Anti-CD47 Antibodies and Their Uses

Abstract

The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-CD47 antibodies and their uses.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-CD47 antibodies and uses thereof. Background Art

[0002] CD47 is a transmembrane glycoprotein widely expressed on the cell surface and belongs to the immunoglobulin superfamily. It can interact with signal regulatory protein α (SIRPα), thrombospondin (TSP1), and integrins to mediate a series of responses such as cell apoptosis, proliferation, and immunity. In the innate immune system, CD47 functions by binding to SIRPα expressed by myeloid cells such as macrophages, neutrophils, and dendritic cells to transmit inhibitory "don't eat me" signals, thereby inhibiting the phagocytosis of target cells expressing CD47 by myeloid cells (especially macrophages). Therefore, the role of CD47's widespread expression under physiological conditions is to protect healthy cells from being eliminated by the innate immune system. However, tumor cells effectively escape immune surveillance by overexpressing CD47. On the other hand, macrophages infiltrating tumor tissues, also known as tumor-associated macrophages (TAMs), often lose their immune effector function of phagocytosis and clearance of tumor cells, and even have immunosuppressive effects, promoting tumor proliferation and invasion. It has been demonstrated that blocking the CD47-SIRPα pathway with anti-CD47 antibodies can effectively mediate phagocytosis of tumor cells, thereby inhibiting the growth of various hematological and solid tumors in vivo. However, CD47 is not only highly expressed on tumor cells, but also on normal cells, such as red blood cells. Therefore, therapies targeting CD47 may cause undesirable side effects.

[0003] Some anti-CD47 antibodies disclosed in the prior art (see, for example, US20160304609A1) bind to red blood cells, which not only causes severe anemia but also requires a dosage of up to 30 mg / kg. These characteristics pose significant challenges to the clinical application of anti-CD47 antibodies. There is an urgent need to develop new therapies and drugs targeting CD47 to expand the application of CD47 as a therapeutic target and macrophages as immune regulatory and effector cells. Summary of the Invention

[0004] In one aspect, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein

[0005] The heavy chain variable region comprises a HCDR1 sequence of SEQ ID NO: 4 or a variant thereof, a HCDR2 sequence of SEQ ID NO: 5 or a variant thereof, and a HCDR3 sequence of SEQ ID NO: 6 or a variant thereof;

[0006] The light chain variable region comprises a LCDR1 sequence of SEQ ID NO: 7 or a variant thereof, a LCDR2 sequence of SEQ ID NO: 8 or a variant thereof, and a LCDR3 sequence of SEQ ID NO: 9 or a variant thereof;

[0007] wherein each of the variants independently comprises a substitution, addition or deletion of 1 amino acid relative to the sequence from which it is derived.

[0008] In some embodiments, 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 HCDR3 sequence shown in SEQ ID NO: 6 or SEQ ID NO: 23;

[0009] The light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:7 or SEQ ID NO:24, the LCDR2 sequence shown in SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:20, and the LCDR3 sequence shown in SEQ ID NO:9 or SEQ ID NO:25.

[0010] In some embodiments, 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 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

[0011] 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 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; or

[0012] 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 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; or

[0013] 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 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.

[0014] In one embodiment, 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; or

[0015] 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

[0016] 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

[0017] 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

[0018] 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.

[0019] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and / or a light chain constant region. In one embodiment, the heavy chain constant region is a human IgG4 heavy chain constant region and / or the light chain constant region is a human kappa light chain constant region.

[0020] 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 an anti-CD47 antibody or an antigen-binding fragment thereof of the present invention.

[0021] In another aspect, the present invention further provides a chimeric antigen receptor comprising the anti-CD47 antibody or antigen-binding fragment thereof of the present invention. Accordingly, the present invention further provides an immune effector cell expressing the chimeric antigen receptor of the present invention on its surface.

[0022] In another aspect, the present invention provides a polynucleotide encoding an anti-CD47 antibody or antigen-binding fragment thereof of the present invention. The present invention also relates to an expression vector comprising the polynucleotide of the present invention. The present invention also relates to a host cell comprising the polynucleotide of the present invention or the expression vector.

[0023] The present invention also provides an antibody conjugate comprising the anti-CD47 antibody or antigen-binding fragment thereof or multispecific antibody of the present invention conjugated to at least one therapeutic agent.

[0024] In another aspect, the present invention also provides a pharmaceutical composition comprising 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.

[0025] 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 drug for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A-1B The binding activity of antibodies A7H3L3, A7-44, A7-28, and A7-47 to CD47 on CCRF-CEM cells is shown.

[0027] Figures 2A-2B The binding activity of antibodies A7H3L3, A7-44, A7-28, and A7-47 to CD47 on erythrocytes is shown.

[0028] Figure 3 Antibodies A7H3L3, A7-44, A7-28, and A7-47 were shown to block the binding activity of human CD47 to SIRPα on CCRF-CEM cells.

[0029] Figures 4A-4B The hemagglutination reactions of antibodies A7, A7H3L3, A7-44, A7-28, and A7-47 on erythrocytes are shown.

[0030] Figure 5 Antibodies A7-28 and A7-47 were shown to promote the phagocytosis of CCRF-CEM cells by macrophages. DETAILED DESCRIPTION

[0031] definition

[0032] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0033] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly 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."

[0034] As used herein, the connection term "and / or" between multiple stated elements should be understood to include both individual and combined options.

[0035] Unless otherwise indicated, any numerical value or numerical range, such as concentration or concentration range, is understood to be modified by the term "about" in any case. Thus, 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. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions within the range, unless the context clearly indicates otherwise.

[0036] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen binding site. In this article, the definition of antibody encompasses antigen binding fragments. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen binding fragments. Antibodies can be synthetic (e.g., produced by chemical coupling or biological coupling), enzymatically treated, or recombinantly produced. The antibodies provided herein include any immunoglobulin type (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). Antibodies can be "monovalent," "divalent," "trivalent," or "tetravalent" or more valent antibodies, meaning that they contain 1, 2, 3, 4, or more antigen binding sites.

[0037] As used herein, "antigen-binding fragment" refers to a portion of a full-length antibody that is less than full-length but comprises at least a portion of the variable region of the full-length antibody (e.g., comprising one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the full-length antibody's ability to specifically bind to an antigen. Antigen-binding fragments may, for example, include antibody derivatives produced by enzymatic treatment of a full-length antibody, synthetically produced derivatives, and recombinantly produced derivatives. Examples of antigen-binding fragments include, but are not limited to, sdAb (e.g., variable domains of heavy chain antibodies), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab')2, diabodies, Fd and Fd' fragments, and other fragments (e.g., fragments comprising modifications).

[0038] As used herein, 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 end) to the C-terminus (carboxyl end). 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. Generally speaking, the heavy chain constant region of a full-length antibody may comprise CH1-hinge region (hinge)-CH2-CH3 from the N-terminus to the C-terminus. In certain immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region may comprise CH1-hinge region-CH2-CH3-CH4 from the N-terminus to the C-terminus.

[0039] The light chain variable region and the heavy chain variable region can each include three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", and are connected from the N-terminus to the C-terminus in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Herein, the CDRs (CDRL or LCDR) of the light chain variable region can be referred to as LCDR1, LCDR2 and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region can be referred to as HCDR1, HCDR2 and HCDR3.

[0040] In the present invention, the amino acid sequences of CDRs are all shown according to the AbM definition rules (the sequences in the claims of the present invention are also shown according to the AbM definition rules). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, 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, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, ACR 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, RM et al., (1996) J. Mol. Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System ColdSpring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described herein.Although the scope of protection requested in the claims of the present invention is based on the sequences shown in the AbM definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

[0041] Thus, when referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0042] As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the terms "framework region," "framework region," or "FR" residues refer to those amino acid residues in the antibody variable region excluding the CDR sequences as defined above.

[0043] An "Fv" fragment, consisting of a single VH and a single VL through non-covalent interactions, is generally considered the smallest antigen-binding fragment containing an antigen-binding site. However, a single variable domain (single-domain antibody) also possesses antigen-binding ability. A "single-chain Fv (scFv)" can be obtained by linking the VH and VL via a peptide linker. By introducing disulfide bonds into an Fv or scFv, a "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained, respectively.

[0044] As used herein, "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). A single-chain "Fab (scFab)" can be obtained by linking the CL and CH1 in "Fab" with a peptide linker. "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 bond in the hinge region of F(ab')2.

[0045] "Hinge region" refers to the portion of an antibody that connects the immunoglobulin Fab and Fc fragments. The hinge region can be a complete hinge region or a portion thereof. For IgG, the Fc region generally includes a portion of the hinge region connected to CH2 and CH3. In this article, when used for chimeric antigen receptors, the hinge region can also refer to any functional equivalent, such as the portion connecting the constant region and the transmembrane domain in a T cell receptor. Those skilled in the art can determine the position of VH, VL, CL, CH1, CH2, CH3 and the hinge region in an antibody based on known algorithms and software. For descriptions of applicable algorithms and software, see, 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.

[0046] As used herein, "diabody" refers to an antibody comprising two scFvs, wherein the VH and VL in each scFv are connected by a short peptide linker (about 5-10 amino acid residues), so that the VH and VL chains are paired (i.e., the VH of the first scFv is paired with the VL of the second scFv, and the VL of the first scFv is paired with the VH of the second scFv) to form an antigen binding site. The diabody can be a bispecific antibody.

[0047] As used herein, "chimeric antibody" refers to an antibody in which a portion (e.g., CDR, FR, variable region, constant region, or a combination thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining portion is identical or homologous to the corresponding sequence in an antibody derived from another species. In some embodiments of the present invention, a chimeric antibody comprises a variable region derived from a non-human species (e.g., mouse) and a constant region derived from a different species (e.g., human). A chimeric antibody may also refer to a multispecific antibody that is specific 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. In particular, chimeric antibodies can be generated by recombinant DNA technology (e.g., see Sambrook, J., et al. (1989). Molecular cloning: a laboratory manual, 2nded. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0048] As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody is modified to increase sequence homology with a human antibody. Humanized antibodies generally retain the antigen-binding ability of the non-human antibody from which they are derived and have lower immunogenicity to the human body. Humanized antibodies can be obtained by engineering any non-human species antibody or an antibody (e.g., a chimeric antibody) comprising a sequence of a non-human species. Non-human species may, for example, include mice, rats, rabbits, alpacas, sharks, or non-human primates. The technology for obtaining humanized antibodies from non-human antibodies is well known to those skilled in the art. For example, the CDR sequences of non-human antibodies (e.g., mouse antibodies) are transplanted into human antibody framework regions. In some cases, in order to maintain the antigen-binding ability and / or stability of the humanized antibody, key amino acid residues of the non-human antibody (e.g., murine antibody) framework sequence can be retained in the human antibody framework region, i.e., "back mutations" are performed (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21): 6851-6855; Neuberger et al. (1984) Nature 312: 604-608).

[0049] As used herein, the term "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 complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0050] As used herein, 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 has improved affinity for the antigen compared to a parent antibody that does not comprise such modifications. 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).

[0051] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has an art-recognized definition and refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This 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.

[0052] As used herein, an amino acid sequence that is "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of a human immunoglobulin may 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% or 100% sequence identity with the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

[0053] Non-critical regions in a polypeptide (e.g., CDR regions of an antibody, non-critical amino acids in the framework region, and amino acids in the constant region) can be modified, for example, by substitution, addition, and / or deletion of one or more amino acids without changing the function of the polypeptide. Those skilled in the art will appreciate that amino acids in non-critical regions of a polypeptide can be substituted with suitable conservative amino acids, and generally do not change their biological activity (see, for example, 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. In some cases, amino acid substitutions are non-conservative substitutions. Those skilled in the art will appreciate that 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 providing active groups for the preparation of antibody conjugates. Antibodies or antigen-binding fragments thereof containing such amino acid mutations or modifications are also encompassed within the scope of the antibodies or antigen-binding fragments thereof of the present invention.

[0054] "Affinity" or "binding affinity" is a measure of the strength of the non-covalent binding between an antibody and an antigen. The magnitude of "affinity" is usually reported as the equilibrium dissociation constant, K. D or EC 50 .K D The equilibrium association constant (ka) and the equilibrium dissociation constant (kd) can be calculated: D= kd / ka. Affinity can be determined using conventional techniques known in the art, such as biofilm interferometry (e.g., using the Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0055] The anti-CD47 antibody or antigen-binding fragment, multispecific antibody or polynucleotide encoding the same of the present invention can be isolated. As used herein, the expression "isolated" means that the substance (e.g., polynucleotide or polypeptide) is separated from its source or environment, i.e., does not substantially contain any other components.

[0056] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, which generally can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0057] In this article, "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. The vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, pox virus vectors, and baculovirus vectors, etc.

[0058] As used herein, the term "expression" refers to the production of RNA and / or polypeptides.

[0059] As used herein, "expression vector" refers to a vector capable of expressing a polynucleotide of interest (including DNA and RNA). For example, in an expression vector, a polynucleotide sequence encoding a polypeptide of interest (including DNA and RNA) can be operably linked to a regulatory sequence (such as a promoter and a ribosome binding site) that can affect the expression of the polynucleotide sequence. The regulatory sequence can include a promoter and a terminator sequence, and optionally can include an origin of replication, a selective marker, an enhancer, a polyadenylation signal, etc. The expression vector can be a plasmid, a phage vector, a recombinant virus, or other vector that, when introduced into an appropriate host cell, results in the expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. Those skilled in the art can prepare the expression vector as a vector that is replicable in a host cell, remains free in the host cell, or is integrated into the host cell genome as needed.

[0060] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. A host cell can also be used to express a polynucleotide or a polypeptide encoded by a vector. The host cell can be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells include Escherichia coli (E. coli) or 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).

[0061] As used herein, the term "treatment" refers to the improvement of a disease / symptom, such as alleviating or eliminating the disease / symptom, preventing or slowing the occurrence, progression and / or worsening of the disease / symptom. Therefore, treatment includes prevention, therapy and / or cure.

[0062] "Effective amount" refers to the amount required to prevent, cure, improve, block or partially block a disease or symptom. For example, for the treatment of tumors, an "effective amount" of the anti-CD47 antibody or its antigen-binding fragment, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention preferably inhibits tumor cell growth or tumor growth 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%, and more preferably at least about 80% relative to an untreated subject. The efficacy of inhibiting tumor growth can be assessed using conventional tumor animal models in the art, such as spontaneous tumors, induced tumors, 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 the antibody or its antigen-binding fragment, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention can reduce tumor size or otherwise alleviate the symptoms of the subject (such as preventing and / or treating metastasis or recurrence). One skilled in the art can determine the effective amount based on factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition or route of administration, etc. The effective amount can be administered in one or more administrations.

[0063] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, sodium chloride, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). 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. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, 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 degradation products thereof (such as lactalbumin hydrolyzate).

[0064] Anti-CD47 antibody or antigen-binding fragment thereof

[0065] In one general aspect, the present invention provides anti-CD47 antibodies or antigen-binding fragments thereof that specifically recognize and bind to CD47.

[0066] As used herein, the term "CD47" refers to the leukocyte surface antigen CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen, or protein MER6. In some embodiments, an anti-CD47 antibody or antigen-binding fragment thereof specifically binds to human CD47. The term "human CD47" refers to CD47 derived from humans. An exemplary amino acid sequence of human CD47 is set forth in GenBank Accession No. NP_001768.1.

[0067] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody, a humanized antibody, a human antibody, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-stabilized Fv (dsFv), or a diabody.

[0068] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention are

[0069] 1) specifically binds to CD47-positive cancer cells but does not bind or substantially does not bind to red blood cells;

[0070] 2) does not cause or barely causes red blood cell agglutination;

[0071] 3) blocking the interaction between CD47 and SIRPα; and / or

[0072] 4) Promote the phagocytosis of CD47-positive tumor cells by macrophages.

[0073] variable region

[0074] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3.

[0075] In some embodiments, the heavy chain variable region comprises a HCDR1 sequence as set forth in SEQ ID NO: 4 (GFNIKDIYIY), a HCDR2 sequence as set forth in SEQ ID NO: 5 (KIDPANGNTK), and a HCDR3 sequence as set forth in SEQ ID NO: 6 (GYGSGFAY). In some embodiments, the light chain variable region comprises a LCDR1 sequence as set forth in SEQ ID NO: 7 (RASQDISNHLN), a LCDR2 sequence as set forth in SEQ ID NO: 8 (YTSRIHS), and a LCDR3 sequence as set forth in SEQ ID NO: 9 (QQGYTLPFT).

[0076] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention are obtained by affinity maturation. Such anti-CD47 antibodies or antigen-binding fragments thereof may comprise one or more variants of the 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), and LCDR3 (SEQ ID NO: 9) sequences described above. In one embodiment, each of the variants independently comprises one amino acid substitution, addition, or deletion relative to the sequence from which it is derived. Preferably, each of the variants independently comprises one amino acid substitution relative to the sequence from which it is derived.

[0077] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0078] The heavy chain variable region comprises a HCDR1 sequence of SEQ ID NO: 4 or a variant thereof, a HCDR2 sequence of SEQ ID NO: 5 or a variant thereof, and a HCDR3 sequence of SEQ ID NO: 6 or a variant thereof;

[0079] The light chain variable region comprises a LCDR1 sequence of SEQ ID NO: 7 or a variant thereof, a LCDR2 sequence of SEQ ID NO: 8 or a variant thereof, and a LCDR3 sequence of SEQ ID NO: 9 or a variant thereof;

[0080] wherein each of the variants independently comprises a substitution, addition or deletion of 1 amino acid relative to the sequence from which it is derived.

[0081] In some embodiments, the HCDR1 sequence comprises the amino acid sequence of SEQ ID NO: 4, wherein the amino acid at position 7 is substituted. In one embodiment, the amino acid at position 7 in SEQ ID NO: 4 is substituted with V (SEQ ID NO: 18; GFNIKDVYIY).

[0082] In some embodiments, the HCDR2 sequence comprises the amino acid sequence of SEQ ID NO: 5, wherein the amino acid at position 10 is substituted. In one embodiment, the amino acid at position 10 in SEQ ID NO: 5 is substituted with H (SEQ ID NO: 19; KIDPANGNTH).

[0083] In some embodiments, the HCDR3 sequence comprises the amino acid sequence of 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).

[0084] In some embodiments, the LCDR1 sequence comprises the amino acid sequence of SEQ ID NO:7, wherein the amino acid at position 10 is substituted. In one embodiment, the amino acid at position 10 in SEQ ID NO:7 is substituted to 1 (SEQ ID NO:24; RASQDISNHIN).

[0085] In some embodiments, the LCDR2 sequence comprises the amino acid sequence of SEQ ID NO:8, wherein the amino acid at position 7 is substituted. In one embodiment, the amino acid at position 7 in SEQ ID NO:8 is substituted with L (SEQ ID NO:16; YTSRIHL). In one embodiment, the amino acid at position 7 in SEQ ID NO:8 is substituted with K (SEQ ID NO:20; YTSRIHK).

[0086] In some embodiments, the LCDR3 sequence comprises the amino acid sequence of SEQ ID NO:9, wherein the amino acid at position 5 is substituted. In one embodiment, the amino acid at position 5 in SEQ ID NO:9 is substituted with H (SEQ ID NO:25; QQGYHLPFT).

[0087] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0088] 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 HCDR3 sequence shown in SEQ ID NO: 6 or SEQ ID NO: 23;

[0089] The light chain variable region comprises the LCDR1 sequence shown in SEQ ID NO:7 or SEQ ID NO:24, the LCDR2 sequence shown in SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:20, and the LCDR3 sequence shown in SEQ ID NO:9 or SEQ ID NO:25.

[0090] In one embodiment, 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 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.

[0091] In one embodiment, 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 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.

[0092] In one embodiment, 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 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.

[0093] In one embodiment, 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 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.

[0094] In some embodiments, the VH as described above further comprises a heavy chain framework region. In some embodiments, the VL as described above further comprises a light chain framework region. In some embodiments, the VH as described above further comprises a heavy chain framework region, and the VL as described above further comprises a light chain framework region. The heavy chain framework region and / or the light chain framework region can each independently be derived from the heavy chain framework region and the light chain framework region of any species immunoglobulin.

[0095] In some embodiments, the VH comprises a heavy chain framework region derived from a murine immunoglobulin, and / or the VL comprises a light chain framework region derived from a murine immunoglobulin.

[0096] In certain preferred embodiments, the VH comprises a heavy chain framework region derived from a human immunoglobulin, and / or the VL comprises a light chain framework region derived from a human immunoglobulin. Thus, in certain preferred embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention are humanized. The heavy chain framework region and / or light chain framework region of the humanized anti-CD47 antibodies or antigen-binding fragments thereof may comprise one or more non-human (e.g., mouse) amino acid residues, for example, the heavy chain framework region and / or light chain framework region may comprise one or more amino acid back mutations, wherein the back mutations comprise corresponding mouse amino acid residues.

[0097] In some embodiments, 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. In some embodiments, 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.

[0098] In some embodiments, the heavy chain variable region comprises an amino acid sequence having 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 26. In some embodiments, the heavy chain variable region comprises an amino acid sequence having one or more (e.g., 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: 10, SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 26. Preferably, the amino acid substitutions, additions, and / or deletions do not occur in the CDR regions.

[0099] In some embodiments, the light chain variable region comprises an amino acid sequence that 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%, or at least 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 27. In some embodiments, the light chain variable region comprises an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, and / or deletions compared to SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 27. Preferably, the amino acid substitutions, additions, and / or deletions do not occur in the CDR regions.

[0100] In some embodiments, the heavy chain variable region comprises: 1) an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 26; 2) an amino acid sequence that 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 26; or 3) an amino acid sequence that has one or more (e.g., 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: 10, SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 26; and

[0101] The light chain variable region comprises: 1) an 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) an amino acid sequence that 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%, or at least 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 27; or 3) an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 27. Preferably, the amino acid substitution, addition and / or deletion does not occur in the CDR region.

[0102] In one embodiment, 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.

[0103] constant region

[0104] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and / or a light chain constant region.

[0105] The heavy chain constant region and the light chain constant region can each independently be derived from the heavy chain constant region and the light chain constant region of the immunoglobulin of any species. The heavy chain constant region can be derived from the heavy chain constant region of the immunoglobulin 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), or a combination thereof. The light chain constant region can be derived from λ (Lambda) light chain or κ (Kappa) light chain constant region.

[0106] Appropriate immunoglobulin constant regions (e.g., CH1 and light chain constant regions, hinge region-CH2-CH3, CH1-hinge region-CH2-CH3 and light chain constant regions or Fc region), as well as types (e.g., IgG, such as IgG1, IgG2, IgG3 and IgG4) can be selected and, optionally, modified to obtain an antibody with the desired properties.

[0107] In preferred embodiments, the heavy chain constant region comprises at least an Fc region. For example, the heavy chain constant region of an IgG may include: 1) all or part of the hinge region - CH2-CH3; or 2) CH1-hinge region-CH2-CH3. In some embodiments, the heavy chain constant region is the heavy chain constant region (e.g., Fc region or CH1-hinge region-CH2-CH3) of a human IgG (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). In one embodiment, the heavy chain constant region is that of a human IgG1. In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the heavy chain constant region is that of a human IgG4. In one embodiment, the heavy chain constant region comprises: 1) an amino acid sequence of SEQ ID NO: 12; or 2) an amino acid sequence having 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%, or at least 99% sequence identity to SEQ ID NO: 12.

[0108] In a preferred embodiment, the light chain constant region is a human kappa light chain constant region. In one embodiment, the light chain constant region comprises: 1) the amino acid sequence of SEQ ID NO: 13; or 2) an amino acid sequence having 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%, or at least 99% sequence identity to SEQ ID NO: 13.

[0109] Antibodies or their antigen-binding fragments can be prepared and produced using methods known in the art. Such methods can include, for example, preparing and separating antibodies or antigen-binding fragments from phage display libraries, yeast display libraries, immortalized B cells (e.g., mouse B cell hybridomas or EBV immortalized B cells). It is also possible to use methods for immunizing animals, such as immunizing animals (e.g., mice) with antigens or DNA encoding antigens, and then separating the B cells expressing the antibodies from the animals after immunization. Preferably, the B cells expressing the antibodies are immortalized, such as prepared into hybridomas or EBV immortalized B cells. It is also possible to separate or prepare polynucleotides encoding antibodies or their antigen-binding fragments from immune animals using chemical synthesis methods, and then construct expression vectors using polynucleotides.

[0110] Multispecific antibodies

[0111] In one aspect, the invention provides a multispecific antibody comprising a first antigen-binding moiety that binds CD47 and a second antigen-binding moiety that binds a second antigen, wherein the first antigen-binding moiety comprises an anti-CD47 antibody of the invention or an antigen-binding fragment thereof.

[0112] As used herein, the term "multispecific antibody" refers to an antibody that can specifically bind to two or more (e.g., 2, 3, 4, 5, or 6) different antigenic epitopes. A multispecific antibody may be, for example, a bispecific, trispecific, or tetraspecific antibody, which can specifically bind to 2, 3, or 4 antigenic epitopes, respectively. As used herein, the term "epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to the antigen-binding site of an antibody. An antigenic epitope is typically composed of chemically active surface groups (e.g., amino acids or sugar side chains) of an antigen and typically has specific three-dimensional structural properties and specific charge properties. The second antigen may be an antigen other than CD47. The second antigen may also be CD47, which binds to a different antigenic epitope on CD47 than the anti-CD47 antibody or antigen-binding fragment thereof of the present invention. Whether the antigenic epitopes bound by the two antibodies are the same can be determined using conventional methods in the art, for example, by measuring competitive binding of the two antibodies to the same antigenic epitope by ELISA, flow cytometry, or surface plasmon resonance.

[0113] A multispecific antibody can be a multivalent (eg, bivalent, trivalent, tetravalent) antibody, ie, it has multiple antigen binding sites. A multispecific antibody can be, for example, a chimeric antibody, a humanized antibody, a scFab, a F(ab')2, or a diabody.

[0114] Methods for constructing multispecific antibodies using antibodies or antigen-binding fragments of interest are well known to those skilled in the art (see, for example, WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121:210; and Traunecker et al., (1991) EMBO, 10:3655-3659). Multispecific antibodies can be produced and isolated using various techniques known in the art. For example, a polynucleotide encoding a multispecific antibody can be obtained by recombinant DNA technology, optionally cloned into an expression vector, and then transformed into a host cell with the polynucleotide or expression vector. The transformed host cell is cultured under appropriate conditions to allow expression of the polynucleotide or expression vector, and finally the multispecific antibody is isolated and purified from the host cell or culture medium. It is also possible to obtain the various portions of the multispecific antibody separately, for example, the first antigen-binding portion and the second antigen-binding portion as described herein, and then enzymatically or chemically coupled to each portion, optionally via a linker, to obtain a multispecific antibody that specifically binds to CD47 and other antigens.

[0115] As used herein, "first antigen-binding portion" and "second antigen-binding portion" refer to amino acid sequences that contain an antigen-binding site and are capable of binding to an antigen epitope, and are defined within the meaning of an antibody or an antigen-binding fragment.

[0116] The first antigen binding portion 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. In some embodiments, the first antigen binding portion comprises an anti-CD47 antibody or antigen binding fragment thereof of the present invention. In one embodiment, the first antigen binding portion comprises VH and VL, which respectively comprise the HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of the anti-CD47 antibody or antigen binding fragment thereof of the present invention as described above. In one embodiment, the first antigen binding portion comprises VH and VL, which respectively comprise the VH and VL of the anti-CD47 antibody or antigen binding fragment thereof of the present invention as described above.

[0117] The second antigen binding portion can be an antibody or antigen binding fragment that binds to any antigenic epitope of interest. In one embodiment, the second antigen is another antigen different from CD47. The antigens that the second antigen binding portion can specifically bind to may include: tumor antigens (e.g., tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors, and immune checkpoint molecules. As used herein, "tumor-associated antigens" refer to antigens that are highly expressed in tumor cells and also present in healthy cells but at a lower expression level. As used herein, "tumor-specific antigens" refer to antigens that are specifically expressed in tumor cells and hardly expressed in healthy cells. Non-limiting examples of tumor antigens can 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, immune activating receptors (e.g., CD27, CD137, CD40, GITR, and OX40) and immunoinhibitory receptors (e.g., BTLA, CTLA4, LAG-3, and PD-1). For example, the second antigen binding moiety can be an agonist antibody to an immunoactivating receptor or an antagonist antibody to an immunoinhibitory receptor.

[0118] In one embodiment, the second antigen binding portion binds to tumor antigens, immunomodulatory receptors and immune checkpoint molecules. In one embodiment, the second antigen binding portion specifically binds 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, folate receptor alpha (FOLR1) and / or other tumor antigens.

[0119] The second antigen binding portion 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 of alpaca or an IgNAR variable domain of shark), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab') 2. In one embodiment, the second antigen binding portion comprises a single variable domain of an immunoglobulin.

[0120] The first antigen binding portion and the second antigen binding portion can optionally be connected by a linker. In some embodiments, the first antigen binding portion and the second antigen binding portion are not connected by a linker. In other embodiments, the first antigen binding portion and the second antigen binding portion are connected by a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen binding portion and the second antigen binding portion are connected by a peptide linker. Exemplary peptide linkers can include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or a combination thereof, such as GGAS, GGGS, GGGSG, or (G4S) n , where n is an integer from 1 to 20.

[0121] In some embodiments, the second antigen is an antigen other than CD47, and the binding affinity of the first antigen-binding moiety to CD47 is weaker than the binding affinity of the second antigen-binding moiety to the other antigen. In such embodiments, the multispecific antibodies of the present invention can preferentially target the other antigen, so that the multispecific antibodies specifically recognize target cells (e.g., cancer cells) that express the other antigen, while the target cells may or may not express CD47. In a specific embodiment, when the target cells simultaneously express the CD47 antigen and the second antigen other than CD47, the multispecific antibodies of the present invention have a stronger binding ability to CD47 than those expressing only CD47, and a stronger ability to block the binding between CD47 and SIRPα.

[0122] CAR and CAR-expressing immune effector cells

[0123] In another aspect, the present invention also provides a chimeric antigen receptor (CAR) comprising an anti-CD47 antibody or antigen-binding fragment thereof of the present invention. CAR is a recombinant receptor that simultaneously provides antigen binding and activates T cell function. CAR structure and engineering are described in, for example, Dotti G. et al., (2014) Immunol Rev. 257 (1): 107-126, which is incorporated herein by reference.

[0124] In an exemplary embodiment, the CAR of the present invention comprises, from N-terminus to C-terminus:

[0125] (a) an extracellular region comprising an anti-CD47 antibody or an antigen-binding fragment thereof of the present invention;

[0126] (b) a transmembrane region that connects the extracellular region and the intracellular signaling region and anchors the CAR to the cell membrane; and

[0127] (c) Intracellular signaling region.

[0128] The extracellular region comprises an anti-CD47 antibody or antigen-binding fragment thereof of the present invention, so that the CAR of the present invention binds to cells (e.g., cancer cells) expressing CD47. In a preferred embodiment, the extracellular region in the CAR is in the form of scFv.

[0129] The transmembrane region connects the extracellular region and the intracellular signaling region and anchors the CAR to the cell membrane.

[0130] When the extracellular region of CAR binds to the antigen it recognizes, the intracellular signaling region transmits T cell receptor (TCR)-like signals into the cell and activates the immune effector cells expressing CAR to exert their effector functions.

[0131] In some embodiments, 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 region.

[0132] In some embodiments, the CAR of the present invention further comprises a spacer connecting the extracellular region and the transmembrane region.In one embodiment, the spacer is derived from the heavy chain constant region of an immunoglobulin.

[0133] In yet another aspect, the present invention also relates to a polynucleotide encoding the CAR of the present invention and an expression vector comprising the polynucleotide.

[0134] On the other hand, the present invention also provides a kind of immune effector cell, which expresses the CAR of the present invention on cell surface.Preferably, the immune effector cell is selected from T lymphocytes (such as cytotoxic T cells (CTL)), natural killer cells (NK) and natural killer T cells (NKT).Immune effector cells can target CD47 positive diseased cells (for example, CD47 positive cancer cells), and are activated to start effector function, for example, causing the death of CD47 positive cancer cells.

[0135] Polynucleotides, vectors and host cells

[0136] In another aspect, the present invention provides a polynucleotide comprising a polynucleotide sequence encoding an anti-CD47 antibody or antigen-binding fragment of the present invention or a multispecific antibody of the present invention.

[0137] The polynucleotides of the present invention can be obtained using methods known in the art. For example, the polynucleotides of the present invention can be isolated from phage display libraries, yeast display libraries, immune animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells) or chemically synthesized. The polynucleotides of the present invention can be codon-optimized for the host cell used for expression.

[0138] In yet another aspect, the present invention also provides an expression vector comprising a polynucleotide of the present invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The polynucleotide of the present invention may be present in one or more expression vectors. In one embodiment, the polynucleotide of the present invention is prepared as a recombinant nucleic acid. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) technology), etc.

[0139] The present invention also provides a host cell comprising a polynucleotide or expression vector of the present invention. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0140] In a preferred embodiment, host cells are used to express the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention or the multispecific antibodies of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., 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 antibody expression.

[0141] The present invention also provides a method for producing the anti-CD47 antibody or antigen-binding fragment thereof or the multispecific antibody of the present invention, comprising the following steps:

[0142] (I) culturing the host cells of the present invention under appropriate conditions to express the anti-CD47 antibody or antigen-binding fragment thereof or the multispecific antibody of the present invention, and

[0143] (II) Isolating the antibody or antigen-binding fragment thereof or the multispecific antibody of the present invention from the host cell or its culture.

[0144] Antibody conjugates

[0145] The present invention also provides an antibody conjugate comprising an anti-CD47 antibody or antigen-binding fragment thereof or a multispecific antibody of the present invention conjugated to at least one therapeutic agent. An antibody-drug conjugate (ADC) is a typical antibody conjugate, wherein the therapeutic agent may be, for example, a cytotoxic agent.

[0146] As used herein, "conjugation" refers to the attachment of two or more moieties to each other through covalent or non-covalent interactions. In a preferred embodiment, the conjugation is covalent conjugation.

[0147] The therapeutic agent can be selected from cytotoxic agents, therapeutic antibodies (e.g., antibodies or antigen-binding fragments thereof that specifically bind to another antigen), radioactive isotopes, oligonucleotides and their analogs (e.g., interfering RNA), biologically active peptides, protein toxins (e.g., diphtheria toxin, ricin toxin), and enzymes (e.g., urease).

[0148] Cytotoxic agents refer to substances that inhibit or reduce the activity, function and / or kill cells. Examples of cytotoxic agents may include, but are not limited to, maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatins, cryptophycins, vinca alkaloids (e.g., vinblastine, vincristine), colchicines, dolastatins, taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin, dihydroxyanthracindione, doxorubicin), cytotoxic antibiotics (e.g., mitomycin, actinomycin), dactinomycin, dactin, dactycin, dactin ...

[0013] Examples of the present invention include, but are not limited to, chloramphenicol, chlortetracycline ...

[0149] The radioisotope may be selected from, for example 212 Bi, 213 Bi, 131 I. 125 I. 111 In, 177 Lu, 186 Re、 188 Re、 153 Sm, 90 Y. Antibodies labeled with radioactive isotopes are also called radioimmunoconjugates.

[0150] In some embodiments, the therapeutic agent is selected from cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs. In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In another preferred embodiment, the therapeutic agent is a radioisotope.

[0151] The therapeutic agent can be conjugated to the antibody or antigen-binding fragment of the present invention or the multispecific antibody of the present invention via a linker using any technique known in the art. The linker can comprise an active group for covalent conjugation, such as an amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, sulfhydryl, or hydroxyl group.

[0152] Pharmaceutical composition

[0153] The present invention also provides a pharmaceutical composition comprising 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.

[0154] Pharmaceutically acceptable carriers may include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, 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, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitan,

[0014] In some embodiments, the carrier may be a surfactant (e.g., a glycerol ...

[0155] The pharmaceutical compositions provided herein can be in various dosage forms, including but not limited to solid, semisolid, liquid, powder or lyophilized forms. Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). For compositions comprising antibodies or antibody conjugates, preferred dosage forms can generally be, for example, injections and lyophilized powders.

[0156] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. The route of administration includes, but is not limited to, parenteral (for example, intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous or intracavitary), local (for example, intratumoral), epidural or mucosal (for example, intranasal, oral, vaginal, rectal, sublingual or topical). It will be understood by those skilled in the art that exact dosage will depend on various factors, such as the metabolic kinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of a specific compound, therapeutic purposes, route of administration and the condition of the subject, such as the patient's age, health status, weight, sex, diet, medical history, and other factors known to the medical field. Methods of administration can be, for example, injection or infusion.

[0157] As a general guide, the dosage range of the anti-CD47 antibodies or antigen-binding fragments thereof, antibody conjugates or multispecific antibodies of the present invention can be about 0.0001 to 100 mg / kg, more typically 0.01 to 20 mg / kg of the subject's body weight. For example, the dosage 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 in the range of 1-20 mg / kg. An exemplary treatment regimen requires administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or with a slightly shorter initial dosing interval and a longer dosing interval later. The administration route can be intravenous drip.

[0158] treat

[0159] In another aspect, the present invention relates to 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.

[0160] The present invention also relates to the anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention, which is used for treating diseases.

[0161] The present invention also provides a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CD47 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, immune effector cell, or pharmaceutical composition of the present invention.

[0162] In some embodiments, the disease is associated with abnormal expression of CD47. The term "abnormal expression" refers to a sample with a high or low protein expression level compared to a normal sample (or a standard sample, such as a sample from a subject not suffering from a disease associated with abnormal expression of CD47). Preferably, the disease is characterized by high expression of CD47. For example, CD47 is highly expressed in tissues (e.g., gastric cancer tissues and adjacent tissues) of subjects suffering from or suspected of having a disease (e.g., gastric cancer), while CD47 is lowly expressed in the corresponding tissues of subjects not suffering from the disease.

[0163] In one embodiment, the disease as described above is cancer. As used herein, "cancer" includes but is not limited to hematologic malignancies and solid tumors. In this article, "hematologic malignancies" refers to cancers of the blood, including leukemia, lymphoma, and myeloma. As a non-limiting example, leukemia can include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and myeloproliferative diseases (such as myelodysplastic syndrome). Lymphoma can include, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma. Myeloma can include, for example, multiple myeloma (MM) and giant cell myeloma. 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. Cancer can also be metastatic cancer. "Metastasis" refers to the spread of cancer cells from their original site to other parts of the body.

[0164] The present invention also relates to an anti-CD47 antibody or its antigen-binding fragment, multispecific antibody, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention for treating cancer. In some embodiments, the cancer is selected from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt 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.

[0165] The anti-CD47 antibodies or antigen-binding fragments thereof of the present invention can be used to treat CD47-positive hematological tumors and solid tumors. In some embodiments, the cancer is selected from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome, myelodysplastic syndrome, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt 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.

[0166] Depending on the second antigen-binding portion, the multispecific antibodies of the invention can be used to treat, for example, a cancer selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt 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, gastric cancer, liver cancer, kidney cancer, leiomyoma, glioma and glioblastoma, among others.

[0167] The anti-CD47 antibodies or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates, immune effector cells, or pharmaceutical compositions of the present invention can be administered in combination with at least one or more therapeutic agents described herein. The manner of combined administration is not limited. For example, all of the above therapeutic agents can be administered at once or separately.

[0168] For cancer treatment, the anti-CD47 antibodies or antigen-binding fragments thereof, multispecific antibodies, antibody conjugates or pharmaceutical compositions of the present invention can be used in combination with other treatment methods, including but not limited to surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition and palliative care.

[0169] In certain embodiments, the anti-CD47 antibody or its antigen-binding fragment, multispecific antibody, antibody conjugate or pharmaceutical composition of the present invention is further used in combination with one or more therapeutic agents selected from the following: 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. As used herein, "tumor-associated antigen" refers to an antigen that is highly expressed in tumor cells and also exists in healthy cells but at a lower expression level. As used herein, "tumor-specific antigen" refers to an antigen that is specifically expressed in tumor cells and is hardly expressed in healthy cells. Other non-limiting examples of therapeutic agents can include, for example, angiogenesis inhibitors, heparin deacetylase (HDAC) inhibitors, hedgehog signaling pathway blockers, mTOR inhibitors, p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, marizomib, oprozomib), and tyrosine kinase inhibitors (e.g., BTK inhibitors).

[0170] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof or multispecific antibody of the present invention is combined with one or more therapeutic agents selected from the group consisting of anti-SIRPα antibody, anti-CD20 monoclonal 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.

[0171] In some embodiments, the anti-CD47 antibodies, or antigen-binding fragments thereof, or multispecific antibodies of the present invention are used in combination with chemotherapeutic agents. In other embodiments, the antibody conjugates of the present invention are used in combination with immune checkpoint inhibitors. In still other embodiments, the antibodies, or antigen-binding fragments thereof, antibody conjugates, or pharmaceutical compositions of the present invention are used in combination with radioactive isotopes.

[0172] Reagent test kit

[0173] The present invention also provides a kit comprising an anti-CD47 antibody or antigen-binding fragment thereof, a multispecific antibody, an antibody conjugate, an immune effector cell, or a pharmaceutical composition of the present invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administration. Typically, the kit also includes a label indicating the intended use and / or method of use of the contents of the kit. The term "label" includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0174] Beneficial effects

[0175] The anti-CD47 antibodies or antigen-binding fragments thereof of the present invention can achieve the following beneficial effects: 1) specifically bind to CD47-positive tumor cells but do not bind or substantially bind to red blood cells; 2) do not cause or substantially do not cause red blood cell agglutination; 3) have a blocking effect on the binding of CD47 to SIRPα; and / or 4) promote phagocytosis of CD47-positive tumor cells by macrophages.

[0176] Example

[0177] The following examples are intended to illustrate the present invention only and therefore should not be construed as limiting the present invention in any way.

[0178] Example 1 Animal immunization, immune library construction and antibody screening

[0179] 1.1 Animal immunization

[0180] Human IgG1 Fc or 6×His tag was fused to the carboxyl terminus (C-terminus) of the extracellular segment CD47 CD47-ECD to construct the CD47 antigen protein CD47-ECD-Fc (SEQ ID NO: 3) or CD47-ECD-His (SEQ ID NO: 2), respectively. CD47-ECD-Fc was used to immunize C57BL / 6 mice (Shanghai Lingchang Biotechnology Co., Ltd.). Subcutaneous multi-point immunization was adopted during immunization, with 50 μg subcutaneously immunized each time, once every two weeks, for a total of four immunizations. After four immunizations, the immune titer was determined by ELISA, in which the ELISA antigen plate was CD47-ECD-His. The test results showed that the immune titer reached 1:600000. At this time, 100 μg CD47-ECD-Fc was used for booster immunization, and the spleen was taken 2-3 days later.

[0181] 1.2 Immune library construction

[0182] B lymphocytes in the spleen of the immunized mouse described in Example 1.1 were isolated, and their RNA was extracted and reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Primers were designed to amplify the coding sequences of the light chain variable region, the heavy chain variable region, and the first constant region (CL and CH1), respectively, and the coding sequence of the M13 phage GIII protein was connected to the 3' end of the coding sequence of CH1, and then cloned into a phage display vector. The vector was then transformed into competent Escherichia coli SS320 cells (Lucigen, MC1061 F) using an electroporator (Bio-Rad, MicroPulser), and after 1 hour of recovery, it was spread on a 2-YT solid plate with ampicillin resistance. By gradient dilution plating, the storage capacity of this immune library was determined to be 1×10 9 The helper phage M13KO7 (NEB) was used for packaging, and the final constructed immune library was displayed in the form of Fab on the coat protein of M13 phage.

[0183] 1.3 Antibody Screening

[0184] In the first round of screening, 4 mL of 50 μg / mL CD47-ECD-His was added to the immunotube and coated overnight at 4°C. The next day, the coating solution was discarded and the tubes were blocked with PBS containing 5% milk powder for 2 h. After rinsing with PBS, the prepared phages were added and incubated for 2 h. After rinsing to remove non-specifically bound phages, 0.8 mL of trypsin digestion solution containing 0.05% EDTA was added to the immunotubes to elute phages that specifically bound to the target antigen. The eluted phages were then used to infect logarithmic-phase Escherichia coli SS320 (Lucigen, 60512-1), incubated at 37°C for 30 min, and then incubated at 220 rpm for 1 h. VSCM13 helper phages were then added and incubated for 30 min. The incubation was continued at 220 rpm for 1 h. The tubes were centrifuged and replaced with C + / K + The cells were cultured overnight in 2-YT medium at 30°C and 220 rpm. The next day, phage was prepared and used for a second round of screening. This process was repeated, with 10 clones randomly selected for sequence analysis in each round. Strains with significant enrichment and high sequence polymorphism in the third round were selected for single-clone plating.

[0185] By ELISA of the induced monoclonal Fab supernatant, positive clones were selected for sequencing analysis. The results showed that this immune library screening obtained more than 60 positive clones with different sequences. After further performing binding experiments on CCRF-CEM cells (a human acute lymphoblastic leukemia T lymphocyte that endogenously expresses CD47, purchased from the Chinese Academy of Sciences, product number TCHu147) and SIRPα binding blocking experiments on the Fab induction supernatant prepared by these clones, some clones were screened to have the function of binding to CCRF-CEM cells and blocking SIRPα binding on cells. Clone A7 is one of them and is a mouse antibody. 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 using the AbM definition 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, respectively.

[0186] Example 2 Antibody Engineering

[0187] The variable regions of humanized antibody A7H3L3 were obtained by cloning and humanizing the variable regions of A7. The variable regions of antibodies A7-44, A7-28, and A7-47 were obtained by affinity maturation of the variable regions of humanized antibody A7H3L3.

[0188] 2.1 Antibody Humanization

[0189] The mouse VH and VL sequences of clone A7 obtained by screening in Example 1 were compared with known human antibody databases to find the human germline gene VH and VL sequences with the highest homology to the mouse VH and VL sequences, respectively. The framework regions of the human germline gene VH and VL sequences were selected (using AbM to define the CDR and framework regions). Then, with the help of computer prediction simulation, the mouse amino acids in the clone A7 framework region that have an important effect on antigen binding were retained by back mutation. Finally, the complementary determining region (CDR) sequence of the germline gene was replaced with the corresponding CDR sequence in clone A7. After humanization of the variable region of clone A7, the variable region of the humanized antibody A7H3L3 was obtained: 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) is shown in SEQ ID NO: 15 (Table 1).

[0190] 2.2 Affinity maturation

[0191] Affinity maturation of the antibody A7H3L3 was performed to improve affinity and biological activity. Affinity maturation was performed using M13 phage display technology. Codon-based primers (a single codon consisting of NNK during primer synthesis) were used to introduce mutations in the CDR regions. Four phage display libraries were constructed: Library 1 and Library 2 contained single-point mutagenesis: Library 1 contained CDRL1+CDRL3+CDRH3 combined mutations, while Library 2 contained CDRL2+CDRH1+CDRH2 combined mutations. Library 3 and Library 4 contained double-point saturation mutagenesis: Library 3 contained double-point saturation mutagenesis of CDRL3, while Library 4 contained double-point saturation mutagenesis of CDRH3. The specific library construction method is as follows: First, primers containing point mutations are synthesized (Genwizhi Biotechnology Co., Ltd.); secondly, the antibody to be modified, A7H3L3, is used as a PCR amplification template to amplify the sequence containing the designed mutation in the CDR region. Fragments containing different CDR mutations are combined by bridge PCR. Then, the point mutation antibody is linked to the phage display vector by double enzyme digestion (Hind III and Not I) and double sticky end ligation. Finally, the antibody sequence with the mutation site is transferred into Escherichia coli SS320 by electroporation. The library capacity calculation, phage library preparation, and library screening procedures are detailed in Example 1. Three anti-CD47 affinity mature antibodies, A7-44, A7-28, and A7-47, were selected (Table 1).

[0192] Table 1 Amino acid sequences of anti-CD47 antibodies (SEQ ID NO:

[0193] 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 21 22 A7-47 4 5 23 24 8 25 26 27

[0194] Example 3 Construction, expression and purification of anti-CD47 antibodies

[0195] The coding sequence for the VH (SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 26) of the anti-human CD47 antibodies described in Examples 1 and 2 was ligated with the coding sequence for the heavy chain constant region of human IgG4 (SEQ ID NO: 12) to construct the heavy chain coding sequence of the anti-CD47 antibody. The coding sequence for the VL (SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 27) of the anti-human CD47 antibody was ligated with the coding sequence for the human kappa constant region (SEQ ID NO: 13) to construct the light chain coding sequence of the anti-CD47 antibody. The heavy and light chain coding sequences were respectively inserted into the expression plasmid pcDNA3.4 (Invitrogen) to construct expression vectors for the heavy and light chains of the anti-CD47 antibody. The heavy chain and light chain expression vectors were transformed into Escherichia coli DH5α, 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 chain and heavy chain plasmids for eukaryotic expression.

[0196] 7-15 days after transfection, the cell culture supernatant expressing the target protein (i.e., anti-CD47 antibody) was centrifuged at 15,000 g for 10 minutes using the ExpiCHO transient expression system (Thermo Fisher, A29133). The resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403). The target protein was then eluted with 100 mM sodium acetate (pH 3.0), neutralized with 1 M Tris-HCl, and finally, the resulting protein was exchanged into PBS buffer using an ultrafiltration concentrator (Millipore, UFC901096). High-purity anti-CD47 antibodies A7H3L3, A7-44, A7-28, and A7-47 were obtained by SDS-PAGE identification and SEC purity testing.

[0197] Example 4 Binding activity of anti-CD47 antibodies to CD47 on CCRF-CEM cells

[0198] Since CD47 at the cellular level is closer to the native conformation than the recombinant protein, this example measured the binding ability of the anti-CD47 antibody of the present invention and the positive control antibody 1F8 (WO2018075857A1, the heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 28, the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO: 29, and the preparation method is shown in Example 3) on CCRF-CEM cells by flow cytometry.

[0199] The specific method is as follows: take 1×10 5 CCRF-CEM cells were centrifuged at low speed (300 g) and the supernatant was removed; the cells at the bottom of the centrifuge tube were rinsed once with prepared FACS buffer (1× PBS buffer containing 2% FBS by volume), and then a gradient dilution of the test antibody was added to the rinsed cells, and incubated at 4°C for 1 hour; then, the cells were rinsed three times with the above-mentioned FACS buffer, and 0.5 μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added, and incubated at 4°C for 1 hour; then, the cells were rinsed three times with FACS buffer and resuspended in 200 μL FACS buffer, and finally, the amount of anti-CD47 antibody bound to the CCRF-CEM cells (expressed as mean fluorescence intensity (MFI)) was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0200] The results are shown in Figure 1A and 1B Among them, antibodies A7H3L3, A7-44, A7-28, and A7-47 bound to CD47 on CCRF-CEM cells with high binding activity, and the binding activity was better than that of 1F8.

[0201] Example 5 Binding activity of anti-CD47 antibodies to CD47 on erythrocytes

[0202] To determine whether the anti-CD47 antibodies of the present invention bind to CD47 on erythrocytes, the binding activity of antibodies A7H3L3, A7-44, A7-28, and A7-47 to erythrocytes was measured by flow cytometry (FACS). For comparison, the binding activity of positive control antibodies 1F8 and F4AM4 (another anti-CD47 antibody developed by the applicant, with the heavy chain amino acid sequence of SEQ ID NO: 30 and the light chain amino acid sequence of SEQ ID NO: 31) to erythrocytes was also measured. Human IgG4 was used as a negative isotype control.

[0203] The specific method is as follows: separate red blood cells from 1 mL of anticoagulated human blood, remove the supernatant after centrifugation, rinse twice with PBS, add 1 mL of PBS and resuspend. Use PBS to dilute the red blood cells to 1×10 7 / mL, 50 μL of red blood cells were aspirated into each well of a 96-well round-bottom cell culture plate. An equal volume of serially diluted test antibodies was then added, mixed thoroughly, and incubated at 4°C for 1 hour. The cells were then rinsed three times with FACS buffer, and 0.5 μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added. The cells were incubated at 4°C for 1 hour. Afterwards, the cells were rinsed three times with FACS buffer and resuspended in 200 μL of FACS buffer. The amount of anti-CD47 antibody bound to the red blood cells (MFI) was determined using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0204] The results are as follows Figures 2A-2B The results showed that antibody A7H3L3 barely binds to CD47 on erythrocytes, and its binding activity is comparable to or weaker than that of the positive control antibody 1F8. Antibodies A7-44 and A7-47 weakly bind to CD47 on erythrocytes, and antibody A7-28 has some binding to CD47 on erythrocytes (but this is still much weaker than the positive control antibody F4AM4). These results indicate that antibodies A7H3L3, A7-44, and A7-47 barely bind to erythrocytes and exhibit a higher specificity for targeting CD47-positive tumor cells.

[0205] Example 6: Anti-CD47 Antibody Blocking Activity of CD47 Binding to SIRPα

[0206] The blocking activity of the anti-CD47 antibodies of the present invention in blocking the binding of CD47 on tumor cells to the receptor SIRPα was determined by flow cytometry (FACS). Human IgG4 was used as an isotype negative control.

[0207] The specific method is as follows: take 1×10 5 CCRF-CEM cells were centrifuged at low speed (300g) and the supernatant removed. The cells at the bottom of the centrifuge tube were rinsed once with prepared FACS buffer (1× PBS containing 2% FBS). Then, serially diluted test antibodies were added to the rinsed cells and incubated for 1 hour. After rinsing the cells twice with FACS buffer, 100 μL of 1 μg / mL SIRPα-mFc (ACRO, SIA-H52A8) was added and incubated at 4°C for 1 hour. After rinsing three times with FACS buffer, 100 μL of PE-conjugated goat anti-mouse Fc secondary antibody (1:200, Abcam, ab98742) was added. After incubation at 4°C for 1 hour, the supernatant was removed and the cells were resuspended in 200 μL of FACS buffer. Finally, the amount of SIRPα-mFc bound to CCRF-CEM cells (MFI) was determined by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0208] The results are shown in Figure 3 Among them, antibodies A7-44, A7-28 and A7-47 effectively blocked the binding of CD47 on tumor cells to SIRPα, and their blocking ability was better than that of the positive control antibody 1F8.

[0209] Example 7 Hemagglutination reaction of red blood cells by anti-CD47 antibodies

[0210] Antibody drugs reach the tumor lesion site through the blood circulation to exert their efficacy, and red blood cells in the blood express CD47 in large quantities. For antibodies that bind to CD47 on the surface of red blood cells with high binding activity, when the antibody concentration reaches a certain level, it may cause aggregation of red blood cells through cross-linking, so that the aggregated red blood cells are cleared by phagocytes, which is one of the important causes of toxic reactions such as anemia to a certain extent. This example compares the hemagglutination reaction of red blood cells by the anti-CD47 antibody of the present invention and the positive control antibodies 1F8 and Hu5F9 (also known as magrolimab, see U.S. patent application US20160304609A1). Human IgG4 is used as an isotype negative control.

[0211] The specific method is as follows: isolate red blood cells from 1 mL of anticoagulated human blood, centrifuge and remove the supernatant, rinse twice with PBS, add 1 mL of PBS and resuspend. Use PBS to dilute the red blood cells 20 times, draw 50 μL of red blood cells per well and add them to a 96-well round-bottom cell culture plate, then add an equal volume of gradient dilution (0.05-100 μg / mL) of the test antibody, mix thoroughly, and incubate in a 37°C incubator for 3 hours. Then take out and observe the degree of hemagglutination reaction. The size of the red blood cell deposition area represents the strength of hemagglutination. The larger the area, the stronger the hemagglutination, and vice versa.

[0212] The results are as follows Figure 4A The results showed that red blood cells treated with antibody A7 aggregated into a small group and sank to the bottom of the circular well, consistent with the negative control results, indicating that it did not undergo hemagglutination. However, red blood cells treated with the positive control antibody Hu5F9 had a large dispersion area, indicating a very severe hemagglutination reaction, which is consistent with the results reported in patent US20160304609A1. This indicates that the chimeric antibody A7 is superior to the positive control antibody Hu5F9 in terms of hemagglutination reaction and is expected to show lower anemia toxicity or side effects in clinical practice.

[0213] The results are as follows Figure 4BThe results showed that red blood cells treated with antibodies A7H3L3, A7-44, and A7-47 aggregated into a small clump and sank to the bottom of the well, consistent with the negative control (IgG4), indicating that they did not induce hemagglutination. However, red blood cells treated with A7-28 exhibited a certain degree of hemagglutination, roughly equivalent to that of antibody 1F8. These results indicate that antibodies A7H3L3, A7-44, and A7-47 are superior to antibody 1F8 in hemagglutination and are expected to exhibit lower anemia toxicity or side effects in clinical practice.

[0214] Example 8 Anti-CD47 Antibodies Promote the Ability of Macrophages to Phagocytose Tumor Cells

[0215] Antibodies that block the binding of CD47 and SIRPα can effectively promote macrophage phagocytosis of CD47-positive tumor cells. This example compares the ability of the anti-CD47 antibodies of the present invention and the positive control antibody 1F8 to promote macrophage phagocytosis of tumor cells. Human IgG4 was used as a negative isotype control.

[0216] The specific method is as follows: First, human peripheral blood mononuclear cells (PBMCs) were isolated and 50 ng / mL rhM-CSF (purchased from Peprotech, 300-25-10) was added to induce cell differentiation into macrophages. After culturing in a cell culture incubator at 37°C for about 8 days, the supernatant and non-adherent cells were removed, and then Accutase was added. TM Cell digestion solution (purchased from Sigma, A6964) was incubated at 37°C for 45 min to digest adherent cells and prepare a uniform cell suspension with complete culture medium (RPMI1640 + 10% FBS). 4 Cells were dispensed into each well of a 96-well plate. Serial dilutions of the test antibody (1 μg / mL-0.002 μg / mL) were added and incubated at 37°C for 30 min. 2.5×10 4 CCRF-CEM cells labeled with CFSE (purchased from Abcam, ab113853) were incubated at 37°C for 1 hour, and then 0.25 μg of APC-labeled anti-CD14 antibody (purchased from eBioscience, 17-0149-42) was added. The cell suspension was then incubated at 4°C for 20 minutes, centrifuged at 500 g for 5 minutes, washed twice with FACS buffer, and finally detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102). + The proportion of macrophages engulfing CFSE-labeled CCRF-CEM cells was the phagocytic rate.

[0217] The results are as follows Figure 5The results showed that antibodies A7-28 and A7-47 promoted the phagocytosis of tumor cells by macrophages in a dose-dependent manner, and their effects in promoting the phagocytosis of tumor cells by macrophages were better than that of antibody 1F8.

[0218] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings disclosed herein, and that such modifications are within the scope of protection of the present invention. The scope of protection of the present invention is given by the appended claims and any equivalents thereof. Sequence Listing <110> Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. <120> Anti-CD47 antibodies and uses thereof <130> I2021TC6176CS <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 heavy chain constant region <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 2 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> CD47-ECD-His <400> 2 Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn 1 5 10 15 Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn 20 25 30 Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 115 120 125 Ser Ala His His His His His His 130 135 <210> 3 <211> 350 <212> PRT <213> Artificial Sequence <220> <223> CD47-ECD-Fc <400> 3 Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn 1 5 10 15 Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn 20 25 30 Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp Glu Pro Lys Ser Cys Asp Lys Thr His Thr 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 165 170 175 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A7-HCDR1 <400> 4 Gly Phe Asn Ile Lys Asp Ile Tyr Ile Tyr 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A7-HCDR2 <400> 5 Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys 1 5 10 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> A7-HCDR3 <400> 6 Gly Tyr Gly Ser Gly Phe Ala Tyr 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> A7-LCDR1 <400> 7 Arg Ala Ser Gln Asp Ile Ser Asn His Leu Asn 1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> A7-LCDR2 <400> 8 Tyr Thr Ser Arg Ile His Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A7-LCDR3 <400> 9 Gln Gln Gly Tyr Thr Leu Pro Phe Thr 1 5 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> A7-VH <400> 10 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Ile Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Ile Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Thr 115 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> A7-VL <400> 11 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Phe Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 heavy chain constant region <400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human light chain constant region <400> 13 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> A7H3L3-VH / A7-44 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> A7H3L3-VL <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> A7-44 LCDR2 <400> 16 Tyr Thr Ser Arg Ile His Leu 1 5 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> A7-44 VL <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Ile His Leu Gly Val Pro Ser Ser Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A7-28 HCDR1 <400> 18 Gly Phe Asn Ile Lys Asp Val Tyr Ile Tyr 1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> A7-28 HCDR2 <400> 19 Lys Ile Asp Pro Ala Asn Gly Asn Thr His 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> A7-28 LCDR2 <400> 20 Tyr Thr Ser Arg Ile His Lys 1 5 <210> 21 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> A7-28 VH <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Val 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr His Tyr Asp Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> A7-28 VL <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Ile His Lys Gly Val Pro Ser Ser Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> A7-47 HCDR3 <400> 23 Gly Tyr Gly Ser Val Phe Ala Tyr 1 5 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> A7-47 LCDR1 <400> 24 Arg Ala Ser Gln Asp Ile Ser Asn His Ile Asn 1 5 10 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A7-47 LCDR3 <400> 25 Gln Gln Gly Tyr His Leu Pro Phe Thr 1 5 <210> 26 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> A7-47 VH <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Ser Val Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> A7-47 VL <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Ile Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr His Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 28 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 1F8 VH <400> 28 Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Arg Lys Thr Asp Gly Glu Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Gly Ser Asn Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ala 115 <210> 29 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 1F8 VL <400> 29 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Asn Gln Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile 65 70 75 80 Ile Ser Ser Leu His Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Thr Pro Pro Leu Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 30 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of F4AM4 <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Thr Asp Gly Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Pro Tyr Tyr Gly Thr Arg Tyr Gly Ser Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 31 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> F4AM4 light chain <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Lys Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An anti-CD47 antibody or an 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 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 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 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; 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 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; 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 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.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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) an amino acid sequence having 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% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:21 or SEQ ID NO:26; or 3) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions as compared with the amino acid sequence of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:21 or SEQ ID NO:

26.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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) an amino acid sequence having 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% or at least 99% sequence identity with SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:27; or 3) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions as compared with SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:

27.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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; 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: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.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is a chimeric antibody, humanized antibody, human antibody, scFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv (dsFv) or diabody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which further comprises a heavy chain constant region and / or a light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the heavy chain constant region is the heavy chain constant region of human IgG4 and / or the light chain constant region is the light chain constant region of human kappa light chain.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain constant region comprises: 1) the amino acid sequence of SEQ ID NO:12; or 2) an amino acid sequence having 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% or at least 99% sequence identity with SEQ ID NO:12; and / or The light chain constant region comprises: 1) the amino acid sequence of SEQ ID NO:13; or 2) an amino acid sequence having 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% or at least 99% sequence identity with SEQ ID NO:

13.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein 1) it specifically binds to CD47-positive cancer cells but does not bind to red blood cells; 2) it does not cause red blood cell agglutination; 3) it blocks the binding of CD47 to SIRPα; and / or 4) it promotes the phagocytosis of CD47-positive tumor cells by macrophages.

10. 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 antibody or antigen-binding fragment thereof according to any one of claims 1-5.

11. The multispecific antibody of claim 10, wherein the second antigen-binding portion specifically binds to a tumor antigen, an immune regulatory receptor or an immune checkpoint molecule.

12. The multispecific antibody of claim 11, wherein the second antigen-binding portion specifically binds to 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, folate receptor α (FOLR1) or other tumor antigens.

13. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5.

14. An immune effector cell that expresses the chimeric antigen receptor of claim 13 on its surface.

15. The immune effector cell of claim 14, which is selected from cytotoxic T cells and natural killer cells.

16. The immune effector cell of claim 14, which is a natural killer T cell.

17. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-9.

18. An expression vector comprising the polynucleotide of claim 17.

19. A host cell comprising the polynucleotide of claim 17 or the expression vector of claim 18.

20. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-9, a multispecific antibody according to any one of claims 10-12, or an immune effector cell according to any one of claims 14-16, and a pharmaceutically acceptable carrier.

21. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-9, a multispecific antibody according to any one of claims 10-12, or the pharmaceutical composition of claim 20 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myelodysplastic syndrome, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma (MM), giant cell myeloma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, gastric cancer, kidney cancer, leiomyoma, and glioma.

22. The use of claim 21, wherein the cancer is selected from Burkitt lymphoma, follicular lymphoma, and glioblastoma.

23. The use of claim 21 or 22, wherein the medicament is used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen-targeted drugs.

Images