WO2022057939A1

WO2022057939A1 - Pharmaceutical composition comprising cd47 antibody and pd-1/pd-l1 inhibitor

Info

Publication number: WO2022057939A1
Application number: PCT/CN2021/119523
Authority: WO
Inventors: Wei Cao; Wenqing Jiang; Chunyan YE; Zhengyi WANG; Bingshi GUO
Original assignee: I-Mab Biopharma Co., Ltd.; I-Mab Biopharma Us Limited
Priority date: 2020-09-21
Filing date: 2021-09-22
Publication date: 2022-03-24

Abstract

Provided is a method of treating a disorder in a subject in need thereof, comprising administrating an anti-CD47 antibody and a PD-1/PD-L1 inhibitor to the subject, also provided is a pharmaceutical composition that includes an anti-CD47 antibody and a PD-1/PD-L1 inhibitor.

Description

Pharmaceutical Composition Comprising CD47 Antibody and PD-1/PD-L1 Inhibitor

Cross-Reference to Related Applications

This application claims priority to PCT Application No. PCT/CN2020/116564, filed September 21, 2020, and PCT/CN2020/116565, also filed September 21, 2020, the contents of which are incorporated herein by reference in their entirety.

Background of the Invention

CD47 (Cluster of Differentiation 47) also known as integrin associated protein (IAP) is a transmembrane protein that in humans is encoded by the CD47 gene. CD47 belongs to the immunoglobulin superfamily, and partners with membrane integrins and also binds the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα) . CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, it plays a key role in immune and angiogenic responses.

CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumor cells. It can act as a don't eat me signal to macrophages of the immune system which has made it a potential therapeutic target in cancers.

Programmed death-1 (PD-1) is a cell surface receptor that functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding of PD-1 to its ligand, programmed death-ligand 1 (PD-L1) , activates downstream signaling pathways and inhibits T cell activation. Moreover, abnormally high PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape. Immunotherapies that target PD-1/PD-L1 signaling pathway have shown unprecedented success in a wide variety of human cancers.

However, the need exists for developing novel combination therapies that activate the immune system for treating cancer.

Summary of the Invention

Pharmaceutical composition and method are provided here to address the need in the field.

In one aspect, provided is a method for treating a disorder in a subject in need thereof, comprising administrating to the subject an anti-CD47 antibody or an antigen binding fragment thereof and a PD-1/PD-L1 signaling inhibitor, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells. In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof binds to human CD47 at an epitope comprising one or more residues selected from 26-31, 34, 35, 74, and 101-103. In some embodiments of the method, the anti-CD47 antibodyor the antigen binding fragment thereof binds to human CD47 at an epitope comprising residues 26-31, 34, 35, 74, and 101-103.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereofis selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and (ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Kabat numbering scheme.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and (ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Chothia numbering scheme.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and (ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to IMGT numbering scheme.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319; (ii) a HCDR2 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320; (iii) HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321; (iv) a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322; (v) a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and (vi) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof comprising: (i) a VH comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77, and (ii) a VL comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.

In some embodiments of the method, the PD-1/PD-L1 signaling inhibitor is an antibody. In some embodiments of the method, the PD-1/PD-L1 signaling inhibitor is an anti-PD-1 antibody or an antigen binding fragment thereof. In some embodiments of the method, the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

In some embodiments of the method, the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting ofpidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, penpulimab, pembrolizumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01. In some embodiments of the method, the anti-PD-1 antibody or the antigen binding fragment thereof is toripalimab.

In some embodiments of the method, the PD-1/PD-L1 signaling inhibitor is an anti-PD-L1 antibodyor an antigen binding fragment thereof. In some embodiments of the method, the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

In some embodiments of the method, the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting ofmanelimab, atezolizumab, avelumab, cosibelimab, durvalumab, envafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003. In some embodiments of the method, the anti-PD-L1 antibody or the antigen binding fragment thereof is B12.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereofis administrated to the subject in need thereof before, after or concurrently with the PD-1/PD-L1 signaling inhibitor.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are administered at the same frequency or at different frequencies.

In some embodiments of the method, the disorder is a proliferative disorder. In some embodiments of the method, the cell proliferative disorder is cancer. In some embodiments of the method, the cancer is selected from the group consisting colorectal cancer, breast cancer, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, gliosarcoma, malignant glioma, peritoneal cancer, fallopian tube cancer, rectal cancer, kidney cancer, Hodgkin's lymphoma, bladder cancer, uveal melanoma, gastric cancer, squamous cell carcinoma, cervical cancer, uterine cancer, chronic lymphocytic leukemia, lymphoma, myeloma, Kaposi's sarcoma, urothelial carcinoma, mesothelioma, malignant fibrous histiocytoma, colon cancer, solid tumor, multiple myeloma, gastrointestinal stromal tumor, head and neck cancer, melanoma, and leiomyosarcoma. In some embodiments of the method, the cancer is solid tumor. In some embodiments of the method, the cancer is relapsed and refractory solid tumor.

In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are formulated together. In some embodiments of the method, the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are formulated separately.

In some embodiments of the method, the administration of anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor result in a synergistic effect in the subject in need thereof.

In another aspect, provided is apharmaceutical composition comprising: (1) an anti-CD47 antibody or an antigen binding fragment thereof, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells; and (2) a PD-1/PD-L1 signaling inhibitor.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof binds to human CD47 at an epitope comprising one or more residues selected from 26-31, 34, 35, 74, and 101-103. In some embodiments of the pharmaceutical composition, the anti-CD47 antibodyor the antigen binding fragment thereof binds to human CD47 at an epitope comprising residues 26-31, 34, 35, 74, and 101-103.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereofis selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; (ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, andwherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Kabat numbering scheme.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; (ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Chothia numbering scheme.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; (ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to IMGT numbering scheme.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319; (ii) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320; (iii) a HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321; (iv) a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322; (v) a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and (vi) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprising: (i) a VH comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and (ii) a VL comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.

In some embodiments of the pharmaceutical composition, the PD-1/PD-L1 signaling inhibitor is an antibody. In some embodiments of the pharmaceutical composition, the PD-1/PD-L1 signaling inhibitor is an anti-PD-1 antibody or an antigen binding fragment thereof. In some embodiments of the pharmaceutical composition, the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

In some embodiments of the pharmaceutical composition, the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting ofpidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, penpulimab, pembrolizumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01. In some embodiments of the pharmaceutical composition, the anti-PD-1 antibody or the antigen binding fragment thereof is toripalimab.

In some embodiments of the pharmaceutical composition, the PD-1/PD-L1 signaling inhibitor is an anti-PD-L1 antibodyor an antigen binding fragment thereof. In some embodiments of the pharmaceutical composition, the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

In some embodiments of the pharmaceutical composition, the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting of Manelimab, atezolizumab, avelumab, Cosibelimab, durvalumab, Envafolimab, Socazolimab, AUNP12, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, KY-1003, CA-170, BMS-986189, B6 and B12-01. In some embodiments of the pharmaceutical composition, the anti-PD-L1 antibody or the antigen binding fragment thereof is B12.

In another aspect, provided herein is an article of manufacture, comprising: (1) an anti-CD47 antibody or an antigen binding fragment thereof, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells; (2) a package insert, wherein the package insert provides instruction of administrating the anti-CD47 antibody or the antigen binding fragment thereof in combination with a PD-1/PD-L1 signaling inhibitor for treating a disorder in a subject in need thereof.

In some embodiments of the article of manufacture, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319; (ii) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320; (iii) a HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321; (iv) a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322; (v) a LCDR2 comprising an amino acid sequence selected from the group consisting of ID NO. 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and (vi) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324.

Brief Description of Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1illustrates antibody-dependent cellular cytotoxicity (ADCC) effects induced by anti-PD-L1 antibody, anti-CD47 antibody and the combination of anti-PD-L1 antibody and anti-CD47 antibody.

FIG. 2 illustrates antibody-dependent cellular phagocytosis (ADCP) effects induced by anti-PD-L1 antibody, anti-CD47 antibody and the combination of anti-PD-L1 antibody and anti-CD47 antibody.

FIG. 3Aand 3B illustrate inhibition of tumor volume and tumorweight by administration of anti-PD-1 antibody, anti-CD47 antibody and a combination of the anti-PD-1 antibody and the anti-CD47 antibody.

FIG. 4A and 4Billustrate inhibition of tumor volume and tumorweight by administration of anti-PD-L1 antibody, anti-CD47 antibody and a combination of the anti-PD-L1 antibody and the anti-CD47 antibody.

FIG. 5illustrates inhibition of tumor volume and tumorweight by administration of anti-PD-L1 antibody, the mouse anti-CD47 antibody and a combination of the anti-PD-L1 antibody and the anti-CD47 antibody.

Detailed Description of the Invention

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

DEFINITION

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a” , “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including” , “includes” , “having” , “has” , “with” , or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising. ”

As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

As used herein, the terms “individual” , “patient” , or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker) .

The term “antigen” as used herein, refers to a molecule or a fragment thereof capable of being bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent such as a receptor. In some cases, the receptor may function as the antigen and the ligand may function as the selective binding agent. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immunological protein (e.g., an antibody) . In some cases, the immunological protein may serve as the antigen and the antigenic molecule may serve as the selective binding agent. An antigen can also refer to a molecule or fragment thereof capable of being used in an animal to produce antibodies capable of binding to that antigen.

As used herein, the term “epitope” and its grammatical equivalentsrefer to a part of an antigen that can be recognized by an antigen binding domain of an antibody, such as CD47 polypeptide or CD47polypeptide fragment, that is capable of being bound by one or more antigen binding regions of an anti-CD47 antibody, and that has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human, that is capable of eliciting an immune response. An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a polypeptide to which an antibody specifically binds as determined by any method well known in the art, for example, by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. A region of a polypeptide contributing to an epitope may be contiguous amino acids of the polypeptide or the epitope may come together from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface feature of the antigen. In certain embodiments, a CD47 epitope is a three-dimensional surface feature of a CD47 polypeptide. In some embodiments, a CD47 epitope is linear feature of a CD47 polypeptide. In some embodiments, the anti-CD47 antibodies provided herein may specifically bind to an epitope of three-dimensional surface feature of CD47. In some embodiments, the anti-CD47 antibodies provided herein may specifically bind to an epitope of linear surface feature of CD47.

As used herein, the term “antibody” , “immunoglobulin” or “Ig” may be used interchangeably and refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F (ab') 2, and Fv fragments) , single chain Fv antibodies (scFvs) , single domain antibodies (sdAbs) , multispecific antibodies such as bispecific antibodies (including dual binding antibodies) , chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. The term “antibody” can also refer to a Y-shaped glycoprotein with a molecular weight of approximately 150 kDa that is made up of four polypeptide chains: two light (L) chains and two heavy (H) chains. There are five types of mammalian Ig heavy chain isotypes denoted by the Greek letters alpha (α) , delta (δ) , epsilon (ε) , gamma (γ) , and mu (μ) . The type of heavy chain defines the class of antibody, i.e., IgA, IgD, IgE, IgG, and IgM, respectively. The γ and α classes are further divided into subclasses on the basis of differences in the constant domain sequence and function, e.g., IgG1, hIgG2, mIgG2A, mIgG2B, IgG3, IgG4, IgA1and IgA2. In mammals, there are two types of immunoglobulin light chains, λ and κ.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively. These domains are generally the most variable parts of the antibody and contain the antigen binding sites. The antibodies described herein may be oligoclonal, polyclonal, monoclonal (including full-length monoclonal antibodies) , camelised, chimeric, CDR-grafted, multi-specific, bi-specific (including dual-binding antibodies) , catalytic, chimeric, humanized, fully human, anti-idiotypic, including antibodies that can be labelled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. An antibody may be from any species. Antibodies described herein can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

As used herein, the term “antigen binding domain” , “antigen binding region” , “antigen binding fragment” , or equivalent thereof refers to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g. the complementarity determining regions (CDRs) ) . Antigen binding fragments described herein can include single-chain Fvs (scFvs) , single domain antibodies (sdAbs) , Fv fragments, Fab fragments, F (ab') fragments, F (ab') ₂ fragments, antibody fragments that exhibit the desired biological activity, disulfide-stabilized variable region (dsFv) , dimeric variable region (diabody) , anti-idiotypic (anti-Id) antibodies (including, e.g. anti-Id antibodies to antibodies) , intrabodies, linear antibodies, singlechain antibodies and multispecific antibodies formed from antibody fragments and epitope-binding fragments of any of the above. In particular, antibodies and antibody fragments described herein can include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site.

Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallize. "Fab" when used herein refers to a fragment of an antibody that includes one constant and one variable domain of each of the heavy and light chains. The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The "Fc fragment" refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with the enzyme, pepsin, results in a F (ab') ₂ fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F (ab') ₂ fragment has the ability to crosslink antigen.

"Fv" when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. 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. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

As used herein, the term “framework” or “FR” residues (or regions) refer to variable domain residues other than the CDR or hypervariable region residues as herein defined. A “human consensus framework” is a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences.

The term “CDR” as used herein refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable domains of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable domains. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991) ) not only provides an unambiguous residue numbering system applicable to any variable domain of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2, and L3 or H1, H2, and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan et al. ( (1995) FASEB J. 9: 133-139) and MacCallum et al. ( (1996) J. Mol. Biol. 262 (5) : 732-745) . Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, including but not limited to Kabat, Chothia or IMGT defined CDRs.

The terms "Kabat numbering" , "Kabat definitions" , and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable domains of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 ; and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and HumanServices, NIH Publication No. 91-3242, which is also available on the world wide web, and is expressly incorporated herein in its entirety by reference. The "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody. As used herein, "Kabat sequence numbering" refers to numbering of the sequence encoding a variable region according to the EU index as in Kabat.

In some embodiments, IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of variable regions can also be used, which is the numbering of the residues in an immunoglobulin variable heavy or light chain according to the methods of the IIMGT, as described in Lefranc, M-P, "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains" , The Immunologist, 7, 132-136 (1999) , and is expressly incorporated herein in its entirety by reference. As used herein, "IMGT sequence numbering" refers to numbering of the sequence encoding a variable region according to the IMGT. For the heavy chain variable domain, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable domain, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g. isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, and are directed against a single antigentic determinant or epitope. In contrast, polyclonal antibody preparations typically include different antibodies directed against different antigenic determinants (or epitopes) . The term "monoclonal antibody" as used herein encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F (ab') 2, Fv) , single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

The monoclonal antibodies herein can include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with 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 (s) is (are) identical with 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 that exhibit the desired biological activity.

The term "humanized antibody" refers to a subset of chimeric antibodies in which a "hypervariable region" from a non-human immunoglobulin (the donor antibody) replaces residues from a hypervariable region in a human immunoglobulin (recipient antibody) . In general, a humanized antibody will include substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the framework regions are those of a human immunoglobulin sequence, although the framework regions may include one or more substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc.

The term "specifically binds to" refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g. by a radioimmunoassay (RIA) .

The term “binding affinity” as used herein refers to strength of the binding interaction between members of a binding pair, for example, an antigen binding molecule and its antigen, or a receptor and its ligand. The binding affinity of a subject antibody for its partner may be characterized by k _on, k _off or K _D. The term “k _on” , as used herein, is intended to refer to the rate constant for association of an antibody to an antigen. The term “k _off” , as used herein, is intended to refer to the rate constant for dissociation of an antibody from the antibody/antigen complex. The term “K _D” , as used herein, is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction. For purposes of the present disclosure, K _D is defined as the ratio of the two kinetic rate constants k _on/k _off. The smaller the equilibrium dissociation constant the tighter the subject antibody and its partner bind to each other.

As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term “nucleotide” , as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) . The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides can include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2’7’-dimethoxy-4’5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N’, N’-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4’dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2’-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) . Specific examples of fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′ -dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP) , biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) , and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) .

The terms “polynucleotide” , “oligonucleotide” , and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase) \. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza- GTP, fluorophores (e.g. rhodamine or fluorescein linked to the sugar) , thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

The term “gene” , as used herein, refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’ ends. In some uses, the term encompasses the transcribed sequences, including 5’ and 3’ untranslated regions (5’ -UTR and 3’ -UTR) , exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .

The terms “derivative” , “variant” and “fragment” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary) , activity (e.g., enzymatic activity) and/or function. Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a referenced polypeptide.

The terms “treatment” and “treating” , as used herein, refer to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. A therapeutic benefit can refer to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

A “therapeutic effect” may occur if there is a change in the condition being treated. The change may be positive or negative. For example, a “positive effect” may correspond to an increase in the number of activated T-cells in a subject. In another example, a ‘negative effect’ may correspond to a decrease in the amount or size of a tumor in a subject. A “change” in the condition being treated, may refer to at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75%, or 100%change in the condition. The change can be based on improvements in the severity of the treated condition in an individual, or on a difference in the frequency of improved conditions in populations of individuals with and without the administration of a therapy. Similarly, a method of the present disclosure may comprise administering to a subject an amount of cells that is “therapeutically effective. ” The term “therapeutically effective” should be understood to have a definition corresponding to ‘having a therapeutic effect.

The term “effective amount” or “therapeutically effective amount” refers to the quantity of a composition, for example a composition comprising antibodies, that is sufficient to result in a desired activity upon administration to a subject in need thereof. The term “therapeutically effective” can refer to a quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

As used herein, the term “PD-1/PD-L1 signaling inhibitor” refers to any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. PD-1/PD-L1 signaling inhibitor that can be used in the combination, method or article of manufacture of the present invention includes but not limited to an antibody, such as a monoclonal antibody (mAb) , or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1. In some embodiments, the antigen binding fragment includes but is not limited topolyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

PHARMACEUTICAL COMPOSITION

In one aspect, provided herein isa pharmaceutical composition comprisingan anti-CD47 antibody or an antigen binding fragment thereofand a PD-1/PD-L1 signaling inhibitor. In some embodiments of the pharmaceutical composition of the present application, the antibody does not cause a significant level of hemagglutination or depletion of red blood cells. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereofis selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

In some embodimentsof the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof binds to human CD47 at an epitope comprising one or more residues selected from 26-31, 34, 35, 74, and 101-103. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof binds to human CD47 at an epitope comprising residues 26-31, 34, 35, 74, and 101-103.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL) , wherein the VH comprises HCDR1, HCDR2 and HCDR3 and the VL comprises LCDR1, LCDR2 and HCDR3. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected fromSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence of any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59. 61, 63, 65, 67, 69, 71, 73, 75, and 77, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence of any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system.

In some embodimentsof the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprisesHCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77; and LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system. In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO: 31; and LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 32, according to Kabat numbering system.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence of any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77; and LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence of anyone selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence of SEQ ID NO: 31; and LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence of SEQ ID NO: 32, according to Kabat numbering system.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of anyone selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of anyone selected from SEQ ID NO: 181, or a variant thereof comprising one or more substitutions, deletions or insertions as compared toSEQ ID NO: 181.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR2 comprising an amino acid sequence of anyone selected from the group consisting of SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR2 comprising an amino acid sequence ofSEQ ID NO: 182, or a variant thereof comprisingone or more substitutions, deletions or insertions as compared toSEQ ID NO: 182.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR3 comprising an amino acid sequence of anyone selected from the group consisting of SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR3 comprising an amino acid sequence ofSEQ ID NO: 183, or a variant thereof comprisingone or more substitutions, deletions or insertions as compared toSEQ ID NO: 183.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a LCDR1 comprising an amino acid sequence of anyone selected from the group consisting of SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a LCDR1 comprising an amino acid sequence ofSEQ ID NO: 184, or a variant thereof comprisingone or more substitutions, deletions or insertions as compared toSEQ ID NO: 184.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a LCDR2 comprising an amino acid sequence of any one selected from the group consisting of SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a LCDR2 comprising an amino acid sequence ofSEQ ID NO: 185, or a variant thereof withone or more substitutions, deletions or insertions as compared toSEQ ID NO: 185.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a LCDR3 comprising an amino acid sequence of any one selected from the group consisting of SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR2 comprising an amino acid sequence ofSEQ ID NO: 186, or a variant thereof withone or more substitutions, deletions or insertions as compared toSEQ ID NO: 186.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (1) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319; (2) a HCDR2 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320; (3) a HCDR3 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321; (4) a LCDR1 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322; (5) a LCDR2 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and (6) a LCDR3 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (1) a HCDR1 comprising an amino acid sequence ofSEQ ID NO: 181, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 181; (2) a HCDR2 an amino acid sequence ofSEQ ID NO: 182, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 182; (3) a HCDR3 comprising an amino acid sequence ofSEQ ID NO: 183, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 183; (4) a LCDR1 comprising an amino acid sequence ofSEQ ID NO: 184, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 184; (5) a LCDR2 comprising an amino acid sequence ofSEQ ID NO: 185, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 185; and (6) a LCDR3 comprising an amino acid sequence ofSEQ ID NO: 186, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 186.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprisesa VH comprising an amino acid sequence of any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO: 31. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a VH comprising an amino acid sequence ofSEQ ID NO: 31.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises aVL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises aVLcomprising an amino acid sequence of any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO: 32. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a VL comprising an amino acid sequence ofSEQ ID NO: 32.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77; and (ii) aVL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO: 31; and (ii) aVL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with ID NO. 32.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a VH comprising an amino acid sequence of any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77; and (ii) aVL comprising an amino acid sequence of any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a VH comprising an amino acid sequence of SEQ ID NO: 31; and (ii) aVL comprising an amino acid sequence of SEQ ID NO: 32.

Any suitable PD-1/PD-L1 signaling inhibitor known in the art can be used in the pharmaceutical composition of the application. In some embodiments, the PD-1/PD-L1 signaling inhibitor includes a small molecule inhibitor of PD-1/PD-L1 signaling. In some embodiments, the PD-1/PD-L1 signaling inhibitor includes an antibodyor an antigen binding fragment thereof, such as an anti-PD-1 antibody, an anti-PD-L1 antibodyor an antigen binding fragment thereof. In some embodiments, the PD-1/PD-L1 signaling inhibitor is an anti-PD-1 antibody or an antigen binding fragment thereof.

Anti-PD-1antibodycan be used in the present application includes but is not limited to polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. Exemplary anti-PD-1 antibody includes but is not limited to pidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, penpulimab, pembrolizumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01. In some embodiments, the anti-PD-1 antibody is toripalimab.

Anti-PD-L1antibody can be used in the present application includes but is not limited to polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. Exemplary anti-PD-L1 antibody includes but is not limited tomanelimab, atezolizumab, avelumab, cosibelimab, durvalumab, envafolimab, socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY- 3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003. In some embodiments, the anti-PD-1 antibody is B12.

In some embodiments, the pharmaceutical composition of the present application further comprises one or more pharmaceutically acceptable excipient. Exemplary pharmaceutically acceptable excipient that can be used in the pharmaceutical composition of the present application includes but is not limited to buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; 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, disacchahdes, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN ^TM, PLURONICS ^TM or polyethylene glycol (PEG) .

In some embodiments, the pharmaceutical composition may be suitable for oral, parenteral, topical administration or for administration by inhalation. Accordingly, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure may be administered parenterally, such as intravenously, or intramuscularly, or subcutaneously. Alternatively, an antibody of the invention may be administered via a non-parenteral route, such as per-orally or topically. In a preferred embodiment, a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

METHOD

In one aspect, provided herein is a method for treating a disorder in a subject in need thereof, comprising administrating to the subject an anti-CD47 antibody or an antigen binding fragment thereof and a PD-1/PD-L1 signaling inhibitor. In some embodiments of the method, the antibody does not cause a significant level of hemagglutination or depletion of red blood cells. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereofis selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.

In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL) , wherein the VH comprises HCDR1, HCDR2 and HCDR3 and the VL comprises LCDR1, LCDR2 and HCDR3. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence of any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59. 61, 63, 65, 67, 69, 71, 73, 75, and 77, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence of any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system.

In some embodimentsof the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59.61, 63, 65, 67, 69, 71, 73, 75, and 77; and LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, according to Kabat numbering system. In some embodiments of the pharmaceutical composition, the anti-CD47 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3as set forth in a VH comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO: 31; and LCDR1, LCDR2 and LCDR3as set forth in a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 32, according to Kabat numbering system.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of anyone selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a HCDR1 comprising an amino acid sequence of anyone selected from SEQ ID NO: 181, or a variant thereof comprising one or more substitutions, deletions or insertions as compared toSEQ ID NO: 181.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319; (ii) a HCDR2 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320; (iii) a HCDR3 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321; (iv) a LCDR1 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322; (v) a LCDR2 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and (vi) a LCDR3 comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises: (i) a HCDR1 comprising an amino acid sequence ofSEQ ID NO: 181, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 181; (ii) a HCDR2 an amino acid sequence ofSEQ ID NO: 182, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 182; (iii) a HCDR3 comprising an amino acid sequence ofSEQ ID NO: 183, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 183; (iv) a LCDR1 comprising an amino acid sequence ofSEQ ID NO: 184, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 184; (v) a LCDR2 comprising an amino acid sequence ofSEQ ID NO: 185, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 185; and (6) a LCDR3 comprising an amino acid sequence ofSEQ ID NO: 186, and a variant thereof with one or more substitutions, deletions or insertions as compared toSEQ ID NO: 186.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises aVL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises aVLcomprising an amino acid sequence of any one selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In some embodiments, the anti- CD47 antibody or the antigen binding fragment thereof comprises a VL comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO: 32. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof comprises a VL comprising an amino acid sequence ofSEQ ID NO: 32.

Any suitable PD-1/PD-L1 signaling inhibitor known in the art can be used in the method of the application. In some embodiments, the PD-1/PD-L1 signaling inhibitor includes a small molecule inhibitor of PD-1/PD-L1 signaling. In some embodiments, the PD-1/PD-L1 signaling inhibitor includes an antibodyor an antigen binding fragment thereof, such as an anti-PD-1 antibody, an anti-PD-L1 antibodyor an antigen binding fragment thereof. In some embodiments, the PD-1/PD-L1 signaling inhibitor is an anti-PD-1 antibody or an antigen binding fragment thereof.

Anti-PD-1antibody can be used in the method of the present application includes but is not limited to polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. Exemplary anti-PD-1 antibody includes but is not limited to pidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, penpulimab, pembrolizumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01.In some embodiments, the anti-PD-1 antibody is toripalimab.

Anti-PD-L1antibody can be used in the method of the present application includes but is not limited to polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb. Exemplary anti-PD-L1 antibody includes but is not limited toManelimab, atezolizumab, avelumab, Cosibelimab, durvalumab, Envafolimab, Socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003.

In some embodiments of the method of the present application, the anti-CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof before, after or concurrently with the PD-1/PD-L1 signaling inhibitor.

In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof before the PD-1/PD-L1 signaling inhibitor such aspembrolizumab ortoripalimab. For example, in some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof beforepembrolizumab ortoripalimab. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof after the PD-1/PD-L1 signaling inhibitor such aspembrolizumab ortoripalimab. For example, in some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof afterpembrolizumab ortoripalimab. In some embodiments, the anti-CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof concurrently with the PD-1/PD-L1 signaling inhibitor such aspembrolizumab ortoripalimab. For example, in some embodiments, the anti- CD47 antibody or the antigen binding fragment thereof is administrated to the subject in need thereof concurrently withpembrolizumab ortoripalimab.

In some embodiments of the method of the present application, the anti-CD47 antibody or antigen-binding fragment thereof and the PD-1/PD-L1 inhibitor are admixed together and administrated to the subject. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof and the PD-1/PD-L1 inhibitor are separately administered within, for example, 1 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, or 20 minutes.

In some embodiments of the method of the present application, anti-C5aR antibody or antigen-binding fragment thereof and the PD-1/PD-L1 inhibitor are administered sequentially. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof is administered before the PD-1/PD-L1 inhibitor. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof is administered after the PD-1/PD-L1 inhibitor. In some embodiments, the anti-CD47 antibody or antigen-binding fragment thereof and the PD-1/PD-L1 inhibitor are separately administered within, for example, 30 minutes, 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or 1 month.

In some embodiments of the method of the present application, the method can be used for treating cancer in a subject in need thereof. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, gliosarcoma, malignant glioma, peritoneal cancer, fallopian tube cancer, rectal cancer, kidney cancer, Hodgkin's lymphoma, bladder cancer, uveal melanoma, gastric cancer, squamous cell carcinoma, cervical cancer, uterine cancer, chronic lymphocytic leukemia, lymphoma, myeloma, Kaposi's sarcoma, urothelial carcinoma, mesothelioma, malignant fibrous histiocytoma, colon cancer, solid tumor, multiple myeloma, gastrointestinal stromal tumor, head and neck cancer, melanoma, and leiomyosarcoma. In some embodiments, the cancer is solid tumor. In some embodiments, the cancer is relapsed and refractory solid tumor.

In some embodiments of the method of the present application, the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are formulated together. In some embodiments of the method of the present application, the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are formulated separately.

Insome embodimentsof the method of the present application, the administration of anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor result in a synergistic effect in the subject in need thereof.

ARTICLE OF MANUFACTURE

In another aspect, provided is an article of manufacture, comprising: (1) an anti-CD47 antibody or an antigen binding fragment thereof, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells; and (2) a package insert, wherein the package insert provides instruction of administrating the anti-CD47 antibody or the antigen binding fragment thereof in combination with a PD-1/PD-L1 signaling inhibitor for treating a disorder in a subject in need thereof.

In some embodiments of the article of manufacture, the anti-CD47 antibody or the antigen binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 as set forth in a variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, andwherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Kabat numbering scheme.

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 the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLES

Example 1. Antibody-dependent cellular cytotoxicity induced by the combination anti-CD47 antibody and PD-L1 antibody

The effects of the combination of anti-CD47 antibody of the application and anti-PD-L1 antibody on human PBMC-mediated cytotoxicitywere evaluated. Briefly, human B cell lymphoma cell line overexpressing PD-L1 (named as “Raji-PD-L1” hereinafter) was labelled with a fluorescence enhancing ligand (DELFIA BATDA reagent) and then co-cultured with human PBMCs in the presence of CD47 mAb (comprising CDRs of SEQ ID NO: 97-102) , PD-L1 mAb B12or the combo of CD47 mAb and PD-L1 mAb at a concentration ranging from 0.0013nM to 20nM. After 2 hours of incubation, the supernatant was collected and the fluorescence was measured and analyzed by using a time-resolved fluorometer. As shown in Fig. 1, the combination of CD47 mAb and PD-L1 mAb showed superior ADCC activity than CD47 mAb or PD-L1 mAb alone.

Example 2. Antibody-dependent cellular phagocytosis induced by the combination of anti-CD47 antibody and PD-L1 antibody

The effects of the combination of anti-CD47 antibody of the application and anti-PD-L1 antibody on human macrophage-mediated phagocytosis of tumor cells were evaluated. Briefly, Raji cells overexpressing PD-L1 were labelled with Carboxyfluorescein Succinimidyl Ester (CFSE) and co-cultured with human differentiated macrophages in the presence of IgG, CD47 mAb (comprising CDRs of SEQ ID NO: 181-186) , PD-L1 mAb (TJ0141H or B12) or combo of CD47 mAb and PD-L1 mAb. After 3 hours of incubation, non-phagocytosed target cells were washed away with PBS and the remaining phagocytes were collected and stained with anti-CD14, followed by flow cytometry analysis. “Phagocytosis index (PI) ” was calculated as percentage of CFSE positive CD14+ cells in the treatment group normalized against that in IgG group at the corresponding concentration.

As shown in FIG. 2, the combination PD-L1 mAb and CD47 mAb induced superior ADCP activity than CD47 mAb or PD-L1 mAb alone. Synergistic effect (Q value) was calculated as: Q=PI _Combo/ (PI _PD-L1 mAbX PI _CD47 mAb) . If Q >1, the combination treatment is considered as synergistic. As shown in Table 1, the combination of CD47 mAband PD-L1 mAb displayed synergistic effect on inducing antibody-dependent cellular phagocytosisat different concentrations.

Table 1. Q value of combination treatment over comparator monotherapy.

Conc.	CD47 mAb+TJ0141H	CD47 mAb+B12
6.67nM	1.44	1.13
0.67nM	1.56	0.85
0.07nM	1.21	1.96
0.01nM	0.91	1.48

Example 3. In vivo anti-tumor efficacy of the combination of anti-CD47 antibody and PD-1 antibody

In vivo efficacy of the combination of Toripalimab (JS001, anti-PD-1 antibody) and anti-CD47 antibody of the present application in the treatment of the subcutaneous MC38-hPD-L1/hCD47 colon carcinoma in humanized B-hPD-1/hPD-L1/hSIRPA/hCD47 mice was evaluated.

Briefly, B-hPD-1/hPD-L1/hSIRPA/hCD47 mice were subcutaneously injected with MC38-hPD-L1/hCD47 tumor cells (2×10 ⁵) in the right front flank for tumor development. When the mean tumor size reached 53 mm ³, tumor-bearing animals were randomly divided into four groups, 8 mice per group, and intraperitoneally administrated with PBS, Toripalimab (1 mg/kg) , CD47 mAb (comprising CDRs of SEQ ID NO: 181-186, 20 mg/kg) , and combo of Toripalimab (1 mg/kg) and CD47 mAb (20 mg/kg) , respectively. The tumor volume and body weight were measured and recorded twice per week. On day 14, animals were euthanized, and tumors were collected, weighed and photographed.

As shown in Figs. 3A and 3B, compared to the treatment with Toripalimab or CD47 mAb alone, treatment with the combo of Toripalimab and CD47 mAb demonstrated significantly stronger anti-tumor activity.

TGI (Tumor growth inhibition) was calculated by the following equation: TGI _TV%= [1- (T _i-T ₀) / (V _i-V ₀) ] ×100%. (Ti: means tumor volume of the treated groups at day i following treatment; T0: means tumor volume of the treated groups at day 0; Vi: means tumor volume of the vehicle groups at day i following treatment; V0: means tumor volume of the vehicle groups at day 0) . TGI was shown in Table 2.

Table 2. TGIin humanized B-hPD-1/hPD-L1/hSIRPA/hCD47 mice with MC38-hPD-L1/hCD47 cells

[1] Synergistic effect of the combo of Toripalimab and CD47 mAb was further analyzed based on TGI according to Kim’s equation: Q = TGI _Combo / (TGI _Toripalimab + TGI _CD47 mAb- TGI _Toripalimab X TGI _CD47 mAb) , in which Q ≥ 1.15 is considered as synergistic; 0.85 ≤ Q < 1 is considered as additive; and Q < 0.85 is considered as antagonistic. As shown in Table 3, the combination of Toripalimab and CD47 mAbachieved synergistic TGIs in mice on day 3, day 10 and day 14.

Table 3. Q value of the combination of CD47 mAb and Toripalimab.

Days post treatment	3	10	14
Q value	1.67	1.40	1.17

Example 4. In vivo anti-tumor efficacy of the combination of anti-CD47 antibody and PD-L1 antibody

[2] In vivo efficacy of the combination of the anti-CD47 antibody of the present application and PD-L1 antibody was evaluated. Briefly, NOD SCID mice were subcutaneously injected with Raji-hPD-L1 tumor cells (3×10 ⁶) in the right front flank for tumor development. When the mean tumor size reached ～146 mm ³, tumor-bearing animals were randomly divided into four groups, 5 mice per group, and intraperitoneally administrated with hIgG, CD47 mAb (comprising CDRs of SEQ ID NO: 97-102, 5 mg/kg) , B12 (5 mg/kg) , combo of theCD47 mAb (5 mg/kg) and B12 (5 mg/kg) at a frequency of twice per week for total of three times.

[3] On day 11, animals wereeuthanized, and tumors were collected, weighed and photographed. As shown in FIGs. 4A and 4B, compared to the treatment with PD-L1 mAb or CD47 mAb alone, treatment with combo of PD-L1 mAb and CD47 mAb demonstrated significantly stronger anti-tumor activity.

[4] TGI (Tumor growth inhibition) was calculated by the following equation: TGI _TV%= [1- (T _i-T ₀) / (V _i-V ₀) ] ×100%. (T _i: means tumor volume of the treated groups at day i following treatment; T ₀: means tumor volume of the treated groups at day 0; V _i: means tumor volume of the vehicle groups at day i following treatment; V ₀: means tumor volume of the vehicle groups at day 0) . TGI was shown in Table 4.

Table 4. TGI inNOD SCID mice with Raji-PD-L1 cells

[5] Synergistic effect of the combo of CD47 mAb and PD-L1 mAb was further analyzed based on TGI according to Kim’s equation: Q = TGI _Combo/ (TGI _PD-L1 mAb + TGI _CD47 mAb-TGI _PD-L1 mAb X TGI _CD47 mAb) , in which Q ≥ 1.15 is considered as synergistic; 0.85 ≤ Q < 1 is considered as additive; and Q < 0.85 is considered as antagonistic. As shown in Table 5, the combination of CD47 mAb and PD-L1 mAb achieved synergistic TGI in mice on

day

4, 7, 9 and 11 of the treatment.

Table 5. Q value of the combination of CD47 mAb and B12

Days post treatment	4	7	9	11
Q value	2.09	2.31	3.90	4.74

Example 5. In vivo anti-tumor efficacy of the combination of anti-CD47 antibody and anti-PD-L1 antibody

[6] In vivo anti-tumor efficacy of the combination of Tecentriq (an anti-PD-L1 antibody) and the anti-CD47 antibody of the present application was evaluated in C57BL/6 mice. Briefly, C57BL/6 mice were subcutaneously injected with MC38 tumor cells (2×10 ⁵) in the right front flank for tumor development. When the mean tumor size reached 85 mm ³, tumor-bearing animals were randomly divided into four groups, 6 mice per group, and administrated with IgG (5 mg/kg) , Tecentriq (5 mg/kg) , CD47 mAb (comprising CDRs of SEQ ID NO: 325-330, 5 mg/kg) , and the combination of Tecentriq (5 mg/kg) and CD47 mAb (5 mg/kg) at a frequency of twice per week. The tumor volume and body weight were measured and recorded twice per week.

[7] On day 18, animals wereeuthanized, and tumors were collected, weighed and photographed. As shown in FIG. 5, compared to the treatment with PD-L1 mAb or CD47 mAb alone, treatment with the combo of PD-L1 mAb and CD47 mAb demonstrated significantly stronger anti-tumor activity.

[8] TGI (Tumor growth inhibition) was calculated by the following equation: TGI _TV%= [1- (T _i-T ₀) / (V _i-V ₀) ] ×100%. (T _i: means tumor volume of the treated groups at day i following treatment; T ₀: means tumor volume of the treated groups at day 0; V _i: means tumor volume of the vehicle groups at day i following treatment; V ₀: means tumor volume of the vehicle groups at day 0) . TGI was shown in Table 6.

Table 6. TGI inC57BL/6 mice with MC38 cells

[9] Synergistic effect of the combo of CD47 mAb and PD-L1 mAb was further analyzed based on TGI according to Kim’s equation: Q = TGI Combo / (TGI _Tecentriq + TGI _CD47 mAb-TGI _Tecentriq X TGI _CD47 mAb) , in which Q ≥ 1.15 is considered as synergistic; 0.85 ≤ Q < 1 is considered as additive; and Q < 0.85 is considered as antagonistic. As shown in Table 7, the combination of CD47 mAb andTecentriq achieved synergistic TGI on

day

4, 6, 8, 13, 15 and 18 after treatment.

Table 7. Q value of the combination of CD47 mAb and Tecentriq

SEQUENCE LISTING

Claims

A method for treating a disorder in a subject in need thereof, comprising administrating to the subject an anti-CD47 antibody or an antigen binding fragment thereof and a PD-1/PD-L1 signaling inhibitor, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells.
The method of claim 1, wherein the anti-CD47 antibody or the antigen binding fragment thereof binds to human CD47 at an epitope comprising one or more residues selected from 26-31, 34, 35, 74, and 101-103.
The method of claim 2, wherein the anti-CD47 antibodyor the antigen binding fragment thereof binds to human CD47 at an epitope comprising residues 26-31, 34, 35, 74, and 101-103.
The method of any one of claims 1-3, wherein the anti-CD47 antibody or the antigen binding fragment thereofis selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.
The method of any one of claims 1-4, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprises:

(i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77;

(ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and

wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Kabat numbering scheme.
The method of any one of claims 1-4, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprises:

(i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319;

(ii) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320;

(iii) a HCDR3 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321;

(iv) a LCDR1 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322;

(v) a LCDR2 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and

(vi) a LCDR3 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324.
The method of any one of claims 1-4, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprising:

(i) a VH comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and

(ii) a VL comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.
The method of any one of claims 1-7, wherein the PD-1/PD-L1 signaling inhibitor is an antibody.
The method of claim 8, wherein the PD-1/PD-L1 signaling inhibitor is an anti-PD-1 antibody or an antigen binding fragment thereof.
The method of claim 9, wherein the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.
The method of claim 9, wherein the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting ofpidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, penpulimab, pembrolizumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK- 103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01.
The method of claim 11, wherein the anti-PD-1 antibody or the antigen binding fragment thereof is toripalimab.
The method of claim 8, wherein the PD-1/PD-L1 signaling inhibitor is an anti-PD-L1 antibodyor an antigen binding fragment thereof.
The method of claim 13, wherein the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.
The method of claim 13, wherein the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting of Manelimab, atezolizumab, avelumab, Cosibelimab, durvalumab, Envafolimab, Socazolimab, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12 and KY-1003.
The method of claim 15, Wherein the anti-PD-L1 antibody or the antigen binding fragment thereof is B12.
The method of any one of claims 1-16, wherein the anti-CD47 antibody or the antigen binding fragment thereofis administrated to the subject in need thereof before, after or concurrently with the PD-1/PD-L1 signaling inhibitor.
The method of any one of claims 1-17, wherein the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are administered at the same frequency or at different frequencies.
The method of any one of claims 1-18, wherein the disorder is a proliferative disorder.
The method of claim 19, wherein the cell proliferative disorder is cancer.
The method of claim 20, wherein the cancer is selected from the group consisting colorectal cancer, breast cancer, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, gliosarcoma, malignant glioma, peritoneal cancer, fallopian tube cancer, rectal cancer, kidney cancer, Hodgkin's lymphoma, bladder cancer, uveal melanoma, gastric cancer, squamous cell carcinoma, cervical cancer, uterine cancer, chronic lymphocytic leukemia, lymphoma, myeloma, Kaposi's sarcoma, urothelial carcinoma, mesothelioma, malignant fibrous histiocytoma, colon cancer, solid tumor, multiple myeloma, gastrointestinal stromal tumor, head and neck cancer, melanoma, and leiomyosarcoma.
The method of claim 21, wherein the cancer is solid tumor.
The method of claim 22, wherein the cancer is relapsed and refractory solid tumor.
The method of any one of claims 1-23, wherein the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are formulated together.
The method of any one of claims 1-23, wherein the anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor are formulated separately.
The method of any one of claims 1-25, wherein the administration of anti-CD47 antibody or the antigen binding fragment thereof and the PD-1/PD-L1 signaling inhibitor result in a synergistic effect in the subject in need thereof.
Apharmaceutical composition comprising:

(1) an anti-CD47 antibody or an antigen binding fragment thereof, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells; and

(2) a PD-1/PD-L1 signaling inhibitor.
The pharmaceutical composition of claim 27, wherein the anti-CD47 antibody or the antigen binding fragment thereof binds to human CD47 at an epitope comprising one or more residues selected from 26-31, 34, 35, 74, and 101-103.
The pharmaceutical composition of claim 28, wherein the anti-CD47 antibodyor the antigen binding fragment thereof binds to human CD47 at an epitope comprising residues 26-31, 34, 35, 74, and 101-103.
The pharmaceutical composition of any one of claims 27-29, wherein the anti-CD47 antibody or the antigen binding fragment thereofis selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.
The pharmaceutical composition of any one of claims 27-30, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprises:

(i) HCDR1, HCDR2 and HCDR3 as set forth ina variable heavy chain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77;

(ii) LCDR1, LCDR2 and LCDR3 as set forth in a variable light chain (VL) comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, and

wherein theHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to Kabat numbering scheme.
The pharmaceutical composition of any one of claims 27-30, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprises:

(i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319;

(ii) a HCDR2 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320;

(iii) a HCDR3 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321;

(iv) a LCDR1 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322;

(v) a LCDR2 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and

(vi) a LCDR3 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324.
The pharmaceutical composition of any one of claims 27-30, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprising:

(i) a VH comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77; and

(ii) a VL comprising an amino acid sequence having at least 80%identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.
The pharmaceutical composition of any one of claims 27-33, wherein the PD-1/PD-L1 signaling inhibitor is an antibody.
The pharmaceutical composition of claim 34, wherein the PD-1/PD-L1 signaling inhibitor is an anti-PD-1 antibody or an antigen binding fragment thereof.
The pharmaceutical composition of claim 35, wherein the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.
The pharmaceutical composition of claim 35, wherein the anti-PD-1 antibody or the antigen binding fragment thereof is selected from the group consisting ofpidilizumab, cemiplimab, sintilimab, cetrelimab, spartalizumab, camrelizumab, tislelizumab, balstilimab, toripalimab, dostarlimab, ABBV-181, penpulimab, pembrolizumab, genolimzumab, retifanlimab, sasanlimab, AMP-224, AB122, F-520, MEDI-3387, MEDI-5771, MEDI-0680, SG-001, nivolumab, BCD-100, BAT-1306, BI-754091, CBT-501, GLS-010, LZM-009, Sym-021, CS-1003, HLX-10, AK-103, AM-0001, ENUM-244C8, ENUM-388D4, JTX-4014, RXI-762, STI-A1110, HLX-20, SSI-361, APL-501, TJ0141H, and SNA-01.
The pharmaceutical composition of claim 37, wherein the anti-PD-1 antibody or the antigen binding fragment thereof is toripalimab.
The pharmaceutical composition of claim 34, wherein the PD-1/PD-L1 signaling inhibitor is an anti-PD-L1 antibodyor an antigen binding fragment thereof.
The pharmaceutical composition of claim 39, wherein the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting of polyclonal antibody, monoclonal antibody, Fab, scFv, diabody, triabody, minibody, VHH and sdAb.
The pharmaceutical composition of claim39, wherein the anti-PD-L1 antibody or the antigen binding fragment thereof is selected from the group consisting ofManelimab, atezolizumab, avelumab, Cosibelimab, durvalumab, Envafolimab, Socazolimab, AUNP12, BGB-A333, CK-301, CS-1001, FAZ-053, APL-502, MDX-1105, IMC-001, KD-005, Gensci-047, LY-3300054, SHR-1316, MSB-2311, AVA-004, CBT-502, JS-003, B12, KY-1003, CA-170, BMS-986189, B6 and B12-01.
The pharmaceutical composition of claim 41, wherein the anti-PD-L1 antibody or the antigen binding fragment thereof is B12.
An article of manufacture, comprising:

(1) an anti-CD47 antibody or an antigen binding fragment thereof, wherein the antibody does not cause a significant level of hemagglutination or depletion of red blood cells; and

(2) a package insert, wherein the package insert provides instruction of administrating the anti-CD47 antibody or the antigen binding fragment thereof in combination with a PD-1/PD-L1 signaling inhibitor for treating a disorder in a subject in need thereof.
The article of manufacture of claim 43, wherein the anti-CD47 antibody or the antigen binding fragment thereof comprises:

(i) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, and 319;

(ii) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, and 320;

(iii) a HCDR3 comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, and 321;

(iv) a LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, and 322;

(v) a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, and 323; and

(vi) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, and a variant thereof comprising one or more substitution, deletion or insertion as compared to SEQ ID NO: 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, and 324.