Novel anti-SIRPA antibodies

Abstract

The present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, an isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and uses thereof.

Description

Field of the Invention

[0001] The present invention generally relates to novel anti-SIRPα antibodies. Background Art

[0002] Signal regulatory protein alpha (SIRPα) is an inhibitory receptor expressed primarily on myeloid cells and dendritic cells. In addition to SIRPα, the SIRP family includes several other transmembrane glycoproteins, including SIRPβ and SIRPγ. Each member of the SIRP family contains three similar extracellular immunoglobulin-like domains with different transmembrane and intracellular domains. CD47 is a ubiquitously expressed transmembrane glycoprotein with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. CD47 acts as a cellular ligand for SIRPα. CD47 binding to SIRPα inhibits phagocytosis by signaling a “don’t eat me” signal, while blocking CD47-mediated binding of SIRPα on phagocytes results in the elimination of live cells with the “eat me” signal. Tumor cells often overexpress CD47 to evade macrophage-mediated destruction. It has been shown that the interaction between CD47 and SIRPα is involved in the regulation of macrophage-mediated phagocytosis (Takenaka et al., Nature Immunol, 8(12): 1313-1323, 2007). In various preclinical models, therapies that block the interaction between CD47 and SIRPα can stimulate phagocytosis of cancer cells in vitro and stimulate anti-tumor immune responses in vivo. Currently, a variety of agents targeting CD47 (anti-CD47 antibodies and SIRPα fusion proteins) have entered clinical trials. However, these agents are associated with hemolytic anemia and thrombocytopenia. In addition to safety issues, the ubiquitous expression of CD47 may also cause antigen sink, resulting in reduced efficacy.

[0003] There remains a need for novel anti-SIRPα antibodies.

[0004] Brief description of the invention

[0005] Throughout this application, the articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an antibody" refers to one antibody or more than one antibody.

[0006] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to human SIRPα, which comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: a) the HCDR1 comprises a sequence selected from the group consisting of RNYWMN (SEQ ID NO: 1), TDYAMH (SEQ ID NO: 2), TX1YAMN (SEQ ID NO: 3), THYSMH (SEQ ID NO: 4), SDYFMT (SEQ ID NO: 5), TNYDIS (SEQ ID NO: 6), and SSYWIH (SEQ ID NO: 7); and b) the HCDR2 comprises a sequence selected from the group consisting of EIX2LKSNTYATHYAESVKG (SEQ ID NO: 8), WKNTETGESTYAEDFKG (SEQ ID NO: 9), X3INTYTGEPTYAX4X5FKG (SEQ ID NO: 10), NO: 10), WINTETAEPTYVDDFKG (SEQ ID NO: 11), NVNYDGRSTYYLDSLKS (SEQ ID NO: 12), VIWTGGDTNFNSAFMS (SEQ ID NO: 13), or LIHPNSGNTDCSETFKN (SEQ ID NO: 14); and c) the HCDR3 comprises a sequence selected from the group consisting of FTKVVADWHLDV (SEQ ID NO: 15), GGYGSNYVMDY (SEQ ID NO: 16), TRGYYDFDGGAFDY (SEQ ID NO: 17), GGLRQGDY (SEQ ID NO: 18), EGSQTPLYAVDY (SEQ ID NO: 19), VQYFGGSYGPMDY (SEQ ID NO: 20), DGASYDWFVH (SEQ ID NO: 21); and d) the LCDR1 comprises a sequence selected from the group consisting of RSSQNIVHSNGNTYLE (SEQ ID NO: 22), NO:22), KASEDIYNRLA (SEQ ID NO:23), X6ASQNVGTHLA (SEQ ID NO:24), SATSSVSASYLY (SEQ ID NO:25), KASQNVGTAVA (SEQ ID NO:26), EASDHINDWLA (SEQ ID NO:27), KSSQSLLYTNGKTYLN (SEQ ID NO:28);and e) the LCDR2 comprises a sequence selected from the group consisting of KX7SNRFS (SEQ ID NO: 29), GATSLET (SEQ ID NO: 30), SAX8YRYI (SEQ ID NO: 31), STSNLAS (SEQ ID NO: 32), LASNRYT (SEQ ID NO: 33), LVSKLDS (SEQ ID NO: 35); and f) the LCDR3 comprises a sequence selected from the group consisting of FQGSHVPFT (SEQ ID NO: 36), QQYWNSPRT (SEQ ID NO: 37), QQYNTYPLT (SEQ ID NO: 38), HQWSSYPYT (SEQ ID NO: 39), QQYSIYPFT (SEQ ID NO: 40), QQYWNTPLT (SEQ ID NO: 41), VQGTHFPRT (SEQ ID NO: 42), NO: 42); wherein X1 is N or D, X2 is S or T, X3 is F or W, X4 is Q or D, X5 is D or G, X6 is K or R, X7 is V or I, and X8 is S or I. ;

[0007] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and / or the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8, and / or the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15, and / or the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, and / or the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 29, and / or the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 36, wherein X2 and X7 are as defined above.

[0008] In some embodiments, the HCDR2 comprises an amino acid sequence selected from the group consisting of EISLKSNTYATHYAESVKG (SEQ ID NO: 48), EITLKSNTYATHYAESVKG (SEQ ID NO: 49), and / or the LCDR2 comprises an amino acid sequence selected from the group consisting of KVSNRFS (SEQ ID NO: 55) and KISNRFS (SEQ ID NO: 56).

[0009] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, and / or the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and / or the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17, and / or the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:24, and / or the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and / or the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:38, wherein X1, X3, X4, X5, X6 and X8 are as defined above.

[0010] In some embodiments, the HCDR1 comprises an amino acid sequence selected from the group consisting of TNYAMN (SEQ ID NO: 43) and TDYAMN (SEQ ID NO: 45), and / or the HCDR2 comprises an amino acid sequence selected from the group consisting of FINTYTGEPTYADDFKG (SEQ ID NO: 50), WINTYTGEPTYAQGFKG (SEQ ID NO: 51), and FINTYTGEPTYAQGFKG (SEQ ID NO: 52), and / or the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 17, and / or the LCDR1 comprises an amino acid sequence selected from the group consisting of KASQNVGTHLA (SEQ ID NO: 53), and RASQNVGTHLA (SEQ ID NO: 54), and / or the LCDR2 comprises an amino acid sequence selected from the group consisting of SASYRYI (SEQ ID NO: 57), and SAIYRYI (SEQ ID NO: 58), and / or the LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 38.

[0011] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: a) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 48, and the HCDR3 comprises the sequence shown in SEQ ID NO: 15; or b) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 49, and the HCDR3 comprises the sequence shown in SEQ ID NO: 15; or c) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the HCDR2 comprises the sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the sequence shown in SEQ ID NO: 16; or d) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 43, the HCDR2 comprises the sequence shown in SEQ ID NO: 50, and the HCDR3 comprises the sequence shown in SEQ ID NO: 51. NO: 17; or e) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence as shown in SEQ ID NO: 43, the HCDR2 comprises the sequence as shown in SEQ ID NO: 51, and the HCDR3 comprises the sequence as shown in SEQ ID NO: 17; or f) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence as shown in SEQ ID NO: 45, the HCDR2 comprises the sequence as shown in SEQ ID NO: 52, and the HCDR3 comprises the sequence as shown in SEQ ID NO: 17; or g) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence as shown in SEQ ID NO: 43, the HCDR2 comprises the sequence as shown in SEQ ID NO: 52, and the HCDR3 comprises the sequence as shown in SEQ ID NO: 17; or h) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence as shown in SEQ ID NO: 4, the HCDR2 comprises the sequence as shown in SEQ ID NO: 11, and the HCDR3 comprises the sequence as shown in SEQ ID NO: 17. NO: 18; or i) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence as shown in SEQ ID NO: 5, the HCDR2 comprises the sequence as shown in SEQ ID NO: 12, and the HCDR3 comprises the sequence as shown in SEQ ID NO: 19;or j) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 6, the HCDR2 comprises the sequence shown in SEQ ID NO: 13, and the HCDR3 comprises the sequence shown in SEQ ID NO: 20; or k) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 7, the HCDR2 comprises the sequence shown in SEQ ID NO: 14, and the HCDR3 comprises the sequence shown in SEQ ID NO: 21. ;

[0012] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises: a) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 55, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36; or b) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 56, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36; or c) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 23, the LCDR2 comprises the sequence shown in SEQ ID NO: 30, and the LCDR3 comprises the sequence shown in SEQ ID NO: 37; or d) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 53, the LCDR2 comprises the sequence shown in SEQ ID NO: 54, and the LCDR3 comprises the sequence shown in SEQ ID NO: 56. NO:57, wherein the LCDR3 comprises the sequence shown in SEQ ID NO:38; or e) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:57, and the LCDR3 comprises the sequence shown in SEQ ID NO:38; or f) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:58, and the LCDR3 comprises the sequence shown in SEQ ID NO:38; or g) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:25, the LCDR2 comprises the sequence shown in SEQ ID NO:32, and the LCDR3 comprises the sequence shown in SEQ ID NO:39; or h) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:26, the LCDR2 comprises the sequence shown in SEQ ID NO: NO:33, wherein the LCDR3 comprises the sequence shown in SEQ ID NO:40; or i) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:27, the LCDR2 comprises the sequence shown in SEQ ID NO:30, and the LCDR3 comprises the sequence shown in SEQ ID NO:41;or j) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 28, the LCDR2 comprises the sequence shown in SEQ ID NO: 35, and the LCDR3 comprises the sequence shown in SEQ ID NO: 42;

[0013] In certain embodiments, in the antibodies or antigen-binding fragments thereof described herein, the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 48, the HCDR3 comprises the sequence shown in SEQ ID NO: 15, the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 55, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36; or the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 49, the HCDR3 comprises the sequence shown in SEQ ID NO: 15, the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 56, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36; or the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 49, the HCDR3 comprises the sequence shown in SEQ ID NO: 15, the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 56, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36. NO:22, the LCDR2 comprises the sequence shown in SEQ ID NO:55, and the LCDR3 comprises the sequence shown in SEQ ID NO:36; or the HCDR1 comprises the sequence shown in SEQ ID NO:2, the HCDR2 comprises the sequence shown in SEQ ID NO:9, and the HCDR3 comprises the sequence shown in SEQ ID NO:16, the LCDR1 comprises the sequence shown in SEQ ID NO:23, the LCDR2 comprises the sequence shown in SEQ ID NO:30, and the LCDR3 comprises the sequence shown in SEQ ID NO:37; or the HCDR1 comprises the sequence shown in SEQ ID NO:43, the HCDR2 comprises the sequence shown in SEQ ID NO:50, the HCDR3 comprises the sequence shown in SEQ ID NO:17, the LCDR1 comprises the sequence shown in SEQ ID NO:53, the LCDR2 comprises the sequence shown in SEQ ID NO:57, and the LCDR3 comprises the sequence shown in SEQ ID NO:38;or the HCDR1 comprises the sequence shown in SEQ ID NO:43, the HCDR2 comprises the sequence shown in SEQ ID NO:51, the HCDR3 comprises the sequence shown in SEQ ID NO:17, the LCDR1 comprises the sequence shown in SEQ ID NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:57, and the LCDR3 comprises the sequence shown in SEQ ID NO:38; or the HCDR1 comprises the sequence shown in SEQ ID NO:45, the HCDR2 comprises the sequence shown in SEQ ID NO:52, the HCDR3 comprises the sequence shown in SEQ ID NO:17, the LCDR1 comprises the sequence shown in SEQ ID NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:57, and the LCDR3 comprises the sequence shown in SEQ ID NO:38; or the HCDR1 comprises the sequence shown in SEQ ID NO:45, the HCDR2 comprises the sequence shown in SEQ ID NO:52, the HCDR3 comprises the sequence shown in SEQ ID NO:17, the LCDR1 comprises the sequence shown in SEQ ID NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:57, and the LCDR3 comprises the sequence shown in SEQ ID NO:38 NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:58, and the LCDR3 comprises the sequence shown in SEQ ID NO:38; or the HCDR1 comprises the sequence shown in SEQ ID NO:43, the HCDR2 comprises the sequence shown in SEQ ID NO:52, the HCDR3 comprises the sequence shown in SEQ ID NO:17, the LCDR1 comprises the sequence shown in SEQ ID NO:54, the LCDR2 comprises the sequence shown in SEQ ID NO:58, and the LCDR3 comprises the sequence shown in SEQ ID NO:38; or the HCDR1 comprises the sequence shown in SEQ ID NO:4, the HCDR2 comprises the sequence shown in SEQ ID NO:11, the HCDR3 comprises the sequence shown in SEQ ID NO:18, the LCDR1 comprises the sequence shown in SEQ ID NO:25, the LCDR2 comprises the sequence shown in SEQ ID NO:32, and the LCDR3 comprises the sequence shown in SEQ ID NO:39;or the HCDR1 comprises the sequence shown in SEQ ID NO:5, the HCDR2 comprises the sequence shown in SEQ ID NO:12, the HCDR3 comprises the sequence shown in SEQ ID NO:19, the LCDR1 comprises the sequence shown in SEQ ID NO:26, the LCDR2 comprises the sequence shown in SEQ ID NO:33, and the LCDR3 comprises the sequence shown in SEQ ID NO:40; or the HCDR1 comprises the sequence shown in SEQ ID NO:6, the HCDR2 comprises the sequence shown in SEQ ID NO:13, the HCDR3 comprises the sequence shown in SEQ ID NO:20, the LCDR1 comprises the sequence shown in SEQ ID NO:27, the LCDR2 comprises the sequence shown in SEQ ID NO:30, and the LCDR3 comprises the sequence shown in SEQ ID NO:41; or the HCDR1 comprises the sequence shown in SEQ ID NO:7, the HCDR2 comprises the sequence shown in SEQ ID NO:14, the HCDR3 comprises the sequence shown in SEQ ID NO:21, and the LCDR1 comprises the sequence shown in SEQ ID NO: NO:28, the LCDR2 comprises the sequence shown in SEQ ID NO:35, and the LCDR3 comprises the sequence shown in SEQ ID NO:42. ;

[0014] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein further comprise one or more of the heavy chain HFR1, HFR2, HFR3 and HFR4, and / or one or more of the light chain LFR1, LFR2, LFR3 and LFR4, wherein: a) the HFR1 comprises QX9QLVQSGSELKKPGASVKVSCX 10 AX 11 GX 12 X 13 (SEQ ID NO: 92) or a homologous sequence having at least 80% sequence identity thereto, b) the HFR2 comprises WVRQAPGQGLEWMG (SEQ ID NO: 93) or a homologous sequence having at least 80% sequence identity thereto, c) the HFR3 comprises RFVFSLDTSVSTAYLQIX 14 SLKAEDTAVYYCAR (SEQ ID NO: 96) or a homologous sequence thereof having at least 80% sequence identity, d) the HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 97) or a homologous sequence thereof having at least 80% sequence identity, e) the LFR1 comprises DIQMTQSPSX 15 LX16 ASVGDRVTITC (SEQ ID NO: 100) or a homologous sequence thereof having at least 80% sequence identity, f) the LFR2 comprises WX 17 QQQKPGKX 18 PKX 19 LIX 20 (SEQ ID NO: 104) or a homologous sequence having at least 80% sequence identity thereto, g) the LFR3 comprises GVPSRFSGSGSGTDFTLTISX 21 LQPEDFATYX 22 C (SEQ ID NO: 108) or a homologous sequence having at least 80% sequence identity thereto, h) the LFR4 comprises FX 23 QGTKLEIKX 24 (SEQ ID NO: 47) or a homologous sequence thereof having at least 80% sequence identity, wherein X9 is I or V, X 10 is R or K, X 11 It is G or R or S, X 12 It is T or S, X 13 It is L or I or F, X 14 It is G or S, X 15 Is S or R, X 16 It is S or G, X 17 Is Y or F, X 18 A or S, X 19 Is S or A, X 20 Is Y or F, X 21 Is S or N, X 22 Is Y or F, X 23 is G or D, X 24 Is R or missing.

[0015] In some embodiments, the HFR1 includes a sequence selected from the group consisting of SEQ ID NOs: 44, 89, 90 and 91, the HFR2 includes a sequence as shown in SEQ ID NO: 93, the HFR3 includes a sequence selected from the group consisting of SEQ ID NOs: 94 and 95, the HFR4 includes a sequence as shown in SEQ ID NO: 97, the LFR1 includes a sequence selected from the group consisting of SEQ ID NOs: 98 and 99, the LFR2 includes a sequence selected from the group consisting of SEQ ID NOs: 101, 102 and 103, the LFR3 includes a sequence selected from the group consisting of SEQ ID NOs: 105, 106 and 107, and the LFR4 includes a sequence selected from the group consisting of SEQ ID NOs: 109 and 46.

[0016] In some embodiments, the heavy chain variable region in the antibody or antigen-binding fragment thereof comprises a sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and homologous sequences having at least 80% sequence identity thereto but still maintaining specific binding affinity to human SIRPα.

[0017] In some embodiments, the light chain variable region in the antibody or antigen-binding fragment thereof comprises a sequence selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, and homologous sequences thereof that have at least 80% sequence identity but still maintain specific binding affinity to human SIRPα.

[0018] In some embodiments, in the antibodies or antigen-binding fragments thereof described in the present invention, the heavy chain variable region comprises the sequence shown in SEQ ID NO: 59, and the light chain variable region comprises the sequence shown in SEQ ID NO: 73; or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 74; or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 61, and the light chain variable region comprises the sequence shown in SEQ ID NO: 75; or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 62, and the light chain variable region comprises the sequence shown in SEQ ID NO: 76; or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 63, and the light chain variable region comprises the sequence shown in SEQ ID NO: 77; or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 64, and the light chain variable region comprises the sequence shown in SEQ ID NO: 78; or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 65, and the light chain variable region comprises the sequence shown in SEQ ID NO: NO:79; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:65, and the light chain variable region comprises the sequence as shown in SEQ ID NO:80; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:66, and the light chain variable region comprises the sequence as shown in SEQ ID NO:81; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:65, and the light chain variable region comprises the sequence as shown in SEQ ID NO:82; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:67, and the light chain variable region comprises the sequence as shown in SEQ ID NO:83; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:68, and the light chain variable region comprises the sequence as shown in SEQ ID NO:82; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:65, and the light chain variable region comprises the sequence as shown in SEQ ID NO:84; or the heavy chain variable region comprises the sequence as shown in SEQ ID NO:69, and the light chain variable region comprises the sequence as shown in SEQ ID NO: NO:85; or the heavy chain variable region comprises the sequence shown in SEQ ID NO:70, and the light chain variable region comprises the sequence shown in SEQ ID NO:86; or the heavy chain variable region comprises the sequence shown in SEQ ID NO:71, and the light chain variable region comprises the sequence shown in SEQ ID NO:87;Or the heavy chain variable region comprises the sequence shown in SEQ ID NO: 72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 88. ;

[0019] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention further comprises one or more amino acid residue substitutions or modifications, but still maintains specific binding affinity to human SIRPα. In some embodiments, at least one of the substitutions or modifications is in one or more CDR sequences and / or one or more non-CDR sequences in the heavy chain variable region or the light chain variable region.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention further comprises an Fc region, optionally comprising an Fc region of a human immunoglobulin (Ig), or optionally comprising an Fc region of a human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgM. In some embodiments, the Fc region is derived from human IgG4. In some embodiments, the Fc region derived from human IgG4 comprises an S228P mutation and / or an L235E mutation.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is humanized. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized bifunctional antibody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody and bivalent domain antibody.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has one or more binding properties to human SIRPα selected from the following group: a) the binding affinity to human SIRPα is no more than 10 -7M, the binding affinity to human SIRPα was determined by Biacore assay, b) with an EC of no more than 1 nM 50 Specifically binds to the extracellular domain (ECD) of human SIRPαv1. 50 As determined by ELISA, c) with an EC no greater than 1 nM 50 Specific binding to human SIRPαv2 ECD 50 Determined by ELISA assay.

[0024] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have one or more binding properties selected from the group consisting of: a) no detectable binding to SIRPγ ECD, b) binding to SIRPγ with an EC of no more than 50 nM, 50 Binding to SIRPγECD, the EC 50 As determined by ELISA, c) with an EC no greater than 1 nM 50 Binding to SIRPβ ECD, the EC 50 d) no detectable binding to SIRPβ ECD by ELISA, e) specific binding to human SIRPα IgV domain by FACS, f) no detectable binding to human SIRPα IgV domain by FACS, g) no detectable binding to human SIRPα IgV domain by no more than 10 -5 M) specifically binds to mouse SIRPα with a binding affinity of 10 nM as determined by Biacore assay, h) specifically binds to cynomolgus monkey SIRPα at a concentration of 10 nM as determined by FACS assay, i) is capable of inducing phagocytosis of target cells expressing CD47 by macrophages at a concentration of 10 nM as determined by phagocytosis assay; and j) does not reduce CD4 + T cells or CD8 + Proliferation of T cells.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention competes with the antibody or antigen-binding fragment thereof provided above for binding to human SIRPα. In some embodiments, the antibody or antigen-binding fragment thereof competes with an antibody comprising a heavy chain variable region and a light chain variable region for binding to human SIRPα, the heavy chain variable region comprising a sequence as shown in SEQ ID NO: 70, and the light chain variable region comprising a sequence as shown in SEQ ID NO: 86. In some embodiments, the antibody or antigen-binding fragment thereof competes with an antibody comprising a heavy chain variable region and a light chain variable region for binding to human SIRPα, the heavy chain variable region comprising a sequence as shown in SEQ ID NO: 72, and the light chain variable region comprising a sequence as shown in SEQ ID NO: 88. In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 62, and the light chain variable region comprising the sequence shown in SEQ ID NO: 76, or competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 69, and the light chain variable region comprising the sequence shown in SEQ ID NO: 85. In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 71, and the light chain variable region comprising the sequence shown in SEQ ID NO: 87.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention is bispecific. In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention can specifically bind to a second antigen other than SIRPα, or can specifically bind to a second epitope on SIRPα. In some embodiments, the second antigen is selected from the group consisting of CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), GPC-3, B7-H3, B7-H4, TROP2, CLDN18.2, EGFR, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

[0027] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are linked to one or more conjugate moieties. In some embodiments, the conjugate moieties include clearance modifiers, chemotherapeutic agents, toxins, radioisotopes, lanthanides, luminescent labels, fluorescent labels, enzyme substrate labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, purification moieties, or other anticancer drugs.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and one or more pharmaceutically acceptable carriers.

[0029] In another aspect, the present invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to the present invention.

[0030] In another aspect, the present invention provides a vector comprising the isolated polynucleotide according to the present invention.

[0031] In another aspect, the present invention provides a host cell comprising the vector according to the present invention.

[0032] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof according to the present invention and / or the pharmaceutical composition according to the present invention, and a second therapeutic agent.

[0033] In another aspect, the present invention provides a method for expressing the antibody or antigen-binding fragment thereof according to the present invention, comprising culturing the host cell according to the present invention under conditions for expressing the vector according to the present invention.

[0034] On the other hand, the present invention provides a method for treating, preventing or alleviating a disease, disorder or condition associated with SIRPα in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the present invention and / or a pharmaceutical composition according to the present invention. In some embodiments, the disease, disorder or condition is cancer, solid tumors, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, nervous system disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction or arthritis. In some embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, In some embodiments, the subject is a human. ... In some embodiments, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anticancer drugs, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0035] In another aspect, the present invention provides a method for regulating SIRPα activity in SIRPα-positive cells, comprising exposing the SIRPα-positive cells to the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition of the present invention. In some embodiments, the cells are phagocytic cells.

[0036] In another aspect, the present invention provides a method for detecting the presence or content of SIRPα in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention, and determining the presence or content of SIRPα in the sample.

[0037] On the other hand, the present invention provides a method for diagnosing a disease, disorder or condition associated with SIRPα in a subject, comprising: a) contacting a sample obtained from the subject with the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention; b) determining the presence or content of SIRPα in the sample; and c) correlating the presence or content of SIRPα with the presence or status of the disease, disorder or condition associated with SIRPα in the subject.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 5, the HCDR2 comprises the sequence shown in SEQ ID NO: 12, the HCDR3 comprises the sequence shown in SEQ ID NO: 19, the LCDR1 comprises the sequence shown in SEQ ID NO: 26, the LCDR2 comprises the sequence shown in SEQ ID NO: 33, and the LCDR3 comprises the sequence shown in SEQ ID NO: 40.

[0039] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof according to the present invention and / or the pharmaceutical composition according to the present invention in the preparation of a medicament for treating, preventing or alleviating a disease, disorder or condition associated with SIRPα.

[0040] On the other hand, the present invention provides the use of the antibody or antigen-binding fragment thereof according to the present invention and / or the pharmaceutical composition according to the present invention in the preparation of a diagnostic reagent for diagnosing a disease, disorder or condition associated with SIRPα. On the other hand, the present invention provides a kit for detecting SIRPα, which comprises the antibody or antigen-binding fragment thereof according to the present invention and / or the pharmaceutical composition according to the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 comprising the sequence shown in SEQ ID NO: 5, the HCDR2 comprising the sequence shown in SEQ ID NO: 12, the HCDR3 comprising the sequence shown in SEQ ID NO: 19, the LCDR1 comprising the sequence shown in SEQ ID NO: 26, the LCDR2 comprising the sequence shown in SEQ ID NO: 33, and the LCDR3 comprising the sequence shown in SEQ ID NO: 40.

[0041] In another aspect, the present invention provides a method of inducing phagocytosis in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof according to the present invention and / or a pharmaceutical composition according to the present invention at a dose effective to induce phagocytosis. In some embodiments, the subject is human. In some embodiments, the subject has a disease, disorder or condition selected from the group consisting of cancer, solid tumors, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, nervous system disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction and arthritis.

[0042] In another aspect, the present invention provides a method for inducing phagocytosis in vitro, comprising contacting a target cell with a SIRPα-positive phagocytic cell sample in the presence of an antibody or antigen-binding fragment thereof according to the present invention and / or a pharmaceutical composition according to the present invention, thereby inducing the phagocytosis of the target cell by the SIRPα-positive phagocytic cell. In some embodiments, the target cell is a cell expressing CD47. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The anti-SIRPα antibody (human IgG4 chimeric antibody with S228P mutation) showed the activity of human SIRPαv1 ECD ( Figure 1 A), human SIRPαv2 ECD ( Figure 1 B) Human SIRPβECD ( Figure 1 C) and human SIPRγECD ( Figure 1 D) ELISA binding specificity of the recombinant protein.

[0045] Figure 2 The anti-SIRPα antibody (human IgG4 chimeric antibody with S228P mutation) was shown to be effective against CHOK1-human SIRPαv1-1B4 cells ( Figure 2 A), CHOK1-cynomolgus monkey SIRPα-2A2 cells ( Figure 2 B) and CHOK1-C57BL / 6 mouse SIRPα-2.22 cells ( Figure 2 C) FACS binding curve.

[0046] Figure 3 The expression of human macrophages on Jurkat cells ( Figure 3 A, 3D), Raji cells ( Figure 3 B) and DLD-1 cells ( Figure 3 C) phagocytosis.

[0047] Figure 4 A illustrates the targeting strategy of B-hSIRPα mouse (Biocytogen). Figure 4 B shows the binding of anti-SIRPα antibody (human IgG4 chimeric antibody with S228P mutation) to B-hSIRPA mouse monocytes.

[0048] Figure 5 A shows the FACS binding curve of humanized antibody hu035.01 to CHOK1-human SIRPαv1-1B4 cells. Figure 5 B shows the ELISA binding of humanized antibody hu035.01 to human SIRPαv2 ECD and mouse SIRPα (C57BL / 6) ECD recombinant protein. Figure 5 C shows the full kinetics of the binding affinity of humanized antibody hu035.01 to human SIRPαv2 determined by surface plasmon resonance.

[0049] Figure 6 The optimized hu035 candidate was shown to be homologous to the human SIRPαv1 ECD ( Figure 6 A), human SIRPαv2 ECD ( Figure 6 B) Human SIRPαv8 ECD ( Figure 6 C) Human SIRPβECD ( Figure 6 D) Human SIRPγECD ( Figure 6 E) and mouse SIRPα(C57BL / 6)ECD( Figure 6 F) ELISA specific binding of recombinant proteins.

[0050] Figure 7 The optimized hu035 candidate is shown to be expressed in CHOK1-human SIRPαv1-1B4 cells ( Figure 7 A), CHOK1-cynomolgus monkey SIRPα-2A2 cells ( Figure 7 B) and CHOK1-C57BL / 6 mouse SIRPα-2.22 cells ( Figure 7 C) FACS binding curve.

[0051] Figure 8 Shown is the detection of CD47 and SIRPα interaction blocking activity of optimized hu035 candidates by competition ELISA assay.

[0052] Fig. 9 shows the effect of human macrophages on Jurkat cells ( Fig. 9 A), DLD1 cells ( Fig. 9B) and Raji cells ( Fig. 9 C) phagocytosis.

[0053] Fig.10 Figure 4 shows the secretion of IFNγ by T cells stimulated by CD3 / CD28 activators in the presence of anti-SIRPα antibody (IgG4 chimeric antibody with S228P mutation) and the optimized hu035 candidate. Fig.10 A) CD4 + T cells ( Fig.10 B) and CD8 + T cells ( Fig.10 C) proliferation rate.

[0054] Fig.11 The results show that in the presence of anti-SIRPα antibody (human IgG4 chimeric antibody with S228P mutation) and the optimized hu035 candidate, the secretion of IFNγ by T cells stimulated by allogeneic dendritic cells ( Fig.11 A) CD4 + T cells ( Fig.11 B) and CD8 + T cells ( Fig.11 C) proliferation rate. DETAILED DESCRIPTION OF THE INVENTION

[0056] The following description of the present invention is only for illustrating various embodiments of the present invention. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A variety of equivalents, variations and modifications can be easily derived by those skilled in the art without departing from the scope of the present invention, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in the present invention, including publications, patents and patent applications, are incorporated by reference in their entirety.

[0057] definition

[0058] The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that can bind to a specific antigen. A natural complete antibody contains two heavy (H) chains and two light (L) chains. The heavy chains of mammals can be divided into α, δ, ε, γ and μ, each of which consists of a variable region (VH) and the first, second, third and fourth (optional) constant regions (CH1, CH2, CH3, CH4, respectively); the light chains of mammals can be divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region. The antibody is "Y"-shaped, and the stem of the "Y"-shaped structure is composed of the second and third constant regions of two heavy chains bound by disulfide bonds. Each arm of the "Y"-shaped structure includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light chain and the heavy chain determine the binding of the antigen. The variable region of each chain usually contains three hypervariable regions, called complementarity determining regions (CDRs) (the light chain CDR includes LCDR1, LCDR2, LCDR3, and the heavy chain CDR includes HCDR1, HCDR2, HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, IMGT, Chothia or Al-Lazikani nomenclature (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3): 651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252): 877-83 (1989); Kabat EA et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, 1986. Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1 (3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). Among them, the three CDRs are separated by flanking parts called framework regions (FR) (light chain FR includes LFR1, LFR2, LFR3 and LFR4, and heavy chain FR includes HFR1, HFR2, HFR3 and HFR4). The framework region is more highly conserved than the CDR and forms a scaffold to support the highly variable loop. The constant regions of the heavy and light chains are not related to antigen binding, but have a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. Antibodies can be divided into five major classes or isotypes, depending on whether they contain α, δ, ε, γ, and μ heavy chains: IgA, IgD, IgE, IgG, and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0059] In certain embodiments, the antibodies provided by the present invention include any antigen-binding fragments thereof. The term "antigen-binding fragment" in the present invention refers to an antibody fragment formed by a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not have a complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment is able to bind to the same antigen as the parent antibody.

[0060] The "Fab" of an antibody refers to a portion of an antibody composed of a single light chain (including a variable region and a constant region) and the variable region and first constant region of a single heavy chain bound together by disulfide bonds.

[0061] "Fab'" refers to the Fab fragment including part of the hinge region.

[0062] "F(ab')2" refers to a dimer of Fab'.

[0063] The "Fc" of an antibody (e.g., IgG, IgA, or IgD isotype) refers to a portion of an antibody consisting of the second and third constant regions of the first heavy chain connected to the second and third constant regions of the second heavy chain via a disulfide bond. The Fc of IgM and IgE isotype antibodies also contains a fourth constant region. The Fc portion of an antibody is responsible for a variety of different effector functions, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0064] The "Fv" of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain.

[0065] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the light chain variable region and the heavy chain variable region are directly linked to each other or connected through a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0066] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc portion of an antibody.

[0067] "Camelized single domain antibody", "heavy chain antibody" or "HCAb (Heavy-chain-only antibody)" all refer to antibodies containing two V H Heavy chain antibodies are antibodies that contain heavy chain domains but not light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies originally came from Camelidae (camels, dromedaries and llamas). Although they lack light chains, camelized antibodies have confirmed full antigen binding function (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al., Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable region (VHH domain) of heavy chain antibodies is the smallest known antigen binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0068] "Nanobody" refers to an antibody fragment that consists of the VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3.

[0069] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments contain V and V linked on the same polypeptide chain. H Domain and V L Domain (V H -V L or V L -V H) (See, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14): 6444-8 (1993); EP404097; WO93 / 11161). The linker between the two domains is short, so that the two domains on the same chain cannot pair with each other, thereby forcing the two domains to pair with the complementary domains of another chain to form two antigen binding sites. The two antigen binding sites can target the same or different antigens (or epitopes). In some embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes).

[0070] "Domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or a light chain. H The domains are covalently linked by a peptide linker to form a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0071] The term "valence" used in the present invention refers to the presence of a specific number of antigen binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen binding fragment having only one antigen binding site. The term "multivalent" refers to an antibody or antigen binding fragment having multiple antigen binding sites. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two binding sites, four binding sites and six binding sites in an antigen binding molecule, respectively. In some embodiments, the antibody or its antigen binding fragment is bivalent.

[0072] In the present invention, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0073] In some embodiments, a "scFv dimer" is a bivalent bifunctional antibody or a bispecific scFv (BsFv) comprising two dimerized V H -V L (connected by a peptide linker) such that one portion of V H With another part of V L The two binding sites can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising V L1 -V H2 (connected by a peptide linker) and V H1 -VL2 (connected by a peptide linker), such that V H1 and V L1 Collaboration, V H2 and V L2 Collaborative, and each collaborating pair has a different antigenic specificity.

[0074] "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain is connected to the variable region of a single heavy chain by a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" contains three peptide chains: two V H The parts are connected by a peptide linker (e.g., a long flexible linker) and are respectively connected to the two V L In some embodiments, dsFv-dsFv' has bispecificity, wherein each pair of heavy chain and light chain paired by disulfide bonds has a different antigen specificity.

[0075] The term "chimeric" used in the present invention refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or a light chain derived from one species, and the remainder of the heavy chain and / or the light chain derived from another different species. In an exemplary example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

[0076] The term "humanized" used in the present invention refers to an antibody or antigen-binding fragment comprising CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable).

[0077] The term "affinity" as used in the present invention refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, antibody) or a fragment thereof and an antigen.

[0078] "Specific binding" or "specifically binds" as used herein refers to a non-random binding reaction between two molecules, for example, a reaction between an antibody and an antigen. Specific binding can be characterized by binding affinity, for example, as measured by K D The value represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on K can be determined by any conventional method known in the art. D , including, but not limited to, surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). ≤10 -6 M (e.g. ≤5x10 -7 M, ≤2x10-7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, or ≤10 -9 M) K D The value may represent specific binding between the antibody or antigen-binding fragment thereof and SIRPα (eg, human SIRPα).

[0079] The ability of "competitive binding to human SIRPα" in the present invention refers to the ability of the first antibody or its antigen-binding fragment to inhibit the binding interaction between human SIRPα and the second anti-SIRPα antibody to any detectable extent. In some embodiments, the antibody or antigen-binding fragment that competes for binding to human SIRPα can inhibit the binding interaction between human SIRPα and the second anti-SIRPα antibody by at least 85% or at least 90%. In some embodiments, such inhibition can be greater than 95% or greater than 99%.

[0080] The term "epitope" used in the present invention refers to a specific atomic group or amino acid on an antigen that binds to an antibody. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitope on the antigen. An epitope may be linear or conformational (i.e., including spaced amino acid residues). For example, if an antibody or its antigen-binding fragment blocks the binding of a reference antibody to an antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or its antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody.

[0081] The term "amino acid" as used herein refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. The amino acid names are also represented in the present invention by standard single-letter or three-letter codes, which are summarized as follows:

[0082] Amino acid name Three letter code Single letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0083] In the present invention, when "conservative substitution" is used for an amino acid sequence, it means that an amino acid residue is replaced with another amino acid residue having a side chain of similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein, and therefore can retain the biological activity of the protein.

[0084] The term "homologous" as used herein refers to a nucleic acid sequence (or its complementary chain) or an amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0085] When "percent (%) sequence identity" is used for an amino acid sequence (or nucleic acid sequence), it means that after the sequence is aligned and, if necessary, intervals are introduced to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, the percentage of amino acid (or nucleic acid) residues identical to those in the reference sequence in the candidate sequence. In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence to be compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). Conservative substitutions of the amino acid residues may or may not be considered identical residues. The sequences can be aligned to determine the percentage sequence identity of the amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see Higgins D G et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0086] As used in the present invention, "effector function" refers to the biological activity of the Fc region of an antibody in combination with its effector (e.g., C1 complex and Fc receptor). Exemplary effector functions include complement-dependent cytotoxicity (CDC) mediated by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the binding of the Fc region of an antibody to Fc receptors on effector cells, and phagocytosis. Effector function can be assessed using various assays (e.g., Fc receptor binding assays, C1q binding assays, and cell lysis assays).

[0087] An "isolated" substance has been artificially altered from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but it may be considered "isolated" if the substance with which it coexists in nature is sufficiently separated and present in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In some embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure, wherein the purity is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0088] The term "vector" in the present invention refers to a vehicle into which a genetic element can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site. The vector can also include components that assist it in entering the cell, including but not limited to, viral particles, liposomes or protein shells. The vector can be an expression vector or a cloning vector. The vector (e.g., expression vector) provided by the present invention contains the nucleic acid sequence encoding the antibody or its antigen-binding fragment described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0089] In the present invention, "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0090] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals (e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles). Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably in the present invention.

[0091] The term "anti-tumor activity" refers to a decrease in tumor cell proliferation, viability or metastatic activity. For example, anti-tumor activity can be shown by a decrease in the growth rate of abnormal cells that occur during treatment or a stabilization or reduction in tumor size, or by a longer survival period due to treatment compared to a control without treatment. Anti-tumor activity can be assessed using an acceptable in vitro or in vivo tumor model (including but not limited to a xenograft model, an allograft model, a mouse mammary tumor virus (MMTV) model, and other known models of anti-tumor activity known in the art).

[0092] As used in the present invention, "treatment" or "therapy" for a disease, disorder or condition includes preventing or alleviating a disease, disorder or condition, reducing the rate of occurrence or development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing or stopping symptoms associated with a disease, disorder or condition, producing complete or partial reversal of a disease, disorder or condition, curing a disease, disorder or condition, or a combination of the above.

[0093] The terms "diagnosis," "diagnose," or "diagnosing" refer to the identification of a pathological state, disease, or condition, such as the identification of a SIRPα-related disease, or the identification of a subject with a SIRPα-related disease who may benefit from a particular treatment regimen. In some embodiments, the diagnosis comprises identifying abnormal levels or activity of SIRPα. In some embodiments, the diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0094] As used in the present invention, the term "biological sample" or "sample" refers to a biological composition obtained from or derived from a target subject, which contains cells and / or other molecular entities to be characterized and / or identified, such as characterized and / or identified based on physical, biochemical, chemical and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs and / or biological fluids of a subject obtained by any method known to those skilled in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal excretions and sputum), peritoneal fluid, pleural fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites or pericardial effusion. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lung, kidney, skin or blood vessel of a subject.

[0095] As used in the present invention, "SIRPα" refers to a regulatory membrane glycoprotein of the signal regulatory protein (SIRP) family, which is mainly expressed by myeloid cells, dendritic cells, and stem cells or neurons. The structure of SIRPα includes an extracellular domain and a cytoplasmic domain. The extracellular domain of SIRPα consists of a membrane distal Ig variable-like (IgV) fold and two membrane proximal Ig constant-like (IgC) folds. The IgV domain of SIRPα is responsible for binding to the extracellular Ig domain of CD47. In certain embodiments, SIRPα is human SIRPα. The gene encoding human SIRPα is a polymorphic gene, and several variants have been described in humans. The most common protein variants are SIRPαv1 and SIRPαv2 (numbers NP_542970 (P78324) and CAA71403). The SIRPα used in the present invention can be from other animal species, such as mice and crab-eating monkeys. Exemplary sequences of Mus musculus (mouse) SIRPα proteins are disclosed in NCBI RefSeq No. NP_031573 or BAA20376.1 or BAA13521.1. Exemplary sequences of Cynomolgus (monkey) SIRPα proteins are disclosed in NCBI Ref Seq No. NP_001271679.

[0096] In addition to SIRPα, the SIRP family includes several other transmembrane glycoproteins, including SIRPβ and SIRPγ. Each member of the SIRP family contains 3 similar extracellular Ig-like domains, but has different transmembrane and cytoplasmic domains. "SIRPβ", encoded by the SIRP beta gene, generates positive signals through intracellular signaling from the cytoplasmic tail by binding to a transmembrane protein called DNAX activating protein 12 or DAP12. The cytoplasmic tail of DAP12 has an immunoreceptor tyrosine-based activation motif (ITAM) that connects SIRPβ1 to the activation machinery. "SIRPγ", also known as SIRPg, is encoded by the SIRPG gene and is highly homologous to the extracellular Ig domains of SIRPα and SIRPβ, but the cytoplasmic tail of SIRPγ is different. SIRPγ has also been shown to bind to CD47, but with lower affinity than SIRPα.

[0097] The term "anti-SIRPα antibody" refers to an antibody that can specifically bind to SIRPα (eg, human or monkey SIRPα). The term "anti-human SIRPα antibody" refers to an antibody that can specifically bind to human SIRPα.

[0098] As used herein, a "SIRPα-associated" disease, disorder or condition refers to any disease or condition caused, aggravated or associated with an increase or decrease in the expression or activity of SIRPα. In some embodiments, a SIRPα-associated disease, disorder or condition is an immune-related disorder, such as an autoimmune disease. In some embodiments, a SIRPα-associated disease, disorder or condition is a disorder associated with excessive cell proliferation, such as cancer. In certain embodiments, a SIRPα-associated disease or condition is characterized by expression or overexpression of a SIRPα gene. In certain embodiments, a SIRPα-associated disease or condition is characterized by expression or overexpression of CD47.

[0099] The term "pharmaceutically acceptable" means that the referred carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient thereof.

[0100] As used herein, the term "SIRPα-positive cells" refers to cells (eg, phagocytes) expressing SIRPα on the cell surface. In some embodiments, "SIRPα-positive cells" may also express SIRPβ or SIRPγ on the cell surface.

[0101] Anti-SIRPα Antibodies

[0102] The present invention provides an anti-SIRPα antibody and an antigen-binding fragment thereof. The anti-SIRPα antibody and the antigen-binding fragment provided by the present invention can specifically bind to SIRPα.

[0103] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present in an amount of no more than 10 -7 M, not more than 8×10 -8 M, not more than 5×10 -8 M, not more than 2×10 -8 M, not more than 8×10 -9 M, not more than 5×10 -9 M, not more than 2×10 - 9 M. No more than 10 -9 M, not more than 8×10 -10 M, not more than 7×10 -10 M or not more than 6×10 -10 M of K D The K D The value is determined by Biacore detection. Biacore detection is based on surface plasmon resonance technology (see, for example, Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006). In certain embodiments, K is determined by the method described in Example 4.3 of the present invention. D value.

[0104] The binding of the antibodies or antigen-binding fragments thereof provided by the present invention to human SIRPα can also be measured by "half maximal effective concentration" (EC 50 ) value, which refers to the antibody concentration at which 50% of the maximum binding of the antibody is observed. EC can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometry assays, and other binding assays). 50 In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention have an EC of no more than 1 nM, no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM, no more than 0.09 nM, no more than 0.08 nM, no more than 0.07 nM, no more than 0.06 nM or no more than 0.05 nM. 50 (i.e. 50% binding concentration) specifically binds to human SIRPα, the EC 50Values ​​were determined by ELISA assay.

[0105] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present at a concentration of no more than 1 nM (e.g., no more than 5 x 10 -10 M, no more than 3x 10 -10 M, not more than 1x 10 -10 EC of M) 50 The value specifically binds to the extracellular domain (ECD) of human SIRPαv1. 50 The value is determined by ELISA. In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are expressed at a concentration of no more than 1 nM (e.g., no more than 5 x 10 -10 M, no more than 3x 10 -10 M, not more than 1x 10 -10 EC of M) 50 The value specifically binds to the extracellular domain (ECD) of human SIRPαv2. 50 Values ​​were determined by ELISA assay.

[0106] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention have an EC no greater than 50 nM (e.g., no greater than 40 nM, no greater than 30 nM, no greater than 20 nM, no greater than 10 nM, no greater than 1 nM). 50 The value specifically binds to human SIRPγECD. 50 Values ​​were determined by ELISA assay.

[0107] An antibody or antigen-binding fragment thereof that "undetectably binds" to SIRPγ ECD is an antibody or antigen-binding fragment thereof that does not detectably bind to SIRPγ or binds to SIRPγ at a level comparable to that of a control antibody under equivalent assay conditions. The control antibody may be any antibody known not to bind to SIRPγ.

[0108] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present at a concentration of no more than 1 nM (e.g., no more than 5 x 10 -10 M, no more than 3x 10 -10 M, not more than 1x 10 -10 EC of M) 50 The value specifically binds to human SIRPβ ECD. 50 The values ​​are determined by ELISA assay. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein do not detectably bind to SIRPβ ECD as determined by ELISA assay.

[0109] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein specifically bind to the human SIRPαIgV domain as determined by FACS detection. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein do not detectably bind to the human SIRPαIgV domain as determined by FACS detection.

[0110] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present in an amount of no more than 10 -5 M (e.g., no more than 5 x 10 -6 M, no more than 3x 10 -6 M, not more than 1x 10 -6 M, no more than 5x 10 -7 M, no more than 3x 10 -7 M, not more than 1x 10 -7 M, no more than 5x 10 -8 M, no more than 3x 10 -8 M, not more than 1x 10 -8 In some embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention specifically bind to cynomolgus monkey SIRPα at a concentration of no more than 10 nM, as determined by FACS detection.

[0111] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are capable of inducing macrophages to phagocytose target cells expressing CD47 at a concentration of no more than 10 nM, as determined by a phagocytosis assay.

[0112] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention do not reduce CD4 + T cells or CD8 + T cell proliferation. It is reported that human T cells co-stimulate T cell proliferation through SIRPγ-CD47 interaction and adhesion of antigen presenting cells. The antibodies and antigen-binding fragments provided by the present invention do not specifically bind to SIRPγ, or do not block SIRPγ-CD47 interaction to reduce CD4 + T cells or CD8 + The extent of T cell proliferation. T cell proliferation can be determined using methods known in the art, for example, by a T cell proliferation assay, such as those described in Example 5.4 of the present invention, for example, by using CellTrace Violet (Life Technologies) markers to determine the amount of proliferation.

[0113] Example Anti-SIRPα Antibodies

[0114] In certain embodiments, the anti-SIRPα antibodies (e.g., anti-human SIRPα antibodies) and antigen-binding fragments thereof provided herein include one or more (e.g., 1, 2, 3, 4, 5 or 6) CDRs, wherein the CDRs include a sequence selected from the group consisting of RNYWMN (SEQ ID NO: 1), TDYAMH (SEQ ID NO: 2), TX1YAMN (SEQ ID NO: 3), THYSMH (SEQ ID NO: 4), SDYFMT (SEQ ID NO: 5), TNYDIS (SEQ ID NO: 6), SSYWIH (SEQ ID NO: 7), EIX2LKSNTYATHYAESVKG (SEQ ID NO: 8), WKNTETGESTYAEDFKG (SEQ ID NO: 9), X3INTYTGEPTYAX4X5FKG (SEQ ID NO: 10), WINTETAEPTYVDDFKG (SEQ ID NO: 11), NVNYDGRSTYYLDSLKS (SEQ ID NO: 12), VIWTGGDTNFNSAFMS (SEQ ID NO: 13), ID NO: 13), or LIHPNSGNTDCSETFKN (SEQ ID NO: 14), FTKVVADWHLDV (SEQ ID NO: 15), GGYGSNYVMDY (SEQ ID NO: 16), TRGYYDFDGGAFDY (SEQ ID NO: 17), GGLRQGDY (SEQ ID NO: 18), EGSQTPLYAVDY (SEQ ID NO: 19), VQYFGGSYGPMDY (SEQ ID NO: 20), DGASYDWFVH (SEQ ID NO: 21), RSSQNIVHSNGNTYLE (SEQ ID NO: 22), KASEDIYNRLA (SEQ ID NO: 23), X6ASQNVGTHLA (SEQ ID NO: 24), SATSSVSASYLY (SEQ ID NO: 25), KASQNVGTAVA (SEQ ID NO: 26), EASDHINDWLA (SEQ ID NO: 27), KSSQSLLYTNGKTYLN (SEQ ID NO: 28), KX7SNRFS (SEQ ID NO: 29), GATSLET (SEQ ID NO: 30), SAX8YRYI (SEQ ID NO: 31), STSNLAS (SEQ ID NO: 32), LASNRYT (SEQ ID NO: 33), LVSKLDS (SEQ ID NO: 35), FQGSHVPFT (SEQ IDNO: 36), QQYWNSPRT (SEQ ID NO: 37), QQYNTYPLT (SEQ ID NO: 38), HQWSSYPYT (SEQ ID NO: 39), QQYSIYPFT (SEQ ID NO: 40), QQYWNTPLT (SEQ ID NO: 41), VQGTHFPRT (SEQ ID NO: 42), wherein X1 is N or D, X2 is S or T, X3 is F or W, X4 is Q or D, X5 is D or G, X6 is K or R, X7 is V or I, and X8 is S or I. In certain embodiments, the present invention further includes antibodies and antigen-binding fragments thereof having no more than one, two or three amino acid residue substitutions for any one of SEQ ID NOs: 1-42, wherein X1 is N or D, X2 is S or T, X3 is F or W, X4 is Q or D, X5 is D or G, X6 is K or R, X7 is V or I, and X8 is S or I.

[0115] The antibody "001" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 59 and the light chain variable region has a sequence as shown in SEQ ID NO: 73.

[0116] The antibody "002" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO: 60 and the light chain variable region has the sequence shown in SEQ ID NO: 74.

[0117] The antibody "022" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 62, and the light chain variable region has a sequence as shown in SEQ ID NO: 76.

[0118] The antibody "032" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 61, and the light chain variable region has a sequence as shown in SEQ ID NO: 75.

[0119] The antibody "035" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 63, and the light chain variable region has a sequence as shown in SEQ ID NO: 77.

[0120] The antibody "050" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 69, and the light chain variable region has a sequence as shown in SEQ ID NO: 85.

[0121] The antibody "055" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 70, and the light chain variable region has a sequence as shown in SEQ ID NO: 86.

[0122] The antibody "060" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 71, and the light chain variable region has a sequence as shown in SEQ ID NO: 87.

[0123] The antibody "074" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 72, and the light chain variable region has a sequence as shown in SEQ ID NO: 88.

[0124] In certain embodiments, the present invention provides anti-SIRPα antibodies and antigen-binding fragments thereof, which include one or more (e.g., 1, 2, 3, 4, 5 or 6) CDR sequences of antibodies 001, 002, 022, 032, 035, 050, 055, 060 or 074.

[0125] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes a sequence selected from the following group: SEQ ID NO: 1-7; the HCDR2 includes a sequence selected from the following group: SEQ ID NO: 8-14; the HCDR3 includes a sequence selected from the following group: SEQ ID NO: 15-21; the LCDR1 includes a sequence selected from the following group: SEQ ID NO: 22-28; the LCDR2 includes a sequence selected from the following group: SEQ ID NO: 29-33 and 35; the LCDR3 includes a sequence selected from the following group: SEQ ID NO: 36-42.

[0126] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 1, the HCDR2 includes the sequence shown in SEQ ID NO: 48, the HCDR3 includes the sequence shown in SEQ ID NO: 15, the LCDR1 includes the sequence shown in SEQ ID NO: 22, the LCDR2 includes the sequence shown in SEQ ID NO: 55, and the LCDR3 includes the sequence shown in SEQ ID NO: 36.

[0127] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 1, the HCDR2 includes the sequence shown in SEQ ID NO: 49, the HCDR3 includes the sequence shown in SEQ ID NO: 15, the LCDR1 includes the sequence shown in SEQ ID NO: 22, the LCDR2 includes the sequence shown in SEQ ID NO: 56, and the LCDR3 includes the sequence shown in SEQ ID NO: 36.

[0128] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 1, the HCDR2 includes the sequence shown in SEQ ID NO: 49, the HCDR3 includes the sequence shown in SEQ ID NO: 15, the LCDR1 includes the sequence shown in SEQ ID NO: 22, the LCDR2 includes the sequence shown in SEQ ID NO: 55, and the LCDR3 includes the sequence shown in SEQ ID NO: 36.

[0129] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 2, the HCDR2 includes the sequence shown in SEQ ID NO: 9, the HCDR3 includes the sequence shown in SEQ ID NO: 16, the LCDR1 includes the sequence shown in SEQ ID NO: 23, the LCDR2 includes the sequence shown in SEQ ID NO: 30, and the LCDR3 includes the sequence shown in SEQ ID NO: 37.

[0130] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 43, the HCDR2 includes the sequence shown in SEQ ID NO: 50, the HCDR3 includes the sequence shown in SEQ ID NO: 17, the LCDR1 includes the sequence shown in SEQ ID NO: 53, the LCDR2 includes the sequence shown in SEQ ID NO: 57, and the LCDR3 includes the sequence shown in SEQ ID NO: 38.

[0131] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 4, the HCDR2 includes the sequence shown in SEQ ID NO: 11, the HCDR3 includes the sequence shown in SEQ ID NO: 18, the LCDR1 includes the sequence shown in SEQ ID NO: 25, the LCDR2 includes the sequence shown in SEQ ID NO: 32, and the LCDR3 includes the sequence shown in SEQ ID NO: 39.

[0132] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 5, the HCDR2 includes the sequence shown in SEQ ID NO: 12, the HCDR3 includes the sequence shown in SEQ ID NO: 19, the LCDR1 includes the sequence shown in SEQ ID NO: 26, the LCDR2 includes the sequence shown in SEQ ID NO: 33, and the LCDR3 includes the sequence shown in SEQ ID NO: 40.

[0133] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 6, the HCDR2 includes the sequence shown in SEQ ID NO: 13, the HCDR3 includes the sequence shown in SEQ ID NO: 20, the LCDR1 includes the sequence shown in SEQ ID NO: 27, the LCDR2 includes the sequence shown in SEQ ID NO: 30, and the LCDR3 includes the sequence shown in SEQ ID NO: 41.

[0134] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 7, the HCDR2 includes the sequence shown in SEQ ID NO: 14, the HCDR3 includes the sequence shown in SEQ ID NO: 21, the LCDR1 includes the sequence shown in SEQ ID NO: 28, the LCDR2 includes the sequence shown in SEQ ID NO: 35, and the LCDR3 includes the sequence shown in SEQ ID NO: 42.

[0135] The CDR amino acid sequences of antibodies 001, 002, 022, 032, 035, 050, 055, 060 and 074 are shown in Table 1 below. CDR boundaries are defined or identified according to the Kabat rules. The amino acid sequences of the heavy chain variable regions and light chain variable regions of antibodies 001, 002, 022, 032, 035, 050, 055, 060 and 074 are shown in Table 2 below.

[0136] Table 1: CDR amino acid sequences of 9 antibodies

[0137]

[0138]

[0139] Table 2: Amino acid sequences of variable regions of 9 antibodies

[0140]

[0141]

[0142] Assuming that antibodies 001, 002, 022, 032, 035, 050, 055, 060 and 074 can all bind to SIRPα, and the antigen binding specificity is mainly provided by the CDR1, CDR2 and CDR3 regions, the HCDR1, HCDR2 and HCDR3 sequences and LCDR1, LCDR2 and LCDR3 sequences of antibodies 001, 002, 022, 032, 035, 050, 055, 060 and 074 can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3) to produce anti-SIRPα binding molecules of the present invention. Such "mixed and matched" antibodies can be tested for SIRPα binding using the binding assays described above and in the Examples. Preferably, when VH CDR sequences are mixed and matched, the HCDR1, HCDR2 and / or HCDR3 sequences from a particular VH sequence are replaced by structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the LCDR1, LCDR2 and / or LCDR3 sequences from a particular VL sequence are preferably replaced by structurally similar CDR sequences. For example, the HCDR1 of antibodies 001 and 035 have some structural similarities and are therefore easily mixed and matched. It is apparent to those skilled in the art that new VH and VL sequences can be generated by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences in the CDR sequences of monoclonal antibodies 001, 002, 022, 032, 035, 050, 055, 060 and 074 disclosed in the present invention.

[0143] It is known that CDR is responsible for antigen binding. However, it has been found that not all 6 CDRs are essential or unchangeable. In other words, one or more CDRs in anti-SIRPα antibodies 001, 002, 022, 032, 035, 050, 055, 060 and 074 can be replaced or changed or modified, but the specific binding affinity to SIRPα is substantially retained.

[0144] In certain embodiments, the antibodies and antigen-binding fragments thereof described in the present invention contain appropriate framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can specifically bind to SIRPα. The CDR sequences shown in Table 1 above are obtained from mouse antibodies, but can be grafted to any suitable FR sequence of any suitable species (e.g., mouse, human, rat, rabbit, and others) using suitable methods known in the art (e.g., recombinant technology).

[0145] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention are humanized. It is expected that humanized antibodies or antigen-binding fragments have reduced immunogenicity in humans. Humanized antibodies or antigen-binding fragments thereof are chimeric in their variable regions because non-human CDR sequences are grafted into human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be accomplished by replacing corresponding human CDR genes with non-human (e.g., mouse) CDR genes on human immunoglobulin genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0146] Suitable human heavy and light chain variable domains can be selected using methods known in the art to achieve this purpose. In an illustrative example, a "best-fit" method can be used, wherein non-human (e.g., rodent) antibody variable domain sequences are screened, or BLAST aligned with a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human framework for grafting non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mot. Biol. 196: 901). Alternatively, a framework derived from the consensus sequence of all human antibodies can be used to graft non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0147] Table 3 below shows the CDR amino acid sequences of 8 humanized antibodies for antibody 035, which are named hu035.01, hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17. CDR boundaries are defined or identified according to the Kabat rule. Table 4 below shows the heavy and light chain variable region amino acid sequences of 8 humanized antibodies hu035.01, hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17. Table 5 below shows the FR amino acid sequences of eight humanized antibodies hu035.01, hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17.

[0148] Table 3: CDR amino acid sequences of 8 humanized antibodies

[0149]

[0150]

[0151] Table 4: Amino acid sequences of variable regions of 8 humanized antibodies

[0152]

[0153]

[0154] Table 5: FR amino acid sequences of 8 humanized antibodies

[0155]

[0156]

[0157]

[0158] In certain embodiments, humanized antibodies or their antigen-binding fragments provided by the invention are substantially composed of all human sequences except non-human CDR sequences. In some embodiments, variable region FR and constant region (if present) are all or substantially from human immunoglobulin sequences. People's FR sequences and people's constant region sequences can be derived from different human immunoglobulin genes, for example, derived from a FR sequence of a human antibody and derived from the constant region of another human antibody. In some embodiments, humanized antibodies or their antigen-binding fragments comprise people's heavy chain HFR1-4 and / or light chain LFR1-4.

[0159] In some embodiments, the FR region derived from people can include the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of people's FR are replaced by corresponding residues from parental non-human antibodies. This is required in certain embodiments, so that humanized antibodies or their fragments are close to non-human parent antibody structures, to optimize binding characteristics (for example, to increase binding affinity). In certain embodiments, humanized antibodies or their antigen-binding fragments of the present invention are included in each human FR sequence and are no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements, or are no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements in all FR sequences of heavy chain variable region or light chain variable region. In some embodiments, the change of this amino acid residue may only be present in the heavy chain FR region, only be present in the light chain FR region, or exist on both chains. In certain embodiments, one or more amino acids of people's FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are back-mutated to the corresponding amino acids of the parent non-human antibody to increase binding affinity.

[0160] In certain embodiments, the present invention also provides a humanized anti-SIRPα antibody and an antigen-binding fragment thereof, which comprises a heavy chain HFR1, a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4, wherein the heavy chain HFR1 comprises the sequence QX9QLVQSGSELKKPGASVKVSCX 10 AX 11 GX 12 X 13 (SEQ ID NO: 92) or a homologous sequence having at least 80% sequence identity thereto, the heavy chain HFR2 comprises the sequence WVRQAPGQGLEWMG (SEQ ID NO: 93) or a homologous sequence having at least 80% sequence identity thereto, the heavy chain HFR3 comprises the sequence RFVFSLDTSVSTAYLQIX 14 SLKAEDTAVYYCAR (SEQ ID NO: 96) or a homologous sequence thereof having at least 80% sequence identity, the heavy chain HFR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 97) or a homologous sequence thereof having at least 80% sequence identity, wherein X9 is I or V, X 10 is R or K, X 11 It is G or R or S, X 12 It is T or S, X 13 It is L or I or F, X 14 It is G or S.

[0161] In certain embodiments, the present invention also provides a humanized anti-SIRPα antibody and an antigen-binding fragment thereof, which comprises a light chain LFR1, a light chain LFR2, a light chain LFR3 and a light chain LFR4, wherein the light chain LFR1 comprises the sequence DIQMTQSPSX 15 LX 16 ASVGDRVTITC (SEQ ID NO: 100) or a homologous sequence thereof having at least 80% sequence identity, wherein the light chain LFR2 comprises the sequence WX 17 QQQKPGKX 18 PKX 19 LIX 20 (SEQ ID NO: 104) or a homologous sequence having at least 80% sequence identity thereto, wherein the light chain LFR3 comprises the sequence GVPSRFSGSGSGTDFTLTISX 21 LQPEDFATYX 22 C (SEQ ID NO: 108) or a homologous sequence having at least 80% sequence identity thereto, wherein the light chain LFR4 comprises the sequence FX 23 QGTKLEIKX 24 (SEQ ID NO: 47) or a homologous sequence thereof having at least 80% sequence identity, wherein X 15 Is S or R, X 16 It is S or G, X 17 Is Y or F, X 18 A or S, X 19 Is S or A, X 20 Is Y or F, X 21 Is S or N, X 22 Is Y or F, X 23 is G or D, X 24 Is R or missing.

[0162] In certain embodiments, the present invention also provides a humanized anti-SIRPα antibody and an antigen-binding fragment thereof, which comprises a heavy chain HFR1, a heavy chain HFR2, a heavy chain HFR3, a heavy chain HFR4, a light chain LFR1, a light chain LFR2, a light chain LFR3 and a light chain LFR4, wherein the heavy chain HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 44, 89, 90 and 91, the heavy chain HFR2 comprises a sequence as shown in SEQ ID NO: 93, the heavy chain HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 94 and 95, the heavy chain HFR4 comprises a sequence as shown in SEQ ID NO: 97, the light chain LFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 98 and 99, the light chain LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 101, 102 and 103, the light chain LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 105, 106 and 107, and the light chain LFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 108, 109 and 110. IDNO:109 and 46.

[0163] In certain embodiments, the present invention also provides humanized anti-SIRPα antibodies and antigen-binding fragments thereof, which include HFR1, HFR2, HFR3 and / or HFR4 sequences contained in the heavy chain variable region selected from the following groups: hu035.01-VH (SEQID NO: 64), hu035.02-VH / hu035.03-VH / hu035.10-VH / hu035.17-VH (SEQ ID NO: 65), hu035.09-VH (SEQ ID NO: 66), hu035.13-VH (SEQ ID NO: 67) and hu035.14-VH (SEQ ID NO: 68).

[0164] In certain embodiments, the present invention also provides humanized anti-SIRPα antibodies and antigen-binding fragments thereof, which include LFR1, LFR2, LFR3 and / or LFR4 sequences contained in a light chain variable region selected from the group consisting of hu035.01-VL (SEQ ID NO: 78), hu035.02-VL (SEQ ID NO: 79), hu035.03-VL (SEQ ID NO: 80), hu035.09-VL (SEQ ID NO: 81), hu035.10-VL / hu035.14-VL (SEQ ID NO: 82), hu035.13-VL (SEQ ID NO: 83) and hu035.17-VL (SEQ ID NO: 84).

[0165] In certain embodiments, the humanized anti-SIRPα antibodies and antigen-binding fragments thereof provided by the present invention include a heavy chain variable region sequence and / or a light chain variable region sequence, wherein the heavy chain variable region sequence includes a sequence selected from the group consisting of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68; the light chain variable region sequence includes a sequence selected from the group consisting of SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84.

[0166] The present invention also provides an exemplary humanized antibody of 035, comprising:

[0167] 1) "hu035.01", which includes the heavy chain variable region shown as hu035.01-VH (SEQ ID NO: 64) and the light chain variable region shown as hu035.01-VL (SEQ ID NO: 78);

[0168] 2) "hu035.02", which includes a heavy chain variable region as shown in hu035.02-VH (SEQ ID NO: 65) and a light chain variable region as shown in hu035.02-VL (SEQ ID NO: 79);

[0169] 3) “hu035.03”, which includes a heavy chain variable region as shown in hu035.03-VH (SEQ ID NO: 65) and a light chain variable region as shown in hu035.03-VL (SEQ ID NO: 80);

[0170] 4) “hu035.09”, which includes a heavy chain variable region as set forth in hu035.09-VH (SEQ ID NO: 66) and a light chain variable region as set forth in hu035.09-VL (SEQ ID NO: 81);

[0171] 5) “hu035.10”, which includes a heavy chain variable region as set forth in hu035.10-VH (SEQ ID NO: 65) and a light chain variable region as set forth in hu035.10-VL (SEQ ID NO: 82);

[0172] 6) “hu035.13”, which includes a heavy chain variable region as set forth in hu035.13-VH (SEQ ID NO: 67) and a light chain variable region as set forth in hu035.13-VL (SEQ ID NO: 83);

[0173] 7) “hu035.14”, which includes a heavy chain variable region as set forth in hu035.14-VH (SEQ ID NO: 68) and a light chain variable region as set forth in hu035.14-VL (SEQ ID NO: 82);

[0174] 8) "hu035.17", which includes the heavy chain variable region shown as hu035.17-VH (SEQ ID NO: 65) and the light chain variable region shown as hu035.17-VL (SEQ ID NO: 84).

[0175] These exemplary humanized anti-SIRPα antibodies retain specific binding ability or affinity for SIRPα and are at least comparable to or even better than the parent mouse antibody 035. For example, data are provided in Example 5.

[0176] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention comprises all or a portion of the heavy chain variable region, and / or all or a portion of the light chain variable region. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention is a single-domain antibody, which consists of all or a portion of the heavy chain variable region of the present invention. More information about single-domain antibodies is known in the art (see, e.g., U.S. Pat. No. 6,248,516).

[0177] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof described in the present invention further comprises an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and / or light chain constant region. In certain embodiments, the heavy chain constant region comprises CH1, a hinge and / or a CH2-CH3 region (or an optional CH2-CH3-CH4 region). In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof described in the present invention comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the light chain constant region comprises Cκ or Cλ. The anti-SIRPα antibody or antigen-binding fragment thereof described in the present invention may be identical to the wild-type constant region sequence or may differ in one or more mutation points.

[0178] In certain embodiments, the heavy chain constant region includes an Fc region. It is known that the Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. The ability of the Fc regions of different Ig isotypes to induce effector functions is different. For example, it has been recognized that the Fc regions of IgG1 and IgG3 are more effective in inducing ADCC and CDC than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention includes an Fc region of an IgG1 or IgG3 isotype, which can induce ADCC or CDC; or includes a constant region of an IgG4 or IgG2 isotype, which has reduced or depleted effector functions. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention includes an Fc region of wild-type human IgG4 or other wild-type human IgG4 alleles. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof of the present invention includes an Fc region of human IgG4, and the Fc region includes an S228P mutation. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof described herein comprises an Fc region of human IgG4, wherein the Fc region comprises an L235E mutation.

[0179] In certain embodiments, the antibodies or fragments thereof described herein have sufficient affinity to specifically bind to human SIRPα for diagnostic and / or therapeutic purposes.

[0180] The antibody or fragment thereof described in the present invention can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a multispecific antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein. Recombinant antibodies are antibodies prepared in vitro using a recombinant method (rather than in an animal).

[0181] In certain embodiments, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment thereof according to the present invention for binding to human SIRPα. In certain embodiments, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence as shown in SEQ ID NO: 70, and the light chain variable region comprising the sequence as shown in SEQ ID NO: 86. In certain embodiments, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence as shown in SEQ ID NO: 72, and the light chain variable region comprising the sequence as shown in SEQ ID NO: 88. In certain embodiments, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 62, and the light chain variable region comprising the sequence shown in SEQ ID NO: 76, or competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 69, and the light chain variable region comprising the sequence shown in SEQ ID NO: 85. In certain embodiments, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which competes for binding to human SIRPα with an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 71, and the light chain variable region comprising the sequence shown in SEQ ID NO: 87.

[0182] In certain embodiments, the present invention provides an anti-SIRPα antibody or an antigen-binding fragment thereof, which competes for binding to human SIRPα with an antibody selected from the following groups: a) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence as shown in SEQ ID NO: 59, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 73; b) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence as shown in SEQ ID NO: 61, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 75; c) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence as shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 74; d) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence as shown in SEQ ID NO: 63, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 77, and wherein the antibody or antigen-binding fragment thereof is not KWAR23, HEFLB, 29-AM4-5, ALX Any one of H21 and 3F9-22.

[0183] "KWAR23" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 111, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 114.

[0184] The "HEFLB" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 112, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 34.

[0185] The "29-AM4-5" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 110, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 113.

[0186] As used in the present invention, "ALX H21" refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 115 and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 117.

[0187] The "3F9-22" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 116, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 118.

[0188] Antibody variants

[0189] The antibodies and antigen-binding fragments thereof provided by the present invention also include various variants of the antibody sequences provided by the present invention.

[0190] In certain embodiments, the antibody variant comprises one or more modifications or substitutions in one or more CDR sequences shown in Tables 1 and 3 above, one or more non-CDR sequences of the heavy chain variable region or light chain variable region sequences of the present invention shown in Tables 2 and 4 above, and / or in the constant region (e.g., Fc region). These antibody variants retain the affinity of their parent for specific binding to SIRPα, but have the desired properties brought about by one or more of the modifications or substitutions. For example, the antibody variant may have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugation (e.g., one or more introduced cysteine ​​residues).

[0191] Parent antibody sequences can be screened to identify suitable or preferred residues to be modified or substituted using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells, (1989) Science, 244: 1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by uncharged or negatively charged amino acids (e.g., alanine or polyalanine), resulting in modified antibodies, and screened for target properties. If a substitution at a particular amino acid position shows a change in the target functionality, the position can be identified as a potential residue for modification or substitution. The potential residue can be further evaluated by replacing it with another residue (e.g., a cysteine ​​residue, a positively charged residue, etc.).

[0192] Affinity variants

[0193] The affinity variants of the antibody may comprise modifications or substitutions in one or more CDR sequences shown in Tables 1 and 3 above, one or more FR sequences shown in Table 5 above, or the heavy or light chain variable region sequences shown in Tables 2 and 4 above. A person skilled in the art can easily determine the FR sequence based on the CDR sequences in Tables 1 and 3 above and the variable region sequences in Tables 2 and 4 above, because it is well known in the art that in the variable region, the CDR region is flanked by two FR regions. The affinity variants retain the affinity of the parent antibody for specific binding to SIRPα, or even have an improved affinity for specific binding to SIRPα relative to the parent antibody. In certain embodiments, at least one (or all) substitutions in the CDR sequence, FR sequence, or variable region sequence comprise conservative substitutions.

[0194] Those skilled in the art will understand that in the CDR sequences provided in Tables 1 and 3 above, and in the variable region sequences provided in Tables 2 and 4 above, one or more amino acid residues may be substituted, and the resulting antibody or antigen-binding fragment still maintains binding affinity or binding ability to SIRPα, or even has improved binding affinity or ability. Various methods known in the art can be used to achieve this goal. For example, an antibody variant library (e.g., Fab or scFv variants) can be generated and expressed using phage display technology, and then screened for affinity for binding to human SIRPα. For another example, computer software can be used to simulate the binding of antibodies to human SIRPα and identify the amino acid residues on the antibody that form the binding interface. These residues can be avoided in the substitution to prevent a reduction in binding affinity, or can be used as targets for substitution to obtain stronger binding.

[0195] In certain embodiments, the humanized antibody or antigen-binding fragment thereof of the present invention comprises one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions in total in the CDR sequences and / or FR sequences.

[0196] In certain embodiments, the anti-SIRPα antibody or its antigen-binding fragment comprises 1, 2 or 3 CDR sequences, which have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequences listed in Tables 1 and 3 above, while maintaining a similar or higher level of specific binding affinity to SIRPα relative to its parent antibody.

[0197] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises one or more variable region sequences, which have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequences listed in Tables 2 and 4 above, while maintaining a similar or higher level of specific binding affinity to SIRPα relative to its parent antibody. In some embodiments, in the sequences of the variable regions listed in Tables 2 and 4 above, a total of 1 to 10 amino acids are substituted, inserted or deleted. In some embodiments, the substitution, insertion or deletion occurs in a region outside the CDR (e.g., in the FR).

[0198] Glycosylation variants

[0199] The anti-SIRPα antibody or antigen-binding fragment thereof of the present invention further comprises a glycosylation variant, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0200] The antibody or its antigen-binding fragment may include one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) may be attached. The glycosylation of an antibody is typically N-connected or O-connected. N-connected refers to the attachment of a carbohydrate moiety to the side chain of an aspartic acid residue, e.g., an aspartic acid residue in a tripeptide sequence, such as aspartic acid-X-serine and aspartic acid-X-threonine, wherein X is any amino acid except proline. O-connected glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly to serine or threonine. Natural glycosylation sites can be easily removed, e.g., by changing the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-connected glycosylation sites) or serine or threonine residues (for O-connected glycosylation sites) present in the sequence is replaced. In a similar manner, new glycosylation sites can be generated by introducing such a tripeptide sequence or a serine or threonine residue.

[0201] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments described herein comprise a mutation at N297 (eg, N297A, N297Q, or N297G) to remove the glycosylation site.

[0202] Cysteine ​​engineered variants

[0203] The anti-SIRPα antibodies and antigen-binding fragments of the present invention also include cysteine ​​engineered variants, which include one or more introduced free cysteine ​​amino acid residues.

[0204] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bond. Cysteine ​​engineered variants can be used to conjugate, for example, cytotoxic compounds and / or imaging compounds, labels or radioisotopes and other substances at the site of the engineered cysteine, for example, via maleimide or haloacetyl groups. Methods for modifying antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are well known in the art, see, for example, WO2006 / 034488.

[0205] Fc variants

[0206] The anti-SIRPα antibodies or antigen-binding fragments thereof described herein also include Fc variants, which include one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region, for example, to provide altered effector functions, such as ADCC and CDC. Methods for altering ADCC activity by antibody engineering have been described in the prior art, see, for example, Shields RL. et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE. et al., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., PNAS, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards JO, et al., Mol Cancer Ther. 2008, 7(8): 2517-27; Shields R Let al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J. Biol. Chem, 2003, 278: 3466-3473.

[0207] The CDC activity of the antibodies or antigen-binding fragments provided herein can also be altered, for example, by improving or reducing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with different amino acid residues to alter C1q binding and / or reduce or eliminate complement dependent cytotoxicity (CDC) (see U.S. Pat. No. 6,194,551 to No. Idusogie et al.). One or more amino acid substitutions can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication No. WO 94 / 29351 to Bodmer et al.).

[0208] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein have reduced effector function and comprise one or more amino acid substitutions in IgG1 at positions selected from the group consisting of 234, 235, 237 and 238, 268, 297, 309, 330 and 331. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and comprise one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S and any combination thereof. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided by the present invention is of IgG2 isotype and comprises one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A and any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided by the present invention is of IgG4 isotype and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A and any combination thereof. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided by the present invention is of IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotyping are described in Rother RP et al., Nat Biotechnol 25: 1256-1264 (2007).

[0209] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and comprise one or more amino acid substitutions at one or more positions of 228 and 235. In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and have an S228P mutation in the Fc region. In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and have an L235E mutation in the Fc region.

[0210] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that can improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such a variant can have a prolonged pharmacokinetic half-life because it binds to FcRn at acidic pH, allowing it to be protected from degradation in lysosomes and subsequently transferred and released outside the cell. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0211] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment comprises one or more amino acid substitutions located at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include introducing a protrusion into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide, wherein the protrusion may be located within the cavity to promote interaction between the first and second Fc polypeptides to form a heterodimer or complex. Methods for producing antibodies with these modifications are well known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0212] Fab

[0213] The present invention also provides anti-SIRPα antigen-binding fragments. Various types of antigen-binding fragments are well known in the art and can be developed based on the anti-SIRPα antibodies described in the present invention, including exemplary antibodies, whose CDRs are shown in Tables 1 and 3 above, whose variable region sequences are shown in Tables 2 and 4 above, and their different variants (e.g., affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered antibodies, etc.).

[0214] In certain embodiments, the anti-SIRPα antigen-binding fragment described in the present invention is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody and bivalent domain antibody.

[0215] A variety of techniques can be used for the production of such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)), recombinant expression by host cells (e.g., E. coli) (e.g., for Fab, Fv, and ScFv antibody fragments), screening of phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form a F(ab')2 fragment (Carter et al., Bio / Technology 10: 163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0216] In certain embodiments, the antigen binding fragment is a scFv. The generation of scFv is described in, for example, WO 93 / 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The scFv can be fused to an effector protein at the amino or carboxyl terminus to obtain a fusion protein (see, for example, Antibody Engineering, Borrebaeck, ed.).

[0217] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided by the present invention are bivalent, tetravalent, hexavalent or multivalent. Any molecule greater than bivalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0218] A bivalent molecule can be monospecific if both binding sites can specifically bind to the same antigen or the same epitope. In certain embodiments, this provides stronger binding to the antigen or epitope than a corresponding monovalent molecule. Similarly, multivalent molecules can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen binding portion, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences but have the same specificity).

[0219] A bivalent can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0220] Bispecific Antibodies

[0221] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is bispecific. In certain embodiments, the antibody or antigen-binding fragment thereof is further linked to a second functional moiety having a different binding specificity than the SIRPα antibody or antigen-binding fragment thereof.

[0222] In certain embodiments, the bispecific antibody or antigen-binding fragment thereof provided by the present invention can specifically bind to a second antigen other than SIRPα or a second epitope on SIRPα. In certain embodiments, the second antigen is selected from the group consisting of CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), GPC-3, B7-H3, B7-H4, TROP2, CLDN18.2, EGFR, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

[0223] Conjugate

[0224] In some embodiments, the anti-SIRPα antibody or its antigen-binding fragment further comprises one or more conjugate parts. The conjugate part can be connected to the antibody or its antigen-binding fragment. The conjugate part is a part that can be attached to the antibody or its antigen-binding fragment. It can be imagined that the antibody or its antigen-binding fragment described in the present invention can be connected to a variety of conjugate parts (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate parts can be connected to the antibody or its antigen-binding fragment by other means such as covalent binding, affinity binding, embedding, coordinate binding, complexation, association, blending or addition. In certain embodiments, the antibody or its antigen-binding fragment is connected to one or more conjugates via a linker.

[0225] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided in the present invention can be engineered to contain specific sites outside the epitope binding portion, which can be used to bind to one or more conjugate moieties. For example, the site can include one or more reactive amino acid residues (e.g., cysteine ​​or histidine residues) to facilitate covalent attachment to the conjugate moiety.

[0226] In some embodiments, the antibody or its antigen binding fragment can be connected to the conjugate part indirectly or through another conjugate part. For example, the antibody or its antigen binding fragment provided by the present invention can be conjugated with biotin and then indirectly conjugated with a second conjugate, which is conjugated with avidin. In some embodiments, the conjugate part includes a scavenging modifier (e.g., a polymer (e.g., PEG) that extends the half-life), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification part or other anticancer drugs.

[0227] A "toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0228] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioactive isotopes (e.g., 123 I. 124 I. 125 I. 131 I. 35 S. 3 H. 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y. 88 Y. 90 Y. 177 Lu, 211 At 186 Re, 188 Re, 153 Sm, 212 Bi、and 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0229] In some embodiments, the conjugate moiety can be a clearance modifier, which helps to increase the half-life of the antibody. Illustrative examples include water-soluble polymers (e.g., PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol copolymers, etc.). The polymer can have any molecular weight and can be branched or unbranched. The number of polymers connected to the antibody can vary, and if the polymer connected is more than one, they can be the same or different molecules.

[0230] In certain embodiments, the conjugate moiety may be a purification moiety, such as a magnetic bead.

[0231] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as the basis of a conjugate.

[0232] Polynucleotides and recombinant methods

[0233] The present invention provides isolated polynucleotides encoding the anti-SIRPα antibody or its antigen-binding fragment. The term "nucleic acid" or "polynucleotide" used in the present invention refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a specific polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0234] DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Encoding DNA can also be obtained synthetically.

[0235] Using recombinant techniques known in the art, the isolated polynucleotide encoding the anti-SIRPα antibody or its antigen-binding fragment can be inserted into a vector for further cloning (amplification of DNA) or for expression. There are a variety of vectors to choose from. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0236] The present invention provides vectors comprising isolated polynucleotides. In certain embodiments, the polynucleotides provided by the present invention encode antibodies or antigen-binding fragments thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0237] The vector comprising the polynucleotide sequence encoding the antibody or its antigen-binding fragment can be introduced into a host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA in the vector described in the present invention is the above-mentioned prokaryotic, yeast or higher eukaryotic cell. Prokaryotic cells suitable for the purposes of the present invention include true bacteria, such as Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae, for example, Escherichia (Escherichia) (for example, Escherichia coli (E.coli)), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (for example, Salmonella typhimurium (Salmonella typhimurium)), Serratia (Serratia) (for example, Serratia marcescens (Serratia marcescans), Shigella, Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces.

[0238] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as suitable cloning or expression hosts for vectors encoding anti-SIRPα antibodies. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly used and suitable for use in the present invention, for example, Schizosaccharomyces pombe; Kluyveromyces hosts, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentali; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus (e.g., A. nidulans and A. niger).

[0239] The host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided in the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculoviral strains and variants thereof and corresponding permissive insect host cells have been found from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV, and the Bm-5 variant of Bombyx mori NPV, which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0240] However, of greatest interest are spinal cells, and their cultivation (tissue culture) has become routine. Examples of mammalian host cells that can be used include SV40-transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse testicular supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In certain embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and derivatives thereof.

[0241] The host cell is transformed with the above-mentioned expression or cloning vector that can produce anti-SIRPα antibodies and cultured in a conventional nutrient medium, which is modified to be suitable for inducing promoters, selecting transformed cells or amplifying genes encoding target sequences. In another embodiment, the antibody can be prepared by homologous recombination methods known in the art. In certain embodiments, the host cell is capable of producing the antibody or antigen-binding fragment thereof of the present invention.

[0242] The present invention also provides a method for expressing the antibody or antigen-binding fragment thereof of the present invention, comprising culturing the host cell provided by the present invention under the condition of expressing the vector of the present invention. The host cell used to produce the antibody or antigen-binding fragment thereof in the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) can be used to culture the host cell. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985 can be used as culture medium for the host cells. These media can be supplemented with necessary hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride, and phosphate), buffers (such as HEPES), nucleotides (such as adenylate and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds whose final concentrations are usually in the micromolar range), and glucose or an equivalent energy source. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH and the like, are those previously used in selecting host cells for expression and are well known to those of ordinary skill.

[0243] When using recombinant technology, antibodies can be produced intracellularly in the periplasmic space, or directly secreted into the matrix. If the antibody is produced intracellularly, the first step is to remove the particle debris (host cell or cracked fragments), for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for separating antibodies secreted into the periplasmic space of Escherichia coli. Briefly, the cell paste is thawed for more than 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antibody is secreted into the matrix, a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device, is usually first used to concentrate the supernatant from the expression system. Any of the aforementioned steps may include a protease inhibitor, such as PMSF, to inhibit proteolysis, and may include antibiotics to prevent the growth of accidental contaminants.

[0244] The anti-SIRPα antibody or antigen-binding fragment thereof prepared from the cells can be purified by a purification method such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out and affinity chromatography, wherein affinity chromatography is a preferred purification technique.

[0245] In certain embodiments, protein A fixed to a solid phase is used for immunoaffinity purification of the antibody and its antigen-binding fragment. The type of the antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is suitable for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, Bakerbond ABX can be used. TM The antibody was purified by HPLC-MS / MS (JT Baker, Phillipsburg, NJ). Other protein purification techniques may also be determined based on the antibody to be obtained, such as fractionation in an ion exchange column, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0246] Following any preliminary purification steps, the mixture comprising the antibody of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using a wash buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25 M salt concentration).

[0247] Pharmaceutical composition

[0248] The present invention further provides a pharmaceutical composition comprising the anti-SIRPα antibody or antigen-binding fragment thereof according to the present invention, and one or more pharmaceutically acceptable carriers.

[0249] The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in the present invention may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending agents / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components well known in the art or various combinations thereof.

[0250] Applicable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Applicable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present invention, one or more antioxidants such as methionine are included in the composition comprising the antibody or its antigen-binding fragment disclosed in the present invention, which can reduce the oxidation of the antibody or its antigen-binding fragment. The reduction of oxidation can prevent or reduce the reduction of binding affinity, thereby improving antibody stability and extending shelf life. Therefore, in certain embodiments, the pharmaceutical composition provided by the present invention comprises one or more antibodies or their antigen-binding fragments described in the present invention and one or more antioxidants such as methionine. The present invention further provides various methods for preventing oxidation of the antibodies or antigen-binding fragments thereof, extending their shelf life and / or improving their activity, for example, by mixing the antibodies or antigen-binding fragments thereof provided in the present invention with one or more antioxidants (e.g., methionine).

[0251] Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinyl pyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents as carriers may be added to the pharmaceutical compositions in multiple dose containers and include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

[0252] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

[0253] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, a liquid solvent, a suspending agent, an emulsifier, or a solid form suitable for producing a liquid solvent, a suspending agent, or an emulsifier. Injectable formulations can include sterile and / or pyrogen-free solutions, sterile dried solubles that are combined with solvents before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions for injection, sterile dried insoluble products that are combined with media before use, and sterile and / or pyrogen-free emulsions. The solvent can be an aqueous phase or a non-aqueous phase.

[0254] In certain embodiments, the unit dose of the injection preparation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for injection should be sterile and pyrogen-free.

[0255] In certain embodiments, a sterile lyophilized powder can be prepared by dissolving the antibody or antigen-binding fragment thereof disclosed in the present invention in a suitable solvent. The solvent may contain a substance that can improve the stability of the powder or a reconstituted solution obtained from the powder, or other pharmacological components that improve the powder or the reconstituted solution. Applicable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other applicable substances. The solvent may contain a buffer, such as a citrate buffer, a sodium phosphate or potassium phosphate buffer or other buffer known to the skilled person, and in one embodiment, the pH of the buffer is neutral. The dissolution is subsequently sterilized by filtration under standard conditions known in the art, and then lyophilized to obtain an ideal formulation. In one embodiment, the resulting solvent is dispensed into small tubes for lyophilization. Each small tube can accommodate a single dose or multiple doses of the anti-SIRPα antibody or antigen-binding fragment thereof or a composition thereof. The amount loaded in each small tube may be slightly higher than that required for each dose or multiple doses (e.g., 10% excess) to ensure accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at a temperature ranging from about 4°C to room temperature.

[0256] The lyophilized powder is reconstituted with water for injection to obtain a preparation for injection. In one embodiment, the lyophilized powder can be added to sterile pyrogen-free water or other applicable liquid carriers for reconstitution. The exact amount is determined by the selected therapy and can be determined based on empirical values.

[0257] Reagent test kit

[0258] In certain embodiments, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof provided by the present invention. In certain embodiments, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof provided by the present invention and a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from chemotherapeutics, anticancer drugs, radiotherapeutics, immunotherapeutics, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0259] If desired, such a kit may further include one or more of the various conventional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc., which will be apparent to those skilled in the art. Instructions (as inserts or labels) indicating the amounts of the components to be administered, instructions for administration, and / or instructions for mixing the components may also be included in the kit.

[0260] How to use

[0261] The present invention also provides a method for treating a disease, disorder or condition associated with SIRPα in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the present invention and / or a pharmaceutical composition according to the present invention. In certain embodiments, the subject is a human.

[0262] In some embodiments, the disease, disorder or condition associated with SIRPα is characterized by expression or overexpression of SIRPα and / or a SIRPα signature gene.

[0263] In certain embodiments, the disease, disorder or condition associated with SIRPα includes, but is not limited to, cancer, solid tumors, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction or arthritis.

[0264] In some embodiments, the cancer is a cancer that expresses SIRPα. In some embodiments, the cancer is a CD47-positive cancer. In some embodiments, the cancer is selected from the group consisting of anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer , prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T or B cell lymphoma, gastrointestinal stromal tumor, soft tissue tumors, hepatocellular carcinoma, and adenocarcinoma.

[0265] In some embodiments, the cancer is a CD47-positive cancer. In some embodiments, the subject to be treated has been identified as having a CD47-positive cancer. "CD47-positive" cancer used in the present invention refers to a cancer characterized by expressing CD47 protein in cancer cells or expressing CD47 in cancer cells at a level significantly higher than that expected for normal cells. The presence and / or content of CD47 in a biological sample of interest can indicate whether the subject from which the biological sample is derived is likely to respond to anti-SIRPα antibodies. A variety of methods can be used to determine the presence and / or content of CD47 in a test biological sample from a subject. For example, a test biological sample can be exposed to an anti-CD47 antibody or an antigen-binding fragment thereof that binds to and detects expressed CD47 protein. Alternatively, CD47 can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc. In some embodiments, the test sample is derived from cancer cells or tissues or tumor-infiltrating immune cells. In certain embodiments, the presence or upregulation of CD47 in the test biological sample indicates the possibility of a response. As used herein, the term "upregulation" refers to an overall increase of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more in the expression level of CD47 in the test sample compared to the expression level of CD47 in the reference sample detected using the same method. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same person from whom the test sample was obtained. For example, the reference sample can be a non-diseased sample adjacent to or near the test sample (e.g., a tumor).

[0266] In another aspect, a method for treating a disease, disorder or condition in an individual who would benefit from modulation of SIRPα activity is provided, the method comprising administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof and / or a pharmaceutical composition as described herein. In certain embodiments, the disease or condition is a disease, disorder or condition associated with SIRPα.

[0267] The therapeutically effective dose of the antibody or antigen-binding fragment thereof of the present invention depends on various factors known in the art, such as body weight, age, past medical history, current treatment, health status of the subject and potential for cross infection, allergy, hypersensitivity and side effects, as well as route of administration and degree of tumor development. A person skilled in the art (e.g., a doctor or veterinarian) may proportionally reduce or increase the dose according to these or other conditions or requirements.

[0268] In certain embodiments, antibodies or antigen binding fragments as described herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the dosage can vary with the course of treatment. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage is adjusted during the course of treatment based on the response of the subject being administered.

[0269] Dosage regimens may be adjusted to achieve the optimum response (eg, therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over a period of time.

[0270] The antibodies or antigen-binding fragments thereof disclosed in the present invention can be administered by administration methods known in the art, such as parenteral administration (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection) or non-parenteral administration routes (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration).

[0271] In some embodiments, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer agent, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine).

[0272] The term "immunotherapy" as used herein refers to a therapy that stimulates the immune system to fight a disease (e.g., cancer) or generally enhances the immune system. Examples of immunotherapy include, but are not limited to, checkpoint regulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0273] "Targeted therapy" is a therapy that acts on specific molecules involved in cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or target molecules in the cancer microenvironment that help cancer grow and survive. Targeted therapy targets the therapeutic agent to the tumor, sparing normal tissue from the effects of the therapeutic agent.

[0274] In some such embodiments, when the antibodies or antigen-binding fragments thereof disclosed in the present invention are used in combination with one or more additional therapeutic agents, they can be administered simultaneously with the one or more additional therapeutic agents. In some such embodiments, the antibodies or antigen-binding fragments thereof and the additional therapeutic agents can be administered simultaneously as part of the same pharmaceutical composition. However, the antibodies or antigen-binding fragments thereof "used in combination" with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "used in combination" in the present invention also includes that the antibodies or antigen-binding fragments thereof administered before or after another therapeutic agent are also considered to be "used in combination" with the therapeutic agent, even if the antibodies or antigen-binding fragments thereof and the second substance are administered by different administration methods. Where possible, the other therapeutic agents used in combination with the antibodies or antigen-binding fragments thereof disclosed in the present invention can be administered according to the methods of the product instructions of the other therapeutic agents, or according to the Surgeon's Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)), or according to other methods known in the art.

[0275] In another aspect, the present invention further provides a method for regulating SIRPα activity in SIRPα-positive cells, comprising exposing SIRPα-positive cells to an antibody or antigen-binding fragment thereof provided by the present invention. In some embodiments, the SIRPα-positive cells are phagocytic cells (eg, macrophages).

[0276] In another aspect, the present invention provides a method for detecting the presence or amount of SIRPα in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof, and determining the presence or amount of SIRPα in the sample.

[0277] On the other hand, the present invention provides a method for diagnosing a disease, disorder or condition associated with SIRPα in an individual, comprising: a) contacting a sample obtained from the individual with the antibody or antigen-binding fragment thereof described in the present invention; b) determining the presence or content of SIRPα in the sample; and c) correlating the presence or content of SIRPα with the presence or condition of the disease, disorder or condition associated with SIRPα in the individual.

[0278] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention, which is optionally conjugated to a detectable moiety, and can be used to detect a disease, disorder or condition associated with SIRPα. The kit may further include instructions for use.

[0279] On the other hand, the present invention also provides the use of the antibody or antigen-binding fragment thereof described in the present invention in the preparation of a medicament for treating, preventing or alleviating a disease, disorder or condition associated with SIRPα in an individual, or in the preparation of a diagnostic reagent for diagnosing a disease, disorder or condition associated with SIRPα.

[0280] On the other hand, the present invention provides a method of inducing phagocytosis in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof provided by the present invention and / or a pharmaceutical composition provided by the present invention at a dose effective to induce phagocytosis. For example, the antibody or antigen-binding fragment thereof provided by the present invention can be administered to induce phagocytosis of cancer cells, inflammatory cells and / or chronically infected cells expressing CD47. In some embodiments, the subject is human. In some embodiments, the subject has a disease, disorder or condition selected from the group consisting of cancer, solid tumors, chronic infections, inflammatory diseases, multiple sclerosis, autoimmune diseases, nervous system diseases, brain damage, nerve damage, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction and arthritis.

[0281] On the other hand, the present invention provides a method for inducing phagocytosis in vitro, comprising contacting target cells with a SIRPα-positive phagocyte sample in the presence of the antibody or antigen-binding fragment thereof according to the present invention and / or the pharmaceutical composition according to the present invention, thereby inducing the phagocytosis of the target cells by the SIRPα-positive phagocytes.

[0282] The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the following specific compositions, materials and methods, in whole or in part, are within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the present invention, but are only intended to illustrate that specific embodiments are within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without creative work and without departing from the scope of the present invention. It should be understood that the various modifications made to the method of the present invention may still be included within the scope of the present invention. The inventor intends to include such changes within the scope of the present invention. Example

[0283] Example 1. Generation of reagents

[0284] 1.1 Generation of reference antibodies

[0285] The DNA sequences encoding the variable regions of the anti-SIRPα reference antibodies 29-AM4-5 (see US20140242095), KWAR23 (see US20170073414A1), HEFLB (see WO2017178653A2), ALX H21 (see US20180105600A1) or 3F9-22 (see US20190359707A1) were cloned into a vector expressing a human IgG constant region. The variable region amino acid sequences of the reference antibodies 29-AM4-5, KWAR23, HEFLB, ALX H21 and 3F9-22 are shown in Table 6 below. Expi293 cells (Invitrogen) transfected with the expression plasmids were cultured at 37°C for one week. The culture medium was then collected and the cell precipitate was removed by centrifugation. The harvested supernatant was purified using a protein A affinity column (Mabselect Sure, GE Healthcare).

[0286] Table 6. Amino acid sequences of the variable regions of five reference antibodies

[0287]

[0288]

[0289] 1.2 Establishment of stable SIRPα expression cell lines in humans, cynomolgus monkeys, and mice

[0290] DNA sequences encoding full-length human SIRPαv1 (NP_542970), cynomolgus monkey SIRPα (NP_001271679), or C57BL / 6 mouse SIRPα (NP_031573) were cloned into pIRES vectors (Clontech). 293F cells (Invitrogen) transfected with human SIRPαv1 expression plasmids were selected and cultured in medium containing 0.5 μg / ml puromycin for 2 weeks. Single cell clones stably expressing human SIRPαv1 were then isolated by limiting dilution and screened by FACS using anti-human SIRPα antibodies (Biolegend, 323802).

[0291] In a similar manner, CHOK1 cells (Invitrogen) transfected with human SIRPαv1, cynomolgus SIRPα, or C57BL / 6 mouse SIRPα expression plasmids were selected and cultured for 2 weeks in a medium containing 6 μg / ml puromycin. Single cell clones that stably expressed human SIRPαv1, cynomolgus SIRPα, or C57BL / 6 mouse SIRPα were then isolated by limiting dilution and screened by FACS using anti-human SIRPα antibodies (Biolegend, 323802) or anti-mouse SIRPα antibodies (Sino Biological, 50956-R001).

[0292] 1.3 Production of recombinant proteins

[0293] The DNA sequence encoding the extracellular region of human CD47 (NP_001768.1, M1-E141), human SIRPαv1 extracellular region (NP_542970, M1-R370), human SIRPαv2 extracellular region (CAA71403.1, M1-R369), human SIRPβ extracellular region (O00241, M1-L371) or human SIRPγ extracellular region (Q9P1W8, M1-P360) was cloned into the pCPC vector (Chempartner) expressing the human IgG Fc region (hFc). Expi293 cells (Invitrogen) transfected with the recombinant plasmid expressing the ECD protein were cultured at 37°C for 1 week. The culture medium was then collected and the cell precipitate was removed by centrifugation. The harvested supernatant was purified using a protein A affinity chromatography column (Mabselect Sure, GE Healthcare).

[0294] The recombinant proteins of 6xHis-tagged human SIRPαv1 ECD and human SIRPαv8 ECD were purchased from Biointron. The recombinant proteins of 6xHis-tagged human CD47 ECD, human SIRPαv2 ECD, and C57BL / 6 mouse SIRPα ECD were purchased from Novoprotein.

[0295] Example 2. Production of antibodies

[0296] 2.1 Preparation of immunogens for protein immunization

[0297] Fc-tagged human SIRPαv1 ECD recombinant protein was used as the immunogen for protein immunization (see Example 1.3).

[0298] 2.2 Preparation of immunogens for cell-mediated immunity

[0299] 293F cells stably expressing human SIRPαv1 were used as the immunogen for cellular immunization (see Example 1.2).

[0300] 2.3 Preparation of immunogens for genetic immunization

[0301] The DNA sequence encoding the full-length human SIRPαv1 protein (NP_542970) was cloned into the pCP vector (Chempartner), and the prepared plasmid was then coated on colloidal gold bullets (Bio-Rad) as an immunogen for genetic immunization.

[0302] 2.4 Immunity

[0303] Balb / c and SJL / J mice (SLAC) were immunized using three different immunization strategies, protein immunization using human SIRPαv1 ECD recombinant protein, cell immunization using 293F cells stably expressing human SIRPαv1, and gene immunization using gold bullets coated with human SIRPαv1 expression plasmid. Serum titers of immunized mice were detected by ELISA analysis using human SIRPαv1 ECD recombinant protein and FACS analysis using 293F cells stably expressing human SIRPαv1. Mice with high serum titers were selected for hybridoma fusion.

[0304] 2.5 Generation of hybridomas

[0305] On the 5th day after the last boost, mice were sacrificed and spleen cells were collected. 1% (v / v) NH4OH was added to the lysed red blood cells. The washed spleen cells were then fused with SP2 / 0 mouse myeloma cells (ATCC) by high-efficiency electrofusion or PEG method. After cell fusion, the fused cells were plated at 2x10 4 The cells were seeded at a density of 10 cells / well into a 96-well plate in 200 μl of DMEM medium containing 20% ​​FBS and 1% HAT.

[0306] 2.6 Screening of hybridomas

[0307] On the 10th to 12th day after fusion, the fusion plate was initially screened by ELISA assay of human SIRPαv1 ECD recombinant protein or Acumen assay (TTP Labtech) of 293F cells with stable expression of human SIRPαv1. The hybridoma cells selected from the positive wells were expanded into 24-well plates for a second screening. In the second screening, the binding activity was evaluated by ELISA assay of human SIRPαv1 ECD recombinant protein and FACS assay of 293F cells with stable expression of human SIRPαv1. The clone with the highest binding activity was selected as a subclone. In addition, the specificity for human SIRPαv2 / β / γ, species cross-reactivity, blocking activity for the interaction between CD47 and SIRPα, and blocking activity for the interaction between CD47 and SIRPβ were also detected in the second hybridoma identification to characterize the hybridoma (see Example 3 for the characterization assay method).

[0308] 2.7 Subcloning of hybridomas

[0309] By limiting dilution, each selected cloned hybridoma cell is inoculated into a 96-well plate at a density of 1 cell / well. The inoculated plate is screened in the same manner as the hybridoma primary screening (see Example 2.6). Positive single clones are selected and characterized by the same method as the hybridoma secondary screening (see Example 2.6). The monoclonal hybridoma cell line with the highest binding activity is then obtained for further hybridoma antibody production, characterization and sequencing.

[0310] A total of 9 antibody clones were identified as functional hits, and the hybridoma antibodies purified from these clones were named 001, 002, 022, 032, 035, 050, 055, 060, and 074, respectively.

[0311] Example 3. Antibody Characterization

[0312] 3.1 Production and purification of hybridoma antibodies

[0313] After about 14 days of culture, the hybridoma cell culture medium was collected and the cells were removed by centrifugation. After filtering through a 0.22 μm PES membrane and adjusting the pH to 7.2, the obtained supernatant was loaded onto a protein A affinity chromatography column (GE). The antibody was eluted with 0.1 M sodium citrate buffer (pH 3.0) and then immediately neutralized with Tris buffer (pH 8.0). After dialysis with PBS buffer, the antibody concentration was determined by Nano Drop (Thermo Fisher). The purity of the protein was evaluated by SDS-PAGE and HPLC-SEC (Agilent). Endotoxin levels were detected with an Endochrome-K kit (Charles River).

[0314] 3.2 Monocyte-derived macrophage phagocytosis assay

[0315] The functional efficacy of purified hybridoma antibodies was evaluated by flow cytometry-based phagocytosis assay. Briefly, human monocyte-derived macrophages were co-cultured with Jurkat and Raji cancer cells expressing CD47 labeled with CellTrace Violet (Life Technologies) in the presence of 50 nM / 2 nM anti-SIRPα antibody. Phagocytosis was analyzed by determining the percentage of macrophages positive for CellTrace Violet dye.

[0316] As shown in Table 7, anti-SIRPα hybridoma antibodies 001, 002, 032, 035, 055, 074, 022, 050 and 060 stimulated potent phagocytosis of Jurkat cells and Raji cells by macrophages at a concentration of 2 nM, while other known anti-SIRPα antibodies 29-AM4-5, KWAR23 and HEFLB had no effect or weak effects. These 9 antibodies are considered to be functional antibodies.

[0317]

[0318] 3.3 Binding specificity detection

[0319] The binding specificity of the purified hybridoma antibodies to SIRP family members was tested by ELISA assay using recombinant proteins of Fc-tagged human SIRPαv1 ECD, human SIRPαv2 ECD, human SIRPβ ECD, and human SIRPγ ECD. Briefly, the antibodies were incubated with antigens coated on ELISA microplates for 1 hour at 37°C. After washing, horseradish peroxidase (HRP)-labeled anti-mouse or anti-human IgG 2 nd Antibody (Sigma) was added and incubated at 37°C for 1 hour. Then, 100 μl / well of TMB solution (Biotechnology) was added. After incubation at room temperature for 15 minutes, the reaction was terminated by adding 50 μl of 1N HCl. OD450nm was read and EC was calculated. 50 . Table 7 summarizes the binding specificity of the 9 functional antibodies. Except for 060 and HEFLB, all the antibodies tested can bind to human SIRPαv1 and SIRPαv2. 060 and HEFLB can bind to human SIRPαv1, but not to v2. Except for 055, all the antibodies tested can bind to human SIRPβ. Compared with other known anti-SIRPα antibodies, only 022, 035 and 050 can bind to human SIRPγ weakly.

[0320] 3.4 Species cross-reactivity detection

[0321] The species cross-reactivity of purified hybridoma antibodies against human, cynomolgus monkey, and mouse SIRPα was tested by flow cytometry using CHOK1-human SIRPαv1-1B4 cells, CHOK1-cynomolgus monkey SIRPα-2A2 cells, and CHOK1-C57BL / 6 mouse SIRPα-2.22 cells that stably express SIRPα protein. 5 The target cells were incubated at 4°C for 1 hour. After washing, fluorescently labeled anti-mouse or anti-human IgG was added. nd Antibodies (Life Technologies) were added and incubated at 4°C for 1 hour. The geometric median of fluorescence intensity was measured and the EC 50 The species cross-reactivity of the nine functional antibodies is summarized in Table 7. Of particular note, compared with other antibodies tested in the same experiment, 060 failed to bind to cynomolgus monkey SIRPα, while 035 had cross-reactivity with C57BL / 6 mouse SIRPα.

[0322] 3.5 Blocking activity detection of CD47 / SIRPα and CD47 / SIRPγ interactions

[0323] Competition ELISA was used to determine whether the purified hybridoma antibodies could block the interaction between CD47 and SIRPα or the interaction between CD47 and SIRPγ. Briefly, to detect the blocking activity on the interaction between CD47 and SIRPα, the antibodies and biotinylated soluble SIRPαv1 ECD recombinant protein were incubated with ELISA microplates coated with human CD47 ECD recombinant protein.

[0324] To detect the blocking activity of the interaction between CD47 and SIRPγ, the antibody and biotin-labeled soluble human CD47 ECD recombinant protein were co-incubated with the ELISA microplate coated with human SIRPγECD recombinant protein. After washing, horseradish peroxidase-labeled streptavidin (HRP-SA, Sigma) was added and incubated at 37°C for 1 hour. Then, 100 μl / well of TMB solution (Biotechnology) was added. After incubation at room temperature for 15 minutes, the reaction was terminated by adding 50 μl of 1N HCl. Read OD 450nm. Calculate the blocking rate and IC 50 Table 7 summarizes the blocking activity of 9 functional antibodies on the interaction between CD47 and SIRPα and the interaction between CD47 and SIRPγ. Compared with other known anti-SIRPα antibodies, 022, 050, 055 and 074 cannot block the interaction between CD47 and SIRPα. In particular, all antibodies of the present invention cannot block the interaction between CD47 and SIRPγ.

[0325] 3.6 Hemagglutination activity

[0326] Anti-CD47 antibodies may promote hemagglutination of red blood cells (RBCs), which may lead to potential safety risks. The hemagglutination activity of purified hybridoma antibodies was tested. Briefly, human RBCs were diluted to 10% in PBS and incubated in 100 nM antibody for 1 hour at 37°C. Hemagglutination was demonstrated by the presence of unsettled RBCs, which appeared as a haze compared to the punctate red dots of unsettled RBCs. The hemagglutination index was determined by quantifying the area of ​​RBC particles in the presence of antibody and normalized to the area in the absence of antibody. As shown in Table 7, all 9 functional antibodies had no hemagglutination activity.

[0327] 3.7 Epitope Binning

[0328] The competitive ELISA method was used to group the epitopes of 9 functional antibodies. In brief, an excess of competitive antibodies and biotin-labeled soluble human SIRPαv1 ECD recombinant protein were incubated with the antibody-coated ELISA microplate. After washing, HRP-SA was added and incubated at 37°C for 1 hour. Then, 100μl / well of TMB solution (Biotechnology) was added. After incubation at room temperature for 15 minutes, the reaction was terminated by adding 50μl of 1N HCl. Read OD 450nm. Calculate the competition rate. Antibodies that can compete with each other for binding to SIRPα have similar binding epitopes.

[0329] As shown in Table 8, the nine anti-SIRPα antibodies belong to five different epitope groups. 001, 002, 032, and 035 belong to the same general category as the reference antibodies 29-AM4-5, KWAR23, and HEFLB, which are all blockers of the interaction between CD47 and SIRPα. Other blockers 060 and non-blockers 055, 074, 022, and 050 belong to four other different unique epitope groups.

[0330] Specifically, anti-SIRPα antibodies 001, 002, 032 and reference antibodies 29-AM4-5 and KWAR23 competed with each other for binding to human SIRPα, indicating that they may bind to the same or closely related epitope, which was classified as Ia, as shown in Table 7. Anti-SIRPα antibody 035 also competed with 001, 002, and 032 for binding to human SIRPα. However, 035 could not be completely competed by reference antibodies 29-AM4-5 and KWAR23, indicating that 035 may have a slightly different epitope, which was classified as Ib, as shown in Table 7. The competition between reference antibody HEFLB and anti-SIRPα antibodies 001, 002, 032, and 035 was not bidirectional. Therefore, the binding epitope of HEFLB was classified as Ic, as shown in Table 7. Ia, Ib, and Ic are considered to be a closely related large group I. Likewise, antibodies 022 and 050 competed with each other for binding to human SIRPα, indicating that they may bind to the same or closely related epitope, which was assigned as IV, as shown in Table 7. Antibodies 055, 074, and 060 did not show competitive binding to human SIRPα with any other antibodies in the assay, indicating that they may each bind to a different epitope, which were assigned as II, III, and V, respectively, as shown in Table 7.

[0331] 3.8 Hybridoma Sequencing

[0332] RNA isolated from monoclonal hybridoma cells was reverse transcribed into cDNA using the SMARTer RACE 5' / 3' kit (Clontech). The heavy and light chain variable regions were amplified using primers from the Mouse Ig-Primer Set (Novagen) using the cDNA as a template. PCR products were analyzed by agarose gel electrophoresis. DNA fragments of the correct size were collected, purified using NucleoSpin Gel and PCR Clear-up Kit (MACHEREY-NAGEL), and then ligated with pMD18-T vector (Takara). The ligated products were transformed into DH5α competent cells. Clones were screened and the inserts were analyzed by DNA sequencing.

[0333] Example 4. Production and characterization of chimeric antibodies

[0334] 4.1 Generation and production of chimeric antibodies

[0335] In order to verify the hybridoma sequencing results, the mouse antibody was converted into a human IgG4 chimeric antibody with an S228P mutation. In short, the DNA sequence encoding the heavy chain variable region was cloned into a pcDNA3.4-hIgG4P vector (Biointron) carrying a human IgG4 heavy chain constant region. The DNA sequence encoding the light chain variable region was cloned into a pcDNA3.4-hIgGk vector (Biointron) carrying a human kappa light chain constant region. The resulting chimeric antibodies are referred to herein as 001c, 002c, 022c, 032c, 035c, 050c, 055c, 060c and 074c, wherein the suffix "c" indicates chimeric.

[0336]

[0337] Expi293 cells (Life Technologies) co-transfected with antibody heavy and light chain expression plasmids were propagated at 37°C for 1 week. The culture medium was then collected and centrifuged to remove the cells. The collected supernatant was loaded onto a protein A affinity chromatography column (Nanomicrotech). The antibody was eluted with 0.1 M sodium citrate buffer (pH 3.4) and then immediately neutralized with Tris buffer (pH 8.0). After dialysis with PBS buffer, the antibody concentration was determined using Nano Drop (ThermoFisher). Protein purity was evaluated using SDS-PAGE and HPLC-SEC (Agilent). Endotoxin levels were detected using an Endochrome-K kit (Charles River).

[0338] 4.2 Characterization of chimeric antibodies

[0339] Purified chimeric antibodies were used for binding specificity analysis and species cross-reactivity analysis (see methods described in Examples 3.3 and 3.4). Figure 1 A to 1D show the binding specificity of anti-SIRPα chimeric antibodies to human SIRPαv1 ECD (A), human SIRPαv2 ECD (B), human SIRPβ ECD (C), and human SIRPγ ECD (D) recombinant proteins.

[0340] All nine chimeric antibodies tested showed subnanomolar EC binding to human SIRPαv1 ECD as measured by ELISA. 50 ( Figure 1 A, Table 9). Reference antibodies 29-AM4-5, KWAR23 and HEFLB also showed similar binding affinities.

[0341] Except for 060c and HEFLB, all other chimeric and reference antibodies showed subnanomolar EC values ​​for binding to human SIRPαv2 ECD as measured by ELISA. 50 ( Figure 1 B, Table 10).

[0342] Except for 055c, all other chimeric and reference antibodies showed subnanomolar EC binding to human SIRPβ ECD as measured by ELISA. 50 ( Figure 1 C, Table 11).

[0343] Table 9

[0344] Antibody <![CDATA[EC 50 (nM)]]> 29-AM4-5 0.11 KWAR23 0.10 HEFLB 0.11 001c 0.12 002c 0.06 022c 0.11 032c 0.12 035c 0.08 050c 0.11 055c 0.11 060c 0.08 074c 0.14

[0345] Table 10

[0346] Antibody <![CDATA[EC 50 (nM)]]> 29-AM4-5 0.08 KWAR23 0.09 HEFLB N / A 001c 0.09 002c 0.06 022c 0.11 032c 0.11 035c 0.08 050c 0.10 055c 0.11 060c N / A 074c 0.14

[0347] Table 11

[0348] Antibody <![CDATA[EC 50 (nM)]]> 29-AM4-5 0.08 KWAR23 0.08 HEFLB 0.08 001c 0.12 002c 0.11 022c 0.08 032c 0.12 035c 0.08 050c 0.08 055c N / A 060c 0.24 074c 0.11

[0349] As measured by ELISA, chimeric antibodies 001c, 002c, 032c, 055c, 060c, and 074c did not show specific binding to SIRPγ ECD ( Figure 1 D, Table 12). As measured by ELISA, chimeric antibodies 022c, 035c and 050c, similar to reference antibodies 29-AM4-5, KWAR23 and HEFLB, all showed specific binding to human SIRPγ ECD ( Figure 1 D, Table 12).

[0350] Table 12

[0351] Antibody <![CDATA[EC 50 (nM)]]> 29-AM4-5 0.11 KWAR23 0.05 HEFLB 15.52 001c N / A 002c N / A 022c 18.73 032c N / A 035c 6.11 050c 0.27 055c N / A 060c N / A 074c N / A

[0352] Figure 2 A to 2C show the species cross-reactivity of anti-SIRPα chimeric antibodies. Figure 2 A shows the FACS binding curve of the antibody against CHOK1-human SIRPαv1-1B4 cells. Figure 2 B and 2C show FACS binding of 10 nM antibody against CHOK1-cynomolgus monkey SIRPα-2A2 cells and CHOK1-C57BL / 6 mouse SIRPα-2.22 cells.

[0353] All nine chimeric antibodies tested showed subnanomolar EC binding to CHOK1-human SIRPαv1-1B4 cells as measured by FACS. 50 ( Figure 2 A, Table 13). Reference antibodies 29-AM4-5, KWAR23 and HEFLB also showed similar binding affinities.

[0354] Table 13

[0355] Antibody <![CDATA[EC 50 (nM)]]> 29-AM4-5 13.6 KWAR23 2.8 HEFLB 11.2 001c 1.7 002c 2.4 022c 1.7 032c 3.0 035c 1.3 050c 2.4 055c 3.4 060c 2.8 074c 5.5

[0356] like Figure 2 As shown in B, the results showed that except for 060c, all antibodies (i.e., 001c, 002c, 022c, 032c, 035c, 050c, 055c, and 074c) had good cross-reactivity to cynomolgus monkey SIRPα. Figure 2 As shown in C, only 035c has cross-reactivity with SIRPα of C57BL / 6 mice.

[0357] The purified chimeric antibodies were also tested in a phagocytosis assay (see the method described in Example 3.2). Figure 3 A to 3D show phagocytosis of Jurkat cells, Raji cells, and DLD-1 cells by human macrophages in the presence of the indicated anti-SIRPα antibodies (human IgG4 chimeric antibodies with S228P mutation).

[0358] like Figure 3 As shown in A to 3D, when used alone, the nine chimeric antibodies stimulated macrophages to attack Jurkat cells ( Figure 3 A, 3D), Raji cells ( Figure 3 B) and DLD-1 cells ( Figure 3 C) showed a strong phagocytic effect in a dose-dependent manner, while reference antibodies 29-AM4-5, KWAR23, and HEFLB had no effect or a weak effect.

[0359] We hypothesized that anti-SIRPα chimeric antibodies could block the interaction between CD47 and SIRPα by binding to the SIRPα IgV domain, which is a key region for SIRPα to interact with CD47. To demonstrate our hypothesis, we examined the binding of anti-SIRPα chimeric antibodies to primary monocytes derived from B-hSIRPA mice (Biocytogen) by FACS ( Figure 4 B).

[0360] like Figure 4As shown in A, exon 2 of the B-hSIRPA mouse Sirpa gene encoding the SIRPα IgV domain that interacts with CD47 was humanized. The humanized mice expressed chimeric SIRPα, including the IgV domain and IgC1 / C2 of human SIRPα, the transmembrane domain and the intracellular domain of mouse SIRPα. Briefly, spleen cells from B-hSIRPA mice were taken and incubated with anti-SIRPα chimeric antibodies at 4°C for 1 hour. After washing, fluorescently labeled anti-human IgG 2 was added. nd Antibodies (Life Technologies) were added and incubated for 1 hour at 4°C. Mouse CD11b and F4 / 80 were also stained to visualize monocytes. The anti-SIRPα positive staining population in the mCD11b and mF4 / 80 double positive subset was counted.

[0361] like Figure 4 As shown in B, the blockers of CD47 and SIRPα interaction 001c, 002c, 032c, 035c, and 060c can bind to primary monocytes derived from B-hSIRPA mice, indicating that they bind to the human SIRPα IgV domain. However, this is not the case for the non-blockers of CD47 and SIRPα interaction 022c, 050c, 055c, and 074c.

[0362] All these characterization data are consistent with the results we obtained from hybridoma antibodies, indicating that the variable region sequences obtained are correct. Table 14 summarizes the characterization data.

[0363]

[0364] 4.3 Binding affinity determined by surface plasmon resonance (SPR)

[0365] Biacore (GE) was used to characterize the binding affinity of anti-SIRPα chimeric antibodies to human SIRPαv1, human SIRPαv2 and C57BL / 6 mouse SIRPα. In brief, the antibodies to be tested were captured into a CM5 chip (GE) using the Human Antibody Capture kit (GE). The antigens of 6xHis-labeled human SIRPαv1, human SIRPαv2 and C57BL / 6 mouse SIRPαECD recombinant proteins were serially diluted in multiple doses and injected at a rate of 30μl / min for 180 seconds. The buffer flow was maintained for 400 seconds of dissociation. The chip was regenerated with 3MMgCl2. The binding curves and dissociation curves were fitted with a 1:1 binding model, and the Ka / Kd / KD values ​​of each antibody were calculated. Tables 15 and 14 summarize the affinity data of anti-SIRPα chimeric antibodies.

[0366] Example 5. Antibody humanization and affinity maturation

[0367] 5.1 Humanization

[0368] The heavy chain and light chain variable region sequences of the 035 antibody were retrieved in the human antibody sequence database. Based on the homology with the original mouse antibody sequence, VH7-4-1 and VK1-16 were selected as humanization templates. The CDRs in the mouse antibody sequence were then grafted onto the template together with the residues to maintain the upper core and central core structure of the antibody. The obtained 035 humanized antibody was named hu035.01, the prefix "hu" means "humanized", and the number in the suffix represents the sequence number of the humanized antibody.

[0369]

[0370] 5.2 Characterization of humanized antibodies

[0371] hu035.01, which is the first version of humanized 035, was characterized by FACS analysis using CHOCK1-human SIRPαv1-1B4 cells, ELISA analysis using Fc-tagged human SIRPαv2 ECD recombinant protein, and SPR analysis using antigen of 6xHis-tagged human SIRPαv2 ECD recombinant protein (refer to the methods described in Examples 3.4, 3.3, and 4.3). Compared with the parent antibody 035c, humanized hu035.01 showed relatively weak binding to CHOK1-human SIRPαv1-1B4 cells in FACS analysis ( Figure 5 A), showed relatively weak binding to human SIRPαv2 ECD recombinant protein in ELISA analysis ( Figure 5 B). SPR analysis using human SIRPαv2 ECD recombinant protein antigen confirmed that the binding affinity of hu035.01 (53.4 nM) was lower than that of 035c (0.61 nM) ( Figure 5 C). In particular, the binding of hu035.01 to SIRPα ECD of C57BL / 6 mice could not be detected in the ELISA assay, probably due to reduced binding activity ( Figure 5 B).

[0372] 5.3 Affinity Maturation

[0373] Due to the reduced binding affinity, hu035.01 was optimized by affinity maturation. In brief, affinity maturation of the first CDR-grafted sequence was completed by random mutation of the heavy and light chains in the scFV single-chain antibody format and screening for better binders to human SIRPα and / or mouse SIRPα. The best binders were sequenced and cloned into a mammalian expression vector, expressed in ExpiCHO cells and purified for further characterization. The humanized antibodies obtained after affinity maturation were named hu035.02, hu035.03, and up to hu035.17, where the prefix "hu" means "humanized" and the number in the suffix represents the sequence number of the humanized antibody.

[0374] 5.4 Characterization of humanized antibodies after affinity maturation

[0375] Finally, 7 humanized mature candidates (named hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17, respectively) were selected for binding specificity analysis and species cross-reactivity analysis (see the methods described in Examples 3.3 and 3.4).

[0376] In the ELISA assay, the optimized hu035 candidate showed a higher affinity for human SIRPαv1 ECD ( Figure 6 A), human SIRPαv2 ECD ( Figure 6 B) Human SIRPαv8 ECD ( Figure 6 C) and human SIRPβ ECD ( Figure 6 D) of the recombinant proteins maintained comparable binding capacity. In particular, in ELISA assays, they were able to bind to human SIRPγECD ( Figure 6 E) and C57BL / 6 mouse SIRPαECD ( Figure 6 F) have different degrees of enhanced binding. Table 17 calculates and summarizes the EC 50 value.

[0377] The optimized hu035 candidate also maintained affinity with human SIRPα ( Figure 7 A), cynomolgus monkey SIRPα ( Figure 7 B) and C57BL / 6 mouse SIRPα ( Figure 7 C) with comparable species cross-reactivity. Consistent with the data obtained from the ELISA assay, they all showed enhanced binding to CHOK1-C57BL / 6 mouse SIRPα-2.22 cells at different levels ( Figure 7 C) EC is calculated and summarized in Table 17 50 value.

[0378] The optimized hu035 candidate was tested for its ability to block the interaction between CD47 and SIRPα ( Figure 8 Compared with the parent antibody 035c, the optimized hu035 candidates hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17 were confirmed to maintain comparable blocking activity on the interaction between CD47 and SIRPα. Table 17 calculates and summarizes the IC 50 value.

[0379] It was further confirmed by SPR analysis that the optimized hu035 candidates hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17 showed comparable binding affinity to human SIRPα alleles and improved binding affinity to C57BL / 6 mouse SIRPα compared to the parent antibody 035c (see the method described in Example 4.3). Tables 16 and 17 summarize the kinetic data.

[0380]

[0381] The optimized hu035 candidates were also tested in a phagocytosis assay for functional evaluation (see the method described in Example 3.2). Fig. 9 As shown, compared with the parent antibody 035c, the optimized hu035 candidates hu035.02, hu035.03, hu035.09, hu035.10, hu035.13, hu035.14 and hu035.17 were able to stimulate macrophages to attack Jurkat cells ( Fig. 9 A), DLD1 cells ( Fig. 9 B) and Raji cells ( Fig. 9 C) produced stronger or comparable phagocytosis, whereas two known anti-SIRPα antibodies, ALX H21 and 3F9-22, showed no or weaker effects.

[0382] It has been reported that human T cells co-stimulate T cell proliferation by adhering to antigen-presenting cells through SIRPγ-CD47 interaction. As these 7 optimized hu035 candidates showed enhanced binding activity to human SIRPγ compared to the parent antibody 035c ( Figure 6 E) to exclude the possibility that disruptions interfered with T cell proliferation, these optimized hu035 candidates as well as some chimeric antibodies were tested in a T cell activation assay. TMPrimary human T cells labeled with CellTrace Violet (Life Technologies) were stimulated for 4 days with human CD3 / CD28 T cell activator (STEMCELL) or allogeneic mature dendritic cells cultured in vitro for 5 days. The indicated antibodies were added at saturating concentrations (10 ug / ml) from the beginning of the assay. Proliferating populations were determined using low-staining CellTrace Violet. IFNγ secretion was detected using the Human IFNγ Kit (Cisbio).

[0383] like Fig.10 As shown, when T cells were stimulated with a CD3 / CD28 T cell activator, the optimized hu035 candidates hu035.02, hu035.03, hu035.09, hu035.10, hu035.14 and hu035.17 and chimeric antibodies 035c, 022c, 032c, 050c, 055c, 060c and 074c had an inhibitory effect on CD4 + T cell proliferation ( Fig.10 B, 10D), CD8 + T cell proliferation ( Fig.10 C, 10D) had no negative effect; when T cells were stimulated with CD3 / CD28 T cell activators, the optimized hu035 candidates hu035.02 and hu035.17 and chimeric antibodies 022c, 032c, 035c, 050c, 055c, 060c and 074c had no negative effect on the secretion of IFNγ ( Fig.10 A). Similarly, when allogeneic dendritic cells were used to stimulate T cells, the optimized hu035 candidates hu035.02, hu035.17 and chimeric antibodies 035c, 022c, 032c, 050c, 055c, 060c and 074c had no significant effects on CD4 + T cell proliferation ( Fig.11 B) CD8 + T cell proliferation ( Fig.11 C) and IFNγ secretion ( Fig.11 A) No negative effects. As expected, the anti-SIRPγ antibody LSR2.20 (Biolegend) is an inhibitor of T cell activation.

[0384] Table 17 summarizes all characterization data to demonstrate successful humanization and affinity maturation.

[0385]

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to human SIRPα, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: a) the amino acid sequence of HCDR1 is shown in SEQ ID NO:43, the amino acid sequence of HCDR2 is shown in SEQ ID NO:50, the amino acid sequence of HCDR3 is shown in SEQ ID NO:17, the amino acid sequence of LCDR1 is shown in SEQ ID NO:53, the amino acid sequence of LCDR2 is shown in SEQ ID NO:57, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:38; or b) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:43, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:51, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:17, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:54, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:57, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:38; or c) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:45, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:52, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:17, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:54, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:57, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:38; or d) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:45, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:52, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:17, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:54, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:58, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:38; or e) the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 43, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 52, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 17, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 54, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 58, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

38.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region is composed of a sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, and homologous sequences having at least 90% sequence identity thereto but still maintaining specific binding affinity to human SIRPα.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region is composed of a sequence selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and homologous sequences having at least 90% sequence identity thereto but still maintaining specific binding affinity to human SIRPα.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein a) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 63, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 77; or b) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 64, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 78; or c) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 79; or d) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 80; or e) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 81; or f) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 82; or g) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 67, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 83; or h) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 82; or i) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

84.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, further comprising an Fc region. 6 . The antibody or antigen-binding fragment thereof according to claim 5 , wherein the Fc region comprises the Fc region of human immunoglobulin (Ig).

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the Fc region comprises the Fc region of human IgG. The antibody or antigen-binding fragment thereof according to claim 5 , wherein the Fc region is derived from human IgG4. 9 . The antibody or antigen-binding fragment thereof according to claim 8 , wherein the Fc region derived from human IgG4 comprises an S228P mutation and / or an L235E mutation.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is humanized.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is a monoclonal antibody, a recombinant antibody or a labeled antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is a multispecific antibody.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is a bispecific antibody.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is a chimeric antibody.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-4 and 6-9, which is a fusion protein.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is a bivalent antibody.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, 6-9, which is a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, single-chain antibody molecule (scFv) or multispecific antibody.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, 6-9, which is a bispecific dsFv, a disulfide-bond stabilized bifunctional antibody or a scFv dimer.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 6 to 9, which is a bivalent, bifunctional antibody.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, 6-9, which has one or more binding properties to human SIRPα selected from the group consisting of: a) The binding affinity to human SIRPα is no more than 10 -7 M, the binding affinity to human SIRPα was determined by Biacore detection method, b) with an EC no greater than 1 nM 50 It specifically binds to the extracellular domain (ECD) of human SIRPα v1. 50 Determined by ELISA, c) with an EC no greater than 1 nM 50 Specifically binds to human SIRPα v2 ECD, which 50 Determined by ELISA assay.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, 6-9, which has one or more binding properties selected from the group consisting of: a) with an EC no greater than 50 nM 50 Binding to SIRPγ ECD, the EC 50 Determined by ELISA, b) with an EC no greater than 1 nM 50 Binding to SIRPβ ECD, the EC 50 Determined by ELISA, c) specific binding to the human SIRPα IgV domain as detected by FACS assay, d) not more than 10 -5 The binding affinity of M specifically binds to mouse SIRPα, as determined by Biacore assay. e) specifically binds to cynomolgus monkey SIRPα at a concentration of 10 nM as determined by FACS detection, f) capable of inducing phagocytosis of target cells expressing CD47 by macrophages at a concentration of 10 nM as measured by a phagocytosis assay; and g) No reduction in CD4 + T cells or CD8 + Proliferation of T cells. 22 . The antibody or antigen-binding fragment thereof according to claim 13 , which is capable of specifically binding to a second antigen other than SIRPα. 23 . The antibody or antigen-binding fragment thereof according to claim 13 , which is capable of specifically binding to a second epitope on SIRPα.

24. The antibody or antigen-binding fragment thereof of claim 22, wherein the second antigen is selected from the group consisting of CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), GPC-3, B7-H3, B7-H4, TROP2, CLDN18.2, EGFR, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

25. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, 6-9, linked to one or more conjugate moieties.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent, a purification moiety or other anti-cancer drug.

27. The antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate moiety comprises a fluorescent label.

28. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the preceding claims and one or more pharmaceutically acceptable carriers.

29. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-27.

30. A vector comprising the isolated polynucleotide according to claim 29.

31. A host cell comprising the vector according to claim 30.

32. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and / or the pharmaceutical composition according to claim 28, and a second therapeutic agent.

33. A method for expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, comprising culturing the host cell according to claim 31 under conditions that express the isolated polynucleotide comprised by the vector according to claim 30.

34. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-27 and / or a pharmaceutical composition according to claim 28 in the preparation of a medicament for treating, preventing or alleviating a disease, disorder or condition associated with SIRPα in a subject, wherein the treatment, prevention or alleviating comprises administering a therapeutically effective amount of the medicament to the subject, wherein the disease, disorder or condition is cancer, wherein the cancer is gastric cancer, lung cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, kidney cancer, bladder cancer, head and neck cancer, endometrial cancer, colorectal cancer, glioblastoma, melanoma, myelodysplastic syndrome, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma or non-Hodgkin lymphoma.

35. The use according to claim 34, wherein the cancer is a CD47 positive cancer.

36. The use according to claim 34, wherein the subject is a human.

37. The use according to claim 34, wherein the administration is via intravenous, subcutaneous or intramuscular administration.

38. The use of claim 34, further comprising administering a therapeutically effective amount of a second therapeutic agent.

39. The use of claim 38, wherein the second therapeutic agent is selected from the group consisting of anticancer drugs, immunotherapeutics, anti-angiogenic agents, targeted therapeutics, cell therapeutics, gene therapeutics, hormone therapeutics, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

40. The use according to claim 39, wherein the anticancer drug is a chemotherapeutic agent or a radiotherapeutic agent.

41. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-27 and / or the pharmaceutical composition according to claim 28 in the preparation of a diagnostic reagent for detecting the presence or content of SIRPα in a sample, wherein the detection comprises contacting the sample with the diagnostic reagent and determining the presence or content of SIRPα in the sample.

42. A kit for detecting SIRPα, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 and / or the pharmaceutical composition according to claim 28.

43. A method for inducing phagocytosis in vitro, comprising contacting target cells with a SIRPα-positive phagocyte sample in the presence of an antibody or antigen-binding fragment thereof according to any one of claims 1-27 and / or a pharmaceutical composition according to claim 28, thereby inducing the phagocytosis of the target cells by the SIRPα-positive phagocytes, wherein the target cells are cells expressing CD47.

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