Anti-CD47 antibodies and uses thereof

Abstract

The present invention provides anti-CD47 antibodies or antigen-binding fragments thereof, methods for their preparation, and their use for treating or preventing CD47-associated diseases.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims priority to CN202010282924.0, filed April 10, 2020, which is incorporated by reference in its entirety for all purposes. [Technical Field]

[0002] The present invention relates to antibodies, in particular anti-CD47 antibodies, and their use for preparing medicaments for the treatment or prevention of CD47-related diseases. [Background technology]

[0003] CD47 (Cluster of Differentiation 47) was first identified in the 1980s as a tumor antigen in human ovarian cancer. Also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-associated antigen, and MER6, CD47 is a multimembrane receptor belonging to the immunoglobulin superfamily, possessing a single immunoglobulin-like domain and five transmembrane domains. As a ligand for signal-regulatory protein α (SIRPα), CD47 binds to the V-like domain at the NH2 terminus of SIRPα. SIRPα is primarily expressed in myeloid cells, including macrophages, granulocytes, dendritic cells (DCs), mast cells, and their precursor cells, such as hematopoietic stem cells. CD47 on normal cells binds to SIRPα on macrophages, releasing a "don't eat me" signal, thereby inhibiting the phagocytic function of macrophages. This is an important mechanism by which macrophages distinguish between self and non-self in the innate immune system. CD47 is widely expressed in human tumor cells and tissues, including acute myeloid leukemia (AML), chronic granulocytic leukemia (CHL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, and other solid tumors. Highly expressed CD47 evades macrophage phagocytosis by binding to SIRPα, which promotes tumor growth, on the macrophage surface. The CD47 immune checkpoint is considered a potentially effective and widely applicable target for tumor immunotherapy. Currently, various specific blocking agents targeting the CD47 / SIRPα interaction are being developed. Numerous preclinical and clinical studies involving agents containing anti-CD47 antibodies and SIRPα fusion proteins are underway for the treatment of diffuse large B-cell lymphoma, acute myeloid leukemia, and advanced solid tumors. These agents are administered alone or in combination with other antitumor drugs. Take the anti-CD47 antibody Hu5F9 developed by Forty Seven as an example. In a phase I clinical trial evaluating the efficacy of Hu5F9 in treating 22 patients with lymphoma, the combination of Hu5F9 with rituximab could induce objective remission in 50% of patients who did not respond to rituximab alone.Clinical data published in 2019 showed that the complete remission rate was up to 36% in 14 patients with relapsed / refractory acute myeloid leukemia treated with Hu5F9 in combination with azacitidine, and the remission rate was up to 55% in 11 patients with myelosuppression syndrome treated with Hu5F9 in combination with azacitidine.

[0004] According to the present invention, a novel anti-CD47 antibody or antigen-binding fragment thereof is provided that has high anti-tumor activity and does not induce significant red blood cell aggregation, thereby meeting more clinical needs. Summary of the Invention

[0005] The present invention provides anti-CD47 antibodies or antigen-binding fragments thereof that bind to CD47 or a fragment thereof, as well as methods for their preparation and use, including methods for treating CD47-related diseases.

[0006] In one aspect, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, comprising one to three HCDRs selected from HCDR1, HCDR2, and HCDR3 of the heavy chain variable region (VH), wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 1, 3, 5, 6, or 7.

[0007] In one embodiment, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises one to three heavy chain complementarity-determining regions selected from HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, 17, or 21.

[0008] In one aspect, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof, comprising one to three selected from LCDR1, LCDR2, and LCDR3 of the light chain variable region (VL), wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 2, 4, 8, 9, or 10.

[0009] In one aspect, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising one to three regions selected from light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 18, or 22, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0010] In some embodiments, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising three CDRs of the heavy chain variable region (VH), namely HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region (VL), namely LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 1, 3, 5, 6, or 7, and the amino acid sequence of the VL is as set forth in SEQ ID NO: 2, 4, 8, 9, or 10.

[0011] In some embodiments, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising three CDRs of a heavy chain variable region (VH), namely HCDR1, HCDR2, and HCDR3, and three CDRs of a light chain variable region (VL), namely LCDR1, LCDR2, and LCDR3, wherein said VH and VL are: (1) a VH comprising the amino acid sequence shown in SEQ ID NO: 1, and a VL comprising the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising the amino acid sequence shown in SEQ ID NO: 3, and VL comprising the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising the amino acid sequence set forth in SEQ ID NO: 5, and VL comprising the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10; (4) VH comprising the amino acid sequence set forth in SEQ ID NO: 6, and VL comprising the amino acid sequence set forth in SEQ ID NO: 9 or 10; and (5) VH comprising the amino acid sequence shown in SEQ ID NO: 7, and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9, or 10 An anti-CD47 antibody or antigen-binding fragment thereof is provided.

[0012] In some embodiments, the present invention provides an anti-CD47 antibody or antigen-binding fragment thereof comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13, 17, or 21, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, 18, or 22, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0013] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0014] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 6, or 7.

[0015] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a light chain variable region (VL), which comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 8, 9, or 10.

[0016] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 6, or 7, and the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 8, 9, or 10.

[0017] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, and the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0018] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are: (1) a VH comprising the amino acid sequence shown in SEQ ID NO: 1, and a VL comprising the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising the amino acid sequence shown in SEQ ID NO: 3, and VL comprising the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising the amino acid sequence set forth in SEQ ID NO: 5, and VL comprising the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10; (4) VH comprising the amino acid sequence set forth in SEQ ID NO: 6, and VL comprising the amino acid sequence set forth in SEQ ID NO: 9 or 10; and (5) VH comprising the amino acid sequence shown in SEQ ID NO: 7, and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9, or 10 is selected from.

[0019] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0020] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:3 and the VL comprises the amino acid sequence set forth in SEQ ID NO:4.

[0021] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:5 and the VL comprises the amino acid sequence set forth in SEQ ID NO:8.

[0022] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:5 and the VL comprises the amino acid sequence set forth in SEQ ID NO:9.

[0023] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:5 and the VL comprises the amino acid sequence set forth in SEQ ID NO:10.

[0024] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:6 and the VL comprises the amino acid sequence set forth in SEQ ID NO:9.

[0025] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:6 and the VL comprises the amino acid sequence set forth in SEQ ID NO:10.

[0026] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:7 and the VL comprises the amino acid sequence set forth in SEQ ID NO:8.

[0027] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:7 and the VL comprises the amino acid sequence set forth in SEQ ID NO:9.

[0028] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:7 and the VL comprises the amino acid sequence set forth in SEQ ID NO:10.

[0029] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain constant region, and the heavy chain constant region is, for example, a human IgG1 constant region, a human IgG4 constant region, a human IgG4P constant region, or a human IgG1™ constant region. The human IgG4P constant region of the present invention is a mutant human IgG4 with an amino acid substitution of S228P (EU numbering). In some embodiments, the human IgG1™ constant region is a mutant human IgG1 constant region with amino acid substitutions of L234F, L235E, and P331S (EU numbering).

[0030] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a light chain constant region, such as a human kappa or lambda constant region.

[0031] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention are monoclonal antibodies. In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention are murine, chimeric, or humanized antibodies. In some embodiments, at least a portion of the framework sequence of the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention is a human consensus framework sequence. In one embodiment, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention are single-chain antibodies such as full-length antibodies, VHHs, Fabs, Fab's, Fab'-SHs, and (Fab')2s, scFvs, Fvs, dAbs (domain antibodies), or bis(multi)specific antibodies.

[0032] In another aspect, the present invention provides an isolated nucleic acid encoding any of the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention, preferably the nucleic acid encoding the heavy or light chain, or heavy or light chain variable region, of an antibody of the invention.

[0033] In another aspect, the present invention provides recombinant or expression vectors comprising one or more nucleic acids provided by the invention, which are suitable for recombinant production of any antibody or antigen-binding fragment thereof provided by the invention. In some embodiments, the vector is an expression vector.

[0034] In another aspect, the invention provides a host cell comprising one or more nucleic acids, or recombinant vectors or expression vectors, provided by the invention.

[0035] In another aspect, the present invention provides an immunoconjugate or immunofusion comprising an anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention.

[0036] In another aspect, the present invention provides a method for the preparation of a composition comprising an anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention, a nucleic acid, a vector, or a host cell provided by the present invention, and optionally at least one pharmaceutically acceptable pharmaceutical excipient, such as a carrier or diluent.

[0037] In another aspect, the present invention provides a method for treating or preventing a CD47-related disease, the method comprising administering to a subject an effective amount of any antibody or antigen-binding fragment thereof described herein, a nucleic acid, a vector, or a host cell provided by the invention, an immunoconjugate or immunofusion provided by the invention, or a pharmaceutical composition provided by the invention.

[0038] In another aspect, the present invention provides use of an anti-CD47 antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament for treating or preventing a CD47-related disease.

[0039] In some embodiments, the CD47-associated disease includes hematological cancers and solid tumors, including, but not limited to, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (chronic) melanoma, liomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer, (benign) melanoma, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, and liver cancer.

[0040] The CD47 antibodies or antigen-binding fragments thereof of the present invention can also be combined with other therapeutic agents or modalities to treat or prevent CD47-associated diseases.

[0041] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used for detecting CD47 protein in a sample or for diagnosing / detecting a CD47-related disease.

[0042] The present invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein, or any combination thereof, is applicable to any and all anti-CD47 antibodies or fragments thereof, methods and uses of the invention described herein. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Raji cells. [Figure 2] FIG. 2 shows the binding activity of antibody HMA02h14-48 to CD47 on the surface of Toledo cells. [Figure 3] FIG. 3 shows the binding affinity of antibody HMA02h14-48 to CD47 on the surface of REC-1 cells. [Figure 4] FIG. 4 shows the activity of antibody HMA02h14-48 in blocking the interaction between human CD47 and SIRPα. [Figure 5]FIG. 5 shows the effect of antibody HMA02h14-48 on the phagocytosis of Raji cells by human MΦ. [Figure 6] FIG. 6 shows the effect of antibody HMA02h14-48 on the phagocytosis of Toledo cells by human MΦ. [Figure 7] FIG. 7 shows the effect of antibody HMA02h14-48 on the phagocytosis of REC-1 cells by human MΦ. [Figure 8] FIG. 8 shows the effect of antibody HMA02h14-48 on the phagocytosis of HL-60 cells by human MΦ. [Figure 9] FIG. 9 shows the effect of antibody HMA02h14-48 on red blood cell aggregation in vitro. [Figure 10] FIG. 10 shows the binding ability of antibody HMA02h14-48 to CD47 on the surface of human erythrocytes. [Figure 11] FIG. 11 shows the inhibition of Toledo tumor growth by Hu5F9 and HMA02h14-48. [Figure 12] FIG. 12 shows the inhibition of REC-1 tumor growth by Hu5F9 and HMA02h14-48. Detailed Description of the Invention

[0044] According to the present invention, there is provided an anti-CD47 antibody or an antigen-binding fragment thereof that can block the interaction between CD47 and SIRPα, has high anti-tumor activity, and does not induce significant red blood cell agglutination.

[0045] The anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention exhibit inhibitory activities such as inhibiting CD47 expression (e.g., inhibiting CD47 expression on the cell surface), CD47 activity and / or signaling, or blocking the interaction between CD47 and SIRPα. The CD47 antibodies provided by the present invention completely or partially reduce or modulate CD47 expression or activity after binding to or interacting with CD47 (e.g., human CD47). After interaction of the antibody with a human CD47 polypeptide and / or peptide, the biological function of CD47 is completely, significantly, or partially reduced or modulated. An antibody is considered to be capable of completely inhibiting CD47 expression or activity if the level of CD47 expression or activity in the presence of the anti-CD47 antibody is reduced by at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) compared to the level of CD47 expression or activity without interaction (e.g., binding) with an anti-CD47 antibody described herein. A CD47 antibody is considered to be capable of significantly inhibiting CD47 expression or activity if the level of CD47 expression or activity in the presence of the anti-CD47 antibody is reduced by at least 50% (e.g., 55%, 60%, 75%, 80%, 85%, or 90% compared to the level of CD47 expression or activity without interaction (e.g., binding) with an anti-CD47 antibody described herein. An antibody is considered to be partially inhibiting CD47 expression or activity if the level of CD47 expression or activity in the presence of the anti-CD47 antibody is reduced by less than 95% (e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90% compared to the level of CD47 expression or activity without interaction (e.g., binding) with an anti-CD47 antibody described herein).

[0046] When the level of interaction between CD47 and SIRPα in the presence of an anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention is compared to the level of interaction between CD47 and SIRPα in the absence of an anti-CD47 antibody or antigen-binding fragment thereof described in the present invention, the anti-CD47 antibody or antigen-binding fragment thereof of the present invention blocks the interaction between CD47 and SIRPα by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 99%.

[0047] The anti-CD47 antibodies or antigen-binding fragments thereof of the invention do not induce significant levels of cell aggregation, e.g., the CD47 antibodies of the invention do not induce significant levels of hemagglutination. In some embodiments, the level of hemagglutination in the presence of the CD47 antibodies of the invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% when compared to the level of hemagglutination in the presence of the CD47 antibody Hu5F9, indicating that the CD47 antibodies of the invention do not induce significant levels of hemagglutination.

[0048] Compared to antibodies known in the art, the antibodies provided by the present invention are also significantly more effective in tumor models. In some embodiments, the antibodies provided by the present invention can significantly inhibit tumor growth. In some embodiments, tumor volume in the presence of an antibody of the present invention is inhibited by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, compared to tumor volume in the absence of the antibody. For example, the ability of macrophages to phagocytose tumor cells in the presence of a CD47 antibody of the present invention is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, compared to the presence of a conventional CD47 antibody.

[0049] The present invention also provides a method for preparing an anti-CD47 antibody or antigen-binding fragment thereof, and use of the antibody or antigen-binding fragment thereof for the treatment or prevention of cancer, etc.

[0050] definition Unless otherwise indicated, the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0051] In order to make the present invention easier to understand, some scientific and technical terms are defined as follows. Unless expressly defined elsewhere herein, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. For definitions and terms in the art, reference can be made to Current Protocols in Molecular Biology (Ausubel) by various experts. Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used in this specification (including the claims), singular forms include the corresponding plural forms unless otherwise specified.

[0052] The term "about" refers to a value or composition that includes an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can refer to a range within 1 or more than 1 standard deviation, in accordance with the conventions in the art. Alternatively, "about" can refer to a range of up to 5%, 10%, or 20% (i.e., ±5%, ±10%, or ±20%).

[0053] When used in conjunction with two or more optional items, the term "and / or" should be understood to mean any one of those optional items or any two or more of those optional items.

[0054] As used herein, the term "comprise" or "include" means including the stated elements, integers, or steps, but does not exclude other elements, integers, or steps. As used herein, when the term "comprise" or "include" is used, unless otherwise indicated, instances consisting of the stated elements, integers, or steps are also encompassed. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass an antibody variable region consisting of that particular sequence.

[0055] The term "integrin-associated protein (IAP)" or "CD47," as used herein, unless otherwise specified, refers to any native CD47 from any vertebrate source, including mammals (such as primates (e.g., humans) and rodents (e.g., mice and rats)). The term encompasses "full-length" unprocessed CD47 and any form of CD47 or any fragment thereof generated by intracellular processing. The term also encompasses naturally occurring CD47 variants, such as splice variants and allelic variants. In some embodiments, CD47 refers to full-length CD47 from humans or a fragment thereof (such as a mature fragment thereof lacking the signal peptide). In some embodiments, human CD47 refers to a CD47 identical to the amino acid sequence set forth in NCBI Accession No. NP_001768.1 or a fragment thereof. In some embodiments, the term also encompasses fusion proteins comprising CD47 or a fragment thereof.

[0056] By "SIRPα" is meant wild-type signal-regulatory protein α, or a recombinant or non-recombinant polypeptide comprising the amino acid sequence of wild-type signal-regulatory protein α, or a native or naturally occurring allelic variant of signal-regulatory protein α.

[0057] The terms "anti-CD47 antibody," "anti-CD47," "CD47 antibody," or "antibody that binds to CD47" refer to an antibody or antigen-binding fragment thereof that can bind to the CD47 protein or a fragment thereof with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent targeting CD47. In some embodiments, the anti-CD47 antibodies provided herein have a dissociation constant (KD) of ≦100 nM, ≦10 nM, ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.9 nM, or ≦0.8 nM. In some embodiments, the antibody or antigen-binding fragment thereof binds to full-length human CD47 or a fragment thereof (particularly an extracellular-binding fragment thereof). In some embodiments, the antibody or antigen-binding fragment thereof binds to a protein comprising full-length CD47 or a fragment thereof. In some other embodiments, the antibody or antigen-binding fragment thereof binds to CD47 or a fragment thereof expressed on the cell surface.

[0058] The term "affinity," as used herein, refers to the strength of the sum of all noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y is generally determined by the dissociation constant (K D ) Examples of assays known in the art for determining binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., ForteBio).

[0059] As used herein, the term "CD47-associated disease" refers to a non-physiological condition associated with CD47 expression, function, or activity, or a non-physiological condition associated with CD47-mediated signaling activity, including, but not limited to, cancer. In some embodiments, the disease would benefit from blocking CD47-associated signaling.

[0060] The terms "immune response" or "immune reaction" may be used interchangeably herein and refer to the effect of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, etc., and the effect of these cells or the liver in producing soluble macromolecules (including antibodies, cytokines, and complement) that result in the selective damage, destruction, or elimination of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues from the human body.

[0061] As used herein, the term "signal transduction" generally refers to a biochemical causal relationship initiated by a protein-protein interaction, such as the binding of CD47 to its receptor, which results in the transmission of a signal from one part of a cell to another part of the cell. Generally, transduction involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues of one or more proteins in a series of reactions that triggers signal transduction. The penultimate process generally involves a nuclear event, which leads to a change in gene expression.

[0062] As used herein, the terms "activity" and "biological activity," or "biological property" and "biological characteristic," are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, ability to neutralize or antagonize CD47 activity in vivo or in vitro, CD47 activity, IC 50These include antibody in vivo stability and enhanced immunogenicity or activation. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide in general, or with other proteins or tissues) and the ability to maintain high levels of antibody expression in mammalian cells. The above-mentioned properties or characteristics can be observed, determined, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance assays, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or other sources).

[0063] The term "antibody" as used herein refers to any form of antibody having the desired biological activity. Thus, the term antibody is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, human antibodies, chimeric antibodies, CrossMab antibodies, or camelized single domain antibodies.

[0064] The terms "whole antibody," "full-length antibody," and "complete antibody" are used interchangeably herein and refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity-determining regions (CDRs)), interspersed with more conserved regions (framework regions (FRs)). A "complementarity-determining region" or "CDR region" or "CDR" is a region within an antibody variable domain that is hypervariable in sequence, forms structurally defined loops ("hypervariable loops"), and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located in an antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, respectively, and the CDRs located in an antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3, respectively. Each VH or VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The constant regions are not directly involved in binding an antibody to an antigen but exhibit multiple effector functions.

[0065] For a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using a variety of well-known schemes, or a combination thereof, including, for example, the Chothia scheme (Chothia et al., "Canonical Structures for the Hypervariable Regions of Immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987)); the Kabat scheme (Kabat et al., "Sequences of Proteins of Immunological Interest," 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University of London); or the North scheme (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)). The boundaries of the CDRs of the anti-CD47 antibodies of the present invention can be determined according to any scheme known in the art or a combination thereof and by personal evaluation.

[0066] The light chains of antibodies can be classified into two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of their constant domains. The heavy chains of antibodies can be classified into five different types: IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of their heavy chain constant regions, and some of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0067] "IgG antibody" refers to the heavy chain constant region of an IgG antibody. For example, an IgG4 antibody means that its heavy chain constant region is derived from IgG4.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the various antibodies comprising the population are identical except for naturally occurring variants that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations generally contain a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring a particular method for producing the antibody.

[0069] As used herein, the term "antigen-binding fragment" of an antibody includes antibody fragments or derivatives. Generally, an antigen-binding fragment contains at least a fragment of the antigen-binding or variable region of an antibody (such as one or more CDRs) and maintains at least some of the antibody's binding properties. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., sc-Fv); and nanobodies and multispecific antibodies formed from antibody fragments. When antigen-binding activity is expressed on a molar basis, binding fragments or derivatives generally maintain at least 10% of the antigen-binding activity of the antibody from which they are derived. Preferably, binding fragments or derivatives maintain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding activity of the antibody from which they are derived.

[0070] It is expected that antibodies or antigen-binding fragments thereof may contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity. In preferred embodiments, the conservative substitutions are from the conservative substitution residues shown in Table A below, preferably the preferred conservative amino acid substitution residues shown in Table A.

[0071] [Table 1]

[0072] An epitope is a region of an antigen to which an antibody binds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0073] As used herein, the term "isolated anti-CD47 antibody or antigen-binding fragment thereof" refers to a purified form of an anti-CD47 antibody or antigen-binding fragment thereof. For example, "isolated" can mean that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, or other substances, such as cellular debris and growth medium. However, as known to those skilled in the art, the term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts, unless such substances are present in amounts that significantly interfere with the experimental or therapeutic application of the antibodies described herein. In some embodiments, an isolated antibody or antigen-binding fragment may have a purity of greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0074] As used herein, the term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, where the first and second antibodies are derived from different species. Generally, the variable domains are obtained from an antibody of a laboratory animal such as a rodent, and the constant domain sequences are obtained from a human antibody, making the resulting chimeric antibody less likely to provoke an adverse immune response in a human subject than antibodies from a laboratory animal.

[0075] The term "humanized antibody" as used herein refers to antibody forms containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody comprises at least one, generally two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions correspond to those of a human immunoglobulin. A humanized antibody may optionally comprise at least a portion of a constant region (Fc) derived from a human immunoglobulin. In some cases, as known to those skilled in the art, amino acid mutations can be introduced into the humanized antibody (e.g., the variable domains, framework regions, and / or constant regions, if present) to, for example, improve certain properties of the antibody; such antibody forms are also included within the scope of the "humanized antibody" of the present invention.

[0076] As known to those skilled in the art, an antibody may have the carbohydrate moiety of the cell in which it is produced. For example, an antibody produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell may contain a mouse carbohydrate moiety. Alternatively, an antibody produced in a rat, a rat cell, or a hybridoma derived from a rat cell may contain a rat carbohydrate moiety.

[0077] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. Native-sequence Fc regions include various naturally occurring immunoglobulin Fc sequences, such as various Ig subtypes and their allogeneic Fc regions (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520). In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0078] The terms "Fc region variant" or "variant Fc region" are used interchangeably herein and are meant to include Fc regions that are modified relative to a native sequence Fc region. Fc region variants of the present invention are defined according to the amino acid modifications to their constituent amino acids.

[0079] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, with which an active substance is administered.

[0080] The term "pharmaceutical composition" refers to a composition that contains an active ingredient contained therein in a form that effectively activates the biological activity of the active ingredient, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0081] As used herein, an "immunoconjugate" is an antibody conjugated to one or more other substances, including, but not limited to, a cytotoxic agent or a label. An "immunofus" is an antibody fused by covalent bonding to one or more other peptides or polypeptides.

[0082] The term "therapeutic agent" as used herein encompasses any substance that is effective in the prevention or treatment of cancer and related diseases.

[0083] The term "cytotoxic agent" as used herein refers to a substance that inhibits the function of cells and / or causes death or destruction of cells.

[0084] "Chemotherapeutic agents" include small chemical molecule drugs useful in the treatment of cancer or immune system disorders.

[0085] The term "small molecule drug" refers to low-molecular-weight organic compounds that can regulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, generally less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules have better cell penetration, are less susceptible to degradation, and are less likely to provoke an immune response than larger molecules.

[0086] As used herein, the term "immunomodulatory agent" refers to a natural or synthetic active agent or agent that modulates (suppresses or enhances) the immune response. The immune response can be a humoral response or a cellular response. Immunomodulatory agents include immunosuppressants. In some embodiments, immunomodulatory agents include active agents or agents that enhance the immune response, for example, active agents or agents that are useful in enhancing anti-cancer immune responses in cancer treatment.

[0087] As used herein, an "immunosuppressant," "immunosuppressive drug," or "immuno-suppressor" is a therapeutic agent used in immunosuppressive therapy to inhibit or prevent the activity of the immune system.

[0088] The terms "carcinoma" and "cancer" refer to or describe a physiological disorder in mammals that is generally characterized by unregulated cell growth. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. "Cancer" includes, but is not limited to, solid tumors and hematological cancers. Examples of various cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

[0089] As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors can induce the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." An expression vector refers to a nucleic acid capable of replicating and expressing a target gene when the vector is transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication, as needed, to ensure maintenance and amplification of the vector within the host.

[0090] As used herein, the term "subject" or "patient" or "individual" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-human mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and other non-mammals.

[0091] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an anti-CD47 antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, effectively prevents or ameliorates one or more symptoms of a disease or condition, or the onset of a disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in symptomatic improvement, such as an amount that treats, cures, prevents, or ameliorates the associated pathology, or an amount that increases the rate of treatment, cure, prevention, or improvement of such a condition. When only the active ingredient is administered to an individual, the therapeutically effective dose refers to the ingredient alone. When administered in combination, the therapeutically effective dose refers to the comprehensive amount of the active ingredients that contribute to the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent increases a diagnostic criterion or parameter by at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0092] As used herein, "treatment" includes 1) therapeutic measures that cure, alleviate, and relieve the symptoms of a diagnosed condition or disease and / or halt the progression of a diagnosed condition or disease, and 2) preventive or prophylactic measures that prevent and / or alleviate the onset of a condition or disease. Thus, subjects receiving treatment include individuals who have suffered from a disease, individuals who are susceptible to a disease, and individuals who wish to prevent a disease. In some embodiments, the invention relates to the treatment of a disease or condition. In some other embodiments, the invention relates to the prevention of a disease or condition.

[0093] In some embodiments of the present invention, "treatment" of a disease or condition refers to ameliorating the disease or condition (i.e., alleviating or preventing or ameliorating the progression of the disease or at least one of its clinical symptoms). In other embodiments, "treatment" refers to alleviating or improving at least one physical parameter, including physical parameters not recognized by the patient. In other embodiments, "treatment" refers to modulating the disease or condition physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. Methods for assessing disease treatment and / or prevention are generally known in the art unless expressly described herein.

[0094] In yet another embodiment of the present invention, "prevention" of a disease or condition includes inhibiting the onset or progression of a disease or condition, or the symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of an agent to a subject prior to the onset of a cancerous condition or symptom, particularly a subject at risk of cancer.

[0095] In some embodiments, after "treating" cancer with the methods of the present invention, an individual patient is considered to have been successfully treated if they exhibit one or more of the following: a reduction in the number of cancer cells or complete disappearance of cancer cells; a reduction in tumor size; a reduction or absence of cancer cell invasion into peripheral organs, including, for example, the spread of cancer cells to soft tissue and bone; a reduction or absence of tumor metastasis; a reduction or absence of tumor growth; an alleviation of one or more symptoms associated with a particular cancer; a reduction in incidence or mortality; an improvement in quality of life; a reduction in the incidence, frequency, or tumorigenicity of tumors; a reduction in the number or frequency of cancer stem cells within the tumor; differentiation of tumor cells to a non-tumorigenic state; or some combination of these effects.

[0096] "Inhibition of tumor growth" refers to any mechanism capable of inhibiting tumor cell growth. In some embodiments, tumor cell growth is inhibited by slowing tumor cell growth. In some embodiments, tumor cell growth is inhibited by halting tumor cell growth. In some embodiments, tumor cell growth is inhibited by killing tumor cells. In some embodiments, tumor cell growth is inhibited by inducing apoptosis of tumor cells. In some embodiments, tumor cell growth is inhibited by inducing differentiation of tumor cells. In some embodiments, tumor cell growth is inhibited by depriving tumor cells of nutrients. In some embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In some embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

[0097] As used herein, " sequence identity " refers to the degree of sequence identity based on each nucleotide or amino acid that is compared in comparison window. " (Percentage) sequence identity " can be calculated as follows: Compare two optimally aligned sequences in comparison window, determine the number of positions that have the same nucleic acid base (for example, A, T, C, G, I) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in comparison window (that is, window size), and multiply the result by 100 to obtain the percentage of sequence identity. Optimal alignment for determining the percentage of sequence identity can be achieved by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequence or across the region of the target sequence being compared. In the present invention, for antibody sequences, the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and then performing optimal alignment according to the Kabat numbering convention in a preferred embodiment.

[0098] As used herein, the term "agglutination" refers to the clumping of cells, and the term "hemagglutination" refers to the clumping of a particular class of cells (i.e., red blood cells). Thus, hemagglutination is a type of agglutination.

[0099] As used herein, the control antibody "Hu5F9" is an anti-CD47 antibody in the form of IgG4P produced by recombinant expression with GenScript according to the variable region sequence of 5F9 disclosed in patent US2015 / 0183874A1. The control antibody "SRF231" is an anti-CD47 antibody in the form of IgG4P produced by recombinant expression with GenScript according to the variable region sequence of 2.3D11 disclosed in patent US20180201677A1.

[0100] Anti-CD47 antibodies and their production The antibodies of the present invention can be produced by any suitable method for producing antibodies. Any suitable form of CD47 can be used as an immunogen (antigen) to produce antibodies. As a non-limiting example, any CD47 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing mouse monoclonal anti-human CD47 antibodies can be produced by methods well known in the art. These methods include, but are not limited to, the hybridoma technology first developed by Kohler et al. (1975) (Nature 256: 495-497). Preferably, mouse spleen cells were isolated and fused with a mouse myeloma cell line by PEG or electrofusion according to standard protocols. Hybridoma cells secreting antibodies with CD47-binding activity were then screened. The DNA sequence of the immunoglobulin variable region of the hybridoma cells of the present invention can be detected by a method based on degenerate primer PCR.

[0101] Antibodies from rodents (such as mice) can cause undesirable antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can induce immune responses against therapeutic antibodies in humans. This type of immune response can lead to at least a loss of therapeutic efficacy and, in severe cases, potentially fatal allergic reactions. One method for reducing the immunogenicity of rodent antibodies is to produce chimeric antibodies in which mouse antibody variable regions are fused with human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, if the complete rodent variable region is retained in the chimeric antibody, it can still cause harmful immunogenicity in patients.

[0102] Grafting CDRs from rodent variable regions onto human frameworks (i.e., humanization) is used to further minimize rodent sequences. For the humanized antibodies of the present invention, mouse CDR regions can be inserted into human germline frameworks using methods known in the art. See U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. However, CDR loop exchange cannot produce antibodies with the same binding characteristics as the original antibody. In humanized antibodies, further modification of framework residues (FRs) (residues involved in supporting the CDR loops) is often necessary to maintain antigen-binding affinity. Kabat et al. (1991) J. Immunol. 147: 170. Briefly, the humanization transformation process involves the following steps: A. aligning the gene sequence of each candidate antibody with that of a human embryonic antibody to find sequences with high homology; B. analyzing and investigating HLA-DR affinity to select human embryonic framework sequences with low affinity; C. analyzing the framework amino acid sequences of the variable region and its surrounding regions using computer simulation technology and molecular docking to examine their spatial and steric binding patterns. By calculating electrostatic force, van der Waals force, hydrophilicity, and entropy, important amino acid individuals that can interact with CD47 and maintain the spatial framework in the candidate antibody gene sequence are analyzed and transplanted into the selected human embryonic gene framework. Based on this, the positions of amino acids that must be maintained in the framework region are marked, and the humanized antibody is synthesized.

[0103] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the present invention can be determined using any of many well-known schemes, such as Kabat, Chothia, AbM, Contact, or North. It should be noted that the CDR boundaries of the variable region of the same antibody obtained by different definition systems may differ. That is, the CDR sequences of the variable region of the same antibody defined by different assignment systems will be different. Therefore, when defining an antibody having a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences. However, due to the application of different schemes (such as different assignment systems or combinations), the claimed CDR boundaries will differ from the specific CDR boundaries defined in the present invention.

[0104] In some embodiments, the CDR boundaries of the anti-CD47 antibody molecules provided by the present invention are determined based on the Kabat assignment system.

[0105] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimal overlap region can be determined using at least two of the Kabat, Chothia, AbM, and North methods to provide a "minimum binding unit" for antigen binding. The minimum binding unit may be a subset of the CDR. As will be understood by those skilled in the art, the remaining residues of the CDR sequence can be determined according to the antibody structure and protein folding. Therefore, any variant of the CDRs shown herein is also contemplated by the present invention. In some embodiments, in variants of the CDRs of the anti-CD47 antibodies or antigen-binding fragments thereof of the present invention, the amino acid residues of the minimum binding unit may remain unchanged, but the remaining residues of the CDRs defined according to Kabat or IMGT may be substituted with conservative amino acid residues.

[0106] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises one to three selected from heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, wherein the HCDR1 comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 11 or has at least one and three or less, two or less, or one amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of SEQ ID NO: 11; the HCDR2 comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 12 or has at least one and three or less, two or less, or one amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of SEQ ID NO: 12; and the HCDR3 comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21 or has at least one and three or less, two or less, or one amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21.

[0107] In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof provided by the present invention comprises one to three selected from light chain complementarity determining region 1 (HCDR1), LCDR2, and LCDR3, wherein the LCDR1 comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 14 or has at least one and no more than three, no more than two, or no more than one amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of SEQ ID NO: 14; the LCDR2 comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 22, or has at least one and no more than three, no more than two, or no more than one amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 22; and the LCDR3 comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 16 or has at least one and no more than three, no more than two, or no more than one amino acid change (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of SEQ ID NO: 16.

[0108] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention include antibodies or antigen-binding fragments thereof that contain a total of at least one and no more than five, four or no more, three or no more, two or no more, or one amino acid change (preferably amino acid substitutions, preferably conservative substitutions) in the three CDRs of the heavy chain variable region compared to the three CDRs specifically disclosed herein, and / or at least one and no more than five, four or no more, three or no more, two or no more, or one amino acid change (preferably amino acid substitutions, preferably conservative substitutions) in the three CDRs of the light chain variable region.

[0109] In some embodiments, anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention include antibodies or antigen-binding fragments thereof having one or more (preferably 10 or fewer, more preferably 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) in the heavy chain variable region and / or light chain variable region compared to the heavy chain variable region and / or light chain variable region of an antibody specifically disclosed herein, and preferably the aforementioned amino acid alterations do not occur in the CDR regions.

[0110] In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 6, or 7. In some embodiments, the anti-CD47 antibodies or antigen-binding fragments thereof provided by the present invention comprise a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 8, 9, or 10.

[0111] In one embodiment of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0112] In preferred embodiments, the amino acid changes of the present invention occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and / or the light chain variable region. In some embodiments, the amino acid changes occur in the heavy chain constant region and / or the light chain constant region.

[0113] In some embodiments, antibodies of the invention containing amino acid alterations have properties that are the same or similar to the specific antibodies disclosed herein.

[0114] In some embodiments, the anti-CD47 antibodies of the invention comprise a CDR, a light chain variable region, a heavy chain variable region, a post-translational modification to the light chain or the heavy chain.

[0115] In some embodiments, the anti-CD47 antibodies provided by the present invention are single-domain antibodies such as full-length antibodies, VHHs, Fab antibodies, Fab' antibodies, Fab'-SH, (Fab')2 antibodies, or single-chain antibodies such as scFvs, Fvs, dAbs (domain antibodies), or bis(multi)specific antibodies.

[0116] In some embodiments, the anti-CD47 antibodies provided by the invention are any antibody in the form of an IgG, such as an antibody in the form of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the anti-CD47 antibodies of the invention are antibodies in the form of IgG4P, i.e., an antibody in which the human IgG4 constant region has been modified in the hinge region, Ser228Pro (S228P, numbering according to EU), to prevent or reduce chain exchange.

[0117] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided by the present invention to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (such as the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid modifications at one or more amino acid positions. For example, the number of modifications to human IgG1 that enhance or reduce FcγR binding and thereby enhance or reduce the corresponding functions are summarized in the paper by Bruhns and Jonsson published in Immunol Rev. 2015 Nov; 268 (1): 25-51, page 44.

[0118] In some embodiments, the anti-CD47 antibodies provided by the present invention comprise an Fc region variant with reduced or absent FcγR binding activity. In some embodiments, the Fc region variant has an amino acid substitution, particularly selected from substitutions of other amino acids at positions E233, L234, L235, N297, and P331 of the immunoglobulin heavy chain. In some embodiments, the amino acid substitution in the Fc region variant is E233P, L234A, L235A, L235E, N297A, N297D, or P331S.

[0119] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites in an antibody can be achieved by altering the amino acid sequence to create or remove one or more glycosylation sites, as appropriate. Glycosylation can be altered, for example, to increase the affinity of the antibody for an "antigen." This can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. This aglycosylation can increase the affinity of the antibody for the antigen. Such methods are described, for example, in U.S. Patent No. 5,426,300. If the antibody contains an Fc region, the carbohydrates attached thereto can be altered. In some applications, modifications to remove unwanted glycosylation sites are useful, such as removing fucose modules to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be made to modify complement dependent cytotoxicity (CDC).

[0120] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0121] In some embodiments, the antibodies provided herein can be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Non-proteinaceous moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dialkanes, poly-1,3,6-trialkanes, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0122] Antibody expression The present invention relates to host cells comprising one or more expression vectors and methods for producing any of the antibodies or antigen-binding fragments thereof of the invention, comprising culturing the host cells and purifying and recovering the antibody or antigen-binding fragment.

[0123] In one embodiment, the present invention provides nucleic acids encoding any of the above-described anti-CD47 antibodies or antigen-binding fragments thereof. For example, the present invention provides nucleic acids encoding the heavy chain, light chain, variable region, or complementarity-determining region described herein. In some embodiments, the nucleic acid encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid set forth in SEQ ID NO: 19. In some embodiments, the nucleic acid encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid set forth in SEQ ID NO: 20.

[0124] In one aspect, one or more vectors containing the nucleic acid are provided. In some embodiments, the vector is an expression vector. The choice of expression vector depends on the host cell in which the vector is intended to be expressed. Generally, the expression vector comprises a promoter and other regulatory sequences (e.g., enhancers) operably linked to the nucleic acid encoding the anti-CD47 antibody chain or antigen-binding fragment thereof. In some embodiments, the expression vector further comprises a sequence encoding an antibody constant region. In some embodiments, the expression vector is a pCDNA vector, such as a PTT5 vector or pCDNA3.4.

[0125] In one aspect, the present invention provides host cells for expressing the recombinant antibodies of the present invention, including prokaryotic or eukaryotic cells. In some embodiments, Escherichia coli is a prokaryotic host that can be used to clone and express the polynucleotides of the present invention. Other suitable microbial hosts include species of the Bacillus genus, such as Bacillus subtilis, and Enterobacteriaceae, such as Salmonea, Serratia, and various Pseudomonas species. Expression vectors containing expression control sequences (e.g., origins of replication) compatible with the host cell can also be prepared for these prokaryotic hosts. In some embodiments, mammalian host cells are used to express and produce the anti-CD47 antibody polypeptides of the present invention. For example, mammalian host cells can be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines harboring exogenous expression vectors, including normal human cells, or immortalized animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, Expire293 cells, HEK293 cells, myeloma cell lines, transformed B cells and hybridomas.

[0126] In one embodiment, the present invention provides a method for preparing an anti-CD47 antibody, comprising introducing an expression vector into mammalian host cells and culturing the host cells for a sufficient period of time to express the antibody within the host cells, or more preferably, to secrete the antibody into the culture medium where the host cells are grown to produce the antibody. The antibody can be recovered from the culture medium using standard protein purification methods. Antibody molecules prepared as described herein can be purified by known and available techniques, such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which will be apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size-exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0127] Antibodies expressed by different cell lines or in transgenic animals may have different glycosylation from each other. However, all antibodies encoded by the nucleic acids provided herein or comprising the amino acid sequences provided herein, regardless of their glycosylation, are components of the present invention.

[0128] How to decide The physical / chemical properties and / or biological activity of the anti-CD47 antibodies provided herein can be identified, screened, or characterized by various determination methods known in the art. In one embodiment, the antigen-binding activity of the antibodies of the present invention is tested by known methods, such as ELISA or Western blot. Binding to CD47 can be determined using methods known in the art, and exemplary methods are disclosed herein.

[0129] The present invention also provides methods for identifying anti-CD47 antibodies with biological activity. Biological activities may include, for example, binding to CD47 (e.g., binding to human CD47), binding to CD47 on the cell surface, blocking the binding of CD47 to its ligand, affecting hemagglutination-promoting activity, and affecting the phagocytosis of tumor cells by human macrophages. Antibodies with such biological activity in vivo and / or in vitro are also provided. In certain embodiments, antibodies of the present invention are tested for such biological activity.

[0130] Cells for use in any of the above in vitro determination methods include cell lines that naturally express CD47, such as tumor cell lines, or cell lines engineered to express CD47, including cell lines that express CD47 and cell lines transfected with DNA encoding CD47 that express CD47 under abnormal circumstances.

[0131] The immunoconjugates or immunofusions of the present invention can be used to replace or supplement anti-CD47 antibodies to perform any of the above-described determination methods.

[0132] It will be appreciated that any of the above determination methods may be performed using a combination of an anti-CD47 antibody and another active agent.

[0133] Immune complexes and immunofusions In some embodiments, the present invention provides an immunoconjugate comprising any of the anti-CD47 antibodies or antigen-binding fragments thereof provided herein and another agent, hi one embodiment, the other agent is a cytotoxic agent, including, for example, any agent that is detrimental to cells.

[0134] In some embodiments, immunofusions comprising any of the anti-CD47 antibodies or antigen-binding fragments thereof provided herein are provided.

[0135] In some embodiments, the immunoconjugates and immunofusions are used to prevent or treat CD47-associated diseases.

[0136] Pharmaceutical Composition The term "pharmaceutical composition" refers to a preparation / formulation that allows the biological activity of the active ingredient contained therein to be present in a form that is effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation / formulation is administered.

[0137] The term "pharmaceutical excipient" refers to a pharmaceutical carrier, diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), or vehicle with which a therapeutic is administered.

[0138] Pharmaceutical compositions of the invention can include an antibody of the invention and a pharmaceutical excipient. These pharmaceutical compositions can be included in kits, such as diagnostic kits.

[0139] As used herein, "pharmaceutical carrier" includes any physiologically compatible solvent, dispersion medium, isotonic agent, absorption delaying agent, etc. Pharmaceutical carriers suitable for the present invention can be sterile liquids such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline, aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injection solutions.

[0140] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, diol, water, ethanol, etc. For details on the application and use of excipients, see also "Handbook of Pharmaceutical Excipients," fifth edition, R.C. Rowe, P.J. Seskey, and S.C. Owen, Pharmaceutical Press, London, Chicago. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0141] The present invention provides pharmaceutical compositions containing one or more monoclonal antibodies that bind to CD47 or an antigen-binding fragment thereof, and a nucleic acid, vector, or host cell, or immunoconjugate or immunofusion of the present invention. The anti-CD47 antibodies, or antigen-binding fragments, nucleic acids, vectors, or host cells thereof, or immunoconjugates or immunofusions thereof provided by the present invention, or pharmaceutical compositions thereof, can be combined with suitable pharmaceutical carriers, excipients, and other reagents in preparations for co-administration to improve transport, delivery, tolerance, etc.

[0142] Pharmaceutical preparations / formulations containing the anti-CD47 antibodies described herein are prepared by mixing an anti-CD47 antibody or antigen-binding fragment thereof of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of an aqueous solution or a lyophilized formulation. Exemplary lyophilized antibody preparations / formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody preparations / formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine acetate buffer.

[0143] The pharmaceutical compositions or preparations / formulations of the present invention may also contain one or more other active ingredients necessary for the treatment of a specific disease, preferably active ingredients with complementary activities that do not adversely affect each other. For example, it may be desirable to include other therapeutic agents. In some embodiments, the other therapeutic agents are chemotherapeutic agents, radiotherapeutic agents, cytokines, vaccines, other antibodies, immunomodulators, or other biopolymer drugs.

[0144] In some embodiments, the pharmaceutical compositions of the invention may also contain a nucleic acid encoding an anti-CD47 antibody or antigen-binding fragment thereof.

[0145] Methods and Uses In one embodiment, the present invention provides a method for preventing, diagnosing, or treating a CD47-related disease in a subject, comprising administering to a patient in need thereof an effective amount of an anti-CD47 antibody or antigen-binding fragment thereof, or an immunoconjugate or immunofusion described herein, or a pharmaceutical composition comprising same, or a nucleic acid, vector, or host cell described herein.

[0146] In one embodiment, the present invention provides the use of an anti-CD47 antibody or antigen-binding fragment thereof in the manufacture or preparation of a medicament for the prevention, diagnosis, or treatment of a CD47-related disease in a subject.

[0147] In one embodiment, the anti-CD47 antibodies and antigen-binding fragments thereof provided by the present invention, as well as pharmaceutical compositions containing them, can be used as therapeutic agents for preventing or treating CD47-related diseases in subjects. For CD47-related diseases in subjects identified using standard methods, anti-CD47 antibodies and antigen-binding fragments thereof, as well as pharmaceutical compositions, immunoconjugates or immunofusions, or nucleic acids, vectors, or host cells disclosed herein containing the same can be administered.

[0148] In some embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent or treatment modality. In some embodiments, the therapeutic agent is, for example, a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, another antibody, an immunomodulator, or another biopolymer drug. In some embodiments, the treatment mode includes surgery; and radiation therapy, local or focal irradiation, etc.

[0149] The combination therapy described above includes combined administration (where two or more therapeutic agents are contained in the same or separate preparations / formulations) and separate administration, where administration of an anti-CD47 antibody or antigen-binding fragment thereof of the present invention can occur prior to, concurrently with, or subsequent to the additional therapeutic agent(s) and / or adjuvant(s) and / or treatment.

[0150] In some embodiments, a CD47-related disease of the present invention refers to a disease associated with abnormal CD47 expression, activity, and / or signaling in a subject, including, but not limited to, cancer. In some embodiments, a CD47-related disease involves increased (level or content) of nucleic acid encoding CD47, increased CD47 expression, increased CD47 protein level or activity, or increased active signaling.

[0151] In some embodiments, treatment of diseases would benefit from inhibition of CD47 at the nucleic acid or protein level, or would benefit from blocking binding of CD47 to its ligand or CD47-mediated signaling.

[0152] In some embodiments, the subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein (e.g., cancer). In certain embodiments, the subject is undergoing or has undergone other treatments, such as chemotherapy and / or radiation therapy.

[0153] In some embodiments, cancers include various blood cancers and solid tumors, as well as metastatic lesions. In one embodiment, an example of a solid tumor is a malignant tumor. The cancer may be in early, mid, or late stage, or may be metastatic. The cancer may be, for example, bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer, (benign) melanoma, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, or liver cancer. In some embodiments, the lymphoma is selected from Burkitt's lymphoma, diffuse large cell lymphoma, or mantle cell lymphoma. In some embodiments, the leukemia is promyelocytic leukemia.

[0154] The antibodies or antigen-binding fragments of the present invention can be administered by any suitable method, including oral administration, parenteral administration, intrapulmonary administration, and intranasal administration, and can be administered intralesionally if localized treatment is required. Parenteral administration includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various administration regimens are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0155] The antibodies or antigen-binding fragments of the present invention are formulated and administered in a manner consistent with good medical practice. Although not required, the antibodies may optionally be formulated with one or more drugs currently used to prevent or treat disease. The effective amount of these other drugs will depend on the amount of antibody present in the preparation / formulation, the type of condition or disease being treated, and other factors discussed above. Regardless of whether the antibody is for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the judgment of the attending physician, the antibodies or antigen-binding fragments of the present invention are administered in appropriate doses to prevent or treat disease, depending on the type of disease being treated, the type of antibody, and the severity and course of the disease. The antibodies are appropriately administered to the patient at one time or over a series of treatments.

[0156] In certain embodiments, any of the anti-CD47 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of CD47 in a sample. In some embodiments, the detection method comprises: (a) contacting the sample with an antibody or antigen-binding fragment thereof, or a conjugate or fusion of the invention; and (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof, or a conjugate or fusion thereof and a CD47 protein; Includes.

[0157] As used herein, the term "detection" includes quantitative or qualitative detection. In certain embodiments, the sample is a blood, serum, or other liquid sample of biological origin. In certain embodiments, the sample comprises cells or tissue. In some embodiments, the sample is from a lesion associated with a hyperproliferative or cancerous lesion.

[0158] In one embodiment, the antibodies or antigen-binding fragments thereof of the present invention can be used to diagnose CD47-related diseases, such as cancer, for example, to evaluate (e.g., monitor) the treatment or progression of the diseases described herein, and to diagnose and / or stage them in individuals. In certain embodiments, labeled anti-CD47 antibodies or antigen-binding fragments thereof are provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties, such as enzymes or ligands, that are indirectly detected, for example, via enzymatic reactions or molecular interactions. In some embodiments, kits for diagnosing CD47-related diseases are provided herein, comprising the antibodies or antigen-binding fragments thereof of the present invention.

[0159] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-CD47 antibody or antigen-binding fragment thereof. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during or after treatment with another therapy.

[0160] The present invention includes any combination of the specific embodiments described herein. While specific details and examples are described to illustrate preferred embodiments of the present invention, it should be understood that these are merely illustrative and are used as examples. The present invention further encompasses embodiments that vary based on the preferred embodiments of the present invention that would be obvious to one skilled in the art. For all purposes, all publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety. [Example]

[0161] Example 1: Production and screening of hybridoma-derived antibodies Anti-CD47 antibodies were obtained using hybridoma technology. Recombinant protein CD47-Fc (ACROBiosystems, catalog number CD7-H5256), which contains the extracellular domain of human CD47 with an Fc tag, was used as an antigen to immunize mice. After mixing and emulsifying the recombinant protein CD47-Fc with complete or incomplete Freund's adjuvant (Sigma-Aldrich), SJL mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) and BALB / c mice (Yangzhou University Medical Center) were immunized. Mice were given one immunization (complete Freund's adjuvant) and two booster immunizations (incomplete Freund's adjuvant), with blood collected after each booster. The binding activity of post-immunization mouse serum to recombinant human CD47-Fc protein (ACROBiosystems, catalog number CD7-H5256) was detected by ELISA assay, and simultaneously, the binding ability of mouse serum to CHO cells overexpressing human CD47 (constructed by GenScript) was detected by flow cytometry (FACS). Spleen cells from mice with the highest serum titers were selected and fused with the myeloma cell line SP2 / 0 (ATCC). Four days before fusion, mice were intraperitoneally injected with recombinant human CD47 extracellular domain protein CD47-Fc for booster immunization. On the day of fusion, mice were euthanized, and then mouse spleen cells were homogenized to obtain a single-cell suspension. Mouse spleen cells were fused with the mouse myeloma cell line SP2 / 0 (constructed by GenScript) at a ratio of 3:1 using an electrofusion device. The fused cells were resuspended in a medium containing HAT (hypoxanthine, aminopterin, and thymidine deoxynucleotides, GIBCO, catalog number 21060016) to screen for successfully fused hybridoma cells. The supernatants of the hybridoma cells were collected and hybridoma cells secreting antibodies that specifically bind to human CD47 were screened by two rounds of ELISA.The activity of the secreted supernatants of the hybridomas was then confirmed by CD47-related functional screening tests (binding specificity to human CD47 or cynomolgus monkey CD47; lack of hemagglutination-inducing activity; promoting phagocytosis of tumor cells by macrophages, etc.). Positive hybridoma clones were then selected and subjected to single or multiple rounds of subcloning to obtain a single clone. After screening, 125G4A4 was finally selected as the hybridoma clone.

[0162] The candidate hybridoma cells 125G4A4 were expanded and cultured for 7-10 days, after which the supernatant was collected, centrifuged, and filtered to remove cells and debris. The supernatant was passed through a Protein A purification column (GenScript), washed and equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0), and then eluted with 0.1 M sodium citrate (pH 3.5). The eluate was immediately neutralized with 1 / 9 volume of 1 M Tris-HCl (pH 9) and then dialyzed against PBS buffer. Finally, the hybridoma-derived antibody 125G4A4 was obtained and further characterized.

[0163] 1.1 Detection of antibody binding activity to CHO-K1 cells overexpressing human CD47 protein by FACS Human CD47 protein (NCBI accession number: NP_001768.1) was overexpressed in the hamster ovary cell line CHO-K1 to establish a CHO-K1 cell line overexpressing human CD47 protein. The cells were incubated with serially diluted antibody 125G4A4 and reference antibody C0774CK230-C (i.e., Hu5F9) (maximum concentration 300 nM, 3-fold dilutions, a total of 12 concentration points) at 4°C for 50 minutes. After washing twice with ice-cold PBS, the cells were incubated with iFluor647-labeled goat anti-mouse IgG (H+L) antibody (Genscript) in the dark at 4°C for 40 minutes. The cells were then washed twice with ice-cold PBS and then detected by flow cytometry using a Calibur (BD Biosciences). A concentration-dependent curve was fitted using GraphPad according to the mean fluorescence intensity (MFI) of the signal, and the EC values ​​were calculated.50 As shown in Table 1, the finally obtained hybridoma-derived antibody 125G4A4 had high binding activity to CHO-K1 cells overexpressing human CD47 protein, and EC 50 was 0.22 nM.

[0164] 1.2 Detection of antibody binding activity against CD47 on tumor cell surface by FACS Human CD47 was endogenously expressed on the cell surface of the human Burkitt's lymphoma cell line, Raji. Antibody 125G4A4 and reference antibody Hu5F9 were serially diluted in PBS containing 2% fetal bovine serum (FBS, Gibco, catalog number 10100147) (maximum concentration 46.3 nM, 3-fold dilutions, total 8 concentration points). The diluted antibodies were mixed with Raji cells (purchased from ATCC) (5 x 10 5 Cells were co-cultured with 1000 μg / well of PBS containing 2% fetal bovine serum (FBS) for 1 hour at 4°C. After washing three times with PBS containing 2% fetal bovine serum (FBS), PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat. No. 409304) was added and incubated for 1 hour in the dark at 4°C. The cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and then the fluorescent signal was detected by Canto II (BD Biosciences) flow cytometry. A concentration-dependent curve was fitted using GraphPad according to the mean fluorescence intensity (MFI) of the signal, and the EC 50 As shown in Table 1, the hybridoma-derived antibody 125G4A4 had an EC 50 The antibody had binding activity to Raji cells.

[0165] 1.3 Blockade of the interaction between human CD47 and SIRPα by anti-CD47 antibodies An ELISA assay was performed to detect the ability of 125G4A4 to block the interaction between human CD47 and SIRPα. A recombinant protein, hCD47-Fc (ACROBiosystems, catalog no. CD7-H5256), containing the extracellular domain of human CD47 fused to the human IgG Fc fragment, was coated onto a 96-well plate and incubated overnight at 4°C. After washing the plate three times with PBST (PBS containing 0.5% Tween-20), 1% BSA was added to the plate for 2 hours to block the plate. After washing the plate three times with PBST, serially diluted antibody 125G4A4 or the reference antibody Hu5F9 (maximum concentration 66.7 nM, 3-fold dilutions, total 8 concentration points) was added along with SIRPα-His recombinant protein (ACROBiosystems, catalog no. SIA-5225) at a final concentration of 2.5 μg / ml and incubated at room temperature for 1 hour. The plate was washed three times with PBST, and horseradish peroxidase-conjugated goat anti-His tag secondary antibody (CWBIO, Cat. No. CW0285M) was added to detect SIRPα captured by the coated CD47 protein. After incubating the 96-well plate at 37°C for 30 minutes, the plate was washed five times with PBST, and TMD (Surmodics, Cat. No. TMBW-1000-01) developer was added and incubated in the dark for 15 minutes. The color reaction was stopped by adding 2N H2SO4. The OD was measured using a microplate reader. 450 The absorbance values ​​reflected the amount of SIRPα bound to CD47. Concentration-dependent curves were fitted using Graphpad to determine the IC of anti-CD47 antibodies to block the binding of CD47 to SIRPα. 50 As shown in Table 1, 125G4A4 had an IC of 3.06 nM. 50 could effectively block the CD47 / SIRPα interaction.

[0166] 1.4 Detection of the activity of anti-CD47 antibodies in inducing human erythrocyte agglutination It is known in the prior art that most anti-CD47 antibodies have the property of inducing hemagglutination. This property is widely believed to be closely related to clinical side effects, such as anemia, that occur during treatment with therapeutic anti-CD47 antibodies. Therefore, we evaluated the anti-CD47 antibodies of the present invention using in vitro hemagglutination experiments to screen for antibodies that do not induce hemagglutination. The method is as follows: fresh human blood from a healthy donor was collected, the cells were washed five times with PBS, and then the cells were diluted to create a suspension containing 10% human red blood cells. The red blood cell suspension was mixed with the experimental antibodies (antibody 125G4A4 and reference antibody Hu5F9, maximum concentration 667 nM, 3-fold dilution, a total of 12 concentration points), and the mixture was then added to a round-bottom 96-well plate. The mixture was incubated at room temperature for 16 hours, then photographed, and the results were determined according to the appearance of cells in the wells. When hemagglutination occurred, cells were seeded into each well like a net, and a larger sheet-like cell layer appeared in wells with a larger diameter than the negative control wells. On the other hand, when hemagglutination did not occur, the red blood cells settled to the bottom of the well, and small, dot-like cell pellet deposits appeared in the well. 125G4A4 did not show any obvious hemagglutination-inducing phenomenon in the experiment.

[0167] 1.5 Measurement of the effect of anti-CD47 antibodies on promoting phagocytosis of tumor cells by human macrophages The ability of the antibody 125G4A4 of the present invention to promote phagocytosis of tumor cells by macrophages was detected using a flow cytometry-based assay. Human blood was freshly collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Catalog No. 17-1440-02). Monocytes were further isolated using a Human Whole Monocyte Isolation Kit (Miltenyi Biotec, Catalog No. 130-096-537). To induce differentiation of monocytes into macrophages, macrophage colony-stimulating factor (M-CSF, R&D Systems, Catalog No. 216-MC) was added, and the monocytes were subjected to adherent culture for 7 consecutive days. On the day of the cell phagocytosis experiment, the differentiated macrophages described above were starved in serum-free medium for 2 hours. Simultaneously, target tumor cells, Raji, were fluorescently labeled with CFSE (eBioscience, Catalog No. 65-0850-85) according to the recommended procedure. CFSE-labeled tumor cells and macrophages were mixed at a 4:1 ratio, and the experimental antibodies were added at a detection concentration and incubated at 37°C for 2 hours. The cells were then washed twice with PBS and digested with trypsin (Gibco, Catalog No. 25200072); APC-labeled anti-CD14 antibody (Biolegend, Catalog No. 325608) was added and incubated on ice in PBS containing 2% fetal bovine serum for 30 minutes in the dark. The cells were washed twice and analyzed by flow cytometry. The percentage of CFSE-positive cells in the CD14-positive macrophage population was calculated. As shown in Table 1, 125G4A4 effectively promoted the phagocytic function of macrophages on tumor cells.

[0168] [Table 2]

[0169] Example 2 Humanization of hybridoma-derived antibodies 2.1 Determination of variable region sequences of hybridoma-derived antibodies According to the hybridoma sequencing method, cells of the hybridoma clone 125G4A4 were expanded; total RNA was extracted with TRIzol (purchased from Ambio) and reverse transcribed into DNA using antibody-specific primers (Takara, PrimerScript 1st Strand cDNA Synthesis Kit); gene fragments encoding mouse immunoglobulin V regions were amplified with antibody-specific primers. The variable region sequences of the hybridoma-derived antibodies were obtained by sequencing analysis. The amino acid sequences of the heavy and light chain variable regions of the 125G4A4 antibody are shown in SEQ ID NOs: 1 and 2, respectively, and the nucleotide sequences are shown in SEQ ID NOs: 19 and 20, respectively.

[0170] 2.2 Construction and Expression of Chimeric Antibodies In accordance with the mechanism of action of CD47, in a specific embodiment of the present invention, the human IgG4 constant region (S228P) was used as the antibody heavy chain constant region, and the human kappa light chain constant region was used as the antibody light chain constant region. The serine-to-proline mutation at position 228 in the IgG4 core hinge region (S228P) strengthened the disulfide bond connection in the core hinge region, reducing IgG4 Fab arm exchange and thereby significantly reducing half-molecule formation. After synthesizing the genes encoding the heavy and light chain constant regions, the heavy and light chain variable region genes were homologously recombined into the PTT5 vector by double digestion with EcoRI and BamHI. After accurate sequencing, the antibody heavy and light chains were co-transfected into HEK293 cells at a molar ratio of 1.5:1. After 120 hours of culture, the supernatant was collected by centrifugation and purified to obtain the chimeric antibody.

[0171] Before humanization, several post-translational modification (PTM) sites in the CDR regions must be mutated to avoid affecting the protein conformation and, therefore, its function. PTM analysis identified two PTM sites in the CDR of 125G4A4, including one NSS glycosylation site in the heavy chain and one DG isomerization site in the light chain. The NSS glycosylation site and the DG isomerization site were mutated to QSS and EG, respectively. The purified chimeric antibody obtained in this example was named Ch-125G4-m35. The amino acid sequences of the heavy and light chain variable regions of the Ch-125G4-m35 antibody are set forth in SEQ ID NOs: 3 and 4, respectively.

[0172] 2.3 Humanized design of chimeric antibodies To select the human antibody backbone sequence with the highest homology to the chimeric antibody 125G4A4m for humanization, the variable region sequence of the chimeric antibody 125G4A4m was aligned using Blast with the PDB antibody database. The heavy chain variable region of 125G4A4m had higher sequence homology with the germline IGHV1-69, and its light chain variable region had higher sequence homology with the human germline IGKV1-16. The amino acid sequences and precise boundaries of the variable region CDRs were then defined using the Kabat assignment system. The CDR segments of the variable region of the mouse antibody were then grafted onto the human backbone sequence to obtain a humanized antibody.

[0173] To maintain the activity of the humanized antibody, the framework amino acid sequences of the variable region and its surrounding regions were analyzed using computer simulation techniques to investigate their steric binding mode. Electrostatic forces, van der Waals forces, hydrophilicity, and entropy were calculated to identify key amino acid sequences that could interact with CD47 and maintain the spatial framework of the candidate antibody gene sequence. These sequences were then grafted onto the selected human antibody gene framework. Meanwhile, amino acid positions in the framework region that needed to be preserved were marked. Through these steps, the humanized antibody was synthesized. Several key sites in the antibody framework region were backmutated into the antibody framework region sequence of the chimeric antibody Ch-125G4-m35. Depending on the number and arrangement of backmutations, a number of different humanized heavy chain variable regions (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7) and light chain variable regions (SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10) were designed (see Table 2). The final humanized antibody of the present invention, Hu-125G4A4-48, is hereinafter designated HMA02h14-48. The amino acid sequences of the heavy and light chain variable regions of the antibody are as shown in SEQ ID NOs: 7 and 8, respectively.

[0174] [Table 3]

[0175] [Table 4]

[0176] [Table 5]

[0177] 2.4 Expression of humanized antibodies The DNA fragments encoding the designed humanized heavy and light chain variable regions were amplified and cloned into a vector containing the constant region for human antibody expression to construct an antibody expression plasmid (pCDNA3.4, purchased from Thermo, catalog number A14697). The heavy and light chain expression vectors were co-transfected into Expire293 cells (Thermo, catalog number A14525). After culturing at 37°C for 6 days, the supernatant was collected. Following the method described above, the recombinant antibody was obtained by Protein A affinity purification for further antibody characterization. The humanized antibody was of the IgG4 S228P (IgG4P) subtype.

[0178] Example 3 Screening of humanized antibodies Highly active humanized antibodies were screened by detecting their binding ability to cynomolgus monkey B cells, phagocytosis of tumor cells by human macrophages, and hemagglutination-inducing ability.

[0179] Detection of the binding ability of humanized antibodies to cynomolgus monkey B cells: Flow cytometry was used to detect the binding of a series of humanized 125G4A4 antibodies to CD47 on the surface of cynomolgus monkey B cells. Peripheral blood mononuclear cells (PBMCs) were isolated from cynomolgus monkey blood (provided by Shanghai Yinuosi Bio-Technology) by density gradient centrifugation using Ficoll-Paque PLUS powder (GE Healthcare, catalog number 17-1440-02). PBMCs were incubated with a series of humanized 125G4A4 antibodies or an isotype control (IgG4P) in PBS containing 2% fetal bovine serum at 4°C for 30 minutes. The cells were then washed three times and incubated with a secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, catalog number 409304) in PBS containing 2% fetal bovine serum at 4°C in the dark for 30 minutes. The cells were washed three times and analyzed by flow cytometry. B cells were labeled with a cynomolgus monkey cross-reactive anti-human CD20 antibody (Brilliant Violet 421™-conjugated anti-human CD20 antibody, Biolegend, Cat. No. 302330) and detected by a Canto II (BD Biosciences) flow cytometer to obtain mean fluorescence intensity (MFI).

[0180] The phagocytosis of tumor cells and the hemagglutination-inducing ability of macrophages were detected according to the methods described in Examples 1.5 and 1.4, respectively.

[0181] As shown in Table 5, a series of humanized 125G4A4 antibodies bound to CD47 expressed on cynomolgus monkey B cells at the tested concentrations. The antibodies promoted phagocytosis of tumor cells, Raji, by macrophages, with Hu-125G4A4m-48 exhibiting the strongest phagocytosis rate at 33 nM. Other activities of Hu-125G4A4m-48 were similar to those of the chimeric antibody Ch-125G4m-m35. Furthermore, the number of reversion mutations was low. Therefore, Hu-125G4A4m-48 was selected for further testing and is hereafter designated HMA02h14-48.

[0182] [Table 6]

[0183] Example 4 Measurement of binding activity of HMA02h14-48 to tumor cells by FACS Human CD47 is endogenously expressed on the surface of human Burkitt's lymphoma cell line Raji cells (Shanghai Institutes for Biological Sciences, SIBS, CCL-86™ / ATCC), human diffuse large cell lymphoma Toledo cells (ATCC® CRL-2631™), and human mantle cell lymphoma REC-1 cells (ATCC® CRL-3004™). Following the detection method described in Example 1.2 above, flow cytometry was used to detect binding of humanized antibody HMA02h14-48 to CD47 on the surface of the aforementioned tumor cell lines. The maximum antibody concentration was 667 nM, and the antibody was serially diluted and tested at a total of eight concentration points.

[0184] As shown in Figures 1-3, both HMA02h14-48 and Hu5F9 bound to CD47 on the surface of tumor cells, including Raji, Toledo, and REC-1. The maximum fluorescence intensity at saturation for HMA02h14-48 was higher than that of Hu5F9, and the EC 50 and maximum fluorescence intensity are shown in Table 6.

[0185] [Table 7]

[0186] The negative control isotype antibody (isotype) used in this and other examples was human IgG4P purchased from Shanghai Chempartner.

[0187] Example 5 Biacore Determination of Binding Affinity of Antibody HMA02h14-48 to Human CD47 Binding kinetic parameters were determined by measuring surface plasmon resonance (SPR) using Biacore. This technique detects the microscopic rate constants of antibody-antigen association (ka) and dissociation (kd). Based on the ka and kd values, the affinity value between the antibody and antigen could be obtained. The Biacore instrument (Biacore T200) and reagents were both purchased from GE Healthcare. Anti-human Fc antibodies were immobilized on a CM5 sensor chip. Purified antibodies (HMA02H14-48 and Hu5F9) were diluted in mobile phase buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.4) and applied to the anti-human Fc antibody-coated CM5 chip. Serially diluted human CD47-His (ACROBiosystems, catalog number CD7-H5227) fusion protein was then passed through the detection chip to measure the binding between the antigen and antibody, and then mobile phase buffer was passed through the chip to detect the dissociation of the antigen from the antibody. The binding and dissociation signal data for the antigen and antibody were collected at different concentrations and fitted 1:1 by the Langmuir model to calculate the affinity between the antigen and antibody.

[0188] As shown in Table 7, HMA02h14-48 has a K of 7.77E-10(M). D It bound to human CD47 with high affinity.

[0189] [Table 8]

[0190] Example 6 Detection of the activity of HMA02h14-48 in blocking the interaction between human CD47 and SIRPα by ELISA The ability of HMA02h14-48 to block the interaction between human CD47 and SIRPα was detected using ELISA, according to the method described in Example 1.3 above. The maximum antibody concentration was 67 nM, and the antibody was serially diluted and tested at a total of 8 concentration points.

[0191] As shown in Figure 4, the antibody of the present invention, HMA02h14-48, exhibited IC 50 = 1.58 nM blocked the interaction between human CD47 and SIRPα.

[0192] Example 7 Effect of HMA02h14-48 on phagocytosis of tumor cells by human macrophages According to the method described in Example 1.5, the effect of HMA02h14-48 on promoting phagocytosis of human Burkitt's lymphoma cell line Raji cells, human diffuse large cell lymphoma Toledo cells, human mantle cell lymphoma REC-1 cells, and human promyelocytic leukemia cell line HL-60 by human macrophages was detected. The maximum antibody concentration was 100 μg / mL, and the antibody was serially diluted and tested at a total of 8 concentration points.

[0193] The results show that HMA02h14-48 effectively promoted the phagocytosis of the human Burkitt's lymphoma cell line Raji by macrophages, compared with the reference antibodies Hu5F9 and SRF231. The maximum phagocytosis rate reached 36.5%, higher than that of Hu5F9 and SRF231 at concentrations ranging from 0.1 to 100 μg / ml. The maximum phagocytosis rate of HMA02h14-48 against the human mantle cell lymphoma REC-1 reached 84.6%, maintaining a phagocytosis rate of approximately 70% even at a low concentration of 0.1 μg / ml, which was higher than that of Hu5F9 and SRF231. HMA02h14-48 promoted the phagocytosis of Toledo cells by macrophages, with a phagocytosis rate of up to 94.2%. HMA02h14-48 also promoted the phagocytosis of tumor cells HL-60 by macrophages, with a maximum phagocytosis rate of 65%.

[0194] [Table 9]

[0195] Example 8: Determination of the effect of HMA02h14-48 on the induction of hemagglutination in vitro Human red blood cells were diluted to 10% with PBS and incubated with CD47 antibody added to a round-bottom 96-well plate at room temperature for 16 hours. The presence of unsedimented red blood cells is evidence of hemagglutination. Compared to the white dots formed by the sedimentation of non-agglutinated red blood cells, the unsedimented red blood cells formed a larger reticulated area than that of the negative control isotype antibody (see Figure 9). The results of the negative control isotype antibody (Isotype) were used as a standard reference.

[0196] Antibody HMA0214-48 was tested to determine whether it induces hemagglutination according to the method described in Example 1.4. The maximum antibody concentration was 667 nM, and the antibody was serially diluted and tested at a total of 12 concentration points.

[0197] As shown in Figure 9, the CD47 antibody Hu5F9 was shown to be able to significantly induce hemagglutination at concentrations of 0.9 nM or higher. In contrast, the antibody of the present invention, HMA02h14-48, did not induce significant hemagglutination of human red blood cells in vitro at concentrations ranging from 0.004 to 667 nM.

[0198] Example 9 Detection of binding activity of HMA02h14-48 to human erythrocytes by FACS It has been reported in the prior art that therapeutic anti-CD47 antibodies often cause side effects, such as anemia, when used clinically. It is generally believed that anti-CD47 antibodies bind to CD47 on the surface of red blood cells, triggering phagocytosis of red blood cells by macrophages. This may be another major cause of anemia. In the present invention, the binding ability of HMA02h14-48 to human red blood cells was detected using flow cytometry to assess the risk of the antibody. Specifically, red blood cells from healthy donors were incubated with diluted HMA02h14-48 (maximum concentration 667 nM, a total of eight test concentration points) in PBS containing 2% fetal bovine serum at 4°C for 30 minutes. The cells were then washed three times and incubated with a secondary antibody (PE-labeled mouse anti-human IgG Fc antibody, Biolegend, catalog number 409304) in PBS containing 2% fetal bovine serum for 30 minutes in the dark at 4°C. The cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and then the fluorescent signals were detected using a Canto II (BD Biosciences) flow cytometer. According to the mean fluorescence intensity (MFI) of the signals, a concentration-dependent curve was fitted using GraphPad, and the EC 50 was calculated.

[0199] As shown in Figure 10, the maximum mean fluorescence intensity of HMA02h14-48 bound to CD47 on the surface of human erythrocytes was lower than that of the control antibody Hu5F9. 50 is shown in Table 9.

[0200] [Table 10]

[0201] Example 10. Inhibition of Toledo tumor growth by humanized antibody HMA02h14-48 Objective: To investigate the antitumor activity of the antibody of the present invention, a Toledo subcutaneous tumor model was established in NOD-Scid mice. Methods: Human diffuse large B-cell lymphoma cells, Toledo (ATCC® CRL-2631™), were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Tumor cells were suspended in RPMI 1640 and injected subcutaneously at 1 × 10 cells per well into the right flank of male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd.). 7 cells / mouse.

[0202] Fifteen days after tumor cell inoculation, the mice were randomly divided into six groups according to tumor volume, and the Hu5F9 antibody and HMA02h14-48 antibody were each diluted with PBS and administered to the mice at a dose of 10 mg / kg according to the schedule shown in Table 10. The negative control isotype antibody (isotype) IgG4P was purchased from Shanghai ChemPartner.

[0203] [Table 11]

[0204] Tumor volume (tumor volume = 0.5 × major axis × minor axis) 2 The tumor volume and body weight of the mice were measured periodically. Changes in tumor volume and body weight were statistically analyzed using the Student's t-test in Excel software, with p<0.05 indicating a significant statistical difference. The tumor regression rate for each antibody treatment group after administration was calculated.

[0205] The formula for calculating the tumor regression rate for each treatment group is [(D mean tumor volume - D t [D0 mean tumor volume] / D0 mean tumor volume] × 100%.

[0206] The formula for calculating the relative body weight of the mice is: (mouse body weight on the day of measurement / mouse body weight at the time of grouping) × 100%.

[0207] result: The experimental results are shown in Table 11 and FIG.

[0208] Tumors in the control isotype antibody group grew well, while in the therapeutic antibody-treated groups, subcutaneous tumor volume gradually decreased until complete regression compared to the initial volume. In the groups treated with various doses of Hu5F9 and HMA02h14-48 antibodies, complete tumor regression (100% regression rate) was achieved when measured on day 11 compared to the control group treated with the control antibody, and the reduction in tumor volume was statistically significant. After discontinuation of treatment, animals were observed up to day 67, but no signs of tumor regrowth were observed. Furthermore, the condition of animals treated with various doses of HMA02h14-48 remained good, with no significant difference in mouse weight on day 21 compared to pre-treatment. The body weight of mice in the high-dose Hu5F9 group on day 21 was reduced by approximately 5% compared with that on day 0, but there was no statistical difference compared with the initial body weight (p>0.05); however, there was no weight loss in the low-dose Hu5F9 group, suggesting a possible dose-related effect of Hu5F9 on body weight.

[0209] Based on the data presented above, antibody treatment with both Hu5F9 and HMA02h14-48 demonstrated highly significant antitumor effects: a single dose of 10 mg / kg of either antibody resulted in complete tumor regression with prolonged duration.

[0210] [Table 12]

[0211] Example 11 Inhibition of REC-1 Tumor Growth by Humanized Antibody HMA02h14-48 Objective: To investigate the antitumor activity of the antibody of the present invention, a REC-1 subcutaneous tumor model was established in NOD-Scid mice. Methods: Human mantle cell lymphoma cells REC-1 (ATCC® CRL-3004™) were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Tumor cells were suspended in RPMI 1640 and injected subcutaneously at 5 × 10 cells per well into the right flank of male NOD-Scid mice (Shanghai Lingchang Biotechnology Co., Ltd.). 6cells / mouse.

[0212] Eleven days after tumor cell inoculation, the mice were randomly divided into five groups according to tumor volume, and the Hu5F9 and HMA02h14-48 antibodies were diluted in PBS and administered to the mice according to the schedule shown in Table 12. The Hu5F9 antibody was prepared by GenScript, and the HMA02h14-48 antibody was prepared according to the method in Example 2. The control isotype antibody (isotype) IgG4p was purchased from Shanghai ChemPartner.

[0213] [Table 13]

[0214] Tumor volume (tumor volume = 0.5 × major axis × minor axis) 2 The tumor inhibition rate and tumor regression rate of the antibody-treated group were calculated 12 days after administration.

[0215] The formula for calculating the tumor inhibition rate is [(mean tumor volume change in the control group - mean tumor volume change in the treatment group) / mean tumor volume change in the control group] × 100%. The changes in tumor volume and body weight were statistically analyzed using Student's t-test in Excel software, and p<0.05 indicates a significant statistical difference.

[0216] The formula for calculating the tumor regression rate for each treatment group is [(D mean tumor volume - D t [D0 mean tumor volume] / D0 mean tumor volume] × 100%.

[0217] The formula for calculating the relative body weight of the mice is (mouse body weight on the day of measurement / mouse body weight at the time of grouping) × 100%.

[0218] result: The experimental results are shown in Table 13 and FIG.

[0219] After 12 days of treatment, tumor growth inhibition was 16.7% (p>0.05) in the group treated with a single dose of 3 mg / kg Hu5F9 compared with the isotype group; tumor growth inhibition rates were 3.8% (p>0.05), 54.7% (p<0.01), and 107.2% (p<0.001) in the groups treated with single doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg HMA02h14-48, respectively. Complete tumor regression (100% regression rate) was achieved by day 10 in the group treated with the high-dose HMA02h14-48 antibody. Furthermore, there was no significant difference in the relative body weight of mice in the different treatment groups.

[0220] In summary, the HMA02h14-48 antibody showed dose-dependent effects in the REC-1 model, with complete tumor regression occurring after a single administration of 10 mg / kg.

[0221] [Table 14] [Table 15-1] [Table 15-2]

Claims

1. An isolated anti-CD47 antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2, and HCDR3 of the heavy chain variable region (VH), and LCDR1, LCDR2, and LCDR3 of the light chain variable region (VL) Including, the VH and VL being: (1) VH comprising the amino acid sequence shown in SEQ ID NO: 1, and VL comprising the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising the amino acid sequence shown in SEQ ID NO: 3, and VL comprising the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising the amino acid sequence shown in SEQ ID NO: 5, and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9, or 10; (4) VH comprising the amino acid sequence shown in SEQ ID NO: 6, and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; and (5) VH comprising the amino acid sequence shown in SEQ ID NO: 7, and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9, or 10. The antibody or antigen-binding fragment thereof is selected from:

2. An isolated anti-CD47 antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 of VH, and VL LCDR1, LCDR2 and LCDR3; (1) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17; and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16; or (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16; The antibody or antigen-binding fragment thereof.

3. An isolated anti-CD47 antibody or antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 of VH, and VL LCDR1, LCDR2 and LCDR3; (1) the HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 12, and the HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 17; and said LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, said LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and said LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 16; or (2) the HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 12, and the HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 13; and the LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 14, the LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 15, and the LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 16; The antibody or antigen-binding fragment thereof. (1) a heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10; (2) a heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; (3) a heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; (4) A heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; and a light chain variable region (VL) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10; or (5) A heavy chain variable region (VH) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; and A light chain variable region (VL) comprising an amino acid sequence identical to or having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9 or 10; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL (1) VH comprising the amino acid sequence shown in SEQ ID NO: 1; and VL comprising the amino acid sequence shown in SEQ ID NO: 2; (2) VH comprising the amino acid sequence shown in SEQ ID NO: 3; and VL comprising the amino acid sequence shown in SEQ ID NO: 4; (3) VH comprising the amino acid sequence shown in SEQ ID NO: 5; and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9, or 10; (4) VH comprising the amino acid sequence shown in SEQ ID NO: 6; and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; and (5) VH comprising the amino acid sequence shown in SEQ ID NO: 7; and VL comprising the amino acid sequence shown in SEQ ID NO: 8, 9, or 10. The antibody or antigen-binding fragment thereof according to claim 4, selected from:

6. The antibody or antigen-binding fragment thereof according to claim 5, comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 7, and the VL comprises the amino acid sequence set forth in SEQ ID NO:

8.

7. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, wherein the antibody comprises a heavy chain constant region.

8. The antibody or antigen-binding fragment thereof described in claim 7, wherein the heavy chain constant region is selected from a human IgG1 constant region, a human IgG1TM constant region, a human IgG4 constant region, and a human IgG4P constant region.

9. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 8, wherein the antibody comprises a light chain constant region.

10. An antibody or antigen-binding fragment thereof described in claim 9, wherein the light chain constant region is a human κ light chain constant region.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which is a murine antibody, a chimeric antibody or a humanized antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which is a full-length antibody, Fab, Fab', Fab'-SH, (Fab')2, single-chain antibody, Fv or bis(multi)specific antibody.

13. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 12.

14. A recombinant or expression vector comprising one or more nucleic acids according to claim 13, suitable for recombinant production of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

15. A host cell comprising one or more of the recombinant vectors or expression vectors of claim 14.

16. An immunoconjugate or immunofusion comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the immunoconjugate or immunofusion according to claim 16.

18. The pharmaceutical composition of claim 17, comprising a pharmaceutically acceptable excipient.

19. 19. The pharmaceutical composition according to claim 17 or 18 for treating or preventing cancer.

20. The pharmaceutical composition of any one of claims 17 to 19, wherein the cancer comprises a blood cancer and a solid tumor.

21. The pharmaceutical composition of claim 20, wherein the cancer is selected from bladder cancer, pancreatic cancer, lymphoma, leukemia, multiple myeloma, (chronic) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, endometrial cancer, kidney cancer, (benign) melanoma, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, liver cancer, colon cancer, ovarian cancer, and urothelial cancer.

22. 1. A method for detecting CD47 protein, comprising: (a) contacting a sample with the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 12, or the immune complex or immunofusion of claim 16; and (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof, or immune complex or immune fusion, and CD47 protein. The method comprising:

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