Novel anti-SIRPA antibodies

Abstract

The present disclosure provides anti-SIRPα antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding them, pharmaceutical compositions comprising them, and uses thereof.

Description

[Technical field]

[0001]

[0001] The present disclosure relates generally to novel anti-SIRPα antibodies. [Background technology]

[0002] Signal regulatory protein alpha (SIRPα) is an inhibitory receptor that is expressed primarily on myeloid and dendritic cells. In addition to SIRPα, the SIRP family also includes several other transmembrane glycoproteins, including SIRPβ and SIRPγ. Each member of the SIRP family contains three similar extracellular Ig-like domains with distinct transmembrane and cytoplasmic domains. CD47 is a widely expressed transmembrane glycoprotein with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. CD47 serves as a cellular ligand for SIRPα. Binding of CD47 to SIRPα delivers a "don't eat me" signal that inhibits phagocytosis, and blocking CD47-mediated engagement of SIRPα on phagocytes can cause removal of live cells bearing the "eat me" signal. Tumor cells often overexpress CD47 to avoid macrophage-mediated destruction. The interaction of CD47 with SIRPα has been shown to be involved in the regulation of macrophage-mediated phagocytosis (Takenaka et al., Nature Immunol., 8(12):1313-1323, 2007). In a wide range of preclinical models, therapies that block the interaction of CD47 with SIRPα stimulate phagocytosis of cancer cells in vitro and antitumor immune responses in vivo. Currently, several drugs targeting CD47 (anti-CD47 antibodies and SIRPα fusion proteins) are progressing into clinical trials. However, these drugs are associated with hemolytic anemia and thrombocytopenia. In addition to safety issues, the ubiquitous expression of CD47 may also cause antigen sink, which results in reduced efficacy. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0003] There remains a need for new anti-SIRPα antibodies. [Means for solving the problem]

[0004]

[0004] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or two or more antibodies.

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to human SIRPα, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and / or a light chain variable region comprising LCDR1, LCDR2, and LCDR3; a) HCDR1 comprises the amino acid sequence of DYYMS (SEQ ID NO: 1); and / or HCDR2 comprises the amino acid sequence of FIKNEANGYTTESSASVKG (SEQ ID NO: 2); and / or HCDR3 comprises the amino acid sequence YDYYGSNYNWYFDA (SEQ ID NO: 3); and / or LCDR1 comprises the amino acid sequence of KASQNVRTAVA (SEQ ID NO: 4); and / or LCDR2 comprises the amino acid sequence of LASKRHT (SEQ ID NO:5); and / or LCDR3 comprises the amino acid sequence of LQHWIHPLT (SEQ ID NO:6); b) HCDR1 is X and / or comprising the amino acid sequence of 1YYMH (SEQ ID NO: 18); HCDR2 is RIDPED X 2E X and / or comprising the amino acid sequence of 3KYAPKFQG (SEQ ID NO: 19); HCDR3 is GX 18 X 4 X and / or comprising the amino acid sequence of 5Y (SEQ ID NO: 20); LCDR1 comprises the amino acid sequence of SASSSVSSSYLY (SEQ ID NO: 10); and / or LCDR2 comprises the amino acid sequence of STSNLAS (SEQ ID NO: 11); and / or LCDR3, X 6QWSSYPYT (SEQ ID NO: 21), c) HCDR1 comprises the amino acid sequence of TYGMS (SEQ ID NO: 22); and / or HCDR2, WINTYSGV X 19 T X 7ADDF X 8G (SEQ ID NO: 38), and / or HCDR3, DPH X 9YG X 10 SPAWF X 11 and / or LCDR1, X 12 ASQ X 13 VGI X 14 and / or comprising the amino acid sequence of VA (SEQ ID NO: 40); LCDR2, SASNR X 15 and / or LCDR3, QQYS X 16 YP X 17 T (SEQ ID NO: 42), d) HCDR1 comprises the amino acid sequence of EYVLS (SEQ ID NO: 43); and / or HCDR2 comprises the amino acid sequence EIYPGTITTYYNEKFKG (SEQ ID NO: 44); and / or HCDR3 comprises the amino acid sequence FYDYDGGWFAY (SEQ ID NO: 45); and / or LCDR1 comprises the amino acid sequence of SASSSVSSSDLH (SEQ ID NO: 46); and / or LCDR2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 47); and / or LCDR3 comprises the amino acid sequence of QQWSGYPWT (SEQ ID NO: 48); X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, X6 is Y or H, X7 is Y or C, X8 is K or Q, X9 is Y or S, and X 10 is N or T or S, and X 11 is P or A or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is Y or F and X 16 is S or T or A, and X 17 is F or L, and X 18 is S or absent, and X 19 is S or P; Antibodies or antigen-binding fragments thereof are provided.

[0006] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a) an HCDR1 comprising the amino acid sequence X1YYMH (SEQ ID NO: 18), and / or b) an HCDR2 comprising the amino acid sequence of RIDPEDX2EX3KYAPKFQG (SEQ ID NO: 19), and / or c) GX 18 HCDR3 comprising the amino acid sequence X4X5Y (SEQ ID NO: 20), and / or d) LCDR1 comprising the amino acid sequence of SASSSVSSSYLY (SEQ ID NO: 10), and / or e) LCDR2 comprising the amino acid sequence of STSNLAS (SEQ ID NO: 11), and / or f) LCDR3 comprising the amino acid sequence of X6QWSSYPYT (SEQ ID NO: 21) X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, X6 is Y or H, 18 is S or absent.

[0007] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a) an HCDR1 comprising the amino acid sequence AYYMH (SEQ ID NO: 7) or DYYMH (SEQ ID NO: 13), and / or b) an HCDR2 comprising an amino acid sequence selected from the group consisting of RIDPEDGESKYAPKFQG (SEQ ID NO: 8), RIDPEDGETKYAPKFQG (SEQ ID NO: 14), and RIDPEDAETKYAPKFQG (SEQ ID NO: 17); and / or c) an HCDR3 comprising the amino acid sequence of GSYEY (SEQ ID NO: 9) or GLAY (SEQ ID NO: 15); and / or d) LCDR1 comprising the amino acid sequence of SASSSVSSSYLY (SEQ ID NO: 10), and / or e) LCDR2 comprising the amino acid sequence of STSNLAS (SEQ ID NO: 11), and / or f) LCDR3 comprising the amino acid sequence YQWSSYPYT (SEQ ID NO: 12) or HQWSSYPYT (SEQ ID NO: 16) Includes.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a) HCDR1 comprising the amino acid sequence of TYGMS (SEQ ID NO: 22), and / or b)WINTYSGVX 19 HCDR2 comprising the amino acid sequence of TX7ADDFX8G (SEQ ID NO: 38), and / or c) DPHX9YGX 10 SPAWFX 11 and / or an HCDR3 comprising the amino acid sequence of Y (SEQ ID NO: 39); d) X 12 ASQX 13 VGIX 14 LCDR1 comprising the amino acid sequence of VA (SEQ ID NO: 40), and / or e) SASNRX 15 LCDR2 comprising the amino acid sequence of T (SEQ ID NO: 41), and / or f) QQYSX 16 YPX 17 LCDR3 comprising the amino acid sequence of T (SEQ ID NO: 42) X7 is Y or C, X8 is K or Q, X9 is Y or S, and X 10 is N or T or S, and X 11 is P or A or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is Y or F, and X 16 is S or T or A, and X 17 is F or L, and X 19 is S or P.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a) HCDR1 comprising the amino acid sequence of TYGMS (SEQ ID NO: 22), and / or b) an HCDR2 comprising an amino acid sequence selected from the group consisting of WINTYSGVSTCADDFKG (SEQ ID NO: 23), WINTYSGVPTYADDFQG (SEQ ID NO: 28), and WINTYSGVPTYADDFKG (SEQ ID NO: 33), and / or c) an HCDR3 comprising an amino acid sequence selected from the group consisting of DPHSYGNSPAWFPY (SEQ ID NO: 24), DPHYYGTSPAWFAY (SEQ ID NO: 29), and DPHYYGSSPAWFVY (SEQ ID NO: 34), and / or d) an LCDR1 comprising an amino acid sequence selected from the group consisting of KASQNVGISVA (SEQ ID NO: 25), KASQIVGIAVA (SEQ ID NO: 30), and EASQIVGIAVA (SEQ ID NO: 35); and / or e) an LCDR2 comprising an amino acid sequence selected from the group consisting of SASNRYT (SEQ ID NO: 26) and SASNRFT (SEQ ID NO: 31); and / or f) LCDR3 comprising an amino acid sequence selected from the group consisting of QQYSSYPLT (SEQ ID NO: 27), QQYSTYPFT (SEQ ID NO: 32), and QQYSAYPFT (SEQ ID NO: 37). Includes.

[0010] In certain embodiments, the heavy chain variable region comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 2, and an HCDR3 comprising the sequence of SEQ ID NO: 3, or b) an HCDR1 comprising the sequence of SEQ ID NO: 7, an HCDR2 comprising the sequence of SEQ ID NO: 8, and an HCDR3 comprising the sequence of SEQ ID NO: 9, or c) an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 17, and an HCDR3 comprising the sequence of SEQ ID NO: 15, or d) HCDR1 comprising the sequence of SEQ ID NO: 22, HCDR2 comprising the sequence of SEQ ID NO: 23, and HCDR3 comprising the sequence of SEQ ID NO: 24, or e) an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 28, and an HCDR3 comprising the sequence of SEQ ID NO: 29, or f) an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 33, and an HCDR3 comprising the sequence of SEQ ID NO: 34, or g) HCDR1 comprising the sequence of SEQ ID NO: 43, HCDR2 comprising the sequence of SEQ ID NO: 44, and HCDR3 comprising the sequence of SEQ ID NO: 45 Includes.

[0011] In certain embodiments, the light chain variable region comprises: a) an LCDR1 comprising the sequence of SEQ ID NO: 4, an LCDR2 comprising the sequence of SEQ ID NO: 5, and an LCDR3 comprising the sequence of SEQ ID NO: 6, or b) an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 11, and an LCDR3 comprising the sequence of SEQ ID NO: 12, or c) an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 11, and an LCDR3 comprising the sequence of SEQ ID NO: 16, or d) an LCDR1 comprising the sequence of SEQ ID NO: 25, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 27, or e) an LCDR1 comprising the sequence of SEQ ID NO: 30, an LCDR2 comprising the sequence of SEQ ID NO: 31, and an LCDR3 comprising the sequence of SEQ ID NO: 32, or f) an LCDR1 comprising the sequence of SEQ ID NO: 35, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 37, or g) LCDR1 comprising the sequence of SEQ ID NO: 46, LCDR2 comprising the sequence of SEQ ID NO: 47, and LCDR3 comprising the sequence of SEQ ID NO: 48 Includes.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 2, and an HCDR3 comprising the sequence of SEQ ID NO: 3, an LCDR1 comprising the sequence of SEQ ID NO: 4, an LCDR2 comprising the sequence of SEQ ID NO: 5, and an LCDR3 comprising the sequence of SEQ ID NO: 6, or b) an HCDR1 comprising the sequence of SEQ ID NO: 7, an HCDR2 comprising the sequence of SEQ ID NO: 8, and an HCDR3 comprising the sequence of SEQ ID NO: 9, an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 11, and an LCDR3 comprising the sequence of SEQ ID NO: 12, or c) an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 17, and an HCDR3 comprising the sequence of SEQ ID NO: 15, an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 11, and an LCDR3 comprising the sequence of SEQ ID NO: 16, or d) an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 23, and an HCDR3 comprising the sequence of SEQ ID NO: 24, an LCDR1 comprising the sequence of SEQ ID NO: 25, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 27, or e) an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 28, and an HCDR3 comprising the sequence of SEQ ID NO: 29, an LCDR1 comprising the sequence of SEQ ID NO: 30, an LCDR2 comprising the sequence of SEQ ID NO: 31, and an LCDR3 comprising the sequence of SEQ ID NO: 32, or f) an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 33, and an HCDR3 comprising the sequence of SEQ ID NO: 34, an LCDR1 comprising the sequence of SEQ ID NO: 35, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 37, or g) HCDR1 comprising the sequence of SEQ ID NO: 43, HCDR2 comprising the sequence of SEQ ID NO: 44, and HCDR3 comprising the sequence of SEQ ID NO: 45, LCDR1 comprising the sequence of SEQ ID NO: 46, LCDR2 comprising the sequence of SEQ ID NO: 47, and LCDR3 comprising the sequence of SEQ ID NO: 48. Includes.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more of heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4, wherein: a) HFR1 is EVQLVQSGAEVKKPGATVKISCKX 20 and / or comprising SGFNIK (SEQ ID NO: 84) or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 74) or a homologous sequence having at least 80% sequence identity thereto; and / or c) The HFR3 sequence is RVTITADTSTX 21 TAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 85) or a homologous sequence having at least 80% sequence identity thereto; and / or d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 76) or a homologous sequence having at least 80% sequence identity thereto; and / or e) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 77) or a homologous sequence having at least 80% sequence identity thereto; and / or f) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 78) or a homologous sequence having at least 80% sequence identity thereto; and / or g) LFR3 is GIPARFSGSGSGTDX 22 TLTISSLEPEDFAVYYC (SEQ ID NO: 86) or a homologous sequence having at least 80% sequence identity thereto; and / or h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 80) or a homologous sequence having at least 80% sequence identity thereto; X 20 is A or V, and X 21 is N or D, and X 22 is Y or F.

[0014] In one particular embodiment, a) HFR1 comprises EVQLVQSGAEVKKPGATVKISCKASGFNIK (SEQ ID NO: 83) or EVQLVQSGAEVKKPGATVKISCKVSGFNIK (SEQ ID NO: 73) or a homologous sequence having at least 80% sequence identity thereto; and / or b) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 74) or a homologous sequence having at least 80% sequence identity thereto; and / or c) the HFR3 sequence comprises RVTITADTSTNTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 75) or RVTITADTSTDTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 82) or a homologous sequence having at least 80% sequence identity thereto; and / or d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 76) or a homologous sequence having at least 80% sequence identity thereto; and / or e) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 77) or a homologous sequence having at least 80% sequence identity thereto; and / or f) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 78) or a homologous sequence having at least 80% sequence identity thereto; and / or g) LFR3 comprises GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO: 79) or GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto; and / or h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 80) or a homologous sequence having at least 80% sequence identity thereto.

[0015]

[0015] In certain embodiments, the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:63, SEQ ID NO:65, and SEQ ID NO:67, as well as homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity for human SIRPα.

[0016]

[0016] In certain embodiments, the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 64, and SEQ ID NO: 66, and homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity for human SIRPα.

[0017] In one particular embodiment, a) the heavy chain variable region comprises the sequence of SEQ ID NO: 49 and the light chain variable region comprises the sequence of SEQ ID NO: 50, or b) the heavy chain variable region comprises the sequence of SEQ ID NO: 51 and the light chain variable region comprises the sequence of SEQ ID NO: 52; or c) the heavy chain variable region comprises the sequence of SEQ ID NO: 53 and the light chain variable region comprises the sequence of SEQ ID NO: 54; or d) the heavy chain variable region comprises the sequence of SEQ ID NO: 55 and the light chain variable region comprises the sequence of SEQ ID NO: 56; or e) the heavy chain variable region comprises the sequence of SEQ ID NO: 57 and the light chain variable region comprises the sequence of SEQ ID NO: 58; or f) the heavy chain variable region comprises the sequence of SEQ ID NO: 59 and the light chain variable region comprises the sequence of SEQ ID NO: 60; or g) the heavy chain variable region comprises the sequence of SEQ ID NO: 61 and the light chain variable region comprises the sequence of SEQ ID NO: 62; or h) the heavy chain variable region comprises the sequence of SEQ ID NO: 63 and the light chain variable region comprises the sequence of SEQ ID NO: 64; or i) the heavy chain variable region comprises the sequence of SEQ ID NO: 63 and the light chain variable region comprises the sequence of SEQ ID NO: 66; or j) the heavy chain variable region comprises the sequence of SEQ ID NO: 65 and the light chain variable region comprises the sequence of SEQ ID NO: 64; or k) the heavy chain variable region comprises the sequence of SEQ ID NO: 67 and the light chain variable region comprises the sequence of SEQ ID NO: 64; or l) the heavy chain variable region comprises the sequence of SEQ ID NO:67 and the light chain variable region comprises the sequence of SEQ ID NO:66.

[0018]

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises a substitution or modification of one or more amino acid residues and still retains specific binding affinity for human SIRPα.

[0019]

[0019] In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of the heavy chain variable region or the light chain variable region and / or in one or more of the non-CDR sequences.

[0020]

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

[0021]

[0021] In certain embodiments, the Fc region is derived from human IgG4.

[0022] In certain embodiments, the Fc region derived from human IgG4 comprises an S228P mutation and / or an L235E mutation.

[0022]

[0023] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are humanized.

[0024] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein 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.

[0023]

[0025] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is a diabody, a Fab, a Fab', a F(ab')2, a Fd, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), a multispecific antibody, a camelized single-chain domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.

[0024]

[0026] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are a) the interaction between SIRP-alpha v1 and CD47 can be completely blocked; b) capable of blocking the interaction between SIRP-alpha v1 and CD47 with an IC50 of 10 nM or less (or 5 nM or less) as measured by competitive ELISA, or with an IC50 of 0.6 nM or less (or 0.5 nM or less) as measured by competitive FACS; c) being able to completely block the interaction between SIRP-alpha v2 and CD47; d) capable of blocking the interaction between SIRP-alpha v2 and CD47 with an IC50 of 10 nM or less (or 5 nM or less) as measured by competitive ELISA, or with an IC50 of 0.8 nM or less (or 0.7 nM or less) as measured by competitive FACS; e) IFNγ secretion by T cells, CD4 + T cell proliferation, or CD8 + having no significant inhibition of T cell proliferation; f) being able to block CD47-mediated recruitment of SHP1 to SIRP alpha; g) It can increase the antibody-dependent cellular phagocytosis (ADCP) effect of the target antibody; h) capable of binding to an epitope comprising an amino acid sequence selected from the group consisting of YNQKEGHFPRVTTVSDL (SEQ ID NO: 36), SGAGTEL (SEQ ID NO: 72), TNVDPVGESVS (SEQ ID NO: 87), and TNVDPVGESVSY (SEQ ID NO: 90); The compound has one or more properties selected from the group consisting of:

[0025]

[0027] In another aspect, the disclosure also provides an antibody or antigen-binding fragment thereof that competes for binding to human SIRPα with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO:53 and a light chain variable region comprising the sequence of SEQ ID NO:54.

[0026]

[0028] In another aspect, the disclosure also provides an antibody or antigen-binding fragment thereof that competes for binding to human SIRPα with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO:55 and a light chain variable region comprising the sequence of SEQ ID NO:56.

[0027]

[0029] In another aspect, the disclosure also provides an antibody or antigen-binding fragment thereof that competes for binding to human SIRPα with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO:61 and a light chain variable region comprising the sequence of SEQ ID NO:62.

[0028]

[0030] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific.

[0031] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second antigen other than SIRPα.

[0029]

[0032] In certain embodiments, the second antigen is a tumor antigen, a tumor surface antigen, an inflammatory antigen, or an antigen of an infectious microorganism.

[0033] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can specifically bind to a second epitope on SIRPα.

[0030]

[0034] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are linked to one or more conjugating moieties.

[0035] In certain embodiments, the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a purification moiety, or other anti-cancer drug.

[0031]

[0036] In another aspect, the present disclosure also provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof provided herein and one or more pharma- ceutically acceptable carriers.

[0037] In another aspect, the present disclosure also provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein.

[0032]

[0038] In another aspect, the present disclosure also provides a vector comprising an isolated polynucleotide provided herein.

[0039] In another aspect, the present disclosure also provides a host cell comprising the vector provided herein.

[0033]

[0040] In another aspect, the disclosure also provides a method of expressing an antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which a vector provided herein is expressed.

[0034]

[0041] In another aspect, the present disclosure also provides a method of 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 provided herein, or a pharmaceutical composition provided herein, optionally in combination with a targeting antibody that specifically binds to a target antigen on the target cell, thereby inducing phagocytosis of the target cell by the SIRPα-positive phagocytic cells.

[0035]

[0042] In another aspect, the present disclosure also provides a method of inducing phagocytosis of a target cell in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, optionally in combination with a targeting antibody that specifically binds to a target antigen on a target cell, in a dose effective to induce phagocytosis of the target cell.

[0036]

[0043] In another aspect, the present disclosure also provides a method for increasing antibody-dependent cellular phagocytosis (ADCP) effect of a targeting antibody against a target cell in a subject, comprising: administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, in combination with a targeting antibody, thereby increasing ADCP of the targeting antibody against a target cell; The targeting antibody binds to a target antigen expressed on a target cell; A method is provided.

[0037]

[0044] In another aspect, the disclosure also provides a method of treating, preventing, or ameliorating a disease, disorder, or condition that can benefit from the induction of phagocytosis of target cells in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, optionally in combination with a targeting antibody that specifically binds to a target antigen on a target cell.

[0038]

[0045] In another aspect, the disclosure also provides a method of treating, preventing, or alleviating a SIRPα-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, optionally in combination with a targeting antibody that specifically binds to a target antigen on a target cell.

[0039]

[0046] In certain embodiments, the target cell is a CD47-expressing cell.

[0047] In certain embodiments, the target cells are cancer cells, inflammatory cells, and / or chronically infected cells.

[0040]

[0048] In certain embodiments, the target antigen is a tumor antigen, a tumor surface antigen, an inflammatory antigen, or an antigen of an infectious microorganism.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 17, an HCDR3 comprising the sequence of SEQ ID NO: 15, an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 11, and an LCDR3 comprising the sequence of SEQ ID NO: 16.

[0041]

[0050] In certain embodiments, the disease, disorder, or condition is cancer, a solid tumor, a chronic infection, an inflammatory disease, multiple sclerosis, an autoimmune disease, a neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, graft dysfunction, or arthritis.

[0042]

[0051] In certain embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct 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, head and neck squamous cell carcinoma, 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, throat 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 (NHL), multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumors, hepatocellular carcinoma, and adenocarcinoma.

[0043]

[0052] In certain embodiments, the cancer is a CD47-positive cancer.

[0053] In certain embodiments, the subject is a human.

[0054] In certain embodiments, administration is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0044]

[0055] In certain embodiments, the methods provided herein further comprise administering a therapeutically effective amount of an additional therapeutic agent.

[0056] In certain embodiments, the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiation therapy agents, immunotherapy agents, anti-angiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormonal therapy agents, anti-viral agents, antibiotics, analgesics, antioxidants, metal chelators, cytokines, anti-infective agents, and anti-inflammatory agents.

[0045]

[0057] In another aspect, the present disclosure also provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, and a targeting antibody that binds to a target antigen expressed on a target cell.

[0046]

[0058] In certain embodiments, the target antigen is a tumor antigen, a tumor surface antigen, or an infectious agent surface antigen.

[0059] In certain embodiments, the kits provided herein further comprise an additional therapeutic agent.

[0047]

[0060] In another aspect, the present disclosure also provides a method for modulating the activity of SIRPα in a SIRPα-positive cell, the method comprising exposing the SIRPα-positive cell to an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein.

[0048]

[0061] In certain embodiments, the cell is a phagocytic cell.

[0062] In another aspect, the present disclosure also provides a method for detecting the presence or amount of SIRPα in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of SIRPα in the sample.

[0049]

[0063] In another aspect, the present disclosure also provides a method for producing a method of making a pharmaceutical composition comprising: i) to treat, prevent, or ameliorate a SIRPα-related disease, disorder, or condition in a subject; ii) to induce phagocytosis of target cells in a subject; ii) to increase the antibody-dependent cellular phagocytosis (ADCP) effect of a targeted antibody against a target cell in a subject The present invention also provides use of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, in the manufacture of a medicament for the treatment of a disease comprising administering to a patient a therapeutically effective amount of the antibody or antigen-binding fragment thereof to a patient.

[0050]

[0064] In another aspect, the present disclosure also provides a method of enhancing a target antibody in the treatment of a disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, in combination with the target antibody, thereby enhancing the target antibody in the treatment of the disease, disorder, or condition in the subject.

[0051]

[0065] In certain embodiments, the disease, disorder, or condition is an immune-related disease or disorder, tumors and cancers, autoimmune diseases, or infectious diseases.

[0066] In certain embodiments, the immune-related disease or disorder is selected from the group consisting of systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), vasculitis, myasthenia gravis, idiopathic pulmonary fibrosis, Crohn's disease, asthma, rheumatoid arthritis, graft-versus-host disease, spondyloarthropathy (e.g., ankylosing spondylitis, psoriatic arthritis, isolated acute enteropathic arthritis with inflammatory bowel disease, reactive arthritis, Behcet's disease, undifferentiated spondyloarthropathy, anterior uveitis, and juvenile idiopathic arthritis), multiple sclerosis, endometriosis, glomerulonephritis, sepsis, diabetes, acute coronary syndrome, ischemia-reperfusion, psoriasis, progressive systemic sclerosis, atherosclerosis, Sjogren's syndrome, scleroderma, or inflammatory autoimmune myositis.

[0052]

[0067] In certain embodiments, the tumors and cancers are solid tumors or hematological malignancies, and optionally include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and negative PTLD, as well as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasms of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis Tumors, brain stem glioma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct 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 tract cancer, skin cancer, prostate cancer, pituitary gland In one embodiment, the cancer is selected from the group consisting of cancers including, but not limited to, cancers of the vagina, thyroid, throat, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma, or metastases thereof. [Brief description of the drawings]

[0053] [Figure 1-1]

[0068] FIG. 1 shows FACS binding curves of anti-SIRPα antibodies 025c, 015c, 042c, 059c, hu1H9G4 (FIG. 1A), 071c, 073c (FIG. 1B) to CHOK1-human SIRPα v1 cells, and 005c (FIG. 1C) to 293F-human SIRPα v1 cells. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 2-1]

[0069] FIG. 2 shows FACS binding curves of anti-SIRPα antibodies 025c, 015c, 042c, 071c, 073c, hu1H9G4 (FIG. 2A), 025c, 059c, 005c, HEFLB (FIG. 2B) to CHOK1-human SIRPα v2 cells. [Figure 2-2] Same as above. [Figure 3-1]

[0070] FIG. 3 shows FACS binding curves of anti-SIRPα antibodies to CHOK1-human SIRPβ cells (FIG. 3C) and their ELISA binding curves to recombinant proteins human SIRPβ ECD (FIGS. 3A and 3B) and human SIRPβ1 ECD (FIGS. 3D and 3E). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1]

[0071] FIG. 4 shows FACS binding curves of anti-SIRPα antibodies to 293F-human SIRPγ cells (FIG. 4A and FIG. 4B) and their ELISA binding curves to recombinant protein cynomolgus monkey SIRPγ ECD (FIG. 4C). [Figure 4-2] Same as above. [Figure 5-1]

[0072] FIG. 5 shows ELISA binding of anti-SIRPα antibodies to recombinant protein C57BL / 6 mouse SIRPα ECD (FIG. 5A) and their FACS binding curves to CHOK1-cynomolgus monkey SIRPα cells (FIGS. 5B and 5C). [Figure 5-2] Same as above. [Figure 6]

[0073] FIG. 6 shows the blocking activity of anti-SIRPα antibodies 025c, 015c, 042c, 059c, 071c, 073c, hu1H9G4 (FIG. 6A), 025c, 005c, 059c (FIG. 6B) against the interaction of CD47 with SIRPα v1 as measured by competitive ELISA assay. [Figure 7]

[0074] FIG. 7 shows the blocking activity of anti-SIRPα antibodies 025c, 059c (FIG. 7A), 025c, 015c, 042c, 071c, 073c, and hu1H9G4 (FIG. 7B) against the interaction of CD47 with SIRPα v2 as measured by competitive ELISA assay. [Figure 8-1]

[0075] FIG. 8 shows the principle of the SHP-1 recruitment assay (FIG. 8A) and the blocking activity of anti-SIRPα antibodies on SHP-1 recruitment measured by this assay (FIG. 8B). [Figure 8-2] Same as above. [Figure 9-1]

[0076] FIG. 9 shows the potential binding epitopes of anti-SIRPα antibodies 025c (FIG. 9A), 042c (FIG. 9B), 073c (FIG. 9C), hu1H9G4 (FIG. 9D), and HEFLB (FIG. 9E) as determined by HDX-MS. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 10-1]

[0077] Figure 10 shows phagocytosis of Raji cells (Figure 10A), DLD1 cells (Figure 10B), and Raji / PD-L1 cells (Figures 10C and 10D) by human macrophages in the presence of the indicated antibodies. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 11-1]

[0078] Figure 11 shows phagocytosis of Raji / PD-L1 cells by human M0 polarized macrophages (Figure 11A) or M1 polarized macrophages (Figure 11B) in the presence of the indicated antibodies. [Figure 11-2] Same as above. [Figure 12-1]

[0079] Figure 12 shows the results of an in vivo syngeneic mouse colon cancer model evaluating the activity of a combination of anti-SIRPα and anti-CLDN18.2 treatments. Figure 12A shows the weight of each tumor at the end of the study, Figure 12B shows the mean tumor volume growth curves for each study group, and Figure 12C shows the individual volume growth curves for each tumor. *p<0.05, **p<0.01, ***p<0.001. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above. [Figure 12-6] Same as above. [Figure 12-7] Same as above. [Figure 13-1]

[0080] FIG. 13 shows IFNγ secretion of T cells (FIG. 13A), proliferation ratios of CD4+ T cells (FIG. 13B) and CD8+ T cells (FIG. 13C) stimulated by allogeneic dendritic cells in the presence of anti-SIRPα antibody. [Figure 13-2] Same as above. [Figure 14-1]

[0081] FIG. 14 shows FACS binding curves of humanized antibodies to CHOK1-human SIRPα v1 cells (FIG. 14A), CHOK1-human SIRPα v2 cells (FIG. 14B), CHOK1-human SIRPβ cells (FIG. 14C), and 293F-SIRPγ cells (FIG. 14D). [Figure 14-2] Same as above. [Figure 15]

[0082] Figure 15 shows the blocking activity of humanized antibodies against the interaction of CD47 with SIRPα, as measured by competitive ELISA assay: (Figure 15A) Blocking of the interaction of human CD47 with human SIRPα v1, (Figure 15B) Blocking of the interaction of human CD47 with human SIRPα v2. [Figure 16]

[0083] Figure 16 shows the blocking activity of humanized antibodies against the interaction of CD47 with SIRPα, as measured by competitive FACS assay: (Figure 16A) Blocking of the interaction of human CD47 with human SIRPα v1, (Figure 16B) Blocking of the interaction of human CD47 with human SIRPα v2. [Figure 17]

[0084] FIG. 17 shows the SHP-1 recruitment blocking activity of humanized antibodies as measured by an SHP-1 recruitment assay. [Figure 18-1]

[0085] Figure 18 shows phagocytosis of Raji / PD-L1 cells by human macrophages in the presence of the indicated antibodies. (Figure 18A, Figure 18C, and Figure 18E) Phagocytosis of Raji / PD-L1 cells by human macrophages from SIRPA homozygous v1 / v1 (A), SIRPA homozygous v2 / v2 (C), or SIRPA heterozygous v1 / v2 (E) donors in the presence of anti-SIRPα antibodies plus anti-PD-L1 antibodies. (Figure 18B and Figure 18D) Phagocytosis of Raji / PD-L1 cells by human macrophages from SIRPA homozygous v1 / v1 (B) or SIRPA homozygous v2 / v2 (D) donors in the presence of anti-SIRPα antibodies plus rituximab. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054]

[0086] Detailed Description of the Invention

[0087] The following description of the present disclosure is intended only to describe various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is clear to those skilled in the art that various equivalents, modifications and alterations can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0055]

[0088] definition

[0089] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, polyvalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A natural intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consisting of a variable region (VH) and a first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively). Mammalian light chains are classified as lambda or kappa, each light chain consisting of a variable region (VL) and a constant region. Antibodies have a "Y" shape, with the axis of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y comprises the variable region and first constant region of one heavy chain bound to the variable and constant regions of one light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, and HCDR3).The boundaries of the CDRs of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, IMGT, Chothia, or Al-Lazikani (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, 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)). The three CDRs are inserted between adjacent sections known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs including HFR1, HFR2, HFR3, and HFR4), which are more highly conserved than the CDRs and form a scaffold supporting the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to a class based on the amino acid sequence of the constant region of their heavy chains.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0056]

[0090] In certain embodiments, the antibodies provided herein encompass any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that includes one or more CDRs, or any other antibody fragment that binds to an antigen but does not include an intact native antibody structure. Examples of antigen-binding fragments include, without limitation, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen that the parent antibody binds.

[0057]

[0091] "Fab," with respect to an antibody, means the portion of the antibody that consists of a single light chain (both the variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0058]

[0092] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0093] "F(ab')2" means a Fab' dimer.

[0094] "Fc" refers to the portion of an antibody (e.g., of an IgG, IgA, or IgD isotype) that consists of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, the Fc further comprises a fourth constant domain. The Fc portion of an antibody is involved in various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0059]

[0095] "Fv" refers to the minimum fragment of an antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0096] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked together directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0060]

[0097] By "single chain Fv-Fc antibody" or "scFv-Fc" is meant an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0098] "Camelized single domain antibodies", "heavy chain antibodies", or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chains and do not contain 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. Patent No. 6,005,079). Heavy chain antibodies are originally derived from the Camelidae family (camels, dromedaries, and llamas). Camelized antibodies have no light chains but have bona fide antigen-binding capacity (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 domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0061]

[0099] "Nanobody" refers to an antibody fragment consisting of a VHH domain and two constant domains, CH2 and CH3, of a heavy chain antibody.

[0100] A "diabody" or "dAb" refers to a small antibody fragment with two antigen-binding sites, arranged in the same polypeptide chain as the V L V linked to domain H Domain Included (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). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0062]

[0101] "Domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0063]

[0102] As used herein, the term "valency" 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 that has only one single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0064]

[0103] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0065]

[0104] In certain embodiments, an "scFv dimer" is a dimer of another V H -V L Dimerized with the V moiety (dimerized via a peptide linker) H -V L and a bivalent diabody or bispecific scFv (BsFv) comprising one part V H V in other parts L In another embodiment, an "scFv dimer" is a combination of a V (linked by a peptide linker) and a V (linked by a peptide linker) to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). L1 -V H2 (also linked by a peptide linker) V H1 -V L2 and a bispecific diabody comprising V H1 and V L1 In cooperation with V H2 and V L2 cooperate, with each cooperated pair having a different antigen specificity.

[0066]

[0105] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" is a Fv fragment that is composed of three peptide chains, i.e., linked by a peptide linker (e.g., a long flexible linker), and each of the two Vs is linked through a disulfide bridge. L Two V's attached to the part H In some embodiments, dsFv-dsFv' are bispecific, in which the respective disulfide-paired heavy and light chains have different antigen specificities.

[0067]

[0106] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy and / or light chain derived from one species and the remainder of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0068]

[0107] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, a constant region derived from a human.

[0069]

[0108] As used herein, the term "affinity" refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0109] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. Specific binding is, for example, the K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on The binding affinity of the ligand can be characterized by its binding affinity, which is expressed by K D may be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). -6 M or less (for example, 5×10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10-9 M or less, or 10 -9 M or less) K D The value can indicate specific binding between the antibody or antigen-binding fragment thereof and SIRPα (eg, human SIRPα).

[0070]

[0110] As used herein, the ability to "compete for binding with human SIRPα" refers to the ability of a first antibody or antigen-binding fragment to inhibit the binding interaction between human SIRPα and a second anti-SIRPα antibody to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment that competes for binding with human SIRPα inhibits the binding interaction between human SIRPα and a second anti-human SIRPα antibody by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 95%, or greater than 99%.

[0071]

[0077] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes in the antigen. Epitopes may be linear or conformational (i.e., involving separated amino acid residues). For example, an antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody if it blocks at least 85%, or at least 90%, or at least 95% of the binding of the reference antibody to the antigen.

[0072]

[0078] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group along with a side chain characteristic of each amino acid. In this disclosure, the names of amino acids may also be represented as standard one-letter or three-letter codes, which are summarized below.

[0073] [Table 1]

[0074]

[0079] "Conservative substitution" in relation to amino acid sequences means replacing an amino acid residue with a different amino acid residue having a side chain with 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). As is known in the art, conservative substitutions usually do not cause significant changes in the conformational structure of a protein, and thus can retain the biological activity of the protein.

[0075]

[0080] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0076]

[0081] "Percent sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps to maximize the number of identical amino acids (or nucleic acids). In other words, the percent sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) with respect to the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for calculating percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, 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 (available from the European Bioinformatics Institute website, see also Higgins DG 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. One skilled in the art may use the default parameters provided by the tool or may customize appropriate parameters for the alignment, for example by selecting a suitable algorithm.

[0077]

[0082] As used herein, "effector function" refers to the biological activity attributable to the binding of the Fc region of an antibody to its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), mediated by the interaction of the 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 an Fc receptor on an effector cell, and phagocytosis. Effector function can be assessed using a variety of assays, such as Fc receptor binding assays, C1q binding assays, and cytolysis assays.

[0078]

[0083] An "isolated" material is altered by the hand of man from its natural state. If an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that occurs naturally in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it exists in a substantially pure state sufficiently separated from the coexisting materials in its natural state. An "isolated nucleic acid sequence" refers to a sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has a purity of 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% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (ion exchange chromatography or reverse phase HPLC).

[0079]

[0084] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted and expression of the genetic element occurs, thereby producing a protein, RNA, or DNA encoded by the genetic element, or replicating the genetic element. A vector may be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries into the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, a vector may contain an origin of replication. A vector may contain substances that aid in the entry of the vector into a cell, including, but not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0080]

[0085] As used herein, the phrase "host cell" means a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0086] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

[0081]

[0087] The term "antitumor activity" refers to a reduction in tumor cell proliferation, viability, or metastatic activity. For example, antitumor activity can be demonstrated by a decrease in the rate of proliferation of abnormal cells during treatment, or a stable or reduced size of the tumor, or prolonged survival with treatment, compared to a control without treatment. Such activity can be evaluated using accepted in vitro or in vivo tumor models, including, but not limited to, xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other models known in the art for studying antitumor activity.

[0082]

[0088] As used herein, "treating" a disease, disorder, or condition or "treatment" thereof includes preventing or alleviating the disease, disorder, or condition, slowing the onset or rate of development of the disease, disorder, or condition, reducing the risk of developing the disease, disorder, or condition, preventing or delaying the onset of symptoms associated with the disease, disorder, or condition, reducing or terminating symptoms associated with the disease, disorder, or condition, completely or partially ameliorating the disease, disorder, or condition, curing the disease, disorder, or condition, or some combination thereof.

[0083]

[0089] 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 having a SIRPα-related disease that may benefit from a particular treatment regimen. In some embodiments, diagnosis includes the identification of an abnormal amount or activity of SIRPα. In some embodiments, diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0084]

[0090] As used herein, the term "biological sample" or "sample" refers to a biological composition containing cells and / or other molecular entities obtained or derived from a subject of interest and characterized and / or identified based on, for example, 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 of skill 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 discharge and sputum), peritoneal fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis, abdominal fluid, peritoneal fluid, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lungs, kidneys, skin, or blood vessels of a subject.

[0085]

[0091] "SIRPα" as used herein refers to a regulatory membrane glycoprotein of the signal-regulatory protein (SIRP) family, which is expressed primarily by myeloid cells, dendritic cells, and also by stem cells or neurons. The structure of SIRPα includes an extracellular domain and a cytoplasmic domain. The extracellular domain of SIRPα consists of one 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 the extracellular Ig domain of CD47. In certain embodiments, SIRPα is human SIRPα. The gene encoding human SIRPα is a polymorphic gene, and multiple variants have been described in the human population. The most common protein variants are SIRPα v1 and SIRPα v2 (accession numbers NP_542970 (P78324) and CAA71403). SIRPα used herein may be derived from other animal species, such as mouse and cynomolgus monkey, among others. Exemplary sequences of Mus musculus (mouse) SIRPα protein are disclosed in NCBI reference sequence number NP_031573 or BAA20376.1 or BAA13521.1. Exemplary sequences of Cynomolgus monkey (monkey) SIRPα protein are disclosed in NCBI reference sequence number NP_001271679.

[0086] In addition to SIRPα, the SIRP family also includes several other transmembrane glycoproteins, including SIRPβ and SIRPγ. Each member of the SIRP family contains three similar extracellular Ig-like domains with distinct transmembrane and cytoplasmic domains. "SIRPβ", encoded by the SIRP beta gene, generates a positive signal by intracellular signaling of its cytoplasmic tail through its association with a transmembrane protein called DNAX-activating protein 12 or DAP12. The cytoplasmic tail of DAP12 has an immunoreceptor tyrosine-based activation motif (ITAM) that links SIRPβ1 to the activation mechanism. "SIRPγ", also named SIRPg, is encoded by the SIRPG gene, and the extracellular Ig domain is highly homologous to SIRPα and SIRPβ, but the cytoplasmic tail of SIRPγ is different. SIRPγ has also been shown to bind to CD47, but with a lower affinity than SIRPα.

[0087]

[0093] The term "anti-SIRPα antibody" refers to an antibody capable of specifically binding to SIRPα (e.g., human or monkey SIRPα). The term "anti-human SIRPα antibody" refers to an antibody capable of specifically binding to human SIRPα.

[0088]

[0094] As used herein, a "SIRPα-associated" disease, disorder, or condition refers to any disease or condition caused, exacerbated, or otherwise associated with increased or decreased expression or activity of SIRPα. In some embodiments, the SIRPα-associated disease, disorder, or condition is an immune-related disorder, such as, for example, an autoimmune disease. In some embodiments, the SIRPα-associated disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as, for example, cancer. In certain embodiments, the SIRPα-associated disease or condition is characterized by expression or overexpression of the SIRPα gene and / or a SIRPα signature gene. In certain embodiments, the SIRPα-associated disease or condition is characterized by expression or overexpression of CD47.

[0089]

[0095] The term "pharmacologically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the recipient thereof.

[0090]

[0096] The term "SIRPα positive cell," as used herein, refers to a cell that expresses SIRPα on its surface (e.g., a phagocytic cell). In some embodiments, a "SIRPα positive cell" may also express SIRPβ or SIRPγ on its surface.

[0091]

[0097] Anti-SIRPα antibody

[0098] The present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof. The anti-SIRPα antibodies and antigen-binding fragments provided herein are capable of specifically binding to SIRPα.

[0092] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are capable of measuring 10 -7 M or less, 8×10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 8×10 -10 M or less, 7×10 -10 M or less, or 6 x 10 -10 M or less, or 5 x 10 -10 M or less, or 4 x 10 -10 K below M D The Octet system specifically binds to human SIRPα at a value of 0.01 μg / mL. The Octet system is based on biolayer interferometry (BLI) technology, see, e.g., Sultana A. et al., Current protocols in protein science, 02 Feb 2015, 79:19.25.1-19.25.26. In certain embodiments, the KD Values ​​are measured by the method described in Example 5.2.5 of this disclosure.

[0093] The binding of the antibodies or antigen-binding fragments thereof provided herein to human SIRPα is measured using a "50% effective concentration" (EC 50 ) value. 50 The values ​​represent the concentration of antibody at which 50% of its maximal binding is observed. EC 50 Values ​​can be measured by binding assays known in the art, for example, direct or indirect binding assays such as enzyme-linked immunosorbent assay (ELISA), flow cytometry assays, and other binding assays. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC of 0.5 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, or 0.05 nM or less as measured by enzyme-linked immunosorbent assay (ELISA). 50 (i.e., 50% binding concentration), it specifically binds to human SIRPα v1 or human SIRPα v2.

[0094] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC50 of 4 nM or less (e.g., 3 nM or less, 2 nM or less, 1.5 nM or less, 1.0 nM or less) as measured by a FACS assay. 50 It specifically binds to human SIRPα v1.

[0095] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC50 of 12.1 nM or less (e.g., 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less) as measured by a FACS assay. 50 and specifically binds to human SIRPα v2.

[0096]

[0103] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein do not have specific binding to mouse SIRPα. An antibody or antigen-binding fragment thereof that "does not have specific binding" to mouse SIRPα is one that does not exhibit detectable binding to mouse SIRPα or one that exhibits binding to mouse SIRPα at a level comparable to that of a control antibody under comparable assay conditions. The control antibody can be any antibody known not to bind to mouse SIRPα.

[0097] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC50 of 40 nM or less (e.g., 30 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.4 nM or less) as measured by a FACS assay. 50 and specifically binds to SIRPβ.

[0098] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC of 3 nM or less (e.g., 2 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.1 nM or less, 0.05 nM or less) as measured by ELISA assay. 50 and specifically binds to the SIRPβ ECD.

[0099] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC50 of 80 nM or less (e.g., 50 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, 0.3 nM or less) as measured by a FACS assay. 50 and binds to SIRPγ.

[0100] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein can completely block the interaction between SIRP-alpha and CD47. "Completely blocking the interaction" between two interacting molecules means that the antibody can inhibit the binding between the two interacting molecules by at least 80% or inhibit the signal transduction induced by the interaction of the two molecules by at least 50%. The signal transduction induced by the interaction between SIRP-alpha and CD47 can be characterized by SHP1 recruitment to the intracellular portion of SIRP-alpha (e.g., the C-terminal tail).

[0101]

[0108] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can completely block the interaction between SIRP-alpha v1 and CD47. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can block at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the binding between SIRP-alpha v1 and CD47 as measured by competitive ELISA assay. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can block at least 97% or at least 98% of the binding between SIRP-alpha v1 and CD47 as measured by competitive FACS assay. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein are capable of blocking the interaction between SIRP-alpha v1 and CD47 with an IC50 of 4 nM or less (or 3 nM or less) as measured by a competitive ELISA assay, or with an IC50 of 0.6 nM or less (or 0.5 nM or less) as measured by a competitive FACS assay.

[0102] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can completely block the interaction between SIRP-alpha v2 and CD47. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can block at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% of the binding between SIRP-alpha v2 and CD47 as measured by competitive ELISA assay. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can block at least 98% or at least 99% of the binding between SIRP-alpha v2 and CD47 as measured by competitive FACS assay. In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein are capable of blocking the interaction between SIRP-alpha v2 and CD47 with an IC50 of 55 nM or less (or 6 nM or less, 5 nM or less, 3 nM or less, or 2 nM or less) as measured by a competitive ELISA assay, or with an IC50 of 3 nM or less (or 2 nM or less) as measured by a competitive FACS assay.

[0103]

[0110] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments provided herein can block at least 50%, 60%, 70%, or 80% of the signal transduction induced by the interaction of SIRP-alpha with CD47.

[0104]

[0111] In certain embodiments, the antibodies may block signal transduction induced by the interaction between SIRP-alpha and CD47, but do not significantly block the binding between SIRP-alpha and CD47. In other words, SIRP-alpha and CD47 can bind to each other in the presence of such anti-SIRP-alpha antibodies, but they are less effective in signal transduction.

[0105]

[0112] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein inhibit IFNγ secretion by T cells, CD4 + T cell proliferation, or CD8 + It has no significant inhibition on T cell proliferation. It has been reported that the attachment of human T cells to antigen-presenting cells through SIRPγ-CD47 interaction costimulates T cell proliferation. T cell proliferation can be determined using methods known in the art, for example, by T cell proliferation assays such as those described in Example 4.2.9 of the present disclosure, for example, by determining the proliferating population using CellTrace Violet (Life Technologies) labeling. As shown in the present disclosure, regardless of the binding activity to human SIRPγ, the antibodies or antigen-binding fragments thereof provided herein can be used to inhibit the proliferation of CD4 + T cells or CD8 + It does not significantly reduce T cell proliferation or affect IFNγ secretion.

[0106]

[0113] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein inhibit IFNγ secretion by T cells, CD4 + T cell proliferation, or CD8 + In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein exhibit inhibition of T cell proliferation by 50% or less (or 40% or less, 30% or less, 20% or less, or 10% or less) compared to control levels obtained with a control antibody (e.g., an antibody that does not bind to SIRPα and is known not to affect T cell proliferation). + T cell proliferation, or CD8 + There is no detectable inhibition of T cell proliferation.

[0107]

[0114] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein as single agents do not induce phagocytosis of certain CD47-expressing cells, e.g., Raji cells.

[0108]

[0115] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided herein can increase the antibody-dependent cellular phagocytosis (ADCP) effect of a target antibody. In certain embodiments, the target antibody binds to a target antigen expressed on a target cell, and the ADCP effect of the target antibody on the target cell is increased. In certain embodiments, the target cell also expresses CD47.

[0109]

[0116] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are capable of binding to an epitope comprising the amino acid sequence of YNQKEGHFPRVTTVSDL (SEQ ID NO: 36).

[0110]

[0117] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are capable of binding to an epitope comprising the amino acid sequence of SGAGTEL (SEQ ID NO: 72) and / or TNVDPVGESVS (SEQ ID NO: 87).

[0111]

[0118] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are capable of binding to an epitope comprising the amino acid sequence of TNVDPVGESVSY (SEQ ID NO: 90).

[0112]

[0119] Exemplary Anti-SIRPα Antibodies

[0120] In certain embodiments, the disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof that comprise one or more (e.g., one, two, three, four, five, or six) CDR sequences of antibodies 005, 015, 025, 042, 071, 073, and / or 059. In certain embodiments, the disclosure provides chimeric antibodies, humanized antibodies, antibody derivatives, and antibody variants of antibodies 005, 015, 025, 042, 071, 073, and / or 059.

[0113]

[0121] As used herein, antibodies "005" and "005c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, that contain a heavy chain variable region having the amino acid sequence of SEQ ID NO:49 and a light chain variable region having the amino acid sequence of SEQ ID NO:50.

[0114]

[0122] As used herein, antibodies "015" and "015c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, that contain a heavy chain variable region having the amino acid sequence of SEQ ID NO:51 and a light chain variable region having the amino acid sequence of SEQ ID NO:52.

[0115]

[0123] As used herein, antibodies "025" and "025c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, that comprise a heavy chain variable region having the sequence of SEQ ID NO:53 and a light chain variable region having the sequence of SEQ ID NO:54.

[0116]

[0124] As used herein, antibodies "042" and "042c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, comprising a heavy chain variable region having the sequence of SEQ ID NO:55 and a light chain variable region having the sequence of SEQ ID NO:56.

[0117]

[0125] As used herein, antibodies "059" and "059c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, that contain a heavy chain variable region having the sequence of SEQ ID NO:57 and a light chain variable region having the sequence of SEQ ID NO:58.

[0118]

[0126] As used herein, antibodies "071" and "071c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, that comprise a heavy chain variable region having the sequence of SEQ ID NO:59 and a light chain variable region having the sequence of SEQ ID NO:60.

[0119]

[0127] As used herein, antibodies "073" and "073c" refer to a monoclonal hybridoma antibody and a chimeric antibody, respectively, that comprise a heavy chain variable region having the sequence of SEQ ID NO:61 and a light chain variable region having the sequence of SEQ ID NO:62.

[0120]

[0128] Table 1 below shows the amino acid sequences of the CDRs of antibodies 005, 015, 025, 042, 071, 073, 059, 005c, 015c, 025c, 042c, 059c, 071c, and 073. The boundaries of the CDRs in Table 1 are defined or identified by the Kabat rules, but one skilled in the art can understand that the CDRs may be defined using other rules, such as IMGT, Chothia, or Al-Lazikani, or in a mixed manner using two or more rules. Table 2 below shows the amino acid sequences of the heavy and light chain variable regions of antibodies 005, 015, 025, 042, 071, 073, 059, 005c, 015c, 025c, 042c, 059c, 071c, and 073.

[0121]

[0129]

[0122] [Table 2-1]

[0123] [Table 2-2]

[0124] X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, X6 is Y or H, X7 is Y or C, X8 is K or Q, X9 is Y or S, and X 10 is N or T or S, and X 11 is P or A or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is Y or F, and X16 is S or T or A, and X 17 is F or L, and X 18 is S or absent, and X 19 is S or P.

[0125]

[0130]

[0126] [Table 3-1]

[0127] [Table 3-2]

[0128]

[0131] Given that each of antibodies 005, 015, 025, 042, 059, 071, 073, 005c, 015c, 025c, 042c, 059c, 071c, and 073 can bind to SIRPα and that antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, antibodies 005, 015, 025, 042, 059, 071, 073, 005c, 015c, 025c, 042c, 059c, 071c, and 073 can bind to SIRPα and that antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions. The HCDR1, HCDR2, and HCDR3 sequences, and LCDR1, LCDR2, and LCDR3 sequences of 1c, and 073 can be "mixed and matched" (i.e., the CDRs of different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3) to create anti-SIRPα binding molecules of the disclosure. SIRPα binding of such "mixed and matched" antibodies can be tested 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 with 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 with structurally similar CDR sequences. It will be readily apparent to one of ordinary skill in the art that novel VH and VL sequences may be created by replacing one or more VH and / or VL CDR region sequences for monoclonal hybridoma antibodies 005, 015, 025, 042, 059, 071, and 073, or chimeric antibodies 005c, 015c, 025c, 042c, 059c, 071c, and 073c with structurally similar sequences from the CDR sequences disclosed herein.

[0129]

[0132] In certain embodiments, the disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 18, 22, and 43, an HCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 17, 19, 23, 28, 33, 38, and 44, and an HCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 20, 24, 29, 34, 39, and 45, and / or an LCDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 25, 30, 35, 40, and 46, an LCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 5, 11, 26, 31, 41, and 47, and an LCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 6, 12, 16, 21, 27, 32, 37, 42, and 48.

[0130]

[0133] In certain embodiments, the present disclosure provides an HCDR1 comprising an amino acid sequence of X1YYMH (SEQ ID NO: 18), an HCDR2 comprising an amino acid sequence of RIDPEDX2EX3KYAPKFQG (SEQ ID NO: 19), an HCDR3 comprising an amino acid sequence of GX 18 X4X5Y (SEQ ID NO: 20), an LCDR1 having an amino acid sequence of SEQ ID NO: 10, an LCDR2 having an amino acid sequence of SEQ ID NO: 11, and an LCDR3 having an amino acid sequence of X6QWSSYPYT (SEQ ID NO: 21), wherein X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, and X6 is Y or H; 18 The present invention provides anti-SIRPα antibodies and antigen-binding fragments thereof, wherein

[0131]

[0134] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising an sequence selected from the group consisting of SEQ ID NOs: 7 and 13, and / or an HCDR2 comprising an sequence selected from the group consisting of SEQ ID NOs: 8, 14, and 17, and / or an HCDR3 comprising an sequence selected from the group consisting of SEQ ID NOs: 9 and 15, and / or an LCDR1 comprising an sequence of SEQ ID NO: 10, and / or an LCDR2 comprising an sequence of SEQ ID NO: 11, and / or an LCDR3 comprising an sequence selected from the group consisting of SEQ ID NOs: 12 and 16.

[0132]

[0135] In certain embodiments, the present disclosure provides an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22, WINTYSGVX 19 HCDR2, DPHX9YGX, comprising the amino acid sequence of TX7ADDFX8G (SEQ ID NO: 38) 10 SPAWFX 11 HCDR3, comprising the amino acid sequence of Y (SEQ ID NO: 39) 12 ASQX 13 VGIX 14 LCDR1, SASNRX, comprising the amino acid sequence of VA (SEQ ID NO: 40) 15 LCDR2 comprising the amino acid sequence of T (SEQ ID NO: 41), and QQYSX 16 YPX 17 X7 is Y or C, X8 is K or Q, and X9 is Y or S; 10 is N or T or S, and X 11 is P or A or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is Y or F and X 16 is S or T or A, and X 17 is F or L, and X 19 is S or P.

[0133]

[0136] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 22, and / or an HCDR2 comprising an sequence selected from the group consisting of SEQ ID NOs: 23, 28, and 33, and / or an HCDR3 comprising an sequence selected from the group consisting of SEQ ID NOs: 24, 29, and 34, and / or an LCDR1 comprising the sequence of SEQ ID NOs: 25, 30, and 35, and / or an LCDR2 comprising an sequence selected from the group consisting of SEQ ID NOs: 31 and 26, and / or an LCDR3 comprising an sequence selected from the group consisting of SEQ ID NOs: 27, 32, and 37.

[0134]

[0137] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:2, an HCDR3 comprising the sequence of SEQ ID NO:3, an LCDR1 comprising the sequence of SEQ ID NO:4, an LCDR2 comprising the sequence of SEQ ID NO:5, and an LCDR3 comprising the sequence of SEQ ID NO:6.

[0135]

[0138] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:7, an HCDR2 comprising the sequence of SEQ ID NO:8, an HCDR3 comprising the sequence of SEQ ID NO:9, an LCDR1 comprising the sequence of SEQ ID NO:10, an LCDR2 comprising the sequence of SEQ ID NO:11, and an LCDR3 comprising the sequence of SEQ ID NO:12.

[0136]

[0139] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 13, an HCDR2 comprising the sequence of SEQ ID NO: 14 or 17, an HCDR3 comprising the sequence of SEQ ID NO: 15, an LCDR1 comprising the sequence of SEQ ID NO: 10, an LCDR2 comprising the sequence of SEQ ID NO: 11, and an LCDR3 comprising the sequence of SEQ ID NO: 16.

[0137]

[0140] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 23, an HCDR3 comprising the sequence of SEQ ID NO: 24, an LCDR1 comprising the sequence of SEQ ID NO: 25, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 27.

[0138]

[0141] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 28, an HCDR3 comprising the sequence of SEQ ID NO: 29, an LCDR1 comprising the sequence of SEQ ID NO: 30, an LCDR2 comprising the sequence of SEQ ID NO: 31, and an LCDR3 comprising the sequence of SEQ ID NO: 32.

[0139]

[0142] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 22, an HCDR2 comprising the sequence of SEQ ID NO: 33, an HCDR3 comprising the sequence of SEQ ID NO: 34, an LCDR1 comprising the sequence of SEQ ID NO: 35, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 37.

[0140]

[0143] In certain embodiments, the present disclosure provides anti-SIRPα antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 43, an HCDR2 comprising the sequence of SEQ ID NO: 44, an HCDR3 comprising the sequence of SEQ ID NO: 45, an LCDR1 comprising the sequence of SEQ ID NO: 46, an LCDR2 comprising the sequence of SEQ ID NO: 47, and an LCDR3 comprising the sequence of SEQ ID NO: 48.

[0141]

[0144] It is known that CDR is involved in antigen binding.However, it has been found that not all six CDRs are essential or invariant.In other words, it is possible to replace, change or modify one or more CDRs in anti-SIRPα antibody 005, 015, 025, 042, 059, 071 and 073, or anti-SIRPα chimeric antibody 005c, 015c, 025c, 042c, 059c, 071c and 073c, while substantially retaining specific binding specificity and / or affinity to SIRPα.

[0142]

[0145] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to SIRPα. The CDR sequences provided in Table 1 above are derived from mouse antibodies, but they can be grafted into any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0143]

[0146] In certain embodiments, the antibody and antigen-binding fragment provided herein are humanized. Humanized antibody or antigen-binding fragment is desirable for its reduced immunogenicity in humans. Humanized antibody is chimeric in its variable region because non-human CDR sequence is grafted to human or substantially human FR sequence. Humanization of antibody or antigen-binding fragment can be essentially performed by replacing non-human (such as mouse) CDR gene with corresponding human CDR gene in human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0144]

[0147] To this end, suitable human heavy and light chain variable domains can be selected using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which the variable domain sequence of a non-human (e.g., rodent) antibody is screened or BLASTed against known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the 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, frameworks derived from the consensus sequence of all human antibodies may be used for grafting of 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).

[0145]

[0148] Table 3 below shows the CDR amino acid sequences of the five humanized antibodies of antibody 025, designated hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060. The boundaries of the CDRs were defined or specified according to the Kabat convention. Table 3 below shows the amino acid sequences of the six CDRs of the five humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060. Table 4 below shows the heavy and light chain variable region amino acid sequences of the five humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060. Table 5 below shows the FR amino acid sequences of the five humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060.

[0146]

[0149]

[0147] [Table 4]

[0148]

[0150]

[0149] [Table 5]

[0150]

[0151]

[0151] [Table 6-1]

[0152] [Table 6-2]

[0153] X 20 is A or V, and X 21 is N or D, and X 22 is Y or F.

[0152] In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein consist of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable regions FR and, if present, the constant region are entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be from different human immunoglobulin genes, e.g., the FR sequences are from one human antibody and the constant region is from another human antibody. In some embodiments, the humanized antibodies or antigen-binding fragments thereof comprise human heavy chains HFR1-4 and / or light chains LFR1-4.

[0154] In some embodiments, the FR region derived from a human may comprise the same amino acid sequence as the original human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residue from the parent non-human antibody. This is desirable in certain embodiments to allow the humanized antibody or fragment thereof to closely approximate the structure of the non-human parent antibody, thereby optimizing the binding properties (e.g., increasing binding affinity). In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in each of the human FR sequences, or the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in all of the FR sequences of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR regions, only in the light chain FR regions, or in both chains. In certain embodiments, one or more amino acids of the human FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequences are backmutated to the corresponding amino acid of the parent non-human antibody to increase binding affinity.

[0155] In certain embodiments, the present disclosure also provides: 20 Heavy chain HFR1 comprising the sequence of SGFNIK (SEQ ID NO: 84) or a homologous sequence having at least 80% sequence identity thereto; heavy chain HFR2 comprising the sequence of WVQQAPGKGLEWIG (SEQ ID NO: 74) or a homologous sequence having at least 80% sequence identity thereto; RVTITADTSTX 21 and a heavy chain HFR3 having the sequence of TAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 85) or a homologous sequence having at least 80% sequence identity thereto, and a heavy chain HFR4 having the sequence of WGQGTLVTVSS (SEQ ID NO: 76) or a homologous sequence having at least 80% sequence identity thereto; 20 is A or V, and X 21 is N or D, and antigen-binding fragments thereof.

[0156]

[0155] In certain embodiments, the present disclosure also provides a light chain LFR1 comprising the sequence of EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 77) or a homologous sequence having at least 80% sequence identity thereto, a light chain LFR2 comprising the sequence of WYQQKPGQAPKLWIY (SEQ ID NO: 78) or a homologous sequence having at least 80% sequence identity thereto, a light chain LFR3 comprising the sequence of GIPARFSGSGSGTDX 22 A light chain LFR3 having the sequence TLTISSLEPEDFAVYYC (SEQ ID NO: 86) or a homologous sequence having at least 80% sequence identity thereto, and a light chain LFR4 having the sequence FGQGTKLEIK (SEQ ID NO: 80) or a homologous sequence having at least 80% sequence identity thereto, 22 is Y or F.

[0157]

[0156] In certain embodiments, the present disclosure also provides humanized anti-SIRPα antibodies and antigen-binding fragments thereof comprising heavy chain HFR1 comprising a sequence selected from the group consisting of SEQ ID NO:73 and 83, heavy chain HFR2 comprising a sequence of SEQ ID NO:74, heavy chain HFR3 comprising a sequence selected from the group consisting of SEQ ID NO:75 and 82, and heavy chain HFR4 comprising a sequence of SEQ ID NO:76, and / or light chain LFR1 comprising a sequence selected from the group consisting of SEQ ID NO:77, light chain LFR2 comprising a sequence selected from the group consisting of SEQ ID NO:78, light chain LFR3 comprising a sequence selected from the group consisting of SEQ ID NO:79 and 81, and light chain LFR4 comprising a sequence selected from the group consisting of SEQ ID NO:80.

[0158]

[0157] In certain embodiments, the present disclosure also provides humanized anti-SIRPα antibodies and antigen-binding fragments thereof comprising HFR1, HFR2, HFR3, and / or HFR4 sequences contained in a heavy chain variable region selected from the group consisting of hu025.021-VH / hu025.023-VH (sequence number 63), hu025.033-VH (sequence number 65), and hu025.059-VH / hu025.060-VH (sequence number 67).

[0159]

[0158] In certain embodiments, the present disclosure also provides humanized anti-SIRPα and antigen-binding fragments thereof, comprising LFR1, LFR2, LFR3, and / or LFR4 sequences contained in a light chain variable region selected from the group consisting of hu025.021-VL / hu025.033-VL / hu025.059-VL (sequence number 64), and hu025.023-VL / hu025.060-VL (sequence number 66).

[0160]

[0159] In certain embodiments, the humanized anti-SIRPα antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO:63, SEQ ID NO:65, and SEQ ID NO:67, and / or a light chain variable domain sequence selected from the group consisting of SEQ ID NO:64 and SEQ ID NO:66.

[0161]

[0160] The present disclosure also provides: 1) the antibody "hu025.021" comprising a heavy chain variable region of SEQ ID NO: 63 and a light chain variable region of SEQ ID NO: 64; 2) the antibody "hu025.023" comprising a heavy chain variable region of SEQ ID NO: 63 and a light chain variable region of SEQ ID NO: 66; 3) the antibody "hu025.033" comprising a heavy chain variable region of SEQ ID NO: 65 and a light chain variable region of SEQ ID NO: 64; 4) the antibody "hu025.059" comprising the heavy chain variable region of SEQ ID NO: 67 and the light chain variable region of SEQ ID NO: 64; and 5) Antibody "hu025.060" comprising a heavy chain variable region of SEQ ID NO: 67 and a light chain variable region of SEQ ID NO: 66 Exemplary humanized antibodies of 025 are provided, including:

[0162]

[0161] These exemplary humanized anti-SIRPα antibodies retained specific binding ability or affinity for SIRPα and were at least as good or better in that respect than the parental murine antibody 025. Detailed information is provided in Example 5.2.

[0163] In some embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-SIRPα antibodies or antigen-binding fragments provided herein are single-domain antibodies consisting of all or a portion of the heavy chain variable domain provided herein. Further information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0164]

[0163] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, optionally including heavy and / or light chain constant regions. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein comprise a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, the light chain constant region comprises Cκ or Cλ. The constant region of the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein may be identical to the sequence of the wild-type constant region or may differ in one or more mutations.

[0165] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype capable of inducing ADCC or CDC, or comprise a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments thereof provided herein comprise a wild-type human IgG4 Fc region or other wild-type human IgG4 alleles.

[0166]

[0165] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided herein has reduced effector function. In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided herein comprises an Fc region of IgG1 isotype and comprises one or more amino acid substitutions to reduce or eliminate effector function. Examples of such substitutions in IgG1 may be 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 antibody or antigen-binding fragment thereof provided herein is of IgG1 isotype and comprises 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.

[0167]

[0166] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are of the IgG2 isotype and contain 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) to reduce or eliminate effector function.

[0168] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof provided herein is of the IgG4 isotype and contains one or more amino acid substitutions to reduce or eliminate effector function. Examples of such substitutions in IgG4 may be at positions selected from the group consisting of 228, 234, 235, 237, 238, 265, 297, 322, 329, and 331. Examples of such substitutions include, without limitation, S228P, L235E, L234A, L235A, N297A, N297Q, N297G, P329G, K322Q, P331S, D265A, G237A, P238S, and any combination thereof.

[0169] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain one or more amino acid substitutions at one or more of 228 and 235. In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain a 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 contain a L235E mutation in the Fc region.

[0170] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are of the IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256-1264 (2007).

[0171]

[0170] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have sufficient specific binding affinity for SIRPα to provide for diagnostic and / or therapeutic use.

[0172]

[0171] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins. Recombinant antibodies are antibodies prepared using recombinant methods in vitro, rather than in an animal.

[0173]

[0172] In certain embodiments, the present disclosure provides an anti-SIRPα antibody, or antigen-binding fragment thereof, that competes with an antibody, or antigen-binding fragment thereof, provided herein for binding to SIRPα.

[0174]

[0173] In certain embodiments, the present disclosure provides an anti-SIRPα antibody or antigen-binding fragment thereof that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 53 and a light chain variable region comprising the sequence of SEQ ID NO: 54 for binding to human SIRPα.

[0175]

[0174] In certain embodiments, the present disclosure provides an anti-SIRPα antibody or antigen-binding fragment thereof that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 55 and a light chain variable region comprising the sequence of SEQ ID NO: 56 for binding to human SIRPα.

[0176]

[0175] In certain embodiments, the present disclosure provides an anti-SIRPα antibody or antigen-binding fragment thereof that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 61 and a light chain variable region comprising the sequence of SEQ ID NO: 62 for binding to human SIRPα.

[0177]

[0176] In certain embodiments, the disclosure provides an anti-SIRPα antibody or antigen-binding fragment thereof that binds to an epitope distinct from that bound by HEFLB or hu1H9G4.

[0178]

[0177] As used herein, "HEFLB" means an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:68 and a light chain variable region having the amino acid sequence of SEQ ID NO:69.

[0179]

[0178] As used herein, "hu1H9G4" refers to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:70 and a light chain variable region having the amino acid sequence of SEQ ID NO:71.

[0180]

[0179] Table 6 shows the amino acid sequences of VH and VL of HEFLB and hu1H9G4.

[0180]

[0181] [Table 7]

[0182] Antibody variants

[0182] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0183] In certain embodiments, antibody variants comprise one or more of the CDR sequences provided in Tables 1 and 3 above, one or more of the non-CDR sequences of the heavy or light chain variable regions provided in Tables 2 and 4 above, and / or one or more modifications or substitutions in the constant region (e.g., Fc region). Such variants retain the binding specificity of their parent antibodies for SIRPα, but have one or more desirable properties conferred by the modifications or substitutions. For example, antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or eliminated effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0184]

[0184] Parent antibody sequences may be screened to identify suitable or preferred residues for modification or substitution 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 with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are produced and screened for the desired properties. If substitution at a particular amino acid position demonstrates a desired change in function, that position can be identified as a potential residue for modification or substitution. Potential residues may be further evaluated by substituting different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0185] Affinity variants

[0186] The affinity variants of the antibodies may include modifications or substitutions in one or more CDR sequences provided in Tables 1 and 3 above, one or more FR sequences provided in Table 5 above, or the heavy or light chain variable region sequences provided in Tables 2 and 4 above. Since it is known in the art that a CDR region is adjacent to two FR regions in the variable region, the FR sequences can be easily identified by those skilled in the art based on the CDR sequences in Tables 1 and 3 above and the variable region sequences in Tables 2 and 4 above. The affinity variants retain the specific binding affinity of the parent antibody to SIRPα, or even have improved specific binding affinity of SIRPα over the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences include conservative substitutions.

[0186]

[0187] Those skilled in the art will understand that in the CDR sequences provided in Tables 1 and 3 above, and 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 may still retain or even have improved binding affinity or capacity to SIRPα. To this end, various methods known in the art may be used. For example, phage display technology may be used to generate and express a library of antibody variants (such as Fab or scFv variants), which are then screened for binding affinity to human SIRPα. As another example, computer software may be used to virtually mimic the binding of an antibody to human SIRPα, and amino acid residues on the antibody that form the binding interface may be identified. Such residues may be avoided in substitutions to prevent a reduction in binding affinity, or may be targeted for substitution to provide stronger binding.

[0187]

[0188] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid residue substitutions in one or more of the CDR sequences and / or in one or more of the FR sequences. In certain embodiments, the affinity variant comprises a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in the CDR sequences and / or in the FR sequences.

[0188]

[0189] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDRs listed in Tables 1 and 3 above, yet retains specific binding affinity for SIRPα at similar or even higher levels than its parent antibody.

[0189]

[0190] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the variable region sequences listed in Tables 2 and 4 above, and retains specific binding affinity for SIRPα at a similar or even higher level than its parent antibody. In some embodiments, a total of 1-10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Tables 2 and 4 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., within the FRs).

[0190]

[0191] Glycosylation variants

[0192] The anti-SIRPα antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants obtained by increasing or decreasing the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0191]

[0193] The antibody or antigen-binding fragment thereof may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of an antibody is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, e.g., an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by modifying the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can likewise be created by introducing such a tripeptide sequence or a serine or threonine residue.

[0192]

[0194] In certain embodiments, the anti-SIRPα antibodies and antigen-binding fragments provided herein contain a mutation at N297 (eg, N297A, N297Q, or N297G) to remove a glycosylation site.

[0193]

[0195] Cysteine ​​engineered variants

[0196] The anti-SIRPα antibodies or antigen-binding fragments thereof provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0194]

[0197] A free cysteine ​​residue is a residue that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugating, for example, with cytotoxic and / or imaging compounds, labels, or especially radioisotopes, at the site of engineered cysteine, for example, by maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0195]

[0198] Fc variants

[0199] The anti-SIRPα antibodies or antigen-binding fragments thereof provided herein also include Fc variants comprising modification or substitution of one or more amino acid residues in the Fc region and / or hinge region to provide altered effector functions, such as, for example, ADCC and CDC. Methods for modifying ADCC activity by antibody engineering have been described in the 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 See 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.

[0196]

[0200] The CDC activity of the antibodies or antigen-binding fragments provided herein can also be modified, for example, by improving or diminishing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent 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 modify C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see, e.g., U.S. Patent No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions can also be introduced to modify the ability of the antibody to fix complement (see, PCT Publication WO 94 / 29351 by Bodmer et al.).

[0197]

[0201] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein can be of the IgG1, IgG2, IgG3, or IgG4 isotype and have reduced effector function, as disclosed herein.

[0198]

[0202] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life because they bind to FcRn at acidic pH, allowing it to escape degradation in lysosomes and then be translocated and released extracellularly. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are 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).

[0199]

[0203] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that facilitate and / or promote heterodimerization at the interface of the Fc region. These modifications include the introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, where the protuberance is located in the cavity to promote the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0200]

[0204] antigen-binding fragment

[0205] Anti-SIRPα antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-SIRPα antibodies provided herein, including, for example, the exemplary antibodies whose CDRs are shown in Tables 1 and 3 above and whose variable sequences are shown in Tables 2 and 4 above, as well as various variants thereof (affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0201]

[0206] In certain embodiments, the anti-SIRPα antigen-binding fragments provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

[0202]

[0207] A variety of techniques can be used to produce such antigen-binding fragments. Illustrative 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 such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0203]

[0208] In certain embodiments, the antigen-binding fragment is an scFv. The generation of scFvs is described, for example, in WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. ScFvs can be fused to effector proteins at the amino or carboxyl terminus to provide fusion proteins (see, for example, Antibody Engineering, edited by Borrebaeck).

[0204]

[0209] In certain embodiments, the anti-SIRPα antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0205]

[0210] A bivalent molecule is monospecific if both binding sites are specific for binding to the same antigen or epitope. In certain embodiments, this provides stronger binding to antigen or epitope than its monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, 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 the same specificity but different sequence).

[0206]

[0211] Bivalent molecules 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 if the two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0207]

[0212] bispecific antibody

[0213] In certain embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is bispecific.

[0208]

[0214] In certain embodiments, the anti-SIRPα antibody or its antigen-binding fragment can specifically bind to a second antigen other than SIRPα.In certain embodiments, the second antigen is a tumor antigen, a tumor surface antigen, or an infectious agent surface antigen.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).

[0209]

[0215] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second epitope on SIRPα.

[0216] Conjugates

[0217] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof further comprises one or more conjugate moieties. The conjugate moieties can be linked to the antibody or antigen-binding fragment thereof. The conjugate moieties are moieties that can bind to the antibody or antigen-binding fragment thereof. It is contemplated that various conjugate moieties can be linked to the antibody or antigen-binding fragment thereof provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among others. In some embodiments, the antibody or antigen-binding fragment thereof can be linked to one or more conjugates via a linker.

[0210]

[0218] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific sites in addition to the epitope-binding moiety that are available for attachment to one or more conjugate moieties. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, that facilitate covalent linkage to the conjugate moiety.

[0211]

[0219] In certain embodiments, the antibody or antigen-binding fragment thereof may be indirectly linked to the conjugate moiety or through another conjugate moiety. For example, the antibody or antigen-binding fragment thereof provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate moiety comprises a clearance modifier (e.g., a polymer such as PEG that extends 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 binder, a purification moiety, or other anti-cancer drug.

[0212]

[0220] A "toxin" can be any agent that is detrimental to or damages or kills a cell. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethate, 5-fluorouracil decarbazine), 5-fluorouracil decarbazine, ... These include, without limitation, cisplatin, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(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 binders.

[0213]

[0221] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, lucerifers, glucoamylase, lysozyme, saccharide oxidases, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14C. 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, plastid moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0214]

[0222] In certain embodiments, the conjugate moiety may be a clearance modifier that helps to extend the half-life of the antibody. Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and others. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and when two or more polymers are attached, they may be the same or different molecules.

[0215]

[0223] In certain embodiments, the conjugated moiety can be a purification moiety, such as a magnetic bead.

[0224] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is used as the basis for a conjugate.

[0216]

[0225] Polynucleotides and Recombinant Methods

[0226] The present disclosure provides an isolated polynucleotide encoding the anti-SIRPα antibody or antigen-binding fragment thereof provided herein. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as encompasses the sequence explicitly indicated. 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)).

[0217]

[0227] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0218]

[0228] The isolated polynucleotide encoding the anti-SIRPα antibody or antigen-binding fragment thereof can be inserted into a vector for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0219]

[0229] The present disclosure provides a vector comprising the isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable 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, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, p GEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, p Includes LexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0220]

[0230] A vector containing a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or DNA expression in a vector herein are prokaryotic cells, yeast, or higher eukaryotic cells as described above. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Escherichia, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0221]

[0231] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-SIRPα antibody-encoding vectors. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, Schizosaccharomyces pombe is capable of inhibiting Kluyveromyces hosts such as 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 (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces Many other genera, species, and strains are commonly available and useful herein, such as Schwanniomyces, e.g., A. occidentalis, and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger.

[0222]

[0232] Suitable host cells for the expression of the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

[0223]

[0233] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, 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 Sertoli 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 carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and human hepatocellular carcinoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and their derivatives.

[0224]

[0234] The host cells are transformed with the above expression or cloning vectors for the production of anti-SIRPα antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, the antibodies may be produced by homologous recombination as known in the art. In certain embodiments, the host cells are capable of producing the antibodies or antigen-binding fragments thereof provided herein.

[0225]

[0235] The present disclosure also provides a method of expressing an antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which a vector of the present disclosure is expressed. The host cells used to produce the antibody or antigen-binding fragment thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. Additionally, 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. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of skill in the art may also be included at appropriate concentrations. Temperature, pH, and other culture conditions will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0226]

[0236] When recombinant techniques are used, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris of host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, for example, a Pellicon ultrafiltration unit from Amicon or Millipore. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of adventitious contaminants.

[0227]

[0237] The anti-SIRPα antibody or antigen-binding fragment thereof prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0228]

[0238] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0229]

[0239] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0230]

[0240] Pharmaceutical Compositions

[0241] The disclosure further provides pharmaceutical compositions comprising an anti-SIRPα antibody, or antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers.

[0231]

[0242] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestrants or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, ingredients known in the art, or various combinations thereof.

[0232]

[0243] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments thereof and conjugates provided herein reduces oxidation of the antibodies or antigen-binding fragments thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving the stability of the antibodies and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that include one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further provided are methods of preventing oxidation, extending shelf life, and / or improving efficacy of the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0233]

[0244] To further illustrate, pharma- ceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride for injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphates or citrates; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as Polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Pharmaceutical compositions in multi-dose containers may contain antimicrobial agents utilized as carriers, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0234]

[0245] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations, or powders. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0235]

[0246] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for producing liquid solution, suspension, emulsion. Preparations for injection may include sterile and / or non-pyrogenic solutions ready for injection, sterile dry soluble products such as lyophilized powders ready for mixing with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products ready for mixing with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solutions may be aqueous or non-aqueous.

[0236]

[0247] In certain embodiments, unit dose preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration should be sterile and nonpyrogenic, as known and practiced in the art.

[0237]

[0248] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological factors of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, at about neutral pH in one embodiment. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-SIRPα antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial by a small amount (e.g., about 10%) more than needed for a dose or set of doses is permissible to facilitate accurate sample removal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0238]

[0249] The lyophilized powder is reconstituted with water for injection to provide a formulation for use in parenteral administration.In one embodiment, for reconstitution, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder.The exact amount depends on the selected therapy given and can be determined empirically.

[0239]

[0250] kit

[0251] In certain embodiments, the present disclosure provides kits comprising an antibody or antigen-binding fragment thereof provided herein.

[0240]

[0252] In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a targeting antibody that binds to a target antigen expressed on a target cell. In certain embodiments, the target cell may be a tumor cell, an inflammatory cell, and / or a chronically infected cell that expresses CD47.

[0241]

[0253] In certain embodiments, the target antigen is a tumor antigen, a tumor surface antigen, or an infectious agent surface antigen.

[0254] In certain embodiments, the kit further comprises an additional therapeutic agent, which may be an anti-cancer therapeutic agent, an anti-inflammatory agent, or an anti-infective agent.

[0242]

[0255] In certain embodiments, the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiation therapy, immunotherapy agents, anti-angiogenic agents, targeted therapy, cell therapy, gene therapy, hormone therapy, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0243]

[0256] Such kits may further include, if desired, for example, a container containing one or more pharma- ceutically acceptable carriers, additional containers, and one or more of a variety of other conventional pharmaceutical kit components, as will be readily apparent to one of skill in the art. Instructions as an insert or label indicating the amounts of the components to be administered, guidelines for administration, and / or guidelines for mixing the components may also be included in the kit.

[0244]

[0257] How to use

[0258] In another aspect, the disclosure provides a method of inducing phagocytosis of a target cell in vitro, the method comprising contacting a target cell with a SIRPα-positive phagocytic cell sample in the presence of an antibody or antigen-binding fragment thereof provided herein, thereby inducing phagocytosis of the target cell by the SIRPα-positive phagocytic cells.

[0245]

[0259] In another aspect, the disclosure provides a method of inducing phagocytosis of a target cell in vitro, the method comprising contacting a target cell with a sample of SIRPα-positive phagocytic cells in the presence of an antibody or antigen-binding fragment thereof provided herein and a targeting antibody that specifically binds to a target antigen on the target cell, thereby inducing phagocytosis of the target cell by the SIRPα-positive phagocytic cells.

[0246]

[0260] In some embodiments, the target cell is a CD47-expressing cell.

[0261] In one aspect, the disclosure provides a method of inducing phagocytosis of a target cell in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in a dose effective to induce phagocytosis of the target cell.

[0247]

[0262] In one aspect, the disclosure provides a method of inducing phagocytosis of a target cell in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in combination with a targeting antibody that specifically binds to a target antigen on a target cell, in a dose effective to induce phagocytosis of the target cell.

[0248]

[0263] In one aspect, the present disclosure provides a method of increasing antibody-dependent cellular phagocytosis (ADCP) action of a targeting antibody against a target cell in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in combination with a targeting antibody having an Fc region, thereby increasing ADCP of the targeting antibody against the target cell, wherein the targeting antibody binds to a target antigen expressed on the target cell. In certain embodiments, the targeting antibody binds to a target antigen expressed on the target cell, and the ADCP action of the targeting antibody against the target cell is increased. The target cell may be a tumor cell, an inflammatory cell, and / or a chronically infected cell that expresses CD47.

[0249]

[0264] In one aspect, the disclosure provides a method of enhancing a target antibody (e.g., an anti-CD20 antibody, an anti-PD-L1 antibody, and an anti-claudin 18.2 antibody) in the treatment of a disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in combination with the target antibody (e.g., an anti-CD20 antibody, an anti-PD-L1 antibody, and an anti-claudin 18.2 antibody), thereby enhancing the target antibody in the treatment of the disease, disorder, or condition in the subject. As used herein, the term "enhance" or "enhancing" means to increase therapeutic efficacy.

[0250]

[0265] In certain embodiments, the targeting antibody has an Fc region. In certain embodiments, the disease, disorder, or condition is an immune-related disease or disorder, tumors and cancers, autoimmune diseases, or infectious diseases. In certain embodiments, the immune-related disease or disorder is selected from the group consisting of systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), vasculitis, myasthenia gravis, idiopathic pulmonary fibrosis, Crohn's disease, asthma, rheumatoid arthritis, graft-versus-host disease, spondyloarthropathy (e.g., ankylosing spondylitis, psoriatic arthritis, sporadic acute enteropathic arthritis with inflammatory bowel disease, reactive arthritis, Behcet's disease, undifferentiated spondyloarthropathy, anterior uveitis, and juvenile idiopathic arthritis), multiple sclerosis, endometriosis, glomerulonephritis, sepsis, diabetes, acute coronary syndrome, ischemia-reperfusion, psoriasis, progressive systemic sclerosis, atherosclerosis, Sjogren's syndrome, scleroderma, or inflammatory autoimmune myositis.

[0251]

[0266] In certain embodiments, the conditions or disorders treatable by the methods provided herein include tumors and cancers. Examples of cancers and tumors include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large infarction, and other hematological malignancies, such as leukemia, lymphoma, myeloma, mycosis fungoides ... B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, as well as EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasms of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis tumors, brain stem gliomas, anal Histological cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct 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 tract cancer, skin cancer, prostate cancer, pituitary gland cancer, vaginal cancer, thyroid cancer, throat cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma, or metastases thereof.

[0252]

[0267] In another aspect, the present disclosure also provides a method of treating a disease, disorder, or condition that can benefit from the induction of phagocytosis of target cells in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein.

[0253]

[0268] In another aspect, the disclosure also provides a method of treating a disease, disorder, or condition that can benefit from the induction of phagocytosis of target cells in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in combination with a targeting antibody that specifically binds to a target antigen on a target cell.

[0254]

[0269] In another aspect, the present disclosure also provides a method of treating a SIRPα-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein.

[0255]

[0270] In another aspect, the present disclosure also provides a method of treating a SIRPα-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in combination with a targeting antibody that specifically binds to a target antigen on a target cell associated with the SIRPα-related disease.

[0256]

[0271] In some embodiments, the target cell is a CD47 expressing cell. In some embodiments, the target cell comprises a cancer cell, an inflammatory cell, and / or a chronically infected cell.

[0272] In certain embodiments, when the antibodies or antigen-binding fragments thereof provided herein are used in combination with a targeting antibody, the antibodies or antigen-binding fragments thereof provided herein can induce selective phagocytosis of target cells over non-target cells (e.g., those that do not express the target antigen).

[0257]

[0273] In some embodiments, the target cell expresses a target antigen. In some embodiments, the target antigen is a tumor antigen, a tumor surface antigen, an inflammatory antigen, or an antigen of an infectious microorganism. In some embodiments, the target antigen can be a tumor antigen (e.g., a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), such as a neoantigen), or an antigen presented on an infected cell (e.g., Hepatitis B surface antigen (HBsAg)).

[0258]

[0274] In some embodiments, the subject is a human. In some embodiments, the subject is SIRPα v1 homozygous. In some embodiments, the subject is SIRPα v2 homozygous. In some embodiments, the subject is SIRPα v1 / v2 heterozygous.

[0259]

[0275] In some embodiments, the subject has a disease, disorder, or condition selected from the group consisting of cancer, a solid tumor, a chronic infection, an inflammatory disease, multiple sclerosis, an autoimmune disease, a neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, graft dysfunction, and arthritis.

[0260]

[0276] 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. As used herein, "CD47-positive" cancer refers to a cancer characterized by expression of CD47 protein in cancer cells or expression of CD47 in cancer cells at a significantly higher level than would be expected in normal cells. The presence and / or amount of CD47 in a biological sample of interest may indicate whether the subject from which the biological sample is derived is likely to respond to an anti-SIRPα antibody. Various methods can be used to determine the presence and / or amount of CD47 in a test biological sample obtained from a subject. For example, the test biological sample may be exposed to an anti-CD47 antibody or an antigen-binding fragment thereof that binds to and detects the expressed CD47 protein. Alternatively, CD47 can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, etc. In some embodiments, the test sample is derived from a cancer cell or tissue, or a tumor-infiltrating immune cell. In certain embodiments, the presence or upregulated level of CD47 in the test biological sample indicates the likelihood of responsiveness. The term "upregulated" as used herein refers to an overall increase in the expression level of CD47 in the test sample of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or more, compared to the expression level of CD47 in a reference sample detected using the same method. The reference sample may be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained. For example, the reference sample may be a non-disease sample adjacent or near the test sample (e.g., tumor). The reference level may be the CD47 expression level found in normal cells of the same tissue type, optionally normalized to the expression level of another gene (e.g., housekeeping gene). Alternatively, the reference level may be the CD47 expression level found in healthy subjects.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 individual from which the test sample is obtained.In some embodiments, the reference is tested and / or determined substantially simultaneously with the test or determination of interest.In some embodiments, the reference is a historical reference, optionally embodied in a tangible medium.Typically, as understood by those skilled in the art, the reference is determined or characterized under conditions or circumstances that are comparable to those that are evaluated.

[0261]

[0277] In certain of these embodiments, an antibody or antigen-binding fragment thereof provided herein that is administered in combination with a targeting antibody or one or more additional therapeutic agents may be administered simultaneously with the targeting antibody or one or more additional therapeutic agents, or in certain of these embodiments, the antibody or antigen-binding fragment thereof and the targeting antibody or additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof that is administered "in combination" with a targeting antibody or additional therapeutic agent may not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof that is administered before or after a targeting antibody or another agent is considered to be administered "in combination" with the agent, as that phrase is used herein, even if the antibody or antigen-binding fragment and the targeting antibody or second agent are administered via different routes. Where possible, the targeted antibody or additional therapeutic agent administered in combination with the antibody or antigen-binding fragment thereof disclosed herein will be administered according to the schedule listed in the product information sheet of that additional therapeutic agent, or according to protocols known in the art, such as those set forth in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)).

[0262]

[0278] In another aspect, methods are provided for treating a disease, disorder, or condition in a subject that would benefit from modulating the activity of SIRPα, comprising administering to a subject in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, and / or a pharmaceutical composition provided herein. In certain embodiments, the disease or condition is a disease, disorder, or condition associated with SIRPα.

[0263]

[0279] The therapeutically effective amount of an antibody or antigen-binding fragment provided herein will depend on a variety of factors known in the art, such as, for example, weight, age, past medical history, current medications, the subject's health status and potential for cross-reactivity, allergies, sensitivities, and adverse side reactions, as well as the route of administration and the extent of disease occurrence. Dosages may be proportionately reduced or increased by the practitioner (e.g., physician or veterinarian) as dictated by these and other circumstances or requirements.

[0264]

[0280] In some embodiments, the antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0265]

[0281] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response), and may for example be administered as a single dose or as several divided doses administered over time.

[0266]

[0282] The antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0267]

[0283] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered alone or in combination with a therapeutically effective amount of an additional therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with an additional therapeutic agent, such as chemotherapeutic agents, anti-cancer drugs, radiation therapy agents, immunotherapy agents, anti-angiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormonal therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0268]

[0284] As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or enhances the immune system in a general manner. Examples of immunotherapies include, without limitation, checkpoint regulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0269]

[0285] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or that are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to the growth and survival of cancer. Targeted therapy targets therapeutic agents to the tumor, thereby sparing normal tissues from the effects of the therapeutic agent.

[0270]

[0286] In another aspect, the disclosure further provides a method of modulating the activity of SIRPα in a SIRPα positive cell, comprising exposing the SIRPα positive cell to an antibody or antigen-binding fragment thereof provided herein, hi some embodiments, the SIRPα positive cell is a phagocytic cell (e.g., a macrophage).

[0271]

[0287] In another aspect, the disclosure provides a method for detecting the presence or amount of SIRPα in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of SIRPα in the sample.

[0272]

[0288] In another aspect, the disclosure provides a method of diagnosing a SIRPα-related disease, disorder, or condition in a subject comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of SIRPα in the sample; and c) correlating the presence or amount of SIRPα with the presence or status of the SIRPα-related disease, disorder, or status in the subject.

[0273]

[0289] In another aspect, the disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety, useful for detecting a SIRPα-related disease, disorder, or condition. The kit may further comprise instructions for use.

[0274]

[0290] In another aspect, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating, preventing, or ameliorating a SIRPα-related disease, disorder, or condition in a subject, and in the manufacture of a diagnostic reagent for diagnosing a SIRPα-related disease, disorder, or condition.

[0275]

[0291] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are within the scope of the invention, either in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention, but merely illustrate specific embodiments within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive ability and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while remaining within the boundaries of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. EXAMPLES

[0276] Example 1. Preparation of Reagents 1.1. Generation of Reference Antibodies

[0261] The DNA sequences encoding the variable regions of anti-SIRPα reference antibodies HEFLB (see US20140242095) or hu1H9G4 (see WO2019 / 023347A1) were cloned into vectors expressing human IgG constant regions. The variable region amino acid sequences of HEFLB and hu1H9G4 are shown in Table 6 of the present disclosure. Expi293 cells (Invitrogen) transfected with the expression plasmids were cultured at 37°C for 5 days. The culture medium was then collected and centrifuged to remove the cell pellet. The collected supernatant was purified by Protein A affinity chromatography. Both HEFLB and hu1H9G4 are human IgG4 monoclonal antibodies with the S228P mutation in the constant region.

[0277] Generation of cell lines stably expressing SIRPα, SIRPβ and SIRPγ

[0263] DNA sequences encoding full-length human SIRPα v1 (NP_542970), human SIRPβ (O00241), cynomolgus monkey SIRPα (NP_001271679), or C57BL / 6 mouse SIRPα (NP_031573) were cloned into pIRES vector (Clontech), respectively. Human SIRPγ (Q9P1W8) expression plasmid was purchased from Sino Biological (HG16111-CF).

[0278]

[0264] 293F cells (Invitrogen) transfected with human SIRPα v1 or human SIRPγ expression plasmids were selectively cultured to obtain and confirm stable clones.

[0279]

[0265] In a similar manner, CHOK1 cells (Invitrogen) transfected with human SIRPα v1, human SIRPβ, cynomolgus monkey SIRPα, or C57BL / 6 mouse SIRPα expression plasmids were selectively cultured and stable clones were obtained and confirmed.

[0280]

[0266] CHOK1 cell line stably expressing exogenous human SIRPα v2 (CAA71403.1) was purchased from KYinno (KC-1720). 1.3. Production of Recombinant Proteins Recombinant proteins human IgG Fc (hFc) tagged human CD47 extracellular domain (ECD, NP_001768.1, M1-E141), human SIRPα v1 ECD (NP_542970, M1-R370), human SIRPα v2 ECD (CAA71403.1, M1-R369), or human SIRPγ ECD (Q9P1W8, M1-P360) were produced by Chempartner. Recombinant proteins 6xHis tagged C57BL / 6 mouse SIRPα ECD, human SIRPβL ECD (NP_001129316.1), and mouse human IgG Fc (mFc) tagged human CD47 ECD, human SIRPα v1 were purchased from Biointron. Recombinant proteins 6xHis-tagged human SIRPα v1 ECD, human SIRPα v2 ECD, human SIRPβ ECD (O00241) were purchased from Sino Biological.

[0281] Example 2. Generation of antibodies 2.1. Preparation of Immunogens for Protein Immunization

[0270] hFc-tagged human SIRPα v1 ECD recombinant protein was used as the immunogen for protein immunization (see Example 1.3).

[0282] 2.2. Preparation of immunogens for cell immunization

[0272] 293F cells stably expressing human SIRPα v1 were used as the immunogen for cell immunization (see Example 1.2).

[0283] 2.3. Preparation of Immunogens for Genetic Immunization

[0274] The DNA sequence encoding the full-length human SIRPα v1 protein (NP_542970) was cloned into the pCP vector (Chempartner). The prepared plasmid was then coated onto a colloidal gold bullet (Bio-Rad) as an immunogen for genetic immunization.

[0284]

[0275] 2.4.Immunization Balb / c and SJL / J mice (SLAC) were immunized by three different strategies: protein immunization using human SIRPα v1 ECD recombinant protein, cell immunization using 293F cells stably expressing human SIRPα v1, and gene immunization using gold bullets coated with human SIRPα v1 expression plasmid. ELISA assay using human SIRPα v1 ECD recombinant protein and FACS assay using CHOK1 cells stably expressing human SIRPα v1 were used to detect the serum titers of immunized mice. Mice with high serum titers were selected for hybridoma fusion.

[0285] 2.5. Generation of Hybridomas Five days after the last booster immunization, mice were sacrificed and splenocytes were harvested. 1% (v / v) NH4OH was added to lyse red blood cells. The washed splenocytes were then fused with SP2 / 0 mouse myeloma cells (ATCC) by high-efficiency electrofusion or PEG method. After cell fusion, the fused cells were diluted with 2×10 4 Cells were seeded into 96-well plates at a density of 100 cells / well.

[0286] 2.6. Hybridoma screening

[0280] 10-12 days after fusion, the fusion plates were screened primarily by ELISA assay using human SIRPα v1 ECD recombinant protein or Acumen assay (TTP Labtech) using CHOK1 cells stably expressing human SIRPα v1 and v2. For secondary screening, the hybridoma cells obtained from the positive wells were expanded and placed into 24-well plates. In secondary screening, the binding activity was evaluated by ELISA assay using human SIRPα v1 and v2 ECD recombinant protein and FACS assay using CHOK1 cells stably expressing human SIRPα v1. The clones with top binding activity to different human SIRPα variants were selected for subcloning. In addition, specificity to human SIRPα / β / γ, interspecies cross-reactivity, blocking activity of the interaction between CD47 and SIRPα were also detected in secondary screening for hybridoma characterization (see Example 3 for characterization assay method).

[0287] 2.7. Hybridoma Subclones

[0282] The hybridoma cells of each selected clone were seeded in 96-well plates at a density of 1 cell / well by limiting dilution. The plates were screened in the same manner as the primary hybridoma screening (see Example 2.6). Positive single clones were selected and characterized in the same manner as the secondary hybridoma screening (see Example 2.6). Then, monoclonal hybridoma cell lines with top binding activity were obtained for further hybridoma antibody production, characterization, and sequencing. A total of seven antibody clones were identified as functional hits, and the hybridoma antibodies purified from these clones were assigned as 005, 015, 025, 042, 059, 071, and 073, respectively (Example 3).

[0288] Example 3. Antibody Characterization 3.1. Hybridoma Antibody Production and Purification After about 14 days of culture, the hybridoma cell culture medium was collected and centrifuged to remove cells. After filtering through a 0.22 μm PES membrane and adjusting the pH to 7.4, the collected 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 immediately neutralized using 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 using an Endochrome-K kit (Charles River).

[0289] 3.2. Detection of binding specificity

[0286] The binding specificity of purified hybridoma antibodies to human SIRPα variants was detected by ELISA assay using recombinant protein Fc-tagged human SIRPα v1 ECD and human SIRPα v2 ECD. Briefly, the antibodies were incubated with antigen coated on ELISA microplates at 37°C for 1 hour. After washing, anti-mouse IgG secondary Ab (Sigma) labeled with horseradish peroxidase (HRP) 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 stopped by adding 50 μl of 1N HCl. OD 450nm was read and EC was calculated using GraphPad Prism 9.0. 50 was calculated. The binding specificity properties of HEFLB and the seven functional antibodies are summarized in Table 8. All antibodies tested, except HEFLB, bind to both human SIRPα v1 and human SIRPα v2. HEFLB can only bind to human SIRPα v1, but not to human SIRPα v2.

[0290] 3.3. Detection of interspecies cross-reactivity

[0288] The cross-species reactivity of purified hybridoma antibodies to human, cynomolgus monkey, and mouse SIRPα was determined by FACS assay using human SIRPα v1, CHOK1-cynomolgus monkey SIRPα, and CHOK1 cells stably expressing C57BL / 6 mouse SIRPα. Briefly, antibodies were diluted at 2×10 5 The cells were incubated with 1000 target cells for 1 h at 4° C. After washing, a fluorescently labeled anti-mouse IgG secondary antibody (Life Technologies) was added and incubated for 1 h at 4° C. The geometric median fluorescence intensity was detected and EC 50 was calculated. The species cross-reactivity properties of HEFLB and the seven functional antibodies are summarized in Table 8. All antibodies tested, except HEFLB, are capable of binding to cynomolgus monkey SIRPα. None of the antibodies tested are capable of binding to C57BL / 6 mouse SIRPα.

[0291] Detection of blocking activity of CD47 / SIRPα interaction A competitive ELISA assay was used to determine whether the purified hybridoma antibodies could block the interaction of CD47 with SIRPα. Briefly, the antibodies and biotin-labeled soluble human SIRPα v1 ECD recombinant protein were co-incubated with human CD47 ECD recombinant protein coated on an ELISA microplate. After washing, HRP-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 stopped by adding 50 μl of 1N HCl. OD 450 nm was read. The blocking ratio was determined by blocking the binding of biotin-labeled human SIRPα v1 ECD recombinant protein to human CD47 ECD recombinant protein coated on an ELISA microplate. IC calculated using GraphPad Prism 9.050 and top blocking ratios are summarized in Table 8. Except for 005, all antibodies tested are able to block the interaction of human CD47 with human SIRPα v1.

[0292]

[0291] 3.5. Detection of hemagglutination activity Anti-CD47 antibodies may promote red blood cell (RBC) aggregation, which may lead to safety risks. Purified hybridoma antibodies were tested for hemagglutination activity. Briefly, human RBCs were diluted to 10% in PBS and incubated at 37°C for 1 hour in the presence of 100 nM antibody. Evidence of hemagglutination is indicated by the presence of loose RBCs, which appear faintly compared to the punctuate red dots of non-hemagglutinated RBCs. Hemagglutination index was determined by quantifying the area of ​​the RBC pellet in the presence of antibody and normalizing it to that in the absence of antibody. As summarized in Table 8, all seven functional antibodies did not show hemagglutination activity.

[0293] 3.6. Detection of SHP-1 Recruitment

[0294] The efficacy of purified hybridoma antibodies to block CD47 / SIRPα-mediated “don’t eat me” signaling was assessed by a cell-based SHP-1 recruitment assay (Figure 8A). Full-length human SIRPα v1 was engineered to have a beta-gal fragment (ED) fused to its C-terminus, and the SH2 domain of SHP-1 was engineered to have a complementary beta-gal fragment (EA). These constructs were stably expressed in K562 cells. Ligand binding through co-culture with human CD47-expressing cells results in phosphorylation of the SIRPα-ED fusion protein, triggering recruitment of SHP-1-EA, which forces the creation of active beta-gal enzyme. This active enzyme hydrolyzes the substrate to generate chemiluminescence as a measure of reporter activity. Blocking ratios were determined by blocking beta-gal enzyme activity. As summarized in Table 8, anti-SIRPα hybridoma antibodies 005, 015, 025, 042, 059, 071, and 073 potently disrupted CD47 / SIRPα-mediated "don't eat me" signaling. These seven antibodies were considered to be functional hits.

[0294]

[0295] 3.7. Sequencing of Hybridomas

[0296] Total RNA isolated from monoclonal hybridoma cells was reverse transcribed into cDNA using either isotype-specific antisense primers or universal primers according to the SMARTScribe reverse transcriptase technical manual. The cDNA was then used as a template to amplify heavy and light chain antibody fragments according to the GenScript rapid amplification of cDNA ends (RACE, rapid amplification of cDNA ends) standard operating procedure (SOP). The amplified antibody fragments were cloned separately into standard cloning vectors. Colony PCR was performed to screen for clones with the correct size insert, and the insert fragments were analyzed by DNA sequencing. Finally, consensus sequences were identified as the heavy and light chain antibody variable regions.

[0295] Example 4. Generation and characterization of chimeric antibodies

[0297] 4.1. Generation and Production of Chimeric Antibodies

[0298] According to the hybridoma sequencing results, the mouse anti-SIRPα functional hits were converted into human IgG4 chimeric antibodies with S228P mutation for characterization. Briefly, the DNA sequence encoding the heavy chain variable region was cloned into pcDNA3.4-hIgG4P vector (Biointron) with human IgG4 heavy chain constant region with S228P mutation. The DNA sequence encoding the light chain variable region was cloned into pcDNA3.4-hIgGk vector (Biointron) with human kappa light chain constant region. Expi293 cells (Life Technologies) co-transfected with antibody heavy and light chain expression plasmids were grown at 37°C for 5 days. The resulting chimeric antibodies are referred to herein as 005c, 015c, 025c, 042c, 059c, 071c, and 073c, where the suffix "c" indicates chimeric.

[0296]

[0299] 4.2. Characterization of Chimeric Antibodies

[0300] 4.2.1 Detection of binding specificity

[0301] The binding activity of the purified chimeric antibodies to human SIRPα variants was detected by FACS assay using CHOK1 cells (Figures 1A and 1B) or 293F cells (Figure 1C) stably expressing human SIRPα v1, and CHOK1 cells (Figures 2A and 2B) stably expressing human SIRPα v2. As shown in Figures 1A, 1B, and 1C, all the tested antibodies strongly bind to cell surface human SIRPα v1. As shown in Figure 2, all the tested antibodies, except HEFLB, bind to cell surface human SIRPα v2. EC calculated using GraphPad Prism 9.0 50 and the top signals are summarized in Table 9.

[0297]

[0302] The binding activity of the purified chimeric antibodies to SIRPβ and SIRPβl was detected by ELISA assay using recombinant proteins human SIRPβ ECD (Figures 3A and 3B), human SIRPβl ECD (Figures 3D and 3E), and by FACS assay using CHOK1 cells stably expressing human SIRPβ (Figure 3C). As shown in Figures 3A-3C, all the tested antibodies bound to human SIRPβ at different levels, among which 042c, 071c, and 073c had weak binding. As shown in Figures 3D and 3E, all the tested antibodies bound strongly to human SIRPβl. EC calculated using GraphPad Prism 9.0 50 and the top signals are summarized in Table 10.

[0298]

[0303] The binding activity of the purified chimeric antibodies to SIRPγ was detected by FACS assay using recombinant protein cynomolgus monkey SIRPγ ECD (Figure 4C) and by FACS assay using 293F cells stably expressing human SIRPγ (Figures 4A and 4B). As shown in Figures 4A and 4B, all the tested antibodies bind to human SIRPγ at different levels, among which 042c, 059c, 071c, and 073c have very weak binding. As shown in Figure 4C, 005c, 042c, and 073c have very weak binding to cynomolgus monkey SIRPγ, which correlates with the binding activity to human SIRPγ. EC calculated using GraphPad Prism 9.0 50 and the top signals are summarized in Table 11.

[0299]

[0304] 4.2.2 Detection of interspecies cross-reactivity

[0305] The species cross-reactivity of the purified chimeric antibodies was detected by ELISA assay using recombinant protein C57BL / 6 mouse SIRPα ECD (Figure 5A) and by FACS assay using CHOK1 cells stably expressing cynomolgus monkey SIRPα (Figures 5B and 5C). All the antibodies tested bind to cynomolgus monkey SIRPα at different levels, but have no species cross-reactivity to C57BL / 6 mouse SIRPα. EC calculated using GraphPad Prism 9.0 50 and the top signals are summarized in Table 12.

[0300]

[0306] 4.2.3. Detection of blocking activity of CD47 / SIRPα interaction

[0307] A competitive ELISA assay was used to determine whether the purified chimeric antibodies could block the interaction of CD47 with SIRPα. Briefly, the antibodies and mFc-tagged human CD47 ECD recombinant protein were co-incubated with human SIRPα v1 ECD (Figures 6A and 6B) or human SIRPα v2 ECD (Figures 7A and 7B) recombinant protein coated on an ELISA microplate. After washing, HRP-labeled anti-mouse Fc secondary 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 stopped by adding 50 μl of 1N HCl. OD 450 nm was read. Blocking ratios were determined by blocking the binding of human CD47 ECD recombinant protein to human SIRPα ECD recombinant protein coated on an ELISA microplate. IC calculated using GraphPad Prism 9.0 50 and top blocking ratios are summarized in Table 13. Except for 005, all tested antibodies are able to block the interaction between human CD47 and different human SIRPα variants.

[0301]

[0308] 4.2.4.SHP-1 Recruitment Assay

[0309] The efficacy of the purified chimeric antibodies in blocking CD47 / SIRPα-mediated "don't eat me" signaling was assessed by a cell-based SHP-1 recruitment assay (Figure 8B, see methods described in Example 3.6). 50 and top blocking ratios were calculated using GraphPad Prism 9.0. As summarized in Table 14, all tested antibodies can disrupt CD47 / SIRPα-mediated "don't eat me" signaling at different levels. Notably, 005c does not block the interaction of CD47 with SIRPα, but can inhibit SHP-1 recruitment to the SIRPα intracellular tail brought about by CD47 binding.

[0302]

[0310] 4.2.5. Affinity detection

[0311] The purified chimeric antibodies were characterized for their binding affinity to human SIRPα v1, human SIRPα v2 using Biolayer Interferometry technology (Octet system). The association and dissociation curves were fitted to a 1:1 binding model, and Ka / Kd / KD values ​​were calculated for each antibody. The affinity data Ka / Kd / KD values ​​for each antibody are summarized in Table 15.

[0303]

[0312] 4.2.6. Epitope Analysis

[0313] A competitive ELISA assay was used for epitope binning of purified chimeric antibodies. Briefly, excess competing antibodies and mFc-tagged human SIRPα v1 ECD recombinant protein were co-incubated with antibodies coated on ELISA microplates. After washing, HRP-labeled anti-mouse Fc secondary 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 stopped by adding 50 μl of 1N HCl. OD 450 nm was read. The competitive ratio was calculated. Antibodies that can compete with each other for binding to SIRPα may have related binding epitopes. As shown in Table 16, 025c did not show competitive binding with 042c, 073c, and hu1H9G4 to human SIRPα, indicating that it may bind to different epitopes. Competition between 042c, 073c, and hu1H9G4 was not bidirectional, indicating that their binding epitopes, although related, may not be completely identical.

[0304]

[0314] Epitope mapping of 025c, 042c, 073c, HEFLB, and hu1H9G4 was further performed using hydrogen deuterium exchange mass spectrometry (HDX-MS). As shown in Figure 9A, 025c binding resulted in low hydrogen deuterium exchange ratios for the region YNQKEGHFPRVTTVSDL of His-tagged human SIRPα v1 ECD, indicating that these amino acids may be important for 025c binding. As shown in Figure 9B, 042c binding resulted in low hydrogen deuterium exchange ratios for two regions SGAGTEL and TNVDPVGESVS of His-tagged human SIRPα v1 ECD, indicating that these amino acids may be important for 042c binding. As shown in Figure 9C, 073c binding resulted in low hydrogen deuterium exchange ratios for the region TNVDPVGESVSY of His-tagged human SIRPα v1 ECD, indicating that these amino acids may be important for 073c binding. Notably, these three regions are not located in the IgV domain of SIRPα ECD where CD47 binds, indicating that 042c and 073c may function as allosteric antibodies that block the interaction of CD47 with SIRPα or that the blocking activity of 042c and 073c is a steric hindrance effect. As shown in Figure 9D, hu1H9G4 binding resulted in a low hydrogen deuterium exchange ratio for the region YNQKEGHFPRVTTVSDL of His-tagged human SIRPα v1 ECD, indicating that these amino acids may be important for hu1H9G4 binding. As shown in Figure 9E, HEFLB binding resulted in a low hydrogen deuterium exchange ratio for the region VGPIQW of his-tagged human SIRPα v1 ECD, indicating that these amino acids may be important for HEFLB binding.

[0305]

[0315] Taking the competitive ELISA data and the HDX-MS data together, it is concluded that 025c, 042c, and 073c may have different binding epitopes, which are also distinct from the reference antibodies hu1H9G4 and HEFLB.

[0306]

[0316] 4.2.7.In Vitro Phagocytosis Assay

[0317] The functional efficacy of purified chimeric antibodies was assessed by a flow cytometry-based phagocytosis assay. Briefly, M0 non-polarized or M1 polarized human monocyte-derived macrophages with different SIRPA genotypes were co-cultured with CellTrace Violet (Life Technologies)-labeled CD47-expressing cancer cells in the presence of the tested antibodies. Phagocytosis was assayed by determining the percentage of macrophages positive for cell trace violet dye. For non-polarized macrophages, peripheral blood mononuclear cells were seeded in 10 cm tissue culture plates in 1640 supplemented with 10% FBS and 50 ng / ml M-CSF for 7–9 days. Adherent cells were harvested as M0 non-polarized macrophages. For M1 polarized macrophages, peripheral blood mononuclear cells were seeded in 10 cm tissue culture plates in 1640 supplemented with 10% FBS and 50 ng / ml GM-CSF for 5 days. 50 ug / ml IFNγ and 100 ug / ml LPS were added for an additional 2-4 days of culture. Adherent cells were harvested as M1 polarized macrophages.

[0307]

[0318] As shown in Figure 10A, 015c, 025c, 042c, 059c, 071c, and 073c did not show single-agent activity in enhancing tumor cell uptake of Raji cells by M0 macrophages obtained from SIRPA heterozygous v1 / v2 individuals. However, in the presence of rituximab (anti-CD20 antibody), all other purified chimeric antibodies tested enhanced macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) of Raji cells, except for 059c, which had weak activity in blocking the interaction between human CD47 and human SIRPα v2.

[0308]

[0319] As shown in Figure 10B, except for 059c, all other purified antibodies tested, regardless of the presence of cetuximab (anti-EGFR antibody), effectively enhanced tumor cell uptake of DLD-1 cells by M0 macrophages obtained from SIRPA homozygous v2 / v2 individuals.

[0309]

[0320] Combinations of SIRPα antibodies plus PD-L1 antibodies were tested in phagocytosis assays using M0 macrophages obtained from SIRPA homozygous v1 / v1 (Figure 10C) or v2 / v2 (Figure 10D) individuals. In the presence of PD-L1 antibodies, 005c, 025c, 042c, and 073c effectively enhanced macrophage-mediated ADCP of Raji cells stably expressing PD-L1.

[0310]

[0321] The combination of 025c plus PD-L1 antibody C71, and 025c plus rituximab were also tested in phagocytosis using M0 non-polarized or M1 polarized macrophages obtained from SIRPA homozygous v1 / v1 individuals. M1 polarized macrophages (Figure 11B) showed weaker phagocytic ability compared to M0 non-polarized macrophages (Figure 11A). Regardless of macrophage polarization status, in the presence of PD-L1 antibody or rituximab, 025c effectively enhanced macrophage-mediated ADCP of Raji cells stably expressing PD-L1. The PD-L1 heavy chain antibody C71 has the VH amino acid sequence shown below.

[0311]

[0322] Anti-PD-L1 heavy chain antibody C71.VH, SEQ ID NO:91:

[0323] EVQVVESGGGLVQSGGSLKLSCAGSGFTESAGFMVWHRQVPGKERELVALIATPSGSTNYADSVKGRFTISRDNGKNTVYLQMNSLKPEDTAVYYCNIRGYWGQGTLVTVSS

[0324] These data suggest that the antibodies or antigen-binding fragments thereof provided herein, when used in combination with an antibody specific for a target antigen of a particular tumor cell, enhance macrophage-mediated ADCP of such tumor cells.

[0312]

[0325] 4.2.8.In Vivo Antitumor Activity

[0326] Human CD47 / human SIRPα double knock-in mice were inoculated with MC38 cells stably expressing human CD47 and human claudin 18.2 (CLDN18.2). Treatment groups included vehicle (PBS), isotype control, 10 mg per kg (mpk) of anti-CLDN18.2 mAb (22E12), and a combination of 10 mpk of anti-CLDN18.2 mAb (22E12) plus 3 or 10 mpk of anti-SIRPα mAb. Tumors were 70–75 mm 3 Treatment was initiated when the tumor reached an average volume of 1000 mg / kg / day. Mice were dosed intraperitoneally (IP) 5 times, twice a week. Tumor volumes were measured twice a week. Three days after the last dose, mice were sacrificed and tumors were weighed. Statistical analysis was performed by one-way or two-way anova comparing the average tumor weight / volume of the different treatment groups with that of the isotype control group. As shown in Figures 12A and 12B, the combination of 10mpk of anti-CLDN18.2 mAb (22E12) plus 10mpk of anti-SIRPα significantly inhibited MC38 tumor growth. Six of six tumors were regressed in the 10mpk 025c combination group, one of six tumors in the 3mpk 042c combination group, and two of six tumors in the 10mpk 042c combination group (Figure 12C). The VH and VL amino acid sequences of anti-CLDN18.2 mAb (22E12) are shown below.

[0313]

[0327] Anti-CLDN18.2 mAb (22E12) VH, SEQ ID NO: 88

[0328] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNWVHWVRQAPGQGLEWMGEINPTNARSNYNEKFKKRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARIYYGNSFAHWGQGTLVTVSS

[0329] Anti-CLDN18.2 mAb (22E12) VL, SEQ ID NO: 89

[0330] DIVMTQSPDSLAVSLGERATINCKSSQSLLNAGNQKNYLTWYQQKPGQPPKLLIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNNYYYPLTFGGGTKLEIK

[0331] Mixed Lymphocyte Reaction Assay (MLR)

[0332] Binding of human T cells to antigen-presenting cells through SIRPγ-CD47 interactions was reported to costimulate T cell proliferation. Since some of the purified chimeric antibodies strongly bind human SIRPγ (FIG. 4), to exclude the possibility of interfering with T cell proliferation and activation, the purified chimeric antibodies were tested in an MLR assay. Briefly, CellTrace Violet-labeled human primary T cells were stimulated with in vitro generated allogeneic mature dendritic cells for 5 days. The indicated antibodies were added at saturating concentrations (100 nM) from the beginning of the study. Low staining of CellTrace Violet was used to determine the proliferating population. IFNγ secretion was determined with a human IFN gamma kit (Cisbio). As shown in FIG. 13, 015c, 025c, 042c, 059c, 071c, and 073c, regardless of their binding activity to human SIRPγ, suppressed IFNγ secretion (FIG. 13A), CD4 + T cell proliferation (Figure 13B), and CD8 + It showed no significant effect on T cell proliferation (Figure 13C). As expected, the anti-SIRPγ antibody LSB2.20 (Biolegend) is a potent inhibitor of T cell activation. In particular, hu1H9G4 showed a clear inhibition of IFNγ secretion and T cell proliferation in this assay.

[0314] Example 5. Antibody Humanization

[0333] 5.1. Humanization

[0334] CDR grafting method was used for humanization of 025c. Briefly, IGHV1-69-2*01 and IGKV3-11*01 were first selected as heavy and light chain humanization templates, respectively, based on their homology to the original mouse antibody sequence. CDRs were then defined using Kabat's definition, except for heavy chain CDR1, which was defined using a combination of Kabat and Chothia's systems. For grafting, CDRs with potential hot spots removed and different combinations of canonical residues from 025c were grafted onto the template, and the resulting variants (human IgG4 antibodies with S228P mutation in the constant region) were expressed by a 96-well high-throughput protein expression system. All variants produced were tested by FACS assay, and top binders to human SIRPα v1 and human SIRPα v2 were selected for further characterization. The resulting humanized antibodies with good binding activity were designated as hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060, where the prefix "hu" indicates "humanized" and the number suffix represents the serial number of the humanized antibody.

[0315]

[0335] 5.2. Characterization of Humanized Antibodies

[0336] 5.2.1. Detection of binding specificity

[0337] The binding activity of the humanized antibodies to human SIRPα variants was detected by FACS assay using CHOK1 cells stably expressing human SIRPα v1 (FIG. 14A), human SIRPα v2 (FIG. 14B), or human SIRPβ (FIG. 14C), and 293F cells stably expressing human SIRPγ (FIG. 14D). All humanized antibodies tested were confirmed to retain binding activity to SIRP family members similar to that of the parent antibody 025c. EC calculated using GraphPad Prism 9.0 50 and the top signals are summarized in Table 17.

[0316]

[0338] 5.2.2. Detection of blocking activity of CD47 / SIRPα interaction

[0339] The humanized antibodies were tested for their ability to block the interaction between CD47 and SIRPα using a competitive ELISA assay (see methods described in Example 4.2.3.). As shown in Figure 15, all humanized antibodies tested were confirmed to retain activity in blocking the interaction between human CD47 and different human SIRPα variants similar to the parent antibody 025c. IC calculated using GraphPad Prism 9.0 50 and upper blocking ratios are summarized in Table 18.

[0317]

[0340] To further compare the blocking activity of humanized and reference antibodies, a competitive FACS assay was also set up. Briefly, the antibodies and mFc-tagged human CD47 ECD recombinant protein were co-incubated with CHOK1 cells stably expressing human SIRPα v1 (FIG. 16A) or human SIRPα v2 (FIG. 16B). After washing, dye-labeled anti-mouse Fc secondary antibody (Sigma) was added and incubated at 37° C. for 1 h. The fluorescence intensity was detected. The blocking ratio was determined by blocking human CD47 ECD recombinant protein binding to CHOK1 cells expressing SIRPα. Hu1H9G4 showed weak activity in blocking the interaction of human CD47 with human SIRPα v2. Notably, HEFLB has no function at all with respect to human SIRPα v2. IC calculated using GraphPad Prism 9.0 50 and upper blocking ratios are summarized in Table 19.

[0318]

[0341] 5.2.4. SHP-1 Recruitment Assay

[0342] The efficacy of the humanized antibodies in blocking CD47 / SIRPα-mediated "don't eat me" signaling was assessed by a cell-based SHP-1 recruitment assay (see FIG. 17, methods described in Example 3.6). All humanized antibodies tested were confirmed to retain activity in blocking SHP-1 recruitment to the SIRPα intracellular tail upon CD47 binding similar to the parent antibody 025c. IC calculated using GraphPad Prism 9.0 50 and upper blocking ratios are summarized in Table 20.

[0319]

[0343] 5.2.5. Affinity detection

[0344] The humanized antibodies were characterized for binding affinity to human SIRPα v2, human SIRPα v1 using surface plasmon resonance technology (Biacore system). Association and dissociation curves were fitted to a 1:1 binding model, and Ka / Kd / KD values ​​were calculated for each antibody. The affinity data Ka / Kd / KD values ​​for each antibody are summarized in Table 21.

[0320]

[0345] 5.2.6.In Vitro Phagocytosis Assay

[0346] For in vitro functional validation, combinations of SIRPα antibodies plus PD-L1 antibodies or rituximab were tested in phagocytosis assays using M0 macrophages obtained from SIRPA homozygous v1 / v1 (Figures 18A and 18B), v2 / v2 (Figures 18C and 18D), or heterozygous v1 / v2 individuals (see methods described in Example 4.2.7). All humanized antibodies tested were confirmed to retain activity in enhancing macrophage-mediated ADCP of Raji cells stably expressing PD-L1 in the presence of PD-L1 antibodies or rituximab, similar to parental antibody 025c. Reference antibodies and 005c were also tested in parallel in these assays. As shown in Figures 18A and 18B, 005c, which can block CD47-binding induced SHP-1 recruitment to the intracellular tail of SIRPα but does not block the interaction of CD47 with SIPRα, effectively enhanced macrophage-mediated ADCP of Raji cells stably expressing PD-L1 in the presence of PD-L1 antibody or rituximab. As shown in Figures 18C, 18D, and 18E, HEFLB, which cannot bind human SIRPα v2, did not function at all with macrophages obtained from SIRPA homozygous v2 / v2 and heterozygous v1 / v2 individuals.

[0321] [Table 8]

[0322] [Table 9]

[0323] [Table 10]

[0324] [Table 11]

[0325]

Table 12

[0326]

Table 13

[0327]

Table 14

[0328]

Table 15

[0329]

Table 16

[0330]

Table 17

[0331]

Table 18

[0332]

Table 19

[0333]

Table 20

[0334]

Table 21

Claims

1. An antibody or antigen-binding fragment thereof that can specifically bind to human SIRPα, comprising a heavy-chain variable region containing HCDR1, HCDR2, and HCDR3 and / or a light-chain variable region containing LCDR1, LCDR2, and LCDR3, a) HCDR1 comprises the amino acid sequence of DYYMS (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence of FIKNEANGYTTESTASVK G (SEQ ID NO: 2), HCDR3 comprises the amino acid sequence of YDYYGSNYNWYFDA (SEQ ID NO: 3), LCDR1 comprises the amino acid sequence of KASQNVRTAVA (SEQ ID NO: 4), LCDR2 comprises the amino acid sequence of LASKRHT (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequence of LQHWIHPLT (SEQ ID NO: 6), b) HCDR1 comprises the amino acid sequence of X1YYMH (SEQ ID NO: 18), HCDR2 comprises the amino acid sequence of RIDPEDX2EX3KYAPKFQG (SEQ ID NO: 19), HCDR3 comprises the amino acid sequence of GX18X4X5Y (SEQ ID NO: 20), LCDR1 comprises the amino acid sequence of SASSSSVSSSYLY (SEQ ID NO: 10), LCDR2 comprises the amino acid sequence of STSNLA S (SEQ ID NO: 11), and LCDR3 comprises the amino acid sequence of X6QWSSYPYT (SEQ ID NO: 21), c) HCDR1 comprises the amino acid sequence of TYGM S (SEQ ID NO: 22), HCDR2 comprises the amino acid sequence of WINTYSGVX19TX7ADD FX8G (SEQ ID NO: 38), HCDR3 comprises the amino acid sequence of DPHX9YGX10SPA WF X11Y (SEQ ID NO: 39), LCDR1 comprises the amino acid sequence of X12ASQX13VGI X14VA (SEQ ID NO: 40), LCDR2 comprises the amino acid sequence of SASSNRX15T (SEQ ID NO: 41), and LCDR3 comprises the amino acid sequence of QQYSX16YPX17T (SEQ ID NO: 42), or d) HCDR1 comprises the amino acid sequence of EYVL S (SEQ ID NO: 43), HCDR2 comprises the amino acid sequence of EIYPGTITTYYNEKFKG (SEQ ID NO: 44), HCDR3 comprises the amino acid sequence of FYDYDG GWFAY (SEQ ID NO: 45), LCDR1 comprises the amino acid sequence of SASSSSVSSSDLH (SEQ ID NO: 46), LCDR2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 47), and LCDR3 comprises the amino acid sequence of QQWSGYPW T (SEQ ID NO: 48), X １ is A or D, and X ２ is G or A, and X ３ is T or S, and X ４ is L or Y, and X ５ is E or A, and X ６ is Y or H, and X ７ is Y or C, and X ８ is K or Q, and X ９ is Y or S, and X １０ is N or T or S, and X １１ is P or A or V, and X １２ is E or K, and X １３ is N or I, and X １４ is S or A, and X １５ is Y or F, and X １６ is S or T or A, and X １７ is F or L, and X １８ is S or absent, and X １９ is S or P, the above antibody or its antigen-binding fragment. **Claim 2** a) HCDR1 comprises the amino acid sequence of AYYMH (SEQ ID NO: 7) or DYYMH (SEQ ID NO: 13), b) HCDR2 comprises an amino acid sequence selected from the group consisting of RIDPEDGESKYAPKFQG (SEQ ID NO: 8), RIDPEDGETKYAPKFQG (SEQ ID NO: 14), and RIDPEDAE TKYAPKFQG (SEQ ID NO: 17), c) HCDR3 comprises the amino acid sequence of GSYEY (SEQ ID NO: 9) or GLAY (SEQ ID NO: 15), d) LCDR1 comprises the amino acid sequence of SASSSSVSSSYLY (SEQ ID NO: 10), e) LCDR2 comprises the amino acid sequence of STSNLAS (SEQ ID NO: 11), and f) LCDR3 comprises the amino acid sequence of YQWSSYPYT (SEQ ID NO: 12) or HQWSSYPYT (SEQ ID NO: 16), The antibody or antigen-binding fragment thereof according to claim 1. **Claim 3** a) HCDR1 comprises the amino acid sequence of TYGM S (SEQ ID NO: 22), b) HCDR2 comprises an amino acid sequence selected from the group consisting of WINTYSGVSTCADDFKG (SEQ ID NO: 23), WINTYSGVPTYADD FQG (SEQ ID NO: 28), and WINTYSGVPTYADD FKG (SEQ ID NO: 33), c) HCDR3 comprises an amino acid sequence selected from the group consisting of DPHSYGNSPAWF PY (SEQ ID NO: 24), DPHYYGTSPAWF AY (SEQ ID NO: 29), and DPHYYGS SPAWF VY (SEQ ID NO: 34), d) LCDR1 comprises an amino acid sequence selected from the group consisting of KASQNVGISVA (SEQ ID NO: 25), KASQIVGI AVA (SEQ ID NO: 30), and EASQIVGI AVA (SEQ ID NO: 35), e) LCDR2 comprises an amino acid sequence selected from the group consisting of SASNRYT (SEQ ID NO: 26) and SASNRF T (SEQ ID NO: 31), and f) LCDR3 comprises an amino acid sequence selected from the group consisting of QQYS SYPLT (SEQ ID NO: 27), QQYS TYPFT (SEQ ID NO: 32), and QQYS AYPFT (SEQ ID NO: 37), The antibody or antigen-binding fragment thereof according to claim 1. **Claim 4** a) The HCDR1 contains the sequence of SEQ ID NO: 1, the HCDR2 contains the sequence of SEQ ID NO: 2, the HCDR3 contains the sequence of SEQ ID NO: 3, the LCDR1 contains the sequence of SEQ ID NO: 4, the LCDR2 contains the sequence of SEQ ID NO: 5, the LCDR3 contains the sequence of SEQ ID NO: 6, or b) The HCDR1 contains the sequence of SEQ ID NO: 7, the HCDR2 contains the sequence of SEQ ID NO: 8, the HCDR3 contains the sequence of SEQ ID NO: 9, the LCDR1 contains the sequence of SEQ ID NO: 10, the LCDR2 contains the sequence of SEQ ID NO: 11, the LCDR3 contains the sequence of SEQ ID NO: 12, or c) The HCDR1 contains the sequence of SEQ ID NO: 13, the HCDR2 contains the sequence of SEQ ID NO: 14 or SEQ ID NO: 17, the HCDR3 contains the sequence of SEQ ID NO: 15, the LCDR1 contains the sequence of SEQ ID NO: 10, the LCDR2 contains the sequence of SEQ ID NO: 11, the LCDR3 contains the sequence of SEQ ID NO: 16, or d) The HCDR1 contains the sequence of SEQ ID NO: 22, the HCDR2 contains the sequence of SEQ ID NO: 23, the HCDR3 contains the sequence of SEQ ID NO: 24, the LCDR1 contains the sequence of SEQ ID NO: 25, the LCDR2 contains the sequence of SEQ ID NO: 26, the LCDR3 contains the sequence of SEQ ID NO: 27, or e) The HCDR1 contains the sequence of SEQ ID NO: 22, the HCDR2 contains the sequence of SEQ ID NO: 28, the HCDR3 contains the sequence of SEQ ID NO: 29, the LCDR1 contains the sequence of SEQ ID NO: 30, the LCDR2 contains the sequence of SEQ ID NO: 31, the LCDR3 contains the sequence of SEQ ID NO: 32, or f) The HCDR1 contains the sequence of SEQ ID NO: 22, the HCDR2 contains the sequence of SEQ ID NO: 33, the HCDR3 contains the sequence of SEQ ID NO: 34, the LCDR1 contains the sequence of SEQ ID NO: 35, the LCDR2 contains the sequence of SEQ ID NO: 26, the LCDR3 contains the sequence of SEQ ID NO: 37, or g) The HCDR1 contains the sequence of SEQ ID NO: 43, the HCDR2 contains the sequence of SEQ ID NO: 44, the HCDR3 contains the sequence of SEQ ID NO: 45, the LCDR1 contains the sequence of SEQ ID NO: 46, the LCDR2 contains the sequence of SEQ ID NO: 47, the LCDR3 contains the sequence of SEQ ID NO: 48, The antibody or an antigen-binding fragment thereof according to claim 1.

5. Further comprising heavy chain HFR1, HFR2, HFR3, and HFR4, and light chain LFR1, LFR2, LFR3, and LFR4, a) HFR1 comprises EVQLVQSGAEVKPGATVKISCKX ２０ SGFNIK (SEQ ID NO: 84) or a homologous sequence having at least 80% sequence identity thereto, b) HFR2 contains the sequence of WVQQAPGKGL EWIG (SEQ ID NO: 74) or a homologous sequence having at least 80% sequence identity thereto, c) The HFR3 sequence is RVTITADTSTX ２１ contains TAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 85) or a homologous sequence having at least 80% sequence identity thereto, d) HFR4 comprises a homologous sequence having at least 80% sequence identity with WGQGTVTVSS (SEQ ID NO: 76), e) LFR1 comprises a homologous sequence having at least 80% sequence identity with EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 77), f) LFR2 comprises a homologous sequence having at least 80% sequence identity with WYQQKPGQAPKLWIY (SEQ ID NO: 78), g) LFR3 is GIPARFSGSGSGTDX ２２ contains TLTLISSLPEPDFAVYYC (SEQ ID NO: 86) or a homologous sequence having at least 80% sequence identity thereto, and h) LFR4 comprises a homologous sequence having at least 80% sequence identity with FGQG TKLEIK (SEQ ID NO: 80), X ２０ is A or V, and X ２１ is N or D, and X ２２ is Y or F, the antibody or antigen-binding fragment thereof according to claim 1. **Claim 6** a) HFR1 comprises a homologous sequence having at least 80% sequence identity with EVQLVQSGAEVK KPGATVKISCKASGFNIK (SEQ ID NO: 83) or EVQLVQSGAEVK KPGATVKISCKVSGFNIK (SEQ ID NO: 73), and / or b) HFR2 comprises a homologous sequence having at least 80% sequence identity with WVQQAPGK GLEWIG (SEQ ID NO: 74), and / or c) The HFR3 sequence comprises a homologous sequence having at least 80% sequence identity with RVTITADTSTNTAYMELSSLRS EDTAVYYCDR (SEQ ID NO: 75) or RVTITADTSTD TAYMELSSLRS EDTAVYYCDR (SEQ ID NO: 82), and / or d) HFR4 comprises a homologous sequence having at least 80% sequence identity with WGQGTVTVSS (SEQ ID NO: 76), and / or e) LFR1 comprises a homologous sequence having at least 80% sequence identity with EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 77), and / or f) LFR2 comprises a homologous sequence having at least 80% sequence identity with WYQQKPGQAPKLWIY (SEQ ID NO: 78), and / or g) LFR3 comprises a homologous sequence having at least 80% sequence identity with GIPARFSGSGSGTDYTLTISSL EPEDFAVYYC (SEQ ID NO: 79) or GIPARFSGSGSGTDFTLTISSL EPEDFAVYYC (SEQ ID NO: 81), and / or h) The antibody or antigen-binding fragment thereof according to claim 5, wherein LFR4 comprises a homologous sequence having at least 80% sequence identity with FGQG TKLEIK (SEQ ID NO: 80). **Claim 7** The heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 65, and SEQ ID NO: 67, and a homologous sequence having at least 80% sequence identity and retaining specific binding affinity for human SIRPα; the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 64 and SEQ ID NO: 66, and a homologous sequence having at least 80% sequence identity and retaining specific binding affinity for human SIRPα. The antibody or antigen-binding fragment thereof according to claim 1.

8. a) the heavy chain variable region comprises the sequence of SEQ ID NO: 49 and the light chain variable region comprises the sequence of SEQ ID NO: 50, or b) the heavy chain variable region comprises the sequence of SEQ ID NO: 51 and the light chain variable region comprises the sequence of SEQ ID NO: 52, or c) the heavy chain variable region comprises the sequence of SEQ ID NO: 53 and the light chain variable region comprises the sequence of SEQ ID NO: 54, or d) the heavy chain variable region comprises the sequence of SEQ ID NO: 55 and the light chain variable region comprises the sequence of SEQ ID NO: 56, or e) the heavy chain variable region comprises the sequence of SEQ ID NO: 57 and the light chain variable region comprises the sequence of SEQ ID NO: 58, or f) the heavy chain variable region comprises the sequence of SEQ ID NO: 59 and the light chain variable region comprises the sequence of SEQ ID NO: 60, or g) the heavy chain variable region comprises the sequence of SEQ ID NO: 61 and the light chain variable region comprises the sequence of SEQ ID NO: 62, or h) the heavy chain variable region comprises the sequence of SEQ ID NO: 63 and the light chain variable region comprises the sequence of SEQ ID NO: 64, or i) the heavy chain variable region comprises the sequence of SEQ ID NO: 63 and the light chain variable region comprises the sequence of SEQ ID NO: 66, or j) the heavy chain variable region comprises the sequence of SEQ ID NO: 65 and the light chain variable region comprises the sequence of SEQ ID NO: 64, or k) the heavy chain variable region comprises the sequence of SEQ ID NO: 67 and the light chain variable region comprises the sequence of SEQ ID NO: 64, or l) the heavy chain variable region comprises the sequence of SEQ ID NO: 67 and the light chain variable region comprises the sequence of SEQ ID NO:

66. The antibody or antigen-binding fragment thereof according to claim 1.

9. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an Fc region, optionally an Fc region of human immunoglobulin (Ig), or optionally an Fc region of human IgG.

10. The Fc region is derived from human IgG4, and optionally, the Fc region derived from human IgG4 comprises the mutation S228P and / or the mutation L235E. The antibody or antigen-binding fragment thereof according to claim 9.

11. a) humanized; b) a monoclonal antibody, bispecific antibody, multispecific antibody, recombinant antibody, chimeric antibody, labeled antibody, bivalent antibody, anti-idiotype antibody, or fusion protein; and / or c) diabody, Fab, Fab’, F(ab’) ２ , Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ２ , bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody; the antibody according to claim 1 or an antigen-binding fragment thereof.

12. bispecific, a) capable of specifically binding to a second antigen other than SIRPα, optionally wherein the second antigen is a tumor antigen, tumor surface antigen, inflammatory antigen, or antigen of an infectious microorganism; or b) capable of specifically binding to a second epitope on SIRPα; the antibody according to claim 1 or an antigen-binding fragment thereof.

13. linked to one or more conjugate moieties, optionally wherein the conjugate moiety comprises a clearance modifier, chemotherapeutic agent, toxin, radioisotope, lanthanide, luminescent label, fluorescent label, enzyme substrate label, DNA alkylating agent, topoisomerase inhibitor, tubulin binder, purification moiety, or other anti-cancer drug, the antibody according to claim 1 or an antigen-binding fragment thereof.

14. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 13 or an antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

15. An isolated polynucleotide encoding the antibody according to any one of claims 1 to 13 or an antigen-binding fragment thereof.

16. A disease, disorder, or condition in which a benefit can be obtained by inducing phagocytosis of target cells in a subject, or the treatment, prevention, or alleviation of an SIRPα-related disease, disorder, or condition in a subject, or the induction of phagocytosis of target cells in a subject, or the increase in the antibody-dependent cellular phagocytosis (ADCP) effect of a target antibody on target cells in a subject, the antibody according to any one of claims 1 to 13 or an antigen-binding fragment thereof, optionally in combination with a target antibody that specifically binds to a target antigen on the target cells.

17. Use according to claim 16, wherein the target cells are CD47-expressing cells.

18. the disease, disorder, or condition is cancer, solid tumor, 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, graft dysfunction, or arthritis, optionally, a) the cancer is anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, lung cancer, bronchial cancer, bone cancer, hilar cholangiocarcinoma, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, ureteropelvic cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal tract cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), multiple myeloma, T or B cell lymphoma, GI stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma; and / or b) the cancer is a CD47-positive cancer; The use according to claim 16.

19. The use according to claim 16, wherein the subject is a human.

20. The use according to claim 16, further comprising the step of administering a therapeutically effective amount of an additional therapeutic agent.

21. The method according to claim 20, wherein the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiation therapy agents, immunotherapy agents, anti-angiogenesis agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, anti-viral agents, antibiotics, analgesics, antioxidants, metal chelating agents, cytokines, anti-infective agents, and anti-inflammatory agents.

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