WO2019042119A1 - 抗人cd47抗体及其用途 - Google Patents

抗人cd47抗体及其用途 Download PDF

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WO2019042119A1
WO2019042119A1 PCT/CN2018/100262 CN2018100262W WO2019042119A1 WO 2019042119 A1 WO2019042119 A1 WO 2019042119A1 CN 2018100262 W CN2018100262 W CN 2018100262W WO 2019042119 A1 WO2019042119 A1 WO 2019042119A1
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antibody
sequence
seq
amino acid
variable region
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French (fr)
Inventor
刘志刚
刘玉兰
郭晶晶
郝小勃
万姝南
胡俊杰
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Genrix (shanghai) Biopharmaceutical Co Ltd
Beijing Wisdomab Biotechnology Co Ltd
Chongqing Genrix Biopharmaceutical Co Ltd
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Genrix (shanghai) Biopharmaceutical Co Ltd
Beijing Wisdomab Biotechnology Co Ltd
Chongqing Genrix Biopharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—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

Definitions

  • the present application relates generally to the field of genetic engineering and antibody drugs; in particular, to the field of anti-human CD47 antibodies and uses thereof.
  • the present application develops a novel anti-human CD47 antibody and provides the use of the antibody in the treatment of CD47 mediated diseases.
  • CD47 belongs to the immunoglobulin superfamily and is a type I transmembrane glycoprotein, including an amino-terminal V-type immunoglobulin-like extracellular domain, a transmembrane domain composed of five highly hydrophobic transmembrane segments and a hydrophilic The carboxy-terminal intracellular domain has a molecular weight between 47 and 55 kD.
  • CD47 was recognized for its interaction with integrin ⁇ v ⁇ 3 , so it is also known as integrin-associated protein (IAP) 1 .
  • CD47 interacts with the ⁇ 3 subunit of integrin ⁇ v ⁇ 3 or ⁇ IIb ⁇ 3 via an extracellular domain, activating the corresponding integrin.
  • SIRP ⁇ Signal regulatory protein ⁇
  • SHPS-1 including Src homology 2 domain-containing protein tyrosine phosphatase substrate-1) /BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs)/CD172a, also a transmembrane glycoprotein with 3 immunoglobulins in the extracellular domain
  • ITIM immunoreceptor tyrosine inhibitory sequence
  • CD47 is a ligand for SIRP ⁇ , which interacts through the extracellular domain to form an intercellular communication complex.
  • CD47 binds to SIRP ⁇ , it induces intracellular ITIM phosphorylation of SIRP ⁇ , and phosphorylation site binds to activate tyrosine phosphatase SHP-1 and SHP-2 containing SH2 (Src homology 2) domain.
  • SHP-1 is mainly expressed in hematopoietic cells, which negatively regulates the function of these cells.
  • SHP-2 is widely expressed, regulating small G proteins Ras and Rho, and positively controlling cell growth and proliferation 3 .
  • CD47-SIRP ⁇ signaling system plays an important role in regulating macrophage phagocytosis of mature blood cells.
  • the CD47 molecule on the surface of normal healthy cells such as erythrocyte 5 or platelet 6 ) interacts with the receptor SIRP ⁇ on macrophages to produce an inhibitory signal, inhibiting its phagocytic activity, and regulating the life cycle of blood cells and their number in the blood. purpose.
  • SIRP ⁇ on monocytes interacts with CD47 on erythrocytes, and Fc ⁇ receptor-dependent phagocytosis is inhibited by myosin-IIA dephosphorylation 7 .
  • the CD47-SIRP ⁇ signaling system inhibits dendritic cell activation and participates in various physiological activities such as nervous system development, neutrophil chemotactic activation and stromal cell-supported hematopoietic cell formation, and induces T cell immune tolerance, activation, and apoptosis.
  • Other aspects also play a variety of regulatory roles 8 .
  • CD47-SIRP ⁇ signaling system has been paid attention to by regulating the phagocytosis of macrophages in tumor immune surveillance.
  • the expression level of CD47 is significantly up-regulated in many malignant tumors such as ovarian cancer 9 , acute myeloid leukemia (AML) 10 , B-cell lymphoma 11 and solid tumor 12 , and the average expression level is about 3.3 times that of normal cells, and This up-regulation is directly related to the poor prognosis of patients with malignant tumors 13 .
  • CD47 In human myeloid leukemia cells (expressing low levels of CD47 endogenous, Rag2-Il2rg- not transplanted mice) Mice of CD47 expression, can be suppressed macrophage phagocytosis of tumor cells and promote tumor cells successfully transplanted 14. It can be inferred that CD47 on tumor cells and SIRP ⁇ on macrophages inhibit the clearance of tumor cells by macrophages and promote the growth and metastasis of tumors in vivo. High expression of CD47 is a common mechanism for tumor cells to evade immune surveillance. Broken CD47-SIRP ⁇ may be a new tumor immunotherapy strategy.
  • high affinity SIRP [alpha] mutant CD172a
  • CD172a can also antagonize the CD47 thereby blocking the CD47-SIRP ⁇ signal pathway, in the AML model can significantly increase the phagocytosis of macrophages AML cells, and inhibition of tumor growth 16 .
  • the application provides an antibody that specifically binds to human CD47, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, wherein
  • the HCDR1 sequence is NYWMH
  • the HCDR2 sequence is VIAPSDNYTNYNQKFQG
  • the HCDR3 sequence is GGKYSMDY
  • the LCDR1 sequence is RSSQSIVHSNGNTYLE
  • the LCDR2 sequence is KVSNRFS
  • the LCDR3 sequence is FQGSHVPFT;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPSSGNTKYAQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSVLYSSNQKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is GQYYAYPIT;
  • HCDR and LCDR sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 23, 28, 30, 32 or 33.
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 24, 29, 31, 34 or 35.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:23, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:24;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 31;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 35;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:35.
  • the application provides an antibody that specifically binds to human CD47, wherein the amino acid sequence of the heavy chain variable region of the antibody has any one of SEQ ID NO: 23, 28, 30, 32 or 33 At least 90% identity, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to any of SEQ ID NO: 24, 29, 31, 34 or 35.
  • the application provides an antibody that specifically binds to human CD47, wherein the binding epitope of the antibody to the human CD47 segment of SEQ ID NO: 1 is a discontinuous epitope, including amino acids Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106, the above residue numbers refer to SEQ ID NO: 1.
  • the antibody is a monoclonal antibody.
  • the antibody is a non-activated antibody.
  • the antibody binds to and neutralizes human CD47, thereby blocking the CD47-SIRP ⁇ signaling pathway.
  • the antibody promotes phagocytosis of tumor cells by macrophages.
  • the antibody inhibits in vivo growth of tumor cells.
  • the antibody does not increase phagocytosis of normal blood cells by macrophages.
  • the antibody has at least one of the following properties:
  • the antibody is a whole antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or comprises a selected from the kappa subtype or the lambda subtype Light chain constant region.
  • the present application provides a nucleic acid molecule encoding the antibody of the first aspect to the third aspect or an antigen binding portion thereof.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first to third aspects and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is for treating a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the application provides the use of the antibodies of the first to third aspects for the preparation of a medicament for the prevention or treatment of a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the present application provides a method of preventing or treating a CD47-mediated disease, comprising administering the antibody of the first aspect to the third aspect or the pharmaceutical composition of the seventh aspect to an individual in need thereof.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • Figure 1 shows the results of the anti-CD47 mAb of the present application competing with hCD172a for binding to hCD47.
  • Figure 3 shows the effect of anti-CD47 mAb of the present application on the phagocytosis of Daudi cells by macrophages by flow cytometry.
  • Figure 4 shows that the anti-CD47 mAb of the present application promotes the phagocytosis of macrophages to Daudi cells.
  • Figure 5 shows the effect of the humanized anti-CD47 mAb of the present application inhibiting the binding of hCD47 to hCD172a.
  • Figure 6 shows the effect of hS2C3 and its variants on inhibition of hCD47 and hCD172a-D1M1 binding.
  • Figure 7 shows the effect of the anti-CD47 mAb of the present application on the degree of red blood cell agglutination.
  • Figure 8 shows that the anti-CD47 mAb of the present application promotes the phagocytosis of macrophages to Daudi cells.
  • Figure 9 shows the effect of the anti-CD47 mAb of the present application on inhibiting tumor growth in mice in vivo.
  • Figure 10 shows the effect of anti-CD47 mAb of the present application on tumor growth of xenograft human myeloma RPMI 8226 in NOD-SCID mice.
  • Figure 11 shows the structure of the CD47-Fab complex obtained by the present application, wherein Panel A shows the overall structure of the CD47-Fab complex; Panel B shows the binding surface of the CD47-Fab complex, and the amino acid residues involved in protein interaction are modeled with a stick. It is indicated that the two main binding areas are marked with dashed lines, the C picture shows the details of the interaction of the area I in the B picture, and the D picture shows the details of the area II interaction in the B picture.
  • SEQ ID NO: 1 shows the amino acid sequence of human (homo sapiens) CD47 extracellular domain D1 (hCD47).
  • SEQ ID NO: 2 shows the amino acid sequence of mouse (mus musculus) CD47 extracellular domain D1 (mCD47).
  • SEQ ID NO: 3 shows the amino acid sequence of the macadam (Macaca mulatta) CD47 extracellular domain D1 (mmCD47).
  • SEQ ID NO: 4 shows the amino acid sequence of the high affinity mutant (hCD172a-D1M1) of the immunoglobulin-like domain of the human CD172a extracellular domain.
  • SEQ ID NO: 5 shows the amino acid sequence of the His tag (His).
  • SEQ ID NO: 6 shows the amino acid sequence of the Fc fragment (Fc) of human antibody IgG1.
  • SEQ ID NO: 8 shows the amino acid sequence of the human IgGl subtype heavy chain constant region.
  • SEQ ID NO: 9 shows the amino acid sequence of the human IgG2 subtype heavy chain constant region.
  • SEQ ID NO: 10 shows the amino acid sequence of the human IgG4 subtype heavy chain constant region.
  • SEQ ID NO: 11 shows the amino acid sequence of the heavy chain constant region of the murine IgGl subtype.
  • SEQ ID NO: 12 shows the amino acid sequence of the heavy chain constant region of the murine IgG2a subtype.
  • SEQ ID NO: 13 shows the amino acid sequence of the human kappa subtype light chain constant region.
  • SEQ ID NO: 14 shows the amino acid sequence of the human lambda subtype light chain constant region.
  • SEQ ID NO: 15 shows the amino acid sequence of the murine kappa subtype light chain constant region.
  • SEQ ID NO: 17 and SEQ ID NO: 18 show the amino acid sequences of the VH and VK sequences of the humanized anti-CD47 mAb Hu5F9-G4, respectively.
  • SEQ ID NO: 19 shows the full-length amino acid sequence of the murine single-chain antibody S4D12
  • SEQ ID NOS: 20 and 21 show the amino acid sequences of its VH and VK sequences, respectively.
  • SEQ ID NO: 22 shows the full-length amino acid sequence of the murine single-chain antibody S2H2
  • SEQ ID NOS: 23 and 24 show the amino acid sequences of the VH and VK sequences, respectively.
  • SEQ ID NO: 25 shows the full length amino acid sequence of the murine single-chain antibody S2C3
  • SEQ ID NOS: 26 and 27 show the amino acid sequences of its VH and VK sequences, respectively.
  • SEQ ID NOS: 28 and 29 show the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of humanized S4D12 (hS4D12), respectively.
  • SEQ ID NOS: 30 and 31 show the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of humanized S2C3 (hS2C3), respectively.
  • SEQ ID NO:32 shows the amino acid sequence of the heavy chain variable region mutant H10C7.
  • SEQ ID NO:33 shows the amino acid sequence of the heavy chain variable region mutant H11E5.
  • SEQ ID NO:34 shows the amino acid sequence of the light chain variable region mutant L25B8.
  • SEQ ID NO: 35 shows the amino acid sequence of the light chain variable region mutant L26A6.
  • a novel anti-human CD47 antibody or antigen-binding fragment thereof obtained novel anti-human CD47 antibodies by antibody engineering techniques.
  • a novel anti-human CD47 antibody or antigen-binding fragment thereof a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector comprising the polynucleotide, comprising the polynucleotide Or a host cell of a vector, a method of making and purifying the antibody, and a medical and biological application of the antibody or antigen-binding fragment thereof.
  • a full-length antibody molecule can be constructed as a medicament for the treatment of a clinically mediated CD47 disease.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, and antibody-containing portions thereof.
  • Fusion proteins humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (eg bispecific antibodies) and any other immunoglobulin containing an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a full or full length antibody comprises two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variant region (VH) and first, second and third constant regions (CH1, CH2 and CH3).
  • Each light chain contains a light chain variant region (VL) and a constant region (CL).
  • the full length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA or IgM (or a subclass of the above), but the antibody does not need to belong to any particular class.
  • Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of the heavy chain.
  • immunoglobulins there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different immunoglobulin classes are referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen binding domain refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variant region (VH), a light chain variant region (VL), or both.
  • VH and VL typically contains three complementarity determining regions, CDR1, CDR2 and CDR3.
  • CDRs complementarity determining regions
  • VH or VL There are two common definitions for the CDR sequences of VH or VL, namely the kabat definition and the Chothia definition.
  • CDR sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, CDR sequences are defined using Kabat.
  • variable region sequences of a given antibody the CDR region sequences in the variable region sequences can be analyzed in a variety of ways, for example, using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab') 2 fragment, which may have two a bivalent fragment of a Fab' fragment joined by a disulfide bridge of the hinge region (ie, a dimer of Fab'); (3) an Fv fragment of the VL and VH domains of the one arm of the antibody; 4) a single-chain Fv (scFv), which may be a single multi-peptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which may comprise two linked by a peptide A coupled VH domain and two VL domains are combined with the two VH domains via a disulfide bridge.
  • a Fab fragment which may be a monovalent fragment having a VL-CL chain and a VH-
  • the term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are identical except that there may be naturally occurring mutations in a small number of individuals.
  • Monoclonal antibodies as used herein specifically include “chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence derived from a particular species or antibody belonging to a particular antibody or subclass, but The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies as long as they exhibit desired Biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • tumor refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • the tumor can be benign, malignant or malignant.
  • a “primary tumor” is a tumor that exists at an initial site in an individual and can be distinguished from a “metastatic tumor” that is present in a distant portion of the individual from the primary tumor.
  • malignant tumor refers to or describes the physiological condition of a mammal, which is typically characterized by unregulated cell growth.
  • exemplary malignancies include: carcinoma, melanoma sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma.
  • malignant tumors include: squamous cell carcinoma (for example, squamous cell carcinoma), including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung cancer of the lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, Gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colon Rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma , brain cancer and head and neck cancer and related metastases.
  • squamous cell carcinoma for example, squamous cell carcinoma
  • gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblast
  • cancer refers to a composition consisting of a variant epithelial cell or a variant cell having an unknown histological occurrence but having specific molecular or histological features associated with epithelial cells, such as the production of cytokeratin or an intercellular bridge.
  • Invasive malignant tumor Exemplary cancers of the present application include ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, gastric cancer, pancreatic cancer, islet tumor, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor.
  • breast cancer, lung cancer, esophageal cancer oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary cancer and bone cancer.
  • lymphomas include: non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( Macroglobulinemia), spleen marginal lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intracranial marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse Large B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, Burkitt's lymphoma, B-cell chronic lymphocytic lymphoma, classic Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia (
  • sarcoma is a malignant tumor derived from a variant cell in one of a variety of tissues developed by the embryonic mesoderm.
  • sarcomas include tumors of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue.
  • osteosarcoma from bone chondrosarcoma from cartilage, liposarcoma from fat, and leiomyosarcoma from smooth muscle.
  • Exemplary sarcomas include: Askin tumor, grape sarcoma, chondrosarcoma, Ewing's sarcoma-PNET, malignant hemangioendothelioma, malignant schwannomas, osteosarcoma, soft tissue sarcoma.
  • Subclasses of soft tissue sarcoma include: soft tissue acinar sarcoma, angiosarcoma, phyllodes cystosarcoma, cutaneous fibrosarcoma, fibroma, profibrotic small round cell tumor, epithelioid sarcoma, extramedullary chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma , vascular epithelioma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma and synovial sarcoma.
  • leukemia is a malignant tumor of blood or bone marrow characterized by an abnormal increase in white blood cells.
  • Leukemia is a broad term that covers a range of diseases. Therefore, leukemia is part of a broader range of diseases known as hematological malignancies.
  • Leukemia is subdivided into several broad categories; the first is the acute and chronic form of leukemia. Acute leukemia is characterized by a rapid increase in the number of immature blood cells. Due to the accumulation of these cells, the bone marrow cannot produce healthy blood cells.
  • Chronic leukemia is characterized by overproduction of relatively mature, but still abnormal, white blood cells.
  • leukemias include: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell lymphocytic leukemia, large granular lymphocytic leukemia, juvenile granulocyte-monocytic leukemia, B-cell lymphoblastic leukemia, Burkitt leukemia, and adult T-cell leukemia.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • T-cell lymphocytic leukemia large granular lymphocytic leukemia
  • juvenile granulocyte-monocytic leukemia B-cell lymphoblastic leukemia
  • Burkitt leukemia Burkitt leukemia
  • R represents A or G
  • Y represents C or T
  • M represents A or C
  • K represents G or T
  • S represents C or G
  • W represents A or T
  • H represents A or C or T
  • B represents C or G or T
  • V represents A or C or G
  • D represents A or G or T
  • N represents A or C or G or T.
  • the application provides an antibody that specifically binds to human CD47, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, wherein
  • the HCDR1 sequence is NYWMH
  • the HCDR2 sequence is VIAPSDNYTNYNQKFQG
  • the HCDR3 sequence is GGKYSMDY
  • the LCDR1 sequence is RSSQSIVHSNGNTYLE
  • the LCDR2 sequence is KVSNRFS
  • the LCDR3 sequence is FQGSHVPFT;
  • the HCDR1 sequence is SYWMH
  • the HCDR2 sequence is TIDRSDSYISYNQKFKG
  • the HCDR3 sequence is GGPYGSKMMDN
  • the LCDR1 sequence is HASQNINVWLS
  • the LCDR2 sequence is KASNLHT
  • the LCDR3 sequence is QQGQSYPLT;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPSSGNTKYAQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPSSGNTKYAQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSVLYSSNQKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is GQYYAYPIT;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSVLYSSNQKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is GQYYAYPIT;
  • HCDR and LCDR sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 23, 28, 30, 32 or 33.
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 24, 29, 31, 34 or 35.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:23, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:24;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 31;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 35;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:35.
  • the application provides an antibody that specifically binds to human CD47, wherein the amino acid sequence of the heavy chain variable region of the antibody has any one of SEQ ID NO: 23, 28, 30, 32 or 33 At least 90% identity, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to any of SEQ ID NO: 24, 29, 31, 34 or 35.
  • the application provides an antibody that specifically binds to human CD47, wherein the binding epitope of the antibody to the human CD47 segment of SEQ ID NO: 1 is a discontinuous epitope, including amino acids Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106, the above residue numbers refer to SEQ ID NO: 1.
  • the antibody is a monoclonal antibody.
  • the antibody binds to and neutralizes human CD47, thereby blocking the CD47-SIRP ⁇ signaling pathway.
  • the antibody promotes phagocytosis of tumor cells by macrophages, inhibits growth of tumor cells in vivo, and does not increase phagocytosis of normal blood cells by macrophages.
  • the antibody has at least one of the following properties:
  • the antibody is a whole antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or comprises a selected from the kappa subtype or the lambda subtype Light chain constant region.
  • the present application provides a nucleic acid molecule encoding the antibody of the first aspect to the third aspect or an antigen binding portion thereof.
  • the nucleic acid molecule is operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first to third aspects and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is for treating a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the pharmaceutical composition may further comprise one or more of the following: a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier; a suspending agent; a preservative, Such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents.
  • a lubricant such as talc, magnesium stearate, and mineral oil
  • a wetting agent such as talc, magnesium stearate, and mineral oil
  • an emulsifier such as benzoic acid, sorbic acid and calcium propionate
  • a preservative such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and / or flavoring agents such as benzoic acid, sorbic acid and calcium propionate
  • the pharmaceutical compositions of the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application can be delivered using any physiologically acceptable administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration. Drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use may be formulated in a lyophilized formulation or as an aqueous solution by mixing an agent having the desired purity with a pharmaceutically acceptable carrier, excipient, or the like, as appropriate. storage.
  • the application provides the use of the antibodies of the first to third aspects for the preparation of a medicament for the prevention or treatment of a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the present application provides a method of preventing or treating a CD47-mediated disease comprising administering the antibody of the first aspect to the third aspect or the pharmaceutical composition of the fifth aspect to an individual in need thereof.
  • the disease is a tumor, such as a malignant tumor.
  • the application further provides a vector comprising an isolated nucleic acid molecule encoding an antibody of the invention or an antigen binding portion thereof, and a host cell comprising the nucleic acid molecule or vector.
  • the present application also provides methods of producing the antibodies of the present application.
  • a method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid.
  • the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.
  • a variety of different recombinant proteins are required for the preparation of anti-CD47 mAb, including human CD47 extracellular domain D1 (hCD47, SEQ ID NO: 1), mouse CD47 extracellular domain D1 (mCD47, SEQ ID NO: 2). , a high affinity mutant of the macaque CD47 extracellular domain D1 (mmCD47, SEQ ID NO: 3) and the human CD172a extracellular domain immunoglobulin-like domain (hCD172a-D1M1, SEQ ID NO: 4). These proteins have post-translational modifications (such as glycosylation or disulfide bonds, etc.), and thus the use of mammalian cell expression systems is more conducive to maintaining the structure and function of the recombinant protein.
  • post-translational modifications such as glycosylation or disulfide bonds, etc.
  • the His tag His, SEQ ID NO: 5
  • the Fc segment of human antibody IgG1 Fc, SEQ ID NO
  • the antibody heavy chain constant region can be a human IgG1 subtype (SEQ ID NO: 8), a human IgG2 subtype (SEQ ID NO: 9), a human IgG4 subtype (SEQ ID NO: 10) or a murine IgG1 subtype (SEQ ID NO: 11), murine IgG2a subtype (SEQ ID NO: 12); light chain constant region may be human kappa subtype (SEQ ID NO: 13), human lambda subtype (SEQ ID NO: 14) or murine kappa Type (SEQ ID NO: 15), murine lambda subtype (SEQ ID NO: 16).
  • the genes of the above various recombinant proteins were designed and synthesized based on the amino acid sequence of the recombinant protein for various purposes of the Uniprot database.
  • the various recombinant protein genes synthesized are cloned into a suitable eukaryotic expression vector (such as invitrogen pcDNA3.1, etc.) using conventional molecular biology techniques, and then using liposomes (such as 293fectin of Invitrogen, etc.) or other cations.
  • the recombinant protein expression plasmid prepared by transfection reagent (such as PEI) is transfected into HEK293 cells (such as HEK293F of Invitrogen) and cultured in serum-free suspension culture for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.
  • the recombinant protein expressed by the His-tag fusion is subjected to one-step purification of the recombinant protein in the culture supernatant by a metal chelate affinity chromatography column (such as GE's HisTrap FF).
  • the recombinant protein expressed by fusion of Fc and mFc was subjected to one-step purification using a Protein A/G affinity chromatography column (e.g., Mabselect SURE, GE).
  • the recombinant protein storage buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (eg, Hitrap desaulting, GE, etc.). If necessary, the antibody samples can be sterilized by filtration and then stored separately at -20 °C.
  • mice of 6-8 weeks old were taken, and the mice were subjected to tail vein blood sampling to leave the background serum before immunization.
  • the hCD47-mFc fusion protein was immunized for the first time and emulsified with complete Freund's adjuvant.
  • Each mouse was intraperitoneally injected with 50 ⁇ g of fusion protein.
  • the immunization was boosted at intervals of two weeks, and the hCD47-mFc fusion protein was emulsified with incomplete adjuvant.
  • Each mouse was intraperitoneally injected with 50 ⁇ g of fusion protein, and blood was taken from the tail before injection for two booster immunizations.
  • the fourth immunization was changed to shock immunization, and the anti-adjuvant hCD47-mFc recombinant antigen was used as an immunogen.
  • Each mouse was intraperitoneally injected with 50 ⁇ g of fusion protein, and the mice were sacrificed 3 days after the immunization to collect spleen cells.
  • mice spleen lymphocytes were separated using mouse lymphocyte separation solution (Dakko, #DKW33-R0100), and the isolated lymphocytes were subjected to total RNA using a total RNA extraction kit (Tiangen, #DP430). extract.
  • a total RNA extraction kit Tiangen, #DP430. extract.
  • the first strand cDNA synthesis kit (Thermo scientific, #K1621) was used to synthesize the heavy chain variable region and the light chain variable region, respectively, and the reverse transcription primers were gene-specific primers, and the primer pairing region was used.
  • the prepared mouse single-chain antibody gene was cloned into the vector pADSCFV-S (experimental technical procedure can be found in Chinese Patent Application No. 201510097117.0) to construct a scFv library.
  • the library capacity of this antibody library reached 1.2 ⁇ 10E7, and the correct rate was about 85%.
  • Phage-ELISA showed that hCD172a-D1M1 could compete with four of the murine monoclonal antibodies (S1F9/S2C3/S2H2/S4D12) for binding to CD47, while the binding of murine monoclonal antibody S2D10 to CD47 was not blocked by hCD172a-D1M1.
  • the murine single-chain antibody S4D12 (SEQ ID NO: 19, VH and VK sequences are SEQ ID NOS: 20 and 21, respectively), S2H2 (SEQ ID NO: 22, VH and VK sequences are SEQ ID NO: 23 and 24, respectively) were selected.
  • S2C3 SEQ ID NO: 25, VH and VK sequences are SEQ ID NOS: 26 and 27, respectively
  • a recombinant human IgG4-kappa form of murine-human chimeric antibody is prepared by molecular biological methods, with reference to US Patent Application No. US9017675B2
  • humanized anti-CD47 mAb Hu5F9-G4 was prepared (VH sequence and VK sequence are SEQ ID NO: 17 and SEQ ID NO: 18, respectively).
  • Recombinant anti-CD47 mAb blocks hCD47 binding to hCD172a
  • the antigen hCD47-mFc was coated, and the S2C3/S2H2/S4D12 chimeric antibody was serially diluted with a fixed concentration of hCD172a-D1M1-His, and CD172a-D1M1- was detected with HRP-mouse-anti-his IgG (Kangwei Century, CW0285M). Binding of His and hCD47-mFc.
  • the results of the ELISA analysis (Fig. 1) showed that the ability of the monoclonal antibodies S2C3, S2H2 and S4D12 to block the interaction of CD47 and CD172a-D1M1 was not much different, and the IC 50 was in the range of 2-5 nM (Table 1).
  • Table 1 Three different anti-CD47 mAb IC 50 inhibition of CD47 and CD172a-D1M1 binding
  • the affinity of the IgG4 chimeric antibody was determined using Biacore X100.
  • Amino coupling kit, human antibody capture kit, CM5 chip and related reagents and consumables such as 10 ⁇ HBS-EP of pH 7.4 were purchased from GE healthcare.
  • the antibody against the human Fc segment was conjugated to the surface of the CM5 chip according to the instructions in the kit, and the antibody protein was diluted to a suitable concentration to ensure that about 300 RU of the antibody was captured by the antibody against human Fc.
  • huCD47-D1-his was placed in a series of concentration gradients (500 nM, 167 nM, 56 nM, 18.5 nM, 6.2 nM) through the surface of the stationary phase, and the affinity of each monoclonal antibody was determined at 25 °C. The results are shown in Table 2.
  • the surface of the red blood cells highly expresses CD47, so the anti-CD47 mAb can specifically bind to CD47 on the surface of red blood cells. Since each antibody has two antigen binding sites, anti-CD47 mAb may cause red blood cell agglutination. Monoclonal antibodies that cause red blood cell agglutination cause side effects such as anemia and decreased red blood cell count in the body. Whether the anti-CD47 mAb causes erythrocyte agglutination depends mainly on the recognition epitope of the mAb on the CD47 molecule. This example utilizes classical hemagglutination experiments to analyze the ability of CD47 monoclonal antibody to cause red blood cell agglutination.
  • the anti-CD47 antibody (40 ⁇ g/ml) selected in the previous examples was diluted with PBS for a series of dilutions, and then mixed with the prepared 1% red blood cell suspension in a microhemagglutination plate and shaken on a micro-oscillator. Mix for 1 min.
  • the micro-hemagglutination reaction plate was allowed to stand at 25 ° C for 1 h, and the degree of erythrocyte agglutination was recorded by photography, and the micro-hemagglutination reaction plate was tilted at an angle of 45° for several minutes, and the degree of red blood cell agglutination was further determined by the rate of red blood cell flow.
  • the degree of erythrocyte agglutination can be divided into 4 grades: 4 condensed into a uniform thin layer, the slant does not flow; 3 condenses into a uniform thin layer, the slant has a slight flow; 2 condenses into a small thin layer, the slanting flow is faster; 1 has no thin layer Formed, the slope is the same as the flow velocity of the control well.
  • the results showed (Fig. 2) that human-mouse chimeric mAbs S2C3 and S2H2 did not cause erythrocyte agglutination, whereas human-mouse chimeric mAb S4D12 and humanized antibody Hu5F9 had strong erythrocyte agglutination.
  • Recombinant anti-CD47 mAb can promote macrophage phagocytosis of tumor cells
  • PBMCs peripheral blood mononuclear cells
  • CD14 + monocytes were sorted from PBMCs by CD14 magnetic beads (anti-human CD14 magnetic particles - DM, BD).
  • CD14 + monocytes were seeded in a 12-well plate at a density of 8 ⁇ 10 5 /well, and induced with 10 ng/ml MCSF for about 7 days to differentiate into mature macrophages.
  • hS2C3 was subjected to in vitro affinity maturation.
  • the main strategy for in vitro affinity maturation is to introduce mutations in the heavy chain CDRs and light chain CDRs in turn, construct a light chain or heavy chain mutation library, and then based on the dual vector phage display system (for specific operations, refer to the Chinese patent application filed by the applicant before).
  • Example 5 of No. 201510097117.0 screens for high affinity mutants of heavy and light chains, respectively.
  • the mutation library of hS2C3 heavy chain variable region was constructed by conventional molecular biology method, and the storage capacity was 6.5 ⁇ 10E5. The correct rate of the mutation library was about 95%.
  • the primers required for introducing mutations into HCDR1 and HCDR2 are shown in Table 5.
  • the hS2C3-HCDR12 mutant library was screened by solid-phase screening strategy for 3 rounds, and finally two mutants H10C7 (SEQ ID NO: 32) and H11E5 (SEQ ID NO:) with further improved affinity were obtained. 33).
  • erythrocyte agglutination experiment showed (Fig. 7) that four humanized anti-CD47 monoclonal antibodies with increased affinity did not cause erythrocyte agglutination in the concentration range of 0-133 nM, and all four humanized mutants could promote macrophage to CD47. + Phagocytosis of tumor cells (Daudi) ( Figure 8).
  • mice subcutaneously inoculated into male NSG mice at a dose of 1 ⁇ 10 4 cells/mouse, and randomly divided into 5 groups with a tumor volume of 100 cm 3 , 8 mice per group (1) vehicle control group; (2) 20 mg/kg H10C7+L26A6 group; (3) 10 mg/kg H10C7+L26A6 group; (4) 3 mg/kg H10C7+L26A6 group; (5) 10 mg/kg hu5F9-IgG4 group . It was administered intraperitoneally twice a week for a total of 3 weeks. Tumor size and animal body weight were measured three times a week during the administration to evaluate the antitumor effect of the anti-CD47 antibody.
  • Fig. 9 The experimental results are shown in Fig. 9.
  • the tumor-bearing mice in which the abscissa is the raji tumor received the drug treatment time, and the ordinate was the tumor volume.
  • the results showed that the humanized anti-CD47 monoclonal antibody H10C7+L26A6 inhibited tumor growth compared with the vehicle control group, and its inhibitory ability was comparable to that of the control antibody hu5F9-IgG4.
  • mice were divided into 5 groups of 8 mice each: (1) vehicle control group (ipBIW*4, poQD*4); (2) 10 mg/kg daratumumab (ipBIW*4) group; (3) 10 mg/kg H10C7+L26A6 (ipBIW*4) group; (4) 50 mg/kg lenolidomide (poQD*4) group; 510 mg/kg dalimumab (ipBIW*4)+ 50 mg/kg lenalidomide (poQD*4) group.
  • Tumor size and animal body weight were measured 3 times a week during the administration.
  • Dalemuzumab is a marketed monoclonal antibody against multiple myeloma
  • lenalidomide is a routine drug for clinical multiple myeloma.
  • Fig. 10 The experimental results are shown in Fig. 10.
  • the abscissa is the time when the NOD-SCID tumor-bearing mice of the subcutaneous xenograft RMPI 8226 tumor were treated with the drug, and the ordinate was the tumor volume, which was humanized compared with the vehicle control group.
  • Anti-CD47 monoclonal antibody H10C7+L26A6 can significantly inhibit tumor growth, and its tumor inhibition ability is significantly higher than the combination of dalimumab, lenalidomide and dalimumab + lenalidomide.
  • HEK293 cells were used to prepare recombinant H10C7+L26A6 Fab fragment and recombinant human CD47 extracellular domain D1 (hCD47, SEQ ID NO: 1) to form Fab- hCD47 complex, forming complex crystals under 2.0M ammonium sulfate + 5% isopropanol, X-ray diffraction experiments on complex crystals, resolution
  • the data (Table 9) and the crystal structure of the CD47 antigen-antibody complex were analyzed by molecular replacement.
  • the resulting structural model includes the CD47 extracellular domain 1-116 aa (SEQ ID NO: 1), the antibody heavy chain 1-219 aa, and the light chain 1-219 aa.
  • the last amino acid residue of the H chain and L chain of the antibody is not visible in the electron density, suggesting that it has no fixed structure in the crystal.
  • CD47 exists in a monomeric form and there is visible glycosylation at the N16, N32, N55 and N93 sites.
  • the interaction area between CD47 and Fab is about
  • the interaction area between CD47 and heavy chain is about
  • the area of interaction with the light chain is approximately
  • the interaction mainly involves the N-terminus of the CD47, the BC loop, the ⁇ -chain F, the FG loop and the ⁇ -strand G, while the HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region, and the LCDR1 and LCDR3 of the light chain variable region are involved.
  • Identification of CD47 ( Figure 11).
  • the interactions include hydrogen bonds, salt bonds, van der Waals forces, and hydrophobic interactions (Table 10).
  • humanized anti-CD47 mAb H10C7+L26A6 recognizes a discontinuous (spatial) epitope of human CD47, mainly including amino acids Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106.
  • the antigen-binding epitope of anti-human CD47 monoclonal antibody hu5F9 monoclonal antibody includes amino acids Q1, E29, K39, K41, E97, T99, R103 and E104; and the epitope of anti-human CD47 monoclonal antibody 2A1 includes amino acid Y37. K39, K41, K43, G44, R45, D46, D51, H90, N93, E97, T99, E104 and E106.
  • the epitopes of the anti-human CD47 mAbs H10C7+L26A6, hu5F9 and 2A1 partially overlap, but they are not identical.
  • BIAcore determined the affinity of humanized anti-CD47 mAb H10C7+L26A6 for these hCD47 mutants and compared it to native hCD47 (Table 11). The results showed that the L101A point mutation resulted in complete no binding, the Q1A mutation resulted in a decrease in affinity of about 100-fold, while the E35A mutation resulted in an approximately 4-fold increase in affinity.
  • SIRPa Signal-regulatory protein a
  • CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.
  • CD47 is upregulated on cir-culating hematopoietic stem cells and leukemia cells to avoid phagocytosis.

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Abstract

本申请公开了特异性结合人CD47的抗体或其抗原结合部分，编码所述抗体或其抗原结合部分的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合部分的应用。

Description

抗人CD47抗体及其用途
相关申请的引用
本申请要求2017年9月1日提交的发明名称为“抗人CD47抗体及其用途”的201710777971.0号中国专利申请以及2018年4月20日提交的发明名称为“抗人CD47抗体及其用途”的201810375653.6号中国专利申请的优先权，在此通过引用的方式将上述两篇专利申请的全部内容并入本文。
技术领域
本申请大体涉及基因工程和抗体药物领域；具体而言，涉及抗人CD47抗体领域及其用途。本申请开发了新的抗人CD47抗体，并提供了该抗体在治疗CD47介导的疾病中的用途。
背景技术
CD47属于免疫球蛋白超家族成员，是I型跨膜糖蛋白，包括氨基端V型免疫球蛋白样细胞外结构域，由5个高度疏水的跨膜片段构成的跨膜结构域和亲水的羧基端细胞内结构域，分子量在47-55kD之间。最初，CD47因为与整合素α vβ 3相互作用而为人们所认识，因此其又称为整合素相关蛋白(IAP) 1。CD47通过细胞外结构域与整合素α vβ 3或α IIbβ 3的β 3亚基相互作用，激活相应的整合素。
SIRPα(信号调节蛋白α(Signal regulatory proteinα))，又称SHPS-1(含Src同源2结构域蛋白酪氨酸激酶底物-1(Src homology 2 domain-containing protein tyrosine phosphatase substrate-1))/BIT(具有酪氨酸的活化基序的脑免疫球蛋白样分子(brain immunoglobulin-like molecule with tyrosine-based activation motifs))/CD172a，也是跨膜糖蛋白，胞外区具有3个免疫球蛋白样结构域，细胞内结构域具有典型的免疫受体酪氨酸抑制性序列(ITIM)，具有4个酪氨酸残基，是潜在的磷酸化位点。
CD47是SIRPα的配体，二者通过胞外结构域相互作用，形成细胞间的通讯复合物。CD47与SIRPα结合后，诱导SIRPα胞内ITIM磷酸化，磷酸化位点结合进而激活含有SH2(Src同源2(Src homology 2))结构域的酪氨酸磷酸酶SHP-1和SHP-2，引发信号传递的级联反应。SHP-1主要表达于造血细胞，负向调节这些细胞的功能 2；而SHP-2表达非常广泛，调节小G蛋白Ras和Rho，正向控制细胞生长和增殖 3。
SIRPα在神经元以及髓系造血细胞(如巨噬细胞和树突细胞)中表达丰富 4，而CD47则表达于多数细胞中。CD47-SIRPα信号系统对调节巨噬细胞对成熟血细胞的吞噬具有重要作用。正常健康细胞(如红细胞 5或血小板 6)表面的CD47分子与巨噬细胞上的受体SIRPα相互作用产生抑制性信号，抑制其吞噬活性，达到调节血细胞的生命周期及其在血液中的数量的目的。单核细胞上的SIRPα与红细胞上的CD47作用，通过肌球蛋白-IIA去磷酸化抑制Fcγ受体依赖的吞噬作用 7。CD47-SIRPα信号系统抑制树突细胞激活，参与神经系统发育、中性粒细胞趋化激活和基质细胞支持的造血细胞生成等多种生理活动，同时在诱导T细胞免疫耐受、活化、凋亡等方面也发挥着多种调节作用 8。
近年来，CD47-SIRPα信号系统通过调节巨噬细胞的吞噬作用在肿瘤免疫监视中发挥的作用受到重视。CD47在许多恶性肿瘤，如卵巢癌 9、急性骨髓性白血病(AML) 10、B细胞淋巴瘤 11和实体瘤 12中的表达水平显著上调，其平均表达水平是对应正常细胞的3.3倍左右，而且这种上调与恶性肿瘤患者的不良预后直接相关 13。在人髓性白血病细胞(低水平表达内源CD47，不能移植于Rag2-Il2rg-小鼠)中表达小鼠CD47，可以抑制巨噬细胞对肿瘤细胞的吞噬作用，促进肿瘤细胞成功移植 14。可以推断，肿瘤细胞上的CD47与巨噬细胞上的SIRPα作用，抑制巨噬细胞对肿瘤细胞的清除，促 进肿瘤在体内的生长和转移，高表达CD47是肿瘤细胞逃避免疫监视的普遍机制，阻断CD47-SIRPα作用，可能是新的肿瘤免疫治疗策略。
抗CD47抗体单独使用 10,12,13或者与其它肿瘤抗原抗体联合应用 11，在人急性骨髓性白血病、非霍奇金淋巴瘤(NHL)以及许多实体瘤的小鼠移植模型上都显示了很好的抑制肿瘤生长的作用。人源化抗CD47单克隆抗体诱导巨噬细胞对人原代AML细胞的吞噬作用，在体内彻底清除人AML细胞，使得移植小鼠长期无病生存；与利妥昔单抗联合作用，能清除NHL肿瘤，治愈异种移植小鼠 15。而且，抗CD47抗体的安全性在猴体内得到证实。除了抗CD47抗体，高亲和力的SIRPα突变体(CD172a)也可以拮抗CD47进而阻断CD47-SIRPα信号通路，在AML模型上能显著增加巨噬细胞对AML细胞的吞噬作用，并抑制肿瘤的生长 16。
新的抗CD47抗体的开发和应用是本领域所需要的。
发明概述
第一方面，本申请提供了一种特异性结合人CD47的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区和含LCDR1、LCDR2和LCDR3序列的轻链可变区，其中
所述HCDR1序列为NYWMH，所述HCDR2序列为VIAPSDNYTNYNQKFQG，所述HCDR3序列为GGKYSMDY，所述LCDR1序列为RSSQSIVHSNGNTYLE，所述LCDR2序列为KVSNRFS，所述LCDR3序列为FQGSHVPFT；或者
所述HCDR1序列为SYWMH，所述HCDR2序列为TIDRSDSYISYNQKFKG，所述HCDR3序列为GGPYGSKMMDN，所述LCDR1序列为HASQNINVWLS，所述LCDR2序列为KASNLHT，所述LCDR3序列为QQGQSYPLT；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPSSGNTKYAQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSLLYSSNKKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为QQFYAYPIS；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPSSGNTKYAQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSVLYSSNQKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为GQYYAYPIT；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPGSGNTRYSQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSLLYSSNKKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为QQFYAYPIS；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPGSGNTRYSQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSVLYSSNQKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为GQYYAYPIT；
其中HCDR和LCDR序列根据Kabat定义。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：23、28、30、32或者33所示。
在一些实施方案中，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：24、29、31、34或者35所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：23所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：29所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：31所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：32所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：34所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：32所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：35所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：33所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：34所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：33所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：35所示。
第二方面，本申请提供了一种特异性结合人CD47的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO：23、28、30、32或者33中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO：24、29、31、34或者35中任何一项具有至少90％的一致性。
第三方面，本申请提供了一种特异性结合人CD47的抗体，其中所述抗体与SEQ ID NO：1所示人CD47区段的结合表位为不连续表位，包括氨基酸Q1，E29，A30，Q31，N32，E35，E97，E100，L101，T102，R103，E104，G105和E106，上述残基编号参照SEQ ID NO：1。
在第一方面至第三方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面至第三方面的一些实施方案中，所述抗体为非激活型抗体。
在第一方面至第三方面的一些实施方案中，所述抗体能结合并中和人CD47，进而阻断CD47-SIRPα信号通路。在一些实施方案中，所述抗体能促进巨噬细胞对肿瘤细胞的吞噬作用。在一些实施方案中，所述抗体抑制肿瘤细胞的体内生长。在一些实施方案中，所述抗体不增加巨噬细胞对正常血细胞的吞噬。
在第一方面至第三方面的一些实施方案中，所述抗体具有至少一项以下性质：
以低于5nM的KD值的亲和力结合重组人CD47胞外区D1(SEQ ID NO：1)；
以低于5nM的IC 50值抑制CD172a高亲和力突变体(SEQ ID NO：4)与人CD47-Fc的结合；
在0-133nM浓度下施用不导致红细胞聚集。
在第一方面至第三方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面至第三方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面至第三方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区和/或包含选自κ亚型或者λ亚型的轻链恒定区。
第四方面，本申请提供了核酸分子，其编码第一方面至第三方面所述的抗体或其抗原结合部分。
第五方面，本申请提供了药物组合物，其包含第一方面至第三方面所述的抗体和药学可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于治疗CD47介导的疾病。
在一些实施方案中，所述疾病为肿瘤，例如恶性肿瘤。
在一些实施方案中，恶性肿瘤为伯基特淋巴瘤或多发性骨髓瘤。
第六方面，本申请提供了第一方面至第三方面所述的抗体在制备用于预防或治疗CD47介导的疾病的药物中的用途。
在一些实施方案中，所述疾病为肿瘤，例如恶性肿瘤。
在一些实施方案中，恶性肿瘤为伯基特淋巴瘤或多发性骨髓瘤。
第七方面，本申请提供了预防或治疗CD47介导的疾病的方法，包括向有需要的个体给予第一方面至第三方面所述的抗体或第七方面的药物组合物。
在一些实施方案中，所述疾病为肿瘤，例如恶性肿瘤。
在一些实施方案中，恶性肿瘤为伯基特淋巴瘤或多发性骨髓瘤。
附图说明
图1显示本申请的抗CD47单抗与hCD172a竞争结合hCD47的结果。
图2显示基于血凝实验分析本申请的抗CD47单抗的致红细胞凝集效应。
图3显示通过流式细胞术，分析本申请的抗CD47单抗促巨噬细胞吞噬Daudi细胞的效果。
图4显示本申请的抗CD47单抗促进巨噬细胞对Daudi细胞的吞噬率。
图5显示本申请的人源化抗CD47单抗抑制hCD47与hCD172a结合的效果。
图6显示hS2C3及其变体抑制hCD47和hCD172a-D1M1结合的效果。
图7显示本申请的抗CD47单抗致红细胞凝集程度的效果。
图8显示本申请的抗CD47单抗促进巨噬细胞对Daudi细胞的吞噬率。
图9显示本申请的抗CD47单抗抑制小鼠体内肿瘤生长的效果。
图10显示本申请的抗CD47单抗抑制NOD-SCID小鼠体内异种移植人骨髓瘤RPMI 8226肿瘤生长的效果。
图11显示了本申请测试得到的CD47-Fab复合物结构，其中A图显示CD47-Fab复合物整体结构；B图显示CD47-Fab复合物结合表面，参与蛋白相互作用的氨基酸残基用棍棒模型表示，两个主要的结合区域用虚线标注，C图显示B图中区域I相互作用的细节；D图显示B图中区域II相互作用的细节。
序列说明
SEQ ID NO：1显示人(homo sapiens)CD47胞外区D1(hCD47)的氨基酸序列。
SEQ ID NO：2显示小鼠(mus musculus)CD47胞外区D1(mCD47)的氨基酸序列。
SEQ ID NO：3显示猕猴(Macaca mulatta)CD47胞外区D1(mmCD47)的氨基酸序列。
SEQ ID NO：4显示人CD172a胞外区免疫球蛋白样结构域的高亲和力突变体(hCD172a-D1M1)的氨基酸序列。
SEQ ID NO：5显示His标签(His)的氨基酸序列。
SEQ ID NO：6显示人抗体IgG1的Fc段(Fc)的氨基酸序列。
SEQ ID NO：7显示鼠抗体IgG2a的Fc段(mFc)的氨基酸序列。
SEQ ID NO:8显示人IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:9显示人IgG2亚型重链恒定区的氨基酸序列。
SEQ ID NO:10显示人IgG4亚型重链恒定区的氨基酸序列。
SEQ ID NO：11显示鼠IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:12显示鼠IgG2a亚型重链恒定区的氨基酸序列。
SEQ ID NO:13显示人κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:14显示人λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:15显示鼠κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO：16显示鼠λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO：17和SEQ ID NO：18分别显示人源化抗CD47单抗Hu5F9-G4的VH序列和VK序列的氨基酸序列。
SEQ ID NO：19显示鼠单链抗体S4D12的全长氨基酸序列，SEQ ID NO：20和21分别显示其VH和VK序列的氨基酸序列。
SEQ ID NO：22显示鼠单链抗体S2H2的全长氨基酸序列，SEQ ID NO：23和 24分别显示其VH和VK序列的氨基酸序列。
SEQ ID NO：25显示鼠单链抗体S2C3的全长氨基酸序列，SEQ ID NO：26和27分别显示其VH和VK序列的氨基酸序列。
SEQ ID NO：28和29分别显示人源化S4D12(hS4D12)的重链可变区氨基酸序列和轻链可变区氨基酸序列。
SEQ ID NO：30和31分别显示人源化S2C3(hS2C3)的重链可变区氨基酸序列和轻链可变区氨基酸序列。
SEQ ID NO：32显示重链可变区突变体H10C7的氨基酸序列。
SEQ ID NO：33显示重链可变区突变体H11E5的氨基酸序列。
SEQ ID NO：34显示轻链可变区突变体L25B8的氨基酸序列。
SEQ ID NO：35显示轻链可变区突变体L26A6的氨基酸序列。
发明详细描述
本申请的发明人通过抗体工程技术得到了新的抗人CD47抗体。在本申请的多个方面，提供了新的抗人CD47抗体或其抗原结合片段，编码该抗体或其抗原结合片段的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合片段的医学和生物学应用。根据本申请提供的抗体的可变区的序列，可构建全长的抗体分子作为药物用于治疗临床上由CD47介导的疾病。
除非另外指明，本发明的实施采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”，是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。标靶包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab'、F(ab') 2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链变异区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链变异区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“抗原结合片段或抗原结合部分”，是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链变异区(VH)、轻链变异区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有 两种常见的定义方式，即kabat定义和Chothia定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab') 2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段；(4)单链Fv(scFv)，其可以是由VH域和VL域经由胜肽连接符组成的单一多胜肽链；以及(5)(scFv) 2，其可以包含两个由胜肽连接符连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(美国专利号4,816,567；和Morrison等，Proc.Natl.Acad.Sci.USA81:6851-6855(1984))。
本文所用术语“肿瘤”指由异常细胞生长形成的赘生物或实体病变。肿瘤可以是良性的、恶变前的或恶性的。
“原发性肿瘤”是在个体中最初位点存在的肿瘤，可以与“转移性肿瘤”相区别，转移性肿瘤存在于个体中距原发性肿瘤的远距离部位。
本文所用术语“恶性肿瘤”是指或描述哺乳动物的生理条件，其典型的特征在于不受调控的细胞生长。示例性恶性肿瘤包括：癌、黑素瘤肉瘤、淋巴瘤、白血病、生殖细胞瘤和胚细胞瘤。恶性肿瘤的更多具体实例包括：鳞状细胞癌(例如，鳞状上皮细胞癌)，包括小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞癌的肺癌，腹膜癌，肝细胞癌，包括胃肠癌的胃癌，胰腺癌，成胶质细胞瘤，子宫颈癌，卵巢癌，肝癌(liver cancer)，膀胱癌，尿路癌，肝细胞瘤，乳腺癌，结肠癌，直肠癌，结肠直肠癌，子宫内膜或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌(hepatic carcinoma)，肛门癌，阴茎癌，黑素瘤，多发性骨髓瘤和B细胞淋巴瘤，脑癌及头颈癌以及相关转移灶。
本文所用术语“癌”指由变异的上皮细胞或具有未知的组织学发生、但具有与上皮细胞有关的特定分子学或组织学特征(如产生细胞角蛋白或细胞间桥)的变异细胞组成的侵袭性恶性肿瘤。本申请的示例性癌包括卵巢癌、阴道癌、子宫颈癌、子宫癌、前列腺癌、肛门癌、直肠癌、结肠癌、胃癌、胰腺癌、胰岛瘤、腺癌、腺鳞癌、神经内分泌肿瘤、乳腺癌、肺癌、食管癌、口腔癌、脑癌、成神经管细胞瘤、神经外胚层瘤、胶质瘤、垂体癌和骨癌。
本文所用术语“淋巴瘤”指免疫系统的淋巴细胞的恶性肿瘤。淋巴瘤通常以实体瘤存在。示例性淋巴瘤包括：非霍奇金淋巴瘤、B细胞淋巴瘤、小淋巴细胞 性淋巴瘤、淋巴浆细胞淋巴瘤、原发性巨球蛋白血症(
Figure PCTCN2018100262-appb-000001
macroglobulinemia)、脾边缘区淋巴瘤、浆细胞瘤、结外边缘区B细胞淋巴瘤、MALT淋巴瘤、结内边缘区B细胞淋巴瘤(NMZL)、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大B细胞淋巴瘤、纵隔(胸腺)大B细胞淋巴瘤、血管内大B细胞淋巴瘤、原发性渗出性淋巴瘤、伯基特淋巴瘤、B细胞慢性淋巴细胞性淋巴瘤、经典霍奇金淋巴瘤、结节性淋巴细胞为主型霍奇金淋巴瘤、成人T细胞淋巴瘤、结外鼻型NK/T细胞淋巴瘤、肠病型T细胞淋巴瘤、肝脾T细胞淋巴瘤、母细胞NK细胞淋巴瘤、蕈样真菌病、赛谢综合征、原发性皮肤CD30阳性T细胞淋巴增生病、原发性皮肤间变性大细胞淋巴瘤、淋巴瘤样丘疹病、血管免疫母细胞性T细胞淋巴瘤、非特指型外周T细胞淋巴瘤和间变性大细胞淋巴瘤。经典霍奇金淋巴瘤的示例性形式包括：结节性硬化型、混合细胞型、淋巴细胞富集型和淋巴细胞消减型或非消减型。
本文所用术语“肉瘤”为来自由胚胎中胚层发育的多种组织之一中的变异细胞的恶性肿瘤。因此，肉瘤包括骨、软骨、脂肪、肌肉、血管和造血组织的肿瘤。例如，来自骨的骨肉瘤、来自软骨的软骨肉瘤、来自脂肪的脂肪肉瘤和来自平滑肌的平滑肌肉瘤。示例性的肉瘤包括：Askin瘤、葡萄状肉瘤、软骨肉瘤、尤文氏肉瘤-PNET、恶性血管内皮瘤、恶性神经鞘瘤、骨肉瘤、软组织肉瘤。软组织肉瘤的亚类包括：软组织腺泡状肉瘤、血管肉瘤、叶状囊肉瘤、皮肤纤维肉瘤硬纤维瘤、促纤维化小圆细胞肿瘤、上皮样肉瘤骨外软骨肉瘤、骨外骨肉瘤、纤维肉瘤、血管外皮细胞瘤、血管肉瘤、卡波济氏肉瘤、平滑肌肉瘤、脂肪肉瘤、淋巴管肉瘤、淋巴肉瘤、恶性纤维组织细胞瘤、神经纤维肉瘤、横纹肌肉瘤和滑膜肉瘤。
本文所用术语“白血病”为血液或骨髓的恶性肿瘤，特征在于白细胞的异常增多。白血病是广义术语，其涵盖了一系列疾病。因此，白血病是更广泛的称为血液肿瘤的疾病种类的一部分。白血病细分为多个大类；第一种分类是白血病的急性和慢性形式。急性白血病的特征在于未成熟的血细胞数量的快速增长。由于这些细胞的积聚使得骨髓不能制造健康的血细胞。慢性白血病的特征在于相对成熟、但仍为异常的白细胞的过度产生。通常通过数月或数年的发展，所述细胞的产生速率大大高于正常细胞，导致血液中有很多的异常白细胞。白血病也可以通过受累的血细胞进行细分。这样的分界将白血病划分为成淋巴细胞或淋巴细胞白血病和骨髓性(myeloid)或骨髓性(myelogenous)白血病。在成淋巴细胞或淋巴细胞白血病中，癌变发生在通常继续形成淋巴细胞的骨髓细胞类型中。在骨髓性(myeloid)或骨髓性(myelogenous)白血病中，癌变发生在通常继续形成红细胞、一些其他类型的白细胞和血小板的骨髓细胞类型中。结合这两种分类提供了总共四种主要类别。在这四种主要分类的每一种中，通常有一些亚类别。还存在分类方案之外的罕见类型。示例性的白血病包括：急性成淋巴细胞性白血病(ALL)、慢性淋巴细胞性白血病(CLL)、急性骨髓性白血病(AML)、慢性髓细胞性白血病(CML)、毛细胞性白血病(HCL)、T细胞幼淋巴细胞性白血病、大颗粒淋巴细胞白血病、幼年型粒-单核细胞白血病、B细胞幼淋巴细胞性白血病、伯基特白血病和成人T细胞性白血病。
在本文给出的核酸序列中涉及兼并碱基(除了A、T、C、G常规碱基之外)的使用，其含义与本领域技术人员通常理解的相同。例如，R代表A或G；Y代表C或T，M代表A或C；K代表G或T；S代表C或G；W代表A或T；H代表A或C或T；B代表C或G或T；V代表A或C或G；D代表A或G或T；N代表A或C或G或T。
第一方面，本申请提供了一种特异性结合人CD47的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区和含LCDR1、LCDR2和LCDR3序列的轻链可变区，其中
所述HCDR1序列为NYWMH，所述HCDR2序列为VIAPSDNYTNYNQKFQG，所述HCDR3序列为GGKYSMDY，所述LCDR1序列为RSSQSIVHSNGNTYLE，所述LCDR2序列为KVSNRFS，所述LCDR3序列为FQGSHVPFT；或者
所述HCDR1序列为SYWMH，所述HCDR2序列为TIDRSDSYISYNQKFKG，所述HCDR3序列为GGPYGSKMMDN，所述LCDR1序列为HASQNINVWLS，所述LCDR2序列为KASNLHT，所述LCDR3序列为QQGQSYPLT；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPSSGNTKYAQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSLLYSSNKKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为QQFYAYPIS；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPSSGNTKYAQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSVLYSSNQKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为GQYYAYPIT；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPGSGNTRYSQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSLLYSSNKKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为QQFYAYPIS；或者
所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPGSGNTRYSQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSVLYSSNQKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为GQYYAYPIT；
其中HCDR和LCDR序列根据Kabat定义。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：23、28、30、32或者33所示。
在一些实施方案中，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：24、29、31、34或者35所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：23所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：29所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：31所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：32所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：34所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：32所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：35所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：33所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：34所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO：33所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：35所示。
第二方面，本申请提供了一种特异性结合人CD47的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO：23、28、30、32或者33中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO：24、29、31、34或者35中任何一项具有至少90％的一致性。
第三方面，本申请提供了一种特异性结合人CD47的抗体，其中所述抗体与SEQ  ID NO：1所示人CD47区段的结合表位为不连续表位，包括氨基酸Q1，E29，A30，Q31，N32，E35，E97，E100，L101，T102，R103，E104，G105和E106，上述残基编号参照SEQ ID NO：1。
在第一方面至第三方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面至第三方面的一些实施方案中，所述抗体为非激活型抗体。
在第一方面至第三方面的一些实施方案中，所述抗体能结合并中和人CD47，进而阻断CD47-SIRPα信号通路。在一些事实方案中，所述抗体能促进巨噬细胞对肿瘤细胞的吞噬作用，抑制肿瘤细胞的体内生长，而不增加巨噬细胞对正常血细胞的吞噬。
在第一方面至第三方面的一些实施方案中，所述抗体具有至少一项以下性质：
以低于5nM的KD值的亲和力结合重组人CD47胞外区D1(SEQ ID NO：1)；
以低于5nM的IC 50值抑制CD172a高亲和力突变体(SEQ ID NO：4)与人CD47-Fc的结合；
在0-133nM浓度下施用不导致红细胞聚集。
在第一方面至第三方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面至第三方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面至第三方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区和/或包含选自κ亚型或者λ亚型的轻链恒定区。
第四方面，本申请提供了核酸分子，其编码第一方面至第三方面所述的抗体或其抗原结合部分。
在一些实施方案中，所述核酸分子可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
第五方面，本申请提供了药物组合物，其包含第一方面至第三方面所述的抗体和药学可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于治疗CD47介导的疾病。
在一些实施方案中，所述疾病为肿瘤，例如恶性肿瘤。
在一些实施方案中，恶性肿瘤为伯基特淋巴瘤或多发性骨髓瘤。
在一些实施方案中，药物组合物还可包含下述中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第六方面，本申请提供了第一方面至第三方面所述的抗体在制备用于预防或治疗CD47介导的疾病的药物中的用途。
在一些实施方案中，所述疾病为肿瘤，例如恶性肿瘤。
在一些实施方案中，恶性肿瘤为伯基特淋巴瘤或多发性骨髓瘤。
第七方面，本申请提供了预防或治疗CD47介导的疾病的方法，包括向有需要的个体给予第一方面至第三方面所述的抗体或第五方面的药物组合物。
在一些实施方案中，所述疾病为肿瘤，例如恶性肿瘤。
在一些实施方案中，恶性肿瘤为伯基特淋巴瘤或多发性骨髓瘤。
在其他方面，本申请还提供包含编码本申请抗体或其抗原结合部分的分离的核酸分子的载体以及包含所述核酸分子或载体的宿主细胞。
在其他方面，本申请还提供产生本申请抗体的方法。在一些实施方案中，产生抗体的方法包括培养宿主细胞以便于表达核酸。在一些实施方案中，产生抗体的方法还包括从宿主细胞培养基中回收抗体。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1：重组蛋白的制备
制备抗CD47单抗的过程中需要用到多种不同的重组蛋白，包括人CD47胞外区D1(hCD47，SEQ ID NO：1)、小鼠CD47胞外区D1(mCD47，SEQ ID NO：2)、猕猴CD47胞外区D1(mmCD47，SEQ ID NO：3)和人CD172a胞外区免疫球蛋白样结构域的高亲和力突变体(hCD172a-D1M1，SEQ ID NO：4)。这些蛋白具有翻译后修饰(如糖基化或者二硫键等)，因而利用哺乳动物细胞表达系统更有利于保持重组蛋白的结构和功能。此外，为了有利于重组蛋白的纯化和单克隆抗体功能的鉴定，在这些重组蛋白的C端添加了His标签(His，SEQ ID NO：5)或者人抗体IgG1的Fc段(Fc，SEQ ID NO：6)或者鼠抗体IgG2a的Fc段(mFc，SEQ ID NO：7)。抗体重链恒定区可以是人IgG1亚型(SEQ ID NO:8)，人IgG2亚型(SEQ ID NO:9)、人IgG4亚型(SEQ ID NO:10)或者鼠IgG1亚型(SEQ ID NO：11)，鼠IgG2a亚型(SEQ ID NO:12)；轻链恒定区可以是人κ亚型(SEQ ID NO:13)、人λ亚型(SEQ ID NO:14)或者鼠κ亚型(SEQ ID NO:15)、鼠λ亚型(SEQ ID NO：16)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His标签或者Fc、mFc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或者其他阳离子转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-4天。然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。Fc和mFc融合表达的重组蛋白用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其他合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2：鼠抗人CD47单抗的制备
1.小鼠免疫及免疫抗体库的制备
取6-8周龄BALB/c小鼠，免疫之前对小鼠进行尾静脉采血留本底血清。首次免疫取hCD47-mFc融合蛋白以费氏完全佐剂乳化，每只小鼠腹腔注射50μg融合蛋白。间隔两周加强免疫，取hCD47-mFc融合蛋白以费式不完全佐剂乳化，每只小鼠腹腔注射50μg融合蛋白，注射前断尾采血，共进行两次加强免疫。第四次免疫改用冲击 免疫，以不加佐剂的hCD47-mFc重组抗原作为免疫原，每只小鼠腹腔注射50μg融合蛋白，冲击免疫后3天处死小鼠，收集脾细胞。
使用小鼠淋巴细胞分离液(达科为，#DKW33-R0100)对小鼠脾脏淋巴细胞进行分离，利用细胞总RNA提取试剂盒(天根，#DP430)，将分离的淋巴细胞进行总RNA的提取。以提取的总RNA为模板，利用第一链cDNA合成试剂盒(Thermo scientific，#K1621)分别合成重链可变区和轻链可变区，反转录引物采取基因特异性引物，引物配对区分别位于抗体重链恒定区和抗体轻链恒定区，具体序列分别为PmCGR:TGCATTTGAACTCCTTGCC和PmCKR:CCATCAATCTTCCACTTGAC。将合成完成的cDNA立即存放于-70℃保存备用。然后，以反转录得到的cDNA为模板，参考文献(Journal of Immunological Methods，201(1997)，35–55)合成引物，并利用PCR分别扩增鼠抗体VH和VK，然后利用重叠延伸PCR技术，构建单链抗体(scFv)。最后将制备的小鼠单链抗体基因克隆至载体pADSCFV-S(实验技术流程可参见中国专利申请201510097117.0号)，构建scFv库。此抗体库的库容达到1.2×10E7，正确率约为85％。
3.小鼠免疫库的筛选和鉴定
按照与中国专利申请201510097117.0号相似的实验流程，以重组huCD47-D1-his为抗原，利用固相筛选策略筛选展示小鼠单链抗体库的噬菌体，共进行三轮筛选，最终获得多株序列不同、但能特异性结合hCD47的鼠抗体，包括克隆S1F9，S2H2，S4D12，S2C3，S2D10。噬菌体-ELISA表明hCD172a-D1M1可以与其中四株鼠单抗(S1F9/S2C3/S2H2/S4D12)竞争结合CD47，而鼠单抗S2D10与CD47的结合不被hCD172a-D1M1阻断。
实施例3：鼠单抗的鉴定
选取鼠单链抗体S4D12(SEQ ID NO：19，VH和VK序列分别为SEQ ID NO：20和21)、S2H2(SEQ ID NO：22，VH和VK序列分别为SEQ ID NO：23和24)和S2C3(SEQ ID NO：25，VH和VK序列分别为SEQ ID NO：26和27)，利用分子生物学方法制备重组人IgG4-κ形式鼠-人嵌合抗体，同时参照美国专利申请US9017675B2的公开内容，制备人源化抗CD47单抗Hu5F9-G4(VH序列和VK序列分别为SEQ ID NO：17和SEQ ID NO：18)。
1.重组抗CD47单抗阻断hCD47结合hCD172a
包被抗原hCD47-mFc，用固定浓度的hCD172a-D1M1-His对S2C3/S2H2/S4D12嵌合抗体进行梯度稀释，用HRP-小鼠-抗his IgG(康为世纪，CW0285M)检测CD172a-D1M1-His和hCD47-mFc的结合。ELISA分析结果(图1)显示，单抗S2C3，S2H2和S4D12阻断CD47和CD172a-D1M1相互作用的能力差异不大，IC 50约在2-5nM的范围(表1)。
表1：三种不同抗CD47单抗抑制CD47与CD172a-D1M1结合的IC 50
S2C3-IgG4 S2H2-IgG4 S4D12-IgG4 Hu5F9-IgG4
IC 50(nM) 5.193 2.077 3.889 9.605
2.重组抗CD47单抗的亲和力分析
利用Biacore X100测定IgG4嵌合抗体的亲和力。氨基偶联试剂盒，人抗体捕获试剂盒，CM5芯片和pH7.4的10×HBS-EP等相关试剂和耗材均购自GE healthcare。根据试剂盒中的说明书，将抗人Fc段的抗体偶联至CM5芯片表面，稀释抗体蛋白至合适浓度，保证300RU左右的抗体被抗人Fc的抗体捕获。将huCD47-D1-his设置一系列的浓度梯度(500nM，167nM，56nM，18.5nM，6.2nM)流经固定相表面， 25℃下测定各单克隆抗体的亲和力。结果如表2所示。
表2.不同抗CD47单抗亲和力常数
K on(1/Ms) K off(1/s) KD(M)
S2C3-IgG4 8.861E+5 2.221E-2 2.506E-8
S2H2-IgG4 7.169E+5 6.959E-3 9.707E-9
S4D12-IgG4 1.414E+6 1.423E-2 1.006E-8
Hu5F9-IgG4 1.317E+6 2.646E-2 2.01E-8
3.重组抗CD47单抗的红细胞凝集能力分析
红细胞表面高表达CD47，因此抗CD47单抗可以和红细胞表面的CD47特异性结合。由于每个抗体有两个抗原结合位点，因而抗CD47单抗可能导致红细胞凝集。导致红细胞凝集的单抗在体内会引起贫血、红细胞数量减少等副反应。抗CD47单抗是否导致红细胞凝集主要取决于单抗在CD47分子上的识别表位。本实施例利用经典的血凝实验对抗CD47单抗导致红细胞凝聚的能力进行了分析。将之前实施例选用的抗CD47抗体(40μg/ml)用PBS稀释进行一系列的倍比稀释，然后在微量血凝反应板中与制备的1％红细胞悬液混合，置于微量振荡器上振荡1min混匀。将微量血凝反应板于25℃室温静置1h后用照相记录红细胞凝集程度，并将微量血凝反应板倾斜45°角持续数分钟，通过红细胞流动速率快慢来进一步确定红细胞凝集程度。红细胞凝集程度可分为4个等级：4凝成均匀薄层，倾斜不流动；3凝成均匀薄层，倾斜有轻微流动；2凝成少量薄层，倾斜流动较快；1未有薄层形成，倾斜后与对照孔流动速度一样。结果显示(图2)，人-鼠嵌合单抗S2C3和S2H2不引起红细胞凝集，而人-鼠嵌合单抗S4D12和人源化抗体Hu5F9有强烈的红细胞凝集作用。
4.重组抗CD47单抗能够促进巨噬细胞吞噬肿瘤细胞
利用Ficoll密度梯度离心法从健康人全血中分离PBMC，然后通过CD14磁珠(抗人CD14磁性颗粒–DM，BD)从PBMC中分选得到CD14 +单核细胞。将CD14 +单核细胞以8×10 5/孔的密度接种于12孔板中，用10ng/ml MCSF诱导7天左右至分化成成熟的巨噬细胞。肿瘤细胞(Daudi淋巴瘤细胞系)预先用CFSE染色后计数重悬，以8×10 5的细胞量和终浓度10μg/ml的不同抗CD47单抗或对照抗体(IgG)共孵育15-30min，随后转移至含巨噬细胞的孔中。共同孵育3h后收集巨噬细胞，利用抗人CD14-APC对巨噬细胞进行染色，30min后PBS洗涤两次，利用流式细胞术(BD Accuri TMC6)检测APC(FL-4)和CFSE(FL-1)双通路，并以巨噬细胞本底的吞噬率作为起始，计算出各个样品的吞噬率。结果显示(图3和图4)，本申请的三种人-鼠嵌合抗CD47单抗和Hu5F9都能够促进巨噬细胞吞噬肿瘤细胞。
实施例4：鼠单抗的人源化及鉴定
1.鼠单抗的人源化改造
对鼠抗体S2C3和S4D12进行人源化研究以降低其免疫原性。将S2C3和S4D12的重链可变区(VH)和轻链可变区(VL)分别与IMGT数据库中的人抗体胚系基因序列相比较，选择合适的胚系基因序列以提供抗体的框架区1至3(FR1+FR2+FR3)，选择合适的J区基因序列以提供框架区4(FR4)。这个模板可以根据例如抗体的相对总长度、CDR的大小、位于抗体框架区(FR)和超变区(CDR)之间连接处的氨基酸残基、序列整体的同源性等等选出，所选的模板可以是多个序列的混合物或者可以是共有模板，目的是尽可能维持亲本互补决定区(CDR)的合适构象。最终确定人源化S4D12(hS4D12)轻、重链可变区氨基酸序列(SEQ ID NO：29，SEQ ID NO：28)和人源化S2C3(hS2C3)的轻、重链可变区氨基酸序列(SEQ ID NO：31，SEQ ID NO： 30)。根据人源化抗体的氨基酸序列设计并合成可变区基因，并制备IgG4版本的人源化抗体hS2C3和hS4D12。
2.人源化抗CD47单抗的活性分析
参照实施例3中的方法分析了人源化抗CD47单抗抑制hCD47与hCD172a结合的能力，结果显示(图5)人源化抗体hS2C3和hS4D12能够有效抑制hCD47与hCD172a的结合，IC 50低于5nM(表3)。利用BIAcore对人源化抗体的亲和力进行了分析，结果显示，人源化抗体基本保持了亲本鼠抗体的亲和力(表4)。
表3：人源化抗CD47单抗抑制CD47与CD172a结合的IC 50
Hu5F9-IgG4 hS4D12-IgG4 hS2C3-IgG4
IC 50 4.161 2.68 3.364
表4：人源化抗D47单抗的亲和力常数
K on K off KD
S4D12 1.285E+6 1.1E-2 8.56E-9
hS4D12 1.225E+6 1.176E-2 9.6E-9
S2C3 1.111E+6 2.055E-2 1.85E-8
hS2C3 1.088E+6 1.099E-2 1.01E-8
Hu5F9-IgG4 2.095E+6 3.142E-2 1.499E-8
实施例5：人源化抗CD47单抗hS2C3的亲和力成熟
为进一步提高人源化抗体的亲和力，对hS2C3进行了体外亲和力成熟。体外亲和力成熟的主要策略是依次在重链CDR和轻链CDR中引入突变，构建轻链或者重链突变库，然后基于双载体的噬菌体展示系统(具体操作可参照申请人之前提交的中国专利申请第201510097117.0号中的实施例5)分别筛选重链和轻链的高亲和力突变体。
1.hS2C3重链突变库的构建及筛选
利用常规分子生物学方法构建hS2C3重链可变区的突变库，库容为6.5×10E5，突变库的正确率约为95％，其中在HCDR1和HCDR2中引入突变所需引物见表5。以重组huCD47-his为抗原，利用固相筛选策略对hS2C3-HCDR12突变库进行了3轮筛选，最终得到亲和力进一步提高的两个突变体H10C7(SEQ ID NO：32)和H11E5(SEQ ID NO：33)。
表5：hS2C3重链可变区突变库扩增所需引物
Figure PCTCN2018100262-appb-000002
2.hS2C3轻链突变库的构建及筛选
类似地，利用表6中的兼并引物，通过重叠延伸PCR技术在hS2C3轻链可变区的LCDR1和LCDR3中引入突变并构建突变库。构建库容量约3.1×10E6，正确率约为90％。以重组huCD47-his为抗原，利用固相筛选策略对hS2C3-LCDR13突变库进行了3轮筛选，最终得到亲和力进一步提高的两个突变体L25B8(SEQ ID NO：34)和L26A6(SEQ ID NO：35)
表6：hS2C3轻链可变区突变库构建所需引物
Figure PCTCN2018100262-appb-000003
3.hS2C3突变体的鉴定
利用筛选到的两种高亲和力轻链突变体和两种高亲和力重链突变体进行组合，制备了四种人源化抗体hS2C3的突变体：利用筛选到的两种高亲和力轻链突变体和两种高亲和力重链突变体进行组合，制备了四种人源化抗体hS2C3的突变体：H10C7+L25B8、H10C7+L26A6、H11E5+L25B8和H11E5+L26A6，并参照实施例3中的方法对四种hS2C3的突变体进行了亲和力和功能分析。
利用BIAcore对四种突变体的亲和力分析显示(表7)：四种突变体较人源化抗体hS2C3的亲和力有5-10倍的提高，而且四种突变体都能有效抑制hCD47结合hCD72a-D1M1(图6)，而且IC 50都低于5nM(表8)。
表7：hS2C3突变体的亲和力常数
抗体 K on K off KD
H10C7+L25B8 1.031E+6 1.151E-3 1.115E-9
H10C7+L26A6 8.33E+5 1.932E-3 2.32E-9
H11E5+L25B8 1.189E+6 9.597E-4 8.072E-10
H11E5+L26A6 9.297E+5 1.7E-3 1.829E-9
hS2C3 1.078E+6 1.029E-2 0.955E-8
表8：hS2C3突变体抑制hCD47和hCD172a-D1M1结合的IC 50
H10C7+L26A6 H11E5+L26A6 H10C7+L25B8 H11E5+L25B8 hS2C3
IC 50 4.25 3.521 4.511 4.129 4.13
红细胞凝集实验显示(图7)四种亲和力提高的人源化抗CD47单抗在0-133nM浓度范围内都不会导致红细胞凝集，而且四种人源化突变体都能够促进巨噬细胞对CD47 +肿瘤细胞(Daudi)的吞噬(图8)。
4.人源化抗CD47单抗体内抑制肿瘤生长
利用人伯基特淋巴瘤raji细胞，以1×10 4细胞/小鼠的量，皮下接种于雄性NSG小鼠，在肿瘤体积长至100cm 3时随机分为5组，每组8只小鼠：(1)介质对照组；(2)20mg/kg H10C7+L26A6组；(3)10mg/kgH10C7+L26A6组；(4)3mg/kg H10C7+L26A6组；(5)10mg/kg hu5F9-IgG4组。每周腹腔给药两次，一共3周。给药过程中，一周3次测量肿瘤大小及动物体重以评价抗CD47抗体的抑瘤作用。实验结果如图9所示， 图9中横坐标为raji肿瘤的荷瘤小鼠接受药物处理的时间，纵坐标为肿瘤体积。结果表明：与介质对照组相比，人源化抗CD47单抗H10C7+L26A6能够抑制肿瘤的生长，其抑制能力与对照抗体hu5F9-IgG4相当。
5.人源化抗CD47单抗在人多发性骨髓瘤RPMI8226细胞皮下异种移植NOD/SCID小鼠动物模型中的抑制肿瘤生长作用
在细胞接种前一天，将小鼠进行60Co照射处理(150rad)。雄性NOD/SCID小鼠于右侧背部皮下接种1×10 7RPMI8226细胞，细胞重悬在PBS与基质胶(matrigel)的1:1混悬液中(0.1ml/只)。待肿瘤平均体积达到80-100mm 3时，称量所有小鼠的体重，测量肿瘤体积后随机分组给药。肿瘤细胞接种当天定义为第0天。共分为5组，每组8只小鼠：(1)介质对照组(i.p.BIW*4，p.o.QD*4)；(2)10mg/kg达雷木单抗(daratumumab)(i.p.BIW*4)组；(3)10mg/kg H10C7+L26A6(i.p.BIW*4)组；(4)50mg/kg来那度胺(lenolidomide)(p.o.QD*4)组；⑤10mg/kg达雷木单抗(i.p.BIW*4)+50mg/kg来那度胺(p.o.QD*4)组。给药过程中，一周3次测量肿瘤大小及动物体重。达雷木单抗为已上市的针对多发性骨髓瘤的单抗药物，来那度胺为临床多发性骨髓瘤常规用药。实验结果如图10所示，图10中横坐标为皮下异种移植RMPI 8226肿瘤的NOD-SCID荷瘤小鼠接受药物处理的时间，纵坐标为肿瘤体积，和介质对照组相比，人源化抗CD47单抗H10C7+L26A6能够显著抑制肿瘤的生长，其抑瘤能力要显著高于达雷木单抗、来那度胺和达雷木单抗+来那度胺的联合用药。
6.人源化抗CD47单抗H10C7+L26A6的CD47结合表位分析
为了研究人源化单抗H10C7+L26A6与人CD47的结合表位，利用HEK293细胞制备重组的H10C7+L26A6 Fab片段以及重组的人CD47胞外区D1(hCD47，SEQ ID NO：1)形成Fab-hCD47复合物，在2.0M硫酸铵+5％异丙醇条件下形成复合物晶体，对复合物晶体进行X射线衍射实验，收集到分辨率
Figure PCTCN2018100262-appb-000004
的数据(表9)，并通过分子置换法解析了CD47抗原抗体复合物的晶体结构。最终得到的结构模型包括CD47胞外区1-116 aa(SEQ ID NO：1)、抗体重链1-219 aa、轻链1-219 aa。抗体H链和L链最后一个氨基酸残基在电子密度中均不可见，提示其在晶体中没有固定结构。在复合物晶体结构中，CD47以单体形式存在，而且在N16、N32、N55和N93位点存在可见的糖基化。
CD47与Fab的相互作用面积约为
Figure PCTCN2018100262-appb-000005
其中CD47与重链的相互作用面积约为
Figure PCTCN2018100262-appb-000006
与轻链的相互作用面积约为
Figure PCTCN2018100262-appb-000007
相互作用主要涉及CD47的N端、BC环、β-链F、FG环和β-链G，而抗体重链可变区的HCDR1、HCDR2、HCDR3，轻链可变区的LCDR1、LCDR3参与了对CD47的识别(图11)。相互作用方式包括氢键、盐键、范德华力以及疏水相互作用(表10)。其中，人源化抗CD47单抗H10C7+L26A6识别人CD47的一个不连续(空间)表位，主要包括氨基酸Q1，E29，A30，Q31，N32，E35，E97，E100，L101，T102，R103，E104，G105和E106。
据文献报道，抗人CD47单抗hu5F9单抗的抗原结合表位包括氨基酸Q1，E29，K39，K41，E97，T99，R103和E104；而抗人CD47单抗2A1的抗原表位包括氨基酸Y37，K39，K41，K43，G44，R45，D46，D51，H90，N93，E97，T99，E104和E106。抗人CD47单抗H10C7+L26A6、hu5F9和2A1的表位都有部分重叠，但又都不完全相同。
为了进一步对抗人CD47单抗H10C7+L26A6的表位进行验证，挑选表位中的部分氨基酸位点，利用常规分子生物学方法制备了三种hCD47的突变体，包括Q1A，E35A和L101A，并利用BIAcore测定了人源化抗CD47单抗H10C7+L26A6与这些hCD47突变体的亲和力，并与天然的hCD47进行了比较(表11)。结果显示：L101A 点突变导致完全不结合，Q1A的突变导致亲和力下降约100倍，而E35A突变导致亲和力提高约4倍。表明这些构成单抗H10C7+L26A6表位的关键氨基酸的突变将直接导致抗体-抗原亲和力的变化(降低或者提高)。同时分析抗人CD47单抗hu5F9与这些突变体的亲和力发现(表11)，只有hCD47中的Q1A突变影响hu5F9与人CD47的亲和力，而hCD47中的E35A和L101A突变对hu5F9的亲和力没有明显的改变。此结果与hu5F9的抗原表位中包括Q1，而不包括E35和L101一致。
表9.复合物晶体的X射线衍射收据收集及统计
Figure PCTCN2018100262-appb-000008
*括号中的数值是对于最高分辨率壳
表10：参与Fab-hCD47结合的氨基酸残基
Figure PCTCN2018100262-appb-000009
表11：基于BIAcore分析不同hCD47突变体与人源化抗CD47单抗的亲和力
Figure PCTCN2018100262-appb-000010
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Claims (11)

  1. 一种特异性结合人CD47的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区和含LCDR1、LCDR2和LCDR3序列的轻链可变区，其中
    所述HCDR1序列为NYWMH，所述HCDR2序列为VIAPSDNYTNYNQKFQG，所述HCDR3序列为GGKYSMDY，所述LCDR1序列为RSSQSIVHSNGNTYLE，所述LCDR2序列为KVSNRFS，所述LCDR3序列为FQGSHVPFT；或者
    所述HCDR1序列为SYWMH，所述HCDR2序列为TIDRSDSYISYNQKFKG，所述HCDR3序列为GGPYGSKMMDN，所述LCDR1序列为HASQNINVWLS，所述LCDR2序列为KASNLHT，所述LCDR3序列为QQGQSYPLT；或者
    所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPSSGNTKYAQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSLLYSSNKKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为QQFYAYPIS；或者
    所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPSSGNTKYAQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSVLYSSNQKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为GQYYAYPIT；或者
    所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPGSGNTRYSQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSLLYSSNKKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为QQFYAYPIS；或者
    所述HCDR1序列为DYYMH，所述HCDR2序列为WIYPGSGNTRYSQKFKD，所述HCDR3序列为REEDYFDY，所述LCDR1序列为KSSQSVLYSSNQKNYLT，所述LCDR2序列为WASTRES，所述LCDR3序列为GQYYAYPIT；
    其中HCDR和LCDR序列根据Kabat定义。
  2. 根据权利要求1所述的抗体，所述抗体的重链可变区的氨基酸序列如SEQ ID NO：23、28、30、32或者33所示。
  3. 根据权利要求1所述的抗体，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：24、29、31、34或者35所示。
  4. 根据权利要求1所述的抗体，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：23所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：24所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：29所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：31所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：32所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：34所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：32所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：35所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：33所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：34所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO：33所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO：35所示。
  5. 一种特异性结合人CD47的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO：23、28、30、32或者33中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO：24、29、31、34或者35中任何一项具有至少90％的一致性。
  6. 一种特异性结合人CD47的抗体，其中所述抗体与SEQ ID NO：1所示人CD47区段的结合表位为不连续表位，包括氨基酸Q1，E29，A30，Q31，N32，E35，E97，E100，L101，T102，R103，E104，G105和E106，上述残基编号参照SEQ ID NO：1。
  7. 根据权利要求1-6中任一项所述的抗体，其中
    所述抗体能以低于5nM的KD值的亲和力结合重组人CD47胞外区D1(SEQ ID NO：1)；和/或
    所述抗体能以低于5nM的IC 50值抑制CD172a高亲和力突变体(SEQ ID NO：4)与人CD47-Fc的结合；和/或
    所述抗体在0-133nM浓度下施用不导致红细胞聚集；和/或
    所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)，优选地，所述抗体为全人源抗体；和/或
    所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区和/或包含选自κ亚型或者λ亚型的轻链恒定区；和/或
    所述抗体为单克隆抗体；和/或
    所述抗体为非激活型抗体；和/或
    所述抗体能结合并中和人CD47，进而阻断CD47-SIRPα信号通路；和/或
    所述抗体能促进巨噬细胞对肿瘤细胞的吞噬作用；和/或
    所述抗体能抑制肿瘤细胞的体内生长；和/或
    所述抗体不增加巨噬细胞对正常血细胞的吞噬。
  8. 核酸分子，其编码权利要求1-7中任一项所述的抗体或其抗原结合部分。
  9. 药物组合物，其包含权利要求1-7中任一项所述的抗体和药学可接受的赋形剂、稀释剂或载体。
  10. 权利要求1-7中任一项所述的抗体在制备用于预防或治疗CD47介导的疾病的药物中的用途，例如所述疾病为肿瘤，例如恶性肿瘤。
  11. 预防或治疗CD47介导的疾病的方法，包括向有需要的个体给予权利要求1-7中任一项所述的抗体或权利要求9所述的药物组合物，例如所述疾病为肿瘤，例如恶性肿瘤。
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