CD47 antibody and its application

Abstract

The present invention relates to CD47 antibodies and their applications, in particular to monoclonal antibodies that recognize CD47, and more specifically to CD47 antibodies that do not cause significant levels of hemagglutination of red blood cells, hemorrhage and thrombocytopenia, methods for preparing these antibodies, and applications of these monoclonal antibodies in preparing drugs for treating cancer or infection.

Description

Technical Field

[0001] The present invention relates to the field of biological immunotechnology, and in particular to a fully human anti-CD47 IgG antibody, more specifically to a CD47 antibody that does not cause significant hemagglutination reaction of human red blood cells, hemoglobin reduction, anemia and / or thrombocytopenia, methods for preparing these antibodies, and use of these monoclonal antibodies in preparing 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 the phagocytosis of macrophages (MatozakiT, et al., Trends Cell Biol.2009). 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's lymphoma, etc., and transmits "Don't eat me" signals to macrophages to escape immune surveillance, and high expression of CD47 is associated with poor clinical prognosis (Willingham et al.Proc NatlAcad Sci USA.2012).

[0003] U.S. patent application 2009 / 0191202 disclosed that Professor Weissman's team 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, while small tumors can be cured. U.S. patent application 2016 / 0251435 disclosed the use of anti-CD47 monoclonal antibodies in tumor treatment. Patent application WO2013056352 describes the use of humanized anti-human SIRPα full-length monoclonal antibodies and antibody fragments derived therefrom for the treatment of hematological tumors, especially leukemia. Blocking the CD47-SIRPα signaling pathway can promote the phagocytosis of tumor cells by macrophages, 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 can recognize and bind to human CD47. The antibodies of the present invention can prevent CD47 from interacting with SIRPα and will 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 multiple desirable properties, such as specifically recognizing human or macaque CD47 by the scheme of non-limiting examples, effectively blocking the interaction between CD47 and its ligand SIRPα without causing significant hemagglutination of erythrocytes, 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] The sequence of SEQ ID NO: 14 has 1 to 3 amino acid substitutions as shown in Table 1;

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

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

[0013] (d) VL CDR1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 30-31;

[0014] (e) VL CDR2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 32-33; and

[0015] (f) VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 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) VH CDR1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 14-16;

[0018] (b) VH CDR2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 17-22;

[0019] (c) VH CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 23-29;

[0020] (d) VL CDR1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 30-31;

[0021] (e) VL CDR2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 32-33; and

[0022] (f) VL CDR3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 34-35.

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

[0024] (a) a VH CDR1 as shown in SEQ ID NO: 14, or a variant of SEQ ID NO: 14, which in SEQ ID NO:

[0025] 14 sequences had 1 to 3 amino acid substitutions as shown in Table 1;

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

[0027] (c) a VH CDR3 as shown in SEQ ID NO: 23, or a variant of SEQ ID NO: 23, which in SEQ ID NO:

[0028] 23 sequences had 1 to 3 amino acid substitutions as shown in Table 1 ;

[0029] (d) any one of the VL CDR1s shown in SEQ ID NOs: 30-31;

[0030] (e) any one of the VL CDR2s shown in SEQ ID NOs: 32-33; or

[0031] (f) any one of the VL CDR3s shown in SEQ ID NOs: 34-35.

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

[0033] (a) any one of the VH CDR1s shown in SEQ ID NOs: 14-16;

[0034] (b) any one of the VH CDR2s shown in SEQ ID NOs: 17-22;

[0035] (c) any one of the VH CDR3s shown in SEQ ID NOs: 23-29;

[0036] (d) any one of the VL CDR1s shown in SEQ ID NOs: 30-31;

[0037] (e) any one of the VL CDR2s shown in SEQ ID NOs: 32-33; or

[0038] (f) any one of the VL CDR3s shown in SEQ ID NOs: 34-35.

[0039] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH, which comprises an amino acid sequence selected from any one of SEQ ID NOs: 11, 36-44, 50 and 56, or an amino acid sequence having at least 90% sequence homology with any one of SEQ ID NOs: 11, 36-44, 50 and 56.

[0040] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region VL, which 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 to any one of SEQ ID NOs: 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-related protein (CD47) and comprises a VH CDR1 as shown in SEQ ID NO: 14, a VH CDR2 as shown in SEQ ID NO: 17, a VH CDR3 as shown in SEQ ID NO: 23, a VL CDR1 as shown in SEQ ID NO: 30, a VL CDR2 as shown in SEQ ID NO: 32, and a 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 comprising one or more amino acid residue mutations according to Kabat numbering selected from the following group:

[0043] (a) R81K, and

[0044] (b)R82aS.

[0045] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region VH, which comprises an amino acid sequence selected from any one of SEQ ID NOs: 11 and 50, or an amino acid sequence having at least 90% sequence homology with any one of SEQ ID NOs: 11 and 50.

[0046] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region VL, which 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 to any one of SEQ ID NOs: 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, wherein 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.

[0048] In some embodiments, the CD47 antibody herein specifically recognizes and binds to human or macaque CD47, and the antibody comprises a heavy chain variable region of an amino acid sequence selected from any one of SEQ ID NOs: 11, 36-44, 50 and / or a light chain variable region of an amino acid sequence selected from any one of SEQ ID NOs: 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, the CD47 antibodies of the invention block 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 compared to the level of interaction between CD47 and SIRPα in the absence of the CD47 antibody.

[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 SEQ ID NO:3 or 4, and the light chain constant region comprises an amino acid sequence selected from 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] QDSKDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0069] In some embodiments, the CD47 antibodies of the present invention do not cause significant cell agglutination, for example, the CD47 antibodies of the present invention do not cause significant hemagglutination of red blood cells. In some embodiments, if the agglutination level in the presence of the CD47 antibody of the present invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% compared to the agglutination level in the presence of the existing CD47 antibody Hu5F9-G4, it is indicated that the CD47 antibody of the present invention does not cause significant agglutination levels. In some embodiments, the CD47 antibody of the present invention does not cause significant cell agglutination when the antibody concentration is between 400 pM and 800 nM.

[0070] In some embodiments, the antibodies of the invention are also significantly effective in tumor models compared to antibodies known in the art. For example, in the presence of the CD47 antibodies of the invention, 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, and the IgG isotype is 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 Ser228 site in the hinge region of IgG4 to Pro to prevent chain exchange; IgG4PE refers to the mutation of Ser228 site in the hinge region of IgG4 to Pro to prevent chain exchange; Leu235 in the constant region is mutated to Glu to change 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 invention is an isolated CD47 antibody or antigen-binding fragment.

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

[0075] In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention can be used in the preparation of a medicament 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 a medicament for treating solid tumors or blood tumors. In some embodiments, the CD47 antibody or antigen-binding fragment of the present invention can be used in the preparation of a medicament for treating non-Hodgkin's lymphoma (NHL), acute lymphocytic 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 a medicament for treating diffuse large B-cell lymphoma or follicular lymphoma.

[0076] In some embodiments, the CD47 antibodies described herein can be used to treat, delay progression of, prevent recurrence of, or alleviate symptoms of cancer or other neoplastic conditions. For example, the CD47 antibodies described herein can be used to treat hematological malignancies and / or tumors, such as hematological malignancies and / or tumors. For example, the CD47 antibodies described herein can be used to treat CD47 +Tumors. As non-limiting examples, the CD47 antibodies described herein can be used to treat non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic 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] In this article, "blood cancer" includes leukemia, lymphoma and myeloma, etc. "Leukemia" means a kind of blood cancer, which makes too many white blood cells that cannot effectively fight infection, thereby excluding other components of blood, such as platelets and red blood cells. Leukemia cases can be divided into acute or chronic. As a non-limiting example, leukemia can include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disease / tumor (MPDS) and myelodysplastic syndrome. "Lymphoma" can include Hodgkin's lymphoma, painless and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma and follicular lymphoma (small cells and large cells), 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, antibodies described herein. Exemplary antibodies include antibodies having a heavy chain variable region selected from any one of the amino acid sequences of SEQ ID NOs: 11, 36-44, 50 and 56 and a light chain variable region selected from any one of the amino acid sequences of SEQ ID NOs: 12, 13, 45-49, 51-55, 65. The antibodies also include antibodies having a heavy chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to at least one of the sequences of SEQ ID NOs: 11, 36-44, 50 and 56 and a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to at least one of the sequences of SEQ ID NOs: 12, 13, 45-49, 51-55, 65. These antibodies showed specificity for human CD47 and were able to block the interaction of CD47 with SIRPα without causing significant hemagglutination of red blood cells.

[0079] In some embodiments, the present invention also provides a biomaterial,

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

[0081] (2) an expression vector comprising a 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 include the antibodies 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 immunologically active fragments as required, wherein the antibody does not cause significant hemagglutination of red blood cells after administration. The dosage of the antibody 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 immunologically active fragment prevents CD47 from interacting with SIRPα.

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

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

[0087] In some embodiments, use of the antibodies or antigen-binding fragments, compositions, and biomaterials of the present invention in the preparation of drugs for treating cancer or infection is provided.

[0088] Those skilled in the art will appreciate that the antibodies of the present invention have a variety of uses. For example, the antibodies of the present invention can be used as therapeutic agents, as reagents in diagnostic kits or as diagnostic tools, or as reagents in competition experiments to generate therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] FIG1 is a set of graphs describing the ability of antibody 17-ScFv to bind to cell surface CD47, using CHO-CD47 cells ( Figure 1A ) or Jurkat cells ( Figure 1B ).

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

[0091] Figure 3 is a group of pictures. Figures 3A-3E Shown is the ability of CD47 antibodies to block SIRPα binding to CD47 antigen assessed by competition ELISA.

[0092] Figure 4 is a group of pictures. Figures 4A-4E The red blood cell (RBC) hemagglutination reaction caused by CD47 antibody is shown. The red blood cells that appear to be flowing in a line do not cause hemagglutination reaction, those that are slightly agglutinated are a dot, and those that are obviously agglutinated are a blurred mass throughout the hole. Figure 4A Antibody 11 showed obvious aggregation. Figure 4B It showed that antibody L12 does not cause hemagglutination. Figure 4C It showed that antibody L12-6 did not cause hemagglutination. Figure 4D Antibodies 17 and Hu5F9-G4 showed obvious hemagglutination, while AB6.12-G1 and SIRPα did not cause hemagglutination. Figure 4EIt was shown that antibody 6Y-G4 does not induce hemagglutination.

[0093] Figure 5 is a group of pictures. Figure 5A-5B Binding of CD47 antibodies to human CD47 or cynomolgus CD47 assessed by ELISA is shown.

[0094] Figure 6 is a group of pictures. Figure 6A-6B Shown is the ability of CD47 antibody to promote phagocytosis of Raji by RAW264.7 as assessed by flow cytometry.

[0095] Figure 7 is a group of pictures. Figures 7A-7D The effects of antibody 6Y-G1 on red blood cells and platelets were analyzed in cynomolgus monkeys; Figure 7A-7B It was shown that antibody 6Y-G1 caused a decrease in hemoglobin and red blood cells, which gradually recovered after about 2 weeks, and the degree of reaction was similar to that of antibodies AB6.12-G1 and Hu5F9-G4; Figure 7C It showed that after each administration of antibody 6Y-G1, platelets fluctuated and recovered after the administration ended; Fig.7D It showed that after each administration of antibody 6Y-G1, the number of reticulocytes increased, and the increase was greater than that in the antibody AB6.12-G1 and Hu5F9-G4 administration groups.

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

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

[0098] The present invention provides antibodies that specifically bind to CD47. In some embodiments, the antibodies of the present invention are capable of specifically binding to human CD47. Herein, these antibodies are collectively referred to as CD47 antibodies. Unless otherwise specified herein, the amino acid residues in the variable and constant regions of antibodies are numbered according to Kabat (EU index in Kabat et al., 1991, Sequences of Proteins of Immunological Interest).

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

[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 antibodies that block SIRPα can cause unwanted hemagglutination side effects. Other existing antibodies (such as 2D3) do not cause hemagglutination, but these antibodies also do not block SIRPα, making them ineffective in promoting phagocytosis. Therefore, cell agglutination is a major limitation of using existing full IgG antibodies to therapeutically target CD47. 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 effect of therapeutic targeting of CD47, and maintaining the ability to block the interaction between CD47 and SIRPα, thereby promoting phagocytosis of cells expressing CD47. In some embodiments, the CD47 antibodies of the present invention (e.g., 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 antibody of the present invention binds to human CD47 and blocks its interaction with SIRPα ( Figures 3A-3E These antibodies do not induce significant hemagglutination of human erythrocytes ( Figures 4A-4E These antibodies can promote macrophages to engulf tumor cells ( Figure 6A-6B In addition, these CD47 antibodies showed potent antitumor activity in a mouse model of human Raji lymphoma ( Fig. 9 ). Thus, 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 of great importance in treating a large number of cancers.

[0104] The antibodies of the present invention bind to the CD47 epitope with an equilibrium dissociation constant (KD) of 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, or 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 regulate, block, inhibit, reduce, antagonize, neutralize or interfere with the functional activity of widely distributed CD47. The functional activity of CD47 includes, for example, signaling 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 a variety of integrins. For example, the CD47 antibody partially or completely regulates, blocks, inhibits, reduces, antagonizes, neutralizes or interferes with the binding of CD47 to SIRPα, thereby completely or partially inhibiting the functional activity of CD47.

[0106] definition

[0107] Unless otherwise defined, the meaning of the scientific and technical terms used in the present invention is the meaning commonly understood by those skilled in the art. Generally, the nomenclature and technology used in cell culture, molecular biology and protein purification as described herein are well known in the art and commonly used. For recombinant DNA, oligonucleotide synthesis and cell culture and transformation (such as electroporation, lipofection), standard techniques are used. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions or the methods commonly used in this area or as described herein. The aforementioned techniques and methods are usually used as described in the multiple comprehensive and more specific documents that are well known in the art and quoted and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual ("Molecular Cloning: 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 "red blood cells" and "erythrocytes" are synonymous and are used interchangeably.

[0109] The term "aggregation" means that cells clump, while the term "hemagglutination" means that a specific type of cells (i.e., red blood cells) clump. Therefore, hemagglutination is a type of agglutination. In the hemagglutination assay described herein, red blood cells form a form that can be a "button", "halo", or an intermediate between the two in a given well. The term "a line" refers to the red blood cell mass flowing into a line after tilting a 96-well plate at 45°. The term "significant hemagglutination activity" refers to the presence of any halo form in the well when the antibodies described herein are added.

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

[0111] It is known that the basic antibody structural unit includes a tetramer. Each tetramer (or "full antibody") is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). 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 function. 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 properties of the heavy chains present in the molecules. Some species 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 population of antibody molecules that contain only one molecular species of antibody molecules consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of monoclonal antibodies are identical in all molecules of the population. MAbs contain an antigen binding site that can immunoreact with a specific epitope of an antigen.

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

[0114] The term "antigen binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly differentiated branches (called "hypervariable regions") in the heavy chain variable region (VH) and the light chain variable region (VL) are located between more conservative branches (called "framework regions" or "FR"). Thus, the term "FR" refers to the amino acid sequence that naturally occurs between or adjacent to the hypervariable regions in an immunoglobulin. 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 chain and light chain are referred to as "complementarity determining regions" or "CDRs". The amino acid alignment of each domain can be consistent with the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health (1987 and 1991)) or the article by Chothia and Lesk (J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989)).

[0115] The term "epitope" as used herein includes any protein determining region that can specifically bind to an immunoglobulin or fragment thereof or a T cell receptor. The epitope determining region is usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and usually has specific three-dimensional structural properties and specific charge properties.

[0116] As used herein, the term "specific binding" refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. 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 requires measuring the rates of formation and dissociation of an antigen binding site / antigen complex, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rate in both directions. Therefore, the "binding rate constant" (k on ) and the “dissociation rate constant” (k off ) Both can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361: 186-87 (1993)). off / k onThe ratio is able to eliminate all parameters not related to affinity and is equal to the equilibrium dissociation constant, KD. (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those skilled in the art. When the equilibrium dissociation constant (KD) is ≦1 μM (e.g., less than or equal to 100 nM, less than or equal to 10 nM, and for example, less than or equal to 1 nM), the antibodies of the present disclosure specifically bind to CD47.

[0117] An "isolated" antibody is one that is separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein 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 measured by the Lowry method; (2) at least 15 residues of N-terminal or internal amino acid sequence are available by using a spinning cup sequenator; or (3) homogeneity is measured by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells. Generally, isolated antibodies will be prepared by at least one or more purification steps. In some embodiments, the purity of the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including the endpoints) between any two of these values ​​or any value therein.

[0118] The term "polypeptide" as used herein is a general term referring to a native protein, fragment or analog of a polypeptide sequence; thus, native protein fragments and analogs are members of the genus polypeptide.

[0119] The term "sequence identity" or "sequence homology" refers to that two polynucleotide or amino acid sequences are identical (i.e., identical on a nucleotide-to-nucleotide or residue-to-residue basis) over a comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and then multiplying the result by 100 to obtain the percentage of sequence identity.

[0120] As used herein, twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, ed., Sinauer Associates, Sunderland 7 Mass. (1991)). Stereoisomers (e.g., D-amino acids), non-natural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid and other unconventional amino acids of twenty conventional amino acids may also be components suitable for use in the polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide representation method used herein, the left-hand direction is the amino terminal direction, and the right-hand direction is the carboxyl 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 indicated, 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 direction of addition from 5' to 3' of a nascent RNA transcript is referred to as the transcription direction; the sequence region on the DNA strand that is identical to the RNA sequence and 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence region on the DNA strand that is identical to the RNA sequence and 3' to the 3' end of the RNA transcript is referred to as 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, such as by the GAP or BESTFIT programs using default gap weights.

[0122] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0123] Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that the identity of the amino acid sequence remains at least 75%, such as at least 80%, 90%, 95% and, for example, 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are substitutions that occur within a family of related amino acids in their side chains. Genetically encoded amino acids are roughly divided into the following categories: (1) acidic amino acids are aspartate and glutamate; (2) basic amino acids are lysine, arginine, and 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, and tyrosine. Other families of amino acids include (i) serine and threonine from the aliphatic-hydroxyl family; (ii) asparagine and glutamine from the amide-containing family; (iii) alanine, valine, leucine, and isoleucine from the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine from the aromatic family. In some embodiments, conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. For example, it is reasonable to predict that a single replacement of leucine with isoleucine or valine, replacement of aspartate with glutamate, replacement of threonine with serine, or similar replacement of an amino acid with a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, particularly where the replacement 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 determinations are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art.

[0124] In some embodiments, the amino acid substitution has the following effects: (1) reducing sensitivity to proteolysis, (2) reducing sensitivity to oxidation, (3) changing binding affinity for forming protein complexes, (4) changing binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. Analogs may include various mutant proteins whose sequences differ from the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain that forms intermolecular contacts). Conservative amino acid substitutions should not significantly change the structural properties of the parent sequence (for example, the substituted amino acid should not tend to disrupt the helical structure present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence). Examples of artificially recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

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

[0126] As used herein, the term "label" or "labeled" refers to a polypeptide that incorporates a detectable label, for example, by incorporation of a radiolabeled amino acid, or attached to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or an enzymatic activity that can be detected by optical methods or calorimetry). In some cases, the label or tag can also be therapeutic. Various methods of 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 markers (e.g., FITC, rhodamine, lanthanide phosphors), enzyme markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl, 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 connected by spacer arms of various lengths to reduce possible steric hindrance.

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

[0128] As used herein, the term "anti-neoplastic agent" refers to an agent that has the functional property of inhibiting the development or progression of a tumor in the human body, especially a malignant (cancerous) lesion such as carcinoma, sarcoma, lymphoma or leukemia. Inhibition of metastasis is a property of anti-neoplastic agents in many cases.

[0129] Other chemical terms are used herein according to conventional usage in the art, as described 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 to 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 assay described in the examples herein.

[0132] Exemplary antibodies of the present invention include antibody 17, antibody L12, antibody 6Y-G1, antibody 6Y-G4, 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 a heavy chain hypervariable region, and VL CDR is a light chain hypervariable region.

[0134] In some embodiments, the present invention replaces one or more amino acid positions in VH CDR1, VH CDR2 and / or VHCDR3 based on antibody 17. In some embodiments, the replaceable positions (parents) of VH CDR1, VH CDR2 and / or VHCDR3 and the amino acids that can be used for replacement are shown in one or more of Table 1. The "position" in Table 1 indicates the amino acid position from left to right of the amino acid sequence shown in SEQ ID NO: 11; for example, position 30 indicates the 30th amino acid "D" from left to right of the amino acid sequence shown in SEQ ID NO: 11.

[0135] Table 1: Heavy chain CDR amino acid substitutions

[0136]

[0137]

[0138] The VH CDRs of antibody L12 and antibody L12-6 have SEQ ID NO: 14 (VH CDR1), SEQ ID NO: 17 (VHCDR2) and SEQ ID NO: 23 (VH CDR3), and the VL CDRs have SEQ ID NO: 30 (VL CDR1), SEQ ID NO: 32 (VLCDR2) and SEQ ID NO: 34 (VL CDR3).

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

[0140] In addition to the changes in CDR, the amino acids in the framework region may also be appropriately changed. 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 produced after two mutations (R81K and R82aS) in the framework region of the heavy chain of antibody L12 are also suitable heavy chain variable regions. In some embodiments, the heavy chain variable region of the CD47 antibody may be a K at position 81, or an S at position 82a (according to Kabat numbering). Other framework region mutations are also embodied 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 antibodies with suitable sequence identity to the sequences of the above antibodies. For example, they 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. In some embodiments, at least the CDRs of these identical sequences do not change.

[0142] In some embodiments, antibody L12 is adapted to a human framework. In some embodiments, the framework mutations of the VH of antibody L12 are shown in Table 3 for the amino acids underlined 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 some embodiments, the VL framework mutations of antibody L12 are shown in Table 3 for the amino acids underlined 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.

[0143] The present invention also includes antibodies that bind to the same epitope as the CD47 antibodies described herein. For example, the antibodies of the present invention specifically bind to an epitope that includes one or more amino acid residues on human CD47 (see, e.g., GenBank Accession No. Q08722.1).

[0144] An exemplary human CD47 ECD amino acid sequence is provided below: SEQ ID NO: 1 (GenBank Accession No. Q08722.1 (GI: 1171879), which is incorporated herein by reference). 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 assessed by methods including measuring CD47- and / or CD47 / SIRPα-mediated signaling in the presence of one or more antibodies described herein. These assays may include competitive binding assays or may measure biological readouts, 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 produced using various methods known in the art (e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain (including CDR) is derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared using trioma technology, human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72), and EBV hybridoma technology to 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 are available 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 in vitro with Epstein Barr Virus (see Cole et al., 1985. In: Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0151] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G, which primarily provides the IgG fraction in immune serum. In addition, the specific antigen or epitope targeted by the immunoglobulin can be fixed on a column to purify the immunospecific antibody by immunoaffinity chromatography. The purification of immunoglobulins can be referenced to the article of D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0152] The CD47 antibody disclosed herein may be a monoclonal antibody. Monoclonal antibodies that regulate, block, inhibit, reduce, antagonize, neutralize or otherwise hinder CD47- and / or CD47 / SIRPα-mediated cell signaling are produced, for example, by immunizing animals with membrane-bound and / or soluble CD47 (e.g., human CD47 or an immunogenic fragment, derivative or variant thereof). Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding CD47, so that CD47 is expressed and associated with the surface of the transfected cells. Alternatively, antibodies are obtained by screening a library containing antibody or antigen binding domain sequences for binding to CD47. The library is prepared as follows: In a phage, a fusion of a protein or peptide fused to a phage coat protein is expressed on the surface of an assembled phage particle, and the encoded DNA sequence is integrated into the phage particle (i.e., a "phage display library"). Screening is then performed by a 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, mice, hamsters or other suitable host animals are usually immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0154] Immunizing agents generally include protein antigens, fragments thereof, or fusion proteins thereof. Generally, if human cells are required, peripheral blood lymphocytes are used, or if non-human mammalian sources are required, spleen cells or lymph node cells are used. Lymphocytes are then fused with an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines generally use rat or mouse myeloma cell lines. Hybridoma cells can be cultured in an appropriate culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cell lacks hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of the hybridoma generally includes hypoxanthine, aminopterin and thymidine ("HAT culture 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. DNA encoding monoclonal antibodies described herein can be separated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells described herein serve as a preferred source of such DNA. Once separated, DNA can be placed in an expression vector, which is then transfected into a host cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) 293 cell, ape COS cell, PER.NS0 cell, SP2 / 0, YB2 / 0, or myeloma cell that does not produce immunoglobulin in addition, thereby obtaining a synthesized monoclonal antibody in a recombinant host cell. The DNA may also be modified, for example, by replacing the homologous mouse sequences with coding sequences for human heavy and light chain constant domains (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides can replace the constant domains of the antibodies described herein, or can replace the variable domains of one antigen-binding site of the antibodies described herein to produce a chimeric bivalent antibody.

[0156] Antibody-producing cell lines can be selected, constructed and cultured using techniques 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, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated herein by reference.

[0157] In some embodiments, the DNA encoding the antibody can be designed and synthesized according to the antibody amino acid sequence described herein in a conventional manner, placed in an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture 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 insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoters of cytomegalovirus, and promoters of other cells such as the actin promoter can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1 (+ / -), pcDNA / Zeo (+ / -), pcDNA3.1 / Hygro (+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

[0158] In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large quantities and 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 shown in SEQ ID NO:57, wherein the underlined portion encodes VH CDR; the amino acid sequence of the encoded heavy chain of antibody 6Y-G4 is 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] ccaagctctatcgagaaaactatttctaaggctaaggacagcctagggaccacaggtgtacaccctgccccctagtcaggagga

[0173] tgactaagaaccaggtctcactgacctgtctggtgaaagggttctatccttcagatattgcagtggagtgggaatccaatggtcagccag

[0174] agaacaattacaagacaactccacccgtgctggacagcgatgggtctttctttctgtattctagactgaccgtggacaaaagtcgctggc

[0175] aggagggtaatgtcttttcttgtagtgtgatgcacgaagccctgcacaaccactacactcagaaaagcctgtcactgtccctgggtaaaSEQ ID NO:60:

[0176] QVQLQESGPGLVKPSETLSLTCTVSGGSLDNYYWSWIRQPPGKGLEWIGYIYYSGNT

[0177] NEW SLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGRFLERYWGQGTLVTVS

[0178] SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

[0179] SSGLIGHTSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDCURVESHYGPPCPPCPAPEFLGG

[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 portion encodes VL CDR; the amino acid sequence of the encoded antibody 6Y-G4 light chain 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 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] NYNPSLKSRVTISSVDTSKNQFSLRLRSVTAADTAVYYCARGGRFLERYWGQGTLVTV

[0214] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

[0215] QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

[0216] LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[0217] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0218] VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[0219] YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0220] In some embodiments, the amino acid sequence of the antibody 17 light chain is shown in SEQ ID NO:64.

[0221] SEQ ID NO:64:

[0222] DIQMTQSPSSSVSASVGDRVTISCRANQAIGTWLAWYQQKPGKAPKLLIYAASTLQSGV

[0223] PSRFSGSGSGTEFTLTISSLQAEDVAVYYCQQYYTTPLTFGGGTKLEIKRTVAAPSVFIFP

[0224] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0225] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0226] In some embodiments, the amino acid sequence of the heavy chain of antibody L12 is shown as SEQ ID NO: 63. In some embodiments, the amino acid sequence of the light chain of antibody L12 is shown as 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 to the administered immunoglobulin in the human.

[0229] CD47 antibodies are produced, for example, by phage display methods using antibodies containing only human sequences. Such methods are well known in the art, for example, WO92 / 01047 and U.S. Patent No. 6,521,404. In this method, a natural or recombinant CD47 source or fragment thereof is used to screen a combinatorial library of phages carrying random light and heavy chain pairs. In another method, CD47 antibodies can be produced by such a method: at least one method step includes immunizing a transgenic non-human animal with a human CD47 protein. In such methods, some transgenic non-human animals have lost their endogenous heavy chain and / or kappa light chain loci, and the rearrangement required to produce genes encoding immunoglobulins that respond to antigens cannot occur. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Therefore, in response to the administered antigen, the human locus is rearranged to provide genes encoding human variable regions that are immunospecific to the antigen. Therefore, upon immunization, the transgenic mouse produces B cells that secrete fully human immunoglobulins.

[0230] The generation of antibodies with reduced immunogenicity is also achieved using appropriate libraries via humanization techniques, chimerization techniques and display techniques. It should be understood that mouse 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. 12125-168 (1992). The antibodies of interest can be engineered by recombinant DNA technology to replace CH1, CH2, CH3, hinge domains and / or backbone domains with 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). It is also known in the art to use Ig cDNA to construct chimeric immunoglobulin genes (Liu et al. PNAS 84: 3439 (1987) and J. Immunol. 139: 3521 (1987)). mRNA is isolated from hybridomas or other cells that produce antibodies and used to produce cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is constructed and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to the human constant region sequence. The sequence of the human constant region gene can be found in Sequences of Proteins of immunological Interest (NIH publication no. 91-3242 (1991)) published by Kabat et al. Human C region genes can be easily obtained from known clones. The choice of isotype will be guided by the desired effector function, such as complement fixation or antibody-dependent cellular toxicity activity. Preferred isotypes are IgG1 and IgG2. Any human light chain constant region, ie, kappa or lambda, can be used and chimeric humanized antibodies can then be expressed by conventional methods.

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

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

[0233] The CD47 antibodies described herein can be expressed by vectors comprising DNA fragments encoding the above antibodies. Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. A suitable vector is typically a vector that encodes a fully functional human heavy chain constant region (CH) or light chain constant region (CL) immunoglobulin sequence, and carries appropriate restriction sites so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing typically 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 occurs at the splice region present in the human CH exon. Polyadenylation and transcription termination occur at the natural chromosomal site downstream of the coding region. The resulting chimeric antibody can be linked to any strong promoter, including retroviral LTR, such as SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3: 280 (1983)), Rous sarcoma virus LTR (Gorman et al. PNAS 79: 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al. Cell 41: 885 (1985)). In addition, natural Ig promoters, etc. can be used.

[0234] Fc modification

[0235] The relevant effector functions of the antibodies described herein can be modified to improve, 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 the effector functions, thereby reducing the potential for killing normal cells.

[0236] In some embodiments, the antibodies described herein are IgG isotypes. In some embodiments, the constant region of the antibody is a human IgG1 isotype having an amino acid sequence as 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 change Fc receptor interactions, such as Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the amino acid Leu234 (Kabat numbering) on ​​the antibody constant region is modified to change Fc receptor interactions, such as Leu234Ala (L234A). In some embodiments, amino acids 234 and 235 on the antibody constant region are modified simultaneously, e.g., 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, amino acid 235 on the human IgG4 constant region is modified to change Fc receptor interaction, such as Leu235Glu (L235E). In some embodiments, the hinge region and amino acid 235 within the human IgG4 constant region 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, amino acids Ser228, Leu235, and Asn297 on the human IgG4 constant region are modified (e.g., S228P / L235E / N297A).

[0239] Use of antibodies against CD47

[0240] In some embodiments, CD47 antibodies including the present invention can be used to diagnose, prognose, monitor, treat, alleviate and / or prevent diseases or lesions associated with abnormal CD47 expression, activity and / or signal transduction in subjects. Treatment regimens can be implemented by identifying subjects (such as human patients) suffering from diseases or conditions associated with abnormal CD47 expression, activity and / or signal transduction (such as cancer or other tumor conditions), or at risk of developing diseases, using standard methods. In some embodiments, methods for treating diseases or conditions involving abnormal CD47 expression, activity and / or signal transduction are provided, comprising administering an effective amount of an antibody described herein or a formulation thereof to a subject in need of treatment of the disease or condition. Administration of antibodies can eliminate or inhibit or interfere with the expression, activity and / or signal transduction function of a target (such as CD47). Administration of antibodies can eliminate or inhibit or interfere with the binding of a target (such as CD47) to an endogenous ligand (such as SIRPα) to which it is bound in its natural state. For example, the antibody binds to the target and regulates, blocks, inhibits, reduces, antagonizes, neutralizes or interferes with the expression, activity and / or signal transduction of CD47.

[0241] In some embodiments, diseases or conditions involving abnormal CD47 expression, activity and / or signal transduction include blood cancers and / or solid tumors. Blood cancers include, for example, leukemias, lymphomas and myelomas. In some embodiments, certain forms of leukemia include acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); myeloproliferative disease / tumor (MPDS); and myelodysplastic syndrome. In some embodiments, certain forms of lymphoma include Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma and follicular lymphoma (small cells and large cells). In some embodiments, certain forms of myeloma include multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence-Jones myeloma. Solid tumors include, for example, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer.

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

[0243] The therapeutically effective amount of the antibody of the present invention generally relates to the amount required to achieve the therapeutic goal. The amount required for administration depends on the binding affinity of the antibody to its specific antigen, the severity of the disease, disorder or condition, the route of administration, the rate at which the antibody administered in the subject receiving administration is exhausted 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 dosage 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 any two values ​​in these numerical values ​​(including the end point). Common dosage frequency ranges are, 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 appropriate physiological samples, for diagnostic methods, for protein imaging, etc.). In a given embodiment, an antibody specific for CD47 or a derivative, fragment, analog or homolog thereof, comprising an antigen binding domain derived from the 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 antibodies specific for CD47 by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) directed against CD47 proteins can be used to detect proteins in biological samples. In some embodiments, CD47 can be detected in biological samples as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineaminofluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S or3 H.

[0246] In 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 their protein fragments) in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody, or more preferably a monoclonal antibody. A complete antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The detection method of the embodiment 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 proteins 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, Vol. 42, JR Crowther Human Press, Totowa, NJ, 1995; Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985). In addition, in vivo detection techniques for analyte proteins include introducing labeled anti-analyte protein antibodies into the subject. For example, the antibody can be labeled with a radioactive label, and the presence and location of the radioactive label in the subject can then be detected by standard imaging techniques.

[0247] Therapeutic administration and formulation of CD47 antibodies

[0248] The antibodies and 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, for example, see 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, Vol. 4, 1991, M. Dekker, New York.

[0249] Such compositions generally comprise an antibody and a pharmaceutically acceptable carrier. In some embodiments, an antibody fragment is used as the smallest 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, bacteriostats, isotonic agents, and absorption delaying agents that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences. Such carriers or diluents may be selected to 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 readily achieved by filtration through sterile filtration membranes.

[0252] The pharmaceutical composition of the embodiment is generally compatible with its 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, fixed oil, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as histidine hydrochloride, acetate, citrate, or phosphate; an osmotic pressure regulator, such as sodium chloride or dextrose; a stabilizer, such as arginine, methionine, trehalose, sucrose, sorbitol; a surfactant, such as Tween 20, Tween 80. pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. The pharmaceutical composition may be packaged in an ampoule, a disposable syringe, or a glass or plastic multidose vial. In some embodiments, pharmaceutical compositions suitable for injection purposes include sterile aqueous solutions (here water-soluble) or dispersions and sterile powders for instant preparation of sterile injections or dispersions. When used, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions and must be able to prevent the contamination of microorganisms such as bacteria and fungi. Prolonged absorption of injection compositions can be achieved by including agents that delay absorption such as aluminum monostearate and gelatin in the composition.

[0253] For transmucosal or transdermal administration, penetrants suitable for permeation barriers are used in the formulation. Such penetrants are generally known in the art and include detergents, bile salts and fusidic acid derivatives such as those used 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 into an ointment, an ointment, a gel, or a cream as is generally known in the art.

[0254] The pharmaceutical composition is formulated in dosage unit form for ease of administration and uniform dosage. As used herein, dosage unit form refers to physically separable units suitable as unit dosages 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 combination with the required pharmaceutical carrier.

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

[0256] The pharmaceutical compositions described herein may also include other active ingredients, preferably those having complementary activities but not negatively affecting each other, depending on the specific conditions to be treated. In some embodiments, the compositions may include agents that enhance their function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such active ingredients are appropriately combined in an amount effective for the intended purpose.

[0257] In one embodiment, one or more active ingredients including CD47 antibodies can be administered in combination therapy, i.e., in combination with other agents such as therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cytostatic agents, etc.). The term "in combination" herein refers to administering the agents substantially simultaneously, concomitantly, or sequentially. In some embodiments, such combination therapy can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies.

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

[0259] The citation of publications and patent documents herein does not constitute an admission that any of the above is relevant prior art, nor does it constitute an admission of its content or date. Now that the present invention has been described in a written specification, it will be appreciated by those skilled in the art that the present invention may be practiced in a variety of embodiments, and the description above and the examples below are intended to illustrate, but not to limit, the claims of the present invention. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0260] Example

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

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

[0263] Jurkat cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences with the catalog number of Clone E6-1.

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

[0265] Macaque CD47-Fc was purchased from Creative BioMart, model number CD47-745C.

[0266] Example 1: Preparation of CD47 antigen

[0267] Preparation of CD47 antigen: A 6×HIS tag (HHHHHH, SEQ ID NO: 2) or human Fc (underlined portion in SEQ ID NO: 3) (refer to R&D system) was added to the C-terminus of the human CD47 extracellular domain (ECD) protein (SEQ ID NO: 1) to construct a CD47 recombinant protein. The ECD region of human CD47 was synthesized with 6×HIS or Fc 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 by immobilized metal affinity chromatography (IMAC) using proteinA 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, where the C-terminus of VH and the N-terminus of VL are connected by a connecting short peptide whose sequence is shown in SEQ ID NO: 10 (GGGGSGGGGSGGGGS); 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 (the C-terminus of 17-VH (as shown in SED ID NO: 11) and the N-terminus of 17-VL (as shown in SED ID NO: 12) are connected by a connecting short peptide as shown in SEQ ID NO: 10 to obtain 17-ScFv).

[0270] The combinations of VH and VL of 30 kinds of IgG1 full antibodies are shown in Table 2; VH and CH constitute the heavy chain of the antibody, and VL and CL constitute 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 antibody heavy chain is composed of 17-VH (as shown in SED ID NO:11) and CH as shown in SED ID NO:3, and the antibody light chain is composed of 17-VL (as shown in SED ID NO:12) and CL as 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 VH and VL sequences corresponding to Table 2 are shown in Table 3, wherein 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. The plasmid was then transiently transfected into HEK293F cells to allow the HEK293F cells to express the antibody of the above amino acid sequence. The CD47 antibody was purified by immobilized metal affinity chromatography (IMAC) using a protein A column (GE Healthcare), and the sequence was consistent with the expected sequence for biological activity 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 CDRs

[0283]

[0284]

[0285] Note: L12X-VH stands for 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 CDRs

[0287]

[0288] Note: , 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 main Fc-dependent functions of antibodies (such as CD47 antibodies) are complement-dependent cytotoxicity (CDC) initiated by C1q binding 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 recognizing opsinized target cells via FcγRI. Each antibody subclass has differences in the ability to mediate Fc-dependent effector activity. In humans, IgG1 and IgG3 subclasses are highly effective for CDC due to binding to C1q. In addition, the IgG1 subclass has the highest affinity for FcγR and is therefore the most effective in ADCC and Fc-dependent ADCP. The IgG4 subclass does not have C1q binding ability and has a greatly reduced FcγR binding affinity and therefore has a significantly weakened effector function.

[0290] Based on antibody 6Y-G1, an IgG4P subclass antibody 6Y-G4 was prepared with a mutation S228P to stabilize the hinge region of the antibody (CH as shown in SEQ ID NO: 3 in 6Y-G1 was replaced with CH as shown in SEQ ID NO: 4, and the other sequences remained unchanged), thereby further weakening the Fc effect of the antibody.

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

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

[0293] Other ScFvs were prepared and purified using a similar method and their sequences were consistent with the expected ones after sequencing.

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

[0295] caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccctcg

[0296] ataattactactggagctggatccggcagcccccagggaagggactggagtggattggatatatctattacagtgggaacaccaactac

[0297] aacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctcgttgaggctgaggtctgtgaccgctgcgga

[0298] cacggccgtgtattactgtgcgagaggagggcgatttttggaacgttatggggccagggaaccctggtcaccgtctcctcaggtgga

[0299] ggcggttcaggcggaggtggctctggcggtggcggatcggacatccagatgacccagtctccatcttccgtgtctgcatctgtaggag

[0300] acagagtcaccatctcttgtcgggcgaatcaggctattggcacttggttagcctggtatcagcaaaagccaggaaaagcccctaagctc

[0301] ctgatctatgcggcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagc

[0302] agcctgcaggctgaagatgtggcagtttattactgtcagcaatattatactactcccctcactttcggcggagggaccaagctggagatc

[0303] aaa

[0304] Example 3: Biological activity of CD47 antibody

[0305] 1. FACS detection of 17-ScFv binding activity

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

[0307] 17-ScFv and Hu5F9-G4 antibodies (the heavy chain of Hu5F9-G4 consists of the variable region shown in SEQ ID NO: 8 and the constant region shown in SEQ ID NO: 4, and the light chain consists of the variable region shown in SEQ ID NO: 9 and the constant region shown in SEQ ID NO: 5) were diluted with PBS at 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 Jurkat cell surface CD47 at 4°C for 30 min, and washed once with PBS; Goat anti-human IgG Fc-PE (eBioscience, Catalog No.: 2183639) fluorescent secondary antibody was added, incubated at 4°C for 15 min, and washed twice with PBS; the average fluorescence signal value of PE-A was detected by upflow cytometry. Figure 1A-1B The results showed that 17-ScFv could bind to cell surface CD47 in a dose-dependent manner.

[0308] 2. SIRP-α blocking activity

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

[0310] Using the ELISA competition 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, washed four times with PBST, and then SIRPα-Fc-Bio (0.1 μg / ml) (refer to Thermo Scientific The ligand (prepared with NHS-Biotin Reagents kit) was incubated with CD47-His antigen at room temperature for 1 hour and washed four times with PBST. The bound SIRPα-Fc-Bio and HRP-conjugated streptavidin secondary antibody produced a chemiluminescent reaction, and the OD450 value was detected by a plate reader. The IC value of the antibody was calculated based on the OD450 value. 50 , thereby determining the SIRPα blocking activity of the antibody. Figures 3A-3E It was shown that all the antibodies tested exhibited dose-dependent inhibition of SIRPα binding to the CD47 antigen.

[0311] 3. Red blood cell agglutination assay

[0312] Detection of the red blood cell agglutination reaction of CD47 antibody. Collect 5 ml of fresh blood from a healthy donor into an anticoagulant tube containing sodium heparin, first separate PBMC with lymphocyte separation solution (for other purposes), then take 1 ml of red blood cells from the bottom of the tube into 6 ml of normal saline, wash repeatedly, centrifuge at 2000 rpm for 5 minutes, wash until the supernatant has no obvious red color, and then resuspend with normal saline to make a 2% red blood cell suspension. At room temperature, 50 μl of CD47 antibody diluted 2 times with PBS (maximum concentration 800 nM, 12 gradients in total) was mixed with 50 μl of 2% red blood cell suspension and added to a 96-well U-shaped plate for incubation for 4 hours. Then evaluate the antibody red blood cell agglutination, tilt the 96-well U-shaped plate 45°, and observe the flow direction of the red blood cell cluster. If it is a "straight line", it means that the red blood cells have not agglutinated. Most of the tested CD47 antibodies induced erythrocyte agglutination at high concentrations (antibody 17 had obvious erythrocyte agglutination), and did not cause erythrocyte agglutination at around 6 nM. Among them, antibody L12, antibody 6Y-G1 (i.e. antibody L12-6), and antibody 6Y-G4 did not cause any erythrocyte agglutination reaction ( Figures 4A-4E). The positive control antibody Hu5F9-G4 causes hemagglutination of red blood cells, while AB6.12-G1 and the ligand SIRPα do not cause hemagglutination of red blood cells. The heavy chain of AB6.12-G1 consists of the variable region shown in SEQ ID NO:6 and the constant region shown in SEQ ID NO:3, and the light chain consists of the variable region shown in SEQ ID NO:7 and the constant region shown in SEQ ID NO:5. The heavy chain of Hu5F9-G4 consists of the variable region shown in SEQ ID NO:8 and the constant region shown in SEQ ID NO:4, and the light chain consists of the variable region shown in SEQ ID NO:9 and the constant region shown in SEQ ID NO:5. See Table 6.

[0313] Table 6: Positive antibody sequence list

[0314]

[0315]

[0316] 4. CD47 binding assay (ELISA):

[0317] The binding activity of CD47 antibody to human CD47 and macaque CD47 was determined by ELISA. Antibody 17, antibody L12, antibody 6Y-G1, antibody 6Y-G4 and antibody Hu5F9-G4 were diluted 3-fold with PBS from 5 μg / ml to 8 concentrations, and then incubated with human or macaque CD47-Fc (2 μg / ml) antigen coated on the ELISA plate at room temperature for 1 hour, washed four times with PBST, and then HRP-anti-kappa (1:10000, sigma) secondary antibody was added and incubated with the antibody at room temperature for 1 hour, and washed four times with PBST. The bound HRP-anti-kappa produced a chemiluminescent reaction with the TMB substrate, and the OD450 value was detected by a plate reader. The EC value of the antibody was converted based on the OD450 value. 50 , thereby determining the binding activity of the antibody to human or macaque CD47. Figure 5A-5B It was shown that all the tested antibodies exhibited dose-dependent binding of the antibodies to the CD47 antigen, and the parameters are shown in Table 7.

[0318] Table 7: Affinity of CD47 antibodies binding to human CD47 and macaque C47

[0319]

[0320] 5. CD47 binding assay (Fortebio)

[0321] The binding activity of 11 antibodies (antibody L12-1, antibody L12-2, antibody L12-3, antibody L12-4, antibody L12-5, antibody L12-6, antibody L12-7, antibody L12-9, antibody L12-10, antibody L12-11, antibody L12) and Hu5F9-G4 to human CD47 was measured by the Fortebio method.

[0322] The Protein A sensor was pre-wetted in PBS for 10 minutes for use. The antibody to be tested was diluted to 10 μg / mL with PBST containing 0.5% BSA at pH 6.8 (hereinafter referred to as dilution buffer), and the PA sensor was solidified in the antibody until the signal was about 1.7 nm. The CD47-His antigen was diluted to 50, 25, 12.5, and 6.25 nM with dilution buffer. The dilution buffer, gradient concentration antigen dilution solution, regeneration buffer (1M MgCl2 at pH 8.4), and neutralization buffer (PBST) were added to the corresponding columns of the 96-well plate in sequence, and the following steps were run: ① Baseline: Detect the baseline in the dilution buffer for 60 seconds; ② Association: Combine in the antigen gradient dilution solution 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 Regeneration and Neutralization were cycled 3 times.

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

[0324] Table 8: Antibody Affinity

[0325]

[0326]

[0327] 6. Phagocytosis

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

[0329] Example 4: Effects of CD47 Antibody on Blood Cells

[0330] Prior to the present invention, known CD47 binding molecules that block SIRPα and contain an Fc domain (e.g., CD47 antibodies and recombinant SIRPα-Fc fusion proteins) all induce erythrocyte and platelet reduction to varying degrees. To this end, the effects of the antibodies of the present invention on blood cells were evaluated in cynomolgus monkeys. A total of 8 cynomolgus monkeys that met the test requirements were selected, of ordinary grade, half female, and the weight ranges of males and females were 3.70-5.30 kg and 2.75-3.55 kg, respectively; they were divided into 4 groups by a simple randomization scheme according to body weight, with 2 animals in each group, half male and half female. The negative control group was given normal saline, and the test substances were antibody 6Y-G1 (i.e., antibody L12-6) and Hu5F9-G4, AB6.12-G1, intravenous infusion of solvent: normal saline, intravenous infusion, once a week, 5 times of medication, each time according to the dose of 1→10→10→30→30 mg / kg, the administration volume was 10 ml / kg. After the end of administration, observation was conducted until the 42nd day. Figures 7A-7D The results showed that the three antibodies all caused a decrease in hemoglobin and red blood cells after administration, which dropped to a low point in about two weeks and then gradually recovered to normal levels; the platelet content fluctuated before and after administration and returned to normal after drug withdrawal; the number of reticulocytes showed an increasing trend after administration, among which 6Y-G1 had a higher increase in reticulocyte count than the other two antibodies.

[0331] Example 5: In vivo anti-tumor activity of Fc variants of CD47 antibodies

[0332] Evaluate the anti-tumor effect of the test drug antibody L12 in the human hematologic tumor MV4-11 system tumor model. NOD.SCID mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., body weight 18g-22g, mouse age 6-8 weeks) were inoculated with MV4-11 cells (ATCC) through the tail vein. About 4 weeks after inoculation, a human hematologic tumor MV4-11 system tumor model was established. The experiment was divided into test AB6.12-G1 0.2mg / mouse, Hu5F9-G4 0.2mg / mouse, antibody L12 0.2mg / mouse and solvent (normal saline) control group, 6 mice in each group, intraperitoneal injection, three times a week, for a total of three weeks. The efficacy was evaluated based on the extended survival time (ILS), and the safety was evaluated based on the weight changes and deaths of animals during the administration period. Figure 8 The results showed that the survival of the test drugs AB6.12-G1 (0.2 mg / mouse) and Hu5F9-G4 (0.2 mg / mouse) was prolonged by 7.4% and 6.5% compared with the vehicle control group, with no statistically significant difference (p=1.000), and failed to show significant anti-tumor effect. The median survival of the antibody L12 (0.2 mg / mouse) was 69 days, which 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 per group, day 0). Group 1: vehicle control (normal saline); Group 2: Hu5F9-G4 (positive control); Group 3: 6Y-G4. When tumors were palpable (50 mm 3 , day 4) were treated with each antibody or vehicle (buffer only) and when the tumor volume reached approximately 3000 mm 3 The mice were sacrificed at 4 dpi. Tumor volumes were measured every 3 days. Antibodies were administered intraperitoneally (IP) at a dose of 100 μg 3 times a week for 3 weeks (9 doses per mouse). Treatment started on day 4 and ended on day 21. Fig. 9 As shown in the figure, the test drugs 6Y-G4 and Hu5F9-G4 treatment groups produced extremely significant anti-tumor effects at a dosage of 0.1 mg / mouse. The growth of tumors was significantly inhibited after the start of administration. By the 17th day after the start of administration (PG-D17), the tumors of most mice disappeared. The average tumor volumes of the 6Y-G4 (0.1 mg / mouse) and Hu5F9-G4 (0.1 mg / mouse) treatment groups were 4 mm 3 (7 out of 8 mice had their tumors disappear), 7mm3 (5 out of 8 mice had their tumors disappear), and the relative tumor inhibition rates TGI were 99.87% and 99.76%, respectively, which were statistically significantly different from the vehicle control group (p values ​​were all < 0.001). Sequence Listing <110> Bio-Thera Pharmaceuticals Co., Ltd. <120> CD47 antibody and its application <130> DSP1F212397JW <160> 65 <170> SIPOSequenceListing 1.0 <210> 1 <211> 139 <212> PRT <213> Homo sapiens <400> 1 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro 130 135 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <400> 2 His His His His His His 1 5 <210> 3 <211> 330 <212> PRT <213> Artificial Sequence <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 4 <211> 327 <212> PRT <213> Artificial Sequence <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence <400> 5 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 6 <211> 120 <212> PRT <213> Artificial Sequence <400> 6 Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Ala Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Pro Asp Gln Gly Asp Thr Glu Tyr Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Arg Asp Arg Ser Met Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Asn Ala Ala Tyr Gly Ser Ser Ser Tyr Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <400> 7 Asn Ile Gln Met Thr Gln Ser Pro Ser Ala Met Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile His Arg Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys His Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <400> 9 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <400> 10 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 11 <211> 116 <212> PRT <213> Artificial Sequence <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Phe Leu Glu Arg Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Asn Gln Ala Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <400> 13 Glu Ile Val Leu Thr Gln Pro Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100 105 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <400> 14 Asp Asn Tyr Tyr Trp Ser 1 5 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <400> 15 Asp Gly Tyr Tyr Trp Ser 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <400> 16 Asn Asp Tyr Tyr Trp Ser 1 5 <210> 17 <211> 16 <212> PRT <213> Artificial Sequence <400> 17 Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <400> 18 Arg Ile Tyr Gly Asn Ser Thr Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <400> 19 Arg Ile Tyr Gly Asn Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 20 <211> 16 <212> PRT <213> Artificial Sequence <400> 20 Arg Ile Tyr Tyr Asn Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> twenty one <211> 16 <212> PRT <213> Artificial Sequence <400> twenty one Arg Ile Tyr Gly Asp Ser Ala Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> twenty two <211> 16 <212> PRT <213> Artificial Sequence <400> twenty two Arg Ile Tyr Asp Gly Ser Gly Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <400> twenty three Gly Gly Arg Phe Leu Glu Arg Tyr 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <400> twenty four Gly Gly Arg Ile Phe Gly Gly Tyr 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <400> 25 Gly Gly Arg Ile Phe Ala Gly Tyr 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <400> 26 Gly Gly Arg Ile Leu Ala Gly Tyr 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <400> 27 Gly Gly Arg Ile Phe Ser Gly Tyr 1 5 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <400> 28 Gly Arg Gly Ile Phe Gly Tyr 1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <400> 29 Gly Gly Arg Ile Phe Gly His 1 5 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <400> 30 Arg Ala Ser Leu Ser Ile Gly Ser Phe Leu Asn 1 5 10 <210> 31 <211> 11 <212> PRT <213> Artificial Sequence <400> 31 Arg Ala Asn Gln Ala Ile Gly Thr Trp Leu Ala 1 5 10 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <400> 32 Ala Ala Ser Ser Leu Arg Ser 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <400> 33 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <400> 34 Gln Gln Thr Tyr Thr Thr Pro Tyr Thr 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <400> 35 Gln Gln Tyr Tyr Thr Thr Pro Leu Thr 1 5 <210> 36 <211> 116 <212> PRT <213> Artificial Sequence <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Gly Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 37 <211> 116 <212> PRT <213> Artificial Sequence <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Ala Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 38 <211> 116 <212> PRT <213> Artificial Sequence <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Gly Asn Ser Thr Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Ala Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 39 <211> 116 <212> PRT <213> Artificial Sequence <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asn Asp Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Gly Asn Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Leu Ala Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 40 <211> 116 <212> PRT <213> Artificial Sequence <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Tyr Asn Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Leu Ala Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 41 <211> 116 <212> PRT <213> Artificial Sequence <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Ser Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 42 <211> 115 <212> PRT <213> Artificial Sequence <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Gly Ile Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 43 <211> 116 <212> PRT <213> Artificial Sequence <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Gly Asp Ser Ala Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Ser Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 44 <211> 115 <212> PRT <213> Artificial Sequence <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Gly His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 45 <211> 107 <212> PRT <213> Artificial Sequence <400> 45 Glu Ile Val Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 107 <212> PRT <213> Artificial Sequence <400> 46 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Pro Gly Thr Arg Leu Asp Ile Lys 100 105 <210> 47 <211> 107 <212> PRT <213> Artificial Sequence <400> 47 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Pro Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 48 <211> 107 <212> PRT <213> Artificial Sequence <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 49 <211> 107 <212> PRT <213> Artificial Sequence <400> 49 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 50 <211> 116 <212> PRT <213> Artificial Sequence <400> 50 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Phe Leu Glu Arg Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 51 <211> 107 <212> PRT <213> Artificial Sequence <400> 51 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 52 <211> 107 <212> PRT <213> Artificial Sequence <400> 52 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 53 <211> 107 <212> PRT <213> Artificial Sequence <400> 53 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 54 <211> 107 <212> PRT <213> Artificial Sequence <400> 54 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 55 <211> 107 <212> PRT <213> Artificial Sequence <400> 55 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 56 <211> 116 <212> PRT <213> Artificial Sequence <400> 56 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Asp Gly Ser Gly Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Phe Gly Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 57 <211> 1329 <212> DNA <213> Artificial Sequence <400> 57 caggtgcagc tgcaggagtc cggccctggc ctggtgaagc cttccgagac cctgtccctg 60 acctgtaccg tgagcggcgg cagcctggat aactattact ggagctggat ccggcagcct 120 cctggcaagg gcctggagtg gatcggctac atctactatt ccggcaacac caattacaac 180 ccttccctga agagccgggt gaccatctcc gtggacacca gcaagaacca gtttagcctg 240 aagctgtcct ccgtgaccgc cgctgatacc gccgtgtact actgtgccag gggcggccgg 300 ttcctggaga gatattgggg ccagggtacc ctcgtgaccg tgtccagcgc tagcaccaag 360 ggcccttccg tgttccccct ggccccctgt agccggtcca cctctgagag caccgctgct 420 ctgggctgtc tggtgaagga ttactttccc gaaccggtga ccgtgtcatg gaactccggg 480 gctctgacat ccggtgtcca cacttttcct gcagtgctgc agtcatccgg cctgtacagc 540 ctgagctctg tggtcacagt cccaagttca tccctgggaa ccaagacata tacttgcaac 600 gtggatcata aacccagcaa tactaaggtc gacaaacgag tggagtctaa gtacggacca ccttgcccac catgtccagc acctgagttc ctgggaggac caagcgtgtt cctgtttcct 720 ccaaagccta aagataccct gatgatcagt cggactcccg aggtcacctg cgtggtcgtg 780 gacgtgtccc aggaggaccc tgaagtccag ttcaactggt acgtggacgg cgtcgaagtg 840. cacaatgcta agacaaaacc tcgagagga cagtttaact ccacataccg tgtcgtgagc gtcctgactg tgctgcatca ggattggctg aacggcaagg agtataagtg caaagtgagc 960 aatagggac tgccaagctc tatcgagaaa actatttcta aggctaaagg acagcctagg gaccacagg tgtacaccct gccccctagt gaccaga tgactaaga ccaggtctca ctgacctgtc tggtgaaagg gttctatcct tcagatattg cagtggagtg ggaatccaat ggtcagccag agaacaatta caagacaact ccacccgtgc tggacagcga tgggtctttc tttctgtatt ctagactgac cgtggacaaa agtcgctggc aggagggtaa tgtcttttct 1320. tgtagtgtga tgcacgaagc cctgcacaac cactacactc agaaaagcct gtcactgtcc ctgggtaaa 1329 <210> 58 <211> 645 <212> DNA <213> Artificial Sequence <400> 58 gagatcgtgc tgacccagtc cccctccagc ctgagcgcca gcgtgggaga ccgggtgacc 60 atcccctgcc gggcttccct gtccatcggc agcttcctga actggtatca gcagaggcct 120 ggcgaggccc ctaagctgct gatctttgcc gcttccagcc tgcggagcgg cgtgcctagc 180 aggttctccg gcagcggctc cggcaccgat ttcaccctga ccatcagcgg cctgcagccc 240 gaggatttcg ccacctacta ctgccagcag acctacacca ccccttacac ctttggccag 300 ggcaccaagg tggacatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga 645 <210> 59 <211> 714 <212> DNA <213> Artificial Sequence <400> 59 60. caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc acctgcactg tctctggtgg ctccctcgat aattactact ggagctggat ccggcagccc 120 180. 180. 180. 180. 180. 180. 180. 180. 180 ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctcgttg aggctgaggt ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aggagggcga 300 360. tttttggac gttactgggg ccaggacc ctggtcaccg tctcctcagg tggaggcggt tcaggcggag gtggctctgg cggtggcgga tcggacatcc agatgaccca gtctccatct 420 tccgtgtctg catctgtagg agacagagtc accatctctt gtcgggcgaa tcaggctatt 480 ggcacttggt tagcctggta tcagcaaaag ccaggaaag cccctaagct cctgatctat 540 gcggcatcca ctttgcaaag tggggtccca tcaaggttca gcggcagtgg atctgggaca 600 gaattcactc tcaccatcag cagcctgcag gctgaagatg tggcagttta ttactgtcag 660 caatattata ctactcccct cactttcggc ggagggacca agctggagat caaa 714 <210> 60 <211> 443 <212> PRT <213> Artificial Sequence <400> 60 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Phe Leu Glu Arg Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 61 <211> 214 <212> PRT <213> Artificial Sequence <400> 61 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 62 <211> 446 <212> PRT <213> Artificial Sequence <400> 62 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Phe Leu Glu Arg Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 63 <211> 446 <212> PRT <213> Artificial Sequence <400> 63 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Asp Asn Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Arg Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Phe Leu Glu Arg Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 64 <211> 214 <212> PRT <213> Artificial Sequence <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Asn Gln Ala Ile Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 65 <211> 107 <212> PRT <213> Artificial Sequence <400> 65 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Leu Ser Ile Gly Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Thr Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100 105

Claims

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

34.

2. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment comprises a heavy chain variable region VH, the amino acid sequence of which is as shown in SEQ ID NO: 11 or 50, or has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 11 or 50.

3. The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that The antibody or antigen-binding fragment comprises a light chain variable region VL, and the amino acid sequence of VL is as shown in SEQ ID NO:65, or has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:

65.

4. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment comprises a heavy chain variable region VH and a light chain variable region VL, the amino acid sequence of the VH is shown in SEQ ID NO: 11 or 50, and the amino acid sequence of the VL is shown in SEQ ID NO:

65.

5. The antibody or antigen-binding fragment according to claim 1, wherein The antibody comprises a heavy chain H and a light chain L; the amino acid sequence of the heavy chain H is shown in SEQ ID NO:60 or 62, and the amino acid sequence of the light chain L is shown in SEQ ID NO:

61.

6. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment is of IgG isotype, selected from IgG1 subtype, IgG2 subtype, IgG3 subtype, or IgG4 subtype.

7. The antibody or antigen-binding fragment of claim 6, which is IgG4P.

8. A composition, characterized in that The composition comprises the antibody or antigen-binding fragment according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. A biomaterial comprising (1) A polynucleotide characterized in that: The polynucleotide encodes the antibody or antigen-binding fragment according to any one of claims 1 to 7; 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 7; or (3) A cell, characterized in that the cell contains one or more polynucleotides encoding the antibody or antigen-binding fragment according to any one of claims 1 to 7.

10. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 7, or the composition according to claim 8, or the biomaterial according to claim 9 in the preparation of a drug for treating cancer, wherein the cancer is a solid tumor or a blood cancer, wherein: The blood cancer is acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myeloproliferative disease / neoplasm, myelodysplastic syndrome, Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma, follicular lymphoma, multiple myeloma, giant cell myeloma, heavy chain myeloma or light chain or Bence-Jones myeloma; The solid tumor is breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, prostate cancer, esophageal cancer, liver cancer or kidney cancer.

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