CD47 antibody and application thereof

Abstract

The invention relates to a CD47 antibody and application thereof, in particular to a monoclonal antibody for recognizing CD47, more particularly to a CD47 antibody which does not cause significant levels of red blood cell agglutination, red blood cell reduction and thrombocytopenia, and relates to a method for preparing the antibodies and application of the monoclonal antibody in preparation of medicines for treating cancers or infections.

Description

Technical Field

[0001] The present invention relates to the field of biological immunology, and more particularly to a fully human anti-CD47 IgG antibody, and more specifically to a CD47 antibody that does not cause significant human red blood cell hemagglutination reaction, hemoglobin reduction, anemia and / or thrombocytopenia, a method for preparing these antibodies, and the use of these monoclonal antibodies in the preparation of drugs. Background Art

[0002] CD47, also known as integrin-associated protein (IAP), is a transmembrane glycoprotein widely expressed on the cell surface and belongs to the immunoglobulin superfamily (Johansen and Brown, J. Biol. Chem. 2007). CD47 binds to its ligand signal regulatory protein α (SIRPα) to inhibit macrophage phagocytosis (Matozaki T, et al., Trends Cell Biol. 2009). Existing studies have confirmed that CD47 is overexpressed in many malignant tumors, such as acute myeloid leukemia (AML), B-cell and T-cell acute leukemia, non-Hodgkin lymphoma, etc., transmitting the "Don't eat me" signal to macrophages to avoid immune surveillance, and high expression of CD47 is associated with poor clinical prognosis (Willingham et al. Proc Natl Acad Sci USA. 2012).

[0003] U.S. Patent Application 2009 / 0191202 discloses that the team of Professor Weissman at Stanford University found through experiments on in situ immunodeficient mouse xenograft animal models that the administration of anti-CD47 monoclonal antibodies can inhibit the growth and metastasis of large tumors, and small tumors can be cured. U.S. Patent Application 2016 / 0251435 discloses the application of anti-CD47 monoclonal antibodies in tumor treatment. Patent Application WO2013056352 describes the use of humanized full-length monoclonal antibodies against human SIRPα and antibody fragments derived therefrom for the treatment of hematological malignancies, especially leukemia. Blocking the CD47-SIRPα signaling pathway can promote macrophage phagocytosis of tumor cells, providing a new means for tumor immunotherapy. Summary of the Invention

[0004] The present invention provides antibodies that recognize and bind to CD47. In some embodiments, the antibodies of the present invention are capable of recognizing and binding to human CD47. The antibodies of the present invention are capable of preventing the interaction between CD47 and SIRPα, and do not cause 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 exhibit a variety of desired properties, such as specifically recognizing human or rhesus macaque CD47 by the methods of non-limiting examples, effectively blocking the interaction between CD47 and its ligand SIRPα, while not causing significant red blood cell hemagglutination, and having 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 1, 2, 3, 4, 5, or 6 of the following VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein:

[0007] (a) VH CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 14, or a variant of SEQ ID NO: 14,

[0008] which has 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 14 sequence;

[0009] (b) VH CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 17, or a variant of SEQ ID NO: 17,

[0010] which has 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 17 sequence;

[0011] (c) VH CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 23, or a variant of SEQ ID NO: 23,

[0012] which has 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 23 sequence;

[0013] (d) VL CDR1 comprises any one of the amino acid sequences as shown in SEQ ID NO: 30-31;

[0014] (e) VL CDR2 comprises any one of the amino acid sequences as shown in SEQ ID NO: 32-33; and

[0015] (f) The VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 34-35.

[0016] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-associated protein (CD47) and comprises 1, 2, 3, 4, 5, or 6 of the following VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein:

[0017] (a) The VH CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO: 14-16;

[0018] (b) The VH CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 17-22;

[0019] (c) The VH CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 23-29;

[0020] (d) The VL CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO: 30-31;

[0021] (e) The VL CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 32-33; and

[0022] (f) The VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 34-35.

[0023] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-associated protein (CD47) and comprises 1, 2, 3, 4, 5, or 6 of the following sequences:

[0024] (a) VH CDR1 as shown in SEQ ID NO: 14, or a variant of SEQ ID NO: 14 that has 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO:

[0025] 14 sequence;

[0026] (b) VH CDR2 as shown in SEQ ID NO: 17, or a variant of SEQ ID NO: 17 that has 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 17 sequence;

[0027] (c) VH CDR3 as shown in SEQ ID NO: 23, or a variant of SEQ ID NO: 23 that has 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO:

[0028] The 23 sequence has 1 to 3 amino acid substitutions as shown in Table 1;

[0029] (d) Any VL CDR1 as shown in SEQ ID NO: 30 - 31;

[0030] (e) Any VL CDR2 as shown in SEQ ID NO: 32 - 33; or

[0031] (f) Any VL CDR3 as shown in SEQ ID NO: 34 - 35.

[0032] In some embodiments, the present invention provides an antibody or antigen - binding fragment that specifically binds to human integrin - associated protein (CD47) and comprises:

[0033] (a) Any VH CDR1 as shown in SEQ ID NO: 14 - 16;

[0034] (b) Any VH CDR2 as shown in SEQ ID NO: 17 - 22;

[0035] (c) Any VH CDR3 as shown in SEQ ID NO: 23 - 29;

[0036] (d) Any VL CDR1 as shown in SEQ ID NO: 30 - 31;

[0037] (e) Any VL CDR2 as shown in SEQ ID NO: 32 - 33; or

[0038] (f) Any VL CDR3 as shown in SEQ ID NO: 34 - 35.

[0039] In some embodiments, the antibody or antigen - binding fragment comprises a heavy - chain variable region VH, and the VH comprises an amino acid sequence selected from any of SEQ ID NO: 11, 36 - 44, 50, and 56, or an amino acid sequence having at least 90% sequence homology with any of the amino acid sequences of SEQ ID NO: 11, 36 - 44, 50, and 56.

[0040] In some embodiments, the antibody or antigen - binding fragment comprises a light - chain variable region VL, and the VL comprises an amino acid sequence selected from any of SEQ ID NO: 12 - 13, 45 - 49, 51 - 55, and 65, or an amino acid sequence having at least 90% sequence homology with any of the amino acid sequences of SEQ ID NO: 12 - 13, 45 - 49, 51 - 55, and 65.

[0041] In some embodiments, the present invention provides an antibody or antigen-binding fragment that specifically binds to human integrin-associated protein (CD47) and comprises VH CDR1 as shown in SEQ ID NO:14, VH CDR2 as shown in SEQ ID NO:17, VH CDR3 as shown in SEQ ID NO:23, VL CDR1 as shown in SEQ ID NO:30, VL CDR2 as shown in SEQ ID NO:32, and VL CDR3 as shown in SEQ ID NO:34.

[0042] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH that comprises one or more amino acid residue mutations according to Kabat numbering selected from the group consisting of:

[0043] (a) R81K, and

[0044] (b) R82aS.

[0045] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH that comprises an amino acid sequence selected from any of SEQ ID NO:11 and 50, or an amino acid sequence having at least 90% sequence homology with an amino acid sequence selected from any of SEQ ID NO:11 and 50.

[0046] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region VL that comprises an amino acid sequence selected from any of SEQ ID NO:13, 45 - 49, 51 - 55, and 65, or an amino acid sequence having at least 90% sequence homology with an amino acid sequence selected from any of SEQ ID NO:13, 45 - 49, 51 - 55, and 65.

[0047] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the VH comprising an amino acid sequence selected from SEQ ID NO:11 or 50, and the VL comprising an amino acid sequence selected from SEQ ID NO:13 or 65.

[0048] In some embodiments, the CD47 antibody herein specifically recognizes and binds to human or cynomolgus CD47, and the antibody comprises a heavy chain variable region having an amino acid sequence selected from any of SEQ ID NO:11, 36 - 44, 50 and / or a light chain variable region having an amino acid sequence selected from any of SEQ ID NO:12, 13, 45 - 49, 51 - 55, 65.

[0049] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain H and a light chain L; the heavy chain H comprises an amino acid sequence selected from any one of SEQ ID NOs: 60 and 62-63, and the light chain L comprises an amino acid sequence selected from any one of SEQ ID NOs: 61 and 64.

[0050] In some embodiments, compared to the level of interaction between CD47 and SIRPα in the absence of the CD47 antibody, the CD47 antibody of the present invention blocks 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% of the interaction between CD47 and SIRPα.

[0051] In some embodiments, the antibody comprises a heavy chain constant region and / or a light chain constant region, the heavy chain constant region comprises an amino acid sequence selected from the sequences shown in SEQ ID NO: 3 or 4, and the light chain constant region comprises an amino acid sequence selected from the sequence shown in SEQ ID NO: 5.

[0052] IgG1 heavy chain constant region sequence (SEQ ID NO: 3):

[0053] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

[0054] SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPEL

[0055] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0056] REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0057] YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0058] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0059] IgG4 heavy chain constant region sequence (SEQ ID NO: 4):

[0060] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0061] SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGP

[0062] SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ

[0063] FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP

[0064] SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT

[0065] VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0066] Light chain constant region sequence (SEQ ID NO: 5)

[0067] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

[0068] QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0069] In some embodiments, the CD47 antibodies of the present invention do not cause significant cell aggregation. For example, the CD47 antibodies of the present invention do not cause significant hemagglutination of red blood cells. In some embodiments, if the aggregation level in the presence of the CD47 antibody of the present invention is decreased 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 aggregation level in the presence of the existing CD47 antibody Hu5F9-G4, it indicates that the CD47 antibody of the present invention does not cause a significant aggregation level. In some embodiments, the CD47 antibodies of the present invention do not cause significant cell aggregation when the antibody concentration is between 400 pM and 800 nM.

[0070] In some embodiments, compared with the antibodies known in the art, the antibodies of the present invention are also significantly effective in tumor models. For example, when the CD47 antibody of the present invention is present, the 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%.

[0071] In some embodiments, the antibody or antigen-binding fragment is an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype. In some embodiments, the antibody or antigen-binding fragment is an IgG isotype selected from IgG4P and IgG4PE. Wherein IgG4P refers to the mutation of the Ser228 site in the IgG4 hinge region to Pro to prevent chain exchange; IgG4PE refers to the mutation of the Ser228 site in the IgG4 hinge region to Pro to prevent chain exchange; and the Leu235 in the constant region is mutated to Glu to alter Fc receptor interaction.

[0072] In some embodiments, the antibody is chimeric, humanized, or fully human. In some embodiments, the antibody binds to human CD47.

[0073] In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention is an isolated CD47 antibody or antigen-binding fragment.

[0074] In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention is a monoclonal antibody or a fragment thereof.

[0075] In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention can be used in the preparation of drugs for treating cancer or infection. In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention can be used in the preparation of drugs for treating solid tumors or hematological tumors. In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention can be used in the preparation of drugs for treating non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid 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 antibody or antigen-binding fragment of the present invention can be used in the preparation of drugs for treating diffuse large B-cell lymphoma or follicular lymphoma.

[0076] In some embodiments, the CD47 antibody described herein can be used to treat, delay the progression of cancer or other tumor diseases, avoid its recurrence, or relieve its symptoms. For example, the CD47 antibody described herein can be used to treat hematological malignancies and / or tumors, such as hematological malignancies and / or tumors. For example, the CD47 antibody described herein can be used to treat CD47 +Tumors. As non-limiting examples, the CD47 antibodies described herein can be used to treat non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid 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, etc. 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.

[0077] As used herein, "blood cancers" include leukemia, lymphoma, and myeloma, etc. "Leukemia" refers to a blood cancer that produces an excessive number of white blood cells that cannot effectively fight infection, thereby crowding out other components that make up the blood, such as platelets and red blood cells. Cases of leukemia can be classified as acute or chronic. As non-limiting examples, leukemia can include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorders / tumors (MPDS), and myelodysplastic syndromes. "Lymphoma" can include Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma, and follicular lymphoma (small cell and large cell), etc. Myeloma can include multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bence-Jones myeloma.

[0078] Exemplary monoclonal antibodies of the invention include, for example, the antibodies described herein. Exemplary antibodies include antibodies having a heavy chain variable region with an amino acid sequence selected from any one of SEQ ID NO: 11, 36-44, 50, and 56 and a light chain variable region with an amino acid sequence selected from any one of SEQ ID NO: 12, 13, 45-49, 51-55, 65. The antibodies also include antibodies having a heavy chain variable region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to at least one of the sequences of SEQ ID NO: 11, 36-44, 50, and 56 and a light chain variable region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to at least one of the sequences of SEQ ID NO: 12, 13, 45-49, 51-55, 65. These antibodies show specificity for human CD47 and are capable of blocking the interaction of CD47 with SIRPα without causing significant hemagglutination of red blood cells.

[0079] In some embodiments, the present invention further provides a biomaterial, which is

[0080] (1) a polynucleotide encoding the antibody or antigen-binding fragment of the present invention; or,

[0081] (2) an expression vector comprising the polynucleotide encoding the antibody or antigen-binding fragment of the present invention; or,

[0082] (3) a cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment of the present invention.

[0083] The pharmaceutical compositions of the present invention may comprise the antibody of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions may be included in a kit, such as a diagnostic kit.

[0084] The present invention also provides a method for alleviating the symptoms of cancer or other tumor disorders by administering to a subject one or more monoclonal antibodies that bind to CD47 or its immunoreactive fragment, wherein the antibody does not cause significant hemagglutination of red blood cells after administration. The dosage of the antibody administered should be sufficient to alleviate the symptoms of cancer or other tumor disorders in the subject. In some embodiments, the subject is a human. In some embodiments, the antibody or its immunoreactive fragment prevents the interaction of CD47 with SIRPα.

[0085] In some embodiments, the CD47 antibody described herein is combined and used in combination with one or more other reagents. Suitable other reagents include existing drugs and / or surgical therapies for specific applications (such as cancer). For example, the CD47 antibody is used in combination with one or more other chemotherapeutic or anti-tumor reagents. Alternatively, the other chemotherapeutic agent is radiotherapy. In some embodiments, the chemotherapeutic agent is a cell death inducer. In some embodiments, the chemotherapeutic agent induces the loss of phospholipid asymmetry across the plasma membrane, for example, causing the 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 the translocation of ER proteins to the cell surface. In some embodiments, the chemotherapeutic agent induces the translocation and cell surface exposure of calreticulin.

[0086] In some embodiments, the CD47 antibody and other reagents are formulated as a single therapeutic composition and the CD47 antibody and other reagents are administered simultaneously. Alternatively, the CD47 antibody and other reagents are independent of each other, for example, formulated as separate therapeutic compositions and administered simultaneously, or the CD47 antibody and other reagents are administered at different times during a treatment regimen. For example, the CD47 antibody is administered before the other reagent, the CD47 antibody is administered after the other reagent, or the CD47 antibody and other reagents are administered in an alternating regimen. Herein, the CD47 antibody and other reagents are administered in a single dose or multiple doses.

[0087] In some embodiments, there is provided the use of the antibody or antigen-binding fragment, composition, biomaterial of the invention in the preparation of a medicament for treating cancer or an infection.

[0088] Those skilled in the art will understand that the antibodies of the invention have various uses. For example, the antibodies of the invention can be used as therapeutic agents, as reagents in diagnostic kits or as diagnostic tools, or as reagents in competitive assays to generate therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] FIG. 1 is a set of graphs depicting the ability of antibody 17-ScFv to bind to cell surface CD47, using CHO-CD47 cells ( Figure 1A ) or Jurkat cells ( Figure 1B ), respectively.

[0090] Figure 2 The ability of antibody 17-ScFv to compete with the ligand SIRPα for binding to CD47 on the surface of CHO was shown by flow cytometry analysis.

[0091] FIG. 3 is a set of graphs, Figures 3A - 3E showing the ability of the CD47 antibody to block the binding of SIRPα to the CD47 antigen as evaluated by competitive ELISA.

[0092] FIG. 4 is a set of graphs, Figures 4A - 4E showing the hemagglutination reaction of red blood cells (RBC) caused by the CD47 antibody. For the appearance, when the red blood cell mass flows in a line, it indicates no hemagglutination reaction; when there is slight aggregation, it is a dot; when the aggregation is obvious, the whole well is blurred. Figure 4A It shows that antibody 11 has obvious aggregation phenomenon, Figure 4B it shows that antibody L12 does not cause hemagglutination reaction, Figure 4C it shows that antibody L12-6 does not cause hemagglutination reaction, Figure 4D it shows that antibodies 17 and Hu5F9-G4 have obvious hemagglutination phenomena, and AB6.12-G1 and SIRPα do not cause hemagglutination reactions, Figure 4EIt is shown that the antibody 6Y-G4 does not cause hemagglutination reaction.

[0093] Figure 5 is a group of figures, Figures 5A - 5B showing the binding of CD47 antibodies to human CD47 or cynomolgus CD47 evaluated by ELISA.

[0094] Figure 6 is a group of figures, Figures 6A - 6B showing the ability of CD47 antibodies to promote the phagocytosis of Raji by RAW264.7 evaluated by flow cytometry.

[0095] Figure 7 is a group of figures, Figures 7A - 7D The effects of the antibody 6Y-G1 on red blood cells and platelets were analyzed in cynomolgus monkeys; Figures 7A - 7B It is shown that the antibody 6Y-G1 causes a decrease in hemoglobin and red blood cells, and gradually recovers approximately 2 weeks later. The degree of reaction is similar to that of the antibodies AB6.12-G1 and Hu5F9-G4; Figure 7C It is shown that the platelets show fluctuating changes after each administration of the antibody 6Y-G1 and recover after the administration ends; Figure 7D It is shown that the number of reticulocytes increases after each administration of the antibody 6Y-G1, and the increase amplitude is greater than that of the antibody AB6.12-G1 and Hu5F9-G4 administration groups.

[0096] Figure 8 The anti-tumor effect of the antibody L12 in the human hematoma MV4-11 system tumor model was shown, using the antibodies AB6.12-G1 and Hu5F9-G4 as positive controls. Mice were treated with a 200 μg antibody dose three times a week.

[0097] Figure 9 The anti-tumor effect of the 6Y-G4 antibody in the Raji tumor model was analyzed; mice were treated with a 100 μg antibody dose three times a week. Detailed implementation mode

[0098] 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. Herein, these antibodies are collectively referred to as CD47 antibodies. Unless otherwise stated herein, the amino acid residues in the antibody variable region and constant region are numbered according to Kabat (EU index in Kabat et al., 1991, Sequences of Proteins of Immunological Interest).

[0099] Some characteristics of the antibodies described herein include: a) specific binding to CD47 (such as human CD47 and rhesus CD47); b) blocking the interaction between CD47 and SIRPα; c) not having significant hemagglutination activity; d) being able to promote the phagocytosis of tumor cells by macrophages; and / or e) showing effective anti-tumor activity in mouse models of human cancers.

[0100] Therefore, the antibodies described herein play an important role in the treatment of various cancers.

[0101] Many existing CD47 antibodies block SIRPα; however, existing SIRPα-blocking antibodies cause unwanted side effects of hemagglutination reactions. Other existing antibodies (such as 2D3) do not cause hemagglutination reactions, but these antibodies also do not block SIRPα, making them unable to effectively promote phagocytosis. Therefore, the agglutination of cells is a major limitation in the therapeutic targeting of CD47 using existing whole IgG antibodies. Therefore, there is an urgent need to obtain CD47 antibodies that can block SIRPα without causing cell agglutination.

[0102] The CD47 antibodies of the present invention avoid unwanted hemagglutination reactions, thereby increasing the effectiveness of therapeutic targeting of CD47 and maintaining the ability to block the interaction between CD47 and SIRPα, thereby promoting the phagocytosis of CD47-expressing cells. In some embodiments, the CD47 antibodies of the present invention (such as antibody L12 and antibody L12-6, etc.) do not agglutinate cells at a significant level. For example, the CD47 antibodies of the present invention do not agglutinate RBCs at a significant level.

[0103] The CD47 antibodies of the present invention bind to human CD47 and block its interaction with SIRPα ( Figures 3A - 3E ). These antibodies do not cause significant hemagglutination of human red blood cells ( Figures 4A - 4E ). These antibodies are able to promote the phagocytosis of tumor cells by macrophages ( Figures 6A - 6B ). In addition, these CD47 antibodies show effective anti-tumor activity in a mouse model of human Raji lymphoma ( Figure 9 ). Therefore, the CD47 antibodies of the present invention overcome a major limiting factor for therapeutic targeting of CD47. Therefore, the CD47 antibodies of the present invention are crucial for the treatment of a large number of cancers.

[0104] The equilibrium dissociation constant (KD) of the antibodies of the present invention binding to the CD47 epitope is less than or equal to 1 μM, such as less than or equal to 100 nM, such as less than or equal to 10 nM, less than or equal to 1 nM. For example, the CD47 antibodies provided herein exhibit a KD value of less than 1 nM.

[0105] The CD47 antibodies of the present invention are used to modulate, block, inhibit, reduce, antagonize, neutralize or interfere with the functional activity of widely distributed CD47. The functional activity of CD47 includes, for example, conducting signals through interaction with SIRPα, regulating (such as increasing) intracellular calcium ion concentration after cell adhesion to the extracellular matrix, interacting with the C-terminal cell-binding domain of thrombospondin, interacting with fibrinogen, and interacting with various integrins. For example, the CD47 antibodies completely or partially inhibit the functional activity of CD47 by partially or completely modulating, blocking, inhibiting, reducing, antagonizing, neutralizing or interfering with the binding of CD47 to SIRPα.

[0106] Definitions

[0107] Unless otherwise defined, the meanings of scientific and technical terms used in the present invention are those commonly understood by those skilled in the art. Generally, the nomenclature and techniques used for cell culture, molecular biology, and protein purification described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (such as electroporation, lipid transfection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or methods commonly used in the art or described herein. The foregoing techniques and methods are generally used as described in many comprehensive and more specific documents well known in the art and cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).

[0108] As used in this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings: The terms "erythrocyte" and "red blood cell" are synonyms and are used interchangeably.

[0109] The term "agglutination" refers to cell clumping, while the term "hemagglutination" refers to the clumping of a specific type of cell, namely erythrocytes. Thus, hemagglutination is a type of agglutination. In the hemagglutination assays described herein, the erythrocytes form in a given well in a form that can be a "button", a "halo", or an intermediate between the two. The term "a line" refers to a linear flow of the erythrocyte mass when the 96-well plate is tilted at 45°. The term "significant hemagglutination activity" refers to the presence of any halo form in the well upon addition of the antibodies described herein.

[0110] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" or "is directed against" means that an antibody reacts with one or more antigenic determinants of a target antigen and does not react with other polypeptides, or binds to other polypeptides with very low affinity (KD > 10 -6 μg / ml). Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single-chain antibodies, Fabs, Fab- and F(ab’)2 fragments, Fvs, and Fab expression libraries.

[0111] It is known that the basic antibody structural unit comprises a tetramer. Each tetramer (or "whole antibody") consists of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, which is mainly responsible for effector functions. Generally speaking, antibody molecules obtained from humans involve any of IgG, IgM, IgA, IgE, and IgD, which are different from each other due to the nature of the heavy chains present in the molecules. Some classes also have subclasses, such as IgG1, IgG2, and others. In addition, in humans, the light chain can be a κ chain or a λ chain.

[0112] As used herein, the term "monoclonal antibody" (mAb) refers to a group of antibody molecules that contain only one molecular species of the antibody molecules composed of a unique light-chain gene product and a unique heavy-chain gene product. Specifically, the complementarity-determining regions (CDRs) of monoclonal antibodies are the same in all molecules of the group. MAbs contain antigen-binding sites capable of immunoreacting with specific epitopes of an antigen.

[0113] The term "single-chain antibody" (scFv) refers to an antibody formed by linking the variable region of the antibody heavy chain (VH) and the variable region of the light chain (VL) by a linker of 15 - 20 amino acids.

[0114] The term "antigen-binding site" or "binding portion" refers to the part of an immunoglobulin molecule that participates in antigen binding. This antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent segments (termed "hypervariable regions") in the heavy-chain variable region (VH) and the light-chain variable region (VL) are located between more conserved segments (termed "framework regions" or "FRs"). Thus, the term "FR" denotes the amino acid sequences in an immunoglobulin that, in the native state, are present between or adjacent to 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 relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are termed "complementary determining regions" or "CDRs". Alignment of the amino acids of each domain can be in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health (1987 and 1991)) or the articles by Chothia and Lesk (J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989)).

[0115] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or its fragment or a T cell receptor. Epitope determinants generally consist of the chemically active surface groups of a molecule (such as amino acids or sugar side chains) and generally have specific three-dimensional structural properties as well as specific charge properties.

[0116] As used herein, the term "specifically binds" refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen specific for the immunoglobulin. The strength or affinity of an immunological binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "association rate constant" (k on ) and the "dissociation rate constant" (k off ) can be determined by calculating concentrations and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). k off / k onThe ratio can eliminate 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 radio-ligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those of skill in the art. The antibodies of the present disclosure specifically bind to CD47 when the equilibrium dissociation constant (KD) ≤ 1 μM (e.g., less than or equal to 100 nM, less than or equal to 10 nM, and yet again less than or equal to 1 nM).

[0117] An “isolated” antibody is one that has been separated and / or recovered from the components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to the extent that: (1) the antibody is greater than 95% by weight, such as greater than 99%, as determined by the Lowry method; (2) at least 15 residues of the N-terminal or internal amino acid sequence can be obtained by use of a spinning cup sequenator; or (3) homogeneity is demonstrated by SDS-PAGE under reducing or non-reducing conditions and visualized by Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells. Ordinarily, an isolated antibody will be prepared by at least one or more purification steps. In some embodiments, the isolated antibody has a purity of 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.

[0118] As used herein, the term “polypeptide” is a generic term that refers to native proteins, fragments, or analogs of polypeptide sequences, and thus native protein fragments and analogs are a species within the genus polypeptide.

[0119] The terms “sequence identity” or “sequence homology” refer to the situation where two polynucleotide or amino acid sequences are identical (i.e., nucleotide-by-nucleotide or residue-by-residue identical) in a comparison window. The term “percent sequence identity” is calculated by: comparing two optimally aligned sequences in a comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in both sequences to yield 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 yield the percent sequence identity.

[0120] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - ASynthesis (2nd Edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Mass. (1991)). Stereoisomers of the twenty conventional amino acids (e.g., D - amino acids), unnatural amino acids (such as α - , α - disubstituted amino acids), N - alkyl amino acids, lactic acid, and other non - conventional amino acids may also be components applicable to the polypeptides of the present disclosure. Examples of non - conventional 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 (such as 4 - hydroxyproline). In the polypeptide representation used herein, the left - hand direction is the amino - terminal direction, and the right - hand direction is the carboxy - terminal direction, consistent with standard usage and convention. Conventional (or natural) 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).

[0121] Similarly, 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 called the transcription direction; the sequence region on the DNA strand that is identical to the RNA sequence and is 5' of the 5' end of the RNA transcript is called the "upstream sequence"; the sequence region on the DNA strand that is identical to the RNA sequence and is 3' of the 3' end of the RNA transcript is called the "downstream sequence". When applied to polypeptides, the term "substantially 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, for example, the GAP or BESTFIT programs with default gap weights.

[0122] In some embodiments, the positions of non - identical residues differ by conservative amino acid substitutions.

[0123] Minor variations 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 at least 75%, such as at least 80%, 90%, 95% and again 99%. In some embodiments, the variation is a conservative amino acid substitution. Conservative amino acid substitutions are substitutions that occur within families of related amino acids in their side chains. The amino acids encoded by genes are generally divided into the following classes: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other families of amino acids include (i) serine and threonine of the aliphatic-hydroxy family; (ii) asparagine and glutamine of the amide-containing family; (iii) alanine, valine, leucine and isoleucine of the aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine of the aromatic family. In some embodiments, the groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. For example, it can be reasonably predicted that substitution of leucine with isoleucine or valine alone, substitution of aspartate with glutamate, substitution of threonine with serine, or substitution of one amino acid with a structurally related amino acid analog will not have a significant effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the 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. Such assays are described in detail herein. Fragments or analogs of an antibody or immunoglobulin molecule can be readily prepared by those of ordinary skill in the art.

[0124] In some embodiments, the amino acid substitutions have the following effects: (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. The analogs can include various mutant proteins with sequences different from the 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 polypeptide portion outside the domain forming intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural properties of the parental sequence (e.g., the substituted amino acid should not tend to disrupt the helical structure present in the parental sequence or disrupt other types of secondary structure characteristic of the parental sequence). Examples of the secondary and tertiary structures of polypeptides identified manually 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, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0125] As used herein, the term "reagent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from a biological material.

[0126] As used herein, the term "labeled" or "label" refers to the incorporation of a detectable label, e.g., by incorporating a radiolabeled amino acid, or a polypeptide attached to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or an enzyme activity detectable by optical or calorimetric methods). In some cases, the label or the labeling can also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (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, the labels are linked by spacer arms of various lengths to reduce possible steric hindrance.

[0127] The term "agent" or "drug" refers to a compound or composition that, when appropriately administered to a patient, is capable of inducing a desired therapeutic effect.

[0128] As used herein, the term "antineoplastic agent" refers to an agent having functional properties that inhibit the development or progression of tumors in a human body, particularly malignant (cancerous) lesions such as carcinomas, sarcomas, lymphomas, or leukemias. Inhibition of metastasis is, in many cases, a property of antineoplastic agents.

[0129] Other chemical terms herein are used according to their conventional usage in the art, such as in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., McGraw-Hill, San Francisco (1985)).

[0130] CD47 antibody

[0131] The antibodies of the present invention have the ability to bind to CD47, inhibit the binding of SIRPα to CD47, reduce CD47-SIRPα-mediated signal transduction, 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 herein.

[0132] Exemplary antibodies of the present invention include antibody 17, antibody L12, antibody 6Y-G1, antibody 6Y-G4, etc., and other similar antibodies having the same or similar CDR regions.

[0133] The VH CDRs of antibody 17 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:31 (VL CDR1), SEQ ID NO:33 (VL CDR2), and SEQ ID NO:35 (VL CDR3). VH CDR is the heavy chain hypervariable region, and VL CDR is the light chain hypervariable region.

[0134] In some embodiments, based on antibody 17, one or more amino acid sites in its VH CDR1, VH CDR2, and / or VH CDR3 are substituted. In some embodiments, the substitutable sites (parents) and the amino acids that can be used for substitution in VH CDR1, VH CDR2, and / or VH CDR3 are one or more of those shown in Table 1. "Position" in Table 1 represents the amino acid site counted 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" counted from left to right in the amino acid sequence shown in SEQ ID NO: 11.

[0135] Table 1: Amino Acid Substitutions in Heavy Chain CDR

[0136]

[0137]

[0138] The VH CDRs of antibody L12 and antibody L12-6 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: 30 (VL CDR1), SEQ ID NO: 32 (VL CDR2), and SEQ ID NO: 34 (VL CDR3).

[0139] Verified by experiments, the VH CDR1 sequence can also be SEQ ID NO: 15 or 16. The VH CDR2 sequence can also be SEQ ID NO: 18, 19, 20, 21, or 22. The VH CDR3 sequence can also be SEQ ID NO: 24, 25, 26, 27, 28, or 29. Other CDR sequences can be achieved by the amino acid substitutions shown in Table 1. In some embodiments, 1, 2, or 3 amino acids are substituted.

[0140] In addition to the changes in the CDRs, the 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 after two mutations (R81K and R82aS) in the framework region of the heavy chain relative to antibody L12 are also suitable heavy chain variable regions. In some embodiments, the heavy chain variable region of the CD47 antibody can be K at position 81 or S at position 82a (according to Kabat numbering). Other framework region mutations are also reflected in different antibody sequences. Therefore, the present invention provides a heavy chain framework region with R81K and / or R82aS (according to Kabat numbering) mutations.

[0141] Exemplary antibodies of the invention include antibodies comprising a heavy chain variable region having a sequence selected from SEQ ID NO: 11, 36 - 44, 50 or 56 and a light chain variable region having a sequence selected from SEQ ID NO: 12 - 13, 45 - 49, 51 - 55, 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 also antibodies having a suitable sequence identity with the sequences of the above - mentioned antibodies. For example, 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, at least the CDRs of these identical sequences do not change.

[0142] In some embodiments, human framework adaptation is performed on antibody L12. In some embodiments, the framework mutations of the VH of antibody L12 are the underlined amino acids in L12 - 1 - VH, L12 - 2 - VH, L12 - 3 - VH, L12 - 6 - 1 - VH, L12 - 6 - VH, L12 - 8 - VH or L12 - 9 - VH in Table 3. In some embodiments, the framework mutations of the VL of antibody L12 are the underlined amino acids in 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 in Table 3.

[0143] The invention also includes antibodies that bind to the same epitope as the CD47 antibodies described herein. For example, the antibodies of the invention specifically bind to an epitope comprising one or more amino acid residues on human CD47 (see, e.g., GenBank accession number Q08722.1).

[0144] An exemplary ECD amino acid sequence of human CD47, SEQ ID NO: 1 (GenBank accession number Q08722.1 (GI: 1171879), which is incorporated herein by reference), is provided below. The signal sequence (amino acids 1 - 18) is underlined.

[0145] SEQ ID NO:1

[0146] MWPLVAALLLGSACCGSA QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVK

[0147] WKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTC

[0148] EVTELTREGETIIELKYRVVSWFSP

[0149] 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 evaluated by methods including measuring CD47- and / or CD47 / SIRPα-mediated signaling in the presence of one or more of the antibodies described herein. These assays can include competitive binding assays or can measure biological indicator readings, such as the ability to promote phagocytosis of cells expressing CD47 by macrophages (as described in Example 3).

[0150] The antibodies described herein can be generated using a variety of 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, N.Y., which is 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 by using the trioma technique, human B cell hybridoma techniques (see Kozbor et al., 1983 Immunol Today 4:72), and EBV hybridoma techniques to produce human monoclonal antibodies (see Cole et al., 1985. In: Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein Barr Virus in vitro (see Cole et al., 1985. In: Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0151] Antibodies can be purified by well-known techniques, such as affinity chromatography using Protein A or Protein G, which mainly provides the IgG fraction in immune sera. Additionally, the specific antigen or its epitope to which the immunoglobulin is targeted can be immobilized on a column to purify immunospecific antibodies by immunoaffinity chromatography. The purification of immunoglobulins can be referred to the article by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0152] The CD47 antibodies of the present disclosure can be monoclonal antibodies. Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise impede CD47- and / or CD47 / SIRPα-mediated cell signaling are produced, for example, by immunizing an animal with a membrane-bound and / or soluble CD47, such as human CD47 or an immunogenic fragment, derivative, or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector comprising a nucleic acid molecule encoding CD47, whereby CD47 is expressed and associated with the surface of the transfected cells. Alternatively, antibodies are obtained by screening a library comprising antibody or antigen-binding domain sequences for binding to CD47. The library is prepared as follows: in phage, a fusion of a protein or peptide fused to a phage coat protein is expressed on the surface of assembled phage particles, and the encoded DNA sequence is integrated within the phage particles (i.e., a "phage display library"). The library is then screened by binding reaction with CD47.

[0153] Monoclonal antibodies are prepared using, for example, the hybridoma method, such as those 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 the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0154] The immunizing agent typically comprises 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 cells are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line typically comprises a rat or mouse myeloma cell line. The hybridoma cells can be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the medium for the hybridomas typically comprises hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0155] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described herein can be isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of murine antibodies). The hybridoma cells described herein serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, simian COS cells, PER.C6 cells, SP2 / 0, YB2 / 0, or myeloma cells that do not otherwise produce immunoglobulins, thereby obtaining synthetic monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by replacing the homologous murine sequences with the coding sequences of the human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking the immunoglobulin coding sequences to all or part of the coding sequences of non-immunoglobulin polypeptides. Such non-immunoglobulin polypeptides can replace the constant domains of the antibodies described herein or can replace the variable domains of one of the antigen-binding sites of the antibodies described herein to produce chimeric bivalent antibodies.

[0156] Cell lines that produce antibodies can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017, the entire content of which including supplementary content is incorporated herein by reference in its entirety.

[0157] In some embodiments, DNA encoding an antibody can be designed and synthesized according to the antibody amino acid sequences described herein by conventional methods, inserted into an expression vector, and then transfected into a host cell. The transfected host cell is cultured in a medium to produce a monoclonal antibody. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. High-efficiency transcription can be achieved through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, or other cell promoters such as the actin promoter. Suitable expression vectors can 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, murine L cells, and CHO cells, etc.

[0158] In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and other selection genes to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into a host cell without the above genes and cultured in a selective medium. The successfully transfected cells grow in large numbers to produce the desired target protein.

[0159] In some embodiments, the DNA sequence encoding the CD47 antibody of the present invention can be obtained by combining the amino acid sequence of the antibody with conventional methods in the art. In some embodiments, the DNA sequence encoding the heavy chain of antibody 6Y-G4 is as shown in SEQ ID NO:57, wherein the underlined part encodes VH CDR; the amino acid sequence of the heavy chain of the encoded antibody 6Y-G4 is as shown in SEQ ID NO:60.

[0160] SEQ ID NO:57 is as follows:

[0161] caggtgcagctgcaggagtccggccctggcctggtgaagccttccgagaccctgtccctgacctgtaccgtgagcggcggcagcct

[0162] g gataactattactggagctggatccggcagcctcctggcaagggcctggagtggatcggc tacatct actattccggcaacaccaatt

[0163] acaacccttccctgaagagc cgggtgaccatctccgtggacaccagcaagaaccagtttagcctgaagctgtcctccgtgaccgccgc

[0164] tgataccgccgtgtactactgtgccagg ggcggccggttcctggagagatat tggggccagggtaccctcgtgaccgtgtccagcgct

[0165] agcaccaagggcccttccgtgttccccctggccccctgtagccggtccacctctgagagcaccgctgctctgggctgtctggtgaagg

[0166] attactttcccgaaccggtgaccgtgtcatggaactccggggctctgacatccggtgtccacacttttcctgcagtgctgcagtcatccgg

[0167] cctgtacagcctgagctctgtggtcacagtcccaagttcatccctgggaaccaagacatatacttgcaacgtggatcataaacccagca

[0168] atactaaggtcgacaaacgagtggagtctaagtacggaccaccttgcccaccatgtccagcacctgagttcctgggaggaccaagcgt

[0169] gttcctgtttcctccaaagcctaaagataccctgatgatcagtcggactcccgaggtcacctgcgtggtcgtggacgtgtcccaggagg

[0170] accctgaagtccagttcaactggtacgtggacggcgtcgaagtgcacaatgctaagacaaaacctcgagaggaacagtttaactccac

[0171] ataccgtgtcgtgagcgtcctgactgtgctgcatcaggattggctgaacggcaaggagtataagtgcaaagtgagcaataagggactg

[0172] ccaagctctatcgagaaaactatttctaaggctaaaggacagcctagggaaccacaggtgtacaccctgccccctagtcaggaggaaa

[0173] tgactaagaaccaggtctcactgacctgtctggtgaaagggttctatccttcagatattgcagtggagtgggaatccaatggtcagccag

[0174] agaacaattacaagacaactccacccgtgctggacagcgatgggtctttctttctgtattctagactgaccgtggacaaaagtcgctggc

[0175] aggagggtaatgtcttttcttgtagtgtgatgcacgaagccctgcacaaccactacactcagaaaagcctgtcactgtccctgggtaaaSEQ ID NO:60:

[0176] QVQLQESGPGLVKPSETLSLTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNT

[0177] NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGRFLERYWGQGTLVTVS

[0178] SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

[0179] SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

[0180] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE

[0181] QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL

[0182] PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL

[0183] TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0184] In some embodiments, the DNA sequence encoding the light chain of antibody 6Y-G4 is as shown in SEQ ID NO:58, wherein the underlined part encodes VL CDR; the amino acid sequence of the encoded light chain of antibody 6Y-G4 is as shown in SEQ ID NO:61.

[0185] SEQ ID NO:58 is as follows:

[0186] gagatcgtgctgacccagtccccctccagcctgagcgccagcgtgggagaccgggtgaccatcccctgc cgggcttccctgtccatc

[0187] ggcagcttcctgaac tggtatcagcagaggcctggcgaggcccctaagctgctgatcttt gccgcttc cagcctgcggagc ggcgtgc

[0188] ctagcaggttctccggcagcggctccggcaccgatttcaccctgaccatcagcggcctgcagcccgaggatttcgccacctactactg

[0189] c cagcagacctacaccaccccttacacc tttggccagggcaccaaggtggacatcaagcgtacggtggctgcaccatctgtcttcatct

[0190] tcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtg

[0191] gaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagc

[0192] agcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgt

[0193] cacaaagagcttcaacaggggagagtgttga

[0194] SEQ ID NO:61:

[0195] EIVLTQSPSSLSASVGDRVTIPCRASLSIGSFLNWYQQRPGEAPKLLIFAASSLRSGVPSR

[0196] FSGSGSGTDFTLTISGLQPEDFATYYCQQTYTTPYTFGQGTKVDIKRTVAAPSVFIFPPS

[0197] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0198] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0199] In some embodiments, the amino acid sequence of the heavy chain of antibody 6Y-G1 is as shown in SEQ ID NO:62.

[0200] SEQ ID NO:62:

[0201] QVQLQESGPGLVKPSETLSLTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNT

[0202] NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGRFLERYWGQGTLVTVS

[0203] SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

[0204] QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

[0205] LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0206] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0207] VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[0208] YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0209] In some embodiments, the amino acid sequence of the light chain of antibody 6Y-G1 is as shown in SEQ ID NO:61.

[0210] In some embodiments, the amino acid sequence of the heavy chain of antibody 17 is as shown in SEQ ID NO:63.

[0211] SEQ ID NO:63:

[0212] QVQLQESGPGLVKPSETLSLTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNT

[0213] NYNPSLKSRVTISVDTSKNQFSLRLRSVTAADTAVYYCARGGRFLERYWGQGTLVTV

[0214] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

[0215] QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

[0216] LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0217] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0218] VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[0219] YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0220] In some embodiments, the amino acid sequence of the light chain of Antibody 17 is as set forth in SEQ ID NO:64.

[0221] SEQ ID NO:64:

[0222] DIQMTQSPSSVSASVGDRVTISCRANQAIGTWLAWYQQKPGKAPKLLIYAASTLQSGV

[0223] PSRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYTTPLTFGGGTKLEIKRTVAAPSVFIFP

[0224] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0225] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0226] In some embodiments, the amino acid sequence of the heavy chain of Antibody L12 is as set forth in SEQ ID NO:63. In some embodiments, the amino acid sequence of the light chain of Antibody L12 is as set forth in SEQ ID NO:61.

[0227] Human antibodies and humanized antibodies

[0228] The antibodies described herein include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without causing an immune response in humans to the administered immunoglobulin.

[0229] CD47 antibodies are produced, for example, by phage display methods using antibodies consisting only of human sequences. Such methods are well known in the art, for example WO92 / 01047 and U.S. Patent No. 6,521,404. In such methods, a natural or recombinant CD47 source or a fragment thereof is used to screen a combinatorial library of phages carrying random pairs of light and heavy chains. In another method, CD47 antibodies can be produced by a method in which at least one method step comprises immunizing a transgenic non-human animal with human CD47 protein. In such methods, the endogenous heavy chain and / or k light chain loci in some transgenic non-human animals have been inactivated and are unable to undergo the rearrangements required to produce genes encoding immunoglobulins in response to an antigen. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Thus, in response to the administered antigen, the human loci rearrange to provide genes encoding human variable regions that are immunologically specific for the antigen. Thus, upon immunization, the transgenic mouse produces B cells that secrete fully human immunoglobulins.

[0230] The production of antibodies with reduced immunogenicity is also achieved using appropriate libraries via humanization techniques, chimerization techniques, and display techniques. It should be understood that murine antibodies or antibodies 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. 12:125 - 168 (1992). Antibodies of interest can be engineered by recombinant DNA techniques to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequences (see WO92102190 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 for constructing chimeric immunoglobulin genes is also known in the art (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from antibody - producing hybridomas or other cells and used to generate cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Patent 4,683,195 and 4,683,202). Alternatively, libraries are constructed and screened to isolate the sequences of interest. Then the DNA sequences encoding the variable regions of the antibody are fused to human constant region sequences. The sequences of human constant region genes can be found in Sequences of Proteins of immunological Interest published by Kabat et al. (N.I.H. publication no. 91 - 3242 (1991)). The human C - region genes can be readily obtained from known clones. The choice of isotype will be guided by the desired effector function, such as complement binding or antibody - dependent cell cytotoxicity activity. Preferred isotypes are IgG1 and IgG2. Either human light - chain constant region, i.e., k or λ, can be used, and then the chimeric humanized antibody is expressed by conventional methods.

[0231] In some embodiments, antibody fragments such as Fv, F(ab’)2, and Fab can be prepared by cleaving the intact protein, for example, by proteases or chemical cleavage. Including but not limited to: (i) digesting the antibody molecule with pepsin to obtain F(ab’)2 fragments; (ii) obtaining Fab fragments by reducing the disulfide bonds of F(ab’)2 fragments; (iii) treating the antibody molecule with papain and a reducing agent to generate Fab fragments, and (iv) Fv fragments.

[0232] The consensus sequence J regions of the heavy and light chains can be used to design oligonucleotides to serve as primers to introduce useful restriction sites within the J regions for subsequent ligation of V region fragments to human C region fragments. The C region cDNA can be modified by site-directed mutagenesis to place a restriction site at a similar position in the human sequence.

[0233] The CD47 antibodies described herein can be expressed by a vector comprising a DNA fragment encoding the above antibodies. Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. A suitable vector is generally one that encodes a functionally intact human heavy chain constant region (CH) or light chain constant region (CL) immunoglobulin sequence and bears appropriate restriction sites such that any VH or VL sequence can be readily inserted and expressed. In such vectors, splicing generally occurs between the splice donor site in the inserted J region and the splice acceptor site in front of the human C region and also at splice regions present within the human CH exons. Polyadenylation and transcriptional termination occur at the natural chromosomal site downstream of the coding region. The resulting chimeric antibody can be linked to any strong promoter, including the retroviral LTR, such as the SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), the Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and the Moloney murine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Additionally, natural Ig promoters, etc., can be used.

[0234] Fc modification

[0235] The effector functions associated with the antibodies described herein can be modified to enhance, for example, the effectiveness of the antibodies in treating diseases and disorders associated with abnormal CD47 signaling. For example, one or more mutations can be introduced into the Fc region of the antibody to silence effector functions, thereby reducing the likelihood of killing normal cells.

[0236] 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 and has the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the amino acid Asn297 (Kabat numbering) on the human IgG1 constant region is modified to avoid glycosylation of the antibody, such as Asn297Ala (N297A). In some embodiments, the amino acid Leu235 (Kabat numbering) on the antibody constant region is modified to alter Fc receptor interaction, such as Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the amino acid Leu234 (Kabat numbering) on the antibody constant region is modified to alter Fc receptor interaction, such as Leu234Ala (L234A). In some embodiments, the amino acids 234 and 235 on the antibody constant region are modified simultaneously, such as Leu234Ala and Leu235Ala (L234A / L235A) (EU index in Kabat et al., 1991, Sequences of Proteins of Immunological Interest).

[0237] In some embodiments, the constant region of the antibody is of the human IgG4 isotype and has the amino acid sequence shown in SEQ ID NO:4.

[0238] In some embodiments, the hinge region within the human IgG4 constant region is modified to avoid or reduce chain exchange, such as Ser228Pro (S228P). In other embodiments, the amino acid 235 on the human IgG4 constant region is modified to alter Fc receptor interaction, such as Leu235Glu (L235E). In some embodiments, the hinge region within the human IgG4 constant region and the amino acid 235 are modified, such as Ser228Pro and Leu235Glu (S228P / L235E). In some embodiments, the amino acid Asn297 (boxed, Kabat numbering) on the human IgG4 constant region is modified to avoid glycosylation of the antibody, such as Asn297Ala (N297A). In some embodiments, the amino acids Ser228, Leu235, and Asn297 on the human IgG4 constant region are modified (such as S228P / L235E / N297A).

[0239] Use of the antibody against CD47

[0240] In some embodiments, the CD47 antibodies of the invention can be used for diagnosing, prognosticating, monitoring, treating, alleviating, and / or preventing diseases or disorders associated with abnormal CD47 expression, activity, and / or signal transduction in a subject. A treatment regimen can be implemented by identifying, using standard methods, a subject (such as a human patient) having a disease or disorder associated with abnormal CD47 expression, activity, and / or signal transduction (such as cancer or other neoplastic disorders), or at risk of developing the disease. In some embodiments, provided are methods of treating a disease or disorder involving abnormal CD47 expression, activity, and / or signal transduction, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or a formulation thereof described herein. Administration of the antibody can eliminate or inhibit or interfere with the expression, activity, and / or signal transduction function of a target (such as CD47). Administration of the antibody can eliminate or inhibit or interfere with the binding of a target (such as CD47) to its endogenous ligand (such as SIRPα) that binds thereto in its native state. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or interferes with the expression, activity, and / or signal transduction of CD47.

[0241] In some embodiments, diseases or disorders involving abnormal CD47 expression, activity, and / or signal transduction include hematologic cancers and / or solid tumors. Hematologic cancers include, for example, leukemia, lymphoma, and myeloma. In some embodiments, certain forms of leukemia include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); myeloproliferative diseases / neoplasms (MPDS); and myelodysplastic syndromes. In some embodiments, certain forms of lymphoma include Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma, and follicular lymphoma (small and large cell). In some embodiments, certain forms of myeloma include multiple myeloma (MM), gigantomyeloma, 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.

[0242] Symptoms associated with cancer and other neoplastic disorders include, for example, inflammation, fever, malaise, pyrexia, pain, often localized to the inflamed area, anorexia, weight loss, edema, headache, fatigue, rash, anemia, myasthenia, muscle fatigue, and abdominal symptoms (such as abdominal pain, diarrhea, or constipation).

[0243] The therapeutically effective amount of the antibody of the present invention generally refers to the amount required to achieve the therapeutic goal. The amount required for administration depends on the binding affinity of the antibody for its specific antigen, the severity of the disease, disorder or condition, the route of administration, the rate at which the antibody administered to the subject depletes from the free volume, etc. In some embodiments, the common range of the therapeutically effective dose of the antibody or antibody fragment of the present invention is from about 0.1 mg / kg to about 100 mg / kg. In some embodiments, the antibody of the present invention is administered to the subject at a dose in the range 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 higher or in the range between any two of these values (including the endpoints). The common range of dose frequencies is, for example, once a day to once a week.

[0244] In other embodiments, antibodies against CD47 can be used in methods known in the art related to the localization and / or quantification of CD47 (e.g., for determining the levels of CD47 and / or both CD47 and SIRPα in a suitable biological sample, for diagnostic methods, for protein imaging, etc.). In a given embodiment, an antibody specific for CD47 or its derivatives, fragments, analogs or homologs, comprising an antigen-binding domain derived from an antibody, is used as a pharmaceutically active compound (hereinafter referred to as a "therapeutic agent").

[0245] In other embodiments, CD47 polypeptides can be isolated using an antibody specific for CD47 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 a biological sample. In some embodiments, CD47 can be detected in a biological sample as part of a clinical testing process, e.g., for determining the efficacy of a given treatment regimen. Conjugating (i.e., physically linking) the antibody to a detectable substance can facilitate detection. Examples of detectable substances include various enzymes, cofactors, 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 cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, fluorescein and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S or3 H.

[0246] 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. The intact antibody or its fragments (e.g., Fab, scFv or F(ab')2) are used. The detection methods of the described embodiments can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for analyte genomic DNA include Southern hybridization. (ELISA: Theory and Practice: Methods in Molecular Biology, Volume 42, J.R. Crowther Human Press, Totowa, N.J., 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 addition, in vivo detection techniques for analyte protein include introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody can be labeled with a radioactive label, and then the presence and location of the radioactive label in the subject can be detected by standard imaging techniques.

[0247] Therapeutic Administration and Formulations of CD47 Antibodies

[0248] The antibodies and their derivatives, fragments, analogs, and homologs described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art. See, for example, Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy, 19th Edition, 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, Volume 4, 1991, M. Dekker, New York.

[0249] Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. In some embodiments, an antibody fragment is a minimal inhibitory fragment that specifically binds to the target protein-binding domain. For example, a peptide based on the variable region sequence of an antibody and retaining the ability to bind to the target protein sequence.

[0250] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, stabilizers, buffers, dispersion media, coatings, antibacterial agents, isotonic agents, absorption delaying agents, and the like that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences. Such carriers or diluents can optionally include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.

[0251] Preparations to be used for in vivo administration must be sterile. This can be easily accomplished by filtration through sterile filtration membranes.

[0252] The pharmaceutical compositions of the described embodiments are generally compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. The pharmaceutical composition may include one or more of the following components: a sterile diluent for injection, such as water, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; bacteriostatic agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffering agents, such as histidine hydrochloride, acetate, citrate, or phosphate; osmotic pressure regulators, such as sodium chloride or dextrose; stabilizers, such as arginine, methionine, trehalose, sucrose, sorbitol; surfactants, such as Tween 20, Tween 80. The pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. The pharmaceutical composition can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water-soluble herein) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. When used, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be preserved against 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.

[0253] For transmucosal or transdermal administration, penetration enhancers suitable for the permeation barrier are used in the formulation. Such penetration enhancers are generally well known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the antibodies can be formulated as ointments, creams, gels, or lotions as is generally known in the art.

[0254] The pharmaceutical compositions are formulated in dosage unit form for ease of administration and uniform dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined quantity of one or more of the antibodies calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0255] The pharmaceutical composition can be placed in a container or dispenser and packaged together with instructions for administration.

[0256] The pharmaceutical compositions described herein may also contain other active ingredients, preferably those with complementary activities and no negative impact on each other, depending on the specific condition to be treated. In some embodiments, the composition may contain reagents that enhance its function, such as cytotoxin reagents, cytokines, chemotherapeutic agents, or growth inhibitors. Such active ingredients are present in appropriate combination in an amount effective for the intended purpose.

[0257] In one embodiment, one or more active ingredients including a CD47 antibody may be administered in combination therapy, i.e., in combination with other reagents such as therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressive agents, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxin or cell growth inhibitors, etc.). The term "combination" as used herein refers to administering the reagents substantially synchronously, simultaneously, or sequentially. In some embodiments, such combination therapy may advantageously utilize lower doses of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various single therapies.

[0258] In some embodiments, the antibodies described herein are used as vaccine adjuvants against autoimmune disorders, inflammatory diseases, etc. The vaccine can be a variety of antigens. The antigens are derived from the targeted autoantigens, i.e., the autoantigens involved in autoimmunity, such as myelin basic protein; inflammatory autoantigens, such as amyloid peptide protein, or transplantation antigens, such as alloantigens. The antigen may include peptides or polypeptides derived from proteins and fragments of any of the following: sugars, proteins, polynucleotides or oligonucleotides, autoantigens, amyloid peptide proteins, transplantation antigens, allergens, or other macromolecular components. In some embodiments, more than one antigen is included in the antigenic composition.

[0259] The citations of publications and patent documents herein do not represent an admission that any of the above is relevant prior art, nor an admission of its content or date. Now that the present invention has been described in a written specification, those skilled in the art should recognize that the present invention can be practiced in various embodiments, and the above specification and the following examples are intended to illustrate rather than limit the claims of the present invention. The entire content of all publications, patents, and patent applications cited herein is incorporated herein by reference for all purposes.

[0260] Examples

[0261] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources or by known methods. In the following examples, antibody L12-6 and antibody 6Y-G1 are the same antibody.

[0262] Procedure for constructing CHO-CD47 cells: Design the HindIII / EcoRI window to introduce the polynucleic acid encoding CD47 (sequence see Uniprot, Q08722) into the plasmid pcDNA3.1(+) (Invitrogen, V790-20), and transfect CHO cells (ATCC#CCL-61) by single digestion with PvuI, and screen to obtain a stable CHO-CD47 cell line.

[0263] Jurkat cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, with the catalog number Clone E6-1.

[0264] The IgG antibody was purchased from BioXCell, with the catalog number BE0297.

[0265] Rhesus CD47-Fc was purchased from CreativeBioMart, with the model number CD47-745C.

[0266] Example 1: Preparation of CD47 antigen

[0267] Preparation of CD47 antigen: Add a 6×HIS tag (HHHHHH, SEQ ID NO:2) or human Fc (the underlined part in SEQ ID NO:3) (refer to R&D Systems) to the C-terminus of the extracellular domain (ECD) protein (SEQ ID NO:1) of human CD47 to construct a CD47 recombinant protein. The ECD region of human CD47 was gene-synthesized with 6×HIS 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) purified by nickel-based immobilized metal affinity chromatography and secreted Fc-tagged human CD47-ECD protein (CD47-Fc) purified via immobilized metal affinity chromatography (IMAC) using a protein A column (GE Healthcare) were obtained.

[0268] Example 2: Preparation of CD47 antibody

[0269] The combinations of VH and VL of 30 single-chain antibodies (scFv) are shown in Table 2, and are linked by a linker peptide with the sequence shown in SEQ ID NO: 10 (GGGGSGGGGSGGGGS) at the C-terminus of VH and the N-terminus of VL; the heavy-chain variable region of the antibody is represented by the antibody number + "-VH", such as 17-VH; the light-chain variable region of the antibody is represented by the antibody number + "-VL", such as 17-VL; the single-chain antibody (scFv) is represented by the antibody number + "-ScFv", such as 17-ScFv (linked by the linker peptide shown in SEQ ID NO: 10 at the C-terminus of 17-VH (shown in SED ID NO: 11) and the N-terminus of 17-VL (shown in SED ID NO: 12) to obtain 17-ScFv).

[0270] The combinations of VH and VL of 30 IgG1 full antibodies are shown in Table 2; VH and CH form the heavy chain of the antibody, and VL and CL form the light chain of the antibody; the sequence of CH is shown in SED ID NO: 3, and the sequence of CL is shown in SED ID NO: 5; the heavy-chain variable region of the antibody is represented by the antibody number + "-VH", such as 17-VH; the light-chain variable region of the antibody is represented by the antibody number + "-VL", such as 17-VL; the IgG1 full antibody is represented by "antibody" + antibody number, such as antibody 17 (the heavy chain of the antibody is composed of 17-VH (shown in SED ID NO: 11) and CH shown in SED ID NO: 3, and the light chain of the antibody is composed of 17-VL (shown in SED ID NO: 12) and CL shown in SED ID NO: 5).

[0271] 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.

[0272] The corresponding VH and VL sequences of Table 2 are shown in Table 3, among which, the heavy-chain CDR is shown in Table 4, and the light-chain CDR is shown in Table 5.

[0273] The plasmid pcDNA3.1(+) (Invitrogen, V790-20) was modified by conventional technical means to carry the DNA sequence encoding the above amino acid sequence. Then the plasmid was transiently transfected into HEK293F cells to make HEK293F cells express the antibody of the above amino acid sequence. The CD47 antibody was purified using a protein A column (GE Healthcare) via immobilized metal affinity chromatography (IMAC), and after sequencing, it was consistent with the expected sequence and was used for bioactivity identification.

[0274] Table 2: Combinations of Variable Regions of Antibodies

[0275]

[0276]

[0277] Table 3: Antibody variable region sequences

[0278]

[0279]

[0280]

[0281]

[0282] Table 4: Antibody VH CDR

[0283]

[0284]

[0285] Note: L12X-VH represents L12-1-VH, L12-2-VH, L12-3-VH, L12-4-VH, L12-5-VH, L12-6-1-VH, L12-6-VH, L12-7-VH, L12-8-VH, L12-9-VH, L12-10-VH or L12-11-VH

[0286] Table 5: Antibody VL CDR

[0287]

[0288] Note: X-VL represents 3-3-VL, 3-6-VL, 3-VL, 6-VL, 10-VL, 11-VL, 16-VL, 18-VL, 24-VL or 25-VL; L12X-VL represents L12-1-VL, L12-2-VL, L12-3-VL, L12-4-VL, L12-5-VL, L12-6-1-VL, L12-6-VL, L12-7-VL, L12-8-VL, L12-9-VL, L12-10-VL or L12-11-VL.

[0289] For target cell clearance, the major Fc-dependent functions of antibodies (such as CD47 antibodies) are complement-dependent cytotoxicity (CDC) initiated by the binding of C1q to the Fc region; antibody-dependent cellular cytotoxicity (ADCC) mediated by the interaction of the Fc region with Fcγ receptors (FcγR), mainly FcγRIIIa, on immune effector cells (such as NK cells and neutrophils); and antibody-dependent cellular phagocytosis (ADCP) by macrophages via the recognition of opsonized target cells through FcγRI. Each antibody subclass differs in its ability to mediate Fc-dependent effector activities. In humans, the IgG1 and IgG3 subclasses have high potency for CDC due to their binding to C1q. In addition, the IgG1 subclass has the highest affinity for FcγR and is thus the most effective in ADCC and Fc-dependent ADCP. The IgG4 subclass does not have the ability to bind C1q and has a substantially reduced FcγR binding affinity and thus has significantly attenuated effector functions.

[0290] An IgG4P subclass antibody 6Y-G4 with a mutated S228P to stabilize the hinge region of the antibody was prepared according to antibody 6Y-G1 (replacing the CH shown in SEQ ID NO:3 in 6Y-G1 with the CH shown in SEQ ID NO:4, with other sequences unchanged), further weakening the antibody Fc effect.

[0291] Taking the preparation of 17-ScFv as an example, the method for preparing ScFv in the present invention is as follows:

[0292] The nucleotide sequence encoding 17-ScFv (as shown in SEQ ID NO:59) was ligated into the pcDNA3.1(+) plasmid vector using the restriction enzymes Hind III / EcoRI. Then the plasmid vector was transiently transfected into HEK293F cells to enable HEK293F cells to express the antibody of the above amino acid sequence. The 17-ScFv antibody was purified using a protein A column via immobilized metal affinity chromatography (IMAC) and was consistent with the expected sequence after sequencing.

[0293] Other ScFvs were prepared and purified in a similar manner and were consistent with the expected sequence after sequencing.

[0294] The sequence of SEQ ID NO:59 is as follows:

[0295] caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccctcg

[0296] ataattactactggagctggatccggcagcccccagggaagggactggagtggattggatatatctattacagtgggaacaccaactac

[0297] aacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctcgttgaggctgaggtctgtgaccgctgcgga

[0298] cacggccgtgtattactgtgcgagaggagggcgatttttggaacgttactggggccagggaaccctggtcaccgtctcctcaggtgga

[0299] ggcggttcaggcggaggtggctctggcggtggcggatcggacatccagatgacccagtctccatcttccgtgtctgcatctgtaggag

[0300] acagagtcaccatctcttgtcgggcgaatcaggctattggcacttggttagcctggtatcagcaaaagccaggaaaagcccctaagctc

[0301] ctgatctatgcggcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagc

[0302] agcctgcaggctgaagatgtggcagtttattactgtcagcaatattatactactcccctcactttcggcggagggaccaagctggagatc

[0303] aaa

[0304] Example 3: Biological Activity of CD47 Antibody

[0305] 1. Detection of the Binding Activity of 17-ScFv by FACS

[0306] Verify the activity of 17-ScFv binding to CD47 on the surface of CHO-CD47 or Jurkat cells ( Figures 1A - 1B ). The experimental operation of the binding activity is as follows:

[0307] The 17-ScFv and Hu5F9-G4 antibodies (the heavy chain of Hu5F9-G4 consists of a variable region shown in SEQ ID NO: 8 and a constant region shown in SEQ ID NO: 4, and the light chain consists of a variable region shown in SEQ ID NO: 9 and a 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, 0.01 μg / ml) and incubated with CHO-CD47 or CD47 on the surface of Jurkat cells at 4°C for 30 min, and then washed once with PBS; Goat anti-human IgG Fc-PE (eBioscience, catalog number: 2183639) fluorescent secondary antibody was added and incubated at 4°C for 15 min, and then washed twice with PBS; the average fluorescence signal value of PE-A was detected by flow cytometry. Figures 1A - 1B It was shown that 17-ScFv could bind to CD47 on the cell surface in a dose-dependent manner.

[0308] 2. SIRP-α blocking activity

[0309] The ability of 17-ScFv to block the binding of SIRPa to CD47 on the surface of CHO-CD47 cells was detected by FACS technology. The 17-ScFv antibody was diluted with PBS to different concentrations (160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625 μg / ml), and Hu5F9-G4 and IgG antibodies (diluted with PBS to different concentrations 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, and then washed once with PBS; SIRPα-Fc-Bio (6.5 μg / ml) was added and incubated at 4°C for 15 min, and then washed once with PBS; then Streptavidin PE (eBioscience, catalog number: 1992345) fluorescent secondary antibody was added and incubated at 4°C for 15 min, and then washed twice with PBS; the average fluorescence signal value of PE-A was detected by flow cytometry. Figure 2 It was shown that 17-ScFv could inhibit the binding of SIRPα to CD47 on the cell surface in a dose-dependent manner.

[0310] Using the ELISA competitive 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), positive control (antibody Hu5F9-G4) and negative control (antibody IgG) were diluted with PBS at 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 the ELISA plate at room temperature for 1 hour. After washing four times with PBST, SIRPα-Fc-Bio (0.1 μg / ml) (prepared with reference to Thermo Scientific NHS-Biotin Reagents kit) ligand was added and incubated with CD47-His antigen at room temperature for 1 hour, followed by washing four times with PBST. The bound SIRPα-Fc-Bio reacted with HRP-conjugated streptavidin secondary antibody to produce chemiluminescence, and the OD450 value was detected by a plate reader. The IC 50 of the antibody was calculated based on the OD450 value, thereby judging the SIRPα blocking activity of the antibody. Figures 3A - 3E It is shown that all the tested antibodies exhibited dose-dependent inhibition of the binding of SIRPα to CD47 antigen.

[0311] 3. Hemagglutination assay

[0312] The hemagglutination reaction of CD47 antibodies was detected. 5 ml of fresh blood was collected from a healthy donor into an anticoagulant tube containing sodium heparin. First, PBMC was separated using lymphocyte separation medium (for other uses), and then 1 ml of red blood cells at the bottom of the tube was taken into 6 ml of physiological saline and washed repeatedly. After centrifugation at 2000 rpm for 5 min, the washing was continued until the supernatant was not significantly red, and then the red blood cells were resuspended with physiological saline to prepare a 2% red blood cell suspension. At room temperature, 50 μl of CD47 antibody serially diluted 2-fold with PBS (highest concentration 800 nM, a total of 12 gradients) was mixed with 50 μl of 2% red blood cell suspension and added to a 96-well U-bottom plate for incubation for 4 hours. Subsequently, the hemagglutination of the antibody was evaluated. The 96-well U-bottom plate was tilted at 45°, and the flow direction of the red blood cell mass was observed. If it was in a "one-line" shape, it indicated that the red blood cells did not agglutinate. Most of the tested CD47 antibodies induced red blood cell agglutination at high concentrations (antibody 17 showed obvious red blood cell aggregation), and did not cause red blood cell agglutination at about 6 nM. Among them, antibody L12, antibody 6Y-G1 (i.e., antibody L12-6), and antibody 6Y-G4 did not cause any red blood cell agglutination reaction ( Figures 4A - 4E)。The positive control antibody Hu5F9-G4 causes red blood cell agglutination, while AB6.12-G1 and the ligand SIRPα do not. The heavy chain of AB6.12-G1 consists of a variable region shown in SEQ ID NO:6 and a constant region shown in SEQ ID NO:3, and the light chain consists of a variable region shown in SEQ ID NO:7 and a constant region shown in SEQ ID NO:5. The heavy chain of Hu5F9-G4 consists of a variable region shown in SEQ ID NO:8 and a constant region shown in SEQ ID NO:4, and the light chain consists of a variable region shown in SEQ ID NO:9 and a constant region shown in SEQ ID NO:5. See Table 6.

[0313] Table 6: Positive Antibody Sequence Table

[0314]

[0315]

[0316] 4. CD47 Binding Assay (ELISA):

[0317] The binding activities of CD47 antibodies to human CD47 and cynomolgus CD47 were determined by ELISA. Antibodies 17, L12, 6Y-G1, 6Y-G4, and Hu5F9-G4 were serially diluted 3-fold with PBS from 5 μg / ml to 8 concentrations and incubated with human or cynomolgus CD47-Fc (2 μg / ml) antigen coated on ELISA plates at room temperature for 1 hour. After washing four times with PBST, HRP-anti-kappa (1:10000, sigma) secondary antibody was added and incubated with the antibodies at room temperature for 1 hour, followed by washing four times with PBST. The bound HRP-anti-kappa reacted with the TMB substrate to produce chemiluminescence, and the OD450 value was detected by a plate reader. The EC 50 of the antibody was calculated based on the OD450 value to determine the binding activity of the antibody to human or cynomolgus CD47. Figures 5A - 5B It shows that all the tested antibodies exhibit dose-dependent binding of the antibody to the CD47 antigen, and the parameters are shown in Table 7.

[0318] Table 7: Affinity of CD47 Antibodies for Human CD47 and Cynomolgus C47

[0319]

[0320] 5. CD47 Binding Assay (Fortebio)

[0321] The binding activities 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, antibody L12) and Hu5F9-G4 to human CD47 were determined by the Fortebio method.

[0322] The Protein A sensor was pre-wetted in PBS for 10 minutes and then used. The antibody to be tested was diluted to 10 μg / mL with PBST (hereinafter referred to as dilution buffer) containing 0.5% BSA at pH 6.8. The PA sensor was immobilized in the antibody until the signal reached about 1.7 nm. The CD47-His antigen was diluted to 50, 25, 12.5, 6.25 nM with the dilution buffer. The dilution buffer, antigen dilutions at gradient concentrations, regeneration buffer (1 M MgCl 2 ) at pH 8.4, and neutralization buffer (PBST) were added to the corresponding columns of a 96-well plate in sequence, and the following steps were performed: ① Baseline: Detect the baseline in the dilution buffer for 60 seconds; ② Association: Bind in the antigen dilutions at gradient concentrations and sample blank (dilution buffer) for 100 seconds; ③ Dissociation: Dissociate in the dilution buffer for 700 seconds; ④ Regeneration: Regenerate in the regeneration buffer for 5 seconds; ⑤ Neutralization: Neutralize in the neutralization buffer for 5 seconds; ⑥ The regeneration and neutralization cycles were performed 3 times.

[0323] The data was processed and analyzed using Data Analysis 8.2. The sample data was fitted after subtracting the reference (sample blank) signal to obtain the affinity constant KD. The statistical results of the antibody affinities are shown in Table 8. The results showed that the affinities of the 11 antibodies binding to the CD47 antigen were similar.

[0324] Table 8: Antibody Affinities

[0325]

[0326]

[0327] 6. Phagocytosis

[0328] An in vitro phagocytosis assay was performed to evaluate whether the CD47 antibody enhanced the phagocytosis of macrophages towards target cells expressing CD47. Briefly, in the presence of the CD47 antibody (0.7 nM), RAW264.7 macrophages (2×10 5cells / mL) (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, 3111C0001CCC000146) and CFSE-labeled Raji cells (4×10 5 cells / mL) (Promega, JA1595) were plated into a 24-well bottom plate at a ratio of 1:2 and incubated for 2 hours at 37°C in the dark. When the incubation was completed, the cells were washed twice with PBS and RAW264.7 cells were digested with trypsin. The digested macrophages were transferred to a 1.5 ml EP tube and centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, and then 100 μl of APC-F4 / 80 fluorescent antibody (eBiosciences) was added to each tube. The cells were incubated for 30 minutes at 4°C in the dark, centrifuged at 2000 rpm for 5 minutes, and washed twice with PBS buffer. Cells were obtained on a BDC6 flow cytometer. The phagocytosis index was analyzed. The calculation method of the phagocytosis index was: phagocytosis index = the number of CFSE-positive macrophages (i.e., the number of macrophages that phagocytosed tumor cells) / every 5000 macrophages. Antibodies 6Y-G1, 6Y-G4, 17, L12, and Hu5F9-G4 showed strong phagocytosis-enhancing ability ( Figures 6A - 6B ). Figure 6B It was shown that the cell phagocytosis reaction induced by 6Y-G4 was slightly weaker than that of 6Y-G1, indicating that the Fc effect of the IgG4 antibody was weaker.

[0329] Example 4: Effects of CD47 antibodies on blood cells

[0330] Before the present invention, known CD47-binding molecules that block SIRPα and contain an Fc domain (such as CD47 antibodies and recombinant SIRPα-Fc fusion proteins) all induced varying degrees of erythrocyte and platelet reduction. Therefore, the effects of the antibodies of the present invention on blood cells were evaluated in cynomolgus monkeys. A total of 8 cynomolgus monkeys meeting the test requirements were selected, ordinary grade, with half males and half females. The body weight ranges of males and females were 3.70 - 5.30 kg and 2.75 - 3.55 kg, respectively. According to body weight, they were divided into 4 groups by a simple randomization scheme, with 2 animals in each group, half males and half females. The negative control group was given normal saline. The test substances were antibody 6Y-G1 (i.e., antibody L12-6), Hu5F9-G4, and AB6.12-G1. The intravenous infusion vehicle was normal saline. Intravenous infusion was administered once a week for 5 times. Each time, the dose was administered according to 1→10→10→30→30 mg / kg, and the administration volume was 10 ml / kg. Observation was continued until the 42nd day after the administration was completed. Figures 7A - 7D It was shown that after administration of the three antibodies, hemoglobin and erythrocytes decreased, reaching a low point approximately two weeks later and then gradually returning to normal levels; the platelet content fluctuated up and down before and after administration and returned to normal after drug withdrawal; the number of reticulocytes showed an increasing trend after administration, and the number of reticulocytes increased higher after administration of 6Y-G1 than the other two antibodies.

[0331] Example 5: In Vivo Antitumor Activity of Fc Variants of CD47 Antibody

[0332] Evaluate the antitumor effect of the test drug antibody L12 in the human hematological tumor MV4-11 systemic tumor model. NOD.SCID mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd., body weight 18 g - 22 g, mouse age 6 - 8 weeks) were inoculated with MV4-11 cells (ATCC) via the tail vein. Approximately 4 weeks after inoculation, a human hematological tumor MV4-11 systemic tumor model was established. The experiment was divided into test groups of AB6.12-G1 at 0.2 mg / animal, Hu5F9-G4 at 0.2 mg / animal, antibody L12 at 0.2 mg / animal, and a solvent (normal saline) control group, with 6 animals in each group. Administration was by intraperitoneal injection three times a week for a total of three weeks. Efficacy was evaluated based on the increase in survival time (ILS), and safety was evaluated based on changes in animal body weight and mortality during administration. Figure 8 It was shown that the survival times of the test drugs AB6.12-G1 (0.2 mg / animal) and Hu5F9-G4 (0.2 mg / animal) were both prolonged by 7.4% and 6.5% compared with the solvent control group, and there were no statistically significant differences (p = 1.000), failing to show a significant antitumor effect. The median survival time of antibody L12 (0.2 mg / animal) was 69 days, and the survival time was prolonged by 27.8% compared with the control group, showing a statistically significant difference (p = 0.007).

[0333] The antitumor activity of antibody 6Y-G4 was evaluated in the Raji model of lymphoma. Raji cells were implanted subcutaneously into NOD.SCID mice and randomly divided into 3 groups (8 mice in each group, day 0). Group 1: Solvent control (normal saline); Group 2: Hu5F9-G4 (positive control); Group 3: 6Y-G4. When the tumors were palpable (50 mm 3 , day 4), each antibody or vehicle (buffer only) was used for treatment, and the mice were sacrificed when the tumor volume reached approximately 3000 mm 3 . The tumor volume was measured every 3 days. The antibodies were administered intraperitoneally (IP) at a dose of 100 μg each, three times a week for 3 weeks (a total of 9 doses per mouse). Treatment started on day 4 and ended on day 21. As Figure 9 shown, the test drugs 6Y-G4 and Hu5F9-G4 treatment groups both produced a highly significant antitumor effect at a dosing concentration of 0.1 mg / animal, significantly inhibiting tumor growth after the start of dosing. By day 17 after the start of dosing (PG-D17), most of the tumors in the mice disappeared. The average tumor volumes of the 6Y-G4 (0.1 mg / animal) and Hu5F9-G4 (0.1 mg / animal) treatment groups were 4 mm 3 (7 out of 8 mice had their tumors disappear) and 7 mm3 (In 8 mice, tumors disappeared in 5), and the relative tumor growth inhibition rates (TGI) were 99.87% and 99.76% respectively, showing significant statistical differences compared with the vehicle control group (both p values < 0.001).

Claims

1. An antibody or antigen-binding fragment, wherein, the antibody or antigen-binding fragment specifically binds to CD47 and comprises one or more 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 shown in SEQ ID NO: 14, or a variant of SEQ ID NO: 14 having 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 14 sequence; (b) VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, or a variant of SEQ ID NO: 17 having 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 17 sequence; (c) VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 23, or a variant of SEQ ID NO: 23 having 1 to 3 amino acid substitutions as shown in Table 1 in the SEQ ID NO: 23 sequence; (d) VL CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO: 30-31; (e) VL CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 32-33; or (f) VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 34-35.

2. An antibody or antigen-binding fragment, wherein, the antibody or antigen-binding fragment specifically binds to CD47 and comprises one or more 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 shown in SEQ ID NO: 14-16; (b) VH CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 17-22; (c) VH CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 23-29; (d) VL CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO: 30-31; (e) VL CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 32-33; or (f) VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 34-35.

3. An antibody or antigen-binding fragment, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region VH, and the VH comprises an amino acid sequence selected from any one of SEQ ID NO: 11, 36-44, 50, and 56, or an amino acid sequence having at least 90% sequence homology with any one of the amino acid sequences of SEQ ID NO: 11, 36-44, 50, and 56.

4. An antibody or antigen-binding fragment, wherein, The antibody or antigen-binding fragment comprises a light chain variable region VL, and the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 12-13, 45-49, 51-55 and 65, or an amino acid sequence having at least 90% sequence homology with any one of the amino acid sequences of SEQ ID NOs: 12-13, 45-49, 51-55 and 65.

5. An antibody or antigen-binding fragment, wherein, the antibody or antigen-binding fragment specifically binds to CD47 and comprises VH CDR1 as shown in SEQ ID NO: 14, VH CDR2 as shown in SEQ ID NO: 17, VH CDR3 as shown in SEQ ID NO: 23, VL CDR1 as shown in SEQ ID NO: 30, VL CDR2 as shown in SEQ ID NO: 32 and VL CDR3 as shown in SEQ ID NO:

34.

6. The antibody or antigen-binding fragment according to claim 1, 2 or 5, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region VH, and the VH comprises one or more amino acid residue mutations numbered according to Kabat selected from the following groups: (a) R81K, and (b) R82aS.

7. The antibody or antigen-binding fragment according to claim 1, 2 or 5, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region VH, and the VH comprises an amino acid sequence selected from SEQ ID NO: 11 or 50, or an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 11 or 50.

8. The antibody or antigen-binding fragment according to claim 1, 2, 5 or 7, wherein, the antibody or antigen-binding fragment comprises a light chain variable region VL, and the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 13, 45-49, 51-55 and 65, or an amino acid sequence having at least 90% sequence homology with any one of the amino acid sequences of SEQ ID NOs: 13, 45-49, 51-55 and 65.

9. An antibody or antigen-binding fragment, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the VH comprises an amino acid sequence selected from SEQ ID NO: 11 or 50, and the VL comprises an amino acid sequence selected from SEQ ID NO: 13 or 65.

10. The antibody or antigen-binding fragment according to any one of claims 1-9, wherein, the antibody comprises a heavy chain H and a light chain L; the heavy chain H comprises an amino acid sequence selected from any one of SEQ ID NOs: 60 and 62-63, and the light chain L comprises an amino acid sequence selected from SEQ ID NO: 61 or 64.

11. The antibody or antigen-binding fragment according to any one of claims 1-9, wherein, The antibody or antigen-binding fragment is of the IgG isotype and is selected from the IgG1 subtype, IgG2 subtype, IgG3 subtype, or IgG4 subtype.

12. The antibody or antigen-binding fragment according to claim 11, wherein the antibody or antigen-binding fragment is IgG4P.

13. A composition, characterized in that the composition comprises the antibody or antigen-binding fragment according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. A biological material, being (1) A polynucleotide, characterized in that the polynucleotide encodes the antibody or antigen-binding fragment according to any one of claims 1 to 12; or, (2) An expression vector, characterized in that the expression vector comprises a polynucleotide encoding the antibody or antigen-binding fragment according to any one of claims 1 to 12; or, (3) A cell, characterized in that the cell comprises one or more polynucleotides encoding the antibody or antigen-binding fragment according to any one of claims 1 to 12.

15. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 12, or the composition according to claim 13, or the biological material according to claim 14 in the preparation of a medicament for treating cancer or infection.

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