 1.34E-09M, about 8.96E-10M, about 7.65E-10M, about 1.89E-08M, about 6.28E-09M, about 9.31E-09M , About 1.71E-08M, about 1.53E-08M, about 7.13E-09M, about 2.04E-10M or about 8.55E-09M.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion has about 1 ng/mL to about 2000 ng/mL, about 1 ng/mL to about 1500 ng/mL, about 1 ng/mL to about 1106 ng to human CD47. /mL, about 1ng/mL to about 699ng/mL, about 1ng/mL to about 340ng/mL, about 30ng/mL to about 340ng/mL, about 50ng/mL to about 340ng/mL, or about 100ng/mL to about 340ng /mL of combined EC50.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion has a human CD47 of about 2000ng/mL or less, about 1500ng/mL or less, about 1106ng/mL or less, about 699ng/mL Binding EC50 of mL or less, or about 340 ng/mL or less.
  • the EC50 of the CD47-binding antigen-binding polypeptides provided herein and the antigen-binding portion thereof are measured by ELISA or FACS.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion block the binding of CD47 to SIRP ⁇ .
  • Signal regulatory protein ⁇ SIRP ⁇
  • SIRP ⁇ Signal regulatory protein ⁇
  • the interaction between CD47 and SIRP ⁇ on macrophages sends a "don't eat me" signal to macrophages.
  • the interaction between SIRP ⁇ and CD47 is blocked with CD47-binding antigen-binding polypeptide or its antigen-binding portion Can promote the host immune system to take up and eliminate cancer cells.
  • the antigen-binding polypeptide (some examples such as chimeric antibodies Xi2B2, Xi2H8, Xi3F10, Xi16E5, Xi14A9) or/and its antigen-binding portion have a blocking IC50 of About 358.5 ng/mL to about 26966 ng/mL, about 358.5 ng/mL to about 4861 ng/mL, about 358.5 ng/mL to about 631.2 ng/mL, or about 358.5 ng/mL to about 1283 ng/mL.
  • the blocking IC50 of the antigen-binding polypeptide or/and its antigen-binding portion is about 26966ng/mL or less, about 15000ng/mL or less, about 10000ng/mL or less, about 8000ng/mL mL or less, about 6000ng/mL or less, about 4861ng/mL or less, about 3000ng/mL or less, about 2500ng/mL or less, about 2000ng/mL or less, about 1500ng/mL or Smaller, about 1283 ng/mL or less, about 1000 ng/mL or less, about 800 ng/mL or less, about 500 ng/mL or less, or about 358.5 ng/mL or less.
  • an antigen-binding polypeptide that binds to CD47 (some examples such as chimeric antibodies Xi2B2, Xi2H8, Xi3F10 or humanized antibodies thereof, Xi16E5 or humanized antibodies thereof, Xi14A9 or humanized antibodies thereof Etc.) or/and the blocking IC50 of its antigen-binding portion is about 4.682 ng/mL to about 15246 ng/mL, about 621.1 ng/mL to about 15246 ng/mL, about 621.1 ng/mL to about 7910 ng/mL, about 621.1 ng /mL to about 2939ng/mL, about 621.1ng/mL to about 2500ng/mL, about 621.1ng/mL to about 2051ng/mL, about 621.1ng/mL to about 1738ng/mL, about 621.1ng/mL to about 1500ng/mL mL,
  • the blocking IC50 of the antigen-binding polypeptide or/and its antigen-binding portion is about 15246 ng/mL or less, about 7910 ng/mL or less, about 2939 ng/mL or less , About 2500ng/mL or less, about 2051ng/mL or less, about 1738ng/mL or less, about 1500ng/mL or less, about 1400ng/mL or less, about 1300ng/mL or less, about 1200ng/mL or less, about 1100ng/mL or less, about 1000ng/mL or less, about 900ng/mL or less, about 800ng/mL or less, about 703.2ng/mL or less, about 621.1 ng/mL or less, about 450ng/mL or less, about 350ng/mL or less, about 250ng/mL or less, about 150ng/mL or less, about 100ng/mL or less, about 50 ng
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion promotes macrophage-mediated phagocytosis of CD47-expressing cells.
  • blocking the binding of SIRP ⁇ and CD47 by binding to the CD47 antigen-binding polypeptide increases the phagocytosis of macrophages on CD47-expressing cells such as cancer cells (specific examples such as HL60 cells, etc.).
  • the phagocytic rate of macrophages is from about 3.5% to about 65.7%, from about 11.5% to about 65.7%, from about 28.8% to about 1 ⁇ g/mL to about 10 ⁇ g/mL of CD47-binding antigen-binding polypeptide concentration.
  • the macrophage phagocytosis rate is about 1.34% to about 81.66%, about 2% to about 81.66%, about 11.95% to about 81.66 at a concentration of about 1 ⁇ g/mL to about 10 ⁇ g/mL antigen-binding polypeptide. %, about 24.19% to about 81.66%, or about 71.48% to about 81.66%.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion does not significantly induce the apoptosis of CD4+ T cells.
  • the detection of apoptosis adopts a flow cytometry method.
  • Obvious apoptosis herein refers to within an acceptable range (for example, no effect on the subject or within a controllable range) or no statistically significant difference compared with the negative control (P ⁇ 0.05), for example, some In the embodiment, there is no significant difference compared with the negative control IgG.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion has the following Tm (melting temperature): about 62° C. or higher, about 63° C. or higher, about 64° C. or higher, about 65° C. °C or higher, about 66 °C or higher, about 67 °C or higher, about 68 °C or higher, about 69 °C or higher, or about 70 °C or higher.
  • the antibody or/and its fragments have the following Tagg (aggregation temperature): about 61°C or higher, about 62°C or higher, about 63°C or higher, about 64°C or higher, About 65°C or higher, about 66°C or higher, about 67°C or higher, about 68°C or higher, about 69°C or higher, about 70°C or higher, about 71°C or higher, about 72°C or higher, or about 73°C or higher.
  • Tagg aggregation temperature
  • the Tm of the antigen-binding polypeptide or/and its antigen-binding portion is about 62°C to about 75°C, about 63°C to about 70.6°C, about 64°C to about 70.6°C, about 65°C to about 70.6°C, about 67°C to about 70.6°C, or about 68°C to about 70.6°C.
  • the Tagg of the antibody or/and its fragment is about 60°C to about 73.81°C, about 61°C to about 73.81°C, about 62°C to about 73.81°C, about 63°C to about 73.81°C, about 64°C to about 73.81°C, about 65°C to about 73.81°C, about 67°C to about 73.81°C, or about 68°C to about 73.81°C.
  • Tm and Tagg are detected by nano DSF.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion does not cause substantial red blood cell reduction, anemia, or agglutination of red blood cells.
  • red blood cell and “red blood cell” are synonymous and can be used interchangeably.
  • agglutination means clumping of cells. Since red blood cells also express CD47, some CD47-binding antigen-binding polypeptides will bind to red blood cells, causing agglutination. Therefore, avoiding or reducing the harmful effects of CD47 antigen-binding polypeptides on red blood cells will be expected to reduce therapeutic side effects.
  • the present invention provides CD47-binding antigen-binding polypeptides and antigen-binding portions thereof that exhibit one or a combination of the following properties:
  • the melting temperature T of the antigen-binding polypeptide or its antigen-binding portion is greater than or equal to 62°C, and the aggregation temperature Tagg is greater than or equal to 61°C.
  • the CD47-binding antigen-binding polypeptide or/and its antigen-binding portion has one, two, three, four, five, or six of the properties (a) to (f).
  • the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), and (c).
  • the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), and (e).
  • the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (c), and (e).
  • the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), (c), and (d). In some embodiments, the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), (c), and (e). In some embodiments, the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), (c), and (f). In some embodiments, the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), (c), (d), and (f). In some embodiments, the antigen-binding polypeptide or antigen-binding portion thereof has the properties (a), (b), (c), (d), (e), and (f).
  • the antigen-binding polypeptide and antigen-binding portion thereof that bind to CD47 is an anti-CD47 antibody or antigen-binding portion thereof.
  • the isolated antigen binding polypeptides provided herein that bind to CD47 are monoclonal antibodies.
  • the isolated antibodies that bind CD47 provided herein are monospecific antibodies.
  • the isolated antibodies that bind CD47 provided herein are multispecific antibodies.
  • multispecific antibodies For example, bispecific antibodies or trispecific antibodies.
  • the invention provides a CD47-binding antigen-binding polypeptide or antigen-binding portion thereof that binds to the same epitope on CD47 as any of the exemplary antibodies provided herein, such as a chimeric antibody or a humanized antibody with 16E5 It binds to the same epitope, for example, it binds to the same epitope as the chimeric antibody or humanized antibody of 3F10, for example, it binds to the same epitope as the chimeric antibody or humanized antibody of 14A9.
  • the antigen-binding polypeptide or antigen-binding portion thereof competes with any of the exemplary antibodies provided herein for binding to CD47, for example, with a chimeric antibody or humanized antibody of 16E5 for binding to CD47, for example with a chimeric antibody of 3F10.
  • the combined antibody or humanized antibody competes for binding to CD47, for example, the chimeric antibody or humanized antibody of 14A9 competes for binding to CD47.
  • the binding to CD47 can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art.
  • the present invention provides some exemplary CD47-binding monoclonal antibodies, including chimeric versions of 2B2, 2H8, 3F10, 16E5 and 14A9 antibodies (Xi2B2, Xi2H8, Xi3F10, Xi16E5 and Xi14A9) and human sources of 3F10, 16E5, and 14A9 ⁇ The variant.
  • the amino acid sequences of the heavy chain CDRs (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) of the exemplary CD47-binding antibodies provided herein are provided in Table S1 below.
  • the amino acid sequence of chain CDR3) is provided in the following table S2, and the amino acid sequence of the variable region and the full-length heavy chain and light chain are provided in the following table S3 and S4.
  • the present invention provides immunoconjugates that bind CD47 antigen-binding polypeptides or antigen-binding portions thereof.
  • the therapeutic agent that can be linked or conjugated to the CD47-binding antigen-binding polypeptide or antigen-binding portion thereof may include, but is not limited to, cytotoxic drugs, radioisotopes, immunomodulators, or antibodies.
  • the CD47-binding antigen-binding polypeptide or antigen-binding portion thereof is directly conjugated to the therapeutic agent.
  • the CD47-binding antigen-binding polypeptide or antigen-binding portion thereof is conjugated to the therapeutic agent via a linker.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated CD47-binding antigen-binding polypeptide or an antigen-binding portion thereof, or a nucleic acid encoding the antibody or fragment, or the immunoconjugate described herein, and also Contains one or more pharmaceutically acceptable carriers.
  • the composition includes any monoclonal antibody among chimeric antibodies and humanized antibodies of 2B2, 2H8, 3F10, 16E5 and 14A9 antibodies, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers or other agents.
  • the present invention provides an isolated hybridoma cell line, which is selected from the group consisting of 2B2, 2H8, 3F10, 16E5 and 14A9.
  • the hybridoma cell lines 2B2, 2H8, 3F10, 16E5, and 14A9 secrete monoclonal antibodies that bind CD47.
  • the present invention provides an isolated nucleic acid comprising a sequence encoding the CD47-binding antigen-binding polypeptide described herein or an antigen-binding portion thereof.
  • the nucleic acid may encode a heavy chain variable comprising the antigen-binding polypeptide described herein. Region or/and the amino acid sequence of the variable region of the light chain.
  • the nucleic acid may encode an amino acid sequence comprising the heavy chain of the antigen-binding polypeptide described herein or/and the light chain of the antigen-binding polypeptide.
  • the sequence listing exemplarily lists the nucleotide sequences of some variable regions, heavy and light chains of the antigen-binding polypeptide.
  • the present invention provides a vector containing the isolated nucleic acid.
  • the vector is a cloning vector; in other embodiments, the vector is an expression vector.
  • the expression vector can optionally be any expression vector capable of expressing the antigen-binding polypeptide described herein or an antigen-binding portion thereof.
  • the present invention provides a host cell comprising the vector.
  • the host cell is a suitable host cell for cloning or encoding an antigen-binding polypeptide that binds CD47 or an antigen-binding portion thereof.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antigen-binding polypeptides or antigen-binding portions thereof.
  • the mammalian cells are, for example, Chinese hamster (CHO) ovary cells.
  • the present invention provides a method of preparing an isolated antigen-binding polypeptide that binds CD47, the method comprising: culturing a nucleic acid encoding the antigen-binding polypeptide under conditions suitable for expression of the antigen-binding polypeptide Host cell, recovering the antigen-binding polypeptide from the host cell or host cell culture medium.
  • the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning or/and expression in host cells.
  • the nucleic acid can be obtained by various methods well known in the art, such as gene cloning, gene splicing, chemical synthesis and the like.
  • the present invention provides the use of an isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47.
  • a therapeutically effective amount of the isolated antigen-binding polypeptide or antigen-binding portion thereof that binds to CD47 herein can be administered to a subject. Reduce or inhibit the growth of tumor cells.
  • administering a therapeutically effective amount of the isolated CD47-binding antigen-binding polypeptide or antigen-binding portion thereof herein can treat cancer.
  • Subjects in need of treatment include those who already have a disease or condition, as well as those who may have a disease or condition and whose purpose is to prevent, delay or attenuate the disease or condition.
  • cancer refers to a physiological condition in mammals that is usually characterized by unregulated cell growth.
  • examples of cancers include, but are not limited to: leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreas Cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, cholangiocarcinoma, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine adenocarcinoma, adrenal gland cancer, pituitary gland cancer, skin cancer, soft tissue cancer, vascular cancer , Brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, and uterine cancer, glioblastoma, medulloblastoma, astrocytoma, glioblastoma
  • an effective amount of the isolated antigen-binding polypeptide or antigen-binding portion thereof that binds CD47 herein is administered to the subject to promote phagocytosis of macrophages in the subject.
  • hCD47-mFc ACRObiosystems, catalog number: CD7-H52A5
  • protein 20 ⁇ g/mouse
  • mice were boosted by intravenous injection of antigen without adjuvant.
  • Spleen cells (1 ⁇ 10 8 ) from immunized mice were fused with SP2/0 myeloma cells (1.5 ⁇ 10 7 ) using polyethylene glycol 1500 (Polyethylene Glycol 1500; Roche, catalog number: 10783641001). After fusion, the cells were dispensed into 96-well plates at a rate of 0.1 ml/well, and incubated in an incubator at 37° C.
  • the cloning of hybridoma cells adopts the limiting dilution method.
  • the selected positive mixed clones were subjected to limiting dilution, and one positive mixed clone was spread on two 96-well plates (100cells/plate, 200cells/plate), and the hybridoma cells were cultured in RPMI 1640 (Hyclone) containing 10% fetal bovine serum. Company, catalog number: SH30809.01). After 10 days, the subclones after limiting dilution were also subjected to the ELISA test to detect the culture supernatant, and the positive subclones were selected. The cloned hybridoma cells are preserved and used for cDNA extraction.
  • RNA extraction kit (Takara, catalog number: 9767) was used to isolate total RNA from the hybridoma cell line that produced hCD47 antibodies. The total RNA was used as a template. According to the manufacturer's instructions, a reverse transcription kit (Thermo Fisher) was used to isolate total RNA. , Catalog number: K1652) Synthesize the first strand cDNA. Then, using the synthesized cDNA and using degenerate mouse IgG primers, the antibody-related sequences were amplified in vitro by PCR reaction.
  • PCR amplified products were separated by electrophoresis in a 1% agarose/Tris-acetate gel.
  • a DNA fragment of the expected size (approximately 450 bp for heavy and light chains) was cut from the gel and purified.
  • 3 ⁇ l of the purified PCR product was cloned into pMD-19T vector (Takara, catalog number: 6013), and transformed into DH5 ⁇ chemically competent E. coli (Takara, catalog number: 9057). In each ligation reaction, 10 positive clones were randomly selected and M13 forward primer was used for DNA sequencing.
  • VH heavy chain variable regions
  • 16E5, 14A9 antibodies
  • the sequence and the light chain variable region (VL) sequence, the sequence information is as follows:
  • the VL region of each mouse antibody was linked to the human kappa chain constant region to construct a chimeric light chain, and the mouse VH region was linked to human IgG4 (EU number S228P )
  • the constant region constructs a chimeric heavy chain.
  • the expression plasmid of the chimeric light chain and the expression plasmid of the chimeric heavy chain were constructed separately by conventional genetic engineering methods, and 100 ⁇ g of each chimeric heavy chain expression plasmid was used for CHO cells (200mL system, at 10 6 cells/mL) Transfected with the chimeric light chain expression plasmid and cultured for 6 days. Then, the chimeric antibody in the supernatant was purified by a protein A column.
  • chimeric antibodies Five chimeric antibodies were constructed and expressed.
  • the full-length sequences of the chimeric heavy chain and the chimeric light chain and their encoding nucleic acids are as follows: Xi2H8 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 89-90; Combined light chain and its encoding nucleic acid: SEQ ID NOs: 91-92), Xi2B2 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 97-98; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 99 ⁇ 100), Xi3F10 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 101 to 102; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 103 to 104), Xi16E5 (chimeric heavy chain and its encoding Nucleic acid: SEQ ID NOs: 93-94
  • Human-mouse chimeric antibodies of 3F10, 14A9, and 16E5 were humanized using a CDR grafting method (see, for example, U.S. Patent No. 5,225,539).
  • the mouse antibodies 3F10, 14A9 and 16E5 were homologously modeled by MOE (Molecular Operating Environment) to generate a protein structure model of the Fv domain.
  • MOE Molecular Operating Environment
  • VH and VL amino acid sequences of the mouse 3F10, 14A9 and 16E5 antibodies were input into the relevant tool website of the International Immune Genetic Information System (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) and selected A human germline antibody sequence with high homology to the variable regions of the mouse 3F10, 14A9 and 16E5 antibodies was produced.
  • the complementarity determining regions (CDRs) in the VH and VL of the murine 3F10, 14A9 and 16E5 antibodies were grafted to the template human antibody.
  • the selected template human VH is a combination of IGHV1-69*01 and IGHJ4*01
  • the selected template human VL is a combination of IGKV1-39*01 and IGKJ2*01.
  • the selected template human VH is a combination of IGHV1-46*01 and IGHJ4*01
  • the selected template human VL is a combination of IGKV1-39*01 and IGKJ2*01.
  • the selected template human VH is a combination of IGHV1-18*01 and IGHJ4*01
  • the selected template human VL is a combination of IGKV1-39*01 and IGKJ2*01.
  • the CDR amino acid sequence of the above template human antibody was replaced by the CDR of hybridoma (mouse) 3F10, 14A9 and 16E5 antibodies.
  • the essential amino acid sequences of VH and VL from mouse 3F10, 14A9 and 16E5 antibodies were used to graft the above template human germline antibody VH and VL frameworks to obtain functional humanized antibodies.
  • VH and VL of the 3F10, 14A9 and 16E5 antibodies several positions of the framework amino acids of the template human antibody were backmutated to the corresponding amino acid sequences in the mouse 3F10, 14A9 and 16E5 antibodies. Individual CDR regions were mutated.
  • the selection and matching of mutation types are from the following positions: the amino acid at position 73 is mutated from Asp (D) to Glu (E); the amino acid at position 74 is from Glu (E) is mutated to Ile(I); the amino acid at position 77 is mutated from Ser(S) to Asn(N), and for the light chain variable region of the humanized 3F10 antibody, the selection and matching of mutation types are as follows Position: The amino acid at position 48 is mutated from Leu (L) to Trp (W); the amino acid at position 71 is mutated from Asp (D) to Ser (S); the amino acid at position 72 is mutated from Phe (F) The mutation is Tyr (Y); the amino acid at position 94 is mutated from Asp (D) to Glu (E).
  • the selection and matching of mutation types come from the following positions: the amino acid at position 24 is mutated from Ala(A) to Val(V); the amino acid at position 72 is from Arg( R) is mutated to Gly(G); the amino acid at position 77 is mutated from Ser(S) to Asn(N); the amino acid at position 97 is mutated from Ala(A) to Thr(T), and for humanization
  • the selection and matching of mutation types come from the following positions: the amino acid at position 63 is mutated from Ser(S) to Ile(I); the amino acid at position 68 is mutated from Gly(G) to Ser(S).
  • the selection and matching of mutation types come from the following positions: the amino acid at position 46 is mutated from Glu (E) to Lys (K); the amino acid at position 63 is from Lys (K) is mutated to Glu(E); the amino acid at position 72 is mutated from Thr(T) to Leu(L); the amino acid at position 77 is mutated from Ser(S) to Arg(R); at position 97
  • the amino acid of is mutated from Ala(A) to Thr(T)
  • the selection and matching of mutation types come from the following positions: the amino acid at position 2 is from Ile(I)
  • the mutation is Ala (A); the amino acid at position 33 is mutated from Asn (N) to Gln (Q).
  • the description of the sequence number of the mutation site in this paragraph starts counting sequentially from the first amino acid of the variable region.
  • sequences of humanized 3F10, 14A9 and 16E5 antibodies and their encoding nucleic acids are as follows: hz3F10-1.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 109 to 110; light chain and its encoding nucleic acid: SEQ ID NOs. 111 to 112 ), hz3F10-2.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 113 to 114; light chain and its encoding nucleic acid: SEQ ID NOs.
  • hz3F10-3.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.117-118; light chain and its encoding nucleic acid: SEQ ID NOs.119-120)
  • hz3F10-4.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 121-122; light chain and its encoding nucleic acid: SEQ ID NOs.123 ⁇ 124)
  • hz3F10-5.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 125-126; light chain and its encoding nucleic acid: SEQ ID NOs.
  • hz3F10-6.1 (heavy chain and its Encoding nucleic acid: SEQ ID NOs. 129 to 130; light chain and its encoding nucleic acid: SEQ ID NOs. 131 to 132)
  • hz3F10-1.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 133 to 134; light chain and its encoding nucleic acid: SEQ ID NOs. 133 to 134)
  • hz3F10-2.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 137 to 138; light chain and its encoding nucleic acid: SEQ ID NOs.
  • hz3F10-3.2 Heavy chain and its encoding nucleic acid: SEQ ID NOs. 141 to 142; Light chain and its encoding nucleic acid: SEQ ID NOs. 143 to 144), hz3F10-4.2 (Heavy chain and its encoding nucleic acid: SEQ ID NOs.
  • hz16E5-1.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.157-158; light chain and its encoding nucleic acid: SEQ ID NOs.159-160)
  • hz16E5-1.3 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 161 to 162; light chain and its encoding nucleic acid: SEQ ID NOs.163 ⁇ 164)
  • hz16E5-1.2 (heavy chain and its Nucleic acid encoding: SEQ ID NOs. 165 to 166; light chain and its encoding nucleic acid: SEQ ID NOs.
  • hz16E5-3.2 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 169 to 170; light chain and its encoding nucleic acid: SEQ ID NOs. 169 to 170)
  • hz16E5-3.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 173 to 174; light chain and its encoding nucleic acid: SEQ ID NOs. 175 to 176), hz16E5-3.3 (Heavy chain and its encoding nucleic acid: SEQ ID NOs. 177-178; Light chain and its encoding nucleic acid: SEQ ID NOs.
  • hz14A9-2.3 Heavy chain and its encoding nucleic acid: SEQ ID NOs. 181-182 ; Light chain and its encoding nucleic acid: SEQ ID NOs. 183 to 184), hz14A9-2.4 (heavy chain and its encoding nucleic acid: SEQ ID NOs. 185 to 186; light chain and its encoding nucleic acid: SEQ ID NOs. 187 to 188 ).
  • CHO cells 200ml system, 10 6 cells/ml
  • ExpiCHO medium Gibco; catalog number: A29100 -01
  • the polyclonal rabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10mM sodium acetate pH 5.0, and fixed to the reference flow cell of the CM5 biosensor chip using an amine coupling kit (GE, BR10050) The experimental flow cell reached about 15000 R ⁇ . At the beginning of each cycle, a diluted test antibody (1.5 ⁇ g/mL) was injected on the experimental flow cell for 1 minute to be captured. These data indicate that anti-CD47 antibodies (chimeric and humanized) bind CD47 as measured by Biacore.
  • ELISA binding analysis is based on human CD47 protein and anti-CD47 chimeric antibody or anti-CD47 humanized antibody.
  • the wells were aspirated and blocked by adding 100 ⁇ L/well blocking solution (PBS with 1% (w/v) bovine serum albumin (BSA, Sigma, catalog number: B2064-100G)) and incubating at 37°C for 1 hour Non-specific binding site.
  • PBS 100 ⁇ L/well blocking solution
  • BSA bovine serum albumin
  • Table 3 ELISA-based EC50 of anti-CD47 chimeric antibody and human CD47 protein binding
  • NA means that the detection limit has not been reached.
  • Table 4 ELISA-based binding EC50 of anti-CD47 humanized antibodies and human CD47 protein
  • CHO-K1-CD47 cells (CHO-K1 cells expressing human CD47 protein) were added to each well of a 96-well plate (Eppendorf, catalog number: C030730.119), 1.2 ⁇ 10 4 cells per well, and combined with anti-CD47 Antibody (20 ⁇ g/ml, 1:4 dilution, dilution buffer is PBS) incubate for 1-2h at room temperature.
  • the cells were washed three times with PBS buffer, and the secondary antibody Alexa Fluor 488 labeled goat anti-human IgG (Jackson ImmunoResearch Inc, catalog number: 109-545-088) was diluted 1:200 with PBS at 100 ⁇ l/well and added to Cells and incubate at room temperature for 30 min.
  • the secondary antibody Alexa Fluor 488 labeled goat anti-human IgG Jackson ImmunoResearch Inc, catalog number: 109-545-088
  • the cells were washed three times with PBS buffer, the nuclear dye Hoechst (Sigma, catalog number: B2261) was diluted with PBS buffer at a dilution ratio of 1:500 and then added to each well at 100 ⁇ l/well for 10 min, washed with PBS 3 times and then passed High content screening system (High content Screening System, manufacturer: PerkinElmer, model specification: Operetta) was analyzed, and the results are shown in Tables 5 and 6. These data indicate that the anti-CD47 antibody binds to CD47 based on the cell-based binding analysis.
  • High content screening system High content Screening System, manufacturer: PerkinElmer, model specification: Operetta
  • Antibody name EC50(ng/mL) Xi2B2 30790 Xi2H8 NA Xi3F10 19803 Xi16E5 2026 Xi14A9 540.7
  • NA means that the detection limit has not been reached.
  • NA means that the detection limit has not been reached.
  • NA means that the detection limit has not been reached.
  • the 96-well plate was washed with washing buffer (PBS with 0.01% (w/v) Tween 20 (Sigma, catalog number: P9416-100ML)). Then add 100 ⁇ l/well of 1 ⁇ g/ml CD47 ligand Human-SIRP ⁇ -mFC (Human SIRP alpha/CD172a Protein, Mouse IgG1Fc Tag, ACRObiosystems, catalog number: SIA-H52A8), incubate at 25 ⁇ 2°C for 1.5h, PBS After washing the 96-well plate with washing buffer, add: anti-CD47 chimeric antibody or anti-CD47 humanized antibody (see Table 8 for chimeric antibody and Table 9 for humanized antibody), starting concentration 20 ⁇ g/mL, 100 ⁇ L/well, Dilute 4 times with PBS buffer, 8 gradients, and incubate at room temperature for 1.5h.
  • washing buffer PBS with 0.01% (w/v) Tween 20 (Sigma, catalog number: P9416-100ML)
  • NA means that the detection limit has not been reached.
  • Antibody name IC50(ng/mL) Antibody name IC50(ng/mL) hz3F10-1.1 1534 hz3F10-6.2 1839 hz3F10-2.1 1612 hz16E5-1.1 NA hz3F10-3.1 1678 hz16E5-3.1 15246 hz3F10-4.1 1952 hz16E5-1.2 2939 hz3F10-5.1 1701 hz16E5-3.2 2051 hz3F10-6.1 1768 hz16E5-1.3 2323 hz3F10-1.2 1689 hz16E5-3.3 1998 hz3F10-2.2 1725 hz14A9-2.3 703.2 hz3F10-3.2 1741 hz14A9-2.4 621.1 hz3F10-4.2 1738 Hu5F9 2398 hz3F10-5.2 1824 To To To To To To To To To To To To To To To To To To To To To To To To To
  • NA means that the detection limit has not been reached.
  • Example 7 Analysis of anti-CD47 antibodies based on phagocytosis of macrophages
  • anti-CD47 antibodies to promote phagocytosis of tumor cells by macrophages containing SIRP ⁇ on the cell surface was determined based on flow cytometry.
  • Monocyte isolation kit (STEMCELL, catalog number: 19058) was used to separate mononuclear cells from human peripheral blood mononuclear cells (PBMC, Cycla Bio, catalog number: 19056), and plated into 24-well plates (Greiner bio -one, catalog number: 662-160), 5 ⁇ 10 5 cells per well, and induced in an environment of 100ng/mL stimulating factor M-CSF (R&D Systems, catalog number: 216-MC-010) for 7 days , Which induces into macrophages.
  • CD14 APC Mouse Anti-Human CD14, BD company, catalog number: 555399
  • PE-CD11b PE Mouse Anti-Human CD11b/Mac-1, BD company, catalog number: 555388 antibody mixture
  • Example 8 The effect of anti-CD47 antibody on CD4+ T cells
  • T cell isolation kit (Miltenyi Biotech, catalog number: 130-094-131) was used to isolate CD4+ T cells from human peripheral blood mononuclear cells (PBMC, Cycla Biotech, catalog number: 19056) and plated with 24 Orifice plate (Greiner bio-one, catalog number: 662106), 5 ⁇ 10 5 cells per well, and anti-CD47 antibodies (see Table 12 for chimeric antibodies and Table 13 for humanized antibodies) to make the final concentration 10 ⁇ g/mL and 1 ⁇ g/mL, respectively, add 1640 complete medium, 37°C, 5% CO 2 incubator for 20 hours, use apoptosis kit (STEMCELL, catalog number: 556547) room temperature staining for 15 minutes, flow cytometer ( BD, model specification: C6) Detect the cell apoptosis rate (%), and calculate the apoptosis rate based on the sum of early and late apoptosis.
  • the results show that anti-CD47 antibodies (see Table 12 for chimeric antibodies and
  • Example 9 Detection of the thermal stability of anti-CD47 antibodies based on nano DSF
  • the high-throughput protein stability analyzer Prometheus NT.48 is a device that obtains various types of data such as protein molecular structure stability, aggregation stability, and colloidal dispersion stability.
  • the device was used to detect the melting temperature (Tm) and polymerization temperature (Tagg) of the anti-CD47 chimeric antibody and the anti-CD47 humanized antibody.
  • Tm melting temperature
  • Tagg polymerization temperature
  • Example 10 The effect of anti-CD47 antibody on erythrocyte agglutination
  • red blood cells contains a certain amount of CD47 protein, which leads to binding with red blood cells after adding certain anti-CD47 antibodies, which may cause agglutination of red blood cells. Therefore, the effect of anti-CD47 antibodies on red blood cell agglutination was tested.
  • Thermo Fisher Scientific catalog number: 14175-095
  • Thermo Fisher Scientific catalog number: 14175-095
  • the red blood cells were diluted 10-fold with PBS buffer to prepare a 10% red blood cell suspension, and 100 ⁇ l/well was placed in a 96-well U-shaped plate (eppendorf, catalog number: 003073119).
  • the red blood cells are all agglutinated, sinking to the bottom, and flattened into a mesh, that is, agglutination; if the red blood cells are not agglomerated, the red blood cells sink to the bottom of the hole and appear as dots.
  • Humanized antibodies hz3F10-3.1, hz3F10-4.1, hz3F10-3.2, hz3F10-4.2 start coagulation from 3.3 ⁇ g/ml, hz16E5-1.1, hz16E5-3.1, hz16E5-1.2, hz16E5-3.2, hz16E5-1.3, hz16E5 -3.3 Blood coagulation starts from 3.3 ⁇ g/ml, and other humanized antibodies have no coagulation phenomenon at all concentrations tested.
  • CD47 Based on a certain amount of CD47 on the surface of red blood cells, it may bind to some anti-CD47 antibodies, so the binding of anti-CD47 antibodies to red blood cells is detected.
  • the separated red blood cells were fixed overnight with 4% paraformaldehyde (Sangong Bioengineering Shanghai Company, catalog number: E672002-0500), and incubated with 10 ⁇ g/ml or 1 ⁇ g/ml anti-CD47 antibody for 1-2h at room temperature the next day, PBS buffer After washing, the secondary antibody Alexa Fluor 488 labeled goat anti-human IgG (Jackson Immuno Research Inc, catalog number: 109-545-088) diluted 1:200 in PBS buffer was used to incubate for 1 hour at room temperature, and then washed with PBS buffer and flow cytometry Instrument (BD company, model specification: C6) detects the binding of red blood cells with the anti-CD47 chimeric antibody (see Table 16) or humanized antibody (see Table 17) with the mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • the Raji cell suspension prepared in PBS buffer has a concentration of 5 ⁇ 10 6 cells/mL, and 0.1 mL/mouse is injected into female NOD/SCID mice through the tail vein. Seven days later, they were randomly divided into 4 groups with 10 animals in each group. Among them, group 1: IgG4 group, group 2: Hu5F9 group, group 3: hz3F10-6.1, group 4: hz14A9-2.3, administered by intraperitoneal injection, and the dose was 10 mg /kg, continuously administered for 21 days. Observe the life of the animal.
  • the anti-CD47 antibodies hz14A9-2.3 and hz3F10-6.1 have anti-tumor activity.
  • the survival rate data of mice showed that all animals in the IgG4 group died on the 17th day, with a median survival time of 13.5 days; the Hu5F9 group died on the 30th day, with a median survival time of 18.5 days; the hz14A9-2.3 group was 34th All animals died on day 21, and the median survival time was 28.5 days; the hz3F10-6.1 group died on the 21st day, and the median survival time was 17.5 days.
  • CD47 is expressed on red blood cells and plays a role in eliminating aging red blood cells, the toxic effects of anti-CD47 antibodies in B-hCD47 mice were compared.
  • the B-hCD47 mice were randomly divided into 3 groups: 3 in the Hu5F9 group, 4 in the hz14A9-2.3 group, and 3 in the hz3F10-6.1 group.
  • a certain dose of Hu5F9 antibody, hz14A9-2.3 antibody or hz3F10-6.1 antibody was administered to B-hCD47 mice through a single tail vein injection at a dose of 10 mg/kg.
  • the detection was performed on 2 days, 4 days, 7 days, and 10 days after administration. Changes in red blood cell count (RBC), hemoglobin (HGB), and body weight on days and 14 days.
  • Table 18 Mouse body weight change rate after administration (vs before administration)
  • mice in the hz14A9-2.3 group continued to increase their body weight.
  • the mice in the Hu5F9 and hz3F10-6.1 groups decreased their body weight first and then increased after the administration; the hz3F10-6.1 group decreased The amplitude is higher than the Hu5F9 group.
  • the RBC and HGB of the three groups of animals decreased after administration, reached the lowest 4 days after administration, and then began to increase.
  • the RBC and HGB in the hz14A9-2.3 group returned to normal 7 days after the administration, and the other two groups returned to normal at least 10 days after the administration.
  • a mouse model of human acute myeloid leukemia cells MOLM-16 cells was used to detect the anti-tumor activity of antibodies.
  • D0 the mice were grouped and administered intravenously, once every 3 days, for a total of 2 doses. The tumor diameter was measured twice a week, and the therapeutic effect was judged based on the tumor volume.
  • V tumor volume
  • T/C(%) (TT 0 )/(CC 0 )*100, where T and C are the average tumor volume at the end of the experiment in the treatment group and the negative control group respectively; T 0 and C 0 are the treatment group and negative respectively The average tumor volume of the control group at the beginning of the experiment.
  • TGI Tumor inhibition rate
  • the results are shown in Table 21 and Table 11.
  • the antibody hz14A9-2.3 can effectively inhibit tumor growth at a dose of 1.5 mg/kg, and the tumor growth inhibition rate (or tumor inhibition rate) on day 17 (D17) reached 93%. At a dose of 5 mg/kg, the tumor growth inhibition rate of the antibody hz14A9-2.3 reached 125%.

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Abstract

提供一种结合CD47的抗原结合多肽或其抗原结合部分，以及编码多肽的核酸、载体、细胞、药物组合物，该多肽可减少肿瘤或抑制肿瘤细胞生长、治疗癌症、促进巨噬细胞吞噬作用等。

Description

结合CD47的抗原结合多肽及用途 技术领域
本发明涉及抗原结合多肽，尤其涉及结合CD47的抗原结合多肽及其抗原结合部分，并且涉及使用这类抗原结合多肽及其抗原结合部分的方法。
背景技术
CD47是一种广泛表达于多个物种和各个组织之间的跨膜糖蛋白，也被称为整合素相关蛋白，是免疫球蛋白超家族中的成员。CD47约50kD，其包含1个胞外类Ig可变结构域、5个高度疏水延伸的跨膜片段以及1个短选择性拼接的羧基端胞质尾区。
抑制性受体信号调节蛋白α(SIRPα)是CD47的配体之一，CD47结合在SIRPα的NH 2末端IgV样结构域。SIRPα主要表达于髓系来源的细胞，包括巨噬细胞、粒细胞、DC细胞、肥大细胞以及它们的前体，包括造血干细胞。例如，CD47可以通过与巨噬细胞表面的蛋白SIRPα结合，抑制巨噬细胞的吞噬功能。有研究显示，阻断CD47介导的与吞噬细胞上的SIRPα结合，或在CD47敲除小鼠中的表达缺失，可引起活细胞和非老化红细胞的去除。对于其中还存在预吞噬信号的那些细胞来说，阻断SIRPα还允许通常不被吞噬的靶标的吞入。
程序性细胞死亡(PCD)和吞噬细胞去除是生物体响应以便除去受损的、癌前或感染的细胞的常见方式。因此，在此生物体响应情况下存活的细胞(癌性细胞、长期感染的细胞等)存在用于逃避PCD和吞噬细胞去除的方式。CD47在各类患病细胞、癌细胞以及受感染细胞中组成性上调，从而允许这些细胞逃避吞噬作用。阻断一种细胞(癌性细胞、长期感染的细胞等)上的CD47与另一种细胞(吞噬细胞)上的SIRPα之间的相互作用的抗CD47化合物，可抵消CD47表达的增加并且促进癌细胞和/或受感染细胞的吞噬作用。
CD47表达和/或活性已经牵涉许多疾病和病症。不同的研究表明，几乎所有的肿瘤细胞和肿瘤组织都高表达CD47。肿瘤细胞表面高表达的CD47通过与巨噬细胞表面SIRPα结合，释放“别吃我”(don’t eat me)的信号，从而导致肿瘤组织浸润区的巨噬细胞不但同肿瘤细胞和谐相处，而且还会通过促进肿瘤内血管增殖，抑制效应T细胞发挥作用，促进肿瘤细胞扩增和生长。因而需要靶向CD47治疗。
发明内容
本发明提供了一种新的分离的与CD47结合的抗原结合多肽及其抗原结合部分，为靶向CD47的治疗提供解决方案。
一方面，本发明提供了结合CD47的分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含以下互补决定区：
重链CDR1，其包含与选自SEQ ID NO：1、2、3、4或5的氨基酸序列具有至少80％同一性的氨基酸序列；
重链CDR2，其包含与选自SEQ ID NO：6、7、8、9或10的氨基酸序列具有至少80％同一性的氨基酸序列；
重链CDR3，其包含与选自SEQ ID NO：11、12、13、14或15的氨基酸序列具有至少80％同一性的氨基酸序列；
轻链CDR1，其包含与选自SEQ ID NO：16、17、18、19或20的氨基酸序列具有至少80％同一性的氨基酸序列；
轻链CDR2，其包含与选自SEQ ID NO：21、22、23、24或25的氨基酸序列具有至少80％同一性的氨基酸序列；和
轻链CDR3，其包含与选自SEQ ID NO：26、27、28、30或32的氨基酸序列具有至少80％同一性的氨基酸序列。
其中，SEQ ID NO：32所示序列为QQFSX 2STWT，X 2为D或E。
另一方面，本发明提供了结合CD47的分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含以下互补决定区：
重链CDR1，其包含选自SEQ ID NO：1、2、3、4或5或者其保守修饰形式的氨基酸序列；
重链CDR2，其包含选自SEQ ID NO：6、7、8、9或10或者其保守修饰形式的氨基酸序列；
重链CDR3，其包含选自SEQ ID NO：11、12、13、14或15或者其保守修饰形式的氨基酸序列；
轻链CDR1，其包含选自SEQ ID NO：16、17、18、19或20或者其保守修饰形式的氨基酸序列；
轻链CDR2，其包含选自SEQ ID NO：21、22、23、24或25或者其保守修饰形式的氨基酸序列；和
轻链CDR3，其包含选自SEQ ID NO：26、27、28、30或32或者其保守修饰形式的氨基酸序列。
另一方面，本发明提供了结合CD47的分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与选自SEQ ID NO：33、35、37、39、41、83、85或87的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与选自SEQ ID NO：34、36、38、40、42、84、86或88的氨基酸序列具有至少80％同一性的氨基酸序列。
其中，SEQ ID NO：83所示序列为：
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGEFIPGSDTTNYAQKFQGRVTITAX 3X 4STX 5TAYMELSSLRSEDTAVYYCARGGLRRMDYWGQGTLVTVSS，其中，X 3为D或E，X 4为I或E，X 5为S或N；
SEQ ID NO：84所示序列为：
DIQMTQSPSSLSASVGDRVTITCRASSSVSSTYLHWYQQKPGKAPKLX 6IYTTSTLASGVPSRFSGSGSGTX 7X 8TLTISSLQPEDFATYYCQQFSX 2STWTFGQGTKLEIK，其中，X 6为L或W，X 7为D或S，X 8为F或Y，X 2为D或E；
SEQ ID NO：85所示序列为：
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLX 9WMGWINTNTGEPTYAQX 10LQGRVTMTX 11DTSTX 12TAYMELRSLRSDDTAVYYCX 13RFSHLRGPMDYWGQGTLVTVSS，其中，X 9为E或K，X 10为K或E，X 11为L或T，X 12为R或S，X 13为A或T；
SEQ ID NO：86所示序列为：
DX 14QMTQSPSSLSASVGDRVTITCRSSQSLVHSNGYTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPPTFGQGTKLEIK，其中，X 14为I或A；
SEQ ID NO：87所示序列为：
QVQLVQSGAEVKKPGASVKVSCKX 15SGFNIEDDYIEWVRQAPGQGLEWMGRIDPANDKTKYAQKFQGRVTMTX 16DTSTX 17TVYMELSSLRSEDTAVYYCX 18RPGLRRYYSMDYWGQGTLVTVSS，其中，X 15为A或V，X 16为R或G，X 17为S或N，X 18为A或T；
SEQ ID NO：88所示序列为：
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFX 19GSGSX 20TDFTLTISSLQPEDFATYYCGQSYSYPLTFGQGTKLEIK，其中，X 19为S或I，X 20为S或G。
另一方面，本发明提供了结合CD47的分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽或其抗原结合部分展现出下述性质中的一种或几种的组合：
(a)以1.89E-08或更小(例如1.53E-08或更小)的K D值与CD47结合；
(b)阻断CD47与SIRPα的结合；
(c)促进巨噬细胞介导的对表达CD47细胞的吞噬作用；
(d)没有明显诱导CD4+T细胞的凋亡；
(e)不引起实质性的红细胞减少、贫血或红细胞凝集；和
(f)所述抗原结合多肽或其抗原结合部分的熔解温度T≥62℃，聚合温度Tagg≥61℃。
另一方面，本发明提供了分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽或其抗原结合部分与本文提供的任何示例性抗原结合多肽或其抗原结合部分结合CD47上的相同表位。
另一方面，本发明提供了分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽或其抗原结合部分与本文提供的任何示例性抗原结合多肽或其抗原结合部分竞争结合CD47，其中所述竞争通过ELISA、流式细胞术或表面等离子体共振测定进行测量。
在本文中，所述抗原结合部分可选自Fab片段、Fab'片段、F(ab') 2片段、Fd片段、Fv片段、dAb片段、分离的互补决定区和纳米抗体，但不限于此。
另一方面，本发明提供了免疫偶联物，其包含本文所述的结合CD47的抗原结合多肽或其抗原结合部分，以及与所述抗原结合多肽或其抗原结合部分连接或缀合的治疗剂。
在本文中，所述抗原结合多肽或其抗原结合部分可通过接头与所述治疗剂连接。接头可以是可裂解的或不可裂解的。
在本文中，所述治疗剂可选自细胞毒性药物、放射性同位素或免疫调节剂，例如化学治疗剂、免疫抑制剂、免疫刺激剂、抗代谢物、烷化剂、抗生素、抗血管生成剂、抗有丝分裂剂、毒素、细胞凋亡剂等。
另一方面，本发明提供了组合物，其包含组分A以及以及药学上可接受的载体，其中组分A为本文所述的结合CD47的抗原结合多肽或其抗原结合部分、或本文所述的免疫偶联物。
另一方面，本发明提供了分离的杂交瘤细胞系，其选自由2B2、2H8、3F10、16E5和14A9组成的组。
另一方面，本发明提供了分离的核酸，其编码本文所述的结合CD47的抗原结合多肽或其抗原结合部分。
另一方面，本发明提供了载体，其包含本文所述的分离的核酸。
另一方面，本发明提供了宿主细胞，其包含本文所述的载体或在其基因组中整合有本文所述的分离的核酸。优选地，所述载体为表达载体。
又一方面，本发明提供了制备结合CD47的抗原结合多肽或其抗原结合部分的方法，包括：在适于表达编码本文所述的结合CD47的抗原结合多肽或其抗原结合部分的核酸的条件下培养本文所述的宿主细胞，分离所表达的所述抗原结合多肽或其抗原结合部分。
又一方面，本发明提供了用于在受试者中减少肿瘤或抑制肿瘤细胞生长的方法，其中所述方法包括向所述受试者施用治疗有效量的本文所述的结合CD47的抗原结合多肽或其抗原结合部分、本文所述的免疫偶联物、或所述的组合物。
又一方面，本发明提供了用于在有需要的受试者中治疗癌症的方法，其中所述方法包括向所述受试者施用治疗有效量的本文所述的结合CD47的抗原结合多肽或其抗原结合部分、本文所述的免疫偶联物、或所述的组合物。
又一方面，本发明提供了促进受试者巨噬细胞的吞噬作用的方法，其中所述方法包括向所述受试者施用有效量的本文所述的结合CD47的抗原结合多肽或其抗原结合部分、本文所述的免疫偶联物、或所述的组合物。
附图说明
图1为抗CD47嵌合抗体对红细胞凝集的影响；其中，相同行样品(抗体或对照)种类相同但是浓度不同，相同列样品浓度相同但是种类不同；由左至右，不同列的样品浓度依次分别为10000ng/ml、3333.33ng/ml、1111.11ng/ml、370.37ng/ml、123.46ng/ml、41.15ng/ml、13.72ng/ml、4.57ng/ml；由上至下，不同行的样品依次分别为Xi2B2、Xi2H8、Xi3F10、Xi16E5、Xi14A9、Xi16E5-2(Xi16E5的另一个重复)、Hu5F9、IgG4、PBS。
图2为部分抗CD47人源化抗体对红细胞凝集的影响；其中，相同列抗体种类相同但是浓度不同，相同行抗体浓度相同但是种类不同；由上至下，不同行的抗体浓度依次分别为10000ng/ml、3333.33ng/ml、1111.11ng/ml、370.37ng/ml、123.46ng/ml、41.15ng/ml、13.72ng/ml、4.57ng/ml；由左至右，第1-12列的抗体依次分别为hz3F10-1.1、hz3F10-2.1、hz3F10-3.1、hz3F10-4.1、hz3F10-5.1、hz3F10-6.1、hz3F10-1.2、hz3F10-2.2、hz3F10-3.2、hz3F10-4.2、hz3F10-5.2、hz3F10-6.2。
图3为部分抗CD47人源化抗体对红细胞凝集的影响；其中，相同列抗体种类相同但是浓度不同，相同行抗体浓度相同但是种类不同；由上至下，不同行的抗体浓度依次分别为10000ng/ml、3333.33ng/ml、1111.11ng/ml、370.37ng/ml、123.46ng/ml、41.15ng/ml、13.72ng/ml、4.57ng/ml；由左至右，第1-8列的抗体依次分别为hz16E5-1.1、hz16E5-3.1、hz16E5-1.2、hz16E5-3.2、hz16E5-1.3、hz16E5-3.3、hz14A9-2.3、hz14A9-2.4。
图4为抗CD47抗体对小鼠生存率的影响。
图5为抗CD47抗体单次给药后小鼠体重随时间的变化。
图6为抗CD47抗体单次给药后不同时间的小鼠体重变化率。
图7为抗CD47抗体单次给药后小鼠RBC随时间的变化。
图8为抗CD47抗体单次给药后不同时间的小鼠RBC变化率。
图9为抗CD47抗体单次给药后小鼠HGB含量随时间的变化。
图10为抗CD47抗体单次给药后不同时间的小鼠HGB变化率。
图11为人急性髓系白血病细胞MOLM-16细胞小鼠模型中，抗CD47抗体给药后平均肿瘤体积随时间的变化。
具体实施方式
以下将对本申请的示例性的实施方式进行描述，但是本领域技术人员将理解的是本申请的保护范围并不限于此，而是可基于本申请的精神和构思进行各种修饰、修改或改变，这些修饰、修改或改变后的内容仍然落在本申请的范围内。
术语
如本文所用，术语“抗原结合多肽”是指具有至少一个抗原结合结构域的多肽和蛋白，抗原结合多肽的实例包括但不限于单克隆抗体、多特异性抗体或融合蛋白。如本文所用，术语抗原结合多肽的“抗原结 合部分”是指抗原结合多肽的一个或多个片段，其保留特异性结合抗原(例如CD47蛋白)的能力。已经显示抗原结合多肽的抗原结合功能可以通过其片段来执行。抗原结合多肽的“抗原结合部分”所涵盖的实例包括：(i)Fab片段，由V L、V H、C L和C H1结构域组成的单价片段；(ii)“Fab'片段”与Fab片段的不同之处在于在重链C H1结构域的羧基末端添加了几个残基，包括来自抗体铰链区的一个或多个半胱氨酸；(iii)F(ab') 2片段，包含通过铰链区二硫桥连接的两个Fab片段的二价片段；(iv)Fd片段，由V H和C H1结构域组成；(v)Fv片段，由抗体单臂的VL和VH结构域组成；(vi)dAb片段(Ward等人(1989)Nature341：544-546)，由V H结构域组成；(vii)分离的互补决定区(CDR)；和(viii)纳米抗体，包含单个可变域的重链可变区和两个恒定域。此外，虽然Fv片段的两个结构域，V L和V H由不同的基因编码，但是可以使用重组方法通过合成的接头将它们连接起来，从而使它们能够制备成单个蛋白链，其中V L和V H区配对形成单价分子(称为单链Fv(scFv)；参见例如Bird等人，(1988)Science 242：423-426；和Huston等人(1988)Proc.Natl.Acad.USA 85：5879-5883)。此类单链抗体也意图涵盖在术语抗原结合多肽中。本文的抗原结合部分可使用本领域技术人员公知的常规技术获得，可以以与完整抗原结合多肽相同的方式对片段筛选功用。
术语“同种型”是指由重链恒定区基因编码的抗体种类。本发明的结合CD47的抗原结合多肽和其抗原结合部分可以衍生自任何物种，其包括但不限于小鼠、大鼠、兔、非人灵长类动物(如黑猩猩、食蟹猴、蜘蛛猴、猕猴)、美洲驼和人。结合CD47的抗原结合多肽和其抗原结合部分可以是嵌合抗体、人源化抗体或完整的人抗体。在一些实施方案中，结合CD47的抗原结合多肽是由源自小鼠的杂交瘤细胞系产生的抗体。因此，在一些实施方案中，结合CD47的抗原结合多肽是鼠类抗体。在另一些实施方案中，结合CD47的抗原结合多肽是嵌合抗体。在一些实施方案中，嵌合抗体是小鼠-人嵌合抗体。在另一些实施方案中，结合CD47的抗原结合多肽是人源化抗体。在另一些实施方案中，结合CD47的抗原结合多肽衍生自鼠类抗体并且是人源化的。
术语“嵌合抗体”是下述抗体：所述抗体具有衍生自一种物种的重链可变区的至少一部分和轻链可变区的至少一部分；以及衍生自另一物种的恒定区的至少一部分。例如，在一些实施方案中，嵌合抗体可以包含鼠类可变区和人恒定区。
“人源化抗体”是下述抗体：所述抗体含有衍生自非人抗体的互补决定区(CDR)；和衍生自人抗体的框架区以及恒定区。例如，本文提供的结合CD47的人源化抗体可以包含衍生自一种或多种鼠类抗体的CDR以及人框架区和恒定区。因此，在一些实施方案中，本文提供的人源化抗体与所述抗体的CDR所衍生自的鼠类抗体结合CD47上的相同表位。本文提供了示例性人源化抗体。包含本文提供的重链CDR和轻链CDR的另外的结合CD47的人源化抗体或其变体可以使用任何人框架序列产生，并且也包括在本发明中。在一些实施方案中，适用于在本发明中使用的框架序列包括在结构上与本文提供的框架序列类似的那些框架序列。可以在框架区中进行另外修饰以改进本文提供的抗体的特性。此类另外的框架修饰可以包括化学修饰，点突变以降低免疫原性或去除T细胞表位，或使突变回复为原始种系序列中的残基。在一些实施方案中，此类修饰包括对应于本文示例的突变的那些修饰，包括对种系序列的回复突变。例如，在一些实施方案中，本文提供的人源化抗体的VH和/或VL的人框架区中的一个或多个氨基酸被回复突变为亲本鼠类抗体中对应的氨基酸。例如，对于人源化3F10、14A9和16E5的VH和VL，上述模板人抗体的框架氨基酸的几个位点被回复突变为小鼠3F10、14A9和16E5抗体中对应的氨基酸序列。在一些实施方案中，轻链可变区的位置2处和/或33处和/或48处和/或63处和/或68处和/或71处和/或72处和/或94处的氨基酸被回复突变为在小鼠3F10、14A9或16E5轻链可变区中的所述位置处发现的对应的氨基酸。在另一些实施方案中，重链可变区的位置24处和/或46和/或63处和/或72处和/或73处和/或74处和/或77处和/或97处的氨基酸被回复突变为在小鼠3F10、14A9或16E5重链可变区中的所述位置处发现的对应的氨基酸。在一些实施方案中，人源化3F10抗体包含重链可变区，其突变类型选自如下位点中的一个或多个：在位置73处的氨基酸从Asp(D)突变为Glu(E)；在位置74处的氨基酸从Glu(E)突变为Ile(I)；以及在位置77处的氨基酸从Ser(S)突变为Asn(N)，并且对于人源化3F10抗体包含轻链可变区，其突变类型选自如下位点中的一个或多个：在位置48处的氨基酸从Leu(L)突变为Trp(W)；在位置71处的氨基酸从Asp(D)突变为Ser(S)；在位置72处的氨基酸从Phe(F)突变为Tyr(Y)；以及在位置94处的氨基酸从Asp(D)突变为Glu(E)。在一些实施方案中，人源化14A9抗体包含重链可变区，其突变类型选自如下位点中的一个或多个：在位置24处的氨基酸从Ala(A)突变为Val(V)；在位置72处的氨基酸从Arg(R)突变为Gly(G)；在位置77处的氨基酸从Ser(S)突变为Asn(N)；以及在位置97处的氨基酸从Ala(A)突变为Thr(T)，并且人源化14A9包含轻链可变区，其突变类型选自如下位点中的一个或多个：在位置63处的氨基酸从Ser(S)突变为Ile(I)；在位置68处的氨基酸从Gly(G)突变为Ser(S)。在一些实施方案中，人源化16E5抗体包含重链可变区，其突变类型选自如下位点中的一个或多个：在位置46处的氨基酸从Glu(E)突变为Lys(K)；在位置63处的氨基酸从Lys(K)突变为Glu(E)；在位置72处的氨基酸从Thr(T)突变为Leu(L)；在位置77处的氨基酸从Ser(S)突变为Arg(R)；以 及在位置97处的氨基酸从Ala(A)突变为Thr(T)，并且人源化16E5抗体包含轻链可变区，其突变类型选自如下位点中的一个或多个：在位置2处的氨基酸从Ile(I)突变为Ala(A)；以及在位置33处的氨基酸从Asn(N)突变为Gln(Q)。如无特殊说明，本文中突变位点序号的描述按照可变区第一个氨基酸开始依次计数。另外的或另选的回复突变可以在本文提供的人源化抗体的框架区中进行以改进抗体的特性。本发明还包括下述人源化抗体，所述人源化抗体结合CD47并且包含对应于本文所述的相对于任何合适的框架序列的示例性修饰的框架修饰，以及以其他方式改进抗体特性的其他框架修饰。
如本文所用，术语“衍生的”当用于指相对于参考抗体或其他结合蛋白的分子或多肽时，意指能够与参考抗体或其他结合蛋白特异性地结合相同表位的分子或多肽。
术语“分离的”是指已经从其天然环境中分离的目标化合物(例如，抗体或核酸)。
如本文所用，术语“EC50”是指有效浓度，抗体的50％最大应答。如本文所用，术语“IC50”是指抑制浓度，抗体的50％最大应答。EC50和IC50两者均可以通过ELISA或FACS分析或本领域已知的任何其他方法进行测量。
术语“K D”在用于本文时指解离常数，以摩尔浓度(M)表示。抗体的K D值可以使用本领域公知的方法测定。一种优选的测定抗体K D的方法是使用表面等离振子共振(surface plasmon resonance)，更优选使用生物传感器系统，例如Biacore系统。
术语“治疗”是指为了以统计学显著的方式治疗、治愈、减轻、缓解、改变、补救、改善、改进或影响疾患(例如疾病)、疾患的症状，或预防或延缓症状、并发症、生化指标的发作，或以其他方式阻滞或抑制疾病、疾患或病症的进一步发展而使用的措施。
如本文所用，术语“治疗有效量”是指向受试者提供治疗性和/或预防性益处所必需的化合物或组合物的量。
如本文所用，术语“受试者”包括任何人类或非人动物。术语“非人动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、绵羊、犬、锚、马、牛、鸡、两栖动物、爬行动物等。优选地，根据本发明的受试者是人。除非标明，术语“患者”或“受试者”可以互换使用。
如本文所用，“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内，其部分取决于如何测量或测定该值，即测量系统的限制。例如，“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，“约”可以表示多至±5％的范围，例如在所给定的具体数值范围±2％范围内、±1％范围内或±0.5％范围内波动。当本申请或权利要求中给出特定值时，除非另有说明，“约”的含义应认为是在该特定值的可接受的误差范围内。在本文中，除非另有说明，步骤参数或条件的值默认均由“约”修饰。
术语“同一性(identity)”，也称一致性。两序列间的百分比同一性为序列共有的相同位置数的函数(即％同一性＝相同位置数/位置总数×100)，其中需考虑产生两序列的最优比对需要引入的缺口数和每个缺口的长度。如下述非限制性实施例所示，可以使用数学算法完成序列的比较和两序列间百分比同一性的测定。可以使用E.Meyers和W.Miller的算法(Comput.Appl.Biosci.,4:11-17(1988))测定两氨基酸序列间的百分比同一性，该算法已收入到ALIGN程序(版本2.0)中，其使用PAM120残基权重表，缺口长度罚分为12，缺口罚分为4。此外，可以使用Needleman和Wunsch的算法(J.Mol.Biol.484-453(1970))测定两氨基酸序列的百分比同一性，该算法已掺入到GCG软件包(可在www.gcg.com获得)中的GAP程序中，其使用Blossum 62矩阵或PAM250矩阵，缺口权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6。
术语“Xn”和“Xaa”等同，是指未指定的氨基酸(Unspecified Amino Acid)，通过相关表述中的后续定义来指定其涵盖的范围。
术语“包括”、“含有”或“包含”及其变体应理解为“包括但不限于”，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
在本文中，除非上下文另有明确规定，否则单数术语涵盖复数指代物，反之亦然。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它已确定的出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本文中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。本发明的各个方面将在下述部分中进一步详细描述。
结合CD47的抗原结合多肽及其抗原结合部分
本文中，术语“CD47”、“整合素相关蛋白”、“IAP(Integrin-associated protein)”、“OA3”、“MER6”之间彼此可以互换的使用。术语“人CD47”和“hCD47”等在本文中可互换使用，并且是指人CD47和 人CD47的变体或同种型。本文中，“结合CD47的抗体”与“抗CD47抗体”之间彼此可以互换的使用。
本发明提供了结合CD47的抗原结合多肽或其抗原结合部分，为靶向CD47的治疗提供新的解决方案。除了与人CD47或/和食蟹猴CD47结合外，本文提供的抗原结合多肽还显示了其他诸多用于靶向治疗的期望的特征。具体的，这些期望的特征例如可以是1.53E-08或更小的K D值与CD47结合、阻断CD47与SIRPα的结合、促进巨噬细胞介导的对表达CD47细胞的吞噬作用、没有明显诱导CD4+T细胞的凋亡、不引起实质性的红细胞减少、贫血或红细胞凝集、所述抗原结合多肽或其抗原结合部分的熔解温度T≥62℃，聚合温度Tagg≥61℃等中的至少一个。在一些实施方案中，结合CD47的抗原结合多肽表现了更好的治疗效果；在一些实施方案中，结合CD47的抗原结合多肽表现了降低的副作用。
一方面，本发明提供了包含特定序列的结合CD47的抗原结合多肽及其抗原结合部分。
在一些实施方案中，本文提供了包含选自由SEQ ID NOs：1～32组成的组的一个或多个CDR的分离的结合CD47的抗原结合多肽或其抗原结合部分。
在一些实施方案中，结合CD47的分离的抗原结合多肽或其抗原结合部分包含重链CDR1序列，所述重链CDR1包含与选自SEQ ID NO：1、2、3、4或5的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽或其抗原结合部分包含重链CDR2，所述重链CDR2包含与选自SEQ ID NO：6、7、8、9或10的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽或其抗原结合部分包含重链CDR3，所述重链CDR3包含与选自SEQ ID NO：11、12、13、14或15的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽或其抗原结合部分包含轻链CDR1，所述轻链CDR1包含与选自SEQ ID NO：16、17、18、19或20的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽或其抗原结合部分包含轻链CDR2，所述轻链CDR2包含与选自SEQ ID NO：21、22、23、24或25的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽或其抗原结合部分包含轻链CDR3，所述轻链CDR3包含与选自SEQ ID NO：26、27、28、30或32的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
本领域技术人员将理解，本文提供的抗原结合多肽的重链CDR和轻链CDR可以独立地进行选择或混合和匹配，以形成包含来自本文提供的抗原结合多肽的任何重链CDR1、CDR2和CDR3；以及任何轻链CDR1、CDR2和CDR3的抗原结合多肽或其抗原结合部分。因此，在一些实施方案中，本发明提供了结合CD47的分离的抗原结合多肽，包含三个重链CDR(重链CDR1、重链CDR2和重链CDR3)和三个 轻链CDR(轻链CDR1、轻链CDR2、轻链CDR3)，其中：
重链CDR1，其包含与选自SEQ ID NO：1、2、3、4或5的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列；
重链CDR2，其包含与选自SEQ ID NO：6、7、8、9或10的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列；
重链CDR3，其包含与选自SEQ ID NO：11、12、13、14或15的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列；
轻链CDR1，其包含与选自SEQ ID NO：16、17、18、19或20的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列；
轻链CDR2，其包含与选自SEQ ID NO：21、22、23、24或25的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列；和
轻链CDR3，其包含与选自SEQ ID NO：26、27、28、30或32的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽，包含以下互补决定区：重链CDR1，其包含选自SEQ ID NO：1、2、3、4或5的氨基酸序列；重链CDR2，其包含选自SEQ ID NO：6、7、8、9或10的氨基酸序列；重链CDR3，其包含选自SEQ ID NO：11、12、13、14或15的氨基酸序列；轻链CDR1，其包含选自SEQ ID NO：16、17、18、19或20的氨基酸序列；轻链CDR2，其包含选自SEQ ID NO：21、22、23、24或25的氨基酸序列；和轻链CDR3，其包含选自SEQ ID NO：26、27、28、30或32的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：1、6和11所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：16、21和26所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含以下互补决定区：包含SEQ ID NO：1的氨基酸序列的重链CDR1；包含SEQ ID NO：6的氨基酸序列的重链CDR2；包含SEQ ID NO：11的氨基酸序列的重链CDR3；包含SEQ ID NO：16的氨基酸序列的轻链CDR1；包含SEQ ID NO：21的氨基酸序列的轻链CDR2；和包含SEQ ID NO：26的氨基酸序列的轻链CDR3。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻 链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：2、7和12所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：17、22和27所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含以下互补决定区：包含SEQ ID NO：2的氨基酸序列的重链CDR1；包含SEQ ID NO：7的氨基酸序列的重链CDR2；包含SEQ ID NO：12的氨基酸序列的重链CDR3；包含SEQ ID NO：17的氨基酸序列的轻链CDR1；包含SEQ ID NO：22的氨基酸序列的轻链CDR2；和包含SEQ ID NO：27的氨基酸序列的轻链CDR3。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：3、8和13所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：18、23和28所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含以下互补决定区：包含SEQ ID NO：3的氨基酸序列的重链CDR1；包含SEQ ID NO：8的氨基酸序列的重链CDR2；包含SEQ ID NO：13的氨基酸序列的重链CDR3；包含SEQ ID NO：18的氨基酸序列的轻链CDR1；包含SEQ ID NO：23的氨基酸序列的轻链CDR2；和包含SEQ ID NO：28的氨基酸序列的轻链CDR3。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：4、9和14所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：19、24和32所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含以下互补决定区：包含SEQ ID NO：4的氨基酸序列的重链CDR1；包含SEQ ID NO：9的氨基酸序列的重链CDR2；包含SEQ ID NO：14的氨基酸序列的重链CDR3；包含SEQ ID NO：19的氨基酸序列的轻链CDR1；包含SEQ ID NO：24的氨基酸序列的轻链CDR2；和包含SEQ ID NO：32的氨基酸序列的轻链CDR3。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：5、10和15所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：20、25和30所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至 少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含以下互补决定区：包含SEQ ID NO：5的氨基酸序列的重链CDR1；包含SEQ ID NO：10的氨基酸序列的重链CDR2；包含SEQ ID NO：15的氨基酸序列的重链CDR3；包含SEQ ID NO：20的氨基酸序列的轻链CDR1；包含SEQ ID NO：25的氨基酸序列的轻链CDR2；和包含SEQ ID NO：30的氨基酸序列的轻链CDR3。
本发明还提供了保守修饰形式的抗原结合多肽及其抗原结合部分。本领域技术人员将认识到，保守的氨基酸取代是用具有类似结构或化学特性(例如像类似的侧链)的另一氨基酸取代一个氨基酸。示例性保守取代在本领域中例如，在Watson等，MolecularBiology of the Gene，The Bengamin/Cummings Publication Company，第4版(1987)中进行了描述。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含：重链CDR1，其包含选自SEQ ID NO：1、2、3、4或5或者其保守修饰形式的氨基酸序列；重链CDR2，其包含选自SEQ ID NO：6、7、8、9或10或者其保守修饰形式的氨基酸序列；重链CDR3，其包含选自SEQ ID NO：11、12、13、14或15或者其保守修饰形式的氨基酸序列；轻链CDR1，其包含选自SEQ ID NO：16、17、18、19或20或者其保守修饰形式的氨基酸序列；轻链CDR2，其包含选自SEQ ID NO：21、22、23、24或25或者其保守修饰形式的氨基酸序列；和轻链CDR3，其包含选自SEQ ID NO：26、27、28、30或32或者其保守修饰形式的氨基酸序列。保守修饰存在于任意一个或多个轻链CDR或重链CDR。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含：重链CDR1，其包含选自SEQ ID NO：3、4或5的氨基酸序列；重链CDR2，其包含选自SEQ ID NO：8、9或10的氨基酸序列；重链CDR3，其包含选自SEQ ID NO：13、14或15的氨基酸序列；轻链CDR1，其包含选自SEQ ID NO：18、19或20的氨基酸序列；轻链CDR2，其包含选自SEQ ID NO：23、24或25的氨基酸序列；和轻链CDR3，其包含选自SEQ ID NO：28、30或32的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1包含SEQ ID NO：1或其保守修饰形式的氨基酸序列，所述重链CDR2包含SEQ ID NO：6或其保守修饰形式的氨基酸序列，所述重链CDR3包含SEQ ID NO：11或其保守修饰形式的氨基酸序列，所述轻链CDR1包含SEQ ID NO：16或其保守修饰形式的氨基酸序列，所述轻链CDR2包含SEQ ID NO：21或其保守修饰形式的氨基酸序列，所述轻链CDR3包含SEQ ID NO：26或其保守修饰形式的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1包含SEQ ID NO：2或其保守修饰形式的氨基酸序列，所述重链CDR2包含SEQ ID NO：7或其保守修饰形式的氨基酸序列，所述重链CDR3包含SEQ ID NO：12或其保守修饰形式的氨基酸序列，所述轻链CDR1包含SEQ ID NO：17或其保守修饰形式的氨基酸序列，所述轻链CDR2包含SEQ ID NO：22或其保守修饰形式的氨基酸序列，所述轻链CDR3包含SEQ ID NO：27或其保守修饰形式的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1包含SEQ ID NO：3或其保守修饰形式的氨基酸序列，所述重链CDR2包含SEQ ID NO：8或其保守修饰形式的氨基酸序列，所述重链CDR3包含SEQ ID NO：13或其保守修饰形式的氨基酸序列，所述轻链CDR1包含SEQ ID NO：18或其保守修饰形式的氨基酸序列，所述轻链CDR2包含SEQ ID NO：23或其保守修饰形式的氨基酸序列，所述轻链CDR3包含SEQ ID NO：28或其保守修饰形式的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1包含SEQ ID NO：4或其保守修饰形式的氨基酸序列，所述重链CDR2包含SEQ ID NO：9或其保守修饰形式的氨基酸序列，所述重链CDR3包含SEQ ID NO：14或其保守修饰形式的氨基酸序列，所述轻链CDR1包含SEQ ID NO：19或其保守修饰形式的氨基酸序列，所述轻链CDR2包含SEQ ID NO：24或其保守修饰形式的氨基酸序列，所述轻链CDR3包含SEQ ID NO：32或其保守修饰形式的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1包含SEQ ID NO：5或其保守修饰形式的氨基酸序列，所述重链CDR2包含SEQ ID NO：10或其保守修饰形式的氨基酸序列，所述重链CDR3包含SEQ ID NO：15或其保守修饰形式的氨基酸序列，所述轻链CDR1包含SEQ ID NO：20或其保守修饰形式的氨基酸序列，所述轻链CDR2包含SEQ ID NO：25或其保守修饰形式的氨基酸序列，所述轻链CDR3包含SEQ  ID NO：30或其保守修饰形式的氨基酸序列。
在一些实施方案中，本文提供的结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，重链和轻链的可变区包含CDR区和框架区FR(包括FR1、FR2、FR3和FR4)，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。
因此，另一方面，本发明提供了结合CD47的分离的抗原结合多肽或其抗原结合部分，所述抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与选自SEQ ID NO：33、35、37、39、41、83、85或87的氨基酸序列具有至少80％、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与选自SEQ ID NO：34、36、38、40、42、84、86或88的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。
其中，SEQ ID NO：83所示序列为：
QVQLVQSGAEVKKPGSSVKVSCKAS GYTFSRYWIEWVRQAPGQGLEWMG EFIPGSDTTNYAQKFQGRVTITAX 3X 4STX 5TAYMELSSLRSEDTAVYYCAR GGLRRMDYWGQGTLVTVSS，其中，X 3为D或E，X 4为I或E，X 5为S或N；
SEQ ID NO：84所示序列为：
DIQMTQSPSSLSASVGDRVTITC RASSSVSSTYLHWYQQKPGKAPKLX 6IY TTSTLASGVPSRFSGSGSGTX 7X 8TLTISSLQPEDFATYYC QQFSX 2STWT FGQGTKLEIK，其中，X 6为L或W，X 7为D或S，X 8为F或Y，X 2为D或E；
SEQ ID NO：85所示序列为：
QVQLVQSGAEVKKPGASVKVSCKAS GYTFTNYGMNWVRQAPGQGLX 9WMG WINTNTGEPTYAQX 10LQGRVTMTX 11DTSTX 12TAYMELRSLRSDDTAVYYCX 13R FSHLRGPMDYWGQGTLVTVSS，其中，X 9为E或K，X 10为K或E，X 11为L或T，X 12为R或S，X 13为A或T；
SEQ ID NO：86所示序列为：
DX 14QMTQSPSSLSASVGDRVTITC RSSQSLVHSNGYTYLHWYQQKPGKAPKLLIY KVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC SQSTHVPPTFGQGTKLEIK，其中，X 14为I或A；
SEQ ID NO：87所示序列为：
QVQLVQSGAEVKKPGASVKVSCKX 15S GFNIEDDYIEWVRQAPGQGLEWMG RIDPANDKTKYAQKFQGRVTMTX 16DTSTX 17TVYMELSSLRSEDTAVYYCX 18R PGLRRYYSMDYWGQGTLVTVSS，其中，X 15为A或V，X 16为R或G，X 17为S或N，X 18为A或T；
SEQ ID NO：88所示序列为：
DIQMTQSPSSLSASVGDRVTITC KASENVVSYVSWYQQKPGKAPKLLIY GASNRYTGVPSRFX 19GSGSX 20TDFTLTISSLQPEDFATYYC GQSYSYPLTFGQGTKLEIK，其中，X 19为S或I，X 20为S或G。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：33、35、37、39、41、83、85或87的氨基酸序列，所述的轻链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：34、36、38、40、42、84、86或88的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：35、39、41、83、85或87的氨基酸序列，所述的轻链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：36、40、42、84、86或88的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含SEQ ID NO：79的氨基酸序列或由SEQ ID NO：79的氨基酸序列组成，所述的轻链可变区包含SEQ ID NO：80的氨基酸序列或由SEQ ID NO：80的氨基酸序列组成。在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含SEQ ID NO：81的氨基酸序列或由SEQ ID NO：81的氨基酸序列组成，所述的轻链可变区包含SEQ ID NO：82的氨基酸序列或由SEQ ID NO：82的氨基酸序列组成。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：33的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％ 同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：34的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，所述的结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：33的氨基酸序列，所述的轻链可变区包含SEQ ID NO：34的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：35的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：36的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，所述的结合抗CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：35的氨基酸序列，所述的轻链可变区包含SEQ ID NO：36的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：37的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：38的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，本文提供的结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：37的氨基酸序列，所述的轻链可变区包含SEQ ID NO：38的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：39的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：40的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，本文提供的结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：39的氨基酸序列，所述的轻链可变区包含SEQ ID NO：40的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：41的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：42的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，本文提供的结合CD47的分离的抗原结合多肽，其中所述的重链可变区包含SEQ ID NO：41的氨基酸序列，所述的轻链可变区包含SEQ ID NO：42的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：83的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至 少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：84的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：83的氨基酸序列，所述的轻链可变区包含SEQ ID NO：84的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：85的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：86的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：85的氨基酸序列，所述的轻链可变区包含SEQ ID NO：86的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：87的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：88的氨基酸序列具有至少80％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一些实施方案中，结合CD47的分离的抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：87的氨基酸序列，所述的轻链可变区包含SEQ ID NO：88的氨基酸序列。
在一些实施方案中，本文所述的重链可变区和轻链可变区中含有本文所述的重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3的序列特征。
在一些实施方案中，本文提供的结合CD47的分离的抗原结合多肽包含重链和轻链，重链和轻链除了包含本文所述的重链可变区和轻链可变区外，还包括恒定区。在一些实施方案中，恒定区是人源化的。在一些实施方案中，恒定区与可变区的框架区FR都是人源化的。通过构建嵌合抗体(例如人源化的恒定区和非人源的可变区)或人源化抗体(例如人源化的恒定区和FR区)降低免疫原性。
在一些实施方案中，抗原结合多肽的轻链恒定区是人κ链恒定区。在一些实施方案中，抗原结合多肽的轻链恒定区是人λ链恒定区。
抗原结合多肽的重链恒定区可来自任何类型的恒定区，例如IgG、IgM、IgD、IgA和IgE；以及任何同种型，例如IgG1、IgG2、IgG3和IgG4。在一些实施方案中，结合CD47的分离的抗原结合多肽是IgG1同种型。在一些实施方案中，结合CD47的分离的抗原结合多肽是IgG4同种型。
在一些实施方案中，抗原结合多肽包含经修饰的恒定区。在一些实施方案中，对人IgG4恒定区内的铰链区进行修饰以避免或减少链交换，例如IgG4型抗体具有根据EU编号索引的Ser228Pro(S228P)突变。
在一些实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，其包含重链可变区和轻链可变区，所述的重链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：33、35、37、39或41的氨基酸序列，所述的轻链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：34、36、38、40或42的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，其包含重链和轻链，所述重链具有与选自SEQ ID NO：89、93、97、101或105所示的氨基酸序列具有至少80％同一性、至少85％同一性、 至少90％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链具有与选自SEQ ID NO：91、95、99、103或107所示的氨基酸序列具有至少80％同一性、至少85％同一性、至少90％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一个实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，包含根据SEQ ID NO：89的重链和根据SEQ ID NO：91的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，包含根据SEQ ID NO：93的重链和根据SEQ ID NO：95的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，包含根据SEQ ID NO：97的重链和根据SEQ ID NO：99的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，包含根据SEQ ID NO：101的重链和根据SEQ ID NO：103的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是嵌合抗体，包含根据SEQ ID NO：105的重链和根据SEQ ID NO：107的轻链。
在一些实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，其包含重链可变区和轻链可变区，所述的重链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：83、85或87的氨基酸序列，所述的轻链可变区包含以下序列或由以下序列组成：选自SEQ ID NO：84、86或88的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，所述重链具有与选自SEQ ID NO：109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181或185所示的氨基酸序列具有至少80％同一性、至少85％同一性、至少90％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列，所述轻链具有与选自SEQ ID NO：111、115、119、123、127、131、135、139、143、147、151、155、159、163、167、171、175、179、183和187所示的氨基酸序列具有至少80％同一性、至少85％同一性、至少90％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、至少99％同一性或100％同一性的氨基酸序列。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：109的重链和根据SEQ ID NO：111的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：113的重链和根据SEQ ID NO：115的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：117的重链和根据SEQ ID NO：119的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：121的重链和根据SEQ ID NO：123的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：125的重链和根据SEQ ID NO：127的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO:129的重链和根据SEQ ID NO：131的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：133的重链和根据SEQ ID NO：135的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：137的重链和根据SEQ ID NO：139的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：141的重链和根据SEQ ID NO：143的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：145的重链和根据SEQ ID NO：147的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：149的重链和根据SEQ ID NO：151的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：153的重链和根据SEQ ID NO：155的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：157的重链和根据SEQ ID NO：159的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：161的重链和根据SEQ ID NO：163的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：165的重链和根据SEQ ID NO：167的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：169的重链和根据SEQ ID NO：171的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：173的重链和根据SEQ ID NO：175的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：177的重链和根据SEQ ID NO：179的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：181的重链和根据SEQ ID NO：183的轻链。在一个实施方案中，结合CD47的分离的抗原结合多肽是人源化抗体，包含根据SEQ ID NO：185的重链和根据SEQ ID NO：187的轻链。
在一个实施方案中，结合CD47的分离的抗原结合多肽包含重链和轻链，所述重链包含以下序列或由以下序列组成：选自SEQ ID NO：93、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181或185的氨基酸序列；所述轻链包含以下序列或由以下 序列组成：选自SEQ ID NO：95、103、107、111、115、119、123、127、131、135、139、143、147、151、155、159、163、167、171、175、179、183或187的氨基酸序列。
在一些实施方案中，结合CD47的分离的抗原结合多肽或/和其抗原结合部分以1.53E-08M或更小的K D值与CD47结合。在一些实施方案中，所述抗原结合多肽或/和其抗原结合部分结合人CD47。在另一些实施方案中，所述抗原结合多肽或/和其抗原结合部分结合食蟹猴CD47。在另一些实施方案中，所述抗原结合多肽或/和其抗原结合部分结合人CD47以及食蟹猴CD47。在一些实施方案中，所述抗原结合多肽或/和其抗原结合部分对人CD47具有下述结合亲和力(K D)：在约1E-12M至约1E-05M、约1E-12M至约1.89E-06M、约1E-12M至约1.89E-07M、约1E-11M至约1.89E-07M、约9.46E-10M至约1.89E-07M、约9.46E-10M至约1.89E-08M、约2.04E-10M至约1.53E-07M或约2.04E-10M至约1.89E-08M的范围内。在一些实施方案中，所述抗原结合多肽或/和其抗原结合部分对人CD47具有下述结合亲和力(K D)：约1E-05M或更小、约1.89E-06M或更小、约1.89E-07M或更小、约1.89E-08M或更小、约1.53E-08M或更小、约9.31E-08M或更小、约9.31E-09M或更小、约8.55E-09M或更小、约2.35E-09M或更小、或者约2.04E-010M或更小。在一些实施方案中，所述抗原结合多肽或/和其抗原结合部分对人CD47具有下述结合亲和力(K D)：约7.48E-09M、约3.50E-10M、约7.67E-10M、约9.06E-09M、约2.35E-09M、约9.46E-10M、约8.36E-10M、约1.53E-09M、约1.42E-09M、约9.60E-10M、约5.92E-10M、约9.04E-10M、约7.79E-10M、约1.15E-09M、约1.34E-09M、约8.96E-10M、约7.65E-10M、约1.89E-08M、约6.28E-09M、约9.31E-09M、约1.71E-08M、约1.53E-08M、约7.13E-09M、约2.04E-10M或约8.55E-09M。在一些实施方案中，通过Biacore来测量本文提供的结合CD47的抗原结合多肽和其抗原结合部分的结合亲和力k D。
在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分对人CD47具有约1ng/mL至约2000ng/mL、约1ng/mL至约1500ng/mL、约1ng/mL至约1106ng/mL、约1ng/mL至约699ng/mL、约1ng/mL至约340ng/mL、约30ng/mL至约340ng/mL、约50ng/mL至约340ng/mL或约100ng/mL至约340ng/mL的结合EC50。在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分对人CD47具有约2000ng/mL或更小、约1500ng/mL或更小、约1106ng/mL或更小、约699ng/mL或更小、或约340ng/mL或更小的结合EC50。在一些实施方案中，通过ELISA或FACS来测量本文提供的结合CD47的抗原结合多肽和其抗原结合部分的EC50。
在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分阻断CD47与SIRPα的结合。信号调节性蛋白α(SIRPα)是结合CD47的配体之一，SIRPα在包括巨噬细胞和树突细胞在内的造血细胞上表达。CD47与巨噬细胞上的SIRPα的相互作用，将“别吃我”信号发送至巨噬细胞，在治疗情况下，用结合CD47的抗原结合多肽或其抗原结合部分阻断SIRPα与CD47的相互作用可以促进宿主免疫系统摄取并清除癌细胞。因此，阻断CD47与SIRPα的相互作用从而使得巨噬细胞吞噬表达CD47的肿瘤细胞的能力是一些结合CD47抗原结合多肽的重要功能特征。在一些实施方案中，基于Jurkat细胞及流式细胞术检测，所述抗原结合多肽(一些示例例如嵌合抗体Xi2B2、Xi2H8、Xi3F10、Xi16E5、Xi14A9)或/和其抗原结合部分的阻断IC50为约358.5ng/mL至约26966ng/mL、约358.5ng/mL至约4861ng/mL、约358.5ng/mL至约631.2ng/mL或约358.5ng/mL至约1283ng/mL。在一些实施方案中，所述抗原结合多肽或/和其抗原结合部分的阻断IC50为约26966ng/mL或更小、约15000ng/mL或更小、约10000ng/mL或更小、约8000ng/mL或更小、约6000ng/mL或更小、约4861ng/mL或更小、约3000ng/mL或更小、约2500ng/mL或更小、约2000ng/mL或更小、约1500ng/mL或更小、约1283ng/mL或更小、约1000ng/mL或更小、约800ng/mL或更小、约500ng/mL或更小、或约358.5ng/mL或更小。
在一些实施方案中，基于ELISA分析，结合CD47的抗原结合多肽(一些示例例如嵌合抗体Xi2B2、Xi2H8、Xi3F10或其人源化抗体、Xi16E5或其人源化抗体、Xi14A9或其人源化抗体等)或/和其抗原结合部分的阻断IC50为约4.682ng/mL至约15246ng/mL、约621.1ng/mL至约15246ng/mL、约621.1ng/mL至约7910ng/mL、约621.1ng/mL至约2939ng/mL、约621.1ng/mL至约2500ng/mL、约621.1ng/mL至约2051ng/mL、约621.1ng/mL至约1738ng/mL、约621.1ng/mL至约1500ng/mL、约621.1ng/mL至约1400ng/mL、约621.1ng/mL至约1300ng/mL、约621.1ng/mL至约1200ng/mL、约621.1ng/mL至约1100ng/mL、约621.1ng/mL至约1000ng/mL、约621.1ng/mL至约900ng/mL、约621.1ng/mL至约800ng/mL、或约621.1ng/mL至约703.2ng/mL。在一些实施方案中，基于ELISA分析，所述抗原结合多肽或/和其抗原结合部分的阻断IC50为约15246ng/mL或更小、约7910ng/mL或更小、约2939ng/mL或更小、约2500ng/mL或更小、约2051ng/mL或更小、约1738ng/mL或更小、约1500ng/mL或更小、约1400ng/mL或更小、约1300ng/mL或更小、约1200ng/mL或更小、约1100ng/mL或更小、约1000ng/mL或更小、约900ng/mL或更小、约800ng/mL或更小、约703.2ng/mL或更小、约621.1ng/mL或更小、约450ng/mL或更小、约350ng/mL或更小、约250ng/mL或更小、约150ng/mL或更小、约100ng/mL或更小、约50 ng/mL或更小、约40ng/mL或更小、约30ng/mL或更小、约20ng/mL或更小、约10ng/mL或更小、或约4.682ng/mL或更小。
在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分促进巨噬细胞介导的对表达CD47细胞的吞噬作用。如前所述，通过结合CD47抗原结合多肽阻断SIRPα与CD47的结合进而增加巨噬细胞对表达CD47细胞如癌细胞(具体的例子如HL60细胞等)的吞噬作用。在一些实施方案中，约1μg/mL至约10μg/mL结合CD47的抗原结合多肽浓度下，巨噬细胞吞噬率为约3.5％至约65.7％、约11.5％至约65.7％、约28.8％至约65.7％、或约44.67％至约65.7％。在一些实施方案中，在约1μg/mL至约10μg/mL抗原结合多肽浓度下，巨噬细胞吞噬率为约1.34％至约81.66％、约2％至约81.66％、约11.95％至约81.66％、约24.19％至约81.66％、或约71.48％至约81.66％。
在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分没有明显诱导CD4+T细胞的凋亡。在一些实施方案中，凋亡检测采用流式细胞术的测定方法。本文明显的凋亡是指在可接受范围内(例如对受试者无影响或在可控范围内)或者与阴性对照相比在统计学意义上无显著差异(P≤0.05)，例如，一些实施方案中，与阴性对照IgG相比无显著差异。
在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分具有如下Tm(熔解温度)：约62℃或更高、约63℃或更高、约64℃或更高、约65℃或更高、约66℃或更高、约67℃或更高、约68℃或更高、约69℃或更高、或约70℃或更高。在一些实施方案中，所述抗体或/和其片段具有如下Tagg(聚集温度)：约61℃或更高、约62℃或更高、约63℃或更高、约64℃或更高、约65℃或更高、约66℃或更高、约67℃或更高、约68℃或更高、约69℃或更高、约70℃或更高、约71℃或更高、约72℃或更高、或约73℃或更高。在一些实施方案中，所述抗原结合多肽或/和其抗原结合部分的Tm为约62℃至约75℃、约63℃至约70.6℃、约64℃至约70.6℃、约65℃至约70.6℃、约67℃至约70.6℃、或约68℃至约70.6℃。在一些实施方案中，所述抗体或/和其片段的Tagg为约60℃至约73.81℃、约61℃至约73.81℃、约62℃至约73.81℃、约63℃至约73.81℃、约64℃至约73.81℃、约65℃至约73.81℃、约67℃至约73.81℃、或约68℃至约73.81℃。在一些实施方案中，Tm、Tagg通过nano DSF检测。
在一些优选的实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分不引起实质性的红细胞减少、贫血或红细胞凝集。术语“血红细胞”和“红细胞”是同义词并可互换使用。术语“凝集”表示细胞结块。由于红细胞也表达CD47，某些结合CD47的抗原结合多肽会与红细胞结合，从而发生凝集现象，因此，避免或降低结合CD47抗原结合多肽对红细胞的伤害作用将有望降低治疗副反应。
因此，另一方面，本发明提供了展现出下述性质中的一种或几种组合的结合CD47的抗原结合多肽及其抗原结合部分：
(a)以1.89E-08(例如1.53E-08)或更小的K D值与CD47结合；
(b)阻断CD47与SIRPα的结合；
(c)促进巨噬细胞介导的对表达CD47细胞的吞噬作用；
(d)没有明显诱导CD4+T细胞的凋亡；
(e)不引起实质性的红细胞减少、贫血或红细胞凝集；和
(f)所述抗原结合多肽或其抗原结合部分的熔解温度T≥62℃，聚集温度Tagg≥61℃。
在一些实施方案中，结合CD47的抗原结合多肽或/和其抗原结合部分具有所述性质(a)～(f)中的一个、两个、三个、四个、五个或六个。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)和(c)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)和(e)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(c)和(e)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)、(c)和(d)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)、(c)和(e)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)、(c)和(f)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)、(c)、(d)和(f)。在一些实施方案中，所述抗原结合多肽或其抗原结合部分具有所述性质(a)、(b)、(c)、(d)、(e)和(f)。
在一些实施方案中，与CD47结合的抗原结合多肽及其抗原结合部分是抗CD47抗体或其抗原结合部分。
在一些实施方案中，本文提供的结合CD47的分离的抗原结合多肽是单克隆抗体。
在一些实施方案中，本文提供的结合CD47的分离的抗体是单特异性抗体。
在一些实施方案中，本文提供的结合CD47的分离的抗体是多特异性抗体。例如，双特异抗体或者三特异性抗体等。
在一些实施方案中，本发明提供了与本文提供的任何示例性抗体结合CD47上的相同表位的结合 CD47的抗原结合多肽或其抗原结合部分，例如与16E5的嵌合抗体或人源化抗体结合相同表位，例如与3F10的嵌合抗体或人源化抗体结合相同表位，例如与14A9的嵌合抗体或人源化抗体结合相同表位。在一些实施方案中，抗原结合多肽或其抗原结合部分与本文提供的用于结合CD47的任何示例性抗体竞争，例如与16E5的嵌合抗体或人源化抗体竞争结合CD47，例如与3F10的嵌合抗体或人源化抗体竞争结合CD47，例如与14A9的嵌合抗体或人源化抗体竞争结合CD47。可以通过ELISA、流式细胞术、表面等离子体共振(SPR)测定或本领域已知的任何其他方法测量与CD47的结合。
本发明提供了一些示例性的结合CD47的单克隆抗体，包括2B2、2H8、3F10、16E5和14A9抗体的嵌合版本(Xi2B2、Xi2H8、Xi3F10、Xi16E5和Xi14A9)以及3F10、16E5、14A9的人源化变体。本文提供的示例性的结合CD47的抗体的重链CDR(重链CDR1、重链CDR2和重链CDR3)的氨基酸序列提供于下表S1中，轻链CDR(轻链CDR1、轻链CDR2和轻链CDR3)的氨基酸序列提供于下表S2中，可变区以及全长重链、轻链的氨基酸序列提供于下表S3、S4中。
表S1：抗CD47抗体的重链的CDR序列
Figure PCTCN2021084802-appb-000001
表S2：抗CD47抗体的轻链CDR序列
Figure PCTCN2021084802-appb-000002
表S3：抗CD47抗体的可变区序列
Figure PCTCN2021084802-appb-000003
Figure PCTCN2021084802-appb-000004
Figure PCTCN2021084802-appb-000005
Figure PCTCN2021084802-appb-000006
表S4：抗CD47抗体的全长重链和全长轻链的序列
Figure PCTCN2021084802-appb-000007
Figure PCTCN2021084802-appb-000008
Figure PCTCN2021084802-appb-000009
Figure PCTCN2021084802-appb-000010
Figure PCTCN2021084802-appb-000011
Figure PCTCN2021084802-appb-000012
Figure PCTCN2021084802-appb-000013
免疫偶联物
在一些实施方案中，本发明提供了结合CD47的抗原结合多肽或其抗原结合部分的免疫偶联物。可以与结合CD47的抗原结合多肽或其抗原结合部分连接或缀合的治疗剂可以包括但不限于细胞毒性药物、放射性同位素、免疫调节剂或抗体。在一些实施方案中，结合CD47的抗原结合多肽或其抗原结合部分与治疗剂直接缀合。在一些实施方案中，结合CD47的抗原结合多肽或其抗原结合部分通过接头与治疗剂缀合。
组合物
本发明提供了药物组合物，所述药物组合物包含分离的结合CD47的抗原结合多肽或其抗原结合部分，或编码所述抗体或片段的核酸，或本文所述的免疫偶联物，并且还包含一种或多种药学上可接受的载体。在一些实施方案中，组合物包括2B2、2H8、3F10、16E5和14A9抗体的嵌合抗体及人源化抗体中的任一单克隆抗体，以及药学上可接受的载体。药学上可接受的载体包括，例如，赋形剂、稀释剂、包封材料、填充剂、缓冲剂或其他试剂。
杂交瘤
本发明提供了分离的杂交瘤细胞系，其选自由2B2、2H8、3F10、16E5和14A9组成的组。杂交瘤细胞系2B2、2H8、3F10、16E5和14A9分泌结合CD47的单克隆抗体。
分离的核酸
本发明提供了分离的核酸，其包含编码本文所述结合CD47的抗原结合多肽或其抗原结合部分的序列，在一些实施方案中，所述核酸可以编码包含本文所述抗原结合多肽重链可变区或/和轻链可变区的氨基酸序列。在另一些实施方案中，所述的核酸可以编码包含本文所述的抗原结合多肽重链或/和抗原结合多肽轻链的氨基酸序列。序列表中示例性的列举了一些可变区、抗原结合多肽重链和轻链的核苷酸序列。
载体
本发明提供了包含所述的分离的核酸的载体。在一些实施方案中，所述的载体为克隆载体；在另一些实施方案中，所述的载体为表达载体。
所述表达载体可选的能够表达本文所述抗原结合多肽或其抗原结合部分的任意表达载体。
宿主细胞
在一些实施方案中，本发明提供包含所述载体的宿主细胞，宿主细胞为用于克隆或编码结合CD47的抗原结合多肽或其抗原结合部分的适当宿主细胞。在一些实施方案中，宿主细胞为原核细胞。在另一些实施方案中，宿主细胞为真核细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳细胞或适用于制备抗原结合多肽或其抗原结合部分的其他细胞。哺乳细胞例如为中国仓鼠(CHO)卵巢细胞。
制备结合CD47抗原结合多肽的方法
在一些实施方案中，本发明提供了制备结合CD47的分离的抗原结合多肽的方法，所述方法包括：在适合所述抗原结合多肽表达的条件下，培养包含编码所述抗原结合多肽的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述抗原结合多肽。为了产生结合CD47的抗原结合多肽，分离编码所述抗体的核酸，并插入一个或多个载体，用于在宿主细胞中进一步克隆或/和表达。所述核酸可以采用基因克隆、基因拼接、化学合成等多种本领域所熟知的方法获取。
用途
本发明提供了结合CD47的分离的抗原结合多肽或其抗原结合部分的用途，一些实施方案中，向受试者施用治疗有效量本文的分离的结合CD47的抗原结合多肽或其抗原结合部分，可以减少或抑制肿瘤细胞生长。一些实施方案中，向受试者施用治疗有效量本文的分离的结合CD47的抗原结合多肽或其抗原结合部分，可以治疗癌症。需要治疗的受试者包括那些已经患有疾病或病状的受试者，以及可能患疾病或病状并且其目的是预防、延迟或减弱疾病或病状的受试者。如本文所用，“癌症”是指哺乳动物中特征通常在 于不受调控的细胞生长的生理病状。癌症的实例包括但不限于：白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、膀胱癌、尿路上皮癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、胃癌、肝细胞癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、宫颈癌、以及子宫癌、成胶质细胞瘤、成神经管细胞瘤、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、急性髓系白血病、骨髓增生异常综合征、以及肉瘤。
一些实施方案中，向所述受试者施用有效量的本文结合CD47的分离的抗原结合多肽或其抗原结合部分，促进受试者巨噬细胞的吞噬作用。
尽管为了清楚理解的目的，已经通过举例说明和实施例相当详细地描述了前述发明，但是根据本发明的教义，本领域的普通技术人员将显而易见的是，可另外对本发明进行某些改变和修改而不背离所附权利要求的精神和范围。以下实施例仅以说明方式提供，而并不起限制作用。本领域的技术人员将容易地识别多种非关键性参数，所述参数可发生改变或修改以产生基本上类似的结果。本文具体实施例中采用的阳性对照Hu5F9，其序列与美国专利US2015/0183874A1中的抗体“5F9”的序列相同，为人源化抗体。
除非特殊说明，否则本发明的实践采用本领域熟知的并且在以下中描述的常规分子生物学、细胞生物学、生物化学和免疫学技术：例如，Methods in Molecular Biology，Humana Press；Molecular Cloning：A Laboratory Manual，第二版(Sambrook等，1989)；Current Protocols in Immunology(J.E.Coligan等编，1991)；Immunobiology(C.A.Janeway和P.Travers，1997)；Antibodies(P.Finch，1997)；Antibodies：a practical approach(D.Catty.编，IRL Press，1988-1989)；Monoclonal antibodies：a practical approach(P.Shepherd和C.Dean编，Oxford University Press，2000)；Phage display：a laboratory manual(C.Barbas III等Cold Spring Harbor Laboratory Press，2001)；以及Using antibodies：a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press，1999)等。
实施例1：抗CD47单克隆抗体的产生
使用CD47蛋白对小鼠的免疫：
为了产生针对CD47的抗体，使用等体积的弗氏完全佐剂(初次免疫)或弗氏不完全佐剂(加强免疫)乳化的重组体hCD47-His Tag蛋白(ACRObiosystems，目录号：CD7-H5227)(50μg/小鼠Balb/c)或hCD47-mFc(ACRObiosystems，目录号：CD7-H52A5)每2周皮下免疫Balb/c小鼠，免疫三次持续6周。第四次免疫采用hCD47-mFc(ACRObiosystems，目录号：CD7-H52A5)蛋白，20μg/只小鼠，肌肉注射(水相佐剂)。细胞融合前三天，通过静脉内注射不含佐剂的抗原来加强小鼠免疫。使用聚乙二醇1500(Polyethylene Glycol 1500；Roche公司，目录号：10783641001)将来自免疫小鼠的脾细胞(1×10 8)与SP2/0骨髓瘤细胞(1.5×10 7)融合。融合后，将细胞以0.1ml/孔分配到96孔板中，并且在37℃、5％CO 2的培养箱中进行孵育。在第1天，通过向每孔添加另外的0.1ml含有血清和补充有2×甲氨蝶呤的HAT培养基来饲养细胞。在第3天和第7天，用0.1ml新鲜的HAT培养基替换来自每孔的0.1ml培养基。通常在第9-14天之间进行筛选，并且通过ELISA针对与hCD47-His Tag(ACRObiosystems公司，目录号：CD7-H5227)反应的抗体测试培养物上清液，筛选阳性孔。
杂交瘤细胞克隆化采用有限稀释法。将挑选出的阳性混合克隆进行有限稀释，一株阳性混合克隆，铺两块96孔板(100cells/板、200cells/板)，将杂交瘤细胞培养在含有10％胎牛血清的RPMI 1640(Hyclone公司，目录号：SH30809.01)中。10天后，待有限稀释后的亚克隆同样经过ELISA试验检测培养物上清，挑取阳性亚克隆。克隆化的杂交瘤细胞保种并用于cDNA的提取。
抗人CD47抗体的cDNA提取和可变区序列获取：
使用RNA提取试剂盒(Takara，目录号：9767)从筛选出的产生hCD47抗体的杂交瘤细胞系中分离总RNA，以总RNA作为模板，根据制造商的说明书，用逆转录试剂盒(Thermo Fisher，目录号：K1652)合成第一链cDNA。然后利用合成的cDNA，使用简并小鼠IgG引物，通过PCR反应对抗体相关序列进行体外扩增。
将PCR扩增产物在1％琼脂糖/Tris-醋酸盐凝胶中进行电泳分离。从凝胶上切下具有预期大小(重链和轻链大约450bp)的DNA片段并且对其纯化。将3μl纯化的PCR产物克隆到pMD-19T载体(Takara，目录号：6013)中，并且转化到DH5α化学感受态大肠杆菌(Takara，目录号：9057)中。每个连接反应中，随机选择10个阳性克隆，使用M13正向引物进行DNA测序。
从对应的杂交瘤克隆(2B2、2H8、3F10、16E5、14A9)扩增、测序得到五个鼠源单克隆抗体(2B2、2H8、3F10、16E5、14A9抗体)的重链可变区(VH)序列和轻链可变区(VL)序列，序列信息如下：
Figure PCTCN2021084802-appb-000014
Figure PCTCN2021084802-appb-000015
嵌合抗体的构建和表达：
通过将小鼠抗体可变区的基因进行化学合成，将每种小鼠抗体的VL区分别连接到人κ链恒定区构建嵌合轻链，将小鼠VH区连接到人IgG4(EU编号S228P)恒定区构建嵌合重链。通过常规基因工程手段分别构建嵌合轻链的表达质粒和嵌合重链的表达质粒，将CHO细胞(200mL体系，在10 6个细胞/mL下)用100μg的每种嵌合重链表达质粒和嵌合轻链表达质粒进行转染并且将其培养6天。然后用蛋白A柱纯化上清液中的嵌合抗体。
构建并表达得到五种嵌合抗体，其嵌合重链和嵌合轻链的全长序列及其编码核酸如下：Xi2H8(嵌合重链及其编码核酸：SEQ ID NOs：89～90；嵌合轻链及其编码核酸：SEQ ID NOs：91～92)、Xi2B2(嵌合重链及其编码核酸：SEQ ID NOs：97～98；嵌合轻链及其编码核酸：SEQ ID NOs：99～100)、Xi3F10(嵌合重链及其编码核酸：SEQ ID NOs：101～102；嵌合轻链及其编码核酸：SEQ ID NOs：103～104)、Xi16E5(嵌合重链及其编码核酸：SEQ ID NOs：93～94；嵌合轻链及其编码核酸：SEQ ID NOs：95～96)、Xi14A9(嵌合重链及其编码核酸：SEQ ID NOs：105～106；嵌合轻链及其编码核酸：SEQ ID NOs：107～108)。
抗体人源化设计：
3F10、14A9和16E5的人鼠嵌合抗体使用CDR移植方法进行人源化(参见，例如，美国专利号5,225,539)。通过MOE(Molecular Operating Environment)对鼠源抗体3F10、14A9和16E5进行同源建模，产生Fv结构域的蛋白质结构模型。分别将鼠源3F10、14A9和16E5抗体的VH和VL氨基酸序列输入到国际免疫遗传信息系统相关工具网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)，从中筛选出与小鼠3F10、14A9和16E5抗体的可变区具有高度同源性的人胚系抗体序列。将鼠源3F10、14A9和16E5抗体的VH和VL中的互补决定区(CDR)嫁接模板人抗体。对于3F10，所选择的模板人VH是IGHV1-69*01和IGHJ4*01的组合，所选择的模板人VL是IGKV1-39*01和IGKJ2*01的组合。对于14A9，所选择的模板人VH是IGHV1-46*01和IGHJ4*01的组合，所选择的模板人VL是IGKV1-39*01和IGKJ2*01的组合。对于16E5，所选择的模板人VH是IGHV1-18*01和IGHJ4*01的组合，所选择的模板人VL是IGKV1-39*01和IGKJ2*01的组合。
上述模板人抗体的CDR氨基酸序列被杂交瘤(小鼠)3F10、14A9和16E5抗体的CDR取代。用来自小鼠3F10、14A9和16E5抗体的VH和VL的必需氨基酸序列嫁接上述模板人胚系抗体VH和VL框架，以得到功能性人源化抗体。对于3F10、14A9和16E5抗体的VH和VL，上述模板人抗体的框架氨基酸的几个位点被回复突变为小鼠3F10、14A9和16E5抗体中对应的氨基酸序列。个别的CDR区进行突变。
对于人源化3F10抗体的重链可变区，突变类型的选择和搭配来自如下位点：在位置73处的氨基酸从Asp(D)突变为Glu(E)；在位置74处的氨基酸从Glu(E)突变为Ile(I)；在位置77处的氨基酸从Ser(S)突变为Asn(N)，并且对于人源化3F10抗体的轻链可变区，突变类型的选择和搭配来自如下位点：在位置48处的氨基酸从Leu(L)突变为Trp(W)；在位置71处的氨基酸从Asp(D)突变为Ser(S)；在位置72处的氨基酸从Phe(F)突变为Tyr(Y)；在位置94处的氨基酸从Asp(D)突变为Glu(E)。对于人源化14A9的重链可变区，突变类型的选择和搭配来自如下位点：在位置24处的氨基酸从Ala(A)突变为Val(V)；在位置72处的氨基酸从Arg(R)突变为Gly(G)；在位置77处的氨基酸从Ser(S)突变为Asn(N)；在位置97处的氨基酸从Ala(A)突变为Thr(T)，并且对于人源化14A9的轻链可变区，突变类型的选择和搭配来自如下位点：在位置63处的氨基酸从Ser(S)突变为Ile(I)；在位置68处的氨基酸从Gly(G)突变为Ser(S)。对于人源化16E5抗体的重链可变区，突变类型的选择和搭配来自如下位点：在位置46处的氨基酸从Glu(E)突变为Lys(K)；在位置63处的氨基酸从Lys(K)突变为Glu(E)；在位置72处的氨基酸从Thr(T)突变为Leu(L)；在位置77处的氨基酸从Ser(S)突变为Arg(R)；在位置97处的氨基酸从Ala(A)突变为Thr(T)，并且对于人源化16E5抗体的轻链可变区，突变类型的选择和搭配来自如下位点：在位置2处的氨基酸从Ile(I)突变为Ala(A)；在位置33处的氨基酸从Asn(N)突变为Gln(Q)。本段中突变位点序号的描述按照可变区第一个氨基酸开始依次计数，
人源化3F10、14A9和16E5抗体的序列及其编码核酸如下：hz3F10-1.1(重链及其编码核酸：SEQ ID NOs.109～110；轻链及其编码核酸：SEQ ID NOs.111～112)、hz3F10-2.1(重链及其编码核酸：SEQ ID NOs.113～114；轻链及其编码核酸：SEQ ID NOs.115～116)、hz3F10-3.1(重链及其编码核酸：SEQ ID NOs.117～118；轻链及其编码核酸：SEQ ID NOs.119～120)、hz3F10-4.1(重链及其编码核酸：SEQ ID NOs.121～122；轻链及其编码核酸：SEQ ID NOs.123～124)、hz3F10-5.1(重链及其编码核酸：SEQ ID  NOs.125～126；轻链及其编码核酸：SEQ ID NOs.127～128)、hz3F10-6.1(重链及其编码核酸：SEQ ID NOs.129～130；轻链及其编码核酸：SEQ ID NOs.131～132)、hz3F10-1.2(重链及其编码核酸：SEQ ID NOs.133～134；轻链及其编码核酸：SEQ ID NOs.135～136)、hz3F10-2.2(重链及其编码核酸：SEQ ID NOs.137～138；轻链及其编码核酸：SEQ ID NOs.139～140)、hz3F10-3.2(重链及其编码核酸：SEQ ID NOs.141～142；轻链及其编码核酸：SEQ ID NOs.143～144)、hz3F10-4.2(重链及其编码核酸：SEQ ID NOs.145～146；轻链及其编码核酸：SEQ ID NOs.147～148)、hz3F10-5.2(重链及其编码核酸：SEQ ID NOs.149～150；轻链及其编码核酸：SEQ ID NOs.151～152)、hz3F10-6.2(重链及其编码核酸：SEQ ID NOs.153～154；轻链及其编码核酸：SEQ ID NOs.155～156)、hz16E5-1.1(重链及其编码核酸：SEQ ID NOs.157～158；轻链及其编码核酸：SEQ ID NOs.159～160)、hz16E5-1.3(重链及其编码核酸：SEQ ID NOs.161～162；轻链及其编码核酸：SEQ ID NOs.163～164)、hz16E5-1.2(重链及其编码核酸：SEQ ID NOs.165～166；轻链及其编码核酸：SEQ ID NOs.167～168)、hz16E5-3.2(重链及其编码核酸：SEQ ID NOs.169～170；轻链及其编码核酸：SEQ ID NOs.171～172)、hz16E5-3.1(重链及其编码核酸：SEQ ID NOs.173～174；轻链及其编码核酸：SEQ ID NOs.175～176)、hz16E5-3.3(重链及其编码核酸：SEQ ID NOs.177～178；轻链及其编码核酸：SEQ ID NOs.179～180)、hz14A9-2.3(重链及其编码核酸：SEQ ID NOs.181～182；轻链及其编码核酸：SEQ ID NOs.183～184)、hz14A9-2.4(重链及其编码核酸：SEQ ID NOs.185～186；轻链及其编码核酸：SEQ ID NOs.187～188)。
人源化3F10、14A9和16E5抗体的构建和表达：
合成编码人源化3F10、14A9和16E5抗体全长轻链和全长重链的DNA并且将所述DNA克隆到表达载体(包括例如CN107001463A披露的pcDNA3.1载体、CN109422811A披露的pCHO1.0载体等)。将CHO细胞(200ml体系，在10 6个细胞/ml下)用100μg的每种人源化抗体重链表达质粒和轻链表达质粒进行转染并且将其用ExpiCHO培养基(Gibco；货号：A29100-01)在37℃、5％CO 2的培养箱中培养6天。离心分离得到上清液后，然后用蛋白A柱纯化上清液中的人源化抗体。
实施例2：抗CD47抗体的基于Biacore的亲和力分析
通过Biacore分别测量人CD47蛋白(Human CD47 Protein,His Tag；ACRObiosystems公司，目录号：CD7-H5227)和食蟹猴CD47蛋白(Cynomolgus/Rhesus macaque CD47 Protein,His Tag；ACRObiosystems公司，目录号：CD7-C52H1)与抗CD47抗体(嵌合的或人源化的)之间的结合动力学，获得平衡解离常数K D(单位M)；所述Biacore分析在25℃下进行并且以1Hz的数据收集速率进行记录。
将多克隆兔抗小鼠IgG(GE，BR-1008-38)用10mM pH 5.0的乙酸钠进行稀释，并且使用胺偶联试剂盒(GE，BR10050)固定到CM5生物传感器芯片的参考流动池和实验流动池上达到约15000RΜ。在每个循环的开始，将稀释的测试抗体(1.5μg/mL)注射在实验流动池上，持续1分钟以被捕获。这些数据表明，如通过Biacore测量的，抗CD47抗体(嵌合的和人源化的)结合CD47。
表1：抗CD47嵌合抗体与人CD47和食蟹猴CD47的结合亲和力
Figure PCTCN2021084802-appb-000016
表2：抗CD47人源化抗体与人CD47和食蟹猴CD47的结合亲和力
Figure PCTCN2021084802-appb-000017
Figure PCTCN2021084802-appb-000018
实施例3：抗CD47抗体的基于ELISA的结合分析
ELISA结合分析基于人CD47蛋白与抗CD47嵌合抗体或抗CD47人源化抗体进行。将96孔板(Costar，目录号：9018)在4℃下用100μl/孔的2μg/ml CD47-His(Human CD47 Protein,His Tag；ACRObiosystems公司，目录号：CD7-H5227)的包被缓冲液PBS包被过夜。抽吸孔，并且通过添加100μL/孔封闭液(具有1％(w/v)牛血清蛋白(BSA，Sigma公司，目录号：B2064-100G)的PBS)并且在37℃下孵育1小时来封闭非特异性结合位点。用洗涤缓冲液(具有0.01％(w/v)吐温20(Sigma公司，目录号：P9416-100ML)的PBS)洗涤96孔板三次后，添加100μl/孔的抗CD47抗体(嵌合抗体见表3，人源化抗体见表4)在封闭液中的1:4连续稀释液(从30μg/mL开始)，并且在室温下孵育2小时。洗涤96孔板，洗涤后再用第二抗体HRP-anti-Human IgG(BD公司，目录号：555788)避光孵育20min。洗涤96孔板后，添加100μl/孔的底物溶液TMB(TIANGEN公司，目录号：PA107-01)并将所述96孔板在室温下温育10min。添加100μl/孔的停止溶液(1M H 2SO 4)以停止反应。产生比色信号并且使用多功能酶标仪(生产厂家：PerkinElmer；型号规格EnVision)在450nm处读取所述比色信号。使用GraphPad Prism5分析数据，并计算EC50。这些数据表明，通过ELISA测量，抗CD47抗体结合CD47，人源化抗体具有良好的表现。
表3：抗CD47嵌合抗体与人CD47蛋白基于ELISA的结合EC50
抗体名称 EC50(ng/mL)
Xi2B2 349.5
Xi2H8 NA
Xi3F10 1106
Xi16E5 87.89
Xi14A9 273.4
注：NA表示未达检测限。
表4：抗CD47人源化抗体与人CD47蛋白基于ELISA的结合EC50
抗体名称 EC50(ng/mL) 抗体名称 EC50(ng/mL)
hz3F10-1.1 70.02 hz3F10-5.2 105.6
hz3F10-2.1 80.94 hz3F10-6.2 129.3
hz3F10-3.1 126.9 hz16E5-1.1 306
hz3F10-4.1 115.1 hz16E5-3.1 139.7
hz3F10-5.1 99.33 hz16E5-1.2 159.5
hz3F10-6.1 56.33 hz16E5-3.2 215.1
hz3F10-1.2 124.8 hz16E5-1.3 138.1
hz3F10-2.2 105.8 hz16E5-3.3 240.9
hz3F10-3.2 165.9 hz14A9-2.3 156.8
hz3F10-4.2 180.7 hz14A9-2.4 339.3
实施例4：抗CD47抗体的基于细胞的结合分析
基于与稳定表达CD47的CHO-K1细胞系的结合进行抗CD47抗体(嵌合的或人源化的)的细胞结合分析。将CHO-K1-CD47细胞(表达人CD47蛋白的CHO-K1细胞)添加到96孔板(Eppendorf，目录号：C030730.119)的各孔中，每孔1.2×10 4个，并与抗CD47抗体(20μg/ml，1:4的稀释度，稀释缓冲液为 PBS)室温孵育1-2h。然后将细胞用PBS缓冲液洗涤三次后，以100μl/孔将二抗Alexa Fluor 488标记山羊抗人IgG(Jackson ImmunoResearch Inc，目录号：109-545-088)按1:200用PBS稀释后添加到细胞，并在室温下孵育30min。将细胞用PBS缓冲液洗涤三次，细胞核染料Hoechst(Sigma公司，目录号：B2261)用PBS缓冲液按稀释比1:500稀释后以100μl/孔加入各孔中染色10min，PBS洗涤3次后通过高内涵筛选仪(High content Screening System，生产厂家：PerkinElmer，型号规格：Operetta)进行分析，结果见表5和6。这些数据表明，基于细胞的结合分析，抗CD47抗体结合CD47。
表5：抗CD47嵌合抗体与CD47基于细胞的结合EC50
抗体名称 EC50(ng/mL)
Xi2B2 30790
Xi2H8 NA
Xi3F10 19803
Xi16E5 2026
Xi14A9 540.7
注：NA表示未达检测限。
表6：抗CD47人源化抗体与CD47基于细胞的结合EC50
抗体名称 EC50(ng/mL) 抗体名称 EC50(ng/mL)
hz3F10-1.1 875.7 hz3F10-6.2 1486
hz3F10-2.1 1068 hz16E5-1.1 3866
hz3F10-3.1 1121 hz16E5-3.1 5883
hz3F10-4.1 1162 hz16E5-1.2 NA
hz3F10-5.1 1480 hz16E5-3.2 NA
hz3F10-6.1 1184 hz16E5-1.3 18845
hz3F10-1.2 1267 Hz16E5-3.3 40479
hz3F10-2.2 1119 hz14A9-2.3 498.6
hz3F10-3.2 1195 hz14A9-2.4 470.7
hz3F10-4.2 1265 Hu5F9 1076
hz3F10-5.2 1260    
注：NA表示未达检测限。
实施例5：抗CD47嵌合抗体基于细胞的抗体阻断分析
基于流式细胞术的检测，对抗CD47嵌合抗体对CD47与SIRPα结合的阻断作用进行评价。
在96孔U型板中加入Jurkat细胞(中国科学院细胞库，目录号：TCHU123)，每孔5×10 5个细胞，每孔加100μl浓度为1μg/mL的CD47配体Human-SIRPα-mFC(Human SIRP alpha/CD172a Protein,Mouse IgG1Fc Tag，ACRObiosystems公司，目录号：SIA-H52A8)的PBS缓冲液稀释液，室温孵育1h。PBS缓冲液洗涤2次后，每孔加入100μl抗CD47嵌合抗体(表7)，起始浓度设置为20μg/ml，以PBS缓冲液进行4倍稀释，8个梯度，室温孵育1-2h，PBS缓冲液洗涤3次后，以Alexa Fluor 488标记的山羊抗小鼠IgG(Alexa Fluor 488-Goat anti mouse IgG，Jackson ImmunoResearch Inc.，目录号：115-545-062)作为检测二抗，每孔加100μl用PBS缓冲液以稀释比1:200稀释的检测二抗，室温1h，PBS缓冲液洗涤3次，通过流式细胞仪(BD公司，型号规格：C6)检测抗CD47嵌合抗体对CD47与SIRPα结合的阻断作用。结果见表7，抗CD47嵌合抗体能够阻断CD47与SIRPα结合，其中Xi3F10、Xi16E5、Xi14A9具有更好的阻断效果。
表7：抗CD47嵌合抗体基于细胞的抗体阻断分析
抗体名称 Xi2B2 Xi2H8 Xi3F10 Xi16E5 Xi14A9
IC50(ng/mL) NA 26966 358.5 4861 1283
注：NA表示未达检测限。
实施例6：抗CD47抗体基于ELISA的抗体阻断分析
基于ELISA的检测，对抗CD47抗体对CD47与SIRPα结合的阻断作用进行评价。
在酶标板96孔板(Costar，目录号：9018)中，以CD47-His(Human CD47 Protein,His Tag；ACRObiosystems公司，目录号：CD7-H5227)为捕获抗原，每孔加入2μg/mL的具有所述捕获抗原的包被缓冲液PBS 100μl，2-8℃过夜，抽吸孔以移除所述包被缓冲液，用100μL/孔的封闭液(具有1％(w/v)牛血清蛋白(BSA，Sigma公司，目录号：B2064-100G)的PBS)在37℃下孵育封闭1h。用洗涤缓冲液(具有0.01％(w/v)吐温20(Sigma，目录号：P9416-100ML)的PBS)洗涤96孔板。然后加入100μl/ 孔的1μg/ml CD47配体Human-SIRPα-mFC(Human SIRP alpha/CD172a Protein,Mouse IgG1Fc Tag，ACRObiosystems公司，目录号：SIA-H52A8)，25±2℃温育1.5h，PBS洗涤缓冲液洗涤96孔板后加入：抗CD47嵌合抗体或抗CD47人源化抗体(嵌合抗体见表8，人源化抗体见表9)，起始浓度20μg/mL，100μL/孔，用PBS缓冲液4倍稀释，8个梯度，室温孵育1.5h。PBS缓冲液洗涤后加入100μl/孔用PBS缓冲液按1:2000稀释的HRP标记的山羊抗小鼠IgG(HRP Goatanti mouse IgG，BD公司，目录号：554002)，25±2℃温育1h，PBS缓冲液洗涤96孔板后再加入100μl/孔的底物溶液TMB(TIANGEN公司，目录号：PA107-01)室温温育显色10min，添加100μl/孔的1M H 2SO 4终止反应。产生比色信号并且使用多功能酶标仪(生产厂家：PerkinElmer；型号规格EnVision)在450nm处读取所述比色信号。使用GraphPad Prism5分析数据，并计算EC50。结果见表8、表9，基于ELISA检测，14A9、16E5、3F10的嵌合抗体(Xi3F10、Xi16E5、Xi14A9)以及人源化抗体具有良好的阻断效果。
表8：抗CD47嵌合抗体基于ELISA的抗体阻断分析
抗体名称 IC50(ng/mL)
Xi2B2 NA
Xi2H8 4.682
Xi3F10 7910
Xi16E5 946.1
Xi14A9 444.4
注：NA表示未达检测限。
表9：抗CD47人源化抗体基于ELISA的抗体阻断分析
抗体名称 IC50(ng/mL) 抗体名称 IC50(ng/mL)
hz3F10-1.1 1534 hz3F10-6.2 1839
hz3F10-2.1 1612 hz16E5-1.1 NA
hz3F10-3.1 1678 hz16E5-3.1 15246
hz3F10-4.1 1952 hz16E5-1.2 2939
hz3F10-5.1 1701 hz16E5-3.2 2051
hz3F10-6.1 1768 hz16E5-1.3 2323
hz3F10-1.2 1689 hz16E5-3.3 1998
hz3F10-2.2 1725 hz14A9-2.3 703.2
hz3F10-3.2 1741 hz14A9-2.4 621.1
hz3F10-4.2 1738 Hu5F9 2398
hz3F10-5.2 1824    
注：NA表示未达检测限。
实施例7：抗CD47抗体基于促使巨噬细胞吞噬作用的分析
基于流式细胞术测定抗CD47抗体促使细胞表面含有SIRPα的巨噬细胞吞噬肿瘤细胞的能力。
使用单核细胞分离试剂盒(STEMCELL，目录号：19058)从人的外周血单个核细胞(PBMC，赛笠生物，目录号：190056)中分离出单核细胞，铺入24孔板(Greiner bio-one，目录号：662-160)中，每孔5×10 5个细胞，并在100ng/mL刺激因子M-CSF(R&D Systems公司，目录号：216-MC-010)的环境诱导7天，从而诱导成巨噬细胞。将1×10 6个靶细胞HL60(上海细胞所，目录号：TCHu23)悬浮于5μmol/L CFSE(BD公司，目录号：565082)荧光染料(荧光染料用PBS缓冲液稀释到所需浓度)中，37℃水浴锅避光孵育15min，PBS缓冲液洗涤细胞，制备成CFSE标记的HL60细胞；将CFSE标记的HL60细胞分别与10μg/mL、1μg/mL抗CD47抗体(嵌合的抗体见表10，人源化的抗体见表11)于37℃、5％CO 2培养箱中孵育1-2h。PBS缓冲液洗涤三次孵育后的HL60细胞，加入到巨噬细胞中，37℃、5％CO 2培养箱中作用2-4h，PBS缓冲液洗涤三次后，每孔加入260μl PBS、20μl抗APC-CD14(APC Mouse Anti-Human CD14，BD公司，目录号：555399)和20μl PE-CD11b(PE Mouse Anti-Human CD11b/Mac-1，BD公司，目录号：555388)抗体的混合液，4℃避光孵育30min，PBS缓冲液洗涤三次，胰酶(Gibco，目录号：12604-013)消化下来用流式细胞仪(BD公司，型号规格：C6)检测巨噬细胞的吞噬率(％)。
抗CD47嵌合抗体和抗CD47人源化抗体对巨噬细胞吞噬作用的影响参见表10和表11，其中嵌合抗体Xi3F10、Xi16E5、Xi14A9以及抗CD47人源化抗体具有良好的促吞噬作用，人源化抗体hz14A9-2.3、hz14A9-2.4表现更优异。
表10：抗CD47嵌合抗体基于促使巨噬细胞吞噬作用的分析(吞噬率，％)
抗体名称 10μg/mL 1μg/mL
Xi2B2 4.1 7.3
Xi2H8 3.5 7.1
Xi3F10 59.8 65.7
Xi16E5 28.8 11.5
Xi14A9 44.67 54.17
表11：抗CD47人源化抗体基于促使巨噬细胞吞噬作用的分析(吞噬率，％)
抗体名称 10μg/mL 1μg/mL 抗体名称 10μg/mL 1μg/mL
hz3F10-1.1 43.10 41.25 hz3F10-6.2 44.19 49.32
hz3F10-2.1 55.23 47.24 hz16E5-1.1 4.02 14.79
hz3F10-3.1 58.72 50.60 hz16E5-3.1 24.19 6.37
hz3F10-4.1 58.26 50.26 hz16E5-1.2 17.83 5.1
hz3F10-5.1 58.46 50.45 hz16E5-3.2 2.03 3.02
hz3F10-6.1 60.91 47.57 hz16E5-1.3 4.33 3.03
hz3F10-1.2 38.86 55.52 hz16E5-3.3 4.7 11.62
hz3F10-2.2 34.72 44.19 hz14A9-2.3 74.93 71.48
hz3F10-3.2 37.09 47.04 hz14A9-2.4 81.66 81.65
hz3F10-4.2 31.67 46.24 Hu5F9 72.35 67.5
hz3F10-5.2 29.6 47.06      
实施例8：抗CD47抗体对CD4+T细胞的影响
基于流式细胞术的测定方法，测定抗CD47抗体对CD4+T细胞的影响。
使用T细胞分离试剂盒(Miltenyi Biotech，目录号：130-094-131)从人的外周血单个核细胞(PBMC，赛笠生物，目录号：190056)中分离出CD4+T细胞，铺入24孔板(Greiner bio-one，目录号：662106)，每孔5×10 5个细胞，同时分别加入抗CD47抗体(嵌合的抗体见表12，人源化的抗体见表13)使终浓度分别为10μg/mL和1μg/mL，加入1640完全培养基，37℃、5％CO 2培养箱中培养20h，使用凋亡试剂盒(STEMCELL，目录号：556547)室温染色15min，流式仪(BD，型号规格：C6)检测细胞凋亡率(％)，以早期凋亡和晚期凋亡总和计算凋亡率。结果表明抗CD47抗体(嵌合的抗体见表12和人源化的抗体见表13)对T细胞凋亡没有明显影响。
表12：抗CD47嵌合抗体对CD4+T细胞的影响(凋亡率％)
抗体名称 10μg/mL 1μg/mL
Xi2B2 10.3 12.1
Xi2H8 10.5 12.1
Xi3F10 13.6 12.6
Xi16E5 23.1 0.4
Xi14A9 1.34 1.42
表13：抗CD47人源化抗体对CD4+T细胞的影响(凋亡率％)
抗体名称 10μg/mL 1μg/mL 抗体名称 10μg/mL 1μg/mL
hz3F10-1.1 1.94 1.82 hz3F10-6.2 1.68 2.18
hz3F10-2.1 1.7 1.38 hz16E5-1.1 1.88 1.62
hz3F10-3.1 1.88 2 hz16E5-3.1 2.04 1.8
hz3F10-4.1 1.86 1.94 hz16E5-1.2 1.66 1.08
hz3F10-5.1 1.74 1.94 hz16E5-3.2 1.8 1.52
hz3F10-6.1 2.12 1.34 hz16E5-1.3 1.28 1.38
hz3F10-1.2 1.74 1.7 hz16E5-3.3 1.54 1.54
hz3F10-2.2 1.82 1.94 hz14A9-2.3 1.56 1.59
hz3F10-3.2 1.84 1.66 hz14A9-2.4 1.89 1.38
hz3F10-4.2 1.84 1.78 Hu5F9 1.35 1.58
hz3F10-5.2 1.94 1.78 IgG4 1.72 1.81
实施例9：基于nano DSF检测抗CD47抗体的热稳定性
高通量蛋白稳定性分析仪Prometheus NT.48是得到蛋白分子结构稳定性、聚集稳定性、胶体分散稳定性等多种类型数据的设备。利用该设备检测抗CD47嵌合抗体以及抗CD47人源化抗体的熔解温度(Tm)和聚合温度(Tagg)，结果见表14和表15，表明抗CD47抗体具有良好的热稳定性。
表14：抗CD47嵌合抗体热稳定性
抗体名称 Tm(℃) Tagg(℃)
Xi2B2 62.99 61.34
Xi2H8 69.77 68.57
Xi3F10 70.51 70.59
Xi16E5 69.89 73.39
Xi14A9 66.73 75.75
表15：抗CD47人源化抗体热稳定性
抗体名称 Tm(℃) Tagg(℃) 抗体名称 Tm(℃) Tagg(℃)
hz3F10-1.1 68.44 72.68 hz14A9-2.4 68.3 68.3
hz3F10-2.1 68.44 72.68 hz3F10-4.2 68.48 61.13
hz3F10-3.1 68.15 61.53 hz3F10-5.2 68.69 62.67
hz3F10-4.1 68.35 62.54 hz3F10-6.2 68.64 61.91
hz3F10-5.1 68.05 62.12 hz16E5-1.1 68.21 61.87
hz3F10-6.1 68.6 68.6 hz16E5-3.1 69.20 61.84
hz3F10-1.2 68.71 61.92 hz16E5-1.2 69.54 61.90
hz3F10-2.2 68.92 62.40 hz16E5-3.2 69.10 61.44
hz3F10-3.2 68.30 61.80 hz16E5-1.3 68.90 73.77
hz14A9-2.3 68.10 73.42 hz16E5-3.3 68.72 73.81
实施例10：抗CD47抗体对红细胞凝集的影响
某些公开的研究表明，红细胞表面含有一定量的CD47蛋白，这导致在加入某些抗CD47抗体后会与红细胞结合，从而可能引起红细胞的凝集现象。因此，测试抗CD47抗体对红细胞凝集的影响。
取抗凝血，加等量生理盐水，混匀，2000rpm/min离心5min，去上清。使用无钙离子、镁离子Hank’s平衡盐溶液(Thermo Fisher Scientific，目录号：14175-095)将红细胞洗涤3次，前2次2000rpm/min离心5min，末次2000rpm/min离心10min，去上清。使用PBS缓冲液将红细胞稀释10倍，制成10％的红细胞悬液，100μl/孔放入96孔U型板(eppendorf，目录号：003073119)中。加入100μl/孔不同浓度的抗CD47抗体，所述抗体的起始浓度设置为10μg/ml，以PBS缓冲液3倍稀释，8个浓度梯度，于37℃中孵育2～6小时。观察拍照，红细胞全部凝集，沉于底部，平铺成网状，即为凝集；不凝集者红细胞沉于孔底呈点状。
抗CD47嵌合抗体对红细胞凝集的影响见图1。抗CD47人源化抗体对红细胞凝集的影响见图2、图3，嵌合抗体Xi3F10、Xi16E5以及Hu5F9均有凝血现象，其中Xi3F10从1111.1ng/ml开始凝血，Xi16E5和Hu5F9从123.46ng/ml开始凝血，嵌合抗体Xi2B2、Xi2H8、Xi14A9在测试的所有浓度下均无凝血现象。人源化抗体hz3F10-3.1、hz3F10-4.1、hz3F10-3.2、hz3F10-4.2从3.3μg/ml开始发生凝血现象，hz16E5-1.1、hz16E5-3.1、hz16E5-1.2、hz16E5-3.2、hz16E5-1.3、hz16E5-3.3从3.3μg/ml开始发生凝血现象，其它人源化抗体在测试的所有浓度下均无凝血现象。
实施例11：抗CD47抗体与红细胞的结合分析
基于红细胞表面一定量的CD47，可能会与某些抗CD47抗体结合，因此检测抗CD47抗体与红细胞的结合情况。
取抗凝血，加等量生理盐水，混匀，2000rpm/min离心5min，去上清。使用无钙离子、镁离子Hank’s平衡盐溶液(Thermo Fisher Scientific，目录号：14175-095)将红细胞洗涤3次，前2次2000rpm/min离心5min，末次2000rpm/min离心10min，去上清。将分离后红细胞用4％多聚甲醛(生工生物工程上海公司，目录号：E672002-0500)固定过夜，次日与10μg/ml或1μg/ml抗CD47抗体室温孵育1-2h，PBS缓冲液清洗后，用PBS缓冲液按1:200稀释的二抗Alexa Fluor 488标记山羊抗人IgG(Jackson ImmunoResearch Inc，目录号：109-545-088)室温孵育1h，PBS缓冲液清洗后用流式细胞仪(BD公司，型号规格：C6)检测，以平均荧光强度(MFI)反映红细胞与抗CD47嵌合抗体(见表16)或人源化抗体(见表17)的结合情况。
表16：抗CD47嵌合抗体与红细胞的结合分析(MFI)
抗体名称 10μg/mL 1μg/mL
Xi2B2 608 656
Xi2H8 5303 952
Xi3F10 61821 36114
Xi16E5 3318 1309
Xi14A9 32831 26477
表17：抗CD47人源化抗体与红细胞的结合分析(MFI)
抗体名称 10μg/mL 1μg/mL 抗体名称 10μg/mL 1μg/mL
hz3F10-1.1 50,463 4,449 hz3F10-6.2 55,886 3,435
hz3F10-2.1 53,218 8,454 hz16E5-1.1 6,461 945
hz3F10-3.1 41,362 6,703 hz16E5-3.1 5,145 945
hz3F10-4.1 41,900 5,976 hz16E5-1.2 4,729 913
hz3F10-5.1 50,634 7,403 hz16E5-3.2 4,204 860
hz3F10-6.1 53,048 7,583 hz16E5-1.3 4,885 894
hz3F10-1.2 56,720 6,247 hz16E5-3.3 3,874 848
hz3F10-2.2 56,086 5,612 hz14A9-2.3 10,979 2,506
hz3F10-3.2 45,305 5,096 hz14A9-2.4 14,954 5,237
hz3F10-4.2 45,616 4,814 IgG4 484 449
hz3F10-5.2 53,329 5,134      
实施例12抗CD47抗体在淋巴瘤Raji细胞小鼠血液瘤模型中的药效活性
使用血液瘤Raji细胞建立小鼠血液瘤模型，并在该模型上应用抗CD47抗体，评价该抗体的药效活性。
PBS缓冲液配制的Raji细胞悬液浓度5×10 6个/mL，0.1mL/每只小鼠通过尾静脉注射到雌性NOD/SCID小鼠中。7天后随机分成4组，每组10只，其中，组1：IgG4组，组2：Hu5F9组，组3：hz3F10-6.1，组4：hz14A9-2.3，腹腔注射给药，给药剂量为10mg/kg，连续给药21天。观察动物生存期。
如图4，在Raji血液瘤模型中，抗CD47抗体hz14A9-2.3和hz3F10-6.1具有抗肿瘤活性。小鼠的生存率数据显示：IgG4组第17天全组动物死亡，中位生存期为13.5天；Hu5F9组第30天全组动物死亡，中位生存期为18.5天；hz14A9-2.3组第34天全组动物死亡，中位生存期为28.5天；hz3F10-6.1组第21天全组动物死亡，中位生存期为17.5天。这些数据表明抗体hz14A9-2.3在延长动物生存期方面的药效优于其他组别，抗体hz3F10-6.1和Hu5F9的效果基本持平。
实施例13抗CD47抗体单次给药的毒性检测
由于CD47在红细胞上表达并在清除老化红细胞方面发挥作用，因此对抗CD47抗体在B-hCD47小鼠中的毒性作用进行比较。
将B-hCD47小鼠随机分成3组：Hu5F9组3只，hz14A9-2.3组4只、hz3F10-6.1组3只。通过单次尾静脉注射给予B-hCD47小鼠一定剂量的Hu5F9抗体、hz14A9-2.3抗体或hz3F10-6.1抗体，给药剂量为10mg/kg，检测给药后2天、4天、7天、10天、14天红细胞计数(RBC)、血红蛋白(HGB)量、体重的变化。
表18：给药后小鼠体重变化率(vs给药前)
给药后天数 Hu5F9 10mpk hz14A9-2.3 10mpk hz3F10-6.1 10mpk
0 0.0％ 0.0％ 0.0％
2 -7.0％ 2.3％ -13.0％
4 -3.2％ 6.3％ -15.8％
7 -5.1％ 2.0％ -2.5％
10 4.8％ 5.9％ -8.4％
14 5.6％ 8.6％ 8.2％
表19：给药后小鼠RBC变化率(vs给药前)
给药后天数 Hu5F9 10mpk hz14A9-2.3 10mpk hz3F10-6.1 10mpk
0 0.0％ 0.0％ 0.0％
2 -52.8％ -13.6％ -56.7％
4 -61.8％ -16.7％ -75.6％
7 -22.2％ -9.8％ -38.7％
10 -21.6％ -8.5％ -6.6％
14 -7.4％ -2.2％ -2.0％
表20：给药后小鼠HGB变化率(vs给药前)
给药后天数 Hu5F9 10mpk hz14A9-2.3 10mpk hz3F10-6.1 10mpk
0 0.0％ 0.0％ 0.0％
2 -52.7％ -13.3％ -57.1％
4 -57.8％ -18.5％ -74.7％
7 -16.5％ -9.9％ -39.2％
10 -12.8％ -10.0％ -6.0％
14 -1.2％ -3.9％ -3.0％
如表18、图5和图6所示，只有hz14A9-2.3组小鼠体重是持续增加的，Hu5F9和hz3F10-6.1两组小鼠在给药后体重先下降，后上升；hz3F10-6.1组下降幅度高于Hu5F9组。如表19-表20、图7-图10所示，给药后三组动物的RBC和HGB均出现下降，给药后4天达最低，随后开始上升。hz14A9-2.3组给药后7天RBC和HGB基本恢复正常，另外两组至少于给药后10天基本恢复正常。给药后4天RBC和HGB的下降幅度，由高到低依次为：hz3F10-6.1＞Hu5F9＞hz14A9-2.3。可知，抗体hz14A9-2.3没有显著的毒性，优于阳性抗体Hu5F9。
实施例14
使用人急性髓系白血病细胞MOLM-16细胞小鼠模型检测抗体的抗肿瘤活性。每只NOD-SCID小鼠皮下接种MOLM-16细胞，待肿瘤长到80-150mm 3后，将动物随机分成5组：①模型组(阴性对照组，给予5mg/kg人IgG4)，n＝10只；②hz14A9-2.3 0.5mg/kg组，n＝6只；③hz14A9-2.3 1.5mg/kg组，n＝6只；④hz14A9-2.3 5mg/kg组，n＝6只；⑤Hu5F9 5mg/kg组，n＝6只。在第0天(D0)小鼠分组，静脉注射给药，每3天给药1次，共给药2次，每周二次测量肿瘤直径，根据肿瘤体积判断疗效。
肿瘤体积(V)计算公式为：V＝(a×b 2)/2，其中a、b分别表示长、宽。
T/C(％)＝(T-T 0)/(C-C 0)*100，其中T、C分别为治疗组、阴性对照组实验结束时的平均肿瘤体积；T 0、C 0分别为治疗组、阴性对照组实验开始时的平均肿瘤体积。
抑瘤率(TGI)(％)＝100％-T/C(％)。
结果见表21和表11。抗体hz14A9-2.3在1.5mg/kg剂量下可以有效抑制肿瘤生长，第17天(D17)的肿瘤生长抑制率(或抑瘤率)达到93％。5mg/kg剂量下，抗体hz14A9-2.3的肿瘤生长抑制率达到125％。
表21
Figure PCTCN2021084802-appb-000019

Claims (26)

  1. 一种结合CD47的分离的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含以下互补决定区：
    重链CDR1，其包含与选自SEQ ID NO：1、2、3、4或5的氨基酸序列具有至少80％同一性的氨基酸序列；
    重链CDR2，其包含与选自SEQ ID NO：6、7、8、9或10的氨基酸序列具有至少80％同一性的氨基酸序列；
    重链CDR3，其包含与选自SEQ ID NO：11、12、13、14或15的氨基酸序列具有至少80％同一性的氨基酸序列；
    轻链CDR1，其包含与选自SEQ ID NO：16、17、18、19或20的氨基酸序列具有至少80％同一性的氨基酸序列；
    轻链CDR2，其包含与选自SEQ ID NO：21、22、23、24或25的氨基酸序列具有至少80％同一性的氨基酸序列；和
    轻链CDR3，其包含与选自SEQ ID NO：26、27、28、30或32的氨基酸序列具有至少80％同一性的氨基酸序列。
  2. 根据权利要求1所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含以下互补决定区：
    重链CDR1，其包含选自SEQ ID NO：1、2、3、4或5的氨基酸序列；
    重链CDR2，其包含选自SEQ ID NO：6、7、8、9或10的氨基酸序列；
    重链CDR3，其包含选自SEQ ID NO：11、12、13、14或15的氨基酸序列；
    轻链CDR1，其包含选自SEQ ID NO：16、17、18、19或20的氨基酸序列；
    轻链CDR2，其包含选自SEQ ID NO：21、22、23、24或25的氨基酸序列；和
    轻链CDR3，其包含选自SEQ ID NO：26、27、28、30或32的氨基酸序列。
  3. 根据权利要求1或2所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽选自由以下各项组成的组：
    (a)包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：1、6和11所示的氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：16、21和26所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (b)包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：2、7和12所示的氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：17、22和27所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (c)包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：3、8和13所示的氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：18、23和28所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (d)包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：4、9和14所示的氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：19、24和32所示的氨基酸序列具有至少80％同一性的氨基酸序列；以及
    (e)包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，所述重链CDR1、重链CDR2和重链CDR3分别包含与如SEQ ID NOs：5、10和15所示的氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链CDR1、轻链CDR2和轻链CDR3分别包含与如SEQ ID NOs：20、25和30所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含以下的互补决定区中的一种：
    (1)
    包含SEQ ID NO：1的氨基酸序列的重链CDR1；
    包含SEQ ID NO：6的氨基酸序列的重链CDR2；
    包含SEQ ID NO：11的氨基酸序列的重链CDR3；
    包含SEQ ID NO：16的氨基酸序列的轻链CDR1；
    包含SEQ ID NO：21的氨基酸序列的轻链CDR2；和
    包含SEQ ID NO：26的氨基酸序列的轻链CDR3；
    (2)
    包含SEQ ID NO：2的氨基酸序列的重链CDR1；
    包含SEQ ID NO：7的氨基酸序列的重链CDR2；
    包含SEQ ID NO：12的氨基酸序列的重链CDR3；
    包含SEQ ID NO：17的氨基酸序列的轻链CDR1；
    包含SEQ ID NO：22的氨基酸序列的轻链CDR2；和
    包含SEQ ID NO：27的氨基酸序列的轻链CDR3；
    (3)
    包含SEQ ID NO：3的氨基酸序列的重链CDR1；
    包含SEQ ID NO：8的氨基酸序列的重链CDR2；
    包含SEQ ID NO：13的氨基酸序列的重链CDR3；
    包含SEQ ID NO：18的氨基酸序列的轻链CDR1；
    包含SEQ ID NO：23的氨基酸序列的轻链CDR2；和
    包含SEQ ID NO：28的氨基酸序列的轻链CDR3；
    (4)
    包含SEQ ID NO：4的氨基酸序列的重链CDR1；
    包含SEQ ID NO：9的氨基酸序列的重链CDR2；
    包含SEQ ID NO：14的氨基酸序列的重链CDR3；
    包含SEQ ID NO：19的氨基酸序列的轻链CDR1；
    包含SEQ ID NO：24的氨基酸序列的轻链CDR2；和
    包含SEQ ID NO：32的氨基酸序列的轻链CDR3；或者
    (5)
    包含SEQ ID NO：5的氨基酸序列的重链CDR1；
    包含SEQ ID NO：10的氨基酸序列的重链CDR2；
    包含SEQ ID NO：15的氨基酸序列的重链CDR3；
    包含SEQ ID NO：20的氨基酸序列的轻链CDR1；
    包含SEQ ID NO：25的氨基酸序列的轻链CDR2；和
    包含SEQ ID NO：30的氨基酸序列的轻链CDR3。
  5. 根据权利要求1所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含与选自SEQ ID NO：33、35、37、39、41、83、85或87的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与选自SEQ ID NO：34、36、38、40、42、84、86或88的氨基酸序列具有至少80％同一性的氨基酸序列。
  6. 根据权利要求5所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含重链可变区和轻链可变区，所述的重链可变区包含选自SEQ ID NO：33、35、37、39、41、83、85或87的氨基酸序列，所述的轻链可变区包含选自SEQ ID NO：34、36、38、40、42、84、86或88的氨基酸序列。
  7. 根据权利要求5所述的分离抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽选自由以下各项组成的组：
    (a)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：33的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：34的氨基酸序列具有至少80％同一性的氨基酸序列；
    (b)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：35的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：36的氨基酸序列具有至少80％同一性的氨基酸序列；
    (c)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：37的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：38的氨基酸序列具有至少80％同一性的氨基酸序列；
    (d)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：39的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：40的氨基酸序列具有至少80％同一性的氨基酸序列；
    (e)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：41的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：42的氨基酸序列具有至少80％同一性的氨基酸序列；
    (f)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：83的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：84的氨基酸序列具有至少80％同一性的氨基酸序列；
    (g)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：85的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：86的氨基酸序列具有至少80％同一性的氨基酸序列；以及
    (h)包含重链可变区和轻链可变区，所述的重链可变区包含与SEQ ID NO：87的氨基酸序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：88的氨基酸序列具有至少80％同一性的氨基酸序列。
  8. 根据权利要求5-7中任一项所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含重链可变区和轻链可变区，其中所述的重链可变区包含SEQ ID NO：33的氨基酸序列，所述的轻链可变区包含SEQ ID NO：34的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：35的氨基酸序列，所述的轻链可变区包含SEQ ID NO：36的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：37的氨基酸序列，所述的轻链可变区包含SEQ ID NO：38的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：39的氨基酸序列，所述的轻链可变区包含SEQ ID NO：40的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：41的氨基酸序列，所述的轻链可变区包含SEQ ID NO：42的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：83的氨基酸序列，所述的轻链可变区包含SEQ ID NO：84的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：85的氨基酸序列，所述的轻链可变区包含SEQ ID NO：86的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：87的氨基酸序列，所述的轻链可变区包含SEQ ID NO：88的氨基酸序列；
    所述的重链可变区包含SEQ ID NO：81的氨基酸序列，所述的轻链可变区包含SEQ ID NO：82的氨基酸序列；或者
    所述的重链可变区包含SEQ ID NO：79的氨基酸序列，所述的轻链可变区包含SEQ ID NO：80的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽包含重链和轻链，所述重链具有与选自SEQ ID NO：89、93、97、101、105、109、113、117、121、125、129、133、137、141、145、149、153、157、161、165、169、173、177、181或185所示的氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链具有与选自SEQ ID NO：91、95、99、103、107、111、115、119、123、127、131、135、139、143、147、151、155、159、163、167、171、175、179、183或187所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的抗原结合多肽或其抗原结合部分，其中，
    所述抗原结合多肽包含根据SEQ ID NO：89的重链和根据SEQ ID NO：91的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：93的重链和根据SEQ ID NO：95的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：97的重链和根据SEQ ID NO：99的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：101的重链和根据SEQ ID NO：103的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：105的重链和根据SEQ ID NO：107的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：109的重链和根据SEQ ID NO：111的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：113的重链和根据SEQ ID NO：115的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：117的重链和根据SEQ ID NO：119的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：121的重链和根据SEQ ID NO：123的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：125的重链和根据SEQ ID NO：127的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：129的重链和根据SEQ ID NO：131的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：133的重链和根据SEQ ID NO：135的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：137的重链和根据SEQ ID NO：139的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：141的重链和根据SEQ ID NO：143的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：145的重链和根据SEQ ID NO：147的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：149的重链和根据SEQ ID NO：151的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：153的重链和根据SEQ ID NO：155的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：157的重链和根据SEQ ID NO：159的轻链
    所述抗原结合多肽包含根据SEQ ID NO：161的重链和根据SEQ ID NO：163的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：165的重链和根据SEQ ID NO：167的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：169的重链和根据SEQ ID NO：171的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：173的重链和根据SEQ ID NO：175的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：177的重链和根据SEQ ID NO：179的轻链；
    所述抗原结合多肽包含根据SEQ ID NO：181的重链和根据SEQ ID NO：183的轻链；或者
    所述抗原结合多肽包含根据SEQ ID NO：185的重链和根据SEQ ID NO：187的轻链。
  11. 根据权利要求1-10中任一项所述的抗原结合多肽或其抗原结合部分，其中所述的抗原结合多肽是嵌合的、人源化的或全人源的。
  12. 根据权利要求1-11中任一项所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽或其抗原结合部分选自由单克隆抗体、融合蛋白、多特异性抗体、Fab片段、Fab'片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区和scFv组成的组。
  13. 根据权利要求1-12中任一项所述的抗原结合多肽或其抗原结合部分，其中，所述抗原结合多肽为IgG1、IgG2、IgG3或IgG4型。
  14. 根据权利要求13所述的抗原结合多肽或其抗原结合部分，其中IgG4型抗原结合多肽具有根据EU编号索引的S228P突变。
  15. 根据权利要求1-14中任一项所述的抗原结合多肽或其抗原结合部分，其中所述抗原结合多肽或其抗原结合部分展现出下述性质中的一种或几种的组合：
    (a)以1.89E-08或更小的K D值与CD47结合；
    (b)阻断CD47与SIRPα的结合；
    (c)促进巨噬细胞介导的对表达CD47细胞的吞噬作用；
    (d)没有明显诱导CD4+T细胞的凋亡；
    (e)不引起实质性的红细胞减少、贫血或红细胞凝集；和
    (f)所述抗原结合多肽或其抗原结合部分的熔解温度T≥62℃，聚集温度Tagg≥61℃。
  16. 一种免疫偶联物，其包含根据权利要求1-15中任一项所述的抗原结合多肽或其抗原结合部分，以及与所述抗原结合多肽或其抗原结合部分连接或缀合的治疗剂。
  17. 根据权利要求16所述的免疫偶联物，其中所述治疗剂为细胞毒性药物、放射性同位素、免疫调节剂或抗体。
  18. 一种组合物，其包含组分A以及药学上可接受的载体，其中组分A为根据权利要求1-15中任一项所述的抗原结合多肽或其抗原结合部分、或根据权利要求16或17所述的免疫偶联物。
  19. 一种分离的核酸，其编码根据权利要求1-15中任一项所述的抗原结合多肽或其抗原结合部分。
  20. 一种载体，其包含根据权利要求19所述的分离的核酸。
  21. 一种宿主细胞，其包含根据权利要求20所述的载体或在其基因组中整合有权利要求19所述的分离的核酸。
  22. 一种制备根据权利要求1-15中任一项所述的抗原结合多肽或其抗原结合部分的方法，包括：在适于表达编码权利要求1-15中任一项的抗原结合多肽或其抗原结合部分的核酸的条件下培养权利要求21所述的宿主细胞，分离所表达的所述抗原结合多肽或其抗原结合部分。
  23. 一种用于在受试者中减少肿瘤或抑制肿瘤细胞生长的方法，其中所述方法包括向所述受试者施用治疗有效量的根据权利要求1-15中任一项所述的分离的抗原结合多肽或其抗原结合部分、根据权利要求16或17所述的免疫偶联物或权利要求18所述的组合物。
  24. 一种用于在有需要的受试者中治疗癌症的方法，其中所述方法包括向所述受试者施用治疗有效量的根据权利要求1-15中任一项所述的抗原结合多肽或其抗原结合部分、根据权利要求16或17所述的免疫偶联物、或权利要求18所述的组合物。
  25. 根据权利要求24所述的方法，其特征在于，所述癌症选自由以下组成的组：白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、结肠癌、直肠癌、膀胱癌、尿路上皮癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、胃癌、肝细胞癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、宫颈癌、以及子宫癌、成胶质细胞瘤、成神经管细胞瘤、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、急性髓系白血病、骨髓增生异常综合征以及肉瘤。
  26. 一种促进受试者巨噬细胞的吞噬作用的方法，其中所述方法包括向所述受试者施用有效量的根据权利要求1-15中任一项所述的抗原结合多肽或其抗原结合部分、根据权利要求16或17所述的免疫偶联物、或权利要求18所述的组合物。
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