WO2023036281A1 - 抗cd47抗体及其用途 - Google Patents

抗cd47抗体及其用途 Download PDF

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WO2023036281A1
WO2023036281A1 PCT/CN2022/118015 CN2022118015W WO2023036281A1 WO 2023036281 A1 WO2023036281 A1 WO 2023036281A1 CN 2022118015 W CN2022118015 W CN 2022118015W WO 2023036281 A1 WO2023036281 A1 WO 2023036281A1
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seq
antibody
antigen
amino acid
variable region
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PCT/CN2022/118015
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French (fr)
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郎国竣
胡宇豪
谭永聪
闫鑫甜
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三优生物医药(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine.
  • the present invention relates to anti-CD47 antibodies and uses thereof.
  • CD47 is a transmembrane glycoprotein widely expressed on the cell surface. It belongs to the immunoglobulin superfamily and can interact with signal regulatory protein ⁇ (SIRP ⁇ ), thrombospondin (TSP1) and integrin (Integrin) to mediate cell A series of reactions such as apoptosis, proliferation, and immunity.
  • SIRP ⁇ signal regulatory protein ⁇
  • TSP1 thrombospondin
  • Integrin integrin
  • CD47 functions by binding to SIRP ⁇ expressed by myeloid cells such as macrophages, neutrophils, and dendritic cells to transmit an inhibitory "don't eat me" signal, thereby inhibiting myeloid cells (especially Phagocytosis of target cells expressing CD47 by macrophages.
  • CD47 the role of ubiquitous expression of CD47 under physiological conditions is to protect healthy cells from elimination by the innate immune system, whereas tumor cells efficiently escape immune surveillance by overexpressing CD47.
  • infiltrating macrophages in tumor tissue also known as tumor-associated macrophages (TAM)
  • TAM tumor-associated macrophages
  • TAM tumor-associated macrophages
  • CD47-SIRP ⁇ pathway by using an anti-CD47 antibody can effectively mediate phagocytosis of tumor cells, thereby inhibiting the growth of various hematological and solid tumors in vivo.
  • CD47 is highly expressed not only on tumor cells, but also on normal cells, such as red blood cells. Therapies targeting CD47 may cause undesired side effects.
  • anti-CD47 antibodies disclosed in the prior art bind to red blood cells, which not only triggers a severe anemia reaction, but also requires a dosage of up to 30 mg/kg in administration. These characteristics give the clinical application of anti-CD47 antibodies posed great challenges. There is an urgent need in this field to develop new therapies and drugs targeting CD47, so as to expand the application of CD47 as a therapeutic target and macrophages as immune regulation and effector cells.
  • the invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 4 or a variant thereof, the HCDR2 sequence of SEQ ID NO: 5 or a variant thereof and the HCDR3 sequence of SEQ ID NO: 6 or a variant thereof;
  • the light chain variable region comprises the LCDR1 sequence of SEQ ID NO: 7 or a variant thereof, the LCDR2 sequence of SEQ ID NO: 8 or a variant thereof and the LCDR3 sequence of SEQ ID NO: 9 or a variant thereof;
  • said variants each independently comprise a substitution, addition or deletion of 1 amino acid relative to the sequence from which it is derived.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4 or SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:5 or SEQ ID NO:19 and the SEQ ID NO: 6 or the HCDR3 sequence shown in SEQ ID NO:23;
  • the light chain variable region comprises an LCDR1 sequence shown in SEQ ID NO:7 or SEQ ID NO:24, an LCDR2 sequence shown in SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:20 and SEQ ID NO: 9 or the LCDR3 sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4, the HCDR2 sequence shown in SEQ ID NO:5 and the HCDR3 sequence shown in SEQ ID NO:6, and the light chain can be
  • the variable region comprises the LCDR1 sequence shown in SEQ ID NO:7, the LCDR2 sequence shown in SEQ ID NO:8 and the LCDR3 sequence shown in SEQ ID NO:9; or
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4, the HCDR2 sequence shown in SEQ ID NO:5 and the HCDR3 sequence shown in SEQ ID NO:6, and the light chain variable region comprises SEQ ID NO LCDR1 sequence shown in :7, LCDR2 sequence shown in SEQ ID NO:16 and LCDR3 sequence shown in SEQ ID NO:9; or
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 18, the HCDR2 sequence shown in SEQ ID NO: 19 and the HCDR3 sequence shown in SEQ ID NO: 6, and the light chain variable region comprises SEQ ID NO LCDR1 sequence shown in :7, LCDR2 sequence shown in SEQ ID NO:20 and LCDR3 sequence shown in SEQ ID NO:9; or
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4, the HCDR2 sequence shown in SEQ ID NO:5 and the HCDR3 sequence shown in SEQ ID NO:23, and the light chain variable region comprises SEQ ID NO LCDR1 sequence shown in :24, LCDR2 sequence shown in SEQ ID NO:8 and LCDR3 sequence shown in SEQ ID NO:25.
  • said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10
  • said light chain variable region comprises the amino acid sequence of SEQ ID NO: 11;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17; or
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:21, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:22; or
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:27.
  • an anti-CD47 antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region and/or a light chain constant region.
  • said heavy chain constant region is a human IgG4 heavy chain constant region and/or said light chain constant region is a human kappa light chain constant region.
  • the present invention also provides a multispecific antibody comprising a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds a second antigen, wherein the first antigen-binding portion comprises the anti-CD47 antibody of the present invention or its Antigen-binding fragments.
  • the present invention also provides a chimeric antigen receptor comprising the anti-CD47 antibody of the present invention or an antigen-binding fragment thereof.
  • the present invention also provides an immune effector cell expressing the chimeric antigen receptor of the present invention on its surface.
  • the invention provides a polynucleotide encoding an anti-CD47 antibody of the invention or an antigen-binding fragment thereof.
  • the invention also relates to expression vectors comprising the polynucleotides of the invention.
  • the invention also relates to host cells comprising the polynucleotides or expression vectors of the invention.
  • the invention also provides an antibody conjugate comprising an anti-CD47 antibody or antigen-binding fragment thereof or a multispecific antibody of the invention conjugated to at least one therapeutic agent.
  • the present invention also provides a pharmaceutical composition, which comprises the anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, immune effector cell or antibody conjugate of the present invention, and a pharmaceutically acceptable carrier agent.
  • the present invention also relates to the use of an anti-CD47 antibody or an antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate or a pharmaceutical composition in the preparation of a medicament for treating cancer.
  • Figures 1A-1B show the binding activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 to CD47 on CCRF-CEM cells.
  • Figures 2A-2B show the binding activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 to CD47 on erythrocytes.
  • Figure 3 shows the activity of antibodies A7H3L3, A7-44, A7-28 and A7-47 in blocking the binding of human CD47 to SIRP ⁇ on CCRF-CEM cells.
  • Figures 4A-4B show the hemagglutination of red blood cells by antibodies A7, A7H3L3, A7-44, A7-28 and A7-47.
  • Figure 5 shows that antibodies A7-28 and A7-47 promote macrophage phagocytosis of CCRF-CEM cells.
  • the expressions “comprising”, “comprising”, “containing” and “having” are open-ended, meaning that listed elements, steps or components are included but other unlisted elements, steps or components are not excluded.
  • the expression “consisting of” does not include any element, step or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the elements, steps or components specified, plus optional elements, steps or components that do not materially affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions “consisting essentially of” and “consisting of” are encompassed within the meaning of the expression “comprising”.
  • any value or range of values such as a concentration or range of concentrations, should in any event be understood to be modified by the term "about". Accordingly, numerical values generally include ⁇ 10% of the stated value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges and all individual values within that range, including integers and fractions within that range, unless the context clearly dictates otherwise.
  • antibody refers to an immunoglobulin or fragment thereof that specifically binds an antigenic epitope through at least one antigen binding site. As used herein, the definition of antibody encompasses antigen-binding fragments.
  • the term “antibody” includes multispecific antibodies (eg, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments. Antibodies can be synthetic (eg, produced by chemical or biological conjugation), enzymatically processed, or recombinantly produced.
  • Antibodies provided herein include any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • An antibody may be "monovalent”, “bivalent”, “trivalent” or “tetravalent” or more valent, meaning that it comprises 1, 2, 3, 4 or more antigen binding sites.
  • antigen-binding fragment refers to a portion of a full-length antibody that is less than full-length, but comprises at least part of the variable region of a full-length antibody (e.g., comprising one or more CDRs and/or one or more antigen-binding site), and thus retain at least part of the full-length antibody's ability to specifically bind the antigen.
  • Antigen-binding fragments may, for example, include antibody derivatives produced by enzymatic treatment of full-length antibodies, synthetically produced derivatives, recombinantly produced derivatives.
  • antigen binding fragments include, but are not limited to, sdAb (e.g., the variable domain of a heavy chain antibody), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab') 2 , diabody, Fd, and Fd ' fragments as well as other fragments (such as those containing modifications).
  • sdAb e.g., the variable domain of a heavy chain antibody
  • a “full-length antibody” generally comprises four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain comprises a “light chain variable region (VL)” and a “light chain constant region (CL)” from the N-terminus (amino acid terminus) to the C-terminus (carboxyl terminus). Each heavy chain comprises a "heavy chain variable region (VH)” and a “heavy chain constant region (CH)” from the N-terminus to the C-terminus.
  • the heavy chain constant region of a full-length antibody may comprise CH1-hinge-CH2-CH3 from N-terminus to C-terminus.
  • the heavy chain constant region may comprise, from N-terminus to C-terminus, CH1-hinge-CH2-CH3-CH4.
  • the light and heavy chain variable regions can each comprise three highly variable "complementarity determining regions (CDRs)" and four relatively conserved “framework regions (FRs)” Sequential linkage of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs of the light chain variable region CDRL or LCDR
  • LCDR1, LCDR2 and LCDR3 the CDRs of the heavy chain variable region
  • the amino acid sequences of CDRs are all shown in accordance with the AbM definition rules (the claims of the present invention are also sequences shown in accordance with the AbM definition rules).
  • the CDR of an antibody can be defined by various methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, for example, Chothia, C. et al., Nature, 342, 877 -883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see e.g. Kabat, E.A. et al.
  • CDR complementarity determining region
  • variable region e.g., variable region
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • a different protocol e.g. Different assignment system rules or combinations
  • framework region and “framework region” are used interchangeably.
  • framework region refers to those amino acid residues in an antibody variable region other than the CDR sequences as defined above.
  • the "Fv" fragment composed of one VH and one VL through non-covalent interaction is the smallest antigen-binding fragment containing an antigen-binding site.
  • single variable domains single variable domain antibodies
  • a "single-chain Fv (scFv)" can be obtained by linking VH and VL via a peptide linker.
  • dsFv disulfide bond-stabilized Fv
  • scdsFv or dsscFv can be obtained by introducing a disulfide bond into Fv or scFv, respectively.
  • Fab comprises a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also referred to as Fd).
  • VL-CL antibody light chain
  • VH-CH1 heavy chain constant region
  • Fab single-chain
  • F(ab') 2 essentially comprises two Fab fragments linked by a disulfide bond in the hinge region.
  • Fab' is half of F(ab') 2 , which can be obtained by reducing the disulfide bonds in the hinge region of F(ab') 2 .
  • “Hinge region” refers to the part of an antibody that connects the Fab and Fc fragments of an immunoglobulin.
  • the hinge region may be the entire hinge region or a portion thereof.
  • the Fc region usually includes part of the hinge region connected to CH2 and CH3.
  • the hinge region when used in reference to a chimeric antigen receptor, the hinge region may also refer to any functional equivalent, such as the part of a T cell receptor that connects the constant region and the transmembrane domain.
  • Those skilled in the art can judge the positions of VH, VL, CL, CH1, CH2, CH3 and the hinge region in the antibody according to known algorithms and software, and the description of applicable algorithms and software can be found in, for example, William R. Strohl, Lila M. Strohl, (2012), Antibody structure–function relationships, In Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pp.37-56.
  • diabody refers to an antibody comprising two scFvs in which VH and VL in each scFv are linked by a short peptide linker (approximately 5-10 amino acid residues) such that The pairing of the VH and VL chains (ie, the VH of the first scFv and the VL of the second scFv, the VL of the first scFv and the VH of the second scFv) forms the antigen binding site.
  • Diabodies can be bispecific antibodies.
  • chimeric antibody refers to an antibody in which a portion (e.g., CDRs, FRs, variable regions, constant regions, or combinations thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining Portions are identical or homologous to corresponding sequences in antibodies derived from another species.
  • chimeric antibodies comprise variable regions derived from a non-human species (eg, mouse) and constant regions derived from a different species (eg, human).
  • Chimeric antibodies can also refer to multispecific antibodies that have specificities for at least two different antigens. Chimeric antibodies can be produced by antibody engineering. Methods of antibody engineering are well known to those skilled in the art.
  • chimeric antibodies can be produced by recombinant DNA techniques (see, for example, Sambrook, J., et al. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y).
  • humanized antibody refers to an antibody in which a non-human antibody has been modified to increase sequence homology to a human antibody.
  • Humanized antibodies generally retain the antigen-binding ability of the non-human antibody from which they were derived and are less immunogenic in humans.
  • Humanized antibodies can be obtained by antibody engineering of any non-human species antibody or an antibody comprising sequences derived from a non-human species (eg, chimeric antibodies).
  • Non-human species can include, for example, mice, rats, rabbits, alpacas, sharks, or non-human primates. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art.
  • the CDR sequences of a non-human antibody are grafted into the framework regions of a human antibody.
  • the key amino acid residues of the framework sequence of the non-human antibody can be retained in the framework region of the human antibody, that is, " Back mutation” (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21):6851-6855; Neuberger et al. (1984) Nature 312:604-608).
  • human antibody refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human.
  • the definition of a human antibody encompasses whole or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
  • an "affinity matured" antibody comprises one or more modifications (e.g., substitutions of amino acid residues) in one or more CDRs such that the affinity matured antibody is less sensitive to Antigen affinity is improved.
  • Methods for affinity maturation of antibodies are known in the art, see, e.g., Marks et al., Bio/Technology 10:779-783 (1992); Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809 -3813 (1994); Scier et al., Gene 169:147-155 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).
  • percentage (%) sequence identity and “sequence identity” of amino acid sequences have definitions recognized in the art, which refer to two sequences determined by sequence alignment (for example, by manual inspection or known algorithms). The percentage of identity between polypeptide sequences. It can be determined using methods known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.
  • amino acid sequence "derived from” or “derived from” a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence.
  • amino acid sequence of a heavy chain constant region derived from a human immunoglobulin may be at least 80%, at least 85%, at least 90%, at least 91% identical to the wild-type sequence of the human immunoglobulin heavy chain constant region from which it is derived , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • Non-critical regions in the polypeptide can be modified, such as one or more amino acid substitutions, additions and/or deletions, without changing the function of the polypeptide .
  • amino acids in non-essential regions of polypeptides may be substituted with suitable conservative amino acids and generally do not alter their biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.co., p.224).
  • suitable conservative substitutions are well known to those skilled in the art.
  • amino acid substitutions are non-conservative substitutions.
  • amino acid mutations or modifications can be made to antibodies or antibody fragments to change their properties, such as changing the type of antibody glycosylation modification, changing the ability to form interchain disulfide bonds, or antibody conjugates
  • the preparation provides reactive groups.
  • Antibodies or antigen-binding fragments thereof comprising such amino acid mutations or modifications are also encompassed within the scope of antibodies or antigen-binding fragments thereof of the present invention.
  • Affinity or "binding affinity” is a measure of the strength of non-covalent binding between an antibody and an antigen.
  • the magnitude of "affinity” can usually be reported as the equilibrium dissociation constant KD or EC50 .
  • Affinity can be determined using conventional techniques known in the art, such as biomembrane interferometry (for example, the Octet Fortebio detection system can be used), radioimmunoassay, surface plasmon resonance, enzyme-linked immunoassay (ELISA) or flow cytometry (FACS )wait.
  • the anti-CD47 antibody or antigen-binding fragment, multispecific antibody, or polynucleotide encoding the same of the invention may be isolated.
  • isolated means that a substance (such as a polynucleotide or polypeptide) is separated from its source or environment in which it exists, ie does not substantially contain any other components.
  • polynucleotide and “nucleic acid” are used interchangeably to denote an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid ( DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a "vector” is a medium for introducing exogenous polynucleotides into host cells, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed.
  • Vectors usually remain episomal, but can be designed to allow integration of a gene, or part thereof, into the chromosome of the genome.
  • the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like.
  • Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, poxviral vectors, baculoviral vectors, and the like.
  • RNA and/or polypeptides As used herein, the term “expression” refers to the production of RNA and/or polypeptides.
  • expression vector refers to a vector capable of expressing a polynucleotide of interest, including DNA and RNA.
  • a polynucleotide sequence including DNA and RNA
  • a polypeptide of interest can be operably linked to regulatory sequences (such as promoters and ribosome binding sites) that can affect the expression of the polynucleotide sequence .
  • regulatory sequences may include promoter and terminator sequences, and optionally may include origins of replication, selectable markers, enhancers, polyadenylation signals, and the like.
  • Expression vectors can be plasmids, phage vectors, recombinant viruses or other vectors which, when introduced into an appropriate host cell, result in the expression of a polynucleotide of interest. Suitable expression vectors are known to those skilled in the art. Those skilled in the art can prepare the expression vector as a vector that can replicate in the host cell, remain free in the host cell, or integrate into the genome of the host cell according to the needs.
  • a "host cell” is a cell used to receive, maintain, replicate or amplify a vector. Host cells can also be used to express polynucleotides or polypeptides encoded by vectors. Host cells can be eukaryotic or prokaryotic. Prokaryotic cells such as Escherichia coli (E.coli) or Bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells (such as S2 Drosophila cells or Sf9), and animal cells (such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or HEK293 cells).
  • E.coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or
  • treatment refers to the improvement of the disease/symptom, such as reducing or disappearing the disease/symptom, preventing or slowing down the occurrence, progression and/or deterioration of the disease/symptom.
  • treatment includes prophylaxis, treatment and/or cure.
  • Effective amount refers to the amount required to prevent, cure, ameliorate, arrest or partially arrest a disease or symptom.
  • an "effective amount" of an anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention is preferred relative to an untreated subject.
  • tumor cell growth or tumor growth is inhibited by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably At least about 70%, more preferably at least about 80%.
  • the efficacy of inhibiting tumor growth can be evaluated using conventional tumor animal models in the art, such as spontaneous tumor, induced tumor, and transplanted tumor animal models. Alternatively, the ability to inhibit cell growth can also be examined using in vitro assays known in the art.
  • An effective amount of an antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell, or pharmaceutical composition of the invention is capable of reducing tumor size, or otherwise alleviating symptoms in a subject (e.g., prophylaxis and / or treatment of metastasis or recurrence). Those skilled in the art can determine the effective amount according to factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition or route of administration. An effective amount can be administered in one or more administrations.
  • the term "pharmaceutically acceptable carrier” refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, reagents for maintaining osmotic pressure , Agents that delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA (Sucrose-Phosphate-Glutamate-Albumin), sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as milk white protein hydrolyzate), etc.
  • SPGA Sudrose-Phosphate-Glutamate-Albumin
  • sugars such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dried whey, albumin, or casein
  • their degradation products such as milk white protein hydrolyzate
  • the invention provides anti-CD47 antibodies or antigen-binding fragments thereof that specifically recognize and bind CD47.
  • CD47 refers to leukocyte surface antigen CD47 (Leukocyte surface antigen CD47), also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-associated antigen or protein MER6.
  • the anti-CD47 antibody or antigen-binding fragment thereof specifically binds human CD47.
  • human CD47 refers to human-derived CD47. An exemplary amino acid sequence of human CD47 is shown in GenBank Accession No. NP_001768.1.
  • the anti-CD47 antibodies or antigen-binding fragments thereof of the invention are chimeric antibodies, humanized antibodies, human antibodies, scFv, Fab, Fab', F(ab') 2 , Fv fragments, disulfide bonds Stable Fv (dsFv) or diabodies.
  • an anti-CD47 antibody of the invention or an antigen-binding fragment thereof is provided.
  • an anti-CD47 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 4 (GFNIKDIYIY), the HCDR2 sequence shown in SEQ ID NO: 5 (KIDPANGNTK) and the HCDR2 sequence shown in SEQ ID NO: 6 (GYGSGFAY) HCDR3 sequence.
  • the light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO: 7 (RASQDISNHLN), the LCDR2 sequence shown in SEQ ID NO: 8 (YTSRIHS) and the LCDR2 sequence shown in SEQ ID NO: 9 (QQGYTLPFT) LCDR3 sequence.
  • the anti-CD47 antibody or antigen-binding fragment thereof of the present invention is engineered through affinity maturation.
  • Such anti-CD47 antibodies or antigen-binding fragments thereof may comprise HCDR1 (SEQ ID NO:4), HCDR2 (SEQ ID NO:5), HCDR3 (SEQ ID NO:6), LCDR1 (SEQ ID NO:7) as described above
  • HCDR1 SEQ ID NO:4
  • HCDR2 SEQ ID NO:5
  • HCDR3 SEQ ID NO:6
  • LCDR1 SEQ ID NO:7
  • each of said variants independently comprises a substitution, addition or deletion of 1 amino acid relative to the sequence from which it was derived.
  • said variants each independently comprise a substitution of 1 amino acid relative to the sequence from which they are derived.
  • an anti-CD47 antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises the HCDR1 sequence of SEQ ID NO: 4 or a variant thereof, the HCDR2 sequence of SEQ ID NO: 5 or a variant thereof and the HCDR3 sequence of SEQ ID NO: 6 or a variant thereof;
  • the light chain variable region comprises the LCDR1 sequence of SEQ ID NO: 7 or a variant thereof, the LCDR2 sequence of SEQ ID NO: 8 or a variant thereof and the LCDR3 sequence of SEQ ID NO: 9 or a variant thereof;
  • said variants each independently comprise a substitution, addition or deletion of 1 amino acid relative to the sequence from which it is derived.
  • the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO: 4, wherein the amino acid at position 7 is substituted.
  • amino acid at position 7 in SEQ ID NO:4 is substituted with V (SEQ ID NO:18; GFNIKDVYIY).
  • the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 5, wherein the 10th amino acid is substituted.
  • the amino acid at position 10 of SEQ ID NO:5 is substituted with H (SEQ ID NO:19; KIDPANGNTH).
  • the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO: 6, wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO:6 is substituted with V (SEQ ID NO:23; GYGSVFAY).
  • the LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO: 7, wherein the 10th amino acid is substituted.
  • amino acid 10 in SEQ ID NO:7 is substituted with I (SEQ ID NO:24; RASQDISNHIN).
  • the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 8, wherein the amino acid at position 7 is substituted.
  • the amino acid at position 7 of SEQ ID NO:8 is substituted with L (SEQ ID NO:16; YTSRIHL).
  • the amino acid at position 7 in SEQ ID NO:8 is substituted with K (SEQ ID NO:20; YTSRIHK).
  • the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO: 9, wherein the 5th amino acid is substituted.
  • the amino acid at position 5 in SEQ ID NO:9 is substituted with H (SEQ ID NO:25; QQGYHLPFT).
  • an anti-CD47 antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4 or SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:5 or SEQ ID NO:19 and the SEQ ID NO:6 or SEQ ID NO: HCDR3 sequence shown in 23;
  • the light chain variable region comprises an LCDR1 sequence shown in SEQ ID NO:7 or SEQ ID NO:24, an LCDR2 sequence shown in SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:20 and SEQ ID NO: 9 or the LCDR3 sequence shown in SEQ ID NO:25.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4, the HCDR2 sequence shown in SEQ ID NO:5 and the HCDR3 sequence shown in SEQ ID NO:6; and the light chain can be The variable region comprises the LCDR1 sequence shown in SEQ ID NO:7, the LCDR2 sequence shown in SEQ ID NO:8 and the LCDR3 sequence shown in SEQ ID NO:9.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4, the HCDR2 sequence shown in SEQ ID NO:5 and the HCDR3 sequence shown in SEQ ID NO:6; and the light chain can be The variable region comprises the LCDR1 sequence shown in SEQ ID NO:7, the LCDR2 sequence shown in SEQ ID NO:16 and the LCDR3 sequence shown in SEQ ID NO:9.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO: 18, the HCDR2 sequence shown in SEQ ID NO: 19 and the HCDR3 sequence shown in SEQ ID NO: 6; and the light chain can be The variable region comprises the LCDR1 sequence shown in SEQ ID NO:7, the LCDR2 sequence shown in SEQ ID NO:20 and the LCDR3 sequence shown in SEQ ID NO:9.
  • the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID NO:4, the HCDR2 sequence shown in SEQ ID NO:5 and the HCDR3 sequence shown in SEQ ID NO:23; and the light chain can be The variable region comprises the LCDR1 sequence shown in SEQ ID NO:24, the LCDR2 sequence shown in SEQ ID NO:8 and the LCDR3 sequence shown in SEQ ID NO:25.
  • a VH as described above further comprises a heavy chain framework region.
  • the VL as described above further comprises a light chain framework region.
  • the VH described above further comprises a heavy chain framework region
  • the VL described above further comprises a light chain framework region.
  • the heavy chain framework region and/or the light chain framework region may each be independently derived from a heavy chain framework region and a light chain framework region of an immunoglobulin of any species.
  • the VH comprises a heavy chain framework region derived from a murine immunoglobulin
  • the VL comprises a light chain framework region derived from a murine immunoglobulin
  • said VH comprises a heavy chain framework region derived from a human immunoglobulin
  • said VL comprises a light chain framework region derived from a human immunoglobulin.
  • the anti-CD47 antibodies or antigen-binding fragments thereof of the invention are humanized.
  • the heavy chain framework region and/or the light chain framework region of the humanized anti-CD47 antibody or antigen-binding fragment thereof may comprise one or more non-human (e.g., murine) amino acid residues, such as the heavy chain framework region and/or
  • the light chain framework region may comprise one or more amino acid backmutations comprising the corresponding murine amino acid residues.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 21 or SEQ ID NO: 26.
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 27.
  • the heavy chain variable region comprises at least 80%, at least 85%, Amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the heavy chain variable region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and/or deletions of the amino acid sequence.
  • said amino acid substitutions, additions and/or deletions do not occur in CDR regions.
  • the light chain variable region comprises at least 80%, at least Amino acid sequences having 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the light chain variable region comprises one or more Amino acid sequences with substitutions, additions and/or deletions of (eg 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids. Preferably, said amino acid substitutions, additions and/or deletions do not occur in CDR regions.
  • the heavy chain variable region comprises: 1) the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 21 or SEQ ID NO: 26; 2) the amino acid sequence of SEQ ID NO: 26; :10, the amino acid sequence of SEQ ID NO:14, SEQ ID NO:21 or SEQ ID NO:26 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity amino acid sequence; or 3) with SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:21 or An amino acid sequence having one or more (eg 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and/or deletions compared to the amino acid sequence of SEQ ID NO: 26 ;and
  • the light chain variable region comprises: 1) the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 27; 2) the amino acid sequence with SEQ ID NO: :11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:27 have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity amino acid sequence; or 3) with SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO :17, SEQ ID NO:22 or SEQ ID NO:27 has one or more (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and and/or missing amino acid sequences.
  • said amino acid substitutions, additions and/or deletions do not occur in CDR regions.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:21 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:22. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:26 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:27.
  • an anti-CD47 antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy and light chain constant regions can each be independently derived from those of an immunoglobulin of any species.
  • the heavy chain constant region can be derived from the immune response of any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • the light chain constant region may be derived from a lambda (Lambda) light chain or a kappa (Kappa) light chain constant region.
  • An appropriate immunoglobulin constant region e.g. CH1 and light chain constant region, hinge-CH2-CH3, CH1-hinge-CH2-CH3 and light chain constant region or Fc region
  • type e.g. IgG, e.g. IgG1, IgG2, IgG3 and IgG4
  • IgG immunoglobulin constant region
  • the heavy chain constant region comprises at least an Fc region
  • the heavy chain constant region of IgG may include: 1) all or part of the hinge region-CH2-CH3; or 2) CH1-hinge region-CH2-CH3 .
  • the heavy chain constant region is that of a human IgG (eg, IgGl, IgG2a, IgG2b, IgG3, or IgG4) heavy chain constant region (eg, Fc region or CH1-hinge-CH2-CH3).
  • the heavy chain constant region is that of a human IgGl.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 1.
  • the heavy chain constant region is that of a human IgG4 heavy chain.
  • the heavy chain constant region comprises: 1) the amino acid sequence of SEQ ID NO: 12; or 2) at least 80%, at least 85%, at least 90%, at least 91%, at least Amino acid sequences having 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the light chain constant region is a human kappa light chain constant region.
  • the light chain constant region comprises: 1) the amino acid sequence of SEQ ID NO: 13; or 2) at least 80%, at least 85%, at least 90%, at least 91%, at least Amino acid sequences having 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • Antibodies or antigen-binding fragments thereof can be prepared and produced using methods known in the art. Such methods can include, for example, preparation and isolation of antibodies or antigen-binding fragments from phage display libraries, yeast display libraries, immortalized B cells (eg, mouse B cell hybridoma cells or EBV immortalized B cells). It is also possible to immunize animals, for example, immunize animals (such as mice) with antigens or DNA encoding antigens, and then isolate antibody-expressing B cells from the immunized animals. Preferably, antibody-expressing B cells are immortalized, eg, produced as hybridomas or EBV immortalized B cells. Polynucleotides encoding antibodies or antigen-binding fragments thereof can also be isolated from immunized animals or prepared by chemical synthesis, and then the polynucleotides can be used to construct expression vectors.
  • methods known in the art can include, for example, preparation and isolation of antibodies or antigen-binding fragments from phage display libraries, yeast display libraries,
  • the invention provides a multispecific antibody comprising a first antigen binding portion that binds CD47 and a second antigen binding portion that binds a second antigen, wherein the first antigen binding portion comprises an anti- CD47 antibody or antigen-binding fragment thereof.
  • multispecific antibody refers to an antibody capable of specifically binding two or more (eg, 2, 3, 4, 5 or 6) different antigenic epitopes.
  • a multispecific antibody may, for example, be a bispecific, trispecific or tetraspecific antibody capable of specifically binding 2, 3 or 4 epitopes, respectively.
  • antigenic epitope or "antigenic determinant” means a region of an antigen that specifically binds to the antigen binding site of an antibody.
  • Antigenic epitopes usually consist of chemically active surface groups of antigens (such as amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.
  • the second antigen may be other antigen than CD47.
  • the second antigen can also be CD47, which binds to a different epitope on CD47 than the anti-CD47 antibody or antigen-binding fragment thereof of the present invention.
  • Conventional methods in the art can be used to determine whether the antigenic epitopes bound by the two antibodies are the same, for example, by ELISA, flow cytometry or surface plasmon resonance to determine the competitive binding of the two antibodies to the same antigenic epitope.
  • Multispecific antibodies may be multivalent (eg, 2, 3, 4) antibodies, ie, they have multiple antigen-binding sites.
  • Multispecific antibodies can be, for example, chimeric antibodies, humanized antibodies, scFabs, F(ab') 2 or diabodies.
  • Multispecific antibodies can be produced and isolated using various techniques known in the art.
  • a polynucleotide encoding a multispecific antibody can be obtained by recombinant DNA technology, optionally, the polynucleotide is cloned into an expression vector, and then the host cell is transformed with the polynucleotide or the expression vector, and under suitable conditions The transformed host cells are cultured to allow expression of the polynucleotide or expression vector, and finally the multispecific antibody is isolated and purified from the host cells or culture medium. It is also possible to obtain the various parts of the multispecific antibody separately, for example obtain the first antigen-binding part and the second antigen-binding part as described herein separately, and then couple the parts by enzymatic or chemical coupling techniques, optionally via linkers. Combined to obtain multispecific antibodies that specifically bind CD47 and other antigens.
  • first antigen-binding portion and second antigen-binding portion mean an amino acid sequence comprising an antigen-binding site capable of binding to an antigenic epitope, and their definitions fall within the meaning of an antibody or antigen-binding fragment .
  • the first antigen binding moiety can be any form of antibody or antigen binding fragment including but not limited to Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab' and F(ab') 2 .
  • the first antigen-binding portion comprises an anti-CD47 antibody or antigen-binding fragment thereof of the invention.
  • the first antigen-binding portion comprises VH and VL comprising HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of the anti-CD47 antibody or antigen-binding fragment thereof of the invention as described above, respectively.
  • the first antigen-binding portion comprises VH and VL, which respectively comprise the VH and VL of an anti-CD47 antibody or antigen-binding fragment thereof of the invention as described above.
  • the second antigen binding moiety can be an antibody or antigen binding fragment that binds any antigenic epitope of interest.
  • the second antigen is an antigen other than CD47.
  • Antigens to which the second antigen-binding moiety can specifically bind may include: tumor antigens (such as tumor-associated antigens and tumor-specific antigens), immune regulatory receptors, and immune checkpoint molecules.
  • tumor antigens such as tumor-associated antigens and tumor-specific antigens
  • immune regulatory receptors such as tumor-associated antigens and tumor-specific antigens
  • immune checkpoint molecules such as tumor-associated antigens and tumor-specific antigens
  • tumor-associated antigen refers to an antigen that is highly expressed in tumor cells and also present but at a lower level in healthy cells.
  • tumor-specific antigen refers to an antigen that is specifically expressed in tumor cells and hardly expressed in healthy cells.
  • Non-limiting examples of tumor antigens may include CD19, CD20, EGFR, GPC3, HER-2, and FOLR1.
  • Non-limiting examples of immune checkpoint molecules can include CTLA-4, LAG-3, PD-1, PD-L1, and TIM-3.
  • Immunomodulatory receptors can include, for example, immunostimulatory receptors (eg, CD27, CD137, CD40, GITR, and OX40) and immunosuppressive receptors (eg, BTLA, CTLA4, LAG-3, and PD-1).
  • the second antigen binding moiety can be an agonist antibody to an immunostimulatory receptor or an antagonist antibody to an immunosuppressive receptor.
  • the second antigen binding moiety binds a tumor antigen, an immune modulatory receptor, and an immune checkpoint molecule. In one embodiment, the second antigen binding moiety specifically binds to SIRP ⁇ , PD-1, PD-L1, LAG3, TIM-3, CTLA-4, VISTA, GPC3, EGFR, HER-2, CD19, CD20, CD33, CD40, CD73, OX40, CD3, DLL-3, TIP-1, folate receptor alpha (FOLR1), and/or other tumor antigens.
  • the second antigen binding moiety can be any form of antibody or antigen binding fragment including, but not limited to, a single variable domain of an immunoglobulin (e.g. comprising a VHH from alpaca or an IgNAR variable domain from a shark), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab' and F(ab') 2 .
  • the second antigen binding portion comprises a single variable domain of an immunoglobulin.
  • the first antigen binding moiety and the second antigen binding moiety may optionally be linked by a linker. In some embodiments, the first antigen binding portion and the second antigen binding portion are not linked by a linker. In other embodiments, the first antigen-binding moiety and the second antigen-binding moiety are linked by a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen binding moiety and the second antigen binding moiety are linked by a peptide linker.
  • Exemplary peptide linkers can include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, or (G 4 S) n , where n is 1 An integer of -20.
  • the second antigen is an antigen other than CD47
  • the binding affinity of the first antigen binding portion to CD47 is weaker than the binding affinity of the second antigen binding portion to the other antigen.
  • the multispecific antibodies of the invention may preferentially target other antigens, such that the multispecific antibodies specifically recognize target cells (e.g., cancer cells) expressing other antigens, which may or may not express CD47 .
  • target cells e.g., cancer cells
  • the multispecific antibody of the present invention has a stronger binding ability to CD47 than that which only expresses CD47, and blocks CD47 and SIRP ⁇ The ability to combine is also stronger.
  • the present invention also provides a chimeric antigen receptor (CAR) comprising an anti-CD47 antibody of the present invention or an antigen-binding fragment thereof.
  • CAR is a recombinant receptor that simultaneously provides antigen binding and activates T cell functions.
  • CAR structure and engineering are described, e.g., in Dotti G. et al., (2014) Immunol Rev. 257(1):107-126, which is incorporated herein by reference.
  • the CAR of the present invention comprises from the N-terminus to the C-terminus:
  • the extracellular region comprises the anti-CD47 antibody of the present invention or an antigen-binding fragment thereof, so that the CAR of the present invention binds to CD47-expressing cells (such as cancer cells).
  • the extracellular region in the CAR is in the form of scFv.
  • the transmembrane region connects the extracellular region and the intracellular signaling region and anchors the CAR to the cell membrane.
  • TCR T cell receptor
  • the intracellular signaling region further comprises at least one co-stimulatory domain.
  • the co-stimulatory domain can promote the activation of CAR-expressing immune effector cells after binding to the antigen targeted by the extracellular domain.
  • the CAR of the invention further comprises a spacer connecting the extracellular region and the transmembrane region.
  • the spacer is derived from a heavy chain constant region of an immunoglobulin.
  • the present invention also relates to a polynucleotide encoding the CAR of the present invention and an expression vector comprising the polynucleotide.
  • the present invention also provides an immune effector cell, which expresses the CAR of the present invention on the cell surface.
  • the immune effector cells are selected from T lymphocytes (eg cytotoxic T cells (CTL)), natural killer cells (NK) and natural killer T cells (NKT).
  • TTL cytotoxic T cells
  • NK natural killer cells
  • NKT natural killer T cells
  • Immune effector cells can target CD47-positive diseased cells (eg, CD47-positive cancer cells) and be activated to initiate effector functions, eg, cause the death of CD47-positive cancer cells.
  • the invention provides a polynucleotide comprising a polynucleotide sequence encoding an anti-CD47 antibody or antigen-binding fragment of the invention or a multispecific antibody of the invention.
  • polynucleotides of the present invention can be obtained using methods known in the art.
  • polynucleotides of the invention can be isolated from phage display libraries, yeast display libraries, immunized animals, immortalized cells (eg, mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemically synthesized.
  • immortalized cells eg, mouse B cell hybridoma cells, EBV-mediated immortalized B cells
  • the polynucleotides of the invention may be codon optimized for the host cell for expression.
  • the present invention also provides an expression vector comprising a polynucleotide of the present invention.
  • Expression vectors may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes.
  • a polynucleotide of the invention may be present in one or more expression vectors.
  • polynucleotides of the invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, recombinant DNA techniques (eg, polymerase chain reaction (PCR) techniques), and the like.
  • PCR polymerase chain reaction
  • the present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the polynucleotide or expression vector of the present invention can be introduced into suitable host cells by various methods known in the art. Such methods include, but are not limited to, lipofection, electroporation, viral transduction, and calcium phosphate transfection, among others.
  • host cells are used to express an anti-CD47 antibody or antigen-binding fragment thereof of the invention or a multispecific antibody of the invention.
  • host cells include, but are not limited to, prokaryotic cells (eg bacteria such as E. coli) and eukaryotic cells (eg yeast, insect cells, mammalian cells).
  • Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expressing antibodies.
  • the present invention also provides a method for producing the anti-CD47 antibody or antigen-binding fragment thereof of the present invention or the multispecific antibody of the present invention, which comprises the following steps:
  • the invention also provides an antibody conjugate comprising an anti-CD47 antibody or antigen-binding fragment thereof of the invention or a multispecific antibody of the invention conjugated to at least one therapeutic agent.
  • Antibody-drug conjugate is a typical antibody conjugate, wherein the therapeutic agent can be, for example, a cytotoxic agent.
  • conjugation refers to the linking of two or more moieties to each other by covalent or non-covalent interactions.
  • the conjugation is covalent conjugation.
  • the therapeutic agent may be selected from cytotoxic agents, therapeutic antibodies (such as antibodies or antigen-binding fragments thereof that specifically bind to another antigen), radioisotopes, oligonucleotides and their analogs (such as interfering RNA), biologically active peptides , protein toxins (eg diphtheria toxin, ricin) and enzymes (eg urease).
  • therapeutic antibodies such as antibodies or antigen-binding fragments thereof that specifically bind to another antigen
  • radioisotopes such as antibodies or antigen-binding fragments thereof that specifically bind to another antigen
  • oligonucleotides and their analogs such as interfering RNA
  • biologically active peptides eg diphtheria toxin, ricin
  • enzymes eg urease
  • Cytotoxic agents refer to substances that inhibit or reduce the activity, function and/or kill cells of cells.
  • examples of cytotoxic agents may include, but are not limited to: maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatins, Nostoc Cryptophycin, vinca alkaloids (eg, vinblastine, vincristine), colchicines, dolastatins, taxanes, paclitaxel, docetaxel, cabazitaxel, enediynes Antibiotics, cytochalasins, camptothecins, anthracyclines (such as daunorubicin, dihydroxyanthracindione, doxorubicin), cytotoxic antibiotics (such as silk mitomycin, actinomycin, duocarmycin (eg CC-1065), auromycin, duomycin, calicheamicin , endomycin, phen
  • the therapeutic agent is selected from cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs.
  • the therapeutic agent is a cytotoxic agent.
  • the therapeutic agent is a radioisotope.
  • a therapeutic agent can be conjugated to an antibody or antigen-binding fragment of the invention or a multispecific antibody of the invention via a linker using any technique known in the art.
  • the linker may comprise reactive groups for covalent conjugation, such as amines, hydroxylamines, maleimides, carboxyls, phenyls, thiols, sulfhydryls or hydroxyls.
  • the present invention also provides a pharmaceutical composition, which comprises the anti-CD47 antibody or antigen-binding fragment, antibody conjugate, CAR-expressing immune effector cell or multispecific antibody of the present invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients Excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, Propyl gallate, ⁇ -tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (for example, water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffer, histidine buffer, and a
  • suitable carriers may be selected from buffers (e.g. citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine buffer, salt buffer), isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (such as polysorbate 80, polysorbate 20, poloxamer) or its combination.
  • buffers e.g. citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine buffer, salt buffer
  • isotonic agents such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose
  • nonionic surfactants such as polysorbate 80, polysorbate 20, poloxamer
  • compositions provided herein can be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion).
  • preferred dosage forms may generally be, for example, injections and lyophilized powders.
  • compositions provided herein can be administered to a subject by any method known in the art, eg, by systemic or topical administration.
  • Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal , rectal, sublingual or topical).
  • the exact dosage administered will depend on various factors, such as the metabolic kinetic properties of the pharmaceutical composition, duration of treatment, rate of excretion of the particular compound, purpose of treatment, route of administration, and subject conditions such as the patient's age, health, weight, sex, diet, medical history, and other factors known in the medical field.
  • the method of administration may be, for example, injection or infusion.
  • an anti-CD47 antibody or antigen-binding fragment thereof, antibody conjugate, or multispecific antibody of the invention may be administered in a dosage range of about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of the subject. person weight.
  • the dose administered can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or within the range of 1-20 mg/kg.
  • Exemplary treatment regimens entail dosing weekly, every two weeks, every three weeks, every four weeks, monthly, every 3 months, every 3-6 months, or an initial dosing interval The interval between dosing is slightly shorter and the later period is longer.
  • the way of administration can be intravenous drip.
  • the present invention relates to the use of the anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease in a subject. use in .
  • the present invention also relates to an anti-CD47 antibody or an antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate, an immune effector cell or a pharmaceutical composition of the present invention for use in the treatment of a disease.
  • the present invention also provides a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD47 antibody or an antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate compounds, immune effector cells or pharmaceutical compositions.
  • the disease is associated with abnormal expression of CD47.
  • abnormal expression means that the protein expression level in the sample is too high or too low compared with a sample in a normal state (or a standard sample, such as a sample of a subject who does not suffer from a disease related to abnormal expression of CD47).
  • the disease is characterized by high expression of CD47.
  • CD47 is highly expressed in tissues (such as gastric cancer tissue and gastric paracancerous tissues) of subjects suffering from or suspected of having a disease (such as gastric cancer), while CD47 is low expressed in corresponding tissues of subjects not suffering from the disease.
  • the disease as described above is cancer.
  • cancer includes, but is not limited to, hematological and solid tumors.
  • blood cancer means cancer of the blood, including leukemia, lymphoma, and myeloma, among others.
  • leukemia may include acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and myeloproliferative disorders such as myeloid dysplastic syndrome).
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • myeloproliferative disorders such as myeloid dysplastic syndrome
  • Lymphoma can include, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma, among others.
  • Myeloma can include, for example, multiple myeloma (MM) and giant cell myeloma, among others.
  • Solid tumors include, for example, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, kidney cancer, leiomyoma, glioma, and Glioblastoma, etc.
  • the cancer can also be metastatic cancer.
  • “Metastasis" is the spread of cancer cells from their original site to other parts of the body.
  • the present invention also relates to anti-CD47 antibodies or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates, immune effector cells, or pharmaceutical compositions of the present invention for use in the treatment of cancer.
  • the cancer is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome , Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, follicular lymphoma, multiple myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, Prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, kidney cancer, leiomyoma, glioma, and gli
  • the anti-CD47 antibody or antigen-binding fragment thereof of the present invention can be used to treat CD47-positive blood tumors and solid tumors.
  • the cancer is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome , myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, follicular lymphoma, multiple myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer , lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, kidney cancer, leiomyoma, glioma and glioblastoma, etc.
  • ALL
  • the multispecific antibody of the invention can be used for the treatment of, for example, a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL ), chronic myelogenous leukemia (CML), myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, follicular lymphoma, multiple myeloma (MM), giant Myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, leiomyoma, colloid tumors and glioblastomas.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CLL
  • An anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell, or pharmaceutical composition of the invention can be administered in combination with at least one or more therapeutic agents described herein.
  • the manner of combined administration is not limited.
  • the above therapeutic agents may be administered all at once or separately.
  • the anti-CD47 antibodies or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates or pharmaceutical compositions of the present invention may be used in combination with other therapeutic methods, including but not limited to: surgery, chemotherapy, radiation therapy, Targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition and palliative care.
  • the anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, or pharmaceutical composition of the present invention is further used in combination with one or more therapeutic agents selected from: chemotherapeutic agents , radioisotopes, immune checkpoint inhibitors, and tumor antigen-targeted drugs.
  • chemotherapeutic agents may include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, microtubule inhibitors.
  • Tumor antigen-targeted drugs include, but are not limited to, drugs targeting tumor-associated antigens and tumor-specific antigens.
  • tumor-associated antigen refers to an antigen that is highly expressed in tumor cells and also present but at a lower level in healthy cells.
  • tumor-specific antigen refers to an antigen that is specifically expressed in tumor cells and hardly expressed in healthy cells.
  • therapeutic agents may include, for example: angiogenesis inhibitors, deacetylase (HDAC) inhibitors, Hedgehog signaling pathway blockers, mTOR inhibitors, p53/mdm2 inhibitors, PARP inhibitors, proteasome Inhibitors (eg, bortezomib, carfilzomib, ixazomib, marizomib, oprozomib) and tyrosine kinase inhibitors (eg, BTK inhibitors).
  • HDAC deacetylase
  • an anti-CD47 antibody or antigen-binding fragment thereof or a multispecific antibody of the invention is used in combination with one or more therapeutic agents selected from the group consisting of: anti-SIRP ⁇ antibody, anti-CD20 antibody, anti-TIM-3 antibody , anti-LAG-3 antibody, anti-EGFR antibody, anti-HER-2 antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD47 antibody, anti-CD73 antibody, anti-DLL-3 antibody, anti-TIP-1 antibody, anti-FOLR1 antibody, anti- CTLA-4 antibody, anti-PD-L1 antibody and anti-PD-1 antibody.
  • one or more therapeutic agents selected from the group consisting of: anti-SIRP ⁇ antibody, anti-CD20 antibody, anti-TIM-3 antibody , anti-LAG-3 antibody, anti-EGFR antibody, anti-HER-2 antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD47 antibody, anti-CD73 antibody, anti-DLL-3 antibody, anti-TIP-1 antibody, anti-FOLR1 antibody, anti- CTLA-4 antibody,
  • an anti-CD47 antibody or antigen-binding fragment thereof or multispecific antibody of the invention is used in combination with a chemotherapeutic agent.
  • the antibody conjugates of the invention are used in combination with immune checkpoint inhibitors.
  • antibodies or antigen-binding fragments thereof, antibody conjugates, or pharmaceutical compositions of the invention are used in combination with radioisotopes.
  • kits which comprises the anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention, and instructions for use.
  • Kits may also comprise suitable containers.
  • the kit further comprises a device for administration.
  • the kit will also include a label indicating the intended use and/or method of use of the kit contents.
  • label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the anti-CD47 antibody or antigen-binding fragment thereof of the present invention can achieve the following beneficial effects: 1) specifically binds to CD47-positive tumor cells but does not bind or substantially does not bind to erythrocytes; 2) does not cause or substantially does not cause erythrocyte agglutination; 3) has no effect on CD47
  • the combination with SIRP ⁇ has a blocking effect; and/or 4) promotes the phagocytosis of CD47-positive tumor cells by macrophages.
  • CD47-ECD-Fc Human IgG1Fc or 6 ⁇ His tag was fused to the carboxy-terminal (C-terminal) of the extracellular segment CD47-ECD of CD47 to construct CD47 antigenic protein CD47-ECD-Fc (SEQ ID NO: 3) or CD47-ECD-His ( SEQ ID NO: 2).
  • CD47-ECD-Fc was used for the immunization of C57BL/6 mice (Shanghai Lingchang Biotechnology Co., Ltd.). The way of immunization was subcutaneous multi-point immunization, each subcutaneous immunization was 50 ⁇ g, and immunization was once every two weeks. Second-rate.
  • the titer of immunization was determined by ELISA, wherein CD47-ECD-His was used for ELISA antigen coating. The measurement results showed that the immune titer reached 1:600000. At this time, 100 ⁇ g CD47-ECD-Fc was used for booster immunization, and the spleen was taken 2-3 days later.
  • the B lymphocytes in the spleen of the immunized mouse described in Example 1.1 were isolated, their RNA was extracted, and reverse-transcribed into cDNA by a reverse transcription kit (TaKaRa, 6210A).
  • the vector was transformed into competent Escherichia coli SS320 cells (Lucigen, MC1061F) by electroporation (Bio-Rad, MicroPulser).
  • the vector was spread on a 2-YT solid plate with ampicillin resistance.
  • the storage capacity of this immune library was determined to be 1 ⁇ 10 9 cfu by serial dilution plating.
  • the helper phage M13KO7 (NEB) was used for packaging, and the finally constructed immune library was displayed on the coat protein of M13 phage in the form of Fab.
  • CCRF-CEM cells a kind of human acute lymphoblastic leukemia T lymphocytes, which endogenously express CD47, purchased from the Chinese Academy of Sciences, Cat. No. TCHu147
  • SIRP ⁇ the binding of SIRP ⁇
  • the amino acid sequence of the heavy chain variable region of clone A7 is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 of clone A7 defined by AbM are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO :7, SEQ ID NO:8 and SEQ ID NO:9.
  • variable region of the humanized antibody A7H3L3 was obtained by cloning the humanized transformation of the variable region of A7.
  • variable regions of antibodies A7-44, A7-28 and A7-47 were obtained by affinity maturation of the variable region of humanized antibody A7H3L3.
  • the murine VH and VL sequences of clone A7 screened in Example 1 were compared with known human antibody databases to find the human germline (germline) gene VH with the highest homology to the murine VH and VL sequences respectively and VL sequences.
  • Select the framework regions of human germline gene VH and VL sequences using AbM to define CDR and framework regions), and then use computer prediction and simulation to retain the mouse-derived amino acids in the framework region of clone A7 that have an important impact on antigen binding through back mutations, Finally, the complementarity determining region (CDR) sequence of the germline gene was replaced with the corresponding CDR sequence in clone A7.
  • CDR complementarity determining region
  • variable region of the humanized antibody A7H3L3 was obtained after the variable region of clone A7 was humanized: the amino acid sequence of the heavy chain variable region (VH) of A7H3L3 is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region (VL) Shown in SEQ ID NO: 15 (Table 1).
  • Antibody A7H3L3 was engineered for affinity maturation to improve affinity and biological activity.
  • the affinity maturation transformation is based on the M13 phage display technology, using codon-based primers (during the primer synthesis process, a single codon consists of NNK) to introduce mutations in the CDR region, and construct 4 phage display libraries: library 1 and library 2 are single-point combinatorial mutations , library 1 is CDRL1+CDRL3+CDRH3 combined mutation, library 2 is CDRL2+CDRH1+CDRH2 combined mutation; library 3 and library 4 are double point saturation mutation, library 3 is double point saturation mutation of CDRL3, library 4 is double point saturation mutation of CDRH3 point saturation mutation.
  • Specific library construction method first, synthesize primers containing point mutations (Jinweizhi Biotechnology Co., Ltd.); secondly, use the antibody A7H3L3 to be transformed as a template for PCR amplification, amplify the CDR region containing the sequence of the designed mutation, and use bridging PCR method, the fragments containing different CDR mutations were combined, and then the point mutant antibody was connected to the phage display vector by double enzyme digestion (HindIII and NotI) and double sticky end ligation, and finally the antibody sequence with the mutation site was electroporated into Escherichia coli SS320.
  • the operating procedures of library capacity calculation, phage library preparation and library screening are detailed in Example 1, and three anti-CD47 affinity maturation antibodies A7-44, A7-28 and A7-47 were selected (Table 1).
  • amino acid sequence (SEQ ID NO:) of table 1 anti-CD47 antibody The amino acid sequence (SEQ ID NO:) of table 1 anti-CD47 antibody
  • Antibody name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VL A7H3L3 4 5 6 7 8 9 14 15 A7-44 4 5 6 7 16 9 14 17 A7-28 18 19 6 7 20 9 twenty one twenty two A7-47 4 5 twenty three twenty four 8 25 26 27
  • the coding sequence of the VH (SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:21 or SEQ ID NO:26) of the anti-human CD47 antibody described in Examples 1 and 2 was combined with the heavy chain of human IgG4
  • the coding sequence of the constant region (SEQ ID NO: 12) was ligated to construct the heavy chain coding sequence of the anti-CD47 antibody, and the VL (SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 17, SEQ
  • the coding sequence of ID NO:22 or SEQ ID NO:27) was connected with the coding sequence of human Kappa light chain constant region (SEQ ID NO:13) to construct the light chain coding sequence of anti-CD47 antibody.
  • the heavy chain and light chain coding sequences were respectively inserted into the expression plasmid pcDNA3.4 (Invitrogen) to construct the expression vectors for the heavy chain and light chain of the anti-CD47 antibody.
  • the heavy chain and light chain expression vectors were respectively transformed into Escherichia coli DH5 ⁇ , cultured overnight at 37°C, and plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody light chains and heavy chains.
  • Strand plasmids for eukaryotic expression were respectively inserted into the expression plasmid pcDNA3.4 (Invitrogen) to construct the expression vectors for the heavy chain and light chain of the anti-CD47 antibody.
  • the heavy chain and light chain expression vectors were respectively transformed into Escherichia coli DH5 ⁇ , cultured overnight at 37°C, and plasmids were extracted using an endotoxin-free plasmid extraction kit (
  • the cell culture supernatant expressing the target protein i.e. anti-CD47 antibody
  • the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10min at high speed, and the obtained supernatant was purified by MabSelect SuRe LX (GE, 17547403) was used for affinity purification, and then the target protein was eluted with 100mM sodium acetate (pH 3.0), then neutralized with 1M Tris-HCl, and finally the resulting protein was replaced by an ultrafiltration concentrator tube (Millipore, UFC901096) into PBS buffer.
  • the anti-CD47 antibodies A7H3L3, A7-44, A7-28 and A7-47 with high purity were obtained by SDS-PAGE identification and SEC purity test.
  • this example measures the anti-CD47 antibody of the present invention and the positive control antibody 1F8 (WO2018075857A1, the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:28, the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO:29, the preparation method is as shown in Example 3) the binding ability on CCRF-CEM cells.
  • the specific method is as follows: take 1 ⁇ 10 5 CCRF-CEM cells, centrifuge at low speed (300g) to remove the supernatant; pass the cells at the bottom of the centrifuge tube through the prepared FACS buffer (1 ⁇ PBS buffer containing 2% FBS by volume percentage) solution) to rinse once, then add the antibody to be tested in serial dilution to the washed cells, and incubate at 4°C for 1 h; then rinse with the above FACS buffer three times, add PE-labeled goat anti-human IgG Fc antibody ( Abcam, ab98596) 0.5 ⁇ g, incubated at 4°C for 1 h; then the cells were washed three times with FACS buffer and resuspended with 200 ⁇ L FACS buffer, and finally CCRF was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102) - Amount of anti-CD47 antibody bound on CEM cells (expressed as mean fluorescence intensity (MFI)).
  • MFI
  • the binding activities of antibodies A7H3L3, A7-44, A7-28 and A7-47 to erythrocytes were measured by flow cytometry (FACS).
  • FACS flow cytometry
  • positive control antibodies 1F8 and F4AM4 another anti-CD47 antibody self-developed by the applicant, the amino acid sequence of the heavy chain is SEQ ID NO: 30, and the amino acid sequence of the light chain is SEQ ID NO: 31
  • red blood cells were also determined. binding activity.
  • Human IgG4 was used as an isotype negative control.
  • the specific method is as follows: separate erythrocytes from 1 mL of anticoagulated human blood, absorb the supernatant after centrifugation, rinse twice with PBS, add 1 mL of PBS and resuspend. Dilute red blood cells to 1 ⁇ 10 7 /mL with PBS, pipette 50 ⁇ L of red blood cells per well and add them to a 96-well round-bottom cell culture plate, then add an equal volume of serially diluted antibody to be tested, mix well, and incubate at 4°C 1h. Then rinse with FACS buffer three times, add 0.5 ⁇ g of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596), and incubate at 4° C.
  • the blocking activity of the anti-CD47 antibody of the present invention to block the binding of CD47 on tumor cells to the receptor SIRP ⁇ was determined by flow cytometry (FACS). Human IgG4 was used as an isotype negative control.
  • the specific method is as follows: take 1 ⁇ 10 5 CCRF-CEM cells, centrifuge at a low speed (300 g) to remove the supernatant. The cells at the bottom of the centrifuge tube were rinsed once with the prepared FACS buffer (1 ⁇ PBS buffer containing 2% FBS), and then a gradient dilution of the antibody to be tested was added to the rinsed cells and incubated for 1 h. After washing the cells twice with FACS buffer, 100 ⁇ L of 1 ⁇ g/mL SIRP ⁇ -mFc (ACRO, SIA-H52A8) was added, and incubated at 4 °C for 1 h.
  • Antibody drugs reach the tumor lesion site through blood circulation to exert their drug effect, and red blood cells in the blood express CD47 in large quantities.
  • red blood cells in the blood express CD47 in large quantities.
  • the aggregation of red blood cells may be caused by cross-linking, so that the aggregated red blood cells are cleared by phagocytes, which is to a certain extent the cause of toxic reactions such as anemia.
  • This example compares the hemagglutination reaction of the anti-CD47 antibody of the present invention and positive control antibodies 1F8 and Hu5F9 (also known as magrolimab, see US patent application US20160304609A1) on red blood cells.
  • Human IgG4 was used as an isotype negative control.
  • the specific method is as follows: separate erythrocytes from 1 mL of anticoagulated human blood, centrifuge and absorb the supernatant, rinse twice with PBS, add 1 mL of PBS and resuspend. Dilute red blood cells 20 times with PBS, draw red blood cells at 50 ⁇ L per well and add them to a 96-well round-bottom cell culture plate, then add an equal volume of the antibody to be tested in a gradient dilution (0.05-100 ⁇ g/mL), mix well, and place at 37 °C incubator for 3h. Then take it out to observe the degree of hemagglutination reaction.
  • the area of red blood cell deposition represents the strength of hemagglutination. The larger the area, the stronger the hemagglutination, and vice versa.
  • Anti-CD47 antibody promotes the ability of macrophages to phagocytose tumor cells
  • Antibodies that block the binding of CD47 and SIRP ⁇ can effectively promote the phagocytosis of CD47-positive tumor cells by macrophages.
  • This example compares the ability of the anti-CD47 antibody of the present invention and the positive control antibody 1F8 to promote the phagocytosis of tumor cells by macrophages.
  • Human IgG4 was used as an isotype negative control.
  • the specific method is as follows: First, isolate human peripheral blood mononuclear cells PBMC, add 50ng/mL rhM-CSF (purchased from Peprotech, 300-25-10) to induce cell differentiation into macrophages, and culture them in a cell culture incubator at 37°C for about After 8 days, aspirate the supernatant and unattached cells, then add Accutase TM cell digestion solution (purchased from Sigma, A6964), incubate at 37°C for 45min, digest adherent cells and prepare with complete medium (RPMI1640+10%FBS) Make a uniform cell suspension, and distribute 5 ⁇ 10 4 cells per well into 96-well plates.
  • rhM-CSF purchased from Peprotech, 300-25-10
  • the phagocytosis rate is the ratio of CCRF-CEM cells phagocytized with CFSE.
  • Antibodies A7-28 and A7-47 dose-dependently promote the phagocytosis of tumor cells by macrophages, and their effects on promoting macrophages to phagocytize tumor cells are better than antibody 1F8.

Abstract

一种抗CD47抗体及其用途。

Description

抗CD47抗体及其用途
本申请要求2021年9月13日提交的，题为“抗CD47抗体及其用途”的第202111067062.0号中国专利申请的优先权，该申请的内容整体援引加入本文。
技术领域
本发明属于生物医药领域。具体地，本发明涉及抗CD47抗体及其用途。
背景技术
CD47是一种广泛表达于细胞表面的跨膜糖蛋白，属于免疫球蛋白超家族，可与信号调节蛋白α(SIRPα)、血小板反应蛋白(TSP1)以及整合素(Integrin)相互作用，介导细胞凋亡、增殖、免疫等一系列的反应。在先天免疫***中，CD47通过结合由髓样细胞诸如巨噬细胞、中性粒细胞和树突细胞表达的SIRPα而传递抑制性“别吃我”信号而发挥功能，从而抑制髓样细胞(尤其是巨噬细胞)对表达CD47的靶细胞的吞噬。因此，CD47在生理条件下广泛表达的作用是保护健康细胞免于被先天免疫***消除，然而，肿瘤细胞则通过过表达CD47而有效逃脱免疫监视。在另一方面，肿瘤组织中浸润的巨噬细胞，又称为肿瘤相关巨噬细胞(TAM)，往往失去吞噬和清除肿瘤细胞的免疫效应功能，甚至具有免疫抑制作用，促进肿瘤增殖和侵袭。已经证实，通过使用抗CD47抗体阻断CD47-SIRPα通路能够有效地介导对肿瘤细胞的吞噬作用，从而在体内抑制各种血液肿瘤和实体肿瘤的生长。然而，CD47不仅在肿瘤细胞上高表达，在正常细胞上，例如红细胞上也有大量CD47的表达，靶向CD47的疗法可能会引发不期望的副作用。
现有技术中公开的一些抗CD47抗体(参见例如US20160304609A1)结合红细胞，这不仅引发严重的贫血反应，而且在给药上需要高达30mg/kg的给药剂量，这些特性给抗CD47抗体的临床应用带来了极大的挑战。本领域亟需开发新的靶向CD47的疗法和药物，以拓展CD47作为治疗靶点以及巨噬细胞作为免疫调节及效应细胞的应用。
发明内容
在一方面，本发明提供一种抗CD47抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
所述重链可变区包含SEQ ID NO:4或其变体的HCDR1序列，SEQ ID NO:5或其变体的HCDR2序列和SEQ ID NO:6或其变体的HCDR3序列；
所述轻链可变区包含SEQ ID NO:7或其变体的LCDR1序列，SEQ ID NO:8或其变体的LCDR2序列和SEQ ID NO:9或其变体的LCDR3序列；
其中所述变体各自相对于其所源自的序列独立地包含1个氨基酸的取代、添加或缺失。
在一些实施方案中，所述重链可变区包含SEQ ID NO:4或SEQ ID NO:18所示HCDR1序列，SEQ ID NO:5或SEQ ID NO:19所示HCDR2序列和SEQ ID NO:6或SEQ ID NO:23所示HCDR3序列；
所述轻链可变区包含SEQ ID NO:7或SEQ ID NO:24所示LCDR1序列，SEQ ID NO:8、 SEQ ID NO:16或SEQ ID NO:20所示LCDR2序列和SEQ ID NO:9或SEQ ID NO:25所示LCDR3序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:6所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:8所示LCDR2序列和SEQ ID NO:9所示LCDR3序列；或者
所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:6所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:16所示LCDR2序列和SEQ ID NO:9所示LCDR3序列；或者
所述重链可变区包含SEQ ID NO:18所示HCDR1序列，SEQ ID NO:19所示HCDR2序列和SEQ ID NO:6所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:20所示LCDR2序列和SEQ ID NO:9所示LCDR3序列；或者
所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:23所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:24所示LCDR1序列，SEQ ID NO:8所示LCDR2序列和SEQ ID NO:25所示LCDR3序列。
在一实施方案中，所述重链可变区包含SEQ ID NO:10的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:11的氨基酸序列；或者
所述重链可变区包含SEQ ID NO:14的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:15的氨基酸序列；或者
所述重链可变区包含SEQ ID NO:14的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:17的氨基酸序列；或者
所述重链可变区包含SEQ ID NO:21的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:22的氨基酸序列；或者
所述重链可变区包含SEQ ID NO:26的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:27的氨基酸序列。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段进一步包含重链恒定区和/或轻链恒定区。在一实施方案中，所述重链恒定区为人IgG4的重链恒定区和/或所述轻链恒定区为人κ轻链恒定区。
本发明还提供一种多特异性抗体，其包含结合CD47的第一抗原结合部分以及结合第二抗原的第二抗原结合部分，其中所述第一抗原结合部分包含本发明的抗CD47抗体或其抗原结合片段。
在另一方面，本发明还提供一种嵌合抗原受体，其包含本发明的抗CD47抗体或其抗原结合片段。相应地，本发明还提供一种免疫效应细胞，其在表面表达本发明的嵌合抗原受体。
在又一方面，本发明提供一种多核苷酸，其编码本发明抗CD47抗体或其抗原结合片段。本发明还涉及包含本发明的多核苷酸的表达载体。本发明还涉及包含本发明的多核苷酸或表达载体的宿主细胞。
本发明还提供一种抗体缀合物，其包含与至少一种治疗剂缀合的本发明的抗CD47抗体或其抗原结合片段或者多特异性抗体。
在另一方面，本发明还提供一种药物组合物，其包含本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、免疫效应细胞或者抗体缀合物，以及药学上可接受的载剂。
本发明还涉及抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物或者药物组合物 在制备用于治疗癌症的药物中的用途。
附图说明
图1A-1B显示抗体A7H3L3、A7-44、A7-28和A7-47与CCRF-CEM细胞上CD47的结合活性。
图2A-2B显示抗体A7H3L3、A7-44、A7-28和A7-47与红细胞上CD47的结合活性。
图3显示抗体A7H3L3、A7-44、A7-28和A7-47阻断CCRF-CEM细胞上人CD47与SIRPα结合的活性。
图4A-4B显示抗体A7、A7H3L3、A7-44、A7-28和A7-47对红细胞的血凝反应。
图5显示抗体A7-28和A7-47促进巨噬细胞吞噬CCRF-CEM细胞的活性。
具体实施方式
定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，表述“包括”、“包含”、“含有”和“具有”是开放式的，表示包括所列举的元素、步骤或组分但不排除其他未列举的元素、步骤或组分。表述“由……组成”不包括未指定的任何元素、步骤或组分。表述“基本上由……组成”是指范围限于指定的元素、步骤或组分，加上不显著影响要求保护的主题的基本和新颖性质的任选存在的元素、步骤或组分。应当理解，表述“基本上由……组成”和“由……组成”涵盖在表述“包括”的含义之内。
如本文所用，多个述及的元素之间的连接术语“和/或”应理解为包括单独的和组合的选项。
除非另有说明，否则任何数值或数值范围，例如浓度或浓度范围，在任何情况下均应理解为由术语“约”修饰。因此，数值通常包括所述值的±10％。例如，1mg/mL的浓度包括0.9mg/mL至1.1mg/mL。同样的，1％至10％(w/v)的浓度范围包括0.9％(w/v)至11％(w/v)。如本文所用，数值范围的使用明确地包括所有可能的子范围，该范围内的所有单个数值，包括该范围内的整数和分数，除非上下文另外明确指出。
如本文所用，“抗体”指免疫球蛋白或其片段，其通过至少一个抗原结合位点特异性结合抗原表位。在本文中，抗体的定义涵盖抗原结合片段。术语“抗体”包括多特异性抗体(例如双特异性抗体)、人抗体、非人抗体、人源化抗体、嵌合抗体、单域抗体以及抗原结合片段。抗体可以是合成的(例如通过化学偶联或生物偶联产生的)、酶促处理得到的或重组产生的。本文所提供的抗体包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)。抗体可以是“单价”、“二价”、“三价”或“四价”或更多价抗体，是指其包含1、2、3、4个或更多个抗原结合位点。
如本文所用，“抗原结合片段”指全长抗体的部分，其少于全长，但是至少包含全长抗体的部分可变区(例如包含一个或多个CDR和/或一个或多个抗原结合位点)，并因此保留全长抗体的至少部分特异性结合抗原的能力。抗原结合片段可以例如包括通过酶促处理全长抗体所产生的抗体衍生物、合成产生的衍生物、重组产生的衍生物。抗原结合片段的实例包括但不限于sdAb(例如重链抗体的 可变结构域)、Fv、scFv、dsFv、scdsFv、Fab、scFab、Fab'、F(ab') 2、双抗体、Fd和Fd'片段以及其他片段(例如包含修饰的片段)。
如本文所用，“全长抗体”通常包含四条多肽：两条重链(HC)和两条轻链(LC)。每条轻链从N端(氨基酸端)到C端(羧基端)包含“轻链可变区(VL)”和“轻链恒定区(CL)”。每条重链从N端至C端包含“重链可变区(VH)”以及“重链恒定区(CH)”。一般而言，全长抗体的重链恒定区从N端至C端可以包含CH1-铰链区(hinge)-CH2-CH3。在某些免疫球蛋白类型(例如IgM和IgE)中，重链恒定区从N端至C端可以包含CH1-铰链区-CH2-CH3-CH4。
轻链可变区和重链可变区各自可以包含三个高度可变的“互补决定区(CDR)”和四个相对保守的“框架区(FR)”，并且从N端至C端以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的次序连接。在本文中，轻链可变区的CDR(CDRL或LCDR)可以称为LCDR1、LCDR2和LCDR3，重链可变区的CDR(CDRH或HCDR)可以称为HCDR1、HCDR2和HCDR3。
在本发明中，CDR的氨基酸序列均是按照AbM定义规则所示出的(本发明的权利要求中也是按照AbM定义规则所示出的序列)。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于抗体的三维结构和CDR环的拓扑学的Chothia(参见例如Chothia,C.et al.,Nature,342,877-883(1989)；和Al-Lazikani,B.et al.,J.Mol.Biol.,273,927-948(1997))、基于抗体序列可变性的Kabat(参见例如Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)、AbM(Martin,A.C.R.and J.Allen(2007)“Bioinformatics tools for antibody engineering,”in S.Dübel(ed.),Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag,pp.95–118)、Contact(MacCallum,R.M.et al.,(1996)J.Mol.Biol.262:732-745)、IMGT(Lefranc,M.-P.,2011(6),IMGT,the International ImMunoGeneTics Information System Cold Spring Harb Protoc.；和Lefranc,M.-P.et al.,Dev.Comp.Immunol.,27,55-77(2003))，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明的权利要求中请求保护的范围是基于AbM定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派***规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
如本文所用，术语“框架区”和“构架区”可以互换使用。如本文中所使用的，术语“框架区”、“构架区”或“FR”残基是指抗体可变区中除了如上定义的CDR序列以外的那些氨基酸残基。
通常认为，由一个VH和一个VL通过非共价作用构成的“Fv”片段为包含抗原结合位点的最小的抗原结合片段。但是单可变结构域(单域抗体)也具有抗原结合能力。可以通过肽接头将VH和VL连接来获得“单链Fv(scFv)”。通过向Fv或scFv中引入二硫键可以分别获得“二硫键稳定的Fv(dsFv)”或“单链二硫键稳定的Fv(scdsFv或dsscFv)”。
如本文所用，“Fab”包含一个完整的抗体轻链(VL-CL)和抗体重链可变区和一个重链恒定区(VH-CH1，也称为Fd)。用肽接头将“Fab”中的CL和CH1连接可以获得单链“Fab(scFab)”。“F(ab') 2”基本上包含通过铰链区的二硫键连接的两个Fab片段。“Fab'”为F(ab') 2的一半，其可以通过还原F(ab') 2铰链区的二硫键获得。
“铰链区”指抗体中连接免疫球蛋白Fab和Fc片段的部分。铰链区可以是完整的铰链区或者其部分。对于IgG而言，Fc区通常包含与CH2连接的部分铰链区以及CH3。在本文中，当对于嵌合抗原受体使用时，铰链区还可以指任何功能等同物，例如T细胞受体中连接恒定区和跨膜结构域的部分。本领域技术人员可以根据已知的算法和软件判断VH、VL、CL、CH1、CH2、CH3和铰链区在抗体中的位置，可以应用的算法和软件的描述可以参见例如William R.Strohl,Lila M.Strohl,(2012),Antibody structure–function relationships,In Woodhead Publishing Series in Biomedicine,Therapeutic Antibody Engineering,Woodhead Publishing,pp.37-56。
如本文所用，“双抗体(diabody)”指这样的抗体，其包含两个scFv，其中每个scFv中的VH和VL之间通过短肽接头(大约5-10个氨基酸残基)连接，使得VH和VL链间配对(即第一scFv的VH和第二scFv的VL配对，第一scFv的VL和第二scFv的VH配对)形成抗原结合位点。双抗体可以是双特异性抗体。
如本文所用，“嵌合抗体”指这样的抗体，其中的一部分(例如CDR、FR、可变区、恒定区或其组合)与衍生自特定物种的抗体中相应序列相同或同源，剩余的部分与衍生自另一物种的抗体中相应序列相同或同源。在本发明的一些实施方案中，嵌合抗体包含衍生自非人物种(例如小鼠)的可变区以及衍生自不同物种(例如人)的恒定区。嵌合抗体还可指对至少两种不同抗原具有特异性的多特异性抗体。嵌合抗体可以通过抗体工程化产生。抗体工程化的方法是本领域技术人员公知的。特别地，可以通过DNA重组技术生成嵌合抗体(例如参加Sambrook,J.,et al.(1989).Molecular cloning:a laboratory manual,2nd ed.Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y)。
如本文所用，术语“人源化抗体”是指非人抗体经修饰以增加与人抗体的序列同源性的抗体。人源化抗体通常保留其所源自的非人抗体的抗原结合能力并且对于人体具有较低的免疫原性。人源化抗体可以通过抗体工程化改造任何非人物种抗体或其中包含非人物种来源序列的抗体(例如嵌合抗体)来获得。非人物种例如可以包括小鼠、大鼠、兔、羊驼、鲨鱼或非人灵长类动物。由非人抗体获得人源化抗体的技术是本领域技术人员熟知的。例如，将非人抗体(例如鼠源抗体)的CDR序列移植到人抗体框架区中。在某些情况下，为了保持人源化抗体的抗原结合能力和/或稳定性，可以在人抗体框架区中保留非人抗体(例如鼠源抗体)框架序列的关键氨基酸残基，即进行“回复突变”(参见，例如Morrison et al.(1984)Proc.Natl.Acad.Sci.81(21):6851-6855；Neuberger et al.(1984)Nature 312:604-608)。
如本文所用，术语“人抗体”是指由人产生的抗体或使用本领域已知的任何技术制备的具有与由人产生的抗体相对应的氨基酸序列的抗体。人抗体的定义涵盖完整或全长抗体、其片段和/或包含至少一种人重链和/或轻链多肽的抗体。
如本文所用，“亲和力成熟”的抗体在一个或多个CDR中包含一个或多个修饰(例如氨基酸残基的取代)，使得亲和力成熟的抗体与不包含这样的修饰的亲本抗体相比，对抗原的亲和力得到改进。对抗体进行亲和力成熟的方法是本领域已知的，参见例如Marks et al.,Bio/Technology 10:779-783(1992)；Barbas et al.,Proc.Nat.Acad.Sci.USA 91:3809-3813(1994)；Scier et al.,Gene 169:147-155(1995)；和Hawkins et al.,J.Mol.Biol.226:889-896(1992)。
如本文所用，氨基酸序列的“百分比(％)序列相同性”、“序列相同性”具有本领域公认的定义，其指通过序列比对(例如通过人工检视或可公知的算法)确定的两个多肽序列之间相同的百分比。可以使用本领域技术人员已知的方法确定，例如使用可公开获得的计算机软件如BLAST、BLAST-2、Clustal Omega和FASTA软件。
在本文中，“源自”或“衍生自”参考氨基酸序列的氨基酸序列与参考氨基酸序列的部分或者全部相同或同源。例如，衍生自人免疫球蛋白的重链恒定区的氨基酸序列可以与其所源自的人免疫球蛋白重链恒定区的野生型序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列相同性。
可以修饰多肽中的非关键区域(例如抗体的CDR区、框架区的非关键氨基酸、恒定区的氨基酸)，例如进行一个或多个氨基酸的取代、添加和/或缺失，而不改变多肽的功能。本领域技术人员应当理解多肽中非关键区域中的氨基酸可以用合适的保守氨基酸取代，并且一般不改变其生物活性(参见，例如Watson et al.,Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。合适的保守取代是本领域技术人员熟知的。在某些情况下，氨基酸取代是非保守取代。本领域即是人员应当理解，可以对抗体或抗体片段进行氨基酸突变或修饰来改变其性能，例如改变抗体糖基化修饰的类型，改变形成链间二硫键的能力，或者为抗体缀合物的制备提供活性基团。包含这类氨基酸突变或修饰的抗体或其抗原结合片段也涵盖在本发明的抗体或其抗原结合片段的范围之内。
“亲和力”或“结合亲和力”用来衡量抗体和抗原之间通过非共价作用相互结合的强度。“亲和力”的大小通常可以报告为平衡解离常数K D或EC 50。K D可以通过测量平衡缔合常数(ka)和平衡解离常数(kd)来计算：K D＝kd/ka。可以用本领域已知的常规技术测定亲和力，例如生物膜干涉技术(可以采用例如Octet Fortebio检测***)、放射免疫法、表面等离子共振法、酶联免疫测定(ELISA)或流式细胞术(FACS)等。
本发明的抗CD47抗体或抗原结合片段、多特异性抗体或者编码其的多核苷酸可以是分离的。如本文所用，表述“分离的”是指物质(例如多核苷酸或多肽)与其存在的来源或环境是分离的，即基本上不包含其他任何成分。
在本文中，术语“多核苷酸”和“核酸”可以互换用于表示包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，通常可以包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。
在本文中，“载体”是用于将外源多核苷酸导入宿主细胞的媒介，当载体转化入适当的宿主细胞时，外源多核苷酸得以扩增或表达。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。如本文所用，载体的定义涵盖质粒、线性化质粒、病毒载体、粘粒、噬菌体载体、噬菌粒、人工染色体(例如，酵母人工染色体和哺乳动物人工染色体)等。病毒载体包括但不限于逆转录病毒载体(包括慢病毒载体)、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体和杆状病毒载体等。
如本文所用，术语“表达”是指产生RNA和/或多肽。
如本文所用，“表达载体”指能够表达感兴趣的多核苷酸(包括DNA和RNA)的载体。例如，在表达载体中，可以将编码感兴趣多肽的多核苷酸序列(包括DNA和RNA)与能够影响多核苷酸序列表达的调控序列(如启动子和核糖体结合位点)可操作地连接。调控序列可以包含启动子和终止子序列，并且任选地可以包含复制起点、选择标记、增强子、多腺苷酸化信号等。表达载体可以是质粒、噬菌体载体、重组病毒或其他载体，当引入适当的宿主细胞时，导致感兴趣的多核苷酸的表达。合适的表达载体是本领域技术人员公知的。本领域技术人员可以根据需要将表达载体制备为在宿主细胞中可复制、在宿主细胞中保持游离或者整合入宿主细胞基因组的载体。
如本文所用，“宿主细胞”是用于接受、保持、复制或扩增载体的细胞。宿主细胞还可以用来表 达多核苷酸或载体所编码的多肽。宿主细胞可以是真核细胞或原核细胞。原核细胞例如大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillus subtilis)，真菌细胞例如酵母细胞或曲霉属、昆虫细胞(如S2果蝇细胞或Sf9)以及动物细胞(如成纤维细胞、CHO细胞、COS细胞、HeLa细胞、NSO细胞或HEK293细胞)。
如本文所用，术语“治疗”指对疾病/症状的改善，例如使疾病/症状减轻或消失、防止或减缓疾病/症状的发生、进展和/或恶化。因此，治疗包括预防、治疗和/或治愈。
“有效量”指防止、治愈、改善、阻滞或部分阻滞疾病或症状所需的量。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞或药物组合物优选地将肿瘤细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的效力可以利用本领域常规的肿瘤动物模型评估，例如自发性肿瘤、诱发性肿瘤、移植性肿瘤动物模型。或者，也可以利用本领域公知的体外检测方法检查抑制细胞生长的能力。有效量的本发明的抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞或药物组合物能够减小肿瘤大小，或以其他方式缓解受试者的症状(如预防和/或治疗转移或复发)。本领域技术人员可以根据例如受试者的年龄、身体状况、性别、症状的严重程度、特定组合物或给药途径等因素来确定有效量。有效量可以在一次或多次施用中给予。
如本文中所使用的，术语“药学上可接受的载剂”是指在药理学和/或生理学上与受试者和活性成分相容的载剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂、表面活性剂、佐剂、离子强度增强剂、稀释剂、维持渗透压的试剂、延迟吸收的试剂、防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子、阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等。维持渗透压的试剂包括但不限于糖、氯化钠及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水、水性缓冲液(如缓冲盐水)、醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞、2-苯氧乙醇、对羟苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠、明胶、SPGA(Sucrose-Phosphate-Glutamate-Albumin)、糖类(如山梨醇、甘露醇、淀粉、蔗糖、乳糖、葡聚糖、或葡萄糖)、氨基酸(如谷氨酸、甘氨酸)、蛋白质(如干燥乳清、白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
抗CD47抗体或其抗原结合片段
在一总的方面，本发明提供抗CD47抗体或其抗原结合片段，其特异性识别并结合CD47。
如本文所用，术语“CD47”是指白细胞表面抗原CD47(Leukocyte surface antigen CD47)，又称为整合素相关蛋白(IAP)、卵巢癌抗原OA3、Rh相关抗原或蛋白MER6。在一些实施方案中，抗CD47抗体或其抗原结合片段特异性结合人CD47。术语“人CD47”是指源自人的CD47。人CD47的示例性氨基酸序列示于GenBank登录号NP_001768.1。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段为嵌合抗体、人源化抗体、人 抗体、scFv、Fab、Fab'、F(ab') 2、Fv片段、二硫键稳定的Fv(dsFv)或双抗体。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段
1)特异性结合CD47阳性癌细胞但不结合或基本不结合红细胞；
2)不引起或基本不引起红细胞凝集；
3)阻断CD47与SIRPα的相互结合；和/或
4)促进巨噬细胞对CD47阳性肿瘤细胞的吞噬。
可变区
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段包含重链可变区和轻链可变区。所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述重链可变区包含SEQ ID NO:4(GFNIKDIYIY)所示HCDR1序列，SEQ ID NO:5(KIDPANGNTK)所示HCDR2序列和SEQ ID NO:6(GYGSGFAY)所示HCDR3序列。在一些实施方案中，所述轻链可变区包含SEQ ID NO:7(RASQDISNHLN)所示LCDR1序列，SEQ ID NO:8(YTSRIHS)所示LCDR2序列和SEQ ID NO:9(QQGYTLPFT)所示LCDR3序列。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段是经过亲和力成熟改造获得的。这类抗CD47抗体或其抗原结合片段可以包含如上所述HCDR1(SEQ ID NO:4)、HCDR2(SEQ ID NO:5)、HCDR3(SEQ ID NO:6)、LCDR1(SEQ ID NO:7)、LCDR2(SEQ ID NO:8)和LCDR3(SEQ ID NO:9)序列的变体中的一个或多个。在一实施方案中，所述变体各自相对于其所源自的序列独立地包含1个氨基酸的取代、添加或缺失。优选地，所述变体各自相对于其所源自的序列独立地包含1个氨基酸的取代。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段包含重链可变区和轻链可变区，其中
所述重链可变区包含SEQ ID NO:4或其变体的HCDR1序列，SEQ ID NO:5或其变体的HCDR2序列和SEQ ID NO:6或其变体的HCDR3序列；
所述轻链可变区包含SEQ ID NO:7或其变体的LCDR1序列，SEQ ID NO:8或其变体的LCDR2序列和SEQ ID NO:9或其变体的LCDR3序列；
其中所述变体各自相对于其所源自的序列独立地包含1个氨基酸的取代、添加或缺失。
在一些实施方案中，HCDR1序列包含SEQ ID NO:4所示的氨基酸序列，其中第7位氨基酸被取代。在一实施方案中，SEQ ID NO:4中第7位氨基酸被取代为V(SEQ ID NO:18；GFNIKDVYIY)。
在一些实施方案中，HCDR2序列包含SEQ ID NO:5所示的氨基酸序列，其中第10位氨基酸被取代。在一实施方案中，SEQ ID NO:5中第10位氨基酸被取代为H(SEQ ID NO:19；KIDPANGNTH)。
在一些实施方案中，HCDR3序列包含SEQ ID NO:6所示的氨基酸序列，其中第5位氨基酸被取代。在一实施方案中，SEQ ID NO:6中第5位氨基酸被取代为V(SEQ ID NO:23；GYGSVFAY)。
在一些实施方案中，LCDR1序列包含SEQ ID NO:7所示的氨基酸序列，其中第10位氨基酸被取代。在一实施方案中，SEQ ID NO:7中第10位氨基酸被取代为I(SEQ ID NO:24；RASQDISNHIN)。
在一些实施方案中，LCDR2序列包含SEQ ID NO:8所示的氨基酸序列，其中第7位氨基酸被取代。在一实施方案中，SEQ ID NO:8中第7位氨基酸被取代为L(SEQ ID NO:16；YTSRIHL)。在一实施方案中，SEQ ID NO:8中第7位氨基酸被取代为K(SEQ ID NO:20；YTSRIHK)。
在一些实施方案中，LCDR3序列包含SEQ ID NO:9所示的氨基酸序列，其中第5位氨基酸被取代。在一实施方案中，SEQ ID NO:9中第5位氨基酸被取代为H(SEQ ID NO:25；QQGYHLPFT)。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段包含重链可变区和轻链可变区，其中
所述重链可变区包含SEQ ID NO:4或SEQ ID NO:18所示HCDR1序列，SEQ ID NO:5或SEQ ID NO:19所示HCDR2序列和SEQ ID NO:6或SEQ ID NO:23所示HCDR3序列；
所述轻链可变区包含SEQ ID NO:7或SEQ ID NO:24所示LCDR1序列，SEQ ID NO:8、SEQ ID NO:16或SEQ ID NO:20所示LCDR2序列和SEQ ID NO:9或SEQ ID NO:25所示LCDR3序列。
在一实施方案中，所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:6所示HCDR3序列；并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:8所示LCDR2序列和SEQ ID NO:9所示LCDR3序列。
在一实施方案中，所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:6所示HCDR3序列；并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:16所示LCDR2序列和SEQ ID NO:9所示LCDR3序列。
在一实施方案中，所述重链可变区包含SEQ ID NO:18所示HCDR1序列，SEQ ID NO:19所示HCDR2序列和SEQ ID NO:6所示HCDR3序列；并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:20所示LCDR2序列和SEQ ID NO:9所示LCDR3序列。
在一实施方案中，所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:23所示HCDR3序列；并且所述轻链可变区包含SEQ ID NO:24所示LCDR1序列，SEQ ID NO:8所示LCDR2序列和SEQ ID NO:25所示LCDR3序列。
在一些实施方案中，如上所述的VH进一步包含重链框架区。在一些实施方案中，如上所述的VL进一步包含轻链框架区。在一些实施方案中，如上所述的VH进一步包含重链框架区，并且如上所述的VL进一步包含轻链框架区。重链框架区和/或轻链框架区可以各自独立地衍生自任何物种免疫球蛋白的重链框架区和轻链框架区。
在一些实施方案中，所述VH包含衍生自鼠免疫球蛋白的重链框架区，和/或所述VL包含衍生自鼠免疫球蛋白的轻链框架区。
在某些优选的实施方案中，所述VH包含衍生自人免疫球蛋白的重链框架区，和/或所述VL包含衍生自人免疫球蛋白的轻链框架区。因此，在某些优选的实施方案中，本发明的抗CD47抗体或其抗原结合片段是人源化的。人源化的抗CD47抗体或其抗原结合片段的重链框架区和/或轻链框架区可以包含一个或多个非人源(例如，鼠源)氨基酸残基，例如重链框架区和/或轻链框架区可以包含一或多个氨基酸回复突变，在这些回复突变中包含相应的鼠源氨基酸残基。
在一些实施方案中，所述重链可变区包含SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列。在一些实施方案中，所述轻链可变区包含SEQ ID NO:11、 SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27的氨基酸序列。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。在一些实施方案中，所述重链可变区包含与SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列相比具有一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失的氨基酸序列。优选地，所述氨基酸的取代、添加和/或缺失不发生在CDR区。
在一些实施方案中，所述轻链可变区包含与SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。在一些实施方案中，所述轻链可变区包含与SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27相比具有一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失的氨基酸序列。优选地，所述氨基酸的取代、添加和/或缺失不发生在CDR区。
在一些实施方案中，所述重链可变区包含：1)SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列；2)与SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列；或者3)与SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列相比具有一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失的氨基酸序列；并且
所述轻链可变区包含：1)SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27的氨基酸序列；2)与SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列；或者3)与SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27相比具有一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失的氨基酸序列。优选地，所述氨基酸的取代、添加和/或缺失不发生在CDR区。
在一实施方案中，所述重链可变区包含SEQ ID NO:10的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:11的氨基酸序列。在一实施方案中，所述重链可变区包含SEQ ID NO:14的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:15的氨基酸序列。在一实施方案中，所述重链可变区包含SEQ ID NO:14的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:17的氨基酸序列。在一实施方案中，所述重链可变区包含SEQ ID NO:21的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:22的氨基酸序列。在一实施方案中，所述重链可变区包含SEQ ID NO:26的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:27的氨基酸序列。
恒定区
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段进一步包含重链恒定区和/或轻链恒定区。
重链恒定区和轻链恒定区可以各自独立地衍生自任何物种的免疫球蛋白的重链恒定区和轻链恒定区。重链恒定区可以衍生自任何亚型(例如IgA、IgD、IgE、IgG和IgM)、类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)的免疫球蛋白的重链恒定区或其组合。轻链恒定区可以衍生自λ(Lambda)轻链或κ(Kappa)轻链恒定区。
可以选择合适的免疫球蛋白恒定区(例如CH1和轻链恒定区、铰链区-CH2-CH3、CH1-铰链区-CH2-CH3和轻链恒定区或者Fc区)，以及类型(例如IgG，例如IgG1、IgG2、IgG3和IgG4)，并且任选地进行修饰，以获得具有期望的特性的抗体。
在优选的实施方案中，重链恒定区至少包含Fc区，例如IgG的重链恒定区可以包括：1)铰链区的全部或部分-CH2-CH3；或者2)CH1-铰链区-CH2-CH3。在一些实施方案中，重链恒定区为人IgG(例如IgG1、IgG2a、IgG2b、IgG3或IgG4)的重链恒定区(例如Fc区或CH1-铰链区-CH2-CH3)。在一实施方案中，重链恒定区为人IgG1的重链恒定区。在一实施方案中，重链恒定区包含SEQ ID NO:1的氨基酸序列。在一优选实施方案中，重链恒定区为人IgG4的重链恒定区。在一实施方案中，重链恒定区包含：1)SEQ ID NO:12的氨基酸序列；或者2)与SEQ ID NO:12具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
在一优选实施方案中，轻链恒定区为人κ轻链恒定区。在一实施方案中，轻链恒定区包含：1)SEQ ID NO:13的氨基酸序列；或者2)与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
可以使用本领域已知的方法制备和生产抗体或其抗原结合片段。这类方法可以包括例如，从噬菌体展示文库、酵母展示文库、永生化的B细胞(例如，小鼠B细胞杂交瘤细胞或EBV永生化的B细胞)制备和分离抗体或抗原结合片段。还可以用免疫动物的方法，例如用抗原或编码抗原的DNA免疫动物(例如小鼠)，然后从免疫后的动物分离表达抗体的B细胞。优选地，将表达抗体的B细胞永生化，例如制备成杂交瘤或EBV永生化的B细胞。还可以从免疫动物中分离或者采用化学合成的方法制备编码抗体或其抗原结合片段的多核苷酸，然后利用多核苷酸构建表达载体。
多特异性抗体
在一方面，本发明提供了一种多特异性抗体，其包含结合CD47的第一抗原结合部分以及结合第二抗原的第二抗原结合部分，其中所述第一抗原结合部分包含本发明的抗CD47抗体或其抗原结合片段。
如本文所用，术语“多特异性抗体”是指能够特异性结合两种或更多种(例如2、3、4、5或6种)不同抗原表位的抗体。多特异性抗体可以例如是双特异性、三特异性或四特异性抗体，其分别能够特异性结合2、3或4种抗原表位。如本文所用，术语“抗原表位”或“抗原决定簇”表示抗原中与抗体的抗原结合位点特异性结合的区域。抗原表位通常由抗原的化学活性表面基团(如氨基酸或糖侧链)组成且通常具有特定的三维结构性质以及特定的电荷性质。第二抗原可以为不同于CD47的其他抗原。第二抗原也可以为CD47，其与本发明的抗CD47抗体或其抗原结合片段结合CD47上不同的抗原表位。可以利用本领域常规的方法确定两种抗体结合的抗原表位是否相同，例如通过ELISA、流式细胞术或表面等离子共振等测定两种抗体对相同抗原表位的竞争结合。
多特异性抗体可以是多价(例如2、3、4价)抗体，即其具有多个抗原结合位点。多特异性抗体 可以例如是嵌合抗体、人源化抗体、scFab、F(ab') 2或双抗体。
利用感兴趣的抗体或抗原结合片段构建多特异性抗体的方法是本领域技术人员熟知的(参见，例如WO 93/08829；Suresh et al.,(1986)Methods in Enzymology,121:210；和Traunecker et al.,(1991)EMBO,10:3655-3659)。可以利用本领域已知的各种技术产生和分离多特异性抗体。例如，可以通过重组DNA技术获得编码多特异性抗体的多核苷酸，任选地将所述多核苷酸克隆入表达载体，然后用所述多核苷酸或表达载体转化宿主细胞，在合适的条件下培养转化的宿主细胞以允许所述多核苷酸或表达载体表达，最后从宿主细胞或培养基中分离和纯化多特异性抗体。还可以分别获得多特异性抗体的各个部分，例如分别获得如本文所述的第一抗原结合部分和第二抗原结合部分，然后通过酶促或化学偶联技术任选地通过接头将各个部分偶联获得特异性结合CD47和其他抗原的多特异性抗体。
如本文所用，“第一抗原结合部分”和“第二抗原结合部分”表示包含抗原结合位点的、能够与抗原表位结合的氨基酸序列，其定义落入抗体或抗原结合片段的含义范围内。
第一抗原结合部分可以是任何形式的抗体或抗原结合片段，包括但不限于Fv、scFv、dsFv、scdsFv、Fab、scFab、Fab'和F(ab') 2。在一些实施方案中，第一抗原结合部分包含本发明的抗CD47抗体或其抗原结合片段。在一实施方案中，第一抗原结合部分包含VH和VL，其分别包含如上所述本发明的抗CD47抗体或其抗原结合片段的HCDR1、HCDR2和HCDR3以及LCDR1、LCDR2和LCDR3。在一实施方案中，第一抗原结合部分包含VH和VL，其分别包含如上所述本发明的抗CD47抗体或其抗原结合片段的VH和VL。
第二抗原结合部分可以是结合任何感兴趣的抗原表位的抗体或抗原结合片段。在一实施方案中，第二抗原为不同于CD47的其他抗原。第二抗原结合部分可以特异性结合的抗原可以包括：肿瘤抗原(例如肿瘤相关抗原和肿瘤特异性抗原)、免疫调节受体和免疫检查点分子。如本文所用，“肿瘤相关抗原”是指在肿瘤细胞中高表达，健康细胞中也存在但是表达水平较低的抗原。如本文所用，“肿瘤特异性抗原”是指肿瘤细胞中特异性表达，健康细胞中几乎不表达的抗原。肿瘤抗原的非限制性实例可以包括CD19、CD20、EGFR、GPC3、HER-2和FOLR1。免疫检查点分子的非限制性实例可以包括CTLA-4、LAG-3、PD-1、PD-L1和TIM-3。免疫调节受体可以包括例如免疫激活性受体(例如CD27、CD137、CD40、GITR和OX40)和免疫抑制性受体(例如BTLA、CTLA4、LAG-3和PD-1)。例如，第二抗原结合部分可以为免疫激活性受体的激动剂抗体或者免疫抑制性受体的拮抗剂抗体。
在一实施方案中，所述第二抗原结合部分结合肿瘤抗原、免疫调节受体和免疫检查点分子。在一实施方案中，第二抗原结合部分特异性结合SIRPα、PD-1、PD-L1、LAG3、TIM-3、CTLA-4、VISTA、GPC3、EGFR、HER-2、CD19、CD20、CD33、CD40、CD73、OX40、CD3、DLL-3、TIP-1、叶酸受体α(FOLR1)和/或其他肿瘤抗原。
第二抗原结合部分可以是任何形式的抗体或抗原结合片段，包括但不限于免疫球蛋白的单可变结构域(例如包含羊驼的VHH或鲨鱼的IgNAR可变结构域)、Fv、scFv、dsFv、scdsFv、Fab、scFab、Fab'和F(ab') 2。在一实施方案中，第二抗原结合部分包含免疫球蛋白的单可变结构域。
第一抗原结合部分和第二抗原结合部分可以任选地通过接头连接。在一些实施方案中，第一抗原结合部分和第二抗原结合部分不通过接头连接。在另一些实施方案中，第一抗原结合部分和第二抗原结合部分通过接头连接，例如肽接头或化学键。优选地，第一抗原结合部分和第二抗原结合部分通过肽接头连接。示例性的肽接头可以包括但不限于聚甘氨酸(G)、聚丙氨酸(A)、 聚丝氨酸(S)或其组合，例如GGAS、GGGS、GGGSG或者(G 4S) n，其中n为1-20的整数。
在一些实施方案中，第二抗原为不同于CD47的其他抗原，并且第一抗原结合部分与CD47的结合亲和力弱于第二抗原结合部分与其他抗原的结合亲和力。在这类实施方案中，本发明的多特异性抗体可以优先靶向其他抗原，从而多特异性抗体特异性识别表达其他抗原的靶细胞(例如癌细胞)，而靶细胞可以表达或不表达CD47。在一具体实施方案中，当靶细胞同时表达CD47抗原和不同于CD47的第二抗原时，本发明的多特异性抗体对CD47的结合能力相对于只表达CD47的更强，阻断CD47和SIRPa结合的能力也更强。
CAR和表达CAR的免疫效应细胞
在另一方面，本发明还提供包含本发明的抗CD47抗体或其抗原结合片段的嵌合抗原受体(CAR)。CAR是同时提供抗原结合并激活T细胞功能的重组受体。CAR结构和工程改造描述于，例如Dotti G.et al.,(2014)Immunol Rev.257(1):107-126，其通过援引加入本文。
在一示例性实施方案中，本发明的CAR从N端至C端包含：
(a)胞外区，其包含本发明的抗CD47抗体或其抗原结合片段；
(b)跨膜区，其连接胞外区和胞内信号区并将CAR锚定在细胞膜上；以及
(c)胞内信号区。
胞外区包含本发明的抗CD47抗体或其抗原结合片段，使得本发明的CAR与表达CD47的细胞(例如癌细胞)结合。在一优选实施方案，CAR中的胞外区为scFv形式。
跨膜区连接胞外区和胞内信号区并将CAR锚定在细胞膜上。
当CAR的胞外区与其识别的抗原结合时，胞内信号区向胞内传导T细胞受体(TCR)样信号并激活表达CAR的免疫效应细胞发挥其效应功能。
在一些实施方案中，胞内信号区还包含至少一个共刺激结构域。共刺激结构域可以促进表达CAR的免疫效应细胞在与胞外区所靶向的抗原结合后的活化。
在一些实施方案中，本发明的CAR还包含连接胞外区和跨膜区的间隔区。在一实施方案中，间隔区衍生自免疫球蛋白的重链恒定区。
在又一方面，本发明还涉及编码本发明的CAR的多核苷酸和包含所述多核苷酸的表达载体。
在另一方面，本发明还提供一种免疫效应细胞，其在细胞表面表达本发明的CAR。优选地，所述免疫效应细胞选自T淋巴细胞(例如细胞毒性T细胞(CTL))、自然杀伤细胞(NK)和自然杀伤T细胞(NKT)。免疫效应细胞可以靶向CD47阳性的病变细胞(例如，CD47阳性的癌细胞)，并被激活以启动效应功能，例如，引起CD47阳性的癌细胞的死亡。
多核苷酸、载体和宿主细胞
在另一方面，本发明提供了一种多核苷酸，其包含编码本发明的抗CD47抗体或抗原结合片段或者本发明的多特异性抗体的多核苷酸序列。
可以利用本领域已知的方法获得本发明的多核苷酸。例如，本发明的多核苷酸可以分离自噬菌体展示文库、酵母展示文库、免疫动物、永生化的细胞(例如，小鼠B细胞杂交瘤细胞、EBV介导的永生化B细胞)或者化学合成。本发明的多核苷酸可以针对用于表达的宿主细胞进行密码子优化。
在又一方面，本发明还提供了包含本发明的多核苷酸的表达载体。表达载体可以进一步包含额外的多核苷酸序列，例如调控序列和抗生素抗性基因。本发明的多核苷酸可以存在于一种或多种表 达载体中。在一实施方案中，本发明的多核苷酸制备为重组核酸。可使用本领域众所周知的技术制备重组核酸，例如化学合成、DNA重组技术(例如聚合酶链式反应(PCR)技术)等。
本发明还提供了一种宿主细胞，其包含本发明的多核苷酸或表达载体。可以采用本领域已知的各种方法将本发明的多核苷酸或表达载体导入合适的宿主细胞中。这类方法包括但不限于脂质体转染、电穿孔、病毒转导和磷酸钙转染等。
在优选的实施方案中，宿主细胞用于表达本发明的抗CD47抗体或其抗原结合片段或者本发明的多特异性抗体。宿主细胞的实例包括但不限于原核细胞(例如细菌，例如大肠杆菌)和真核细胞(例如酵母、昆虫细胞、哺乳动物细胞)。适合于抗体表达的哺乳动物宿主细胞包括但不限于骨髓瘤细胞、HeLa细胞、HEK细胞(例如HEK 293细胞)、中国仓鼠卵巢(CHO)细胞和其他适于表达抗体的哺乳动物细胞。
本发明还提供了一种产生本发明的抗CD47抗体或其抗原结合片段或者本发明的多特异性抗体的方法，其包括以下步骤：
(Ⅰ)在合适条件下培养本发明宿主细胞以表达本发明的抗CD47抗体或其抗原结合片段或者本发明的多特异性抗体，以及
(Ⅱ)从宿主细胞或其培养物分离所述抗体或其抗原结合片段或者本发明的多特异性抗体。
抗体缀合物
本发明还提供了一种抗体缀合物，其包含与至少一种治疗剂缀合的本发明的抗CD47抗体或其抗原结合片段或者本发明的多特异性抗体。抗体-药物缀合物(ADC)是一种典型的抗体缀合物，其中所述治疗剂可以例如为细胞毒性剂。
如本文所用，“缀合”是指两个或多个部分通过共价或非共价作用相互连接。在优选的实施方案中，所述缀合是共价缀合。
治疗剂可以选自细胞毒性剂、治疗性抗体(例如与另外的抗原特异性结合的抗体或其抗原结合片段)、放射性同位素、寡核苷酸及其类似物(例如干扰RNA)、生物活性肽、蛋白毒素(例如白喉毒素、蓖麻毒素)和酶(例如脲酶)。
细胞毒性剂是指抑制或降低细胞的活性、功能和/或杀死细胞的物质。细胞毒性剂的实例可以包括但不限于：类美登素(maytansinoid)(例如美登素(maytansine)、奥利斯他汀类(例如MMAF、MMAE、MMAD)、多司他汀(duostatin)、念珠藻环肽(cryptophycin)、长春花生物碱类(例如长春碱、长春新碱)、秋水仙碱类、海兔毒素类、紫杉烷、紫杉醇、多西他赛、卡巴他赛、烯二炔类抗生素、细胞松弛素类、喜树碱类、蒽环类抗生素(例如道诺霉素(daunorubicin)、二羟基蒽二酮(dihydroxyanthracindione)、多柔比星(doxorubicin))、细胞毒性抗生素(例如丝裂霉素(mitomycin)、放线菌素(actinomycin)、倍癌霉素(duocarmycin)(例如CC-1065)、金霉素(auromycin)、多霉素(duomycin)、卡奇霉素(calicheamicin)、内孢霉素(endomycin)、酚霉素(phenomycin))、阿霉素、柔红霉素、卡奇霉素、顺铂(cisplatin)、溴化乙锭(ethidiumbromide)、博来霉素、丝裂霉素、光辉霉素、普拉地内酯、鬼臼毒素、依托泊苷(etoposide)、米托蒽醌、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨、奈拉滨、卡莫司汀、洛莫司汀、甲氨蝶呤、苯丙氨酸氮芥、替尼泊苷(tenoposide)、糖皮质激素等。
放射性同位素可以选自例如 212Bi、 213Bi、 131I、 125I、 111In、 177Lu、 186Re、 188Re、 153Sm、 90Y。用放射性同位素标记的抗体又可称为放射免疫缀合物。
在一些实施方案中，治疗剂选自细胞毒性剂、化疗剂、放射性同位素、免疫检查点抑制剂、靶向肿瘤特异性抗原的抗体和其他抗肿瘤药物。在优选一实施方案中，治疗剂为细胞毒性剂。在又一优选实施方案中，治疗剂为放射性同位素。
可以利用本领域已知的任何技术将治疗剂与本发明的抗体或抗原结合片段或者本发明的多特异性抗体通过接头缀合。所述接头可以包含用于共价缀合的活性基团，例如胺、羟胺、马来酰亚胺基、羧基、苯基、硫醇、巯基或羟基。
药物组合物
本发明还提供药物组合物，其包含本发明的抗CD47抗体或抗原结合片段、抗体缀合物、表达CAR的免疫效应细胞或者多特异性抗体，以及药学上可接受的载剂。
药学上可接受的载剂可以包括但不限于：稀释剂、粘合剂和胶粘剂、润滑剂、崩解剂、防腐剂、媒介物、分散剂、助流剂、甜味剂、包衣、赋形剂、防腐剂、抗氧化剂(如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠、抗坏血酸棕榈酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚、柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等)、增溶剂、胶凝剂、软化剂、溶剂(例如，水、酒精、乙酸和糖浆)、缓冲剂(例如，磷酸盐缓冲剂、组氨酸缓冲剂和乙酸盐缓冲剂)、表面活性剂(例如非离子表面活性剂，例如聚山梨酯80、聚山梨酯20、泊洛沙姆或聚乙二醇)、抗细菌剂、抗真菌剂、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、吸收延迟剂、螯合剂和乳化剂。对于包含抗体或者抗体缀合物的组合物而言，合适的载剂可以选自缓冲剂(例如柠檬酸盐缓冲液、乙酸盐缓冲液、磷酸盐缓冲液、组氨酸缓冲液、组氨酸盐缓冲液)、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、非离子表面活性剂(例如聚山梨酯80、聚山梨酯20、泊洛沙姆)或其组合。
本文提供的药物组合物可以为多种剂型，包括但不限于固体、半固体、液体、粉末或冻干形式。优选地，所述药物组合物适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。对于包含抗体或者抗体缀合物的组合物而言，优选的剂型通常可以为例如注射液和冻干粉。
可通过本领域已知的任何方法，例如通过全身或局部施用，将本文提供的药物组合物给药于受试者。给药途径包括但不限于肠胃外(例如，静脉内、腹膜内、皮内、肌肉内、皮下或腔内)、局部(例如瘤内)、硬膜外或粘膜(例如鼻内、口服、***、直肠、舌下或局部)。本领域技术人员应当理解，确切的给药剂量将取决于各种因素，例如药物组合物的代谢动力学性质、治疗的持续时间、特定化合物的***速率、治疗目的、给药途径和受试者的状况，例如患者的年龄、健康状况、体重、性别、饮食、病史，以及医学领域公知的其他因素。给药方法可以为例如注射或输注。
作为一般性指导，本发明的抗CD47抗体或其抗原结合片段、抗体缀合物或者多特异性抗体的给药剂量范围可以为约0.0001至100mg/kg，更通常为0.01至20mg/kg受试者体重。例如，给药剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重，10mg/kg体重或20mg/kg体重，或在1-20mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短后期给药间隔加长。给药方式可以是静脉滴注。
治疗
在又一方面，本发明涉及本发明的抗CD47抗体或其抗原结合片段、多特异性抗体抗、抗体缀合物、免疫效应细胞或者药物组合物在制备用于治疗受试者中疾病的药物中的用途。
本发明还涉及本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞或者药物组合物，其用于治疗疾病。
本发明还提供一种治疗受试者中疾病的方法，所述方法包括向所述受试者给药治疗有效量的本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞或者药物组合物。
在一些实施方案中，所述疾病与CD47异常表达相关。术语“异常表达”指与正常状态的样品(或标准样品，例如未患有与CD47异常表达相关疾病的受试者的样品)相比，样品中蛋白表达水平过高或过低。优选地，所述疾病以CD47高表达为特征。例如，患有或怀疑患有疾病(例如胃癌)的受试者的组织(例如胃癌组织和胃癌旁组织)中高表达CD47，而未患有所述疾病的对象的相应组织中低表达CD47。
在一实施方案中，如上所述的疾病为癌症。如本文所用，“癌症”包括但不限于血液瘤和实体瘤。在本文中，“血液癌”表示血液的癌症，包括白血病、淋巴瘤和骨髓瘤等。作为非限制性示例，白血病可以包括急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)和骨髓性增生疾病(例如骨髓增生异常综合征)。淋巴瘤可以包括例如霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特(Burkitt)淋巴瘤和滤泡性淋巴瘤等。骨髓瘤可以包括例如多发性骨髓瘤(MM)和巨细胞骨髓瘤等。实体瘤包括例如乳腺癌、卵巢癌、肺癌、胰腺癌、***癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌、肾癌、平滑肌瘤、胶质瘤和成胶质细胞瘤等。癌症还可以是转移性癌。“转移”是指癌细胞从其原始部位扩散到身体的其他部分。
本发明还涉及本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞、或者药物组合物用于治疗癌症。在一些实施方案中，所述癌症选自急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)、骨髓增生异常综合征、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、滤泡性淋巴瘤、多发性骨髓瘤(MM)、巨细胞骨髓瘤、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌、肾癌、平滑肌瘤、胶质瘤和成胶质细胞瘤。
本发明的抗CD47抗体或其抗原结合片段可以用于治疗CD47阳性的血液瘤和实体瘤。在一些实施方案中，所述癌症选自急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)、骨髓增生异常综合征、脊髓发育不良综合征、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、滤泡性淋巴瘤、多发性骨髓瘤(MM)、巨细胞骨髓瘤、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌、肾癌、平滑肌瘤、胶质瘤和成胶质细胞瘤等。
取决于第二抗原结合部分，本发明的多特异性抗体可以用于治疗例如选自以下的癌症：急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)、骨髓增生异常综合征、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、滤泡性淋巴瘤、多发性骨髓瘤(MM)、巨细胞骨髓瘤、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、胃癌、肝癌、肾癌、平滑肌瘤、胶质瘤和成胶质细胞瘤等。
本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞或者药 物组合物可以与至少一种或多种本文所述的治疗剂联合施用。联合施用的方式没有限制。例如，可以将上述治疗剂全部在一次施用或分开施用。
对于癌症治疗，本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物或者药物组合物可以与其他治疗方法联合使用，包括但不限于：手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、血管生成抑制和姑息治疗。
在某些实施方案中，本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物或者药物组合物进一步与选自以下的一种或多种治疗剂联合使用：化疗剂、放射性同位素、免疫检查点抑制剂和肿瘤抗原靶向药物。化疗剂可以包括例如：抗代谢药、烷化剂、细胞毒性剂、拓扑异构酶抑制剂、微管抑制剂。肿瘤抗原靶向药物包括但不限于靶向肿瘤相关抗原和肿瘤特异性抗原的药物。如本文所用，“肿瘤相关抗原”是指在肿瘤细胞中高表达，健康细胞中也存在但是表达水平较低的抗原。如本文所用，“肿瘤特异性抗原”是指肿瘤细胞中特异性表达，健康细胞中几乎不表达的抗原。治疗剂的其他非限制性实例可以包括例如：血管生成抑制因子、脱乙酰化酶(HDAC)抑制因子、Hedgehog信号通路阻滞剂、mTOR抑制剂、p53/mdm2抑制剂、PARP抑制剂、蛋白酶体抑制剂(例如bortezomib、carfilzomib、ixazomib、Marizomib、Oprozomib)以及酪氨酸激酶抑制剂(例如BTK抑制剂)。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段或者多特异性抗体与选自以下的一种或多种治疗剂联用：抗SIRPα抗体、抗CD20抗体、抗TIM-3抗体、抗LAG-3抗体、抗EGFR抗体、抗HER-2抗体、抗CD19抗体、抗CD33抗体、抗CD47抗体、抗CD73抗体、抗DLL-3抗体、抗TIP-1抗体、抗FOLR1抗体、抗CTLA-4抗体、抗PD-L1抗体和抗PD-1抗体。
在一些实施方案中，本发明的抗CD47抗体或其抗原结合片段或者多特异性抗体与化疗剂联合使用。在另一些实施方案中，本发明的抗体缀合物与免疫检查点抑制剂联合使用。在又一些实施方案中，本发明的抗体或其抗原结合片段、抗体缀合物或者药物组合物与放射性同位素联合使用。
试剂盒
本发明还提供试剂盒，其包含本发明的抗CD47抗体或其抗原结合片段、多特异性抗体、抗体缀合物、免疫效应细胞或者药物组合物，以及使用说明。试剂盒还可以包含合适的容器。在某些实施方案中，试剂盒还包含给药的装置。通常，试剂盒还包括标签，其用于表明试剂盒内容物的预期用途和/或使用方法。术语“标签”包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
有益效果
本发明的抗CD47抗体或其抗原结合片段可以实现以下有益效果：1)特异性结合CD47阳性肿瘤细胞但不结合或基本不结合红细胞；2)不引起或基本不引起红细胞凝集；3)对CD47与SIRPα的结合具有阻断效果；和/或4)促进巨噬细胞对CD47阳性肿瘤细胞的吞噬。
实施例
以下实施例旨在仅对本发明进行举例说明，因此并不应被视为以任何方式限制本发明。
实施例1动物免疫、免疫库构建和抗体筛选
1.1动物免疫
在CD47的胞外区段CD47-ECD的羧基端(C端)融合人IgG1Fc或者6×His标签，分别构建CD47抗原蛋白CD47-ECD-Fc(SEQ ID NO:3)或CD47-ECD-His(SEQ ID NO:2)。将CD47-ECD-Fc用于C57BL/6小鼠(上海灵畅生物科技有限公司)的免疫，免疫时采取皮下多点免疫的方式，每次皮下免疫50μg，每两星期免疫一次，共免疫四次。免疫四次后通过ELISA测定免疫的效价，其中ELISA抗原包板采用CD47-ECD-His。测定结果显示免疫效价达到1:600000，此时采用100μg CD47-ECD-Fc进行加强免疫，2-3天后取脾脏。
1.2免疫库构建
分离实施例1.1所述经免疫的小鼠脾脏中的B淋巴细胞，抽取其RNA，并通过反转录试剂盒(TaKaRa，6210A)反转录成cDNA。设计引物分别扩增轻链可变区和重链可变区以及第一个恒定区(CL和CH1)的编码序列，并且在CH1的编码序列的3’端连接M13噬菌体GIII蛋白的编码序列，然后克隆至噬菌体展示用载体上。然后通过电转仪(Bio-Rad，MicroPulser)将载体转化至感受态大肠杆菌SS320细胞(Lucigen，MC1061F)中，复苏1小时后，涂布于具有氨苄抗性的2-YT固体平板。通过梯度稀释铺板，测定此免疫库的库容为1×10 9cfu。采用辅助噬菌体M13KO7(NEB)进行包装，最终构建的免疫库以Fab的形式展示在M13噬菌体的外壳蛋白上。
1.3抗体筛选
第一轮筛选时，在免疫管中加入4mL的50μg/mL CD47-ECD-His，4℃包被过夜；第二天弃去包被液，加入含5％奶粉的PBS封闭2h；用PBS润洗后加入制备好的噬菌体孵育2h；润洗以去除非特异性结合的噬菌体，然后向免疫管中加入0.8mL含0.05％EDTA的胰酶消化液，用于洗脱特异性结合目标抗原的噬菌体；接着用洗脱下来的噬菌体侵染对数期的大肠杆菌SS320(Lucigen，60512-1)，37℃静置30min，然后220rpm条件下培养1h，再加入VSCM13辅助噬菌体静置30min，继续在220rpm条件下培养1h；离心并置换至C +/K +2-YT培养基中，并于30℃、220rpm环境下继续培养过夜。第二天制备成噬菌体后继续用于第二轮的筛选，如此反复，同时对每轮随机挑选10个克隆进行序列分析。选择第三轮富集较明显并且序列多态性较高的菌进行单克隆涂布制备。
通过诱导的单克隆Fab上清的ELISA，选择阳性克隆进行测序分析。结果发现，此次免疫库筛选得到了超过60个具有不同序列的阳性克隆。通过进一步将这些克隆制备的Fab诱导上清进行CCRF-CEM细胞(一种人急性淋巴细胞白血病T淋巴细胞，内源性表达CD47，购自中国科学院，货号TCHu147)上的结合实验和SIRPα的结合阻断实验之后，筛选得到部分克隆既结合CCRF-CEM细胞又具有可以阻断SIRPα在细胞上结合的功能。克隆A7是其中之一，为鼠源抗体。克隆A7的重链可变区的氨基酸序列示于SEQ ID NO:10，轻链可变区的氨基酸序列示于SEQ ID NO:11。采用AbM定义的克隆A7的HCDR1、HCDR2和HCDR3的氨基酸序列分别示于SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6，LCDR1、LCDR2和LCDR3的氨基酸序列分别示于SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9。
实施例2抗体工程改造
通过克隆A7可变区的人源化改造获得人源化抗体A7H3L3的可变区。通过对人源化抗体A7H3L3的可变区进行亲和力成熟改造获得抗体A7-44、A7-28和A7-47的可变区。
2.1抗体的人源化
将实施例1筛选得到的克隆A7的鼠源VH和VL序列和已知的人源抗体数据库进行比对，找到分别与鼠源VH和VL序列同源性最高的人种系(germline)基因VH和VL序列。选择人种系基因VH和VL序列的框架区(采用AbM定义CDR和框架区)，然后借助计算机预测模拟，通过回复突变的方式保留克隆A7框架区中对抗原结合具有重要影响的鼠源氨基酸，最后将该种系基因的互补决定区(CDR)序列替换为克隆A7中对应的CDR序列。克隆A7可变区经人源化后获得人源化抗体A7H3L3的可变区：A7H3L3重链可变区(VH)氨基酸序列示于SEQ ID NO:14，轻链可变区(VL)氨基酸序列示于SEQ ID NO:15(表1)。
2.2亲和力成熟改造
对抗体A7H3L3进行亲和力成熟改造，用于提高亲和力和生物学活性。亲和力成熟改造是基于M13噬菌体展示技术，采用codon-based引物(引物合成过程中，单个密码子由NNK组成)引入CDR区突变，构建4个噬菌体展示文库：文库1和文库2为单点组合突变，文库1为CDRL1+CDRL3+CDRH3组合突变，文库2为CDRL2+CDRH1+CDRH2组合突变；文库3和文库4为双点饱和突变，文库3为CDRL3的双点饱和突变，文库4为CDRH3的双点饱和突变。具体的建库方法：首先合成包含点突变的引物(金唯智生物科技有限公司)；其次以待改造的抗体A7H3L3为PCR扩增模板，扩增CDR区包含设计突变的序列，通过桥连PCR的方法，将包含不同CDR突变的片段进行组合，然后通过双酶切(HindⅢ和NotⅠ)和双粘端连接将点突变抗体连接到噬菌体展示载体中，最后通过电转将带有突变位点的抗体序列转入大肠杆菌SS320中。库容计算、噬菌体文库制备和文库筛选操作过程详见实施例1，选择了3个抗CD47亲和力成熟抗体A7-44、A7-28和A7-47(表1)。
表1抗CD47抗体的氨基酸序列(SEQ ID NO:)
抗体名称 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VL
A7H3L3 4 5 6 7 8 9 14 15
A7-44 4 5 6 7 16 9 14 17
A7-28 18 19 6 7 20 9 21 22
A7-47 4 5 23 24 8 25 26 27
实施例3抗CD47抗体的构建、表达和纯化
将如实施例1和2所述的抗人CD47抗体的VH(SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26)的编码序列与人IgG4的重链恒定区(SEQ ID NO:12)的编码序列连接构建获得抗CD47抗体的重链编码序列，抗人CD47抗体的VL(SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27)的编码序列与人的Kappa轻链恒定区(SEQ ID NO:13)的编码序列连接构建获得抗CD47抗体的轻链编码序列。将重链和轻链编码序列分别***表达质粒pcDNA3.4(Invitrogen)以构建抗CD47抗体的重链和轻链的表达载体。将重链和轻链表达载体分别转化到大肠杆菌DH5α中，37℃过夜培养，利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的抗体轻链和重链质粒以供真核表达使用。
通过ExpiCHO瞬转表达***(Thermo Fisher，A29133)表达的，在转染7-15天后，将表达有目的蛋白(即抗CD47抗体)的细胞培养上清于15000g高速离心10min，所得上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH 3.0)洗脱目的蛋白，接着用1M Tris- HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白置换至PBS缓冲液中。经SDS-PAGE鉴定、SEC纯度检测，获得纯度较高的抗CD47抗体A7H3L3、A7-44、A7-28和A7-47。
实施例4抗CD47抗体与CCRF-CEM细胞上CD47的结合活性
由于细胞水平的CD47相对于重组蛋白更接近天然构象，本实施例通过流式细胞术测定了本发明的抗CD47抗体和阳性对照抗体1F8(WO2018075857A1，重链可变区(VH)氨基酸序列示于SEQ ID NO:28，轻链可变区(VL)氨基酸序列示于SEQ ID NO:29，制备方法如实施例3所示)在CCRF-CEM细胞上的结合能力。
具体方法如下：取1×10 5个CCRF-CEM细胞，低速离心(300g)去上清；将离心管底部的细胞通过配制好的FACS缓冲液(含体积百分比为2％FBS的1×PBS缓冲液)润洗一次，然后向润洗后的细胞中加入梯度稀释的待测抗体，在4℃孵育1h；接着再用上述FACS缓冲液润洗三次，加入PE标记的山羊抗人IgG Fc抗体(Abcam，ab98596)0.5μg，在4℃孵育1h；然后将细胞用FACS缓冲液润洗三次后用200μL FACS缓冲液重悬，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测CCRF-CEM细胞上结合的抗CD47抗体的量(表示为平均荧光强度(MFI))。
结果显示在图1A和1B中，抗体A7H3L3、A7-44、A7-28和A7-47以高结合活性结合CCRF-CEM细胞上的CD47，并且结合活性优于1F8。
实施例5抗CD47抗体与红细胞上CD47的结合活性
为了测定本发明的抗CD47抗体是否结合红细胞上的CD47，通过流式细胞术(FACS)测定了抗体A7H3L3、A7-44、A7-28和A7-47与红细胞的结合活性。作为比较，还测定阳性对照抗体1F8和F4AM4(本申请人自研的另一抗CD47抗体，重链的氨基酸序列为SEQ ID NO:30，轻链的氨基酸序列为SEQ ID NO:31)与红细胞的结合活性。人IgG4用作同种型阴性对照。
具体方法如下：从1mL抗凝处理的人血液中分离红细胞，离心后吸去上清，PBS润洗两次后,加入1mL PBS并重悬。使用PBS将红细胞稀释至1×10 7/mL，以每孔50μL吸取红细胞加入到96孔圆底细胞培养板中，然后等体积加入梯度稀释的待测抗体，充分混匀，置于4℃孵育1h。接着再用FACS缓冲液润洗三次，加入PE标记的山羊抗人IgG Fc抗体(Abcam，ab98596)0.5μg，在4℃孵育1h。其后，经FACS缓冲液润洗三次，并向细胞中加入200μL FACS缓冲液重悬细胞，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测红细胞上结合的抗CD47抗体的量(MFI)。
结果如图2A-2B显示，抗体A7H3L3与红细胞上的CD47几乎不结合，且结合活性与阳性对照抗体1F8相当或更弱；抗体A7-44和A7-47与红细胞上的CD47有微弱结合；抗体A7-28与红细胞上的CD47有一定的结合(但也远远弱于阳性对照抗体F4AM4)。这些结果表明抗体A7H3L3、A7-44、A7-47基本不与红细胞结合，同时展现出更高的靶向CD47阳性肿瘤细胞的特异性。
实施例6抗CD47抗体阻断CD47与SIRPα结合的阻断活性
通过流式细胞术(FACS)测定了本发明的抗CD47抗体阻断肿瘤细胞上的CD47与受体SIRPα结合的阻断活性。人IgG4用作同种型阴性对照。
具体方法如下：取1×10 5个CCRF-CEM细胞，低速离心(300g)去上清。将离心管底部的细胞通过配制好的FACS缓冲液(含2％FBS的1×PBS缓冲液)润洗一次，然后向润洗后的细胞中加入梯度稀释的待测抗体，孵育1h。用FACS缓冲液润洗细胞两次后加入100μL的1μg/mL的SIRPα-mFc(ACRO，SIA-H52A8)，在4℃孵育1h。经FACS缓冲液润洗三次，加入100μL PE标记的羊抗鼠Fc二抗(1:200，Abcam，ab98742)，在4℃孵育1h后离心去上清，向细胞中加入200μL FACS缓冲液重悬细胞，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测CCRF-CEM细胞上结合的SIRPα-mFc的量(MFI)。
结果显示在图3中，抗体A7-44、A7-28和A7-47有效阻断肿瘤细胞上的CD47与SIRPα的结合，并且阻断能力优于阳性对照抗体1F8。
实施例7抗CD47抗体对红细胞的血凝反应
抗体药通过血液循环到达肿瘤病灶部位从而发挥药效，而血液中的红细胞大量表达CD47。对于以高结合活性结合红细胞表面CD47的抗体，当抗体浓度达到一定水平时可能通过交联作用引起红细胞的聚集，从而使聚集的红细胞被吞噬细胞清除，一定程度上是造成贫血等毒性反应的重要原因之一。本实施例比较了本发明的抗CD47抗体和阳性对照抗体1F8、Hu5F9(又名magrolimab，可参见美国专利申请US20160304609A1)对红细胞的血凝反应。人IgG4用作同种型阴性对照。
具体方法如下：从1mL抗凝处理的人血液中分离红细胞，离心后吸去上清，PBS润洗两次后，加入1mL PBS并重悬。使用PBS将红细胞稀释20倍，以每孔50μL吸取红细胞加入到96孔圆底细胞培养板中，然后等体积加入梯度稀释(0.05-100μg/mL)的待测抗体，充分混匀，置于37℃培养箱孵育3h。之后取出观察血凝反应程度，红细胞沉积的面积大小代表了血凝的强度，面积越大代表血凝越强，反之越弱。
结果如图4A显示，抗体A7处理的红细胞聚集成一小团沉在圆孔底部，与阴性对照结果一致，表示其不具有血凝反应，而阳性对照抗体Hu5F9处理的红细胞分散面积大，显示血凝反应非常严重，这与专利US20160304609A1中报道结果是一致的。这表明嵌合抗体A7在血凝反应方面相对于阳性对照抗体Hu5F9更为优异，预期在临床上会显示出较低的贫血毒性或者副作用。
结果如图4B显示，抗体A7H3L3、A7-44和A7-47处理的红细胞聚集成一小团沉在圆孔底部，与阴性对照(IgG4)结果一致，表示其不引发血凝反应，而A7-28处理的红细胞有一定程度的血凝，基本与抗体1F8相当。以上结果表明，抗体A7H3L3、A7-44和A7-47在血凝反应方面相对于抗体1F8更为优异，预期在临床上会显示出较低的贫血毒性或者副作用。
实施例8抗CD47抗体促进巨噬细胞吞噬肿瘤细胞的能力
阻断CD47和SIRPα结合的抗体可以有效促进巨噬细胞对CD47阳性肿瘤细胞的吞噬。本实施例比较了本发明的抗CD47抗体和阳性对照抗体1F8促进巨噬细胞吞噬肿瘤细胞的能力。人IgG4用作同种型阴性对照。
具体方法如下：首先分离人外周血单个核细胞PBMC，加入50ng/mL rhM-CSF(购自Peprotech，300-25-10)用于诱导细胞分化为巨噬细胞，37℃细胞培养箱中培养约8天后，吸除上清和未贴壁的细胞，然后加入Accutase TM细胞消化液(购自Sigma，A6964)，在37℃孵育45min，消化贴壁细胞并用完全培养基(RPMI1640+10％FBS)制备成均一的细胞悬液，按5×10 4个细胞每孔分装至96孔板中。加入梯度稀释的待测抗体(1μg/mL-0.002μg/mL)，在37℃孵育30min，然后再加入2.5×10 4个 CFSE(购自Abcam，ab113853)标记的CCRF-CEM细胞，在37℃孵育1h后加入0.25μg APC标记的抗CD14抗体(购自eBioscience，17-0149-42)。然后将细胞悬液于4℃孵育20min，然后500g离心5min，用FACS缓冲液将细胞洗两遍，最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)进行检测，其中CD14 +巨噬细胞中吞噬有CFSE标记的CCRF-CEM细胞的比例即为吞噬率。
结果如图5显示，抗体A7-28和A7-47剂量依赖性地促进巨噬细胞对肿瘤细胞的吞噬，且其促进巨噬细胞吞噬肿瘤细胞的效果均优于抗体1F8。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据本文公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的保护范围由所附权利要求及其任何等同物给出。
序列表
Figure PCTCN2022118015-appb-000001
Figure PCTCN2022118015-appb-000002
Figure PCTCN2022118015-appb-000003

Claims (20)

  1. 一种抗CD47抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
    所述重链可变区包含SEQ ID NO:4或其变体的HCDR1序列，SEQ ID NO:5或其变体的HCDR2序列和SEQ ID NO:6或其变体的HCDR3序列；
    所述轻链可变区包含SEQ ID NO:7或其变体的LCDR1序列，SEQ ID NO:8或其变体的LCDR2序列和SEQ ID NO:9或其变体的LCDR3序列；
    其中所述变体各自相对于其所源自的序列独立地包含1个氨基酸的取代、添加或缺失。
  2. 权利要求1的抗体或其抗原结合片段，其中
    所述重链可变区包含SEQ ID NO:4或SEQ ID NO:18所示HCDR1序列，SEQ ID NO:5或SEQ ID NO:19所示HCDR2序列和SEQ ID NO:6或SEQ ID NO:23所示HCDR3序列；
    所述轻链可变区包含SEQ ID NO:7或SEQ ID NO:24所示LCDR1序列，SEQ ID NO:8、SEQ ID NO:16或SEQ ID NO:20所示LCDR2序列和SEQ ID NO:9或SEQ ID NO:25所示LCDR3序列。
  3. 权利要求1或2的抗体或其抗原结合片段，其中
    所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:6所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:8所示LCDR2序列和SEQ ID NO:9所示LCDR3序列；或者
    所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:6所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:16所示LCDR2序列和SEQ ID NO:9所示LCDR3序列；或者
    所述重链可变区包含SEQ ID NO:18所示HCDR1序列，SEQ ID NO:19所示HCDR2序列和SEQ ID NO:6所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:7所示LCDR1序列，SEQ ID NO:20所示LCDR2序列和SEQ ID NO:9所示LCDR3序列；或者
    所述重链可变区包含SEQ ID NO:4所示HCDR1序列，SEQ ID NO:5所示HCDR2序列和SEQ ID NO:23所示HCDR3序列，并且所述轻链可变区包含SEQ ID NO:24所示LCDR1序列，SEQ ID NO:8所示LCDR2序列和SEQ ID NO:25所示LCDR3序列。
  4. 权利要求1-3中任一项的抗体或其抗原结合片段，其中
    所述重链可变区包含：1)SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列；2)与SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列；或者3)与SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:21或SEQ ID NO:26的氨基酸序列相比具有一个或多个氨基酸的取代、添加和/或缺失的氨基酸序列。
  5. 权利要求1-4中任一项的抗体或其抗原结合片段，其中
    所述轻链可变区包含：1)SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27的氨基酸序列；2)与SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27具有至少80％、至少85％、至少90％、至少91％、至少92％、至少 93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列；或者3)与SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:22或SEQ ID NO:27相比具有一个或多个氨基酸的取代、添加和/或缺失的氨基酸序列。
  6. 权利要求1-5中任一项的抗体或其抗原结合片段，其中
    所述重链可变区包含SEQ ID NO:10的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:11的氨基酸序列；或者
    所述重链可变区包含SEQ ID NO:14的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:15的氨基酸序列；或者
    所述重链可变区包含SEQ ID NO:14的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:17的氨基酸序列；或者
    所述重链可变区包含SEQ ID NO:21的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:22的氨基酸序列；或者
    所述重链可变区包含SEQ ID NO:26的氨基酸序列，并且所述轻链可变区包含SEQ ID NO:27的氨基酸序列。
  7. 权利要求1-6中任一项的抗体或其抗原结合片段，其为嵌合抗体、人源化抗体、人抗体、scFv、Fab、Fab'、F(ab') 2、Fv片段、二硫键稳定的Fv(dsFv)或双抗体。
  8. 权利要求1-7中任一项的抗体或其抗原结合片段，其进一步包含重链恒定区和/或轻链恒定区；
    优选地，所述重链恒定区为人IgG4的重链恒定区和/或所述轻链恒定区为人κ轻链恒定区；
    更优选地，所述重链恒定区包含：1)SEQ ID NO:12的氨基酸序列；或者2)与SEQ ID NO:12具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列；和/或所述轻链恒定区包含：1)SEQ ID NO:13的氨基酸序列；或者2)与SEQ ID NO:13具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
  9. 权利要求1-8中任一项的抗体或其抗原结合片段，其
    1)特异性结合CD47阳性癌细胞但不结合或基本不结合红细胞；
    2)不引起或基本不引起红细胞凝集；
    3)阻断CD47与SIRPα的相互结合；和/或
    4)促进巨噬细胞对CD47阳性肿瘤细胞的吞噬。
  10. 一种多特异性抗体，其包含结合CD47的第一抗原结合部分以及结合第二抗原的第二抗原结合部分，其中所述第一抗原结合部分包含权利要求1-7中任一项的抗体或其抗原结合片段。
  11. 权利要求10的多特异性抗体，其中所述第二抗原结合部分特异性结合肿瘤抗原、免疫调节受体和免疫检查点分子；
    优选地，所述第二抗原结合部分特异性结合SIRPα、PD-1、PD-L1、LAG3、TIM-3、CTLA-4、VISTA、GPC3、EGFR、HER-2、CD19、CD20、CD33、CD40、CD73、OX40、CD3、DLL-3、TIP-1、叶酸受体α(FOLR1)和/或其他肿瘤抗原。
  12. 一种嵌合抗原受体，其包含权利要求1-7中任一项的抗体或其抗原结合片段。
  13. 一种免疫效应细胞，其在表面表达权利要求12嵌合抗原受体；优选地，所述免疫效应 细胞选自细胞毒性T细胞、自然杀伤细胞和自然杀伤T细胞。
  14. 一种多核苷酸，其编码权利要求1-9中任一项的抗体或其抗原结合片段。
  15. 一种表达载体，其包含权利要求14的多核苷酸。
  16. 一种宿主细胞，其包含权利要求14的多核苷酸或权利要求15的表达载体。
  17. 一种抗体缀合物，其包含与至少一种治疗剂缀合的权利要求1-9中任一项的抗体或其抗原结合片段或者权利要求10或11的多特异性抗体。
  18. 一种药物组合物，其包含权利要求1-9中任一项的抗体或其抗原结合片段、权利要求10或11的多特异性抗体、权利要求13的免疫效应细胞或者权利要求17的抗体缀合物，以及药学上可接受的载剂。
  19. 权利要求1-9中任一项的抗体或其抗原结合片段、权利要求10或11的多特异性抗体、权利要求17的抗体缀合物或者权利要求18的药物组合物在制备用于治疗癌症的药物中的用途；优选地，所述癌症为CD47阳性的血液瘤或实体瘤；较优选地，所述癌症选自急性淋巴细胞性白血病(ALL)、急性骨髓性白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性骨髓性白血病(CML)、骨髓增生异常综合征、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、滤泡性淋巴瘤、多发性骨髓瘤(MM)、巨细胞骨髓瘤、乳腺癌、卵巢癌、肺癌、胰腺癌、***癌、黑素瘤、结直肠癌、肺癌、头颈癌、膀胱癌、食道癌、肝癌、胃癌、肾癌、平滑肌瘤、胶质瘤和成胶质细胞瘤。
  20. 权利要求19的用途，其中所述药物与选自以下的一种或多种治疗剂联合使用：化疗剂、放射性同位素、免疫检查点抑制剂和肿瘤抗原靶向药物。
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