阅读说明：本技术 包含cd47抗体和细胞因子的融合蛋白 (Fusion protein comprising CD47 antibody and cytokine ) 是由 王正毅 曹巍 方磊 郭炳诗 于 2019-04-30 设计创作，主要内容包括：本发明提供含有细胞因子和新的CD 47抗体或其免疫活性片段的融合蛋白,以及含有这种融合蛋白的药物组合物,可用于治疗由CD 47介导的疾病或抑制吞噬或血小板聚集。这些融合蛋白在人体中具有低的免疫原性,并引起低水平的或不引起红血细胞清除或血细胞凝聚。(The present invention provides fusion proteins comprising a cytokine and a novel CD47 antibody or immunologically active fragment thereof, and pharmaceutical compositions comprising such fusion proteins, useful for treating CD 47-mediated diseases or inhibiting phagocytosis or platelet aggregation. These fusion proteins have low immunogenicity in humans and cause low or no red blood cell clearance or hemagglutination.)

1. A fusion protein comprising an isolated monoclonal antibody or immunologically active fragment thereof that binds human CD47 and a cytokine, wherein the monoclonal antibody or immunologically active fragment thereof is fused to the N-terminus of the cytokine with or without a linker between the monoclonal antibody or fragment thereof and the cytokine.

2. The fusion protein of claim 1, wherein the isolated monoclonal antibody or immunologically active fragment thereof comprises:

A Variable Heavy (VH) chain sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, 69, 71, 73, 75, and 77; and

A Variable Light (VL) chain sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, 70, 72, 74, 76, and 78.

3. The fusion protein of claim 2, wherein the isolated monoclonal antibody, or immunologically active fragment thereof, comprises a VH/VL sequence pair comprising VH and VL chain sequences at least 95% identical to a VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NO 1 and SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, and 77 and 78.

4. The fusion protein of claim 2, wherein the isolated monoclonal antibody or immunologically active fragment thereof comprises a VH/VL sequence pair, wherein the VH/VL sequence pair comprises VH and VL chain sequences, and wherein the sequence pair is selected from the group consisting of: SEQ ID NO 1 and SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76, and SEQ ID NO:77 and SEQ ID NO: 78.

5. The fusion protein of claim 2, wherein the isolated monoclonal antibody or immunologically active fragment thereof is chimeric or humanized.

6. The fusion protein of claim 2, wherein the isolated monoclonal antibody or immunologically active fragment thereof prevents the interaction of human CD47 with signal-regulated protein a (sirpa).

7. The fusion protein of claim 2, wherein the isolated monoclonal antibody or immunologically active fragment thereof promotes macrophage-mediated phagocytosis of a CD 47-expressing cell.

8. The fusion protein of claim 2, wherein the isolated monoclonal antibody or immunologically active fragment thereof does not cause a significant level of erythrocyte agglutination or clearance of red blood cells.

9. The fusion protein of claim 2, wherein the isolated monoclonal antibody or immunologically active fragment thereof does not cause erythrocyte agglutination or clearance of red blood cells.

10. The fusion protein of any one of claims 1-9, wherein the cytokine comprises an immunoglobulin (Ig), a hematopoietic growth factor, an interferon, a tumor necrosis factor, an interleukin-17 receptor, or a wild-type or variant of a monomeric glycoprotein.

11. The fusion protein of claim 10, wherein the cytokine is a wild-type or variant of a monomeric glycoprotein.

12. The fusion protein of claim 11, wherein the cytokine is a wild-type or variant of granulocyte macrophage colony stimulating factor (GM-CSF).

13. The fusion protein of any one of claims 1-12, wherein the monoclonal antibody or immunologically active fragment thereof is fused to the cytokine with or without a linker selected from the group consisting of: (G4S)3, (G4S)6, (GS)9, IGD (F30), IGD (F64), IGD (R30), IGN (R64), IGD (R30-Cys), and IGD (R64-Cys).

14. The fusion protein of any one of claims 1-13, wherein the fusion protein inhibits the interaction of human CD47 and human sirpa.

15. The fusion protein of any one of claims 1-14, further comprising a small molecule therapeutic agent or label, and wherein the small molecule therapeutic agent or label is conjugated to the monoclonal antibody or immunologically active fragment thereof, or to a cytokine.

16. The fusion protein of claim 15, wherein the small molecule therapeutic agent is an anti-cancer or anti-inflammatory agent; the label is a biomarker or a fluorescent label.

17. The fusion protein of claim 12, wherein the isolated monoclonal antibody or immunologically active fragment thereof comprises a VH/VL sequence pair at least 95% identical to a pair of VH and VL amino acid sequences selected from the group consisting of: 3 and 4, and 31 and 32; and the cytokine is a wild-type or variant of granulocyte macrophage colony stimulating factor (GM-CSF).

18. The fusion protein of claim 17, wherein the fusion protein comprises:

A Variable Light (VL) chain expression vector at least 95% identical to an amino acid sequence selected from the group consisting of: 108 and 116 SEQ ID NO; and

A Variable Heavy (VH) chain expression vector at least 95% identical to an amino acid sequence selected from the group consisting of: 109, 110, 111, 112, 113, 114, 115, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 147 and 158.

19. The fusion protein of claim 18, wherein the fusion protein comprises a VH/VL pair that is at least 95% identical to a VH and VL amino acid sequence pair selected from the group consisting of seq id nos: 108 and 109, 108 and 110, 108 and 114, 108 and 115, 108 and 117, 108 and 118, 108 and 119, 108 and 120, 108 and 121, 108 and 122, 108 and 123, 108 and 124, 108 and 125, 108 and 126, 108 and 127, 108 and 128, 108 and 132, 108 and 133, 108 and 134, 108 and 135, 108 and 136, 108 and 137, 108 and 138, 108 and 139, 140, 108 and 141, 108 and 142, 108 and 143, 108 and 144 SEQ ID NO, 108 and 145 SEQ ID NO, 108 and 146 SEQ ID NO, and 108 and 147 SEQ ID NO; 116 and 109, 116 and 110, 116 and 114, 116 and 115, 116 and 117, 116 and 120, 116 and 121, 116 and 122, 116 and 124, 116 and 125, 116 and 126, 116 and 127, 116 and 128, 116 and 129, 116 and 130 SEQ ID NO, 116 and 131 SEQ ID NO, 116 and 132 SEQ ID NO, 116 and 133 SEQ ID NO, 116 and 134 SEQ ID NO, 116 and 135 SEQ ID NO, 116 and 136 SEQ ID NO, 116 and 137 SEQ ID NO, 116 and 138 SEQ ID NO, 116 and 139 SEQ ID NO, 116 and 140 SEQ ID NO, 116 and 141 SEQ ID NO, 116 and 142 SEQ ID NO, 116 and 143 SEQ ID NO, 116 and 144 SEQ ID NO, 116 and 145 SEQ ID NO, 116 and 146 SEQ ID NO, 116 and 147, 116 and 148, 116 and 149, 116 and 150, 151, 116 and 152, 116 and 153, 116 and 154, 116 and 155, and 116 and 156.

20. A pharmaceutical composition comprising the fusion protein of any one of claims 1-19, and a pharmaceutically acceptable carrier.

21. A method of treating a disease in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 1-16, or the pharmaceutical composition of claim 17; wherein the disease is cancer, a fibrotic disease, a disease associated with inhibition of phagocytosis, or a disease associated with platelet aggregation.

22. The method of claim 21, wherein the cancer is selected from the group consisting of: ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck tumors, bladder cancer, colorectal cancer, pancreatic cancer, non-hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, Hairy Cell Leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, endometrial cancer, kidney cancer, (benign) fetal tumor, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, renal cancer, colorectal carcinoma, leukemia, Liver cancer, and solid tumors; the fibrotic disease is selected from the group comprising: myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis, bronchitis, and asthma; diseases associated with inhibition of phagocytosis are cardiovascular diseases; diseases associated with platelet aggregation are thrombocytopenia, prolonged bleeding time, Immune Thrombocytopenia (ITP), von willebrand disease (vWD).

23. The method of claim 22, wherein the cardiovascular disease is selected from the group consisting of: atherosclerosis, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral artery disease, and venous thrombosis.

Background

CD47 (differentiation group 47) was first identified as a tumor antigen for human ovarian cancer in the 80's 20 th century. Since then, CD47 was found to be expressed on a variety of human tumor types, including Acute Myeloid Leukemia (AML), chronic myeloid leukemia, Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), bladder cancer, and other solid tumors. High levels of CD47 allow cancer cells to avoid phagocytosis despite the higher level of calreticulin-dominant pro-phagocytic signals on the surface of cancer cells.

CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-associated antigen and MER6, is a multi-spanning transmembrane receptor belonging to the immunoglobulin superfamily. Its expression and activity has been linked to a number of diseases and disorders. It is a widely expressed transmembrane glycoprotein with an immunoglobulin-like domain and five transmembrane domains, which acts as a cellular ligand for SIRP α by signaling NH of protein α (SIRP α)₂The terminal V-like domain binds sirpa. Sirpa is expressed primarily in bone marrow cells, including macrophages, granulocytes, Dendritic Cells (DCs), mast cells, and their precursor cells, including hematopoietic stem cells.

Macrophages scavenge pathogens and damaged or aged cells from the bloodstream by phagocytosis. CD47 on the cell surface binds to its receptor sirpa on macrophages, thereby inhibiting phagocytosis of normal, healthy cells by macrophages. Sirpa inhibits phagocytosis of host cells by macrophages, wherein sirpa on macrophages binds to CD47 expressed on target cells of the host, producing SHP-1 mediated inhibitory signals that negatively regulate phagocytosis.

Consistent with CD47 inhibiting phagocytosis of normal cells, there is evidence that CD47 is transiently upregulated before and during the metastatic phase of Hematopoietic Stem Cells (HSCs) and progenitor cells, and that the level of CD47 on these cells determines the probability of these cells being phagocytosed in vivo.

CD47 is also constitutively upregulated on many cancers, including myeloid leukemia. Overexpression of CD47 on myeloid leukemia lines increases its pathogenicity by allowing it to escape phagocytosis. It has been concluded that during inflammation-mediated mobilization, upregulation of CD47 is an important mechanism to provide protection for normal HSCs, and leukemic progenitors select for this ability to evade killing by macrophages.

Certain CD47 antibodies have been shown to restore phagocytosis and prevent atherosclerosis. Please refer, for example, to Kojima et al, nature, volume 36, 86-90 (8 months and 4 days 2016). The present invention provides a novel CD47 antibody or immunologically active fragment thereof that has low immunogenicity in humans and results in low levels of red blood cell depletion or no red blood cell clearance. Those skilled in the art know that such antibodies may also be referred to as "anti-CD 47 antibodies".

Cytokines are a broad and loose class of small proteins (-5-20 kDa) that play an important role in cell signaling. Their release has an effect on the behavior of the surrounding cells. It can be said that cytokines are involved in autocrine signals, paracrine signals and endocrine signals as immunomodulators. Their clear distinction from hormones remains part of ongoing research. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although some overlap in terms). Cytokines are produced by a variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a particular cytokine may be produced by a variety of cells. They act through receptors, which are particularly important in the immune system; cytokines regulate the balance between humoral and cellular immune responses and regulate the maturation, growth and reactivity of specific cell populations. Some cytokines enhance or inhibit the action of other cytokines in a complex manner. They are of great importance in health and disease, particularly in the host's response to infection, immune response, inflammation, trauma, sepsis, cancer and reproduction.

Granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine, is a well-known immunostimulating factor that promotes innate immunity and adaptive immune responses and is used clinically for myeloid lineage reconstitution. It specifically activates macrophages and transfers the macrophage phenotype from M2 to M1.

To date, no fusion protein of a CD47 antibody and a cytokine has been reported, or even suggested.

Disclosure of Invention

In one aspect, the invention provides isolated monoclonal antibodies and immunologically active fragments thereof that bind to human CD 47. For brevity, these isolated monoclonal antibodies and immunologically active fragments thereof that bind CD47 are hereinafter referred to as "CD 47 antibodies". The CD47 antibodies of the invention are capable of modulating, e.g., blocking, inhibiting, reducing, antagonizing, neutralizing or otherwise interfering with the expression, activity and/or signaling of CD47, or the interaction between CD47 and sirpa. Importantly, the CD47 antibodies of the invention do not typically cause significant levels of clearance or agglutination of human red blood cells, and surprisingly, in many cases, do not cause clearance or agglutination of human red blood cells at all. In addition, the CD47 antibody of the present invention exhibits potent anti-tumor activity.

In some embodiments, the CD47 antibodies of the invention comprise (a) a Variable Heavy (VH) chain sequence having at least 90% (e.g., at least 95%) identity to an amino acid sequence selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, 69, 71, 73, 75, and 77; and (b) a Variable Light (VL) chain sequence having at least 90% (e.g., at least 95%) identity to an amino acid sequence selected from the group consisting of: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, 70, 72, 74, 76, and 78.

In some other embodiments, the CD47 antibodies of the invention comprise paired VH/VL chain sequences that have at least 90% (e.g., at least 95%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to paired VH and VL amino acid sequences selected from the group consisting of: SEQ ID NO 1 and SEQ ID NO 2 (i.e., 1A1), SEQ ID NO 3 and SEQ ID NO 4 (i.e., 1F8), SEQ ID NO 5 and SEQ ID NO 6 (i.e., 2A11), SEQ ID NO 7 and SEQ ID NO 8 (i.e., 2C2), SEQ ID NO 9 and SEQ ID NO 10 (i.e., 2D7), SEQ ID NO 11 and SEQ ID NO 12 (i.e., 2G4), SEQ ID NO 13 and SEQ ID NO 14 (i.e., 2G11), SEQ ID NO 15 and SEQ ID NO 16 (i.e., 6F4), SEQ ID NO 17 and SEQ ID NO 18 (i.e., 5H1), SEQ ID NO 19 and SEQ ID NO 20 (i.e., 5F6), SEQ ID NO 21 and SEQ ID NO 22 (i.e., 1F3), SEQ ID NO 23 and SEQ ID NO 24 (i.e., 2A4), SEQ ID NO:25 and SEQ ID NO:26 (i.e., 2B12), SEQ ID NO:27 and SEQ ID NO:28 (i.e., 13A11), SEQ ID NO:29 and SEQ ID NO:30 (i.e., 15E1), SEQ ID NO:31 and SEQ ID NO:32 (i.e., 13H3), SEQ ID NO:33 and SEQ ID NO:34 (i.e., 14A8), SEQ ID NO:35 and SEQ ID NO:36 (i.e., 16H3), SEQ ID NO:37 and SEQ ID NO:38 (i.e., 1A1), SEQ ID NO:39 and SEQ ID NO:40 (i.e., 1A1-A), SEQ ID NO:41 and SEQ ID NO:42 (i.e., 1A1-Q), SEQ ID NO:43 and SEQ ID NO:44 (i.e., 1A2), SEQ ID NO:45 and SEQ ID NO:46 (i.e., 1A8), SEQ ID NO:47 and SEQ ID NO:47 (i.e., 1A 3648, 1B1) SEQ ID NO:49 and SEQ ID NO:50 (i.e., 1B2), SEQ ID NO:51 and SEQ ID NO:52 (i.e., 1H3), SEQ ID NO:53 and SEQ ID NO:54 (i.e., 1H3-Q), SEQ ID NO:55 and SEQ ID NO:56 (i.e., 1H3-A), SEQ ID NO:57 and SEQ ID NO:58 (i.e., 2A2), SEQ ID NO:59 and SEQ ID NO:60 (i.e., 2A3), SEQ ID NO:61 and SEQ ID NO:62 (i.e., 2A6), SEQ ID NO:63 and SEQ ID NO:64 (i.e., 2A10), SEQ ID NO:65 and SEQ ID NO:66 (i.e., 2B1), SEQ ID NO:67 and SEQ ID NO:68 (i.e., 2C6), SEQ ID NO:69 and SEQ ID NO:70 (i.e., 2E7), SEQ ID NO:72 and SEQ ID NO:72 (i.e., 2C6), 2E9) SEQ ID NO:73 and SEQ ID NO:74 (i.e., 2F1), SEQ ID NO:75 and SEQ ID NO:76 (i.e., 2F3), and SEQ ID NO:77 and SEQ ID NO:78 (i.e., 34C 5). In some instances, the CD47 antibodies of the invention comprise paired VH and VL chain sequences selected from the group consisting of: SEQ ID NO.1 and SEQ ID NO. 2 (i.e., 1A1), SEQ ID NO. 3 and SEQ ID NO. 4 (i.e., 1F8), SEQ ID NO. 5 and SEQ ID NO. 6 (i.e., 2A11), SEQ ID NO. 7 and SEQ ID NO. 8 (i.e., 2C2), SEQ ID NO. 9 and SEQ ID NO. 10 (i.e., 2D7), SEQ ID NO. 11 and SEQ ID NO.12 (i.e., 2G4), SEQ ID NO. 13 and SEQ ID NO. 14 (i.e., 2G11), SEQ ID NO. 15 and SEQ ID NO. 16 (i.e., 6F4), SEQ ID NO. 17 and SEQ ID NO. 18 (i.e., 5H1), SEQ ID NO. 19 and SEQ ID NO. 20 (i.e., 5F6), SEQ ID NO. 21 and SEQ ID NO. 22 (i.e., 1F3), SEQ ID NO. 23 and SEQ ID NO. 24 (i.e., 2A4), SEQ ID NO:25 and SEQ ID NO:26 (i.e., 2B12), SEQ ID NO:27 and SEQ ID NO:28 (i.e., 13A11), SEQ ID NO:29 and SEQ ID NO:30 (i.e., 15E1), SEQ ID NO:31 and SEQ ID NO:32 (i.e., 13H3), SEQ ID NO:33 and SEQ ID NO:34 (i.e., 14A8), SEQ ID NO:35 and SEQ ID NO:36 (i.e., 16H3), SEQ ID NO:37 and SEQ ID NO:38 (i.e., 1A1), SEQ ID NO:39 and SEQ ID NO:40 (i.e., 1A1-A), SEQ ID NO:41 and SEQ ID NO:42 (i.e., 1A1-Q), SEQ ID NO:43 and SEQ ID NO:44 (i.e., 1A2), SEQ ID NO:45 and SEQ ID NO:46 (i.e., 1A8), SEQ ID NO:47 and SEQ ID NO:47 (i.e., SEQ ID NO:47, 1B1) SEQ ID NO:49 and SEQ ID NO:50 (i.e., 1B2), SEQ ID NO:51 and SEQ ID NO:52 (i.e., 1H3), SEQ ID NO:53 and SEQ ID NO:54 (i.e., 1H3-Q), SEQ ID NO:55 and SEQ ID NO:56 (i.e., 1H3-A), SEQ ID NO:57 and SEQ ID NO:58 (i.e., 2A2), SEQ ID NO:59 and SEQ ID NO:60 (i.e., 2A3), SEQ ID NO:61 and SEQ ID NO:62 (i.e., 2A6), SEQ ID NO:63 and SEQ ID NO:64 (i.e., 2A10), SEQ ID NO:65 and SEQ ID NO:66 (i.e., 2B1), SEQ ID NO:67 and SEQ ID NO:68 (i.e., 2C6), SEQ ID NO:69 and SEQ ID NO:70 (i.e., 2E7), SEQ ID NO:72 and SEQ ID NO:72 (i.e., 2C6), 2E9) SEQ ID NO:73 and SEQ ID NO:74 (i.e., 2F1), SEQ ID NO:75 and SEQ ID NO:76 (i.e., 2F3), and SEQ ID NO:77 and SEQ ID NO:78 (i.e., 34C 5).

The CD47 antibodies of the invention can be chimeric or humanized. They may prevent or significantly reduce human CD47 interaction with sirpa, or promote macrophage-mediated phagocytosis of CD 47-expressing cells.

The CD47 antibodies of the present invention do not cause significant or significant levels of erythrocyte aggregation or clearance of red blood cells, and in many cases do not cause erythrocyte aggregation or clearance of red blood cells at all.

In another aspect, the invention provides an isolated bispecific monoclonal antibody. The isolated bispecific monoclonal antibody comprises a first arm comprising the first monoclonal antibody or immunologically active fragment thereof described above that binds to human CD47 and a second arm comprising a second monoclonal antibody that does not bind to human CD 47.

In some embodiments, the second arm of the isolated bispecific monoclonal antibody is capable of binding to a cancer cell.

In other embodiments, the bispecific monoclonal antibody inhibits the interaction between human CD47 and human sirpa.

The present invention also provides fusion proteins, each comprising an isolated monoclonal antibody or immunologically active fragment thereof and a cytokine, wherein the monoclonal antibody or immunologically active fragment thereof binds to human CD47, the monoclonal antibody or immunologically active fragment thereof is fused to the cytokine at the N-terminus, and there is or is no linker between the monoclonal antibody or fragment thereof and the cytokine.

In some embodiments, the isolated monoclonal antibody or immunologically active fragment thereof comprises:

A Variable Heavy (VH) chain sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, 69, 71, 73, 75, and 77, and

A Variable Light (VL) chain sequence having at least 95% identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, 70, 72, 74, 76, and 78.

In some other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof comprises a VH/VL sequence pair comprising VH and VL chain sequences at least 95% identical to a VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NO 1 and SEQ ID NO 2 (i.e., 1A1), SEQ ID NO 3 and SEQ ID NO 4 (i.e., 1F8), SEQ ID NO 5 and SEQ ID NO 6 (i.e., 2A11), SEQ ID NO 7 and SEQ ID NO 8 (i.e., 2C2), SEQ ID NO 9 and SEQ ID NO 10 (i.e., 2D7), SEQ ID NO 11 and SEQ ID NO 12 (i.e., 2G4), SEQ ID NO 13 and SEQ ID NO 14 (i.e., 2G11), SEQ ID NO 15 and SEQ ID NO 16 (i.e., 6F4), SEQ ID NO 17 and SEQ ID NO 18 (i.e., 5H1), SEQ ID NO 19 and SEQ ID NO 20 (i.e., 5F6), SEQ ID NO 21 and SEQ ID NO 22 (i.e., 1F3), SEQ ID NO 23 and SEQ ID NO 24 (i.e., 2A4), SEQ ID NO:25 and SEQ ID NO:26 (i.e., 2B12), SEQ ID NO:27 and SEQ ID NO:28 (i.e., 13A11), SEQ ID NO:29 and SEQ ID NO:30 (i.e., 15E1), SEQ ID NO:31 and SEQ ID NO:32 (i.e., 13H3), SEQ ID NO:33 and SEQ ID NO:34 (i.e., 14A8), SEQ ID NO:35 and SEQ ID NO:36 (i.e., 16H3), SEQ ID NO:37 and SEQ ID NO:38 (i.e., 1A1), SEQ ID NO:39 and SEQ ID NO:40 (i.e., 1A1-A), SEQ ID NO:41 and SEQ ID NO:42 (i.e., 1A1-Q), SEQ ID NO:43 and SEQ ID NO:44 (i.e., 1A2), SEQ ID NO:45 and SEQ ID NO:46 (i.e., 1A8), SEQ ID NO:47 and SEQ ID NO:47 (i.e., 1A 3648, 1B1) SEQ ID NO:49 and SEQ ID NO:50 (i.e., 1B2), SEQ ID NO:51 and SEQ ID NO:52 (i.e., 1H3), SEQ ID NO:53 and SEQ ID NO:54 (i.e., 1H3-Q), SEQ ID NO:55 and SEQ ID NO:56 (i.e., 1H3-A), SEQ ID NO:57 and SEQ ID NO:58 (i.e., 2A2), SEQ ID NO:59 and SEQ ID NO:60 (i.e., 2A3), SEQ ID NO:61 and SEQ ID NO:62 (i.e., 2A6), SEQ ID NO:63 and SEQ ID NO:64 (i.e., 2A10), SEQ ID NO:65 and SEQ ID NO:66 (i.e., 2B1), SEQ ID NO:67 and SEQ ID NO:68 (i.e., 2C6), SEQ ID NO:69 and SEQ ID NO:70 (i.e., 2E7), SEQ ID NO:72 and SEQ ID NO:72 (i.e., 2C6), 2E9) SEQ ID NO:73 and 74 (i.e., 2F1), SEQ ID NO:75 and 76 (i.e., 2F3), SEQ ID NO:77 and 78 (i.e., 34C 5).

In some other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof comprises a VH/VL sequence pair, wherein the VH/VL sequence pair comprises VH and VL chain sequences, and wherein the sequence pair is selected from the group consisting of: SEQ ID NO 1 and SEQ ID NO 2 (i.e., 1A1), SEQ ID NO 3 and SEQ ID NO 4 (i.e., 1F8), SEQ ID NO 5 and SEQ ID NO 6 (i.e., 2A11), SEQ ID NO 7 and SEQ ID NO 8 (i.e., 2C2), SEQ ID NO 9 and SEQ ID NO 10 (i.e., 2D7), SEQ ID NO 11 and SEQ ID NO 12 (i.e., 2G4), SEQ ID NO 13 and SEQ ID NO 14 (i.e., 2G11), SEQ ID NO 15 and SEQ ID NO 16 (i.e., 6F4), SEQ ID NO 17 and SEQ ID NO 18 (i.e., 5H1), SEQ ID NO 19 and SEQ ID NO 20 (i.e., 5F6), SEQ ID NO 21 and SEQ ID NO 22 (i.e., 1F3), SEQ ID NO 23 and SEQ ID NO 24 (i.e., 1F 3623 and SEQ ID NO 24, 2A4) SEQ ID NO 25 and SEQ ID NO 26 (i.e., 2B12), SEQ ID NO 27 and SEQ ID NO 28 (i.e., 13A11), SEQ ID NO 29 and SEQ ID NO 30 (i.e., 15E1), SEQ ID NO 31 and SEQ ID NO 32 (i.e., 13H3), SEQ ID NO 33 and SEQ ID NO 34 (i.e., 14A8), SEQ ID NO 35 and SEQ ID NO 36 (i.e., 16H3), SEQ ID NO 37 and SEQ ID NO 38 (i.e., 1A1), SEQ ID NO 39 and SEQ ID NO 40 (i.e., 1A1-A), SEQ ID NO 41 and SEQ ID NO 42 (i.e., 1A1-Q), SEQ ID NO 43 and SEQ ID NO 44 (i.e., 1A2), SEQ ID NO 45 and SEQ ID NO 46 (i.e., 1A8), SEQ ID NO 48 and SEQ ID NO 47 (i.e., 1A 3647), 1B1) SEQ ID NO:49 and SEQ ID NO:50 (i.e., 1B2), SEQ ID NO:51 and SEQ ID NO:52 (i.e., 1H3), SEQ ID NO:53 and SEQ ID NO:54 (i.e., 1H3-Q), SEQ ID NO:55 and SEQ ID NO:56 (i.e., 1H3-A), SEQ ID NO:57 and SEQ ID NO:58 (i.e., 2A2), SEQ ID NO:59 and SEQ ID NO:60 (i.e., 2A3), SEQ ID NO:61 and SEQ ID NO:62 (i.e., 2A6), SEQ ID NO:63 and SEQ ID NO:64 (i.e., 2A10), SEQ ID NO:65 and SEQ ID NO:66 (i.e., 2B1), SEQ ID NO:67 and SEQ ID NO:68 (i.e., 2C6), SEQ ID NO:69 and SEQ ID NO:70 (i.e., 2E7), SEQ ID NO:72 and SEQ ID NO:72 (i.e., 2C6), 2E9) SEQ ID NO:73 and SEQ ID NO:74 (i.e., 2F1), SEQ ID NO:75 and SEQ ID NO:76 (i.e., 2F3), or SEQ ID NO:77 and SEQ ID NO:78 (i.e., 34C5), or a combination having at least 90% (e.g., at least 95%) identity to the above-described pair of sequences.

In other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof is chimeric or humanized.

In other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof prevents the interaction of human CD47 with signal-regulated protein alpha (sirpa).

In other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof does not cause a significant level of erythrocyte agglutination or clearance of the red blood cells.

In other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof does not cause erythrocyte agglutination or clearance of red blood cells.

In other embodiments, the cytokine comprises an immunoglobulin (Ig), hematopoietic growth factor, interferon, tumor necrosis factor, interleukin-17 receptor, or a wild-type form of a monomeric glycoprotein or variant thereof.

In other embodiments, the cytokine is a wild-type form of a monomeric glycoprotein or a variant thereof. Further, in some embodiments, the cytokine is a wild-type of granulocyte macrophage colony stimulating factor (GM-CSF) or a variant thereof.

In other embodiments, the monoclonal antibody or immunologically active fragment thereof is fused to a cytokine with or without a linker selected from the group consisting of: (G4S)3, (G4S)6, (GS)9, IGD (F30), IGD (F64), IGD (R30), IGN (R64), IGD (R30-Cys), and IGD (R64-Cys).

In other embodiments, the fusion protein inhibits the interaction of human CD47 and human sirpa.

In other embodiments of the fusion protein, the isolated monoclonal antibody or immunologically active fragment thereof promotes macrophage-mediated phagocytosis of CD 47-expressing cells.

In other embodiments, the fusion protein further comprises a small molecule therapeutic agent or label, and the small molecule therapeutic agent or label is conjugated to the monoclonal antibody or immunologically active fragment thereof, or to a cytokine. The small molecule therapeutic agent is an anti-cancer or anti-inflammatory agent; the label is a biomarker or a fluorescent label.

In other embodiments, the isolated monoclonal antibody or immunologically active fragment thereof comprises a VH/VL sequence pair that is at least 90% (e.g., at least 95%) identical to a pair of VH and VL amino acid sequences selected from the group consisting of SEQ ID NOs: 3 and SEQ ID NO:4, and SEQ ID NO:31 and SEQ ID NO: 32; the cytokine is a wild-type granulocyte-macrophage colony stimulating factor (GM-CSF) or a variant thereof.

In yet other embodiments, the fusion protein comprises a Variable Light (VL) chain expression vector that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 108 and SEQ ID NO:116 are at least 90% (e.g., at least 95%) identical in amino acid sequence; and a variable heavy chain (VH) expression vector that is at least 90% (e.g., at least 95%) identical to an amino acid sequence selected from the group consisting of seq id nos: 109, 110, 111, 112, 113, 114, 115, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 147 and 158.

In yet other embodiments, the fusion protein comprises a VH/VL pair that is at least 90% (e.g., at least 95%) identical to a pair of VH and VL amino acid sequences selected from the group consisting of: 108 and 109, 108 and 110, 108 and 114, 108 and 115, 108 and 117, 108 and 118, 108 and 119, 108 and 120, 108 and 121, 108 and 122, 108 and 123, 108 and 124, 108 and 125, 108 and 124, 108 and 126, 108 and 127, 108 and 128, 108 and 132, 108 and 133, 108 and 134, 108 and 135, 108 and 136, 108 and 137, 108 and 138, 108 and 139, 140, 108 and 141, 108 and 142, 108 and 143, 108 and 144 SEQ ID NO, 108 and 145 SEQ ID NO, 108 and 146 SEQ ID NO, and 108 and 147 SEQ ID NO; 116 and 109, 116 and 110, 116 and 114, 116 and 115, 116 and 117, 116 and 120, 116 and 121, 116 and 122, 116 and 124, 116 and 125, 116 and 126, 116 and 127, 116 and 128, 116 and 129, 116 and 130 SEQ ID NO, 116 and 131 SEQ ID NO, 116 and 132 SEQ ID NO, 116 and 133 SEQ ID NO, 116 and 134 SEQ ID NO, 116 and 135 SEQ ID NO, 116 and 136 SEQ ID NO, 116 and 137 SEQ ID NO, 116 and 138 SEQ ID NO, 116 and 139 SEQ ID NO, 116 and 140 SEQ ID NO, 116 and 141 SEQ ID NO, 116 and 142 SEQ ID NO, 116 and 143 SEQ ID NO, 116 and 144 SEQ ID NO, 116 and 145 SEQ ID NO, 116 and 146 SEQ ID NO, 116 and 147, 116 and 148, 116 and 149, 116 and 150, 151, 116 and 152, 116 and 153, 116 and 154, 116 and 155, and 116 and 156.

In another aspect, the invention provides a pharmaceutical composition comprising a fusion protein of the invention, and a pharmaceutically acceptable carrier or excipient.

As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that can be used to prepare a pharmaceutical composition or formulation, is generally safe, non-toxic, and is neither biologically nor otherwise undesirable. The carrier or excipient employed is typically one suitable for administration to a human or other mammal. In preparing the compositions, the active ingredient is typically mixed with, diluted with, or enclosed by a carrier or excipient. When the carrier or excipient serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient of the antibody.

The invention also includes a method of treating a disease in a human subject in need thereof, and the method includes administering to the subject a therapeutically effective amount of the fusion protein of the invention or the pharmaceutical composition of the invention, the disease being cancer, a fibrotic disease, or any disease associated with inhibition of phagocytosis. In some cases, the cancer is selected from the group comprising: ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck tumors, bladder cancer, colorectal cancer, pancreatic cancer, non-hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, Hairy Cell Leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, endometrial cancer, kidney cancer, (benign) fetal tumor, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, renal cancer, colorectal carcinoma, leukemia, Liver cancer, and solid tumors; the fibrotic disease may be selected from the group comprising: myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis, bronchitis, and asthma. Examples of solid tumors include, for example, endometrial, thyroid, cervical, gastric, breast, ovarian, lung, pancreatic, prostate, (malignant) melanoma, (benign) fetal, colorectal, lung, head and neck, bladder, esophageal, liver and kidney tumors and neuroblastoma-derived CNS tumors. The disease associated with inhibition of phagocytosis may be a cardiovascular disease (e.g. atherosclerosis, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral arterial disease or venous thrombosis).

As used herein, the term "effective amount" refers to an amount of CD47 antibody, as described herein, that is sufficient or required to affect the treatment, prognosis or diagnosis of a CD 47-dependent signaling-related disease when administered to a subject. When used alone or in combination, a therapeutically effective amount of an antibody provided herein will vary depending on the relative activity of the antibody (e.g., promoting macrophage-mediated phagocytosis of CD 47-expressing cancer cells) and the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art.

As described herein, the term "isolated" prior to an antibody described herein (e.g., a CD47 antibody) means that the antibody is substantially free of other cellular material. In one embodiment, the isolated antibody is substantially free of other proteins from the same species. In another embodiment, the isolated antibody is expressed by a cell from a different species and is substantially free of other proteins from the different species. Proteins that are substantially free of naturally associated components (or components associated with the cellular expression systems used to produce the antibodies) can be isolated using protein purification techniques well known in the art. In one embodiment, the antibody or antigen binding fragment of the invention is isolated.

The term "biomolecule" as used herein is meant to include synthetic antibodies (monoclonal or bispecific), peptides, and biomimetic molecules. The term "biomimetic molecule" refers to a molecule designed or developed to have a structure or property similar or analogous to a naturally occurring large compound, such as a protein or nucleotide, and which has a molecular weight of, for example, at least 3000, at least 5000, or at least 10000.

All references cited herein are incorporated by reference in their entirety.

Drawings

FIG.1 shows the dose effect of the binding of CD47 antibody to monomeric CD 47-ECD.

Fig.2a and 2b show the dose effect of CD47 antibody binding to dimer CD 47-ECD.

Fig.3a, fig.3b, and fig.3c show the dose effect of CD47 antibody blocking CD47 and sirpa binding.

Fig.4a and 4b show the dose effect of CD47 antibody binding to CD47+ Raji cells; and fig.4c, 4d and 4e show the binding kinetics of the CD47 antibody and its data measured by Biacore analysis.

Fig.5a and 5b show phagocytosis of tumor cells by human M Φ and CD47 antibodies.

FIGS. 6a-6c show macrophage-mediated phagocytosis of various human blood cancer cell lines by the CD47 antibody.

Fig.7a and 7b show the activity of the CD47 antibody to induce Red Blood Cell (RBC) aggregation at different doses.

Fig.8a, 8b, 8c and 8d show the activity of CD47 antibody at different and higher doses to bind RBCs and induce RBC aggregation.

Fig.9a, 9b, 9c and 9d show RBC binding activity of the CD47 antibody.

FIG.10 shows the results of erythrocyte aggregation induction by the CD47 antibody for different sources of humans.

FIG.11 shows the binding activity of CD47 antibody and SIRPa-Ig fusion, respectively, to human platelets, in which CD61 was stained as a surface marker for platelets.

FIGS. 12a and 12b show the results of in vitro assays for the induction of hemagglutination of Cyno monkeys by the CD47 antibody and SIRPa-Ig fusion protein, respectively.

Figure 13 shows the results of tests for CD47 antibody and control antibody binding and phagocytosis by AML cells.

FIGS. 14a and 14b show the therapeutic effect of CD47 antibody and control antibody on luciferase-Raji xenografted mice.

FIG.15 shows the CD47 antibody and control antibody induced macrophage polarization in tumor-bearing mice.

Fig.16 shows CD47 expression profiles of PDX samples using various human cancer types.

Figure 17 shows the results of a safety drug study (hematology) on cynomolgus monkeys.

Fig.18 shows competition between antibodies 1F8 and 5F9, and between antibodies 1F8 and 2a1 in CD47 binding due to their different epitopes and the structure of the 5F9/CD47 complex and the 1F8/CD47 complex.

Fig.19a, 19b, 19c, 19d, 19e, 19f, 19g and 19H show the changes in erythrocytes, hemoglobin, platelets and lymphocytes of CD47 antibody 13H3 in a single-dose and multi-dose model of cynomolgus monkeys, respectively.

FIG.20 shows strong binding affinity of 34C5 to recombinant CD 47-ECD.

Fig.21 shows the strong binding affinity of 34C5 to Raji cells carrying CD 47.

FIG.22 shows that 34C5 is able to effectively block the binding of CD47 to SIRPa, EC₅₀Was 0.30 nM.

Figure 23 shows that antibody 34C5 promotes phagocytosis of tumor cells by human M Φ.

Figure 24 shows that antibody 34C5 does not cause RBC agglutination in vitro.

Figure 25 shows that binding of antibody 34C5 to RBCs decreases with decreasing antibody concentration.

FIG.26 shows that 1F8-GMCSF fusion protein caused a greater relative fold change in the percentage of CD14+ cells phagocytic cells compared to IgG control, 1F8 treated, and GM-CSF treated groups.

Figure 27 shows that the fusion protein 1F8-GMCSF has a stronger binding affinity to the human GMCSF receptor than recombinant human GMCSF.

Figure 28 shows that 1F8-GMCSF has similar activity as GMCSF itself in inducing STAT5 phosphorylation.

FIG.29 shows that fusion protein 1F8-GMCSF has a greater ability to stimulate TF-1 proliferation than GMCSF.

FIG.30(a), FIG.30(b), FIG.30(c) and FIG.30(d) show the production of IL-6, IL-12, TNF- α and CD80 by M1 macrophage activation in the presence of IgG, 1F8, GMCSF or 1F8-GMCSF fusion protein.

FIG.31 shows that the 1F8-GMCSF fusion protein shows the best efficacy in all five treatment regimens of the Raji xenograft model.

FIG.32 shows the dose effect of fusion protein 13H3-GMCSF on binding to CD47+ Raji cells.

FIG.33 shows the dose effect of the fusion protein 13H3-GMCSF blocking the binding of CD47 to SIRP α.

FIG.34 shows the effect of 13H3-GMCSF on phagocytosis of Raji cells by human M Φ.

FIG.35 shows the activity of the fusion protein 13H3-GMCSF in inducing Red Blood Cell (RBC) coagulation at different doses.

FIG.36 shows the dose effect of fusion protein 13H3-GMCSF binding to the GMCSF receptor.

FIG.37 shows the dose effect of fusion protein 13H3-GMCSF on stimulation of STAT5 phosphorylation.

FIG.38 shows the dose effect of fusion protein 13H3-GMCSF in stimulating TF-1 proliferation.

FIG.39 shows the therapeutic effect of the fusion protein 13H3-GMCSF and control on the luciferase-Raji xenograft mouse model.

FIG.40 shows the concentration of serum levels of 13H3-GMCSF versus time after a single 20mg/kg dose in cynomolgus monkeys.

FIGS. 41a and 41b show the levels of erythrocytes and platelets after multiple administrations of 13H3-GMCSF or IgG in cynomolgus monkeys at a dose of 20 mg/kg.

FIG.42a, FIG.42b and FIG.42c show the levels of leukocytes, neutrophils and monocytes after multiple dosing of 13H3-GMCSF or IgG in cynomolgus monkeys at a dose of 20 mg/kg.

Figure 43 shows the dose-dependent response of the 13H3-GMCSF variant in stimulating STAT5 phosphorylation.

FIG.44 shows the dose-dependent response of 13H3-GMCSF variant in stimulating TF-1 proliferation.

FIG.45 shows the dose-dependent response of 13H3-GMCSF variant in stimulating the production of IL-6 by macrophages.

FIG.46 shows the effect of fusion protein 13H3-GMCSF variants on phagocytosis of Raji cells by human M Φ.

Figures 47a, 47b and 47c show the dose-dependent response of 13H3-GMCSF variant that blocks binding of CD47 to sirpa.

Figure 48 shows the dose-dependent response of 13H3-GMCSF variant binding to erythrocytes.

Figure 49 shows the activity of inducing Red Blood Cell (RBC) coagulation when RBC coagulation assays were performed using the 13H3-GMCSF variant.

FIG.50 shows the effect of 13H3-GMCSF with the IgG1N297A version on phagocytosis of Raji cells by human M Φ.

Figure 51 shows the effect of 13H3-GMCSF variant with IgG1N297A on phagocytosis of Raji cells by human M Φ.

FIG.52 shows the dose-dependent response of deglycosylated 13H3-GMCSF in the induction of STAT5 phosphorylation.

FIG.53 shows the dose-dependent response of deglycosylated 13H3-GMCSF in stimulating TF-1 proliferation.

FIG.54 shows the effect of deglycosylated 13H3-GMCSF on phagocytosis of Raji cells by human M Φ.

FIG.55 shows the dose-dependent response of deglycosylated 13H3-GMCSF variant in stimulating TF-1 proliferation.

Figure 56 shows a concentration-time curve of the serum levels of the fusion protein 13H3-GMCSF variant after a single dose of 10mg/kg in cynomolgus monkeys.

FIGS. 57a, 57b and 57c show the effect of fusion protein 13H3-GMCSF variants on peripheral levels of neutrophils, monocytes and leukocytes.

FIGS. 58a, 58b and 58c show the effect of fusion protein 13H3-GMCSF variants on peripheral levels of erythrocytes, hemoglobin and platelets.

Figure 59 shows the dose-dependent response of 13H3-gmcsf variant in the induction of STAT5 phosphorylation.

FIG.60 shows the dose-dependent response of 13H3-mGMCSF variant in stimulating FDC-P1 proliferation.

Detailed Description

The present invention provides novel isolated monoclonal CD47 antibodies that can prevent human CD47 from interacting with sirpa or promote macrophage-mediated phagocytosis of CD 47-expressing cells. These CD47 antibodies do not cause significant or significant levels of erythrocyte aggregation or clearance of red blood cells, and in many cases, do not cause erythrocyte aggregation or clearance of red blood cells at all.

Illustratively, the CD47 antibodies of the invention comprise (a) a Variable Heavy (VH) chain sequence having at least 90% (e.g., at least 95%) identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, 69, 71, 73, 75, and 77; and (b) a Variable Light (VL) chain sequence having at least 90% (e.g., at least 95%) identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, 70, 72, 74, 76, and 78. Further, in some instances, a CD47 antibody of the invention includes a bound VH/VL chain sequence that has at least 90% (e.g., at least 95%) identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 1 and SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, and 77 and 78.

As used herein, the term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. "antibodies" (or "Abs") and "immunoglobulins" (or "Igs") are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include antibodies and other antibody-like molecules that lack antigen specificity. The latter polypeptides are produced, for example, at low levels by the lymphatic system and at high levels by myeloma.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen that binds to the paratope of an antibody. Epitopic determinants generally consist of chemically active surface groups of the molecule, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics.

As used herein, the terms "natural antibody and immunoglobulin" generally refer to a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond (also referred to as a "VH/VL pair"), while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has a regular arrangement of intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a plurality of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. See, e.g., Clothia et al, journal of molecular biology, 186: 651 (1985); novotny and Haber, proceedings of the american academy of sciences, 82: 4592(1985).

As used herein, the term "variable" refers to the fact that certain portions of the sequences of the variable domains of antibodies vary strongly and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is unevenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domains are called the Framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely in a β -sheet configuration, connected by three CDRs, forming a loop junction, and in some cases forming part of a β -sheet structure. The CDRs in each chain are held in close proximity by the FR region and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. Please refer, for example, to Kabat et al, immunological protein sequences, fifth edition, national institutes of health, besesda, maryland (1991). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity. The study of variable region sequences included humanized variable region sequences of the provided CD47 antibody. For example, 1a1 includes SEQ ID NO:1 (heavy chain) and SEQ ID NO:2 (light chain), 1F8 comprises SEQ ID NO:3 (heavy chain) and SEQ ID NO:4 (light chain), 2a11 comprising SEQ ID NO:5 (heavy chain) and SEQ ID NO:6 (light chain).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. Pepsin produces F (ab') 2 fragments that have two antigen binding sites and are still capable of cross-linking antigens. "Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In single chain Fv species (scFv), one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can bind in a "dimeric" structure similar to that of a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. Please refer, for example, to Pluckthun, monoclonal antibody pharmacology, Vol 113, Rosenburg and Moore eds, Schpringer, New York, p.269-315 (1994).

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy Chain (CH)₁). Fab' fragments differ from Fab fragments by the presence of the heavy chain CH₁The carboxy terminus of the domain has several residues added, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab', in which the cysteine residues of the constant domains carry a free sulfhydryl group. F (ab')₂Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

There are five main types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

As used herein, the term "antibody fragment" and all grammatical variants thereof is defined as encompassing antigen binding of an intact antibodyA portion of an intact antibody of site or variable region, wherein the portion does not contain the constant heavy chain domains (i.e., CH 2, CH 3 and CH 4, depending on the antibody isotype) in the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab ', Fab ' -SH, F (ab ')₂And Fv fragments; a double body; any antibody fragment, which is a polypeptide having a primary structure consisting of an uninterrupted sequence of one contiguous amino acid residue (referred to herein as a "single chain antibody fragment" or a "single chain polypeptide"), including, but not limited to, (1) a single chain antibody (scFv) molecule, (2) a single chain polypeptide comprising only one light chain variable domain or a fragment thereof comprising three CDRs of a light chain variable domain, without an associated heavy chain portion, and (3) a single chain polypeptide comprising only one heavy chain variable region or a fragment thereof comprising three CDRs of a heavy chain variable region, without an associated light chain portion; and multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g. CH1 in the IgG isotype), and/or may contain any hinge region sequence found in an intact antibody, and/or may contain a leucine zipper sequence fused to or located within the hinge region sequence or heavy chain constant region sequence of the heavy chain.

Unless specifically stated to the contrary, the term "conjugated" as used herein is defined as one or more antibody fragments bound to one or more polymer molecules by covalent bonds to form a heterogeneous molecule, wherein the heterogeneous molecule is water-soluble, i.e., soluble in physiological fluids such as blood, and wherein the heterogeneous molecule does not contain any structured aggregates. A commonly used conjugate is polyethylene glycol (PEG). In the context of the foregoing definitions, the term "structured aggregate" refers to (1) any aggregate of molecules in aqueous solution having a sphere or sphere shell structure, such that the heterogeneous molecules are not in a micelle or other emulsion structure and are not immobilized to a lipid bilayer, vesicle or liposome; and (2) any aggregates of molecules in solid or insoluble form, such as a chromatography bead matrix, which do not release heterogeneous molecules into solution upon contact with an aqueous phase. Thus, the term "conjugate" as defined herein encompasses the aforementioned heterogeneous molecules that may be in a precipitate, sediment, bioerodible matrix, or other solid capable of releasing the heterogeneous molecule into an aqueous solution upon hydration of the solid.

As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible small amounts of natural mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the invention may be prepared in immortalized B cells or hybridomas thereof, or may be prepared by recombinant DNA methods.

monoclonal antibodies described herein include hybrid and recombinant antibodies produced by splicing the variable (including hypervariable) domain of the CD47 antibody to a constant domain (e.g., "humanized" antibodies), or the light and heavy chains, or the chains from one species to another, or to a heterologous protein, regardless of the species of origin or immunoglobulin class or subclass name and antibody fragment (e.g., Fab, F (ab')₂And Fv) as long as they exhibit the desired biological activity.

Monoclonal antibodies described herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

As used herein, an "isolated" antibody is an antibody that has been recognized and isolated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that interfere with diagnostic or therapeutic uses for antibodies, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody will be purified: (1) the weight of the purified antibody is greater than 75%, and most preferably greater than 80%, 90% or 99%, as determined by the Lowry method, or (2) the antibody is rendered homogeneous by SDS-PAGE electrophoresis under reducing or non-reducing conditions using coomassie blue, or, preferably, silver staining. The isolated antibody includes an in situ antibody within a recombinant cell because at least one component of the antibody's natural environment is not present. Typically, however, the isolated antibody is prepared by at least one purification step.

As used herein, the term "epitope-tagged" refers to a CD47 antibody fused to an "epitope tag". The epitope tag polypeptide has enough residues that it can provide an epitope for making an antibody, and it is short enough so as not to interfere with the activity of the CD47 antibody. The epitope tag is preferably sufficiently unique that antibodies specific for the epitope do not substantially cross-react with other epitopes. Suitable tag polypeptides typically have at least 6 amino acid residues and typically about 8-50 amino acid residues (preferably about 9-30 residues). Examples include the C-myc tag protein and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereon (see, e.g., Evan et al, molecular and cellular biology, 5(12): 3610-; and the herpes simplex virus glycoprotein D (gD) tag and its antibodies (see, e.g., Paborsky et al, protein engineering, 3 (6): 547-553 (1990)).

As used herein, the term "label" refers to a detectable compound or composition conjugated directly or indirectly to an antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical reaction of a detectable substrate compound or composition.

As used herein, the term "solid phase" refers to a non-aqueous matrix to which the antibodies of the present invention can adhere. Examples of solid phases contemplated herein include solid phases formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other cases, it is a purification column (e.g., an affinity chromatography column). The term also includes a discontinuous solid phase of discrete particles. See, for example, U.S. Pat. No. 4,275,149.

The invention also provides pharmaceutical compositions comprising the above CD47 antibodies, and methods of treating a disease in a subject with the above CD47 antibodies or pharmaceutical compositions.

As used herein, the terms "treatment" or "treating" refer to both the therapeutic treatment and the prophylactic or preventative measures of a disease, such as cancer or a fibrotic disease. Persons in need of treatment include those already with the disease as well as those preventing the disease.

The cancer is selected from, but not limited to, ovarian cancer, colon cancer, breast cancer, lung cancer, head and neck tumors, bladder cancer, colorectal cancer, pancreatic cancer, non-hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, (malignant) melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, myeloma, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, endometrial cancer, kidney cancer, (benign) fetal tumor, prostate cancer, thyroid cancer, cervical cancer, gastric cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, cervical cancer, and pancreatic cancer, Liver cancer, and solid tumors; the fibrotic disease may be, for example, myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis, bronchitis, and asthma.

As used herein, the term "subject" for use as a subject of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports or pet animals, such as dogs, horses, cats, cattle, and the like. Preferably, the mammal is a human.

The CD47 antibodies of the invention can also be used in vitro and in vivo to monitor the course of treatment of CD47 diseases. Thus, for example, by measuring an increase or decrease in the number of cells expressing CD47, particularly cancer cells expressing CD47, it can be determined whether a particular therapeutic regimen aimed at ameliorating the disease is effective.

The CD47 antibodies of the invention can be used in immunoassays in vitro, where they can be used in liquid phase or bound to a solid support. In addition, the CD47 antibody in these immunoassays may be detectably labeled in various ways. Examples of the types of immunoassays which can utilize the monoclonal antibodies of the present invention are flow cytometry, e.g., FACS, MACS, immunohistochemistry, direct and indirect forms of competitive or non-competitive immunoassays. Detection of antigens using the CD47 antibodies of the present invention can be accomplished using immunoassays that are run in a forward, reverse, or simultaneous mode, including immunohistochemical assays on physiological samples. Other immunoassay formats will be known or can be readily identified by those skilled in the art without undue experimentation.

The CD47 antibodies of the invention can be conjugated to a variety of different vectors and used to detect the presence of CD47 expressing cells. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble. Those skilled in the art will know of other suitable vectors for binding monoclonal antibodies, or will be able to determine such using routine experimentation.

Many different markers and methods of labeling are known to those of ordinary skill in the art, which can be used as tracers in therapeutic methods, in diagnostic methods, and the like. For diagnostic purposes, the label may be covalently or non-covalently linked to an antibody or fragment thereof of the invention, including fragments consisting of or comprising CDR sequences. Examples of labels useful in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds and bioluminescent compounds. One of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibodies of the invention, or can determine using routine experimentation. In addition, binding of these labels to the monoclonal antibodies of the invention can be accomplished using standard techniques common to those of ordinary skill in the art.

In some embodiments, the CD47 antibodies of the invention are attached to nanoparticles, e.g., for imaging. Useful nanoparticles are those known in the art, including, for example, but not limited to, raman-silica-gold-nanoparticles (R-Si-Au-NPs). The R-Si-Au-NP is composed of Raman organic molecules, has a narrow band spectrum characteristic and is adsorbed on a gold core. Since raman organic molecules can be varied, each nanoparticle can carry its own characteristics, allowing multiple nanoparticles to be detected independently at the same time through multiple passes. The entire nanoparticle is encapsulated in a silica shell to hold the raman organic molecule on the gold nanocore. R-Si-Au-NPs are conjugated with optional polyethylene glycol (PEG), increasing their bioavailability and providing a functional "handle" for attachment of targeting moieties. Please refer, for example, to Thakor et al (2011), transform medicine, 3(79):79ra 33; jokerst et al (2011) Small, 7(5) 625-33; gao et al (2011) biomaterials, 32(8): 2141-8.

For one of the purposes of the present invention, CD47 in vivo or in vitro in a biological fluid or on a tissue can be detected by the CD47 antibodies provided herein. Can be used for any sample containing a detectable amount of CD 47. The sample may be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or the sample may be a solid or semi-solid such as tissue, stool and the like, or may be a solid tissue such as those commonly used in histological diagnosis.

Another labeling technique that can improve sensitivity includes antibodies that bind to low molecular weight haptens. These haptens can be specifically detected by the second reaction. For example, haptens such as biotin, which react with avidin, or dinitrophenol, vitamin B, or fluorescein, are commonly used, and the hapten can react with a specific anti-hapten antibody.

The CD47 antibodies provided by the present invention can be conveniently used in a kit, i.e., a kit of combined reagents consisting of a plurality of predetermined amounts of reagents according to the instructions for the diagnostic assay to be performed. Wherein the antibody is labeled with an enzyme, and further comprising a substrate required for the enzyme and a cofactor (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, the kit may include other additives such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents may be varied over a wide range so that the concentrations of the various reagents in the solution optimize the sensitivity of the assay. In particular, the agent may be a dry powder, typically a lyophilized powder, comprising excipients which, when dissolved, provide a solution of the agent with the appropriate concentration.

Therapeutic formulations comprising one or more antibodies provided herein are prepared by mixing an antibody of the invention of the desired purity with any physiologically acceptable carrier, vehicle or stabilizer (see, e.g., Remington's pharmaceutical Sciences,16th edition, Osol, A.Ed. (1980)) to form a lyophilized formulation or aqueous solution for easy storage. The antibody compositions may be formulated, dosed, and administered with good medical practice. Factors to be considered herein include the particular disorder being treated, the cause of the disorder, the site of delivery of the agent, the mode of administration, the plan of administration, and other factors known to the treating physician. The "therapeutically effective amount" of the antibody administered is governed by these considerations and is the minimum amount required to prevent the disease associated with CD 47.

The therapeutic dose can be at least about 0.01 μ g/kg body weight, at least about 0.05 μ g/kg body weight, at least about 0.1 μ g/kg body weight, at least about 0.5 μ g/kg body weight, at least about 1 μ g/kg body weight,. 2.5 μ g/kg body weight, at least about 5 μ g/kg body weight, and no more than about 100 μ g/kg body weight. Those skilled in the art will recognize that these guidelines require adjustment based on the molecular weight of the active agent, e.g., using antibody fragments, or using antibody conjugates. The dosage may also vary depending on local administration, e.g., intranasal, inhalant, etc., or on the manner of systemic administration, e.g., intraperitoneal (I.P.), intravenous (i.v.), intradermal (i.d.), intramuscular (I.M), and the like.

The CD47 antibodies provided herein do not require, but may be optionally formulated with, one or more agents that enhance activity, or enhance therapeutic efficacy. These are usually used in the same dosage and route of administration indicated above or about from 1% to 99% of the dosage used before.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages or concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; quaternary ammonium chloride hexahydrocarbonates; algaecide; benzethonium chloride; phenol, butyl or benzylethanol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; counter ions of salt formation such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as TWEEN^TM，PLURONICS^TMOr polyethylene glycol (PEG). Formulations for in vivo administration must be sterile preparations. This can be conveniently accomplished by filtration through sterile filtration membranes.

The active ingredient containing the CD47 antibody may also be encapsulated in microcapsules, for example, prepared by demulsification techniques or interfacial polymerization, for example, hydroxymethylcellulose or gel-microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, emulsions, nanoparticles and nanocapsules) or in emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

The CD47 antibodies or pharmaceutical compositions provided herein can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral injection includes intramuscular, intravenous, arterial, intraperitoneal or subcutaneous administration. In addition, the anti-CD 47 antibodies are suitable for administration by pulsed infusion, particularly low dose antibodies.

For the prevention or treatment of disease, the appropriate dosage of antibody depends on the type of disease being treated, as indicated above, the severity and course of the disease, whether the antibody is used for prophylactic purposes, early treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibodies are suitable for administration to a patient at one time or over a series of treatments.

In another embodiment of the invention, an article of manufacture containing a material for treating the above-described disorders is provided. The article includes a container and a label. Suitable containers include, for example, bottles, vials, pipettes, and test tubes. The container may be made of various materials such as glass or plastic. The container contains a composition effective to treat the condition and may have a sterile interface (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the anti-CD 47 antibody. The label on or with the container indicates that the composition is used to treat the selected condition. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, a combination sodium chloride solution, and a glucose solution. It may further include other materials that are commercially and user-desired, including other buffers, diluents, filters, needles, syringes, and packaging containing instructions for use.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what is claimed or to represent that the embodiments are all or the only embodiments. Efforts have been made to obtain accurate values for use (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The present invention has been described with respect to particular embodiments found or suggested by the inventors, including preferred embodiments of the present invention. Those skilled in the art, having the benefit of this disclosure, will be able to affect numerous modifications to, and departures from, the specific embodiments disclosed herein without departing from the scope of the present invention. For example, the following DNA sequence can be altered without affecting the protein sequence due to excessive codons. Moreover, due to the equivalence of biological functions, the protein structure can be altered without affecting the kind or effect of the biological function. All such modifications are intended to be included within the scope of the present invention as defined in the appended claims.

Construction of phage libraries

CD47 is a 50kDa membrane receptor with an extracellular N-terminal IgV region, five transmembrane regions, and a short C-terminal intracellular tail. Human CD47-IgV region protein conjugated to human Fc or biotinylated human CD47-IgV region protein (ACROBIOSystems) were used as antigens for phage library screening.

Phage libraries were constructed using phagemid vectors, including amplified antibody gene fragments from spleen or bone marrow cells of more than 50 healthy humans. The antibody is a single chain variable fragment (VH + linker + VL). The library capacity was 1.1X 10¹⁰The diversity of the sequences was analyzed as follows. Further sequencing of 62 clones extracted from the library, 16 sequences with truncated, frameshifted or amber codons; the 46 sequences had a full-length scFv, in which all HCDR3 sequences were identical. In 46 full lengthsIn the scFv, 13 sequences had lambda light chains and 33 sequences had k light chains.

Phage panning and clonal screening

To obtain phage clones that specifically bind to the human CD47-IgV region, two methods of phage panning were used. 1. Phage library immunotube panning based on human CD47-IgV

In this method, the phage library developed above was first incubated in a casein-coated immune tube for 2 hours. Human CD47-IgV-Fc fusion protein was used for the first panning round. Unbound phage were washed away by washing 5-20 times with PBST. Bound phage were eluted with freshly prepared 100mM triethylamine solution and neutralized by adding Tris-hydrochloride buffer as the first pool of exported phage. The first pool of exported phage was rescued by infection and expansion of TG-1 cells of E.coli, followed by a second round of panning using biotinylated human CD47-IgV as antigen. Bound phage were eluted in the same procedure as a second pool of exported phage, which was then rescued, and then a third round of panning was performed using human CD47-IgV-Fc fusion protein as the antigen. Bound phage then became the third pool of export phage and were subjected to a fourth panning run with biotinylated human CD 47-IgV.

2. Phage library liquid phase panning based on human CD47-IgV

In this second method, the phage library is first incubated in 100. mu.L of streptavidin magnetic beads blocked with casein to deplete the streptavidin magnetic bead binding sites. The streptavidin magnetic beads and AG0084-huIgG1/k were used for enrichment. The enriched library was rescued, followed by a second round of panning with biotinylated human CD47-IgV as antigen and further enrichment with casein-blocked streptavidin magnetic beads. Unbound phage were washed away with PBST 5-20 times. Bound phage were eluted with freshly prepared 100mM triethylamine solution and neutralized with Tris-HCl buffer, followed by rescue, followed by a third round of panning with human CD47-IgV-Fc fusion protein and enrichment with AG0084-huIgG 1/k. The bound phage then became the third pool of exported phage, and was subjected to a fourth round of panning with biotinylated human CD47-IgV and enrichment with casein-blocked streptavidin magnetic beads.

After this step, a number of phage clones that specifically bind to the human CD47-IgV region were obtained and enriched. The phage clones were then diluted and plated to grow at 37 ℃ for 8 hours and reacted overnight with anti-kappa antibody coated filters. Biotinylated human CD47-IgV (50nM) and avidin-AP conjugate (1:1000 dilution) were used in the filter to detect positively bound phage clones. Positive phage plates were picked and eluted into 100. mu.L phage eluate. Approximately 10-15. mu.L of eluted phage was used to infect 1mL XL1 Blue cells to prepare high titer phage (HT) for phage spot ELISA (SPE). The positive monoclonal picked off the filter was bound to human CD47-IgV-Fc fusion protein and enzymatically-cleaved human CD47-IgV domain protein. VH and VL genes were sequenced for these positive monoclonals. All positive sites of the unique VH and VL genes were cloned into the expression vectors pFUSE2ss-CLIg-hk (light chain, InvivoGen, Cat No. pfuse2ss-hclk) and pFUSES-CHIg-hG 1 (heavy chain, InvivoGen, Cat No. pFUSEss-hchg 1). These antibodies were expressed in HEK293 cells and purified from protein a and agarose.

Affinity maturation of CD47 antibody

The binding affinity of the CD47 antibodies provided herein can be increased by in vitro affinity maturation, for example, by random variation at specific sites, which results in variant sequences that are also within the scope of the present invention.

For example, BiaCore analysis of 1F8, a CD47 antibody provided herein, showed a binding affinity (KD) of 2.8nM with a high dissociation rate of 1.04E-031/s, which can be increased by in vitro affinity maturation. CDR sequence epitaxy analysis of the heavy and light chains of 1F8 demonstrated that several residues of the HCDR1 and LCDR1 regions were capable of random variation. Thus, random variation libraries can be constructed and specific residues introduced to generate a wide variety of new sequences. The CDR variation library was panned with biotinylated soluble CD47 ECD in solution phase under equilibrium conditions. After multiple rounds of panning with reduced antigen concentration, the enriched output binders were selected for binding to ELISA experiments and subsequently converted to intact IgG, which was the subject of BiaCore analysis, to specifically select for sequences with improved dissociation rates. Through the screening process, the antibody molecule provided by the invention can be constructed into clinically applied antibodies with comprehensive optimal performance.