CD47 antibodies and their applications

Abstract

The present invention relates to CD47 antibodies and their applications, particularly to monoclonal antibodies that recognize CD47, more particularly to CD47 antibodies that do not cause significant levels of hemagglutination, erythropenia, and thrombocytopenia, as well as methods for preparing these antibodies and the applications of these monoclonal antibodies in preparing drugs for treating cancer or infectious diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of bioimmunology, in particular to fully human anti-CD47 IgG antibodies, more particularly to CD47 antibodies that do not cause significant human erythrocyte hemagglutination, hemoglobin depletion, anemia, and / or thrombocytopenia, methods for preparing these antibodies, and the application of these monoclonal antibodies in the preparation of drugs. [Background technology]

[0002] CD47, also known as integrin-associated protein (IAP), is a transmembrane glycoprotein widely expressed on cell surfaces and a member of the immunoglobulin superfamily (Johansen and Brown, J. Biol. Chem. 2007). CD47 inhibits macrophage phagocytosis by binding to its ligand, signal regulatory protein α (SIRPα) (Matozaki, T. et al., Trends Cell Biol. 2009). CD47 is overexpressed in many malignancies, including acute myeloid leukemia (AML), B-cell and T-cell acute leukemia, and non-Hodgkin's lymphoma, and studies have shown that it transmits a "don't eat me" signal to macrophages, allowing them to escape immune surveillance. High CD47 expression has also been associated with poor clinical prognosis (Willingham et al., Proc. Natl. Acad. Sci. USA. 2012).

[0003] In U.S. Patent Application No. 2009 / 0191202, Professor Weissman and his team at Stanford University demonstrated through experiments with xenograft animal models in orthotopic immunodeficient mice that administration of anti-CD47 monoclonal antibodies inhibits the growth and metastasis of large tumors and cures small tumors. U.S. Patent Application No. 2016 / 0251435 discloses the use of anti-CD47 monoclonal antibodies in tumor treatment. Patent Application No. WO2013056352 describes full-length humanized anti-human SIRPα monoclonal antibodies and antibody fragments derived therefrom for use in the treatment of hematological tumors, particularly leukemia. Blocking the CD47-SIRPα signaling pathway promotes phagocytosis of tumor cells by macrophages, providing a new approach to tumor immunotherapy. Summary of the Invention [Means for solving the problem]

[0004] The present invention provides antibodies that recognize and bind to CD47. In some embodiments, the antibodies of the present invention can recognize and bind to human CD47. The antibodies of the present invention can block the interaction between CD47 and SIRPα and do not induce significant hemagglutination of red blood cells. The antibodies provided by the present invention are collectively referred to as "CD47 antibodies."

[0005] In some embodiments, the CD47 antibodies of the present invention have been shown to have several required properties, including, by way of non-limiting examples, specific recognition of human or macaque CD47, effective inhibition of the interaction of CD47 with its ligand SIRPα while not inducing significant red blood cell hemagglutination, and effective anti-tumor activity.

[0006] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-associated protein (CD47) and comprises one, two, three, four, five, or six of the following VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein: (a) VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14 or a variant of SEQ ID NO: 14 having 1 to 3 amino acid substitutions in the sequence of SEQ ID NO: 14 as set forth in Table 1; (b) VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant of SEQ ID NO: 17 having 1 to 3 amino acid substitutions in the sequence of SEQ ID NO: 17 as set forth in Table 1; (c) VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23 or a variant of SEQ ID NO: 23 having 1 to 3 amino acid substitutions in the sequence of SEQ ID NO: 23 as set forth in Table 1; (d) VL CDR1 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 30 to 31; (e) VL CDR2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 32 to 33, and (f) VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 34 to 35.

[0007] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-associated protein (CD47) and comprises one, two, three, four, five, or six of the following VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein: (a) VH CDR1 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 14 to 16; (b) VH CDR2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 17 to 22; (c) VH CDR3 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 23 to 29; (d) VL CDR1 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 30 to 31; (e) VL CDR2 comprises any one of the amino acid sequences set forth in SEQ ID NOs: 32 to 33, and (f) VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 34 to 35.

[0008] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-related protein (CD47) and comprises one, two, three, four, five, or six of the following sequences: (a) VH CDR1 shown in SEQ ID NO: 14, or a variant of SEQ ID NO: 14 having 1 to 3 amino acid substitutions in the sequence of SEQ ID NO: 14 as shown in Table 1; (b) VH CDR2 as set forth in SEQ ID NO: 17, or a variant of SEQ ID NO: 17 having 1 to 3 amino acid substitutions in the sequence of SEQ ID NO: 17 as set forth in Table 1; (c) a VH CDR3 as set forth in SEQ ID NO: 23, or a variant of SEQ ID NO: 23 having 1 to 3 amino acid substitutions as set forth in Table 1 in the sequence of SEQ ID NO: 23; (d) any one of VL CDR1s shown in SEQ ID NOs: 30 to 31; (e) any one of VL CDR2s shown in SEQ ID NOs: 32 to 33, or (f) any one of the VL CDR3s shown in SEQ ID NOs: 34 to 35.

[0009] In some embodiments, the present invention provides antibodies or antigen-binding fragments that specifically bind to human integrin-related protein (CD47) and include: (a) any one of VH CDR1s shown in SEQ ID NOs: 14 to 16; (b) any one of VH CDR2s shown in SEQ ID NOs: 17 to 22; (c) any one of VH CDR3s shown in SEQ ID NOs: 23 to 29; (d) any one of VL CDR1s shown in SEQ ID NOs: 30 to 31; (e) any one of VL CDR2s shown in SEQ ID NOs: 32 to 33, or (f) any one of the VL CDR3s shown in SEQ ID NOs: 34 to 35.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH comprising any one of the amino acid sequences selected from SEQ ID NOs: 11, 36 to 44, 50, and 56, or an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 11, 36 to 44, 50, and 56.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region VL comprising any one of the amino acid sequences selected from SEQ ID NOs: 12 to 13, 45 to 49, 51 to 55, and 65, or an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 12 to 13, 45 to 49, 51 to 55, and 65.

[0012] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-associated protein (CD47) and comprises a VH CDR1 set forth in SEQ ID NO: 14, a VH CDR2 set forth in SEQ ID NO: 17, a VH CDR3 set forth in SEQ ID NO: 23, a VL CDR1 set forth in SEQ ID NO: 30, a VL CDR2 set forth in SEQ ID NO: 32, and a VL CDR3 set forth in SEQ ID NO: 34.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region, VH, comprising an amino acid residue mutation chosen from one or more of the following, numbered according to Kabat: (a) R81K, and (b) R82aS.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH comprising any one of the amino acid sequences selected from SEQ ID NOs: 11 and 50, or an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 11 and 50.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region VL comprising any one of the amino acid sequences selected from SEQ ID NOs: 13, 45 to 49, 51 to 55, and 65, or an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 13, 45 to 49, 51 to 55, and 65.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 11 or 50, and a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO: 13 or 65.

[0017] In some embodiments, the CD47 antibodies described herein specifically recognize and bind to human or macaque CD47, and the antibodies comprise a heavy chain variable region having any one of the amino acid sequences selected from SEQ ID NOs: 11, 36-44, and 50, and / or a light chain variable region having any one of the amino acid sequences selected from SEQ ID NOs: 12, 13, 45-49, 51-55, and 65.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain H comprising any one of the amino acid sequences selected from SEQ ID NOs: 60 and 62-63, and a light chain L comprising any one of the amino acid sequences selected from SEQ ID NOs: 61 and 64.

[0019] In some embodiments, the CD47 antibodies of the present invention inhibit 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% compared to the level of interaction between CD47 and SIRPα in the absence of the CD47 antibody.

[0020] In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:3 or 4, and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:5.

[0021] IgG1 heavy chain constant region sequence (SEQ ID NO: 3): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0022] IgG4 heavy chain constant region sequence (SEQ ID NO: 4): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0023] Light chain constant region sequence (SEQ ID NO: 5) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0024] In some embodiments, the CD47 antibodies of the invention do not induce significant cell aggregation, e.g., they do not induce significant hemagglutination of red blood cells. In some embodiments, a CD47 antibody of the invention does not induce significant aggregation when the level of aggregation in the presence of a CD47 antibody 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% compared to the level of aggregation in the presence of the conventional CD47 antibody Hu5F9-G4. In some embodiments, the CD47 antibodies of the invention do not induce significant cell aggregation when the antibody concentration is between 400 pM and 800 nM.

[0025] In some embodiments, the antibodies of the invention are also significantly more effective in tumor models than antibodies known in the art, e.g., the phagocytic ability of macrophages to phagocytose tumor cells 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% in the presence of a CD47 antibody of the invention.

[0026] In some embodiments, the antibody or antigen-binding fragment is an IgG isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, the antibody or antigen-binding fragment is an IgG isotype selected from IgG4P and IgG4PE. IgG4P refers to mutating Ser228 in the IgG4 hinge region to Pro to prevent chain exchange. IgG4PE refers to mutating Ser228 in the IgG4 hinge region to Pro to prevent chain exchange and mutating Leu235 in the constant region to Glu to alter Fc receptor interaction.

[0027] In some embodiments, the antibody is chimeric, humanized, or fully human. In some embodiments, the antibody binds to human CD47.

[0028] In some embodiments, the CD47 antibody or antigen-binding fragment of the invention is an isolated CD47 antibody or antigen-binding fragment.

[0029] In some embodiments, the CD47 antibody or antigen-binding fragment of the invention is a monoclonal antibody or fragment thereof.

[0030] In some embodiments, the CD47 antibodies or antigen-binding fragments of the present invention can be used to prepare medicaments for treating cancer or infectious diseases. In some embodiments, the CD47 antibodies or antigen-binding fragments of the present invention can be used to prepare medicaments for treating solid tumors or hematological tumors. In some embodiments, the CD47 antibodies or antigen-binding fragments of the present invention can be used to prepare medicaments for treating non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, breast cancer, ovarian cancer, lung cancer, prostate cancer, melanoma, colorectal cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the CD47 antibodies or antigen-binding fragments of the present invention can be applied to the preparation of a medicament for treating diffuse large B-cell lymphoma or follicular lymphoma.

[0031] In some embodiments, the CD47 antibodies described herein can be applied to treat, delay progression, prevent recurrence, or alleviate symptoms of cancer or other neoplastic conditions. For example, the CD47 antibodies described herein can be used to treat hematological malignancies and / or tumors, such as hematological malignancies and / or tumors. For example, the CD47 antibodies described herein can be used to treat CD47 +They can be used to treat tumors. As a non-limiting example, the CD47 antibodies described herein can be used to treat non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and the like. Solid tumors include, for example, breast cancer, ovarian cancer, lung cancer, prostate cancer, melanoma, colorectal cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer. For example, the CD47 antibodies described herein can be used to treat diffuse large B-cell lymphoma or follicular lymphoma.

[0032] As used herein, "blood cancer" includes leukemia, lymphoma, and myeloma. "Leukemia" refers to a blood cancer that produces too many white blood cells that cannot effectively fight infection and crowds out other components of the blood, such as platelets and red blood cells. Leukemia cases can be classified as acute or chronic. As non-limiting examples, leukemia can include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative disorders / neoplasias (MPDS), and myelodysplastic syndromes. "Lymphoma" can include Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). Myeloma can include multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bence-Jones myeloma.

[0033] Exemplary monoclonal antibodies of the present invention include, for example, antibodies described herein, including antibodies having a heavy chain variable region of any one of the amino acid sequences selected from SEQ ID NOs: 11, 36-44, 50, and 56, and a light chain variable region of any one of the amino acid sequences selected from SEQ ID NOs: 12, 13, 45-49, 51-55, and 65. The above antibodies further include antibodies having a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to at least one of the sequences of SEQ ID NOs: 11, 36 to 44, 50, and 56, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to at least one of the sequences of SEQ ID NOs: 12, 13, 45 to 49, 51 to 55, and 65. These antibodies exhibit specificity for human CD47 and can block the interaction of CD47 with SIRPα without inducing significant hemagglutination of erythrocytes.

[0034] In some embodiments, the present invention further provides a biomaterial, as follows: (1) a polynucleotide encoding an antibody or antigen-binding fragment according to the present invention; or (2) an expression carrier comprising a polynucleotide encoding an antibody or antigen-binding fragment according to the invention; or (3) A cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment of the present invention.

[0035] The pharmaceutical compositions of the present invention can comprise the antibody of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be included in a reagent kit, such as a diagnostic reagent kit.

[0036] The present invention further provides methods for alleviating symptoms of cancer or other neoplastic diseases by administering to a subject in need thereof one or more monoclonal antibodies that bind to CD47 or an immunologically active fragment thereof without causing significant red blood cell hemagglutination after administration. The amount of antibody administered should be sufficient to alleviate symptoms of cancer or other neoplastic conditions in the subject. In some embodiments, the subject is human. In some embodiments, the antibody or immunologically active fragment thereof inhibits the interaction of CD47 with SIRPα.

[0037] In some embodiments, the CD47 antibodies described herein are used in combination with one or more other agents. Suitable other agents include existing drugs and / or surgical therapies used in particular applications (e.g., cancer). For example, the CD47 antibodies are used in combination with one or more other chemotherapeutic agents or anti-tumor agents. Alternatively, the other chemotherapeutic agent is radiation therapy. In some embodiments, the chemotherapeutic agent is a cell death-inducing agent. In some embodiments, the chemotherapeutic agent induces asymmetric loss of phospholipids across the plasma membrane, causing, for example, cell surface exposure of phosphatidylserine (PS). In some embodiments, the chemotherapeutic agent induces endoplasmic reticulum (ER) stress. In some embodiments, the chemotherapeutic agent is a proteasome inhibitor. In some embodiments, the chemotherapeutic agent induces translocation of ER proteins to the cell surface. In some embodiments, the chemotherapeutic agent induces the translocation and cell surface exposure of calreticulin.

[0038] In some embodiments, the CD47 antibody and other reagent are formulated as a single therapeutic composition and administered simultaneously. Alternatively, the CD47 antibody and other reagent are formulated independently, e.g., as separate therapeutic compositions, and administered simultaneously, or at different times during a therapeutic regimen. For example, the CD47 antibody is administered before the other reagent, or after the other reagent, or the CD47 antibody and other reagent are administered alternately. As used herein, the CD47 antibody and other reagent are administered in a single dose or multiple doses.

[0039] In some embodiments, there is provided the application of the antibodies or antigen-binding fragments, compositions, and biomaterials of the present invention in the preparation of a medicament for treating cancer or an infectious disease.

[0040] Those skilled in the art will appreciate that the antibodies of the present invention have a variety of uses, for example, they can be used as therapeutic agents, as reagents or diagnostic tools in diagnostic reagent kits, or as reagents in competitive experiments to generate therapeutic agents. [Brief explanation of the drawings]

[0041] [Figure 1] 1A is a series of graphs showing the ability of antibody 17-ScFv to bind to CD47 on the cell surface using CHO-CD47 cells (FIG. 1A) or Jurkat cells (FIG. 1B), respectively. [Figure 2] 1 shows a flow cytometric analysis of the ability of antibody 17-ScFv and the ligand SIRPα to compete for binding to CD47 on the surface of CHO. [Figure 3] Figures 3A-3E are a series of graphs showing the assessment of the ability of CD47 antibodies to block the binding of SIRPα to the CD47 antigen by competitive ELISA. [Figure 4]Figures 4A-4E are a series of graphs showing hemagglutination of red blood cells (RBCs) by CD47 antibodies. The appearance shows that red blood cell clumps flowing in a line do not cause hemagglutination, slight agglutinations are dots, and clear agglutinations are agglutinations throughout the well. Figure 4A shows that antibody 11 causes clear agglutination, Figure 4B shows that antibody L12 does not cause hemagglutination, Figure 4C shows that antibody L12-6 does not cause hemagglutination, Figure 4D shows that antibodies 17 and Hu5F9-G4 cause clear hemagglutination, but AB6.12-G1 and SIRPα do not cause hemagglutination, and Figure 4E shows that antibody 6Y-G4 does not cause hemagglutination. [Figure 5] 5A-5B are a series of graphs showing the ELISA assessment of binding of CD47 antibodies to human CD47 or macaque CD47. [Figure 6] Figures 6A-6B are a series of graphs showing flow cytometric assessment of the ability of CD47 antibodies to promote phagocytosis of RAW264.7 against Raji. [Figure 7] Figures 7A to 7D are a series of graphs analyzing the effects of antibody 6Y-G1 on red blood cells and platelets in cynomolgus monkeys. Figures 7A to 7B show that antibody 6Y-G1 causes a decrease in hemoglobin and red blood cells, which gradually recovers after about two weeks, with the degree of response being similar to that of antibodies AB6.12-G1 and Hu5F9-G4. Figure 7C shows that platelets fluctuate after each administration of antibody 6Y-G1, recovering after the end of administration. Figure 7D shows that the number of reticulocytes increases after each administration of antibody 6Y-G1, and the increase is greater than that in the antibody AB6.12-G1 and Hu5F9-G4 groups. [Figure 8]

[0023] Figure 1 shows the antitumor effect of antibody L12 in the human hematological tumor MV4-11 systemic tumor model, using antibodies AB6.12-G1, Hu5F9-G4 as positive controls. All mice are treated with an antibody dose of 200 μg three times a week. [Figure 9]The antitumor effect of the 6Y-G4 antibody in a large tumor model was analyzed. Mice were treated with a 100 μg antibody dose three times a week. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention provides antibodies that specifically bind to CD47. In some embodiments, the antibodies of the present invention can specifically bind to human CD47. These antibodies are collectively referred to herein as anti-CD47 antibodies. Unless otherwise specified herein, amino acid residues in the variable and constant regions of antibodies are numbered according to Kabat (Kabat et al., 1991, EU Index of Sequences of Proteins of Immunological Interest).

[0043] Some properties of the antibodies described herein include a) specific binding to CD47 (e.g., human CD47 and macaque CD47), b) blocking the interaction of CD47 with SIRPα, c) lacking significant hemagglutination activity, d) promoting phagocytosis of tumor cells by macrophages, and / or e) demonstrating potent anti-tumor activity in mouse models of human cancer.

[0044] Thus, the antibodies described herein have an important place in the treatment of various cancers.

[0045] Many existing CD47 antibodies block SIRPα, but existing antibodies that block SIRPα cause the undesirable side effect of hemagglutination. Other existing antibodies (e.g., 2D3) do not cause hemagglutination, but these antibodies also do not block SIRPα, and therefore cannot effectively promote phagocytosis. Therefore, cell aggregation is a major limitation of therapeutically targeting CD47 using existing full IgG antibodies. Therefore, there is an urgent need to obtain CD47 antibodies that can block SIRPα without causing cell aggregation.

[0046] The CD47 antibodies of the invention enhance the effectiveness of therapeutic targeting of CD47 by avoiding undesirable hemagglutination and retain the ability to block the interaction of CD47 with SIRPα, thereby promoting phagocytosis of CD47-expressing cells. In some embodiments, the CD47 antibodies of the invention (e.g., antibody L12 and antibody L12-6) do not significantly agglutinate cells. For example, the CD47 antibodies of the invention do not significantly agglutinate RBCs.

[0047] The CD47 antibodies of the present invention bind to human CD47 and block its interaction with SIRPα (Figures 3A-3E). These antibodies do not induce significant hemagglutination of human erythrocytes (Figures 4A-4E). These antibodies can promote phagocytosis of tumor cells by macrophages (Figures 6A-6B). Furthermore, these CD47 antibodies exhibit effective antitumor activity in a mouse model of human Raji lymphoma (Figure 9). Thus, the CD47 antibodies of the present invention overcome a major limiting factor in the therapeutic targeting of CD47. Therefore, the CD47 antibodies of the present invention are highly important in the treatment of numerous cancers.

[0048] The equilibrium dissociation constant (KD) of antibodies of the invention that bind to a CD47 epitope is 1 μM or less, e.g., 100 nM or less, e.g., 10 nM or less, 1 nM or less. For example, the CD47 antibodies provided herein exhibit KD values ​​of less than 1 nM.

[0049] The CD47 antibodies of the present invention are used to modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activities of the widely distributed CD47. Functional activities of CD47 include, for example, signal transduction via interaction with SIRPα, regulation (e.g., increase) of intracellular calcium ion concentration following cell adhesion to the extracellular matrix, interaction with the C-terminal cell-binding domain of thrombospondin, interaction with fibrinogen, and interaction with various integrins. For example, the CD47 antibodies completely or partially inhibit the functional activities of CD47 by partially or completely modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or interfering with the binding of CD47 to SIRPα.

[0050] definition Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques used in cell culture, molecular biology, and protein purification described herein are those known and commonly used in the art. Standard techniques were used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's instructions or methods commonly used in the art or described herein. The techniques and methods described above are generally used as known in the art and described in several comprehensive and relatively specific publications referenced and described herein. See Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring, NY (1989)).

[0051] As used in this disclosure, unless otherwise explained, the following terms shall be understood to have the following meanings: The terms "red blood cells" and "erythrocytes" are synonymous and may be used interchangeably.

[0052] The term "agglutination" refers to the clumping of cells, and the term "hemagglutination" refers to the clumping of a particular type of cell (i.e., red blood cells). Thus, hemagglutination is a type of agglutination. In the hemagglutination assays described herein, red blood cells may form in a given well as a "button," a "halo," or an intermediate form between the two. The term "line" refers to the linear flow of red blood cell clumps after tilting a 96-well plate 45°. The term "significant hemagglutination activity" refers to the presence of any halo formation in a well when an antibody described herein is added.

[0053] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "antibody" refers to an antibody that reacts with one or more antigenic determinants of a target antigen without reacting with other polypeptides, or with very low affinity (KD > 10 -6 Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab, Fab- and F(ab')2 fragments, Fv and Fab expression libraries.

[0054] The basic structural unit of an antibody is known to comprise a tetramer. Each tetramer (also called a "whole antibody") is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Antibody molecules obtained from humans generally belong to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another depending on the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, and others. In humans, light chains can be classified as either kappa or lambda chains.

[0055] As used herein, the term "monoclonal antibody" (mAb) refers to a population of antibody molecules that contain only one type of antibody molecule that consists of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a specific epitope of an antigen.

[0056] The term "single-chain antibody" (scFv) refers to an antibody in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody are linked by a short linker peptide of 15 to 20 amino acids.

[0057] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent branches (called "hypervariable regions") of the heavy chain variable region (VH) and light chain variable region (VL) are located between more conserved branches (called "framework regions" or "FR"). The term "FR" therefore refers to immunoglobulin amino acid sequences naturally occurring between or adjacent to the hypervariable regions. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and all three hypervariable regions of each heavy and light chain are referred to as "complementarity-determining regions" or "CDRs." The amino acid alignment of each domain can be in accordance with the definitions in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health (1987 and 1991)) or Chothia and Lesk (J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989)).

[0058] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof or a T-cell receptor. Epitope determinants generally consist of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains) and generally have specific three-dimensional structural characteristics and specific charge characteristics.

[0059] As used herein, the term "specific binding" refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and the immunoglobulin's specific antigen. The strength or affinity of an immunological binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where the smaller the KD, the higher the affinity. The immunobinding properties of a selected polypeptide may be quantified by methods well known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect this rate equally in two directions. Thus, the "association rate constant" (k on ) and "dissociation rate constant" (k off Both the k and k values ​​can be measured by calculating the concentration and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). off / k on The ratio can exclude all parameters unrelated to affinity and is equal to the equilibrium dissociation constant, KD. (See generally Davies et al (1990) Annual Rev Biochem 59:439-473.) Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those of skill in the art, and an antibody of the present disclosure specifically binds to CD47 if the equilibrium dissociation constant (KD) is ≦1 μM (e.g., ≦100 nM, ≦10 nM, or ≦1 nM).

[0060] An "isolated" antibody is one that has been separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, monoamines, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to the following extent: (1) the antibody is greater than 95%, e.g., greater than 99%, by weight as determined by the Lowry method; (2) at least 15 residues of N-terminal or internal amino acid sequence can be obtained using a spinning cup sequenator; or (3) homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include in situ antibodies within recombinant cells. Typically, isolated antibodies are prepared by at least one or more purification steps. In some embodiments, the purity of the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0061] The term "polypeptide" as used herein is a general term that refers to naturally occurring proteins, fragments, or analogs of polypeptide sequences; thus, naturally occurring protein fragments and analogs are a species of the polypeptide genus.

[0062] The term "sequence identity" or "sequence homology" means that two polynucleotide or amino acid sequences are identical (i.e., identical on a nucleotide-by-nucleotide or residue-by-residue basis) over a comparison window. The term "percent sequence identity" is calculated by the following method: comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the percent sequence identity.

[0063] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd ed., ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. 7, 1991). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids (such as α- and α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Conventional (or naturally occurring) amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0064] Analogously, unless otherwise specified, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of a nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand that is identical to the RNA sequence and is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand that is identical to the RNA sequence and is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence." When applied to polypeptides, the term "essentially identical" means that two peptide sequences share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity, when optimally aligned using default gap weights, such as by the GAP or BESTFIT programs.

[0065] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0066] Any minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the amino acid sequence identity is maintained at least 75%, e.g., at least 80%, 90%, 95%, or 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly classified as follows: (1) acidic amino acids, which are aspartate and glutamate; (2) basic amino acids, which are lysine, arginine, and histidine; (3) nonpolar amino acids, which are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, which are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) the aliphatic-hydroxyl family (serine and threonine), (ii) the amide-containing family (asparagine and glutamine), (iii) the aliphatic family (alanine, valine, leucine, and isoleucine), and (iv) the aromatic family (phenylalanine, tryptophan, and tyrosine). In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. For example, individual substitutions of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar substitutions of amino acids with structurally related amino acids can be reasonably predicted without significantly affecting the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a binding site. Whether an amino acid change results in a functional peptide can be readily determined by measuring the specific activity of the polypeptide derivative, as described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art.

[0067] In some embodiments, amino acid substitutions have the effect of (1) reducing the protein's susceptibility to hydrolysis, (2) reducing its susceptibility to oxidation, (3) altering its binding affinity for forming protein complexes, (4) altering its binding affinity, or (5) conferring or improving other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins whose sequences differ from those of naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt the helical structure present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of artificially recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (edited by Creighton, W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (edited by C. Branden and J. Tooze, Garland Publishing, New York, NY (1991)), and Thornton et al., Nature 354:105 (1991).

[0068] As used herein, the term "reagent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from biological materials.

[0069] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, for example, by incorporating a radioactively labeled amino acid or a polypeptide attached to a biotin moiety that is detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or having enzymatic activity detected by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0070] The term "pharmaceutical agent" or "drug" refers to a chemical compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0071] As used herein, the term "antineoplastic agent" refers to an agent that has the functional property of inhibiting the development or progression of a tumor, particularly a malignant (cancerous) lesion such as a carcinoma, sarcoma, lymphoma, or leukemia, in humans. Inhibition of metastasis is often a property of an anti-neoplastic agent.

[0072] Other chemical terms herein are used according to conventional practice in the art, e.g., The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).

[0073] CD47 antibody The antibodies of the present invention have the ability to bind to CD47, inhibit the binding of SIRPα to CD47, reduce CD47-SIRPα-mediated signaling, promote phagocytosis, and inhibit tumor growth and / or metastasis. For example, the inhibitory effect can be determined using the cell experiments described in the Examples section of the present specification.

[0074] Exemplary antibodies of the invention include antibody 17, antibody L12, antibody 6Y-G1, antibody 6Y-G4, etc., and other similar antibodies having the same or similar CDR regions.

[0075] The VH CDRs of antibody 17 have SEQ ID NO: 14 (VH CDR1), SEQ ID NO: 17 (VH CDR2) and SEQ ID NO: 23 (VH CDR3), and the VL CDRs have SEQ ID NO: 31 (VL CDR1), SEQ ID NO: 33 (VL CDR2) and SEQ ID NO: 35 (VL CDR3). The VH CDRs are heavy chain hypervariable regions, and the VL CDRs are light chain hypervariable regions.

[0076] In some embodiments, the present invention substitutes one or more amino acid positions in VH CDR1, VH CDR2, and / or VH CDR3 of antibody 17. In some embodiments, the substitutable positions (parent) and amino acids available for substitution in VH CDR1, VH CDR2, and / or VH CDR3 are shown in one or more of Table 1. The "position" in Table 1 represents the amino acid position from left to right in the amino acid sequence shown in SEQ ID NO: 11; for example, position 30 represents the 30th amino acid, "D," from left to right in the amino acid sequence shown in SEQ ID NO: 11.

[0077] [Table 1]

[0078] The VH CDRs of antibody L12 and antibody L12-6 are SEQ ID NO: 14 (VH CDR1), SEQ ID NO: 17 (VH CDR2) and SEQ ID NO: 23 (VH CDR3), and the VL CDRs are SEQ ID NO: 30 (VL CDR1), SEQ ID NO: 32 (VL CDR2) and SEQ ID NO: 34 (VL CDR3).

[0079] Upon experimental validation, the VH CDR1 sequence may be SEQ ID NO: 15 or 16. The VH CDR2 sequence may be SEQ ID NO: 18, 19, 20, 21, or 22. The VH CDR3 sequence may be SEQ ID NO: 24, 25, 26, 27, 28, or 29. Other CDR sequences can be realized with the amino acid substitutions shown in Table 1. In some embodiments, one, two, or three amino acids are substituted.

[0080] In addition to altering the CDRs, amino acids in the framework regions can also be appropriately altered. For example, L12-1-VH, L12-2-VH, L12-3-VH, L12-6-1-VH, L12-6-VH, L12-8-VH, or L12-9-VH, generated by two mutations (R81K and R82aS) in the framework regions of the heavy chain of antibody L12, are also suitable heavy chain variable regions. In some embodiments, the heavy chain variable region of a CD47 antibody can have a K at position 81 or an S at position 82a (numbered according to Kabat). Mutations in other framework regions are also reflected in different antibody sequences. Thus, the present invention provides heavy chain framework regions with R81K and / or R82aS (numbered according to Kabat) mutations.

[0081] Exemplary antibodies of the present invention include antibodies having a heavy chain variable region whose sequence is selected from SEQ ID NOs: 11, 36-44, 50, or 56, and a light chain variable region whose sequence is selected from SEQ ID NOs: 12-13, 45-49, 51-55, and 65. In particular, exemplary antibodies include antibody 17, antibody 3-3, antibody 3-6, antibody 3, antibody 6, antibody 10, antibody 11, antibody 16, antibody 18, antibody 24, antibody 25, antibody L1, antibody L2, antibody L5, antibody L12-1-1, antibody L12, antibody L24, antibody L26, antibody L12-1, antibody L12-2, antibody L12-3, antibody L12-4, antibody L12-5, antibody L12-6-1, antibody L12-6, antibody L12-7, antibody L12-8, antibody L12-9, antibody L12-10, antibody L12-11, and antibodies having suitable sequence identity with the sequences of the above antibodies, e.g., sharing at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity. In some embodiments, these sequences that share identity have at least one CDR that is unchanged.

[0082] In some embodiments, human framework adaptations are performed on antibody L12. In some embodiments, the VH framework mutations of antibody L12 are shown in Table 3 at the underlined amino acids labeled L12-1-VH, L12-2-VH, L12-3-VH, L12-6-1-VH, L12-6-VH, L12-8-VH, or L12-9-VH. In some embodiments, the VL framework mutations of antibody L12 are shown in Table 3 at the underlined amino acids labeled L12-1-1-VL, L12-1-VL, L12-2-VL, L12-3-VL, L12-4-VL, L12-5-VL, L12-6-1-VL, L12-7-VL, L12-8-VL, L12-9-VL, L12-10-VL, or L12-11-VL.

[0083] The present invention also includes antibodies that bind to the same epitope as the CD47 antibodies described herein, for example, antibodies of the present invention that specifically bind to an epitope comprising one or more amino acid residues of human CD47 (see, e.g., GenBank Accession No. Q08722.1).

[0084] An exemplary human CD47 ECD amino acid sequence, SEQ ID NO: 1, is provided below (GenBank Accession No. Q08722.1 (GI:1171879), incorporated herein by reference). The signal sequence (amino acids 1-18) is underlined.

[0085] SEQ ID NO: 1 MWPLVAALLLGSACCGSA QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP

[0086] The ability of the antibodies described herein to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with CD47- and / or CD47 / SIRPα-mediated signaling can be assessed by methods including, for example, measuring CD47- and / or CD47 / SIRPα-mediated signaling in the presence of one or more antibodies described herein. These measurements may include competitive binding assays, or may measure biological measurements, such as the ability to promote phagocytosis of CD47-expressing cells by macrophages (e.g., as described in Example 3).

[0087] The antibodies described herein can be produced by various methods known in the art (e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). A fully human antibody is an antibody molecule in which the entire sequences of both the light and heavy chains (including the CDRs) are derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to generate human monoclonal antibodies (see Cole et al., 1985. In: Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies are available and can be produced using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr Virus (see Cole et al., 1985. In: Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0088] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction in immune serum. Alternatively, specific antigens targeted by immunoglobulins or their epitopes can be immobilized on a column to purify immune-specific antibodies by immunoaffinity chromatography. For details on immunoglobulin purification, see D. Wilkinson's article (The Scientist, The Scientist, Inc., Philadelphia, Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0089] The CD47 antibodies of the present disclosure may be monoclonal antibodies. Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with CD47- and / or CD47 / SIRPα-mediated cell signaling can be generated, for example, by immunizing an animal with membrane-bound and / or soluble CD47 (e.g., human CD47 or an immunogenic fragment, derivative, or variant thereof). Alternatively, CD47 can be expressed and associated with the surface of the transfected cells by immunizing an animal with cells transfected with a carrier containing a nucleic acid molecule encoding CD47. Alternatively, antibodies that bind to CD47 can be obtained by screening a library containing antibody or antigen-binding domain sequences. This library is prepared in phage by expressing protein or peptide fusions fused to phage coat proteins on the surface of assembled phage particles and incorporating the encoded DNA sequences into the phage particles (i.e., a "phage display library"). Antibodies are then screened for binding to CD47.

[0090] For example, monoclonal antibodies are prepared by the hybridoma method, as described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0091] The immunizing agent typically contains a protein antigen, a fragment thereof, or a fusion protein thereof. Typically, peripheral blood lymphocytes are used if human cells are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is typically a rat or mouse myeloma cell line. The hybridoma cells can be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxanthine, aminopterin, and thymine ("HAT medium"), which prevents growth of HGPRT-deficient cells.

[0092] Monoclonal antibodies can also be prepared by recombinant DNA techniques, e.g., as described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies described herein can be isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells described herein are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vehicle and transfected into host cells that do not otherwise produce immunoglobulins, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, monkey COS cells, PER.NS0 cells, SP2 / 0, YB2 / 0, or myeloma cells, to obtain the monoclonal antibody synthesized in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently joining the coding sequence for an immunoglobulin to the coding sequence for all or part of a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of the antibodies described herein, or for the variable domains of one antigen-binding site of the antibodies described herein, to generate a chimeric bivalent antibody.

[0093] Antibody-producing cell lines can be selected, constructed, and cultured by techniques known to those skilled in the art, all of which are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FMWurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including any supplements, are incorporated by reference in their entirety.

[0094] In some embodiments, antibody-encoding DNA can be designed and synthesized using conventional methods based on the antibody amino acid sequences described herein, inserted into an expression vehicle, transfected into host cells, and cultured in culture to produce monoclonal antibodies. In some embodiments, the expression vehicle contains at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and RNA splicing donor and acceptor sites on either side of the insertion sequence. Highly efficient transcription can be achieved using the early and late promoters of SV40, long terminal repeats from retroviruses, such as RSV, HTLV-1, HIV-1, and the early promoter of cytomegalovirus, and several other cellular promoters, such as the actin promoter, can also be used. Suitable expression vehicles may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0095] In some embodiments, the inserted gene fragment must contain a screening marker; common screening markers include screening genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, and hygromycin resistance, to facilitate screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the gene and cultured in a selective medium; successfully transfected cells grow in large quantities and produce the desired target protein.

[0096] In some embodiments, DNA sequences encoding CD47 antibodies of the present invention can be obtained by combining the amino acid sequences of the antibodies using conventional methods in the art. In some embodiments, the DNA sequence encoding the heavy chain of antibody 6Y-G4 is set forth in SEQ ID NO:57, in which the underlined parts encode the VH CDRs, and the amino acid sequence encoding the heavy chain of antibody 6Y-G4 is set forth in SEQ ID NO:60.

[0097] SEQ ID NO: 57 is as follows: caggtgcagctgcaggagtccggccctggcctggtgaagccttccgagaccctgtccctgacctgtaccgtgagcggcggcagcctg gataactattactggagc tggatccggcagcctcctggcaagggcctggagtggatcggc tacatctactattccggcaacaccaattacaacccttccctgaagagc cgggtgaccatctccgtggacaccagcaagaaccagtttagcctgaagctgtcctccgtgaccgccgctgataccgccgtgtactactgtgccagg ggcggccggttcctggagatat

[0098] SEQ ID NO: 60 is as follows: QVQLQESGPGLVKPSETLSLTTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGRFLERYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0099] In some embodiments, the DNA sequence encoding the light chain of antibody 6Y-G4 is set forth in SEQ ID NO: 58, in which the underlined portion encodes the VL CDR, and the amino acid sequence encoding the light chain of antibody 6Y-G4 is set forth in SEQ ID NO: 61.

[0100] SEQ ID NO: 58 is as follows: gagatcgtgctgacccagtccccctccagcctgagcgccagcgtgggagaccgggtgaccatcccctgc cgggcttccctgtccatcggcagcttcctgaac tggtatcagcagaggcctggcgaggcccctaagctgctgatcttt gccgcttccagcctgcggagc ggcgtgcctagcaggttctccggcagcggctccggcaccgatttcaccctgaccatcagcggcctgcagcccgaggatttcgccacctactactgc cagcagacctacaccaccccttacacctttggccagggcaccaaggtggacatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccagagaggccaaagtacagtggaaggtggataacgccctccaatcg ggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga

[0101] SEQ ID NO: 61 is as follows: EIVLTQSPSSLSASVGDRVTIPCRASLSIGSFLNWYQQRPGEAPKLLIFAASSLRSGVPSRFSGSGSGTDFLTISGLQPEDFATYYCQQTYTTPYTFGQGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0102] In some embodiments, the amino acid sequence of the antibody 6Y-G1 heavy chain is set forth in SEQ ID NO:62.

[0103] SEQ ID NO: 62 is as follows: QVQLQESGPGLVKPSETLSLTTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGRFLERYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] In some embodiments, the amino acid sequence of the antibody 6Y-G1 light chain is set forth in SEQ ID NO:61.

[0105] In some embodiments, the amino acid sequence of the antibody 17 heavy chain is set forth in SEQ ID NO:63.

[0106] SEQ ID NO: 63 is as follows: QVQLQESGPGLVKPSETLSLTTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNTNYNPSLKSRVTISVDTSKNQFSLRLRSVTAADTAVYYCARGGRFLERYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0107] In some embodiments, the amino acid sequence of the antibody 17 light chain is set forth in SEQ ID NO:64.

[0108] SEQ ID NO: 64 is as follows: DIQMTQSPSSVSASVGDRVTISCRANQAIGTWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYTTPLTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0109] In some embodiments, the amino acid sequence of the antibody L12 heavy chain is set forth in SEQ ID NO: 63. In some embodiments, the amino acid sequence of the antibody L12 light chain is set forth in SEQ ID NO: 61.

[0110] Human and humanized antibodies The antibodies described herein include fully human or humanized antibodies, which are suitable for administration to humans without eliciting a human immune response against the administered immunoglobulin.

[0111] CD47 antibodies can be generated, for example, by phage display using antibodies containing only human sequences. Such methods are well known in the art, e.g., WO 92 / 01047 and U.S. Patent No. 6,521,404. In this method, a combinatorial library of phage containing random light and heavy chain pairs is screened using native or recombinant CD47 or fragments thereof. In another method, CD47 antibodies can be generated by immunizing a transgenic non-human animal with human CD47 protein in at least one step. In such methods, some transgenic non-human animals lose their endogenous heavy and / or light chain loci and are unable to undergo the rearrangement necessary to generate genes encoding immunoglobulins that respond to antigens. Additionally, at least one human heavy chain locus and at least one human light chain locus are stably transfected into the animal. Thus, in response to an administered antigen, the human loci rearrange to provide genes encoding human variable regions with immunospecificity for the antigen. Thus, upon immunization, the transgenic mice produce B cells that secrete fully human immunoglobulins.

[0112] The production of antibodies with reduced immunogenicity can also be achieved by humanization, chimerization, and display techniques using appropriate libraries. It should be understood that murine antibodies or antibodies derived from other species can be humanized or primatized using techniques well known in the art. See, for example, Winter and Harris, Immunol Today 14:43-46 (1993) and Wright et al., Crit. Reviews in Immunol. 12125-168 (1992). An antibody of interest can be engineered by recombinant DNA technology to replace the CH1, CH2, CH3, hinge domain, and / or scaffold domain with the corresponding human sequence (see WO 92102190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). The use of Ig cDNA to construct chimeric immunoglobulin genes is also known in the art (Liu et al., PNAS 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from hybridomas or other cells producing the antibody and used to generate cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Patents 4,683,195 and 4,683,202). Alternatively, the sequence of interest can be isolated by constructing and screening a library. DNA sequences encoding the antibody variable region are then fused to human constant region sequences. The sequences of human constant region genes are described in Sequences of Proteins of Immunological Interest by Kabat et al. (NIH publication no. 91-3242 (1991)). Human C region genes can be readily obtained from known clones. The choice of isotype is guided by the desired effector functions, such as complement fixation or antibody-dependent cellular cytotoxicity activity. Preferred isotypes are IgG1 and IgG2.Any human light chain constant region, ie, k or λ, can be used and then the chimeric humanized antibody can be expressed by conventional methods.

[0113] In some embodiments, antibody fragments such as Fv, F(ab')2, and Fab can be prepared by cleavage of the intact protein, such as by protease or chemical cleavage, including, but not limited to, (i) digestion of antibody molecules with pepsin to obtain F(ab')2 fragments, (ii) reduction of disulfide bonds of F(ab')2 fragments to obtain Fab fragments, (iii) treatment of antibody molecules with papain and a reducing agent to generate Fab fragments, and (iv) Fv fragments.

[0114] The J regions of the heavy and light chain consensus sequences may be used to design oligonucleotides to be used as primers to ligate V region fragments to human C region fragments after introducing useful restriction sites into the J regions. The C region cDNA may be modified by site-directed mutagenesis to place restriction sites at analogous positions in the human sequence.

[0115] The CD47 antibodies described herein may be expressed from a carrier containing a DNA fragment encoding the antibody. Expression carriers include plasmids, retroviruses, YACs, EBV-induced adducts, and the like. Suitable carriers typically encode functionally complete human immunoglobulin sequences for the heavy (CH) or light (CL) chain constant regions and contain appropriate restriction sites to allow for the easy insertion and expression of any VH or VL sequence. In such carriers, splicing typically occurs between a splice donor site within the inserted J region and a splice acceptor site preceding the human C region, as well as at a splice region present within the human CH exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The resulting chimeric antibody can be linked to any strong promoter, including retroviral LTRs, such as the SV-40 early promoter (Okayama et al., Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al., PNAS 79:6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl et al., Cell 41:885 (1985)). Alternatively, natural Ig promoters can be used.

[0116] Fc modification The relevant effector functions of the antibodies described herein can be modified to improve the effectiveness of the antibodies in, for example, treating diseases and disorders associated with aberrant CD47 signaling. For example, one or more mutations can be introduced into the Fc region of the antibody to silence the effector function, thereby reducing the likelihood of killing normal cells.

[0117] In some embodiments, the antibodies described herein are of the IgG isotype. In some embodiments, the constant region of the antibody is of the human IgG1 isotype having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, amino acid Asn297 (numbered according to Kabat) on the human IgG1 constant region is modified to avoid glycosylation of the antibody, e.g., Asn297Ala (N297A). In some embodiments, amino acid Leu235 (numbered according to Kabat) on the antibody constant region is modified to alter Fc receptor interaction, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, amino acid Leu234 (numbered according to Kabat) on the antibody constant region is modified to alter Fc receptor interaction, e.g., Leu234Ala (L234A). In some embodiments, amino acids 234 and 235 on the antibody constant region are modified simultaneously, for example Leu234Ala and Leu235Ala (L234A / L235A) (Kabat et al., 1991, EU Index of Sequences of Proteins of Immunological Interest).

[0118] In some embodiments, the constant region of the antibody is a human IgG4 isotype having the amino acid sequence set forth in SEQ ID NO:4.

[0119] In some embodiments, the hinge region in the human IgG4 constant region is modified to prevent or reduce chain exchange, for example, Ser228Pro (S228P). In other embodiments, amino acid 235 on the human IgG4 constant region is modified to alter Fc receptor interaction, for example, Leu235Glu (L235E). In some embodiments, the hinge region and amino acid 235 on the human IgG4 constant region are modified, for example, Ser228Pro and Leu235Glu (S228P / L235E). In some embodiments, amino acid Asn297 (boxing, numbered according to Kabat) on the human IgG4 constant region is modified to prevent glycosylation of the antibody, for example, Asn297Ala (N297A). In some embodiments, amino acids Ser228, Leu235, and Asn297 on the human IgG4 constant region are modified (for example, S228P / L235E / N297A).

[0120] Use of antibodies against CD47 In some embodiments, CD47 antibodies, including those of the present invention, can be used in the diagnosis, prognosis, monitoring, treatment, mitigation, and / or prevention of diseases or pathologies associated with aberrant CD47 expression, activity, and / or signaling in a subject. Standard methods can be used to identify subjects (human patients) suffering from or at risk of developing a disease or condition associated with aberrant CD47 expression, activity, and / or signaling (e.g., cancer or other neoplastic conditions), and then administer a treatment regimen. In some embodiments, a method for treating a disease or condition involving aberrant CD47 expression, activity, and / or signaling is provided, comprising administering to a subject in need of treatment an effective amount of an antibody or formulation thereof described herein. Administration of the antibody can neutralize, inhibit, or interfere with the expression, activity, and / or signaling function of a target (e.g., CD47). Administration of the antibody can neutralize, inhibit, or interfere with the binding of a target (e.g., CD47) to its naturally-bound endogenous ligand (e.g., SIRPα). For example, the antibody binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes or interferes with CD47 expression, activity and / or signaling.

[0121] In some embodiments, the disease or condition implicated in aberrant CD47 expression, activity, and / or signaling includes hematological cancers and / or solid tumors. Hematological cancers include, for example, leukemia, lymphoma, and myeloma. In some embodiments, forms of leukemia include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative disorders / neoplasias (MPDS), and myelodysplastic syndromes. In some embodiments, forms of lymphoma include Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). In some embodiments, forms of myeloma include multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bence-Jones myeloma. Solid tumors include, for example, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer.

[0122] Symptoms associated with cancer and other neoplastic conditions include, for example, inflammation, fever, general malaise, fever, pain, often localized to the inflamed area, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue, and abdominal symptoms (e.g., abdominal pain, diarrhea, or constipation).

[0123] A therapeutically effective amount of an antibody of the present invention generally relates to the amount necessary to achieve a therapeutic goal. The amount required for administration depends on factors such as the binding affinity of the antibody to its specific antigen, the severity of the disease, disorder, or condition, the route of administration, and the rate at which the administered antibody depletes free volume in the administered subject. In some embodiments, a therapeutically effective amount of an antibody or antibody fragment of the present invention generally ranges from about 0.1 mg / kg to about 100 mg / kg. In some embodiments, an antibody of the present invention is administered to a subject at a dose of about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, or more, or a range between any two of these values ​​(including the endpoints). Typical administration frequencies are, for example, once daily to once weekly.

[0124] In some other embodiments, antibodies to CD47 can be applied in methods known in the art related to the localization and / or quantification of CD47 (e.g., used to measure levels of CD47 and / or both CD47 and SIRPα in appropriate physiological samples, used in diagnostic methods, used in protein imaging, etc.). In certain embodiments, antibodies specific for CD47 or derivatives, fragments, analogs, or homologs thereof, including antigen-binding domains derived from the antibodies, are used as pharmaceutically active compounds (hereinafter referred to as "therapeutic agents").

[0125] In some other embodiments, CD47 polypeptides can be isolated using CD47-specific antibodies by standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) against the CD47 protein can be used to detect the protein in biological samples. In some embodiments, CD47 can be detected in biological samples as part of a clinical trial procedure, for example, to determine the effectiveness of a given therapeutic regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylfluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131 I, 35 S or 3 Contains H.

[0126] In some other embodiments, the antibodies of the present disclosure can be used as reagents for detecting the presence of CD47 and / or both CD47 and SIRPα proteins (or protein fragments thereof) in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody, or more preferably, a monoclonal antibody. An intact antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The detection methods of the above embodiments can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include northern hybridization and in situ hybridization. In vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. (ELISA: Theory and Practice: Methods in Molecular Biology, Vol. 42, J.R.Crowther Human Press, Totowa, NJ, 1995; Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985). In vivo detection techniques for analyte proteins also involve introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody can be labeled with a radioactive label, whose presence and location within the subject can then be detected by standard imaging techniques.

[0127] Therapeutic Administration and Formulations of CD47 Antibodies The antibodies and derivatives, fragments, analogs, and homologs thereof described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in the preparation of such compositions and guidance for component selection are well known in the art; see, e.g., Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy, 19th ed., Mack Pub. Co., Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; Peptide And Protein Drug Delivery, Advances In Parenteral Sciences, Vol. 4, 1991, M. Dekker, New York.

[0128] Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. In some embodiments, the antibody fragment used is the smallest inhibitory fragment that specifically binds to the binding domain of the target protein, such as a peptide based on the variable region sequence of the antibody and that retains the ability to bind to the target protein sequence.

[0129] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, stabilizers, buffers, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, and the like, compatible with drug administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences. Such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin.

[0130] Preparations to be used for internal administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0131] The pharmaceutical composition of the above embodiments will generally be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Pharmaceutical compositions can include one or more of the following components: a sterile injectable diluent such as water, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as histidine hydrochloride, acetate, citrate, or phosphate; an agent for adjusting tonicity such as sodium chloride or dextrose; a stabilizer such as arginine, methionine, trehalose, sucrose, or sorbitol; or a surfactant such as Tween 20 or Tween 80. pH can be adjusted with an acid or base, e.g., hydrochloric acid or sodium hydroxide. The pharmaceutical compositions can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. When used, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms, such as bacteria and fungi. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0132] For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used in transmucosal administration. Transmucosal administration can be achieved by using nasal drops or suppositories. For transdermal administration, one or more of the above antibodies can be formulated into ointments, ointments, gels, or creams that are generally known in the art.

[0133] The pharmaceutical composition is prepared in dosage unit form, which is easy to administer and has uniform dosage.As used herein, dosage unit form refers to a physically separable unit suitable as a unit dose for use in a subject to be treated, each unit containing a predetermined amount of one or more of the above antibodies, which are calculated to be bound to the required drug carrier to produce the desired therapeutic effect.

[0134] The pharmaceutical composition can be placed in a container or dispenser and packaged with instructions for administration.

[0135] The pharmaceutical compositions described herein can also contain other active ingredients depending on the specific condition requiring treatment, preferably with complementary activities that do not adversely affect each other. In some embodiments, the composition can include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such active ingredients are present in appropriate combinations in amounts effective for the intended purpose.

[0136] In one embodiment, one or more active ingredients, including CD47 antibodies, can be administered in combination therapy, i.e., in combination with other agents, e.g., therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell growth inhibitors, etc.). The term "combination," as used herein, refers to essentially synchronous, simultaneous, or sequential administration of agents. In some embodiments, such combination therapy advantageously allows for the use of lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies.

[0137] In some embodiments, the antibodies described herein are used as vaccine adjuvants for autoimmune diseases, inflammatory diseases, and the like. The vaccines can be various antigens. The antigens are derived from targeted autoantigens, i.e., autoantigens involved in autoimmunity such as myelin basic protein, inflammatory autoantigens such as amyloid peptide proteins, or transplantation antigens such as alloantigens. Antigens can include peptides or polypeptides derived from proteins, as well as fragments of any one of sugars, proteins, polynucleotides or oligonucleotides, autoantigens, amyloid peptide proteins, transplantation antigens, allergens, or other macromolecular components. In some embodiments, one or more antigens are included in an antigenic composition.

[0138] Citation of publications and patent documents herein is not an admission that any of the material therein is pertinent prior art, nor is it an admission as to the content or date thereof. While the present invention has been described in terms of a written specification, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing specification and the following examples are intended to illustrate, but not limit, the scope of the invention as claimed. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. [Example]

[0139] Unless otherwise specified, all materials, reagents, etc. used in the following examples are available from commercial sources or by known methods. In the following examples, antibody L12-6 and antibody 6Y-G1 are the same antibody.

[0140] CHO-CD47 cell construction process: A HindIII / EcoRI window was designed, and the polynucleotide encoding CD47 (sequence is from Uniprot, Q08722) was introduced into the pcDNA3.1(+) (Invitrogen, V790-20) plasmid, and CHO cells (ATCC#CCL-61) were transfected with a PvuI single cut. After screening, a stable CHO-CD47 cell line was obtained.

[0141] Jurkat cells were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, with the product number Clone E6-1.

[0142] The IgG antibody was purchased from BioXCell and had the product number BE0297.

[0143] Macaque CD47-Fc was purchased from Creative BioMart and had the model number CD47-745C.

[0144] Example 1: Preparation of CD47 antigen CD47 antigen preparation: CD47 recombinant proteins were constructed by adding a 6xHIS tag (HHHHHH, SEQ ID NO: 2) or human Fc (the underlined portion of SEQ ID NO: 3) (see R&D systems) to the C-terminus of the human CD47 extracellular domain (ECD) protein (SEQ ID NO: 1). The ECD region of human CD47 was genetically synthesized with 6xHIS or Fc, respectively, and subcloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen, V790-20). After transient transfection of HEK293F cells, His-tagged human CD47-ECD protein (CD47-His) was purified by nickel-based immobilized metal affinity chromatography, and secreted Fc-tagged human CD47-ECD protein (CD47-Fc) was purified by immobilized metal affinity chromatography (IMAC) using a protein A column (GE Healthcare).

[0145] Example 2: Preparation of CD47 antibodies The VH and VL combinations of 30 single-chain antibodies (scFv) are shown in Table 2. The C-terminus of VH and the N-terminus of VL are linked by a linker short peptide of the sequence (GGGGSGGGGSGGGGS) shown in SEQ ID NO: 10, with the antibody heavy chain variable region represented by the antibody number + "-VH," such as 17-VH, the antibody light chain variable region represented by the antibody number + "-VL," such as 17-VL, and the single-chain antibody (scFv) represented by the antibody number + "-ScFv," such as 17-ScFv (the C-terminus of 17-VH (shown in SEQ ID NO: 11) and the N-terminus of 17-VL (shown in SEQ ID NO: 12) were linked by the linker short peptide shown in SEQ ID NO: 10 to obtain 17-ScFv).

[0146] The VH and VL combinations of 30 IgG1 complete antibodies are shown in Table 2. VH and CH constitute the antibody heavy chain, and VL and CL constitute the antibody light chain, with the CH sequence shown in SEQ ID NO: 3 and the CL sequence shown in SEQ ID NO: 5. The antibody heavy chain variable region is represented by the antibody number (e.g., 17-VH) + "-VH," and the antibody light chain variable region is represented by the antibody number (e.g., 17-VL) + "-VL," with IgG1 complete antibodies represented by "antibody" + antibody number (e.g., antibody 17) (the antibody heavy chain is composed of 17-VH (shown in SEQ ID NO: 11) and CH shown in SEQ ID NO: 3, and the antibody light chain is composed of 17-VL (shown in SEQ ID NO: 12) and CL shown in SEQ ID NO: 5).

[0147] The amino acid sequence of the heavy chain of antibody 6Y-G4 is shown in SEQ ID NO:60, and the amino acid sequence of the light chain is shown in SEQ ID NO:61.

[0148] The VH and VL sequences corresponding to Table 2 are shown in Table 3, where the heavy chain CDRs are shown in Table 4 and the light chain CDRs are shown in Table 5.

[0149] Plasmid pcDNA3.1(+) (Invitrogen, V790-20) was modified by conventional techniques to contain a DNA sequence encoding the above amino acid sequence. The plasmid was then transiently transfected into HEK293F cells to express the antibody with the above amino acid sequence. The CD47 antibody was purified by immobilized metal affinity chromatography (IMAC) using a protein A column (GE Healthcare). The sequence was confirmed to be consistent with the predicted sequence after sequencing and was used to identify its biological activity.

[0150] [Table 2]

[0151] [Table 3A]

[0152] [Table 3B]

[0153] [Table 4]

[0154] [Table 5]

[0155] The main Fc-dependent functions of antibodies (e.g., CD47 antibodies) for target cell clearance are complement-dependent cytotoxicity (CDC), initiated by C1q binding to the Fc region; antibody-dependent cellular cytotoxicity (ADCC), mediated by interaction of the Fc region with Fcγ receptors (FcγR), primarily FcγRIIIa, on immune effector cells (e.g., NK cells and neutrophils); and antibody-dependent cellular phagocytosis (ADCP), mediated by macrophage recognition of opsinized target cells via FcγRI. Each antibody subclass differs in its ability to mediate Fc-dependent effector activities. In humans, the IgG1 and IgG3 subclasses are highly potent in CDC due to their binding to C1q. Furthermore, the IgG1 subclass has the highest affinity for FcγR, making it the most effective in ADCC and Fc-dependent ADCP. The IgG4 subclass lacks C1q binding ability and has significantly reduced FcγR binding affinity, resulting in significantly reduced effector function.

[0156] Based on the antibody 6Y-G1, an IgG4P subclass antibody 6Y-G4 was prepared with the mutation S228P to stabilize the hinge region of the antibody (the CH shown in SEQ ID NO: 3 in 6Y-G1 is replaced with the CH shown in SEQ ID NO: 4, and the other sequences remain unchanged), further reducing the antibody Fc effector.

[0157] Taking the preparation of 17-ScFv as an example, the method for preparing the ScFv of the present invention is as follows: The nucleotide sequence encoding 17-ScFv (shown in SEQ ID NO:59) was ligated into the pcDNA3.1(+) plasmid carrier using the restriction endonucleases Hind III / EcoRI. The plasmid carrier was then transiently transfected into HEK293F cells so that the HEK293F cells would express an antibody with the above amino acid sequence. The 17-ScFv antibody was purified by immobilized metal affinity chromatography (IMAC) using a protein A column, and sequencing confirmed that the sequence was consistent with the predicted sequence.

[0158] Other ScFvs were prepared by similar methods, purified, and sequenced to match the predicted sequences.

[0159] The sequence of SEQ ID NO: 59 is as follows: caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccctcgataattactactggagctggatccggcagcccccagggaagggactggagtggattggatatatctattacagtgggaacaccaactaca acccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctcgttgaggctgaggtctgtgaccgctgcggacacggccgtgtattactgtgcgagaggagggcgattttggaacgttactggggccagggaaccctggtcaccgtctctcctcaggtggaggc ggttcaggcggaggtggctctggcggtggcggatcggacatccagatgacccagtctccatcttccgtgtctgcatctgtaggagacagagtcaccatctcttgtcgggcgaatcaggctattggcacttggttagcctggtatcagcaaaagccaggaaaagcccctaagctcctga tctatgcggcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaatattatactactcccctcactttcggcggagggaccaagctggagatcaaa

[0160] Example 3: Biological activity of CD47 antibodies 1. Detection of binding activity of 17-ScFv by FACS The binding activity of 17-ScFv to CD47 on the surface of CHO-CD47 or Jurkat cells was examined (Figures 1A-1B). The experimental procedure for binding activity was as follows: 17-ScFv and Hu5F9-G4 antibody (Hu5F9-G4 has a heavy chain composed of the variable region shown in SEQ ID NO: 8 and the constant region shown in SEQ ID NO: 4, and a light chain composed of the variable region shown in SEQ ID NO: 9 and the constant region shown in SEQ ID NO: 5) were diluted with PBS to different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02, and 0.01 μg / mL) and incubated with CD47 on the surface of CHO-CD47 or Jurkat cells at 4°C for 30 minutes. After washing once with PBS, a goat anti-human IgG Fc-PE (eBioscience, product code 2183639) fluorescent secondary antibody was added, and the cells were incubated for 15 minutes at 4°C and washed twice with PBS. The PE-A mean fluorescence signal was detected using an upflow cytometer. 1A-1B show that 17-ScFv can bind to CD47 on the cell surface in a dose-dependent manner.

[0161] 2. SIRP-α blocking activity The ability of 17-ScFv to block SIRPa binding to CD47 on the surface of CHO-CD47 cells was detected by FACS technology. 17-ScFv antibody was diluted to different concentrations in PBS (160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625 μg / mL). Hu5F9-G4 and IgG antibodies (diluted to different concentrations in PBS: 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 μg / mL) were incubated with CD47 on the surface of CHO-CD47 cells at 4°C for 30 min, washed once with PBS, and then SIRPα-Fc-Bio (6.5 μg / mL) was added. The cells were incubated at 4°C for 15 min, washed once with PBS, and then streptavidin was added. PE (eBioscience, product number 1992345) fluorescent secondary antibody was then added, incubated at 4°C for 15 min, washed twice with PBS, and the PE-A mean fluorescence signal was detected using an upflow machine. Figure 2 shows that 17-ScFv can inhibit SIRPα binding to CD47 on the cell surface in a dose-dependent manner.

[0162] Using a competitive ELISA method, 17-ScFv or 18 antibodies (Antibody 3, Antibody 3-3, Antibody 3-6, Antibody 6, Antibody 10, Antibody 11, Antibody 16, Antibody 18, Antibody 24, Antibody 25, Antibody L12, Antibody L1, Antibody L5, Antibody L24, Antibody L26, Antibody 17, Antibody 6Y-G1, Antibody 6Y-G4), a positive control (Antibody Hu5F9-G4), and a negative control (Antibody IgG) were diluted in PBS to different concentrations (12, 6, 3, 2, 1.5, 1.2, 0.75, 0.375, 0.188, 0.094 μg / mL) and incubated with CD47-His (2 μg / mL) antigen coated on an ELISA plate at room temperature for 1 hour, washed four times with PBST, and then incubated with SIRPα-Fc-Bio (0.1 μg / mL) (Thermo Scientific EZ-Link® NHS-Biotin) for 1 hour. The ligand was then added (see Preparation with Reagent Kit) and incubated with the CD47-His antigen at room temperature for 1 hour, followed by washing four times with PBST. The bound SIRPα-Fc-Bio and HRP-conjugated streptavidin secondary antibody reacted with each other via chemiluminescence, and the OD450 value was detected using a plate reader. The IC value of the antibody was calculated based on the OD450 value. 50 The SIRPα-blocking activity of the antibodies was determined by converting the SIRPα-blocking activity of the antibodies. Figures 3A to 3E show that all of the antibodies tested showed dose-dependent inhibition of SIRPα binding to the CD47 antigen.

[0163] 3. Hemagglutination Assay We detected hemagglutination of CD47 antibodies. Five milliliters of fresh blood was collected from a healthy human donor into an anticoagulant tube containing sodium heparin. PBMCs were first isolated using lymphocyte separation buffer (for other purposes). Next, 1 mL of red blood cells was collected from the bottom of the tube and added to 6 mL of saline. Repeated washing was performed, and the mixture was centrifuged at 2000 rpm for 5 minutes until the supernatant was no longer noticeably red. The red blood cells were then resuspended in saline to create a 2% red blood cell suspension. At room temperature, 50 μL of CD47 antibodies serially diluted 2-fold in PBS (maximum concentration 800 nM, a total of 12 gradients) was mixed with 50 μL of the 2% red blood cell suspension and added to a 96-well U-shaped plate for 4 hours. The antibody-red blood cell agglutination was then assessed by tilting the 96-well U-shaped plate 45° and observing the flow direction of the red blood cell clots. A "straight line" indicated no red blood cell agglutination. Most of the CD47 antibodies tested induce red blood cell agglutination at high concentrations (antibody 17 causes obvious red blood cell agglutination), but do not induce red blood cell agglutination at concentrations around 6 nM. Among them, antibodies L12, 6Y-G1 (i.e., antibody L12-6), and 6Y-G4 do not induce any red blood cell agglutination (Figures 4A-4E). The positive control antibody Hu5F9-G4 can induce red blood cell agglutination, while AB6.12-G1 and the ligand SIRPα do not induce red blood cell agglutination. AB6.12-G1 has a heavy chain consisting of the variable region set forth in SEQ ID NO:6 and the constant region set forth in SEQ ID NO:3, and a light chain consisting of the variable region set forth in SEQ ID NO:7 and the constant region set forth in SEQ ID NO:5. Hu5F9-G4 has a heavy chain consisting of the variable region set forth in SEQ ID NO:8 and the constant region set forth in SEQ ID NO:4, and a light chain consisting of the variable region set forth in SEQ ID NO:9 and the constant region set forth in SEQ ID NO:5. As shown in Table 6.

[0164] [Table 6]

[0165] 4. CD47 binding assay (ELISA): The binding activity of CD47 antibodies to human CD47 and macaque CD47 was measured by ELISA. Antibodies 17, L12, 6Y-G1, 6Y-G4, and Hu5F9-G4 were diluted 3-fold from 5 μg / mL to 8 μg / mL in PBS and then incubated with human or macaque CD47-Fc (2 μg / mL) antigen coated on an ELISA plate for 1 hour at room temperature. The plate was then washed four times with PBST. HRP-anti-kappa (1:10,000, Sigma) secondary antibody was added and incubated with the antibody for 1 hour at room temperature. The plate was then washed four times with PBST. The bound HRP-anti-kappa and TMB substrate reacted chemiluminescently, and the OD450 value was detected using a plate reader. The EC values ​​of the antibodies were determined based on the OD450 value. 50 The binding activity of the antibodies to human or macaque CD47 was determined by converting the values. Figures 5A and 5B show that all of the antibodies tested showed dose-dependent binding to the CD47 antigen. Each parameter is shown in Table 7.

[0166] [Table 7]

[0167] 5. CD47 binding assay (Fortebio) The binding activity of 11 antibodies (antibody L12-1, antibody L12-2, antibody L12-3, antibody L12-4, antibody L12-5, antibody L12-6, antibody L12-7, antibody L12-9, antibody L12-10, antibody L12-11, and antibody L12) and Hu5F9-G4 to human CD47 was measured using the Fortebio method.

[0168] The Protein A sensor was pre-wetted with PBS for 10 minutes before use. The antibody to be tested was diluted to 10 μg / mL in PBST, pH 6.8, containing 0.5% BSA (hereafter referred to as the dilution buffer). The PA sensor was then immobilized with the antibody until the signal reached approximately 1.7 nm. The CD47-His antigen was diluted to 50, 25, 12.5, and 6.25 nM in the dilution buffer. The dilution buffer, gradient concentrations of antigen dilutions, regeneration buffer (1 M MgCl2, pH 8.4), and neutralization buffer (PBST) were added sequentially to the corresponding columns of a 96-well plate, and the following steps were performed: (1) Baseline: A baseline was detected in the dilution buffer for 60 seconds. (2) Association: Association was performed for 100 seconds in the gradient dilutions of antigen and a sample blank (dilution buffer). (3) Dissociation: Dissociation was performed for 700 seconds in the dilution buffer. (4) Regeneration: Regeneration was performed for 5 seconds in the regeneration buffer. (5) Neutralization: Neutralization was performed in neutralization buffer for 5 seconds. (6) Regeneration and neutralization cycles were performed three times.

[0169] Data was processed and analyzed using Data Analysis 8.2, and the sample data was fitted after subtracting the reference (sample blank) signal to obtain the affinity constant KD. The antibody affinity result statistics are shown in Table 8. The results show that the 11 antibodies bound to the CD47 antigen with similar affinities.

[0170] [Table 8]

[0171] 6, phagocytosis To assess whether CD47 antibodies enhance the phagocytosis of CD47-expressing target cells by macrophages, an in vitro phagocytosis assay was performed. Briefly, RAW264.7 macrophages (2 × 10) were cultured in the presence of CD47 antibodies (0.7 nM). 5cells / mL) (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, 3111C0001CCC000146) and CFSE-labeled Raji cells (4 × 10 5 RAW264.7 cells were seeded at a 1:2 ratio (cells / mL) onto a 24-well bottom plate and incubated at 37°C in the dark for 2 hours. After incubation, the cells were washed twice with PBS. RAW264.7 cells were digested with trypsin, and the digested macrophages were transferred to 1.5 mL EP tubes and centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, and 100 μL of APC-F4 / 80 fluorescent antibody (eBiosciences) was added to each tube. The cells were incubated at 4°C in the dark for 30 minutes, centrifuged at 2000 rpm for 5 minutes, and washed twice with PBS buffer. Cells were acquired using a BD C6 flow cytometer. The phagocytic index was analyzed and calculated as follows: phagocytic index = number of CFSE-positive macrophages (i.e., number of macrophages that phagocytosed tumor cells) / 5000 macrophages. Antibodies 6Y-G1, 6Y-G4, 17, L12, and Hu5F9-G4 exhibited potent phagocytosis-enhancing abilities (Figures 6A-6B). Figure 6B shows that the phagocytic response of 6Y-G4 was slightly weaker than that of 6Y-G1, indicating that the Fc effector of IgG4 antibodies is weaker.

[0172] Example 4: Effect of CD47 antibodies on blood cells Prior to the present invention, known CD47-binding molecules that block SIRPα and contain an Fc domain (e.g., CD47 antibodies and recombinant SIRPα-Fc fusion proteins) all induced varying degrees of reduction in red blood cells and platelets. Therefore, the effects of the antibodies of the present invention on blood cells were evaluated in cynomolgus monkeys. A total of eight cynomolgus monkeys meeting the test requirements were selected, half male and half female, of standard grade, with weight ranges of 3.70-5.30 kg and 2.75-3.55 kg, respectively. They were randomly assigned according to weight to four groups, with two animals per group, each half male and half female. The negative control group received normal saline. The test substances were antibodies 6Y-G1 (i.e., antibody L12-6), Hu5F9-G4, and AB6.12-G1. Normal saline was used as a vehicle and administered intravenously once weekly at doses of 1, 10, 10, 30, and 30 mg / kg, five times in a 10 mL / kg administration volume. After administration, the subjects were observed for up to 42 days. As shown in Figures 7A-7D, all three antibodies caused a decrease in hemoglobin and red blood cell counts after administration, reaching a nadir approximately two weeks later and then gradually returning to normal levels. Platelet counts fluctuated before and after administration and returned to normal after a drug withdrawal. Reticulocyte counts tended to increase after administration in all three groups, with the reticulocyte count increasing more with 6Y-G1 than with the other two antibodies.

[0173] Example 5: In vivo anti-tumor activity of Fc variants of CD47 antibody The antitumor activity of the test antibody L12 was evaluated in a human hematologic tumor MV4-11 systemic tumor model. MV4-11 cells (ATCC) were inoculated via the tail vein into NOD.SCID mice (Jiangsu Jixi Yaokang Biotechnology Co., Ltd., weighing 18-22g, aged 6-8 weeks). Four weeks after inoculation, the human hematologic tumor MV4-11 systemic tumor model was established. The study was divided into test groups: AB6.12-G1 0.2mg / mouse, Hu5F9-G4 0.2mg / mouse, antibody L12 0.2mg / mouse, and a vehicle (saline) control group. Each group consisted of six mice. The treatment was administered intraperitoneally three times a week for a total of three weeks. Therapeutic efficacy was assessed based on time to survival (ILS), and safety was assessed based on changes in body weight and mortality during the treatment period. As shown in Figure 8, the test drugs AB6.12-G1 (0.2 mg / mouse) and Hu5F9-G4 (0.2 mg / mouse) extended survival time by 7.4% and 6.5% compared to the vehicle control group, respectively, which was not statistically significant (p = 1.000), demonstrating no significant antitumor effect. The median survival time for antibody L12 (0.2 mg / mouse) was 69 days in both groups, extending survival time by 27.8% compared to the control group, demonstrating a statistically significant difference (p = 0.007).

[0174] The antitumor activity of the antibody 6Y-G4 was evaluated in a Raji model of lymphoma. Raji cells were implanted subcutaneously into NOD.SCID mice, which were randomly divided into three groups (8 mice per group, day 0): Group 1: vehicle control (physiological saline), Group 2: Hu5F9-G4 (positive control), and Group 3: 6Y-G4. When tumors became palpable (50 mm 3 On day 4), treatment with each antibody or vehicle (buffer only) was performed until the tumor volume reached approximately 3000 mm 3Mice were sacrificed when tumor volume reached 100 μg. Tumor volume was measured every 3 days. Antibodies were administered intraperitoneally (IP) at a dose of 100 μg, three times a week for a total of 3 weeks (a total of 9 doses per mouse). Treatment began on day 4 and ended on day 21. As shown in Figure 9, both the 6Y-G4 and Hu5F9-G4 treatment groups at a dose of 0.1 mg / mouse exhibited highly significant antitumor effects, clearly inhibiting tumor growth after the start of treatment. By day 17 after the start of treatment (PG-D17), most of the mouse tumors had disappeared. The mean tumor volumes of the 6Y-G4 (0.1 mg / mouse) and Hu5F9-G4 (0.1 mg / mouse) treatment groups were 4 mm, respectively. 3 (7 tumors disappeared in 8 mice), 7mm 3 The relative tumor inhibition rates (TGI) were 99.87% and 99.76%, respectively (tumor disappearance in 5 of 8 mice), both of which were statistically significant compared to the vehicle control group (all p values ​​< 0.001).

Claims

1. an antibody or antigen-binding fragment, which specifically binds to CD47 and comprises a VH CDR1 set forth in SEQ ID NO: 14, a VH CDR2 set forth in SEQ ID NO: 17, a VH CDR3 set forth in SEQ ID NO: 23, a VL CDR1 set forth in SEQ ID NO: 30, a VL CDR2 set forth in SEQ ID NO: 32, and a VL CDR3 set forth in SEQ ID NO: 34; An antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment comprises a heavy chain variable region VH comprising an amino acid residue mutation selected from one or more of the following, numbered according to Kabat: The antibody or antigen-binding fragment of claim 1, (a) R81K, and (b) R82aS.

3. The antibody or antigen-binding fragment comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 11 or 50, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 or 50.

3. The antibody or antigen-binding fragment of claim 1 or 2.

4. the antibody or antigen-binding fragment comprises a light chain variable region VL comprising any one of the amino acid sequences selected from SEQ ID NOs: 13, 45 to 49, 51 to 55, and 65, or an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 13, 45 to 49, 51 to 55, and 65; The antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. An antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprising a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 11 or 50, and a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 13 or 65; An antibody or antigen-binding fragment thereof.

6. The antibody comprises a heavy chain H comprising any one of the amino acid sequences selected from SEQ ID NOs: 60 and 62 to 63, and a light chain L comprising the amino acid sequence shown in SEQ ID NO:

61. The antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. the antibody or antigen-binding fragment is an IgG isotype selected from the IgG1 subtype, the IgG2 subtype, the IgG3 subtype, or the IgG4 subtype; The antibody or antigen-binding fragment according to any one of claims 1 to 6.

8. the antibody or antigen-binding fragment is IgG4P; The antibody or antigen-binding fragment of claim 7.

9. 9. A method for the preparation of a medicament for the treatment of a rheumatoid arthritis, comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 8 and a pharmaceutically acceptable carrier. A composition characterized by:

10. (1) A polynucleotide encoding the antibody or antigen-binding fragment according to any one of claims 1 to 8, or (2) An expression carrier comprising a polynucleotide encoding the antibody or antigen-binding fragment according to any one of claims 1 to 8, or (3) A cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment according to any one of claims 1 to 8. A biomaterial.

11. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 8, or a composition according to claim 9, or a biomaterial according to claim 10, in the preparation of a medicament for treating cancer or an infectious disease.

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