WO2024125330A1

WO2024125330A1 - ANTIBODIES AGAINST SIRPα AND USES THEREOF

Info

Publication number: WO2024125330A1
Application number: PCT/CN2023/136008
Authority: WO
Inventors: Ying Lei; Puwei YUAN; Junli Liu; Bingshi GUO; Fan Liu
Original assignee: Beijing Neox Biotech Ltd
Current assignee: Beijing Neox Biotech Ltd
Priority date: 2022-12-16
Filing date: 2023-12-04
Publication date: 2024-06-20
Anticipated expiration: 2025-06-16

Abstract

This present application relates to an isolated antibody or antigen binding fragment thereof binding to SIRPα protein or the extracellular domain thereof, wherein the antibody is capable to induce phagocytosis of tumor cells by macrophages independently, without disruption of the SIRPγ/CD47 interaction. Also provided herein are the pharmaceutical composition and use of the antibody or the antigen binding fragment thereof.

Description

ANTIBODIES AGAINST SIRPα AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to antibodies or antigen binding fragments thereof binding to SIRPα protein and the use thereof in the treatment of diseases.

BACKGROUND OF THE INVENTION

Signal regulatory protein α (SIRPα) is an innate immune checkpoint receptor expressed primarily on myeloid cells, including monocyte, macrophages, dendritic cells, and neutrophils (Ho CCM, et al., J. Biol. Chem., 2015; 290 (20) : 12650–63) . An SIRPα suppresses innate immunity upon interaction with its ligand, CD47. The CD47/SIRPα interaction regulates macrophage and dendritic cell phagocytosis of tumor cells, sending an inhibitory “do-not-eat-me” signal to the phagocyte. CD47 is upregulated by virtually all human tumors to escape macrophage recognition and programmed cell removal (Willingham SB, et al., Proc. Natl. Acad. Sci., 2012; 109 (17) : 6662–67) . Targeting of the CD47/SIRPα signaling axis is emerging as a promising therapeutic intervention.

Numerous agents blocking the CD47/SIRPα innate immune checkpoint have been developed thus far including anti-CD47 and anti-SIRPα antibodies, and soluble SIRPαFc, of which several are currently being evaluated in clinical trials. However, due to the broad expression of CD47 on virtually all normal cells, including red blood cells and platelets, the systemic use of anti-CD47 antibody is thought to be hampered, which is manifested by severe depletion of RBCs and platelets, leading to acute anemia and thrombocytopenia in treated patients (Advani R, et al., N. Engl. J. Med., 2018; 379 (18) : 1711–21; Sikic BI, et al., J. Clin. Oncol., 2019; 37: 946–53) . Therefore, developing an anti-SIRPα antibody may display a favorable safety profile due to the more restricted expression of SIRPα. Additionally, relatively low doses of an anti-SIRPα antibody are anticipated to be necessary to inhibit CD47/SIRPα signaling in tumors, in contrast to CD47 targeting agents, which require higher doses to effectively saturate the pathway.

SIRPα belongs to the SIRP family of immunoreceptors that include the highly homologous activating receptor SIRPβ1 and the decoy receptor SIRPγ (Matlung HL, et al., Immunol. Rev., 2017; 276 (1) : 145–64) . Both SIRPα and SIRPβ are expressed in myeloid lineage cells, while SIRPγ is expressed on T-cells, NK cells and NKT cells. SIRPβ1 has no extracellular binding interaction with CD47, whereas SIRPγ binds to CD47 albeit with a 10-fold weaker affinity than SIRPα (Brooke G, et al., J. Immunol., 2004; 173 (4) : 2562–70) . CD47/SIRPγ appears to be important for mediating adhesion between T cell and APC and for T cell functions including proliferation and activation. Human SIRPα is highly polymorphic and SIRPα V1, SIRPα V2 and SIRPα V8 are the most prominent haplotypes present among the human population (Erik Voets, et al., J. Immunother. Cancer, 2019; 7 (1) : 340-354) .

Despite of several anti-SIRPα antibodies in early clinical (OSE-172) or preclinical development, some defects of them are yet to be overcome and there remains a significant need for novel SIPRα antibodies with improved properties, such as binding to different SIRPα variants, cross-reaction with other animals, and inducing phagocytosis as single agents, as well as therapeutic and diagnostic uses thereof.

SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen binding fragments thereof binding to SIRPα protein and the use thereof.

In one aspect, provided is an antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment bind to SIRPα protein or the extracellular domain thereof. In some embodiments, the SIRPα protein comprises the amino acid sequence as shown by SEQ ID NO: 22, or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the amino acid sequence as shown by SEQ ID NO: 22.

The antibody provided herein comprises or consists of: (i) a light chain variable region (VL) comprising the identical LCDR1, LCDR2, and LCDR3 with those in any one of SEQ ID NO: 11 to SEQ ID NO: 14 or SEQ ID NO: 16; and (ii) a heavy chain variable region (VH) comprising the identical HCDR1, HCDR2, and HCDR3 with those in any one of SEQ ID NO: 7 to SEQ ID NO: 10 and SEQ ID NO: 15; wherein the HCDRs and LCDRs are defined by any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition, or defined by a hybrid scheme that combines any two, three or four of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition. In some embodiments, the heavy chain variable region comprises:

HCDR1 sequence comprising or consisting of the amino acid sequence as shown by SYYIH (SEQ ID NO: 1) , HCDR2 sequence comprising or consisting of the amino acid sequence as shown by WIDPGNLNTKYNEKFTG (SEQ ID NO: 2) , and HCDR3 sequence comprising or consisting of the amino acid sequence as shown by LNYYGNYGDF (SEQ ID NO: 3) ; and the light chain variable region comprises LCDR1 sequence comprising or consisting of the amino acid sequence as shown by KSSQSLLNSGNQRNYLA (SEQ ID NO: 4) , LCDR2 sequence comprising or consisting of the amino acid sequence as shown by GASIRES (SEQ ID NO: 5) , and LCDR3 sequence comprising or consisting of the amino acid sequence as shown by QHDHSYPLT (SEQ ID NO: 6) ; wherein the CDR sequences are defined according to the Kabat CDR definition.

In some embodiments, the antibody is a chimeric antibody, humanized antibody, or human antibody. In some embodiments, the antibody is an antibody of other species.

In some embodiments, in the antibody provide herein, the heavy chain variable region and the light chain variable region further comprise a human acceptor framework. In some embodiments, the human acceptor framework derives immunoglobulin variable regions. In some embodiments, the human acceptor framework comprises human consensus framework. In some embodiments, the human acceptor framework of the heavy chain variable region derives from any immunoglobulin heavy chain variable region germline selected from the group consisting of: IGHV1-3, IGHV1-46, IGHV1-2, and IGHV1-69, and variants thereof. In some embodiments, the human acceptor framework of the heavy chain variable region comprises one or more amino acid residues selected from the group consisting of: 27Y, 60N, 61E, 64T, 71A, 73K and 78A, and wherein the amino residues are numbered according to the Kabat numbering system. In some embodiments, the human acceptor framework of the heavy chain variable region derives from IGHV1-69, and comprises one or more amino acid residues selected from the group consisting of: 27Y, 60N, 61E, 64T, 71A, 73K and 78A, and wherein the amino residues are numbered according to the Kabat numbering system. In some embodiments, the human acceptor framework of the heavy chain variable region is generated by introducing amino acid substitutions G27Y, A60N, Q61E, and Q64T to the frame work region of IGHV1-69, and wherein the amino residues are numbered according to the Kabat numbering system. In some embodiments, the human acceptor framework of the light chain variable region derives from an immunoglobulin Kappa chain. In some embodiments, the human acceptor framework of the light chain variable region derives from any immunoglobulin Kappa chain variable region germline selected from the group consisting of: IGKV3-15, IGKV1-39, IGKV2-28, and IGKV4-1, and variants thereof. In some embodiments, the human acceptor framework of the light chain variable region comprises the amino acid residue 83L, and wherein the amino residues are numbered according to the Kabat numbering system. In some embodiments, the human acceptor framework of the light chain variable region derives from IGKV1-39 and comprises the amino acid residue 83L, and wherein the amino residues are numbered according to the Kabat numbering system. In some embodiments, the human acceptor framework of the light chain variable region is generated by introducing an amino acid substitution F83L to the frame work region of IGKV1-39, and wherein the amino residues are numbered according to the Kabat numbering system.

In some embodiments, the heavy chain variable region comprises or consists of any one amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, and SEQ ID NO: 15, or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any one amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, and SEQ ID NO: 15. In some embodiments, the light chain variable region comprises or consists of any one amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 14, and SEQ ID NO: 16, or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 14, and SEQ ID NO: 16.

In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVS S (SEQ ID NO: 7) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 11) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 11. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQRNYLAWYQQKPGQAPRLLIYGASIRES GIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 12) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 12. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQRNYLAWYLQKPGQSPQLLIYGASIRESG VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 13) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 13. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSISTAYMELSRLRSDDTAVYYCARLNYYGNYGDFWGQGTMVTVS S (SEQ ID NO: 9) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 11) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 11. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSISTAYMELSRLRSDDTAVYYCARLNYYGNYGDFWGQGTMVTVS S (SEQ ID NO: 9) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 10) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 11) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 11. In some embodiments, the antibody provide herein comprises a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 10) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14.

In some embodiments, the antibody provided herein further comprises a heavy chain constant region and/or a light chain constant region. In some embodiments, the heavy chain constant region is an IgG heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG4 heavy chain constant region comprising the amino acid residue 228P numbered according to the EU numbering system. In some embodiments, the heavy chain constant region comprises or consists of an amino acid sequence as shown by SEQ ID NO: 17 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 17. In some embodiments, the light chain constant region is a light chain kappa constant region. In some embodiments, the light chain constant region comprises or consists of an amino acid sequence as shown by SEQ ID NO: 18 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 18. In some embodiments, the antibody provided herein further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is an IgG4 heavy chain constant region comprising the amino acid residue 228P numbered according to the EU numbering system, and the light chain constant region is a light chain kappa constant region. In some embodiments, the antibody provided herein further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or consists of an amino acid sequence as shown by SEQ ID NO: 17 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 17, and the light chain constant region comprises or consists of an amino acid sequence as shown by SEQ ID NO: 18 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 18. In some embodiments, the antibody provided herein comprises or consists of a heavy chain comprising or consisting of the amino acid sequence as shown by SEQ ID NO: 19 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 19 and a light chain sequence comprising or consisting of the amino acid sequence as shown by SEQ ID NO: 20 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 20. In some embodiments, the antibody provided herein comprises or consists of a heavy chain comprising or consisting of the amino acid sequence as shown by SEQ ID NO: 19 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 21 and a light chain sequence comprising or consisting of the amino acid sequence as shown by SEQ ID NO: 20 or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 20.

In some embodiments, the antigen binding fragment provided herein comprises a Fab, F (ab') 2, Fab', scFv, Fv, Fd or dAb of the antibody provided herein. In some embodiments, the antigen binding fragment provided herein is a Fab, F (ab') 2, Fab', scFv, Fv, Fd or dAb of the antibody provided herein. In some embodiment, the antibody provided herein is a nanobody, a diabody, a minibody or an IgG. In some embodiment, the antibody provided herein is a monoclonal antibody.

In the second aspect of the present application, a bispecific or multispecific antibody is provided, wherein the bispecific or multispecific antibody comprises the antibody or antigen binding fragment in the first aspect described above, and a second or several more antibodies or antigen binding fragments thereof. In some embodiments, the second or several more antibodies or antigen binding fragments thereof bind to the same antigen as the antibody or antigen binding fragment in the first aspect described above. In some embodiments, the second or several more antibodies or antigen binding fragments thereof bind to one or more antigens, wherein some or all of the antigens are different from that of the antibody or antigen binding fragment in the first aspect. In some embodiments, the second or several more antibodies or antigen binding fragments thereof bind to one or more tumor antigens. In some embodiments, the tumor antigens are expressed on the surface of a tumor cell. In some embodiments, the tumor antigens are one or more selected from the group consisting of A33; ADAM-9; ALCAM; BAGE; beta-catenin; BCMA; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD30; CD33; CD36; CD40/CD154; CD45; CD46; CD47, CD5; CD56; CD70; CD79a/CD79b; CD123, CD133, CD138; PSCA; Claudin; CLL-1; CDK4; CEA; CTLA4; PD-L1, Cytokeratin 8; LeY; Ov-γ; NKG2D; EGF-R; IL13Rα2; EphA2; ErbB1; ErbB3; ErbB4; EpCAM; GAGE-1; GAGE-2; GD2/GD3/GM2; HER-2/neu; GPC3; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A) ; LUCA-2; MAGE-1; MAGE-3; MART; Meso; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M; pl5; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-a receptor; TNF-γ receptor; Transferrin Receptor; and VEGF receptor.

In the third aspect of the present application, provided is a nucleic acid comprising a sequence encoding the antibody or antigen binding fragment thereof in the first aspect or an antisense strand thereof. In some embodiments, the nucleic acid is single stranded. In some embodiments, the nucleic acid is double stranded. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an RNA. In some embodiments, the nucleic acid is a hybrid of DNA and RNA. In some embodiments, the nucleic acid is linear. In some embodiments, the nucleic acid is circular. In some embodiments, the nucleic acid is chemically modified. In some embodiments, the nucleic acid comprises a bulge or a hairpin. In some embodiments, the nucleic acid is integrated in a eukaryotic genome. In some embodiments, the nucleic acid is integrated in a procaryotic genome. In some embodiments, the nucleic acid is integrated in a viral genome. In some embodiments, the nucleic acid further comprises a promoter and/or other regulatory sequence which enables the nucleic acid to express in a host cell. In some embodiments, the nucleic acid is wrapped in lipid.

Also provided is a vector comprising the nucleic acid in the third aspect is provided. In some embodiments, the vector is an expression vector. In some embodiment, the vector is a plasmid. In some embodiment, the vector is a virus vector.

In the fourth aspect of the present application, provided is a host cell comprising the antibody or the antigen binding fragment thereof in the first or second aspect or the nucleic acid in the third aspect. In some embodiments, the host cell is a procaryotic cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the antibody or the antigen binding fragment thereof is expressed in the cell.

Thus, also provided herein is a method for preparing an antibody or antigen binding fragment thereof binding to SIRPα protein or the extracellular domain thereof, comprising: (a) growing the host cell of described above under conditions so that the host cell expresses the antibody or antigen binding fragment thereof; and (b) purifying the antibody or antigen binding fragment thereof.

In the fifth aspect of the present application, provided is a pharmaceutical composition comprising the antibody or antigen binding fragment thereof in the first aspect or the nucleic acid in the third aspect. In some embodiments, the pharmaceutical composition comprises the antibody or antigen binding fragment thereof in the first aspect and/or the nucleic acid in the third aspect, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an antineoplastic drug. In some embodiments, the antineoplastic drug comprises one or more antibodies binding to one or more tumor antigens selected from the group consisting of A33; ADAM-9; ALCAM; BAGE; beta-catenin; BCMA; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD30; CD33; CD36; CD40/CD154; CD45; CD46; CD47, CD5; CD56; CD70; CD79a/CD79b; CD123, CD133, CD138; PSCA; Claudin; CLL-1; CDK4; CEA; CTLA4; PD-L1, Cytokeratin 8; LeY; Ov-γ; NKG2D; EGF-R; IL13Rα2; EphA2; ErbB1; ErbB3; ErbB4; EpCAM; GAGE-1; GAGE-2; GD2/GD3/GM2; HER-2/neu; GPC3; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A) ; LUCA-2; MAGE-1; MAGE-3; MART; Meso; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M; pl5; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-a receptor; TNF-γ receptor; Transferrin Receptor; and VEGF receptor. In some embodiment, the antineoplastic drug comprises a chemotherapeutic drug, a cytotoxic agent, and/or a radionuclide. In some embodiment, the antineoplastic drug comprises an agonist of stimulator of interferon genes (STING) receptor and/or a cytokine.

Also provided herein is a method for treatment of a cancer, comprising administrating an effective amount of the pharmaceutical composition described above to a subject having the cancer. In some embodiments, the cancer is any one selected from the group consisting of: leukemia, lymphoma, colorectal adenocarcinoma, pancreatic cancer, breast cancer, bladder cancer, renal cell cancer, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer; renal pelvis cancer, spinal tumors, glioma, pituitary adenoma, Kaposi’s sarcoma, and combinations and metastatic lesions thereof.

Besides, provided herein is the above mentioned antibody, the above mentioned conjugate, the above mentioned nucleic acid, the above mentioned vector, the above mentioned host cell, or the above mentioned pharmaceutical composition for use in the treatment of a cancer. In some embodiments, the cancer is any one selected from the group consisting of: leukemia, lymphoma, colorectal adenocarcinoma, pancreatic cancer, breast cancer, bladder cancer, renal cell cancer, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer; renal pelvis cancer, spinal tumors, glioma, pituitary adenoma, Kaposi's sarcoma, and combinations and metastatic lesions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, 1B, and 1C demonstrate representative binding curves of hSIRPα V2, V8, and mkSIRPα for antibodies of the present disclosure.

FIG. 2 show all anti-SIRPα mAbs binding to SIRPα V2 expressing THP-1 cells in a concentration-dependent manner.

FIG. 3 shows there’s hardly any binding affinity of the humanized anti-SIRPα mAbs and the reference OSE-172 to hSIRPγ.

FIG. 4 shows all anti-SIRPα mAbs exhibiting no binding to hSIRPγ on Jurkat cells even at high concentrations.

FIG. 5 shows in the competitive ELISA assay, 3R-2A10 exhibiting no inhibition to the human CD47 and SIRPα interaction even at high antibody concentrations.

FIG. 6 shows 3R-2A10 exhibiting no inhibition to the CD47 and SIRPγ interaction even at high antibody concentrations.

FIG. 7 shows the competitive ELISA assay result for 3R-2A10 binding to hSIRPα, compared with the reference antibody OSE-172.

FIG. 8A to 8D demonstrate representative M0 macrophage phagocytosis curves of the anti-SIRPα mAbs. FIG. 8E to8F demonstrate representative M2 macrophage phagocytosis curves of the anti-SIRPα mAbs.

FIG. 9 shows the humanized mAb and the chimeric mAb binding to human macrophages with comparable EC₅₀.

FIG. 10A to 10B show both chimeric and humanized mAbs exhibiting no binding toCD3⁺ T cells (FIG. 10A) or activated CD3⁺ T cells (FIG. 10B) .

FIG. 11 shows the result of human T cell proliferation assay exhibiting that 3R-2A10 does not affect T cell proliferation.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present invention is not limited to the aspects described herein, as such may, of course, vary. It is also to be understood that the terminology used herein is for describing particular aspects only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Those skilled in the art will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual. MONOCLONAL ANTIBODIES: A PRACTICAL APPROACH (Shepherd, P. et al. Eds., 2000) Oxford University Press, USA, New York N.Y.

The term “SIRPα” or “Signal regulatory protein α” , also known as CD172 antigen-like family member A (CD172a) , SHP substrate 1 (SHPS-1) , or Myeloid/Dendritic-1 (MyD-1) , is a member of the signal-regulatory-protein (SIRP) family, and also belongs to the immunoglobulin superfamily. SIRPα is a transmembrane protein with an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine–based inhibition motifs that mediate binding of the protein tyrosine phosphatases SHP1 and SHP2 (Matozaki T, et al., Trends Cell Biol., 2009; 19 (2) : 72–80; Barclay AN and Van den Berg TK, Annu. Rev. Immunol., 2014; 32: 25–50) . Unless otherwise specified, SIRPα can refer to any allelic variants of an SIRPα protein (including but are not limited to SIRPαV1, SIRPαV2, and SIRPαV8) or an SIRPα protein from any species (e.g., human and non-human primates) . Exemplary SIRP α protein is encoded by the gene with the Gene ID: 140885 or 717811 in NCBI data base, or any post translationally modified variants, conformation variants or homologous proteins thereof.

The term “SIRPγ” or “Signal regulatory protein γ” , also known as SIRPβ2 and CD172g, is a transmembrane glycoprotein with extracellular immunoglobulin-like domains. SIRPγ also belongs to the signal-regulatory protein (SIRP) family and the immunoglobulin superfamily. Unless otherwise specified, SIRPγ can refer to any variants or subtypes of an SIRPγ protein, or an SIRPγ protein from any species (e.g., human and non-human primates) . Exemplary SIRPγ protein is encoded by the gene with the Gene ID: 55423 in NCBI data base, or any post translationally modified variants, conformation variants, or homologous proteins thereof.

The term “CD47” , is a membrane protein, also known as Integrin Associated Protein (IAP) , Antigenic Surface Determinant Protein OA3, OA3, CD47 Antigen, Rh-Related Antigen, Integrin-Associated Signal Transducer, Antigen Identified by Monoclonal Antibody 1D8, CD47 glycoprotein) . Unless otherwise specified, CD47 can refer to any allelic variants of a CD47 protein or a CD47 from any species (e.g., human and non-human primates) . Exemplary CD47 protein is encoded by the gene with the Gene ID: 961 in NCBI data base, or is any post translationally modified variants, conformation variants, or homologous proteins thereof.

As used herein, “IGHV1-3” , “IGHV1-46” , “IGHV1-2” , “IGHV1-69” , “IGKV3-15” , “IGKV1-39”, “IGKV2-28” , and “IGKV4-1” respectively represent the immunoglobulin heavy chain variable regions or light chain variable regions encoded by the genes of the Gene IDs: 28473, 28465, 28474, 28461, 28913, 28930, 28921, and 28908 in NCBI (National Center of Biotechnology Information) database in sequence.

As used in the present invention, the term “antibody” , also called “immunoglobulin” , covers antibodies with structural characteristics of a native antibody and antibody-like molecules having structural characteristics different from a native antibody but exhibiting binding specificity to a certain antigen molecule. For example, an SIRPα antibody, an antibody for SIRPα, or an antibody specifically binds to SIRPα protein refers to an antibody exhibiting binding specificity to SIRPα. The term antibody is intended to encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The terms “heavy chain” ( “CH” ) , “light chain” ( “CL” ) , “light chain variable region” ( “VL” ) , “heavy chain variable region” ( “VH” ) , “framework region” ( “FR” ) , refer to domains in naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies) . The basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is a tetramer having two light chains and two heavy chains. The amino-terminal ( “N” ) portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal ( “C” portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains and a hinge region. Thus, the structure of the light chains of a naturally occurring IgG molecule is N-VL-CL-C and the structure of IgG heavy chains is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region) . The variable region of an IgG molecule consists of the complementarity determining regions (CDRs) , which contain the residues in contact with antigen and non-CDR segments, referred to as framework segments, which maintain the structure and determine the positioning of the CDR loops. Thus, the VL and VH domains have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.

The term of “antigen binding fragment” of an antibody (or simply “antibody fragment” ) , as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., SIRPα protein, such as SIRPα V1) . An “antigen binding fragment” , and all grammatical variants thereof, are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody which, in certain instances, is free of the constant heavy chain domains (i.e., CH2, CH3, and/or CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab’, Fab’-SH, F (ab’) ₂, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide” ) , including without limitation (1) single-chain Fv (scFv) molecules, (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety, and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi-specific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain (s) can contain any constant domain sequence (e.g., CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain (s) .

Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and a residual Fc fragment, whose name reflects its ability to crystallize readily. The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. “Fab'” fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. “Fab'-SH” is the designation for Fab' in which the cysteine residue (s) of the constant domains have a free thiol group. “F (ab') ” fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the “F (ab') 2” which is pepsin digestion product. “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy-and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv) , one heavy-and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. See, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .

A “Fd” fragment consists of the VH and CH1 domains. A “dAb” fragment (Ward et al., (1989) Nature 341: 544-546) consists of a VH or VL domain. An isolated complementarity determining region (CDR) and a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH -VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404, 097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-48 (1993) .

These antibody fragments are obtained using conventional techniques known to those with skill in the art, for example, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

An “human acceptor framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. A human acceptor framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 1-10, 2-9, 3-8, 4-7 or 5-6.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3. In certain embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al, supra. In certain embodiments, for the VH, the subgroup is subgroup III as in Kabat et al, supra.

As used herein, the term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. As used herein, the 3 CDRs from N terminal to C terminal on a heavy chain is referred to as HCDR1, HCDR2, and HCDR3, while the 3 CDRs from N terminal to C terminal on a light chain is referred to as LCDR1, LCDR2, and LCDR3. And CDR1 to CDR3 represents LCDR1 to LCDR3 or HCDR1 to HCDR3 respectively. Correspondingly, the more highly conserved portions of variable domains are called the framework (FR) . The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. As used herein, the 4 FRs from N terminal to C terminal on a heavy chain is referred to as HFR1, HFR2, HFR3, and HFR4, while the 4 FRs from N terminal to C terminal on a light chain is referred to as LFR1, LFR2, LFR3 and LFR4. And FR1 to FR4 represents LFR1 to LFR4 or HFR1 to HFR4 respectively. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991) . The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. Variable region sequences of interest include the humanized variable region sequences for SIRPα antibodies described in detail elsewhere herein.

The term “complementarity determining region (CDR) ” or “hypervariable region (HVR) ” may refer to the subregions of the VH and VL domains characterized by enhanced sequence variability and/or formation of defined loops. These include three CDRs in the VH domain (H1, H2, and H3) and three CDRs in the VL domain (L1, L2, and L3) . H3 is believed to be critical in imparting fine binding specificity, with L3 and H3 showing the highest level of diversity. See Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N. J., 2003) .

A number of CDR/HVR definitions /delineations are known. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) . Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987) ) . The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs/CDRs are noted below. “Framework” or “FR” residues are those variable domain residues other than the HVR/CDR residues.

¹Residue numbering follows the nomenclature of Kabat et al., supra
²Residue numbering follows the nomenclature of Chothia et al., supra
³Residue numbering follows the nomenclature of MacCallum et al., supra
⁴Residue numbering follows the nomenclature of Lefranc et al., supra
⁵Residue numbering follows the nomenclature of Honegger and Plückthun, supra

Extended CDRs are also known: 24-36 or 24-34 (L1) , 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1) , 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH (Kabat numbering) .

“Numbering according to Kabat” may refer to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. The actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR/HVR of the variable domain. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Typically, the Kabat numbering is used when referring to a residue in the variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) , whereas the EU numbering system or index (e.g., the EU index as in Kabat, numbering according to EU IgG1) is generally used when referring to a residue in the heavy chain constant region.

As used herein, a “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., substantially identical but allowing for minor levels of background mutations and/or modifications. “Monoclonal” denotes the substantially homogeneous character of antibodies, and does not require production of the antibody by any particular method. In some embodiments, a monoclonal antibody is selected by its CDR/HVR, VH, and/or VL sequences and/or binding properties, e.g., selected from a pool of clones (e.g., recombinant, hybridoma, or phage-derived) . A monoclonal antibody may be engineered to include one or more mutations, e.g., to affect binding affinity or other properties of the antibody, create a humanized or chimeric antibody, improve antibody production and/or homogeneity, engineer a multispecific antibody, resultant antibodies of which are still considered to be monoclonal in nature. A population of monoclonal antibodies may be distinguished from polyclonal antibodies as the individual monoclonal antibodies of the population recognize the same antigenic site. A variety of techniques for production of monoclonal antibodies are known; see, e.g., the hybridoma method (e.g., Kohler and Milstein, Nature, 256: 495-97 (1975) ; Hongo et al., Hybridoma, 14 (3) : 253-260 (1995) , Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) ; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) ) , recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) , phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991) ; Marks et al., J. Mol. Biol. 222: 581-597 (1992) ; Sidhu et al., J. Mol. Biol. 338 (2) : 299-310 (2004) ; Lee et al., J. Mol. Biol. 340 (5) : 1073-1093 (2004) ; Fellouse, Proc. Natl. Acad. Sci. USA 101 (34) : 12467-12472 (2004) ; and Lee et al., J. Immunol. Methods 284 (1-2) : 119-132 (2004) , and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993) ; Jakobovits et al., Nature 362: 255-258 (1993) ; Bruggemann et al., Year in Immunol. 7: 33 (1993) ; U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992) ; Lonberg et al., Nature 368: 856-859 (1994) ; Morrison, Nature 368: 812-813 (1994) ; Fishwild et al., Nature Biotechnol. 14: 845-851 (1996) ; Neuberger, Nature Biotechnol. 14: 826 (1996) ; and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995) .

As used herein the phrase “bispecific antibody” or “bispecific antigen binding antibody” or “bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.

As used herein, the term “native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond (also termed a “VH/VL pair” ) , while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-and heavy-chain variable domains. See, e.g., Chothia et al., J. Mol. Biol., 186: 651 (1985) ; Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A., 82: 4592 (1985) .

“Chimeric antibodies” may refer to an antibody with one portion of the heavy and/or light chain from a particular isotype, class, or organism and another portion from another isotype, class, or organism. In some embodiments, the variable region will be from one source or organism, and the constant region will be from another.

“Humanized antibodies” may refer to antibodies with predominantly human sequence and a minimal amount of non-human (e.g., mouse or chicken) sequence. In some embodiments, a humanized antibody has one or more CDR sequences (bearing a binding specificity of interest) from an antibody derived from a non-human (e.g., mouse or chicken) organism grafted onto a human recipient antibody framework (FR) . In some embodiments, non-human residues are further grafted onto the human framework (not present in either source or recipient antibodies) , e.g., to improve antibody properties. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. See Jones et al., Nature 321: 522-525 (1986) ; Riechmann et al., Nature 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992) .

A “human antibody” may refer to an antibody having an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) ; preparation of human monoclonal antibodies as described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) ; Boerner et al., J. Immunol., 147 (1) : 86-95 (1991) ; and by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE^TM technology) or chickens with human immunoglobulin sequence (s) (see, e.g., WO2012162422, WO2011019844, and WO2013059159) .

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) , e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

As used herein, the term “epitope” means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

A “conjugate” is an antibody conjugated to one or more heterologous molecule (s) , including but not limited to a cytotoxic agent.

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

The term "isolated" as used herein refers to molecules or biological or cellular materials being substantially free from other materials. For example, a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.

“Affinity” refers to the total strength of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) . Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) . Affinity can be measured by common methods known in the art, including, for example, Biacore, radioimmunoassay (RIA) and ELISA.

The affinity of a molecule X for its partner Y can generally be represented by equilibrium dissociation constant (K_D) , calculated as the ratio koff/kon (kd/ka) . See, e.g., Chen, Y., et al., (1999) J. MoI Biol 293: 865-881. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. In one embodiment of the invention, the “dissociation rate (kd) ” is measured by using surface plasmon resonance assays. An “on-rate” or “rate of association” or “association rate (ka) ” or “kon” according to this invention can also be determined with the same surface plasmon resonance technique and calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software) by simultaneous fitting the association and dissociation sensor gram.

The term “EC50” , as used herein, refers to the concentration of an antibody or an antigen-binding fragment thereof, which binds to the antigen and/or induces a response, either in an in vitro or an in vivo assay, which is 50%of the maximal binding or response, i.e., halfway between the maximal binding or response and the baseline.

The terms “cancer” or “neoplasm” and “tumor” can be used interchangeably in the present invention, referring to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells that makes them pathological to the host organism. In some embodiments, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites. In some embodiments, the cancer is associated with a specific cancer antigen.

As used herein, “treating” or “treatment” of a disease in a subject refers to an approach for obtaining beneficial or desired results, including one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease) , stabilized (i.e., not worsening) state of a condition (including disease) , delay or slowing of condition (including disease) , progression, amelioration or palliation of the condition (including disease) , states and remission (whether partial or total) , whether detectable or undetectable.

A “pharmaceutically acceptable carrier” is a carrier with which an active ingredient constitutes a pharmaceutical formulation. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “package insert” is used to refer to instructions customarily included in commercial package of a therapeutic product. Generally, there is information about the use of the therapeutic product on the package insert such as indications, usage, dosage, administration, combination therapy, contraindications and/or warnings.

An “effective amount” is at least the minimum amount required to affect a measurable improvement or prevention of a particular disease (e.g., cancer) . An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of a therapeutic agent (or combination of therapeutic agents) to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

As used herein, the term “subject” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

As used herein, percent of “homology” or “identity” is used in the context of two or more nucleic acids or polypeptide sequences, referring to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 80%identity, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein) . Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi. nlm. nih. gov/cgi-bin/BLAST.

The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

It is understood that aspects and embodiments of the present disclosure include “comprising, ” “consisting, ” and “consisting essentially of” aspects and embodiments. The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. The term “comprising” as used in the present description and claims does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g., “a” or “an” , “the” , this includes a plural of that noun unless something else is specifically stated. All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

Anti-SIRPα Antibody and Methods for Preparing the Same

This invention encompasses isolated anti-SIRPα antibodies or fragments thereof, polynucleotides or nucleic acid comprising sequences encoding the anti-SIRPα antibodies or fragments thereof.

The isolated anti-SIRPα antibodies or fragments thereof bind to SIRPα molecule expressed on cells (for example, macrophage and dendritic cell) with high affinity and facilitating effective immune response against cancer cells. The antibody and its immunoreactive fragments provided herein are capable of enhancing the activation of the immune system, and thus providing important therapeutic agents for use in targeting pathological conditions associated with expression and/or activity of SIRPα and/or CD47 molecule. In some embodiments, the isolated anti-SIRPα antibodies or fragments thereof induce or enhance immune response (e.g., phagocytosis of tumor cells) independently without disrupting the CD47/SIRPγ interaction or T cell function (e.g., T cell proliferation) . In some embodiments, the isolated anti-SIRPα antibodies or fragments thereof induce or enhance immune response independently without disrupting the CD47/SIRPγ interaction, CD47/SIRPα interaction or T cell function.

In one aspect, the present invention provides an isolated antibody or antigen binding fragment thereof, wherein the antibody comprises a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to an extracellular domain of SIRPα with a sufficient affinity. In some embodiments, the antibody or antigen binding fragment thereof binds to any one or more selected from the group consisting of: human SIRP α variant 1 (hSIRPα V1) , human SIRP α variant 2 (hSIRPα V2) , human SIRP α variant 8 (hSIRPα V8) , Cyno Monkey SIRPα (mkSIRPα) . In some embodiments, the antibody or antigen binding fragment thereof binds to human SIRP α variant 1 (hSIRPα V1) , human SIRP α variant 2 (hSIRPα V2) , human SIRP α variant 8 (hSIRPα V8) and Cyno Monkey SIRPα (mkSIRPα) . In some embodiments, the antibody or antigen binding fragment thereof binds to SIRPα protein with an affinity constant (KD) about or better than 2E-08 M as determined by SPR analysis, for example about or better than 1.5E-08M, 1E-08M, 2E-09M, 1.5E-09M, 1E-09M, 8E-10M, 6E-10M, or 5E-10M. In some embodiments, the antibody or antigen binding fragment thereof binds to hSIRPα V1 with an affinity constant (KD) about or better than 2E-08 M as determined by SPR analysis, for example about or better than 1.5E-08M, 1E-08M, 2E-09M, 1.5E-09M, 1E-09M, 8E-10M, 6E-10M or 5E-10M.

In some embodiments, the antibody or antigen binding fragment thereof binds to isolated hSIRPα V2 protein with a K_D about or better than 3E-09M as determined by SPR analysis, for example about or better than 2.5E-09M, 2E-09M, 8E-10M, 6E-10M, 4E-10M, 3E-10M, 2E-10M, 1E-10M, 1E-11M, 8E-12M, 6E-12M, 5E-12M, 4E-12M, 3E-12M, or 2E-12M as determined by SPR analysis. In some embodiments, the antibody or antigen binding fragment thereof binds to isolated hSIRPα V2 protein with an EC₅₀ about or better than 0.1 μg/mL as determined by ELISA, for example about or better than 0.09 μg/mL, 0.08 μg/mL, 0.07 μg/mL, 0.06 μg/mL, 0.05 μg/mL, 0.04 μg/mL, 0.03 μg/mL, 0.02 μg/mL, or 0.01 μg/mL. In some embodiments, the antibody or antigen binding fragment thereof binds to isolated hSIRPα V8 protein with a K_D about or better than 3E-09M as determined by SPR, for example about or better than 2.5E-09M, 2E-09M, 1.5E-09, 2.5E-09M, 2E-09M, 8E-10M, 6E-10M, 4E-10M, 3E-10M, 2E-10M, 1E-10M, 1E-11M, 8E-12M, 6E-12M, 5E-12M, 4E-12M, 3E-12M, or 2E-12M as determined by SPR analysis. In some embodiments, the antibody or antigen binding fragment thereof binds to isolated hSIRPα V8 protein with an EC50 about or better than 0.1 μg/mL as determined by ELISA, for example about or better than 0.09 μg/mL, 0.08 μg/mL, 0.07 μg/mL, 0.06 μg/mL, 0.05 μg/mL, 0.04 μg/mL, 0.03 μg/mL, 0.02 μg/mL, or 0.01 μg/mL. In some embodiments, the antibody or antigen binding fragment thereof binds to isolated mkSIRPα protein with a K_D about or better than 3E-09M as determined by SPR, for example about or better than 3E-09M, 2E-09M, 1E-09M, 2E-10M, 1E-10M, 1E-11M, 8E-12M, 6E-12M, 5E-12M, 4E-12M, 3E-12M, or 2E-12M. In some embodiments, the antibody or antigen binding fragment thereof binds to isolated mkSIRPα protein with an EC50 about or better than 0.1 μg/mL as determined by ELISA, for example about or better than 0.09 μg/mL, 0.08 μg/mL, 0.07 μg/mL, 0.06 μg/mL, 0.05 μg/mL, 0.04 μg/mL, 0.03 μg/mL, 0.02 μg/mL, or 0.01 μg/mL.

In some embodiments, the antibody or antigen binding fragment thereof doesn’t block the human CD47 and SIRPα interaction. In some embodiments, the antibody or antigen binding fragment thereof doesn’t block the human CD47 and SIRPγ interaction. In some embodiments, the antibody or antigen binding fragment thereof block neither the human CD47 and SIRPα interaction nor the human CD47 and SIRPγ interaction. In some embodiments, the antibody or antigen binding fragment thereof doesn’t block the human CD47 and SIRPγ interaction even when the antibody concentration is up to 50 μg/mL.

In some embodiments, the antibody or antigen binding fragment thereof binds to SIRPα at a different epitope from the OSE-172 antibody. In some embodiments, the antibody or antigen binding fragment thereof binds to hSIRPα V1 at a different epitope from the OSE-172 antibody. As used herein, the OSE-172 antibody is an anti-SIRPα antibody in early clinical (see e.g., US20190127477A1) . In some embodiments the antibody or antigen binding fragment thereof induces immune response independently. In some embodiments the antibody or antigen binding fragment thereof binds to human macrophages. In some embodiments the antibody or antigen binding fragment thereof induces the phagocytosis of tumor cells by macrophages independently. In some embodiments the tumor cells express CD47. In some embodiments the tumor cells are colorectal cancer cells and/or hematologic tumor cells. In some embodiments, the macrophages are human macrophages. In some embodiments, the antibody or antigen binding fragment thereof hardly binds to human CD3⁺ T cells. In some embodiments the binding affinity of the antibody or antigen binding fragment thereof to human CD3⁺ T cells is not strong. In some embodiments, the antibody or antigen binding fragment thereof doesn’t inhibit T cell proliferation. In some embodiments, the antibody or antigen binding fragment thereof doesn’t inhibit allogeneic dendritic cell-induced T cell proliferation.

In certain embodiments, the present invention provides an antibody or antigen binding fragment thereof, wherein the antibody comprises at least:

(i) a light chain variable region (VL) comprising the identical LCDR1, LCDR2, and LCDR3 with those in any one of SEQ ID NO: 11 to SEQ ID NO: 14 and SEQ ID NO: 16; and

(ii) a heavy chain variable region (VH) comprising the identical HCDR1, HCDR2, and HCDR3 with those in any one of SEQ ID NO: 7 to SEQ ID NO: 10 and SEQ ID NO: 15;

wherein the HCDRs and LCDRs are defined by any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition, or defined by a hybrid scheme that combines any two, three or four of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition.

In certain embodiments, the antibody is a chimeric antibody, humanized antibody, or human antibody. In certain embodiments, the antibody or antigen binding fragment thereof of the present invention further comprises a human acceptor framework. In certain embodiments, the human acceptor framework is derived from a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework comprises subgroup kappa I framework sequences for VL, and subgroup III framework sequences for VH. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3. In certain embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al, supra. In certain embodiments, for the VH, the subgroup is subgroup III as in Kabat et al, supra. In some embodiments, the human acceptor framework of the heavy chain variable region derives from any immunoglobulin heavy chain variable region germline selected from the group consisting of: IGHV1-3, IGHV1-46, IGHV1-2, and IGHV1-69, and variants thereof. In some embodiments, the human acceptor framework of the light chain variable region derives from any immunoglobulin Kappa chain variable region germline selected from the group consisting of: IGKV3-15, IGKV1-39, IGKV2-28, and IGKV4-1, and variants thereof.

In certain embodiments, the antibody or antigen binding fragment thereof comprises human consensus framework. In some embodiments, the antibody or antigen binding fragment thereof comprises human consensus framework with amino acid sequence changes, for example, 1-15, 1-10, 2-9, 3-8, 4-7 or 5-6 amino acid changes.

In some embodiments, the heavy chain variable region comprises or consists of any amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, or from SEQ ID NO: 15, or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, or from SEQ ID NO: 15. In some embodiments, the light chain variable region comprises or consists of any amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 14, or from SEQ ID NO: 16, or an amino acid sequence having at least 80%, 82%, 83%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 14, or from SEQ ID NO: 16.

In certain embodiments, the antibody is an IgG isotype, for example, IgG1, IgG2 or IgG4 isotype. In certain embodiments, the antigen binding fragment is selected from the group consisting of Fab, F (ab') 2, Fab', scFv, and Fv. In certain embodiments, the antibody or antigen binding fragment thereof of the invention is a blocking antibody or an antagonist antibody which inhibits or reduces biological activity of the SIRPα molecule it binds. Preferred the blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the SIRPα molecule.

The anti-SIRPα antibodies of the invention in the first aspect is preferably monoclonal. Also encompassed within the scope of the invention are Fab, Fab', Fab'-SH and F (ab') 2 fragments of the anti-SIRPα antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. The anti-SIRPα antibodies and fragments thereof are useful for the therapeutic purposes, including therapy of cancers.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of different antibodies. The monoclonal anti-SIRPα antibodies of the invention can be made using the hybridoma method or recombinant DNA methods (U.S. Patent No. 4,816,567) .

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized by a whole SIRPα molecule or part of the molecule, for example, a polypeptide comprising the extracellular domain of SIRPα, together with an adjuvant. A SIRPα molecule or a polypeptide comprising the extracellular domain of A SIRPα molecule may be prepared using methods well-known in the art. In one embodiment, animals are immunized with a polypeptide that contains the extracellular domain (ECD) of SIRPα fused to the Fc portion of an immunoglobulin heavy chain. In one embodiment, animals are immunized with an SIRPα-IgG1 fusion protein. The immunizations were performed one or more times and the interval duration among each immunization two weeks. 7 to 14 days later, animals are bled and the serum is assayed for anti-SIRPα titer. Animals are boosted until titer plateaus. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) ) .

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as SP-2/0 or X63-Ag8-653 cells. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133: 3001 (1984) ; Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) ) .

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against SIRPα. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) .

The binding affinity of the monoclonal antibody can then be determined by conventional methods in the art. After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) ) . Suitable culture media for this purpose include, for example, HT containing medium, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures.

The anti-SIRPα antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution. Any of the anti-SIRPα antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-SIRPα antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols. 1-3.

Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al, Ann. Rev. Immunol, 12: 433-455 (1994) . Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al, EMBO J, 12: 725-734 (1993) . Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. MoI Biol, 227: 381-388 (1992) .

The antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity, but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries. For example, mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989) ) in the method of Hawkins et al., J. MoL Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992) . Additionally, affinity maturation can be performed by randomly mutating one or more CDRs, e.g., using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol, 10: 779-783 (1992) .

It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for SIRPα. However, random mutation of a selected antibody (e.g., as performed in some of the affinity maturation techniques described above) is likely to give rise to many mutants, most binding to antigen, and a few with higher affinity. To retain all the higher affinity mutants, phages can be incubated with excess biotinylated SIRPα, but with the biotinylated SIRPα at a concentration of lower molarity than the target molar affinity constant for SIRPα. The high affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity.

Anti-SIRPα clones may be selected based on performance of activity. In one embodiment, the invention provides anti-SIRPα antibodies that block the binding between SIRPα and its ligand. Anti-SIRPα antibodies of the invention possessing the properties described herein can be obtained by screening anti-SIRPα hybridoma clones for the desired properties by any convenient method. For example, if an anti-SIRPα monoclonal antibody that blocks or does not block the binding of SIRPα to SIRPα ligand is desired, the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, wherein plate wells are coated with SIRPα, and a solution of antibody in an excess of SIRPα is layered onto the coated plates, and bound antibody is detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated anti-Ig antibody or biotinylated anti-Ig antibody and developing the HRP color reaction., e.g. by developing plates with streptavidin-HRP and/or hydrogen peroxide and detecting the HRP color reaction by spectrophotometry at a certain wavelength with an ELISA plate reader.

Isolated Polynucleotides, Vectors, Host Cells and Recombinant Methods

Provided herein are isolated polynucleotides, vectors, or host cells comprising the coding sequence of the anti-SIRPα antibodies or fragments thereof of the present disclosure described above. In some embodiments, the anti-SIRPα antibody is the hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention. In some embodiments, hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template) . Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells.

DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g., the appropriate DNA sequences can be obtained from Kabat et al, supra) to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. A Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence (s) for "hybrid" , full length heavy chain and/or light chain is included in the definition of "chimeric" and "hybrid" antibody as used herein. In a preferred embodiment, a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence (s) for all human, full or partial length heavy and/or light chains.

DNA encoding anti-SIRPα antibodies derived from a hybridoma of the invention can also be modified, for example, by substituting the coding sequence for human heavy-and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone (e.g., as in the method of Morrison et al, Proc. Natl Acad. Sci. USA, 81: 6851-6855 (1984) ) . DNA encoding a hybridoma or Fv clone-derived antibody or fragment can be further modified by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of the Fv clone or hybridoma clone-derived antibodies of the invention.

For recombinant production of an antibody of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

Conjugates and Methods for Preparing the Same

Conjugates of the anti-SIRPα antibody or fragment thereof of the present invention and one or more additional moiety are also contemplated herein. Such additional moiety includes, without limitation, small molecule compounds, peptides, nucleic acids, toxins, PEG, lipids, cytokines, antigen-binding molecules, and radioactive isotopes.

Antibody Fragments and Methods for Preparing the Same

The present invention encompasses antibody fragments. The antibody fragments are the immunoreactive fragments of the anti-SIRPα antibody of the present disclosure. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic 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) ) . However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab') 2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992) ) . According to another approach, F (ab') 2 fragments can be isolated directly from recombinant host cell culture. Fab and F (ab') 2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) . See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only known species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody" , e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies and Human Antibodies

The anti-SIRPα antibodies of the present invention in some embodiments are humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al (1986) Nature 321: 522-525; Riechmann et al (1988) Nature 332: 323-327; Verhoeyen et al (1988) Science 239: 1534-1536) , by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. MoI. Biol. 196: 901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for SIRPα, is achieved.

Transgenic animals (e.g., mice) that are also capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Nature, 362: 255 (1993) ; Bruggermann et al, Year in Immunol, 7: 33 (1993) .

Gene shuffling can also be used to derive human antibodies from non-human, e.g., rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called "epitope imprinting" , either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, i.e., the epitope governs (imprints) the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published April 1, 1993) . Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin.

Bispecific Antibodies and Methods for Preparing the Same

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for SIRPα and the other is for any other antigen. Exemplary bispecific antibodies may bind to two different epitopes of the SIRPα protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express SIRPα, in this case, antibodies possess an SIRPα-binding arm and an arm which binds the cytotoxic agent.

In some embodiments, the bispecific antibodies possess an SIRPα-binding arm which comprising the anti-SIRPα antibody or fragment thereof of the present disclosure and an arm which binds to a tumor antigen or an immune checkpoint protein. In some embodiments the tumor antigen is selected from the group consisting of A33; ADAM-9; ALCAM; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40/CD154; CD45; CD46; CD5; CD56; CD79a/CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2/GD3/GM2; HER-2/neu; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A) ; LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M; pl5; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-a receptor; TNF-γ receptor; Transferrin Receptor; and VEGF receptor. In some embodiments, the immune checkpoint protein is selected from the group consisting of 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; Galectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRP α; TIM-3; TIGIT; VSIG8.

Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F (ab') 2 bispecific antibodies) . Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) , containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific antibody provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et ah, Methods in Enzymology, 121: 210 (1986) .

Pharmaceutical Compositions

Therapeutic formulations comprising the anti-SIRPα antibodies fragments, polynucleotides (or nucleic acid) , vectors, host cells, conjugates or bispecific antibodies of the present disclosure are prepared for storage by mixing the anti-SIRPα antibodies, fragments, polynucleotides (or nucleic acid) , vectors, host cells, conjugates or bispecific antibodies of the present disclosure with the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000) ) , in the form of aqueous solutions, lyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes; and/or non-ionic surfactants such as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG) .

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly- (methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsule.

Pharmaceutical and Detective Uses of Anti-SIRPα antibodies

In one aspect, the invention provides methods for treating cancers or immune disorder comprise administering an effective amount of an anti-SIRPα antibody or fragments thereof specifically binding SIRPα to a subject in need of such treatment. The antibodies of the present invention can be used to treat, inhibit, delay progression of, prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or conditions associated with expression and/or activity of one or more antigen molecules including SIRPα or CD47 molecule, or increased expression and/or activity of one or more antigen molecules including SIRPα and CD47 molecule.

For treatment use of the anti-SIRPα antibody or fragments thereof of the present invention, the appropriate dosage of an antibody of the invention (when used alone or in combination with other agents will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one or multiple times. Depending on the type and severity of the disease, in some embodiments, about 1 μg/kg to 15 mg/kg (e.g., 0.1mg/kg-10mg/kg) of antibody is a propriate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.

Antibodies of the invention can be used either alone or in combination with other compositions in a therapy. For instance, an antibody of the present invention may be co-administered with another antibody, steroids (such as inhalable, systemic or cutaneous steroids) , chemotherapeutic agent (s) (including cocktails of chemotherapeutic agents) , other cytotoxic agent (s) , anti-angiogenic agent (s) , cytokines, and/or growth inhibitory agent (s) . Such combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations) , and separate administration, in which case, the anti-SIRPα antibody or fragment thereof of the present invention can be administrated prior to, during and/or following administration of one or more other agents. The effective amounts of therapeutic agents administered in combination depend on such factors as the type of therapeutic agent to be used and the specific patient being treated. and will generally be at the physician's or veterinarian's discretion.

In one aspect, based on specific binding of the anti-SIRPα antibodies or fragments thereof disclosed herein to SIRPα, the antibodies of the present invention can be used in detection and quantitation of a SIRPα polypeptide in physiological samples, such as blood and biopsy samples. Thus, the anti-SIRPα antibodies disclosed herein can be used diagnostically to monitor SIRPα levels in tissues, e.g., to determine the efficacy of a given treatment regimen. A skilled person in the art knows that the SIRPα antibodies disclosed herein can be coupled with detectable materials to facilitate the detection. In certain embodiments, the anti-SIRPα antibody or fragment thereof disclosed herein is bound to a solid support to facilitate the detection.

In another aspect, based on the specific binding of the antibodies disclosed herein to SIRPα, the antibodies of the present invention can be used in, for example, isolating by affinity chromatography methods or immunoprecipitation methods, analyzing or sorting cells by flow cytometry methods, and detecting a SIRPα polypeptide within fixed tissue samples or cell smear samples by immunohistochemistry, cytology analysis, ELISA, or immunoprecipitation methods.

In certain embodiments, the SIRPα molecule to be detected, quantified or analyzed is human SIRPα protein or fragments thereof. In certain embodiments, the SIRPα protein or fragment thereof is disposed in a solution, such as a lysis solution or a solution containing a sub-cellular fraction of a fractionated cell, or present on surface of SIRPα-positive cells, or in complexes containing SIRPα and other cellular components.

The detection method of the present invention can be used to detect expression levels of SIRPα polypeptides in a biological sample in vitro as well as in vivo. In vitro techniques for detection of SIRPα polypeptides include enzyme linked immunosorbent assays (ELISAs) , Western blots, flow cytometry, immunoprecipitations, radioimmunoassay, and immunofluorescence (e.g., IHC) . Furthermore, in vivo techniques for detection of SIRPα polypeptides include introducing into a subject a labeled anti-SIRPα antibody. By way of example only, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA) . Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agents and fluorescent labels, such as fluorescein and rhodamine, and biotin.

The SIRPα antibodies or fragments thereof disclosed herein can be used as diagnostic or detective reagents for any kind of biological sample. In one aspect, the SIRPα antibodies disclosed herein are useful as diagnostic reagents for human biological samples. SIRPα antibodies can be used to detect SIRPα polypeptides in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, flow cytometry, IHC and immunometric assays.

The present invention also provides for prognostic (or predictive) uses of the anti-SIRPα antibodies and fragments thereof for determining whether a subject is at risk of developing a medical disease or condition associated with increased SIRPα polypeptide expression or activity (e.g., detection of a precancerous cell) . Thus, the anti-SIRPα antibodies and fragments thereof disclosed herein can be used for prognostic or predictive purpose to prophylactically treat an individual prior to the onset of a medical disease or condition (for example cancer or immune disorder) characterized by or associated with increased SIRPα polypeptide expression or activity.

Another aspect of the present invention provides methods for determining SIRPα expression in a subject to thereby screen therapeutic or prophylactic compounds for a medical disease or condition (for example cancer or immune disorder) characterized by or associated with increased SIRPαpolypeptide expression or activity.

In certain embodiments, the medical disease or condition characterized by or associated with SIRPα polypeptide expression or activity or increased SIRPα polypeptide expression or activity stated above is precancerous condition, cancer or immune disorder. In certain embodiments, the prognostic assays can be utilized to identify a subject having or at risk for developing a cancer or immune disorder. Thus, the present invention provides a method for identifying a disease or condition (for example cancer) associated with increased SIRPα polypeptide expression levels in which a test sample is obtained from a subject and the SIRPα polypeptide detected, wherein the presence of increased levels of SIRPα polypeptides compared to a control sample is predictive for a subject having or at risk of developing a disease or condition (for example cancer or immune disorder) associated with increased SIRPα polypeptide expression levels.

In another aspect, the present invention provides methods for determining whether a subject can be effectively treated with a therapeutic agent for a disorder or condition (for example cancer or immune disorder) associated with increased SIRPα polypeptide expression wherein a biological sample is obtained from the subject and the SIRPα polypeptide is detected using the SIRPα antibody. The expression level of the SIRPα polypeptide in the biological sample obtained from the subject is determined and compared with the SIRPα expression levels found in a biological sample obtained from a subject who is free of the disease. Elevated levels of the SIRPα polypeptide in the sample obtained from the subject suspected of having the disease or condition compared with the sample obtained from the healthy subject is indicative of the SIRPα -associated disease or condition (for example cancer or immune disorder) in the subject being tested.

In one aspect, the present invention provides for methods of monitoring the treatment efficacy of agents on the expression of SIRPα polypeptides. Such assays can be applied in drug screening and in clinical trials. For example, the effectiveness of an agent to decrease SIRPα polypeptide levels can be monitored in clinical trials of subjects exhibiting elevated expression of SIRPα, e.g., patients diagnosed with cancer or immune disorder. An agent that affects the expression of SIRPα polypeptides can be identified by administering the agent and observing a response. In this way, the expression pattern of the SIRPα polypeptide can serve as a marker, indicative of the physiological response of the subject to the agent.

The foregoing are merely exemplary assays for using the anti-SIRPα antibodies and fragments thereof of the present invention. Other methods now or hereafter developed that use the antibodies or fragments thereof for the determination of SIRPα are included within the scope hereof.

Kits and Articles of Manufacture

The present invention provides diagnostic methods for determining the expression level of SIRPα. In one particular aspect, the present invention provides kits for determining the expression level of SIRPα. The kit comprises the anti-SIRPα antibody or fragment thereof disclosed herein and instructions about how to use the kit, for example, instructions for collecting samples and/or performing the detection, and/or analyzing the results. The kits are useful for detecting the presence of SIRPα polypeptides in a biological sample e.g., any body fluid including, but not limited to, e.g., sputum, serum, lymph, cystic fluid, urine, stool, cerebrospinal fluid, acitic fluid or blood and including biopsy samples of body tissue. The test samples may also be an immune cell, a normal cell corresponding to the tumor tissue type, a blood cell, a peripheral blood lymphocyte, or combinations thereof.

In certain embodiments, the kit may further comprise one or more other SIRPα antibodies apart from the anti-SIRPα antibody of the present invention, which are capable of binding a SIRPαpolypeptide in a biological sample. The one or more of the SIRPα antibodies may be labeled. In certain embodiments, the kit comprises a first antibody, e.g., attached to a solid support, which binds to a SIRPα polypeptide; and, optionally; 2) a second, different antibody which binds to either the SIRPα polypeptide or the first antibody and is conjugated to a detectable label.

The kit can also comprise, e.g., a buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions written on a package insert about how to use the kit, for example, instructions for collecting samples and/or performing the detection, and/or analyzing the results.

In another aspect, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided by the present invention. The article of manufacture comprises a container and a label or package insert on or associated with the container with written instructions of, for example, indications to be treated, administration regimens and warnings. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition comprising the anti-SIRPα antibody or fragment thereof of the present invention, which is by itself or when combined with other composition (s) effective for treating, preventing and/or diagnosing the medical disease or condition characterized by or associated with increased expression and/or activity of one or more molecules including SIRPα polypeptide (e.g., cancers) .

The article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second, third or fourth container with a composition comprising another active ingredient. Additionally, the article of manufacture may further comprise a container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Treatment methods

The anti-SIRPα antibodies or fragments thereof of the present invention can be used in certain treatment methods. The present invention further encompasses the antibody-based therapies which involve administering effective amount of the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention described above to a patient, for example human patient or non-human primate, for treating one or more of the disorders or conditions described herein.

In some embodiments, the patient is a patient with tumor. In some embodiments, the patient is with an infection. In one embodiment, the patient has tumor cells or infected cells with the overexpression of SIRPα ligands, for example CD47. In some embodiments, the patient is with an immune disorder.

Non-limiting examples of cancers include colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, thyroid cancer, leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ) , polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease) , multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma. And in some embodiments, the infection is viral, bacterial, fungal, or parasite infection.

A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the anti-SIRPα antibodies or fragments thereof of the present invention used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

In some embodiments, the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention is administered in combination with an antineoplastic agent, an antiviral agent, antibacterial or antibiotic agent or antifungal agents. Any of these agents known in the art may be administered in the compositions of the current disclosure.

In another embodiment, the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the disclosure include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin) ; antiestrogens (e.g., tamoxifen) ; antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine) ; cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate) ; hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone) ; nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa) ; steroids and combinations (e.g., bethamethasone sodium phosphate) ; and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide) .

In an additional embodiment, the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention is administered in combination with cytokines, wherein the cytokines include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-l0, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α. In additional embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

The antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention can be used, in some embodiments, together with an immune checkpoint inhibitor. Immune checkpoints are molecules in the immune system that either turn up a signal (co-stimulatory molecules) or turn down a signal. Many cancers protect themselves from the immune system by inhibiting the T cell signal. An immune checkpoint inhibitor can help stop such a protective mechanism. An immune checkpoint inhibitor may target any one or more of the following checkpoint molecules, 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; Galectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; B7-H1; TIM-3; SIRPα; VSIG8.

A CD47 inhibitor that attenuates the interaction between CD47 and SIRPα can be administrated in combination with the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate of the present invention. In some embodiments, a CD47 inhibitor is an anti-CD47 antibody. In some embodiments, a CD47 inhibitor suppresses the expression of CD47, for example a shRNA, miRNA, siRNA or antisense oligonucleotide regulating CD47 expression. Exemplary CD47 inhibitors are described in US-20200289440, WO-2019027903, WO-2019091473, WO-2019086573, WO-2019173420, WO-2019217145, WO-2018160739, WO-2020185677, WO-2019157843, WO-2021102376, WO-2017053423, WO-2018210793, WO-2019040791, WO-2019109876, WO-2020247572, WO-2016109415, WO-2016081423, WO-2019086573, WO-2019226973, WO-2018215835, WO-2020200256, WO-2020151258, WO-2019228345, WO-2021137230, WO-2021005599, WO-2019042285, WO-2021119832, WO-2010130053, WO-2014087248, WO-2010130053, WO-2021113596, WO-2019109357, WO-2021124073, WO-2020095256, WO-2020259605, WO-2012078992, WO-2020088580, WO-2018205936, WO-2018157162, WO-2016023040; WO-2017027422, WO-2021043220, WO-2014149477, and WO-2015191861. Programmed T cell death 1 (PD-1) is a trans-membrane protein found on the surface of T cells, which, when bound to programmed T cell death ligand 1 (PD-L1) on tumor cells, results in suppression of T cell activity and reduction of T cell-mediated cytotoxicity. Thus, SIRPα and PD-L1 are immune down-regulators or immune checkpoint "off switches" . Example SIRPα inhibitor include, without limitation, nivolumab, (Opdivo) (BMS-936558) , pembrolizumab (Keytruda, pidilizumab, AMP-224, MEDI0680 (AMP-514, PDR001, MPDL3280A, MEDI4736, BMS-936559 and MSB0010718C. Programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans encoded by the CD274 gene. Nonlimiting examples of PD-L1 inhibitor include Atezolizumab (Tecentriq) , Durvalumab (MEDI4736) , Avelumab (MSB0010718C) , MPDL3280A, BMS935559 (MDX-Ll05) and AMP-224. CTLA-4 is a protein receptor that downregulates the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-0l0, MDX-l0l) and tremelimumab (formerly ticilimumab, CP-675, 206) . Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor on the cell surface works to suppress an immune response by action to Tregs as well as direct effects on CD8⁺ T cells. LAG-3 inhibitors include, without limitation, LAG525 and BMS-986016. CD28 is constitutively expressed on almost all human CD4⁺ T cells and on around half of all CD8 T cells. prompts T cell expansion. Non-limiting examples of CD28 inhibitors include TGN1412. CD122 increases the proliferation of CD8⁺ effector T cells. Non-limiting examples include NKTR-214. 4-IBB (also known as CD137) is involved in T-cell proliferation. CD137-mediated signaling is also known to protect T cells, and in particular CD8⁺ T cells from activation-induced cell death. PF-05082566, Urelumab (BMS-663513) and lipocalin are example CD137 inhibitors.

For any of the above combination treatments, the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention can be administered simultaneously or separately from the other anticancer agent.

In one embodiment, a method of treating or inhibiting infection in a patient in need thereof is provided, comprising administering to the patient an effective amount of the antibody or antigen binding fragment thereof, the bispecific antibody, the polypeptide, the conjugate, the composition, the article of manufacture or kit of the present invention.

EXAMPLES

The following illustrative examples are representative of embodiments of compositions and methods described herein and are not meant to be limiting in any way. The experimental methods without specific conditions in the following examples usually follow the conventional conditions as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, 2002, or follow the conditions suggested by the manufacturers.

Example 1. Generation of Anti-Human SIRPα Antibodies

Immunization

Anti-human SIRPα monoclonal antibodies were produced by immunizing mice. BABL/c mi ce, female, 6-8 weeks old, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Animal production license number: SCXK (Zhejiang) 2019-0001) . The mice were acclima ted to laboratory housing for about 7 days with a 12-hour light/12-hour dark cycle adjustment, at roo m temperatures of 20-25℃ with a relative humidity of 40-60%. The mice having adapted to the envir onment were immunized in accordance with the following protocol. The immunizing antigen was pur chased from ACROBiosystems (Cat. #SIA-H5225) , which is a His-tagged human SIRPα extracellula r domain fusion protein (SIRPα V1 (31-370aa) -His, with the amino acid sequence: EEELQVIQPDK SVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMD FSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHT VSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVIC EVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLE NGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLK VSAHPKEQGSNTAAENTGSNER, numbered as SEQ ID NO: 22) . The mice were immunized with the 50 μg antigen and adjuvant per mouse. The ratio of antigen and adjuvant was 1: 1, and the antigen and adjuvant were inoculated after being mixed quickly and thoroughly. The immunizations were pe rformed three times and the interval duration among each immunization two weeks. The blood was c ollected at 7 days after the last immunization. The titers of the antibody in mice serum were determin ed by ELISA assay. Mice with high antibody titers in the serum and titers tended to plateau were sele cted for splenocyte fusion. A booster immunization was performed by intraperitoneal injection of the antigen formulated in saline three days before the splenocyte fusion.

Hybridoma Generation

3 days after the final boost, mice were sacrificed, and the spleen cells were collected. After lysis of erythrocytes, the spleen cells were fused with Sp2/0 myeloma cells (National Collection of Authenticated Cell Cultures) in the presence of poly-ethylene glycol solution (Sigma, Cat. #P7181) . The fused hybridoma cells were cultured using a single step cloning method (HAT selection) . This method uses a semi-solid methylcellulose-based HAT selective medium to combine the hybridoma selection and cloning into one step. The hybridoma cells grow to form monoclonal colonies on the semi-solid media. 10 days after the fusion event, the hybridoma cells were seeded to 96-well plates and grown in HT containing medium for 2-4 days.

Hybridoma Screening

The fusion plates were primarily screened by ELISA assay with human SIRPα V1 (ACROBiosystems, Cat. #SIA-H5225) . The hybridoma cells from the positive wells were amplified into 48-well plates for 2^nd round screening. In the 2^nd round screening, binding activity was assessed by ELISA assay with human SIRPα V1. Clones with positive binding activity were selected for subclones. Thereafter, the specificity against human SIRPα V2 (ACROBiosystems, Cat. #SI2-H52H9) , SIRPα V8 (ACROBiosystems, Cat. #SI8-H52H5) , and SIRPγ (ACROBiosystems, Cat. #SIG-H5253) , species cross reactivity, and blocking activity of the human CD47 (ACROBiosystems, Cat. #CD7-H82E9) and SIRPα interaction were detected. The cell-based binding activities of U937, THP-1, and Jurkat cells (ATCC) were also determined by flow cytometry. The positive clones in both protein binding and cell binding assays were expanded immediately, frozen for cryopreservation, and sub-cloned two to three times to obtain a single-cell clone. Each selected clone was seeded into 96-well plates at the density of single cell per well by limiting dilution. Subsequently, one lead antibody 3R-2A10 with higher binding affinity to SIRPα variants and no binding to SIRPγ, and stronger functional potency was identified. The variable heavy chain (mVH) and variable light chain of 3R-2A10 are shown below:

mVH:

mVL:

Example 2. Humanization of Anti-Human SIRPα Murine Monoclonal Antibodies

The CDR regions of the heavy (VH) and light (VL) chains of the murine monoclonal antibody 3R-2A10 were identified and shown below in Table 1:

Table 1:

The amino acid sequences of the VH and VL of 3R-2A10 were compared against the available database of human Ig gene sequences to find the overall best-matching human germline Ig gene sequences. The CDR regions of 3R-2A10 were grafted into the best-matching human antibody frameworks to form a variable region sequence in the order of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 or LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. Back mutation was performed to achieve better affinity. The amino acid residues were identified and annotated by the Kabat numbering system.

As a result, four preferred humanized VH and four humanized VL versions were designed, respectively. The preferred VHs and VLs are randomly combined for further test. The amino acid sequences of the humanized antibodies are provided in table 1 below. The immunoglobulin heavy chain variable (IGHV) region or the immunoglobulin Kappa chain variable (IGKV) region, as the origin of the framework regions for each variable region, and the back mutations in the framework regions are shown as FR graft germline + FR back mutations.

Table 2. Exemplary humanized VHs and VLs.

Example 3. Chimeric Antibodies and Humanized Antibodies

In the following examples, the heavy chain variable regions in table 2 were recombinantly expressed with the human heavy chain IgG4 constant region to obtain full-length heavy chains, and the light chain variable regions in table 2 were recombinantly expressed with human light chain kappa constant region to obtain full-length light chains. S228P mutation (EU nomenclature, S241P in Kabat nomenclature) was introduced into the Fc segment to increase the stability of the IgG4 antibody. As is well known to those skilled in the art, the above heavy and light chain variable regions can also be recombined with other heavy and light chain constant regions of the IgG family or constant regions of mutated IgG family commonly known in the art, forming the complete heavy and light chains of the antibodies.

Exemplary heavy and light chain constant regions employed in the Examples of the present invention are as follows:

Sequence of mutated human IgG4 heavy chain constant region: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG (with mutation S228P, SEQ ID NO: 17, abbreviated as CH1-Fc herein) .

Sequence of light chain kappa constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18, abbreviated as CLκ herein) .

The humanized VH and VL sequence variants were combined individually with each other, resulting 9 VH-VL as shown in Table 3.

Table 3. Variable regions of exemplary humanized mAbs.

The synthesized VH and VL genes were inserted into corresponding vectors to be recombinantly expressed with the constant region encoding sequence respectively.

In this example, full-length heavy chains are formed by substituting the variable region of CH1-Fc with a VH region shown in table 3, and each full-length light chain is formed by substituting the variable region of CLκ with the a VL region shown in table 3. The full-length heavy and light chains of an exemplary antibody are shown in table 4. The sequences of CH1-Fc and CLκ are shown above in this Example. In table 4, variable regions are underlined, while constant regions are not.

Table 4: Full-length heavy and light chains of an exemplary antibody

The recombinant constructs were transfected into HEK293/CHO cells. After 4 days incubation, the expression supernatant was collected and purified by Protein A affinity chromatography column. The antibodies were eluted with a glycine buffer (pH3.0-3.5) followed by immediately neutralization with 1 M Tris-HCl buffer (pH8.0-9.0) . The purified proteins were replaced with PBS buffer by dialysis, and the aliquoted and frozen for later use.

Example 4. Biosensor Affinity Determination for Human SIRPα V1

The binding affinities of anti-SIRPα monoclonal antibodies (mAbs) including one chimeric mAb (with variable regions of 3R-2A10 and CH1-Fc and CLκ as the constant regions) , and humanized mAbs to human SIRPα (hSIRPα) V1 were determined by surface plasmon resonance (SPR) method.

Anti-SIRPα mAbs were immobilized into a Protein A sensor chip (Cytiva) at 2μg/mL (250-300 RU) and his tagged recombinant hSIRPα V1 (ACROBiosystems, Cat. #SIA-H5225) was applied at 100 nM with a flow rate of 30 μL/min. Analysis was performed with a BIAcore 8K (Cytiva) . Values were measured after an association period (ka) of 120 s followed by a dissociation period of 400 s (kd) to determine affinity constant (K_D) .

As shown in Table 5 all anti-SIRPα mAbs and the chimeric mAb 3R-2A10 have a strong affinity (K_D) for hSIRPα V1 from 2.37E-10 M to 5.26E-10 M. All humanized mAbs are equivalent to the affinity of the chimeric mAb.

Example 5. Binding Activity to Human SIRPα Variants and Cyno Monkey SIRPα

The binding activities of anti-SIRPα monoclonal antibodies (mAbs) of the present invention including one chimeric mAb, and humanized mAbs to human SIRPα (hSIRPα) V2/V8 and cynomolgus monkey SIRPα (mkSIRPα) were determined by ELISA assay. OSE-172, a known anti-SIRPα antibody was used as a reference antibody (see e.g., US20190127477A1) .

His tagged recombinant hSIRPα V2/V8 (ACROBiosystems, Cat. #SI2-H52H9/SI8-H52H5) and mkSIRPα (ACROBiosystems, Cat. #SIA-C52H7) were adsorbed to high-binding 96-well plates at a concentration of 2 μg/mL diluted in PBS overnight at 4℃. The coating solution is removed, the wells were washed with PBST and then blocked with 5%milk for 2 hours at room temperature while shaking. Blocking solution was removed, the wells were washed with PBST and incubated for 60 min at room temperature while shaking with either chimeric or humanized mAbs diluted in 5%milk at a starting concentration of 10 or 20 μg/mL and reducing the concentration in 4-fold serial dilutions. The wells were washed with PBST and incubated for 60 min at room temperature while shaking with an HRP-labeled goat anti-human IgG (Fc) antibody (Jackson ImmunoResearch Laboratories, Cat. #109-035-098) diluted 1: 10,000 in PBST. The wells were washed with PBST. TBM was developed for 8 min and stopped with 2M HCl. The absorbance at 450 nm was measured. EC₅₀ was calculated using a non-linear fit model (GraphPad Prism 9.3.0)

As shown in Table 6, all anti-SIRPα mAbs bound to hSIRPα V2/V8 and had similar EC₅₀ with mkSIRPα, while the reference antibody OSE-172 could not bind to hSIRPα V2/V8 and mkSIRPα.

Example 6. Binding Activity to THP-1 Cells Expressing SIRPα V2

The binding activities of anti-SIRPα monoclonal antibodies (mAbs) including one chimeric mAb, and humanized mAbs to THP-1 cells which express SIRPα V2, but not SIRPγ, were determined by flow cytometry.

THP-1 cells (ATCC) incubated for 60 min at 4℃ with increasing concentration of the anti-SIRPα mAbs diluted in PBS with 1%BSA. Cells were then washed with PBS with 1%BSA and incubated for additional 30 min with R-PE-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat. #109-116-098) diluted 1: 200 in PBS with 1%BSA. Cells were washed and binding analyzed using a Beckman CytoFLFX Flow Cytometer. EC₅₀ was calculated using a non-linear fit model (GraphPad Prism 9.3.0) .

As shown in Table 7 and FIG. 2, all anti-SIRPα mAbs bound to SIRPα V2 expressing THP-1 cells in a concentration-dependent manner. Additionally, the humanized mAbs have lower EC₅₀ than the chimeric mAb and the reference.

Example 7. Binding Activity to Human SIRPγ

The binding activities of anti-SIRPαmAbs including one chimeric mAb and humanized mAbs to human SIRPγ (hSIRPγ) were determined by ELISA assay.

Biotinlylated recombinant hSIRPγ (ACROBiosystems, Cat. #SIG-H82E3) was adsorbed to high-binding 96-well plates at a concentration of 2 μg/mL diluted in PBS overnight at 4℃. The coating solution is removed, the wells were washed with PBST and then blocked with 5%milk for 2 hours at room temperature while shaking. Blocking solution was removed, the wells were washed with PBST and incubated for 60 min at room temperature while shaking with either chimeric or humanized mAbs diluted in 5%milk at a starting concentration of 20 or 50 μg/mL and reducing the concentration in 4-fold serial dilutions. The wells were washed with PBST and incubated for 60 min at room temperature while shaking with an HRP-labeled goat anti-human IgG (Fc) antibody (Jackson ImmunoResearch Laboratories, Cat. #109-035-098) diluted 1: 10,000 in PBST. The wells were washed with PBST. TBM was developed for 8 min and stopped with 2M HCl. The absorbance at 450 nm was measured.

As shown in FIG. 3, the humanized anti-SIRPα mAbs did not appreciably bind to hSIRPγ at the antibody concentrations up to 50 μg/mL, while the reference OSE-172 weakly bound to hSIRPγ at the antibody concentrations up to 10 μg/mL.

Example 8. Binding Activity to Jurkat T Cells Expressing SIRPγ

The binding activities of anti-SIRPα monoclonal antibodies (mAbs) including one chimeric mAb and humanized mAbs to Jurkat cells which express SIRPγ, were determined by flow cytometry.

Jurkat cells (ATCC) incubated for 60 min at 4℃ with increasing concentration of the anti-SIRPα mAbs diluted in PBS with 1%BSA. Cells were then washed with PBS with 1%BSA and incubated for additional 30 min with R-PE-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat. #109-116-098) diluted 1: 200 in PBS with 1%BSA. Cells were washed and binding analyzed using a Beckman CytoFLFX Flow Cytometer.

As shown FIG. 4, all anti-SIRPα mAbs exhibited no binding to hSIRPγ on Jurkat cells at the antibody concentrations up to 200 μg/mL.

Example 9. Human CD47/SIRPα Blocking Activity

To assess the ability of the chimeric anti-SIRPα mAb 3R-2A10 to block the binding of human CD47 to SIRPα, the following method was employed using competitive ELISA assay.

His tagged recombinant hSIRPα (ACROBiosystems, Cat. #SIA-H5225) was adsorbed to high-binding 96-well plates at a concentration of 2 μg/mL diluted in PBS overnight at 4℃. The coating solution is removed, the wells were washed with PBST and then blocked with 4%BSA for 2 hours at room temperature while shaking. Blocking solution was removed, the wells were washed with PBST. Different concentrations of 3R-2A10 diluted in 2%BSA were mixed with a 0.15 μg/mL final concentration of biotinylated human CD47 protein (ACROBiosystems, Cat. #CD7-H82A3) diluted in 2%BSA, and then added into the wells and incubated for 60 min at room temperature while shaking. 3R-2A10 was evaluated at a starting concentration of 50 μg/mL and reducing the concentration in 3-fold serial dilutions. The wells were washed with PBST and incubated for 60 min at room temperature while shaking with an HRP-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Cat. #115-035-164) diluted 1: 10,000 in 2%BSA. The wells were washed with PBST. TBM was developed for 8 min and stopped with 2M HCl. The absorbance at 450 nm was measured.

As shown in FIG. 5, 3R-2A10 did not blocked the human CD47 and SIRPα interaction at the antibody concentration up to 50 μg/mL, while the reference blocked the binding of human SIRPα to CD47.

Example 10. Human CD47/SIRPγ Blocking Activity

To assess the ability of the chimeric anti-SIRPα mAb 3R-2A10 to block the binding of human CD47 to SIRPγ, the following method was employed using competitive ELISA assay.

Recombinant streptavidin (Beyotime Biotechnology, Cat. #P5084) was adsorbed to high-binding 96-well plates at a concentration of 5 μg/mL diluted in PBS overnight at 4℃. The coating solution is removed, the wells were washed with PBST and then blocked with 4%BSA for 2 hours at room temperature while shaking. Blocking solution was removed, the wells were washed with PBST and incubated for 60 min at 37℃ with 1 μg/mL of biotinylated human SIRPγ protein (ACROBiosystems, Cat. #SIG-H82E3) diluted in 2%BSA. The wells were washed with PBST. Different concentrations of 3R-2A10 diluted in 2%BSA were mixed with a 0.25 μg/mL final concentration of biotinylated human CD47 protein (ACROBiosystems, Cat. #CD7-H82A3) diluted in 2%BSA, and then added into the wells and incubated for 60 min at room temperature while shaking. 3R-2A10 was evaluated at a starting concentration of 50 μg/mL and reducing the concentration in 3-fold serial dilutions. 3R-2A10 was evaluated at a starting concentration of 50 μg/mL and reducing the concentration in 3-fold serial dilutions. The wells were washed with PBST and incubated for 60 min at room temperature while shaking with an HRP-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Cat. #115-035-164) diluted 1: 10,000 in 2%BSA. The wells were washed with PBST. TBM was developed for 10 min and stopped with 2M HCl. The absorbance at 450 nm was measured.

As shown in FIG. 6, 3R-2A10 did not blocked the human CD47 and SIRPγ interaction at the antibody concentration up to 50 μg/mL, as well as the reference OSE-172.

Example 11. Biosensor Affinity Determination for Human SIRPα V2/V8, Cyno Monkey SIRPα, and Human SIRPγ

The binding affinities of anti-SIRPα monoclonal antibodies (mAbs) including one chimeric mAb and humanized mAbs to human SIRPα V2/V8, cyno monkey SIRPα, and human SIRPγ were determined by surface plasmon resonance (SPR) method.

Recombinant hSIRPα V2/V8 (ACROBiosystems, Cat. #SI2-H52H9/SI8-H52H5) , mkSIRPα (ACROBiosystems, Cat. #SIA-C52H7) , and hSIRPγ (ACROBiosystems, Cat. #SIG-H82E3) were immobilized into a Series S Sensor Chip NTA (Cytiva) at 0.5 μg/mL and anti-SIRPα mAbs were applied at different concentrations (0.78-50 nM) with a flow rate of 30 μL/min. Analysis was performed with a BIAcore 8K (Cytiva) . Values were measured after an association period (ka) of 150 s followed by a dissociation period of 600 s (kd) to determine affinity constant (KD) .

As shown in Table 8, the humanized mAb hu2A1044 has a strong affinity (KD) for hSIRPα variants and similar affinity for mkSIRPα from 1.68E-12 M to 3.76E-10 M, which is equivalent to the affinity of the chimeric mAb 3R-2A10. The humanized mAb has much lower affinity for hSIRPγ, but a little higher than the chimeric mAb.

Example 12. Epitope Binning

To assess the binding epitope of the anti-SIRPα mAb 3R-2A10 compared to the reference OSE-172, competitive ELISA assay was applied for epitope binning.

His tagged recombinant hSIRPα (ACROBiosystems, Cat. #SIA-H5225) was adsorbed to high-binding 96-well plates at a concentration of 2 μg/mL diluted in PBS overnight at 4℃. The coating solution is removed, the wells were washed with PBST and then blocked with 4%BSA for 2 hours at room temperature while shaking. Blocking solution was removed, the wells were washed with PBST. OSE-172 diluted in 2%BSA was added into the wells with a starting concentration of 20 μg/mL and reducing the concentration in 4-fold serial dilutions and incubated for 30 min at room temperature while shaking. Biotinylated 3R-2A10 diluted in 2%BSA with a final concentration 0.1μg/mL was then added into the wells and incubated for 60 min at room temperature while shaking. The wells were washed with PBST and incubated for 60 min at room temperature while shaking with high-sensitive HRP labeled streptavidin (Thermo Scientific, Cat. #21134) diluted 1: 400 in 2%BSA. The wells were washed with PBST. TBM was developed for 4 min and stopped with 2M HCl. The absorbance at 450 nm was measured. Competition ratio was calculated.

As shown in FIG. 7 and Table 9, 3R-2A10 bound to hSIRPα even when the concentration of the competitor OSE-172 was increased to 20 μg/mL, indicating 3R-2A10 binds to a different unique epitope from the reference OSE-172.

Example 13. Human Macrophage Mediated Phagocytosis on Tumor Cells

To assess the effect of the anti-SIRPα mAbs including one chimeric mAb and humanized mAbs on phagocytosis of tumor cells by human macrophages, in vitro the following method was employed using flow cytometry.

Human monocytes were isolated from PBMCs and were induced to differentiate into M0 macrophages with 50 ng/mL M-CSF (PeproTech, Cat. #AF-300-25) or M2 macrophages with 50 ng/mL M-CSF, 50 ng/mL TGF-β1 (PeproTech, Cat. #AF-100-21) , and 50 ng/mL IL-10 (PeproTech, Cat. #AF-200-10) for later use. The tumor cells DLD1 and Jurkat were labeled with CFSE and were mixed with M0 or M2 macrophages at a ratio of 1: 1. Then 5-fold serial dilutions of the anti-SIRPαmAbs at a starting concentration of 1 or 2 μg/ml were added. After incubating for 4 h at room temperature, cells were washed and CD11b flow staining was performed. The ratios of respective cell subpopulations were detected by flow cytometer. Phagocytosis%= CFSE⁺CD11b⁺ /CD11b⁺ x 100%.

As shown in Table 10, both chimeric and humanized mAbs induced strong phagocytosis against solid tumor cells (DLD1) and hematologic tumor cells (Jurkat) by human M0 and/or M2 macrophages as compared to a hIgG4 isotype control. In contrast, OSE-172 did not induced the phagocytosis of DLD1 nor Jurkat cells. FIG. 8A-8D demonstrate representative M0 macrophage phagocytosis curves of the anti-SIRPα mAbs. FIG. 8E-8F demonstrate representative M2 macrophage phagocytosis curves of the anti-SIRPα mAbs.

Example 14. Human Macrophages Binding Activity

The binding activity of anti-SIRPα mAbs including one chimeric mAb and humanized mAbs to human macrophages were determined by flow cytometry.

Human monocytes were isolated from PBMCs and were induced to differentiate into M0 macrophages with 50 ng/mL M-CSF (PeproTech, Cat. #AF-300-25) for later use. Human Fc receptor binding inhibitor (BioLegend, Cat. #422302) was first added for 10 min at room temperature to block human Fc receptors on human macrophages to reduce background. Then, 5-fold serial dilutions of the anti-SIRPα mAbs at a starting concentration of 10 μg/ml were incubated for 30 min at 4℃ and washed before stained 30 min at 4℃ with an R-PE conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat. #109-116-098) . Samples were analyzed on a Beckman CytoFLFX Flow Cytometer.

As shown in Table 11 and FIG. 9, chimeric and humanized mAbs bound to human macrophages with comparable EC₅₀.

Example 15. Human CD3⁺ T Cells Binding Activity

The binding activity of anti-SIRPα mAbs including one chimeric mAb and humanized mAbs to human CD3⁺ T cells were determined by flow cytometry.

Human CD3⁺ T cells were isolated from PBMCs and were incubated with 4-fold serial dilutions of the anti-SIRPα mAbs at a starting concentration of 10 μg/ml for 30 min at 4℃. Then the cells were washed and stained 30 min at 4℃ with an R-PE conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat. #109-116-098) . Samples were analyzed on a Beckman CytoFLFX Flow Cytometer. For activated T cells, prior to the binding assay CD3⁺ T cells were activated for 72 hours in a plate coated with anti-CD3 (BioLegend, Cat. #317326) in the presence of anti-CD28 (BioLegend, Cat. #302934) and IL-2 (R&D Systems, Cat. #202-IL-050) .

As shown in FIG. 10A (CD3⁺ T cells) and FIG. 10B (activated CD3⁺ T cells) , both chimeric and humanized mAbs did not bind to humanor activated CD3⁺ T cells.

Example 16. Human T Cell Proliferation

To assess the effect of the chimeric anti-SIRPα mAbs 3R-2A10 on allogeneic dendritic cell-induced T cell proliferation, in vitro the following method was employed using flow cytometry.

Human CD3⁺ T cells were isolated from PBMCs. Effect of anti-SIRPα mAbs antibodies on proliferation of T cells was determined by activating CFSE Cell Division Tracer kit (BioLegend, Cat. #423801) labelled human CD3⁺ T cells with allogeneic human matured dendritic cells at a 1: 10 DC : T cell ratio in the presence of 10 ug/ml anti-SIRPα mAbs. Flow cytometry was used to determine the percentage of proliferated CD3⁺ T cells following 7-day coculture.

As shown in FIG. 11, 3R-2A10 showed no effect on T cell proliferation, as well as OSE-172.

Claims

An isolated antibody or antigen binding fragment thereof binding to SIRPα protein or the extracellular domain thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable region comprises

HCDR1 sequence comprising the amino acid sequence as shown by SYYIH (SEQ ID NO: 1) ,

HCDR2 sequence comprising the amino acid sequence as shown by WIDPGNLNTKYNEKFTG (SEQ ID NO: 2) , and

HCDR3 sequence comprising the amino acid sequence as shown by LNYYGNYGDF (SEQ ID NO: 3) ; and

(b) the light chain variable region comprises

LCDR1 sequence comprising the amino acid sequence as shown by KSSQSLLNSGNQRNYLA (SEQ ID NO: 4) ,

LCDR2 sequence comprising the amino acid sequence as shown by GASIRES (SEQ ID NO: 5) , and

LCDR3 sequence comprising the amino acid sequence as shown by QHDHSYPLT (SEQ ID NO: 6) ; wherein the CDR sequences are defined according to the Kabat CDR definition.
The antibody or antigen binding fragment thereof of claim 1, wherein the antibody is a chimeric antibody, humanized antibody, or human antibody.
The antibody or antigen binding fragment thereof of claim 1 or 2, wherein the heavy chain variable region and the light chain variable region further comprise a human acceptor framework.
The antibody or antigen binding fragment thereof of claim 3, wherein the human acceptor framework of the heavy chain variable region derives from any immunoglobulin heavy chain variable region germline selected from the group consisting of: IGHV1-3, IGHV1-46, IGHV1-2, and IGHV1-69, and variants thereof.
The antibody or antigen binding fragment thereof of claim 3 or 4, wherein the human acceptor framework of the heavy chain variable region comprises one or more amino acid residues selected from the group consisting of: 27Y, 60N, 61E, 64T, 71A, 73K and 78A, and wherein the amino residues are numbered according to the Kabat numbering system.
The antibody or antigen binding fragment thereof of claim 3, wherein the human acceptor framework of the light chain variable region derives from any immunoglobulin Kappa chain variable region germline selected from the group consisting of: IGKV3-15, IGKV1-39, IGKV2-28, and IGKV4-1, and variants thereof.
The antibody or antigen binding fragment thereof of claim 3 or 6, wherein the human acceptor framework of the light chain variable region comprises the amino acid residue 83L, and wherein the amino residues are numbered according to the Kabat numbering system.
An isolated antibody or antigen binding fragment thereof binding to SIRPα protein or the extracellular domain thereof, wherein the antibody comprises: (i) a light chain variable region (VL) comprising the identical LCDR1, LCDR2, and LCDR3 with those in any one of SEQ ID NO: 11 to SEQ ID NO: 14 and SEQ ID NO: 16; and (ii) a heavy chain variable region (VH) comprising the identical HCDR1, HCDR2, and HCDR3 with those in any one of SEQ ID NO: 7 to SEQ ID NO: 10 and SEQ ID NO: 15; wherein the HCDRs and LCDRs are defined by any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition, or defined by a hybrid scheme that combines any two, three or four of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition.
The antibody or antigen binding fragment thereof of any one of claims 1-8, wherein the heavy chain variable region comprises any one amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 15, and an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any one amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, and SEQ ID NO: 15.
The antibody or antigen binding fragment thereof of any one of claims 1-9, wherein the light chain variable region comprises any one amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 14, SEQ ID NO: 16, and an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with any one amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 14, and SEQ ID NO: 16.
The antibody or antigen binding fragment thereof of any one of claims 1-10, comprising:

1) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVS S (SEQ ID NO: 7) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14;

2) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 11) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 11,

3) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQRNYLAWYQQKPGQAPRLLIYGASIRES GIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 12) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 12;

4) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQRNYLAWYLQKPGQSPQLLIYGASIRESG VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 13) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 13;

5) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEWMGWIDPGNLNTK YNEKFTGRVTITADKSASTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 8) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14;

6) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSISTAYMELSRLRSDDTAVYYCARLNYYGNYGDFWGQGTMVTVS S (SEQ ID NO: 9) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 11) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 11;

7) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSISTAYMELSRLRSDDTAVYYCARLNYYGNYGDFWGQGTMVTVS S (SEQ ID NO: 9) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14;

8) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 10) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 11) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 11;

9) a heavy chain variable region comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS (SEQ ID NO: 10) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIK (SEQ ID NO: 14) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 14;

10) a heavy chain variable region comprising the amino acid sequence as shown by QVQLQQSGPELVKPGASVRISCKASGYTFTSYYIHWVRQRPGQGLEWIGWIDPGNLNTKYN EKFTGKATLTADKSSSTAYMQFSSLTSEDSAVYFCARLNYYGNYGDFWGQGTTLTVSS (SEQ ID NO: 15) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence as shown by DIVMTQSPSSLSVSAGEKVTLSCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIYGASIRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQHDHSYPLTFGAGTKLELK (SEQ ID NO: 16) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 16.
The antibody or antigen binding fragment thereof of any one of claims 1 to 11, further comprising a heavy chain constant region and a light chain constant region.
The antibody or antigen binding fragment thereof of claim 12, wherein the heavy chain constant region is an IgG heavy chain constant region.
The antibody or antigen binding fragment thereof of claim 13, wherein the heavy chain constant region is an IgG4 heavy chain constant region, optionally, the IgG4 heavy chain constant region comprises the amino acid residue 228P numbered according to the EU numbering system.
The antibody or antigen binding fragment thereof of claim 14, wherein the heavy chain constant region comprises an amino acid sequence as shown by ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG (SEQ ID NO: 17) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 17.
The antibody or antigen binding fragment thereof of claim 12, wherein the light chain constant region is a light chain kappa constant region.
The antibody or antigen binding fragment thereof of claim 16, wherein the light chain constant region comprises an amino acid sequence as shown by RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 18.
The antibody or antigen binding fragment thereof of any of claims 1-17, comprising:

1) the heavy chain comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTMTADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG (SEQ ID NO: 19) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 19, and the light chain sequence comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 20; or

2) the heavy chain comprising the amino acid sequence as shown by QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIDPGNLNTK YNEKFTGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLNYYGNYGDFWGQGTMVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG (SEQ ID NO: 21) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 21, and the light chain sequence comprising the amino acid sequence as shown by DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQRNYLAWYQQKPGKAPKLLIYGASIRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQHDHSYPLTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20) or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with SEQ ID NO: 20; .
The antibody or antigen binding fragment thereof of any of claims 1-18, wherein the antigen binding fragment is Fab, F (ab') 2, Fab', scFv, Fv, Fd, dAb, or diabody.
A bispecific antibody comprising the antibody or antigen binding fragment thereof of any one of the claims 1-19 and a second antibody or antigen binding fragment thereof.
The bispecific antibody of claim 20, wherein the second antibody or antigen binding fragment thereof binds to a tumor antigen expressed on the surface of a tumor cell, wherein the tumor antigen is selected from the group consisting of A33; ADAM-9; ALCAM; BAGE; beta-catenin; BCMA; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD30; CD33; CD36; CD40/CD154; CD45; CD46; CD47, CD5; CD56; CD70; CD79a/CD79b; CD123, CD133, CD138; PSCA; Claudin; CLL-1; CDK4; CEA; CTLA4; PD-L1, Cytokeratin 8; LeY; Ov-γ; NKG2D; EGF-R; IL13Rα2; EphA2; ErbB1; ErbB3; ErbB4; EpCAM; GAGE-1; GAGE-2; GD2/GD3/GM2; HER-2/neu; GPC3; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A) ; LUCA-2; MAGE-1; MAGE-3; MART; Meso; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M; pl5; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-a receptor; TNF-γ receptor; Transferrin Receptor; and VEGF receptor.
A conjugate, comprising the antibody or antigen binding fragment thereof of any one of the claims 1-19, or the bispecific antibody of claim 20 or 21.
A nucleic acid comprising a sequence encoding the antibody or antigen binding fragment thereof of any one of the claims 1-19, or an antisense strand thereof.
A vector comprising the nucleic acid of claim 23.
A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of the claims 1-19, the bispecific antibody of claim 20 or 21, the conjugate of claim 22 or the nucleic acid of claim 23.
A host cell comprising the nucleic acid of claim 23.
A method for preparing an antibody or antigen binding fragment thereof binding to SIRPα protein or the extracellular domain thereof, comprising: (a) growing the host cell of claim 26 under conditions so that the host cell expresses the antibody or antigen binding fragment thereof; and (b) purifying the antibody or antigen binding fragment thereof.
A method for treating a cancer, comprising administrating an effective amount of the antibody or antigen binding fragment thereof of any one of the claims 1-19, the bispecific antibody of claim 20 or 21, the conjugate of claim 22, the nucleic acid of claim 23, or a pharmaceutical composition of claim 25 to a subject having the cancer.
The method of claim 28, wherein the cancer is any one selected from the group consisting of: leukemia, lymphoma, colorectal adenocarcinoma, pancreatic cancer, breast cancer, bladder cancer, renal cell cancer, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer; renal pelvis cancer, spinal tumors, glioma, pituitary adenoma, Kaposi's sarcoma, and combinations and metastatic lesions thereof.