Polypeptide, fusion protein, bispecific antibody and application thereof

Abstract

The invention relates to an SIRP alpha polypeptide, an immunoglobulin fusion protein, a monospecific antibody, a bispecific antibody and a multispecific antibody, which are used for developing a new tumor targeting drug, and the tumor targeting drug can specifically target and treat tumors, activate and enhance the immune function in the body of a patient, and improve the tumor targeting effect. On the premise of ensuring the safety, the tumor treatment effect is obviously improved, and meanwhile, the medication cost of a patient is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a SIRPα polypeptide, an immunoglobulin fusion protein, a monospecific antibody, a bispecific antibody, a multispecific antibody, and their use in the development of targeted drugs. Background Technology

[0002] CD47 is a transmembrane protein widely distributed on the surface of normal cells, with a molecular weight of approximately 50 kDa. It belongs to the immunoglobulin superfamily and its main biological functions include cell adhesion, cell migration, regulation of inflammatory responses, and inhibition of macrophage phagocytosis. Three known natural ligands for CD47 are integrin, platelet-1, and SIRPα.

[0003] In tumor tissues, macrophages can clear tumor cells through phagocytosis, but this phagocytosis is inhibited by the CD47-SIRPα immune checkpoint pathway. Furthermore, macrophages themselves express CD47 molecules, which act cis-on SIRPα on the same cell surface. Extensive research data indicates that cis-acting CD47 molecules elicit stronger intracellular SIRPα signals compared to trans-acting molecules, thus triggering a stronger inhibitory effect on themselves. Therefore, due to the aforementioned immune checkpoint role of CD47-SIRPα in macrophages, it can serve as a pathway for tumor treatment. This target can be used to treat various cancers, and the effectiveness of CD47 blockade has been demonstrated in mouse xenograft tumor transplantation models. Therefore, CD47 has become a novel target for cancer immune checkpoint therapy (Vonderheide R H. CD47blockade as another immune checkpoint therapy for cancer. Nature Medicine, 2015, 21(10):1122-1123).

[0004] According to statistics, as of 2026, there are 39 antibody / fusion protein drugs targeting the CD47-SIRPα pathway in clinical research stages (see Table 1).

[0005] Table 1: Antibody / fusion protein drugs targeting the CD47-SIRPα pathway in clinical trials

[0006] Drug Name The highest level of R&D in the world Original research company Letaplimab (IBI-188) Phase III clinical trials Innovent Biologics (Suzhou) Co., Ltd. Timdarpacept Phase III clinical trials Emin-Onco Biopharmaceutical Technology (Shanghai) Co., Ltd. Evorpacept (ALX148) Phase III clinical trials Alexo Therapeutics; Magrolimab (Hu5F9-G4) Phase III clinical trials Stanford University; Gilead Sciences; Ono Pharmaceutical; Roche; Merck; Lemzoparlimab (TJ-011133) Phase III clinical trials I-Mab Biotech (Shanghai) Co., Ltd.; Newbridge Biotech; AbbVie Biopharmaceuticals; DSP-107 Phase II clinical trials Kahr Medical Ligufalimab (AK-117) Phase III clinical trials Zhongshan Kangfang Biomedical Co., Ltd. Maplirpacept (TTI-622) Phase II clinical trials Pfizer Pharmaceuticals; Ontorpacept Phase II clinical trials Pfizer Pharmaceuticals; BI 770371 Phase II clinical trials OSE Immunotherapeutics; Boehringer Ingelheim Pharmaceuticals HCB101 Phase II clinical trials Hankang Biotechnology Co., Ltd.; Shanghai Henlius Biotech Co., Ltd.; LM-101 Phase II clinical trials China Biopharmaceutical Co., Ltd. TTI-621 Phase II clinical trials University Health Network; The Hospital For Sick Children; IMM 01 Phase II clinical trials Yiming Angke Biomedical Technology (Shanghai) Co., Ltd. BSI-082 Phase I clinical trial Bio-Sens Biotechnology (Nanjing) Co., Ltd.; JY47 Phase I clinical trial Hangzhou Jiuyuan Gene Biopharmaceutical Co., Ltd. FP002 Phase I clinical trial Guangdong Feipeng Pharmaceutical Co., Ltd.; ADU-1805 Phase I clinical trial Sairopa; Exelixis; Chinook Therapeutics; DS-1103a Phase I clinical trial AstraZeneca Pharmaceuticals; Daiichi Sankyo Co., Ltd.; Kobe University BYON4228 Phase I clinical trial Byondis HMPL-A83 Phase I clinical trial Hutchison MediPharma F527 Phase I clinical trial Shandong New Era Pharmaceutical Co., Ltd. lumistobart Phase I clinical trial Zhejiang Borui Biopharmaceutical Co., Ltd. LD002 Phase I clinical trial Zhejiang Landun Pharmaceutical Co., Ltd. STI-6643 Phase I clinical trial Sorrento Therapeutics sB24M Phase I clinical trial SWISS BIOPHARMA MED TQB2928 Phase I clinical trial Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Epacmarstobart Phase I clinical trial Gilead Sciences Anzurstobart Phase I clinical trial Bristol-Myers Squibb Pharmaceuticals SHR-1603 Phase I clinical trial Jiangsu Hengrui Medicine Co., Ltd. urabrelimab Phase I clinical trial Novartis Pharmaceuticals gentulizumab Phase I clinical trial Changchun Jinsai Pharmaceutical Co., Ltd.; SG404 Phase I clinical trial Hangzhou Shangjian Biotechnology Co., Ltd.; AL-008 Phase I clinical trial Alector; ZL-1201 Phase I clinical trial Zai Ding Pharmaceuticals (Shanghai) Co., Ltd.; MIL-95 Phase I clinical trial Beijing Tianguangshi Biotechnology Co., Ltd.; Con诺亚BioMedtech (Chengdu) Co., Ltd.; IMC-002 Phase I clinical trial Immuneoncia Therapeutics; CC-90002 Phase I clinical trial Inhibrx; Bristol-Myers Squibb; Inhibrx Biosciences BI-765063 Phase I clinical trial Boehringer Ingelheim Corp;

[0007] The aforementioned anti-CD47 antibodies and SIRPα-Fc fusion proteins, among other investigational drugs, exhibit certain anti-tumor activity by blocking the CD47-SIRPα signaling pathway and relieving the inhibitory effect of CD47 on immune cells. However, because erythrocytes also express a large amount of CD47 protein, anti-CD47 antibody therapy, which binds to CD47 protein with high affinity, can cause toxic side effects such as erythrocyte agglutination and anemia (Mccracken MN, et al. Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 “Don't Eat Me” Signals. Clinical Cancer Research, 2015, 21(16):3597-3601). This makes the clinical development of anti-CD47 antibody drugs extremely difficult, so no anti-CD47 antibody drugs have been approved for marketing to date. Furthermore, current clinical trial results indicate that systemic monotherapy for blocking the CD47-SIRPα pathway in cancer patients has negligible efficacy, and CD47 blockade can lead to numerous side effects such as anemia and thrombocytopenia. Therefore, given the current structure of clinical research, eliminating the side effects of the target will be a key focus of future research.

[0008] Therefore, given the potential safety risks (such as anemia, hemagglutination, etc.) of monovalent or multivalent antibodies or recombinant proteins targeting CD47, there is a need to develop new tumor-targeting drugs that can specifically target and treat tumors while activating and enhancing the patient's immune function, significantly improving the efficacy of tumor treatment while ensuring safety and reducing the cost of medication for patients. Summary of the Invention

[0009] This invention specifically describes a SIRPα polypeptide, a SIRPα immunoglobulin fusion protein, a monospecific antibody, a bispecific antibody, a multispecific antibody, and their applications.

[0010] Specifically, the present invention provides a polypeptide comprising a SIRPαV2 D1 domain mutant, wherein the SIRPαV2 D1 domain mutant comprises an amino acid mutation at position 31, 37, 52, 96 or 80 relative to the wild-type SIRPαV2 D1 domain.

[0011] In some specific embodiments, the present invention provides a polypeptide comprising a SIRPαV2 D1 domain mutant, wherein the SIRPαV2 D1 domain mutant comprises, relative to the wild-type SIRPαV2 D1 domain, (1) a Q37H mutation and an N80Q mutation, or (2) an I31L mutation, a Q37H mutation and an N80Q mutation, wherein the wild-type SIRPαV2 D1 domain comprises the amino acid sequence shown in SEQ ID NO:1.

[0012] The amino acid sequence of the wild-type SIRPαV2 D1 domain is shown below (SEQ ID NO:1): EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0013] In some specific embodiments, the SIRPαV2 D1 domain mutant comprises the following amino acid sequence (SEQ ID NO:2): EEELQVIQPDKSVSVAAGESAILHCTVTSLX1PVGPIX2WFRGAGPARELIYNX3KEGHFPRVTTVSESTKRENMDFSISISX4ITPADAGTYYCVKFRX5GSPDTEFKSGAGTELSVRAKPS

[0014] Where X1 represents the mutation at position 31, X2 represents the mutation at position 37, X3 represents the mutation at position 52, X4 represents the mutation at position 80, and X5 represents the mutation at position 96, and:

[0015] X1 is selected from I or L;

[0016] The X2 is selected from Q, H, or R;

[0017] The X3 is selected from Q, F, or H;

[0018] The X4 is selected from N or Q;

[0019] The X5 mentioned is selected from K or R.

[0020] In some specific embodiments, X1 is L, X2 is H, X3 is Q, X4 is Q, and X5 is K, and the amino acid sequence of the SIRPαV2 D1 domain mutant is shown in SEQ ID NO:3: EEELQVIQPDKSVSVAAGESAILHCTVTSLLPVGPIHWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISQITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0021] In some specific embodiments, X1 is I, X2 is H, X3 is Q, X4 is Q, and X5 is K. The amino acid sequence of the SIRPαV2 D1 domain mutant is shown in SEQ ID NO:4: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIHWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISQITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0022] In some specific embodiments, X1 is I, X2 is R, X3 is Q, X4 is Q, and X5 is K. The amino acid sequence of the SIRPαV2 D1 domain mutant is shown in SEQ ID NO:5: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIRWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISQITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0023] In some specific embodiments, X1 is I, X2 is Q, X3 is Q, X4 is Q, and X5 is R. The amino acid sequence of the SIRPαV2 D1 domain mutant is shown in SEQ ID NO:6: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISQITPADAGTYYCVKFRRGSPDTEFKSGAGTELSVRAKPS

[0024] In some specific embodiments, X1 is I, X2 is Q, X3 is H, X4 is Q, and X5 is K, and the amino acid sequence of the SIRPαV2 D1 domain mutant is shown in SEQ ID NO:7: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNHKEGHFPRVTTVSESTKRENMDFSISISQITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0025] In some specific embodiments, X1 is I, X2 is Q, X3 is F, X4 is Q, and X5 is K. The amino acid sequence of the SIRPαV2 D1 domain mutant is shown in SEQ ID NO:8: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNFKEGHFPRVTTVSESTKRENMDFSISISQITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

[0026] The present invention also provides the use of the polypeptides described in any of the above claims in the preparation of immunoglobulin fusion proteins, monospecific antibodies, bispecific antibodies and multispecific antibodies.

[0027] The present invention also provides a SIRPα immunoglobulin fusion protein comprising the following portions directly or indirectly linked, wherein the indirect linking comprises being linked via a linker fragment, a flexible sequence, or a hinge region sequence:

[0028] (1) The above-mentioned polypeptides; and

[0029] (2) Immunoglobulin homeostasis domain or fragments thereof.

[0030] In some specific embodiments, the polypeptide portion of the SIRPα immunoglobulin fusion protein is located at the N-terminus of the immunoglobulin constant region domain or a fragment thereof.

[0031] In some specific embodiments, the immunoglobulin is a human immunoglobulin or a mutant thereof.

[0032] In some specific embodiments, the human immunoglobulin is IgG1, IgG2, IgG3 or IgG4; preferably, the immunoglobulin is IgG1 or IgG4.

[0033] In some specific embodiments, the immunoglobulin constant region domain or a fragment thereof includes an immunoglobulin hinge region domain and an immunoglobulin Fc domain.

[0034] In some specific embodiments, the amino acid sequence of the immunoglobulin hinge region is shown in SEQ ID NO:22: EPKSCDKTHTCPPCP

[0035] In some specific embodiments, the amino acid sequence of the immunoglobulin hinge region is shown in SEQ ID NO:23: ESKYGPPCPSCP

[0036] In some specific embodiments, the amino acid sequence of the immunoglobulin hinge domain mutant is shown in SEQ ID NO:24: ESKYGPPCPPCP

[0037] In some specific embodiments, the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO:25: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0038] In some specific embodiments, the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO:26: APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0039] In some specific embodiments, the amino acid sequence of the SIRPα immunoglobulin fusion protein is shown in SEQ ID NO:9: EEELQVIQPDKSVSVAAGESAILHCTVTSLL PVGPI H WFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0040] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids.

[0041] In other specific embodiments, the amino acid sequence of the SIRPα immunoglobulin fusion protein is shown in SEQ ID NO:10: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI H WFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0042] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids.

[0043] In other specific embodiments, the amino acid sequence of the SIRPα immunoglobulin fusion protein is shown in SEQ ID NO:11: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI R WFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0044] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids.

[0045] In other specific embodiments, the amino acid sequence of the SIRPα immunoglobulin fusion protein is shown in SEQ ID NO:12: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYYCVKFR R GSPDTEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0046] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids.

[0047] In other specific embodiments, the amino acid sequence of the SIRPα immunoglobulin fusion protein is shown in SEQ ID NO:13: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYN H KEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0048] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids.

[0049] In other specific embodiments, the amino acid sequence of the SIRPα immunoglobulin fusion protein is shown in SEQ ID NO:14: EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYN F KEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0050] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids.

[0051] The present invention also provides the use of the fusion protein described in any of the above claims in the preparation of monospecific antibodies, bispecific antibodies or multispecific antibodies.

[0052] The present invention also provides a bispecific antibody comprising:

[0053] (1) A first binding domain comprising: the above-described polypeptide or the above-described SIRPα immunoglobulin fusion protein, and

[0054] (2) The second binding domain includes: an antibody or an antigen-binding fragment thereof targeting the second antigen;

[0055] Furthermore, the first bonding domain is directly connected to or connected to the C-end, N-end, or hinge area of ​​the second bonding domain through a connecting segment. Preferably, the connecting segment is (GGGGS)n, where n is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0056] In some specific embodiments, the second binding domain is a complete antibody, and the first binding domain is directly connected to the C-terminus or N-terminus of the complete antibody heavy chain or connected through a linker fragment, or the first binding domain is directly connected to the C-terminus or N-terminus of the complete antibody light chain or connected through a linker fragment.

[0057] In some specific embodiments, the second antigen is a tumor cell antigen; preferably, it is selected from: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACA M5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GP C-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, integrin-4, Notch 2. Notch 1, PD-1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA or CD138.

[0058] In some specific embodiments, the antibody targeting the second antigen or its antigen-binding fragment is selected from anti-CD20 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, or anti-PD-L1 antibody or its antigen-binding fragment.

[0059] In some specific embodiments, the bispecific antibody includes the SIRPαV2 D1 domain mutant fusion protein or the SIRPαV2 D1 domain mutant as described in any of the above-mentioned claims of the present invention, as well as the complete sequence or functional fragment of an antibody that binds to a second antigen.

[0060] In some specific embodiments, the antibodies targeting the second antigen include, but are not limited to: anti-CD20 antibodies (including, but not limited to, rituximab, offatumumab, ofbinutuzumab, ixemomab tiuxetan, occaratuzumab, occrelizumab, and tositumomab I-131). I-131), ututuximab, vetuzumab, anti-PD-1 antibodies (including but not limited to nivolumab, pembrolizumab, cimiplimab, zimberelimab, tislelizumab, sintilimab, camrelizumab, toripalimab, penpulimab) (Pianpulimab), Dostarlimab), anti-PD-L1 antibodies (including but not limited to Atezolizumab, Avelumab, Durvalumab, Envafolimab), and anti-EGFR antibodies (including but not limited to Cetuximab, Panitumumab, Nimotuzumab, Matuzumab, Fentuximab, Modotuximab, Ingatuzumab, and Nexituzumab).

[0061] In some specific embodiments, the bispecific antibody comprises: i) CDR-H1 shown in SEQ ID NO:43, CDR-H2 shown in SEQ ID NO:44 and CDR-H3 shown in SEQ ID NO:45, and ii) CDR-L1 shown in SEQ ID NO:46, CDR-L2 shown in SEQ ID NO:47 and CDR-L3 shown in SEQ ID NO:48 (see Table 2 for sequence information).

[0062] Table 2: CDR sequences of bispecific antibodies

[0063] Description SEQ ID NO Amino acid sequence VH-CDR1 43 SYNMH VH-CDR2 44 AIYPGNGDTSYNQKFKG VH-CDR3 45 STYYGGDWYFNV VL-CDR1 46 RASSSVSYIH VL-CDR2 47 ATSNLAS VL-CDR3 48 QQWTSNPPT

[0064] Note: CDR sequences are numbered according to the Kabat system.

[0065] In some specific embodiments, the bispecific antibody comprises: i) CDR-H1 shown in SEQ ID NO:49, CDR-H2 shown in SEQ ID NO:50 and CDR-H3 shown in SEQ ID NO:51, and ii) CDR-L1 shown in SEQ ID NO:52, CDR-L2 shown in SEQ ID NO:53 and CDR-L3 shown in SEQ ID NO:54 (see Table 3 for sequence information).

[0066] Table 3: CDR sequences of bispecific antibodies

[0067] Description SEQ ID NO Amino acid sequence VH-CDR1 49 NSGMH VH-CDR2 50 VIWYDGSKRYYADSVKG VH-CDR3 51 NDDY VL-CDR1 52 RASQSVSSYLA VL-CDR2 53 DASNRAT VL-CDR3 54 QQSSNWPRT

[0068] Note: CDR sequences are numbered according to the Kabat system.

[0069] In some specific embodiments, the bispecific antibody comprises: i) the amino acid sequence of the heavy chain variable domain VH shown in SEQ ID NO:55, and ii) the amino acid sequence of the light chain variable domain VL shown in SEQ ID NO:56 (see Table 4 for sequence information).

[0070] Table 4: Variable region sequence of bispecific antibodies

[0071] Description SEQ ID NO Amino acid sequence VH 55 QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA 50IYPGNGDTSY NQKFKGKATLTADKSSSTAY MQLSSLTSED SAVYYCARST 100YYGGDWYFNV WGAGTTVTVS A 121 VL 56 QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT 50SNLASGVPVR FSGSGSGTSYSLTISRVEAE DAATYYCQQW TSNPPTFGGG 100TKLEIK 106

[0072] In some specific embodiments, the bispecific antibody comprises: i) the amino acid sequence of the heavy chain variable domain VH shown in SEQ ID NO:57, and ii) the amino acid sequence of the light chain variable domain VL shown in SEQ ID NO:58 (see Table 5 for sequence information).

[0073] Table 5: Variable region sequence of bispecific antibodies

[0074] Description SEQ ID NO Amino acid sequence VH 57 QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV 50IWYDGSKRYY ADSVKGRFTISRDNSKNTLF LQMNSLRAED TAVYYCATND 100DYWGQGTLVT VSS 113 VL 58 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD 50ASNRATGIPA RFSGSGSGTDFTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ 100GTKVEIK 107

[0075] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:3, and the second binding domain comprises: i) CDR-H1 shown in SEQ ID NO:43, CDR-H2 shown in SEQ ID NO:44, and CDR-H3 shown in SEQ ID NO:45, and ii) CDR-L1 shown in SEQ ID NO:46, CDR-L2 shown in SEQ ID NO:47, and CDR-L3 shown in SEQ ID NO:48.

[0076] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:4, and the second binding domain comprises: i) CDR-H1 shown in SEQ ID NO:43, CDR-H2 shown in SEQ ID NO:44, and CDR-H3 shown in SEQ ID NO:45, and ii) CDR-L1 shown in SEQ ID NO:46, CDR-L2 shown in SEQ ID NO:47, and CDR-L3 shown in SEQ ID NO:48.

[0077] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:3, and the second binding domain comprises: i) CDR-H1 shown in SEQ ID NO:49, CDR-H2 shown in SEQ ID NO:50, and CDR-H3 shown in SEQ ID NO:51, and ii) CDR-L1 shown in SEQ ID NO:52, CDR-L2 shown in SEQ ID NO:53, and CDR-L3 shown in SEQ ID NO:54.

[0078] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:4, and the second binding domain comprises: i) CDR-H1 shown in SEQ ID NO:49, CDR-H2 shown in SEQ ID NO:50, and CDR-H3 shown in SEQ ID NO:51, and ii) CDR-L1 shown in SEQ ID NO:52, CDR-L2 shown in SEQ ID NO:53, and CDR-L3 shown in SEQ ID NO:54.

[0079] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:3, and the second binding domain comprises: i) the amino acid sequence of the heavy chain variable domain VH shown in SEQ ID NO:55, and ii) the amino acid sequence of the light chain variable domain VL shown in SEQ ID NO:56.

[0080] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:4, and the second binding domain comprises: i) the amino acid sequence of the heavy chain variable domain VH shown in SEQ ID NO:55, and ii) the amino acid sequence of the light chain variable domain VL shown in SEQ ID NO:56.

[0081] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:3, and the second binding domain comprises: i) the amino acid sequence of the heavy chain variable domain VH shown in SEQ ID NO:57, and ii) the amino acid sequence of the light chain variable domain VL shown in SEQ ID NO:58.

[0082] In some specific embodiments, the first binding domain of the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:4, and the second binding domain comprises: i) the amino acid sequence of the heavy chain variable domain VH shown in SEQ ID NO:57, and ii) the amino acid sequence of the light chain variable domain VL shown in SEQ ID NO:58.

[0083] In some specific embodiments, the bispecific antibody includes a constant region domain, which contains the amino acid sequence shown in SEQ ID NO:59 or SEQ ID NO:60 (see Table 6 for sequence information).

[0084] Table 6: Sequence information of the constant region of bispecific antibodies

[0085]

[0086] In some specific embodiments, the amino acid sequence of the bispecific antibody is as follows:

[0087] (1) Contains the heavy chain amino acid sequence shown in SEQ ID NO:29 and the light chain amino acid sequence shown in SEQ ID NO:30; or

[0088] (2) Contains the heavy chain amino acid sequence shown in SEQ ID NO:31 and the light chain amino acid sequence shown in SEQ ID NO:30; or

[0089] (3) Contains the heavy chain amino acid sequence shown in SEQ ID NO:36 and the light chain amino acid sequence shown in SEQ ID NO:37; or

[0090] (4) Contains the heavy chain amino acid sequence shown in SEQ ID NO:38 and the light chain amino acid sequence shown in SEQ ID NO:37; or

[0091] (5) Contains the heavy chain amino acid sequence shown in SEQ ID NO:39 and the light chain amino acid sequence shown in SEQ ID NO:37; or

[0092] (6) Contains the heavy chain amino acid sequence shown in SEQ ID NO:40 and the light chain amino acid sequence shown in SEQ ID NO:37.

[0093] Methods for generating bispecific antibodies are described in the literature, such as in USPN 5989830 and USPN 5798229, which are incorporated herein by reference. Holliger and Hudson (2005) Nature Biotechnology 23:1126-1136 describe higher levels of specificity, such as trispecific antibodies.

[0094] The present invention also provides a pharmaceutical composition for treating diseases, comprising the above-mentioned SIRPα immunoglobulin fusion protein, polypeptide, monospecific antibody, bispecific antibody or multispecific antibody and pharmaceutically acceptable carrier thereof.

[0095] In some specific embodiments, the pharmaceutical composition further comprises at least one additional agent, which is an antibody or non-antibody therapeutic agent.

[0096] The present invention also provides a nucleic acid encoding the aforementioned SIRPα immunoglobulin fusion protein, polypeptide, monospecific antibody, bispecific antibody, or multispecific antibody.

[0097] The present invention also provides a vector comprising the nucleic acid described above.

[0098] The present invention also provides a host cell comprising the nucleic acid or vector described above.

[0099] The present invention also provides a SIRPα immunoglobulin fusion protein, a monospecific antibody, a bispecific antibody, or a multispecific antibody, characterized in that it comprises a CD47 binding domain, wherein the CD47 binding domain comprises the amino acid sequence of the polypeptide described above.

[0100] The present invention also provides the use of the above-described fusion proteins, peptides, monospecific antibodies, bispecific antibodies, multispecific antibodies, pharmaceutical compositions, nucleic acids, vectors, or host cells in the preparation of drugs for treating diseases, wherein the diseases are cancer, autoimmune diseases, or inflammatory diseases.

[0101] The present invention also provides a method for treating a disease, comprising administering to a subject in need an effective amount of the aforementioned polypeptide, fusion protein, monospecific antibody, bispecific antibody, multispecific antibody, pharmaceutical composition, nucleic acid, vector, or host cell, wherein the disease is cancer, an autoimmune disease, or an inflammatory disease.

[0102] The present invention also provides the use of the aforementioned polypeptides, fusion proteins, monospecific antibodies, bispecific antibodies, multispecific antibodies, pharmaceutical compositions, nucleic acids, vectors, or host cells in the preparation of drugs for the treatment of cancer, autoimmune diseases, or inflammatory diseases.

[0103] The beneficial effects of this disclosure are that the optimized peptides screened out in this disclosure, and the fusion proteins developed based on the peptides in the examples do not produce blood agglutination, hardly bind to red blood cells, and exhibit good antigen binding ability and the ability to block the binding of CD47 and SIRPα, thus improving the phagocytic effect. At the same time, the bispecific antibodies developed based on the peptides show the same excellent effects, which is conducive to simultaneous verification in animal models, effectively inhibiting the growth of tumor cells and having high safety. Attached Figure Description

[0104] Figure 1 Non-denatured SDS-PAGE electrophoresis image of the supernatant from the cellular expression of the target protein;

[0105] Figure 2 This is a non-denaturing SDS-PAGE electrophoresis image of the purified target protein;

[0106] Figure 3 This is a denatured SDS-PAGE electrophoresis image of the purified target protein.

[0107] Figure 4 The results of the SIRPα immunoglobulin fusion protein coagulation assay;

[0108] Figure 5 The results show the binding of SIRPα immunoglobulin fusion protein to erythrocytes;

[0109] Figure 6 The results of enzyme-linked immunosorbent assay (ELISA) show the binding affinity of the SIRPα immunoglobulin fusion protein.

[0110] Figure 7The result is that the SIRPα immunoglobulin fusion protein blocks the binding of human SIRPα and human CD47 to enzyme-linked immunosorbent assay (ELISA).

[0111] Figure 8 The experimental results of THP-1 cells phagocytizing Raji cells mediated by SIRPα immunoglobulin fusion protein;

[0112] Figure 9 The results are from a coagulation test using bispecific antibodies.

[0113] Figure 10 The results are from a coagulation test using bispecific antibodies.

[0114] Figure 11 The results are from a coagulation test using bispecific antibodies.

[0115] Figure 12 This is the result of the binding of bispecific antibodies to red blood cells;

[0116] Figure 13 The result is the enzyme-linked immunosorbent assay (ELISA) of SIRPα-anti-CD20 bispecific antibody binding to CD47;

[0117] Figure 14 The result is an enzyme-linked immunosorbent assay (ELISA) showing the blocking effect of SIRPα-anti-CD20 bispecific antibody on the binding of SIRPα to CD47.

[0118] Figure 15 Results of tumor growth inhibition rate of bispecific antibody against Raji cell growth;

[0119] Figure 16 The trend of tumor volume change for Raji cells inhibited by bispecific antibodies;

[0120] Figure 17 Changes in mouse volume due to bispecific antibody inhibition of Raji cell growth. Detailed Implementation

[0121] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0122] As used herein, unless otherwise specified, the singular forms “a / an” and “the” include plural referents.

[0123] The term "SIRPα" in this invention refers to the amino acid sequence of wild-type signal regulatory protein α, or a recombinant or non-recombinant polypeptide having the amino acid sequence of wild-type signal regulatory protein α, or a mutant of naturally occurring signal regulatory protein α. In humans, 10 mutant forms of SIRPα have been found. The amino acid sequence of one mutant form (mutant 1 or V1) is listed as NCBI RefSeqNP_001035111.1 (residues 31-504 constitute the mature form). Another form (mutant 2 or V2) differs from mutant 1 or V1 by 13 amino acids, and its amino acid sequence is listed in GenBank as CAA71403.1. These two forms of SIRPα constitute approximately 80% of the various types of SIRPα present in humans.

[0124] The term "immunoglobulin" refers to an animal protein with antibody activity, preferably a human immunoglobulin. In some embodiments, "immunoglobulin" can be IgG, IgM, IgE, IgD, or IgA, with IgG being the preferred immunoglobulin and IgG1 or IgG4 being more preferred. As an alternative embodiment, the Fc fragment of the immunoglobulin may incorporate one or more amino acid alterations (e.g., mutations, additions, or deletions), typically no more than about five such alterations, including mutations, additions, or deletions of amino acids affecting certain Fc properties. As an alternative embodiment, the hinge region of the immunoglobulin may incorporate one or more amino acid alterations (e.g., mutations, additions, or deletions), typically no more than about five such alterations, including mutations, additions, or deletions of amino acids affecting certain Fc properties. In some specific embodiments, the SIRPα portion of the immunoglobulin fusion protein is fused to the C-terminus of the immunoglobulin fragment, while in other specific embodiments, the SIRPα portion is fused to the N-terminus of the immunoglobulin fragment. The SIRPα portion may be fused to the immunoglobulin fragment via an optional linker or directly without a linker.

[0125] The term "mutation" means that at least one existing amino acid residue is replaced by another different amino acid residue (e.g., a substitute amino acid residue). For example, the mutation Q37H indicates that the amino acid residue glutamine at position 37 of the SIRPα polypeptide is replaced by the amino acid residue histidine (histidine-substituted glutamine) (numbered according to the EU index numbering system). "Natural amino acid residues" refers to amino acid residues from the following groups: alanine (three-letter code: Ala, single-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0126] The term "rituximab" is an INN for rituximab, and its amino acid sequence can be found at https: / / www.who.int / publications / m / item / inn-rl-94.

[0127] The term "nivolumab" has the INN name nivolumab, and its amino acid sequence can be found at https: / / www.who.int / publications / m / item / inn-pl-111.

[0128] The term "bispecific antibody" or "multispecific antibody" refers to an antibody that recognizes two or more different antigens by having a first antigen-binding domain specific to a first antigen, a second antigen-binding domain specific to a second antigen, and additional antigen-binding domains specific to additional antigens. Bispecific antibodies bind specifically to two target antigens and are therefore a class of multispecific antibodies. Multispecific antibodies can be generated by recombinant DNA methods, or, including but not limited to, antibodies chemically generated by any convenient method. Bispecific antibodies include all antibodies or antibody conjugates or polymeric forms capable of recognizing two different antigens. Bispecific antibodies include antibodies that have been simplified and reformed to retain their bivalent characteristics, and chemically conjugated antibodies that may have multiple antigen recognition sites against each antigen.

[0129] The term "specificity" refers to an antigen-binding protein or antibody that selectively recognizes a specific epitope of an antigen. For example, natural antibodies are monospecific. As used herein, the terms "bispecific" or "multispecific" indicate that an antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen.

[0130] The term "Fc domain" generally includes the hinge region, the CH2 domain of the heavy chain constant region, and the CH3 domain of the heavy chain constant region (i.e., Hinge-CH2-CH3). The Fc domain often forms a dimer via disulfide bonds. Currently, the most commonly used fusion partner is the Fc segment of immunoglobulin IgG. The antibody Fc is part of the antibody constant region (including the hinge region -CH2-CH3, but excluding the CH1 portion of the antibody constant region). Fusion with the Fc segment increases the molecular weight and enhances the stability of the fusion molecule through FcRn-mediated recycling mechanisms, prolonging its in vivo half-life. Simultaneously, the Fc segment can mediate various biological functions such as ADCC and CDC. However, such fusion proteins lack the variable region of the antibody; their pharmacology and efficacy primarily depend on the functional molecule fused to the Fc segment. Human immunoglobulin G has four subtypes, each with varying biological activities. Currently, IgG1 antibodies are the most widely used. In recent years, with the expansion of new indications and the emergence of antibody drugs with new mechanisms of action, the IgG2 and IgG4 subtypes, which have lower cytotoxicity, have gained attention. In this invention, the "Fc domain" is preferably the Fc domain of an IgG1 antibody or the Fc domain of an IgG4 antibody.

[0131] The term “link” or “connection” refers to a connection or union of two or more components held together by a bond, link, force or binding, encompassing direct or indirect links, such as in the case of directly binding a first polypeptide to a second polypeptide or material, and in the case of placing one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) between the first polypeptide and the second polypeptide or material (linking).

[0132] The term "vector" is not particularly limited and can refer to any vector capable of replicating and / or expressing polynucleotides in eukaryotic or prokaryotic cells, including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), plant cells, yeast cells, insect cells, and bacterial cells (e.g., E. coli).

[0133] The term "host cell" includes prokaryotic and eukaryotic cells, including (but not limited to) bacterial cells such as *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*; yeast cells; fungal cells such as *Pichia pastoris*; insect cells such as *Drosophila melanoma* or *Noctua sf.* Sf9 cells; animal cells such as CHO-K1 (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma cells), human lymphoblastoid blasts, COS, NSO, 293T, Bowes melanoma cells, HT-1080, BHK (young hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc.; and plant cells. Any cell known to those skilled in the art as a mammalian host cell may be used in this field.

[0134] Pharmaceutically acceptable carriers should not induce antibodies harmful to the individual receiving the composition, and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, peptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral particles. They can also be pharmaceutically acceptable salts, such as inorganic acid salts like hydrochloride, hydrobromide, phosphate, and sulfate, or organic acid salts like acetate, propionate, malonate, and benzoate.

[0135] Pharmaceutically acceptable carriers may also contain liquids such as water, saline, glycerin, and ethanol. Additionally, excipients (such as wetting agents, emulsifiers, or pH buffers) may be present in the composition. These carriers enable the pharmaceutical composition to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, pastes, and suspensions for patient ingestion.

[0136] The pharmaceutical compositions of the present invention can be prepared in various forms. For example, the compositions can be prepared as injectable solutions or suspensions, or as pre-injection solid forms suitable for dissolving in liquid carriers (e.g., lyophilized compositions, reconstituted with preservative-containing sterile water). The compositions can be prepared for topical administration, such as as ointments, creams, or powders. The compositions can be prepared for oral administration, such as as tablets or capsules, as sprays, or as syrups (optionally flavored). The compositions can be prepared for pulmonary administration, such as as inhalants, in the form of fine powders or sprays. The compositions can be prepared as suppositories or uterine pessaries. The compositions can be prepared for nasal, ear, or ocular administration, such as as drops. The compositions can be in the form of kits designed to reconstitute mixed compositions prior to administration to a patient. For example, lyophilized antibodies in kit form, along with sterile water or sterile buffer, can be provided.

[0137] The pharmaceutical compositions of the present invention can be administered via any number of routes, including but not limited to: oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intracardiac, transdermal, transcutaneous, topical, subcutaneous, intranasal, intraenteral, sublingual, vaginal, or rectal routes. A needle-free injector (Hypospray) can also be used to administer the pharmaceutical components of the present invention. Typically, the therapeutic compositions can be prepared as injectable liquid solutions or suspensions. They can also be prepared as pre-injection solid forms suitable for dissolving in liquid carriers.

[0138] Direct delivery of drug compositions is typically accomplished via injection, subcutaneous injection, intraperitoneal injection, intravenous injection, or intramuscular injection. Therapeutic doses can be administered in single-dose or multiple-dose regimens. Known fusion protein-based drugs provide instructions regarding dosing frequency, such as whether the drug needs to be administered daily, weekly, or monthly. Frequency and dosage also depend on the severity of symptoms.

[0139] The pharmaceutical compositions of the present invention can be administered in pharmaceutically effective amounts. "Pharmaceutically effective amount" refers to an amount sufficient to treat a disease and a reasonable benefit / risk ratio suitable for any medical treatment. The effective dose level of the composition can be determined based on the subject's type, disease severity, age and sex, drug activity, drug sensitivity, time of administration, route of administration, excretion rate, treatment duration, drugs used in combination with the composition, and other known factors in the medical field. The pharmaceutical compositions of the present invention can be used alone or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. The compositions can be administered in one or more dosage forms. Considering all the above factors, it is crucial to administer the composition at the minimum amount that will produce the maximum effect without causing side effects, which can be readily determined by those skilled in the art.

[0140] The term “treatment” refers to the treatment of diseases in mammals (such as humans) such as cancer, autoimmune diseases, or inflammatory diseases. This includes: (a) suppressing the disease, i.e., stopping its development; and (b) alleviating the disease, i.e. causing the disease state to subside.

[0141] In some preferred embodiments, the cancer includes any form of cancer, including but not limited to solid tumor cancers (e.g., lung cancer, prostate cancer, breast cancer, bladder cancer, colon cancer, ovarian cancer, pancreatic cancer, kidney cancer, liver cancer, glioblastoma, medulloblastoma, leiomyosarcoma, squamous cell carcinoma of the head and neck, melanoma, neuroendocrine carcinoma, etc.), and liquid cancers (e.g., hematologic cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemia; lymphomas; and brain cancers, including minimal residual disease, and including both primary and metastatic tumors. Any cancer in which cancer cells express CD47 (e.g., in some cases, cancer cells show increased CD47 expression compared to non-cancer cells) is a suitable cancer to be treated by the subject method and composition.

[0142] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0143] Example 1: Construction and expression of SIRPα immunoglobulin fusion protein

[0144] Human SIRPαV2 (amino acids 31-148) was synthesized using the gene sequence of human SIRPαV2 (Genebank sequence number: CAA71403.1) and ligated into the target vector pcDNA3.1 containing the hinge region-Fc fragment of immunoglobulin IgG4 (the hinge region contains the S228P mutation). Primers with corresponding amino acid site mutations were designed based on the nucleotide sequence of the synthesized SIRPαV2. Circular PCR was performed using the vector containing the synthesized SIRPαV2-Fc fragment as a template. After PCR, Dpn I enzyme was added for digestion at 37°C for 1 hour. The target fragment was recovered by 1% agarose gel electrophoresis and then transformed into TOP10 competent cells. After colony growth the next day, single clones were picked and sequenced. Nucleotide sequences completely identical to the target sequence were selected for plasmid extraction for cell transfection. During cell transfection, the plasmid and transfection reagent ExpiFectamine™ 293 were mixed at a ratio of 1:2.7 and allowed to stand for 20 minutes. Then, the mixture was added to 293SQ cells in the logarithmic growth phase and cultured in a cell culture shaker at 37°C, 80% humidity, 8% CO2, and 220 rpm. Enhancers were added the next day, and the cell culture supernatant was collected after 4 days. The target protein was obtained by affinity chromatography using Protein A magnetic beads. The target protein described in Table 7 was obtained.

[0145] The cell secretion supernatant and the target protein purified from Protein A were subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 1 , Figure 2 , Figure 3 As shown. Among them, Figure 1 This is a non-denatured SDS-PAGE electrophoresis image of the supernatant from the cellular secretion of the target protein. Figure 2 This is a non-denaturing SDS-PAGE electrophoresis image of the target protein after purification with Protein A. Figure 3 This is a denaturing SDS-PAGE electrophoresis image of the target protein after purification with Protein A. Figures 1-3 As can be seen, the protein expression processes of target proteins #6, #13, #14, #17, #18, #21, and #28 are all uneven, while the protein expression processes of target proteins #37, #38, #39, #40, #41, and #42 are uniform.

[0146] Table 7: Sequence information of SIRPα immunoglobulin fusion protein

[0147] serial number mutation SEQ ID NO sequence #6 I31LS228P 15 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSL L PVGPIQWFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISINITPADAGTYYCVKFRKGSPD100TEFKSGAGTELSVARKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSG 346<!-- 14 --> ]]> #13 Q37RS228P 16 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI R WFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSG 346<!-- 15 --> ]]> #14 Q37HS228P 17 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI H WFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSG 346<!-- 16 --> ]]> #17 Q52FS228P 18 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIY50N F KEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD100TEFKSGAGTELSVARKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSG 346<!-- 17 --> ]]> #18 Q52HS228P 19 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIY50N H KEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD100TEFKSGAGTELSVARKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSG 346<!-- 18 --> ]]> #21 K96RS228P 20 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFR R GSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346 <!-- 19 -->]]> #28 I31LQ37HS228P 21 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSL L PVGPI H WFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSG 346<!-- 20 --> ]]> #37 I31LQ37HN80QS228P 9 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSL L PVGPI H WFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYY CVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346 <!-- 21 -->]]> #38 Q37HN80QS228P 10 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI H WFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYY CVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346 <!-- 22 -->]]> #39 Q37RN80QS228P 11 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI R WFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYY CVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346 <!-- 23 -->]]> #40 K96RN80QS228P 12 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIY50NQKEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYY CVKFR R GSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346 <!-- 24 -->]]> #41 Q52HN80QS228P 13 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIY50N H KEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYY CVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346 <!-- 25 -->]]> #42 Q52FN80QS228P 14 <![CDATA[EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIY50N F KEGHFPRVTTVSESTKRENMDFSISIS Q ITPADAGTYY CVKFRKGSPD100TEFKSGAGTELSVRAKPSESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDT150LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY200RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT250LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS300DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 346]]>

[0148] Note: The amino acids in bold and underlined in the sequence are the mutated amino acids. S228P is numbered according to the EU system, and I31L, Q37H, Q37R, Q52H, Q52F, K96R, and N80Q are numbered according to sequence.

[0149] Example 2 Blood Agglutination

[0150] Human erythrocytes were diluted to a 4% concentration using PBS, and 25 μL / well was added to a 96-well plate. The test samples and controls were added, with the test samples being target proteins #37, #38, #39, #40, #41, and #42, and the controls being TTI-622 analogs and Hu5F9 analogs (sequence information is shown in Table 8). The plates were incubated together at 37°C for 3 hours and then photographed.

[0151] Figure 4 The results of the coagulation test showed that the Hu5F9 analogue, which has been reported to produce blood agglutination, produced a blood agglutination reaction, while the TTI-622 analogue and #37, #38, #39, #40, #41 and #42 did not produce a blood agglutination reaction.

[0152] Table 8: Sequence information of TTI-621 analog, TTI-622 analog and Hu5F9 analog reference standards

[0153]

[0154] Example 3: Binding with red blood cells

[0155] Human erythrocytes were diluted to a concentration of 0.25% with PBS and added to 96-well plates. Then, serially diluted test samples / controls were added. The test samples were target proteins #37, #38, #39, #40, #41, and #42, and the controls were TTI-622 analogs and Hu5F9 analogs, 100 μL / well. The plates were incubated together at 4°C for 1 hour. After washing twice with 1% BSA, 100 μL / well of fluorescently labeled goat anti-human IgG Fc antibody was added, and the plates were reacted at 4°C for 40 minutes. After washing twice with 1% BSA, the fluorescence values ​​were read using a flow cytometer (BD Lyric). The binding curves of the test samples and erythrocytes were fitted to the RS904 plate with the fluorescence values ​​as the Y-axis.

[0156] from Figure 5 As can be seen, the positive control Hu5F9 analog strongly binds to red blood cells, #37 weakly binds to red blood cells, and the negative control TTI-622 analog and #38, #39, #40, #41, and #42 hardly bind to red blood cells.

[0157] Example 4 Binding affinity of SIRPα immunoglobulin fusion protein

[0158] Human CD47 antigen (purchased from Sino Biological, catalog number 12283-HCCH) was coated onto microplates at a concentration of 0.5 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with 2% BSA blocking buffer at room temperature for 1 hour, 200 μL / well. After washing with PBST, 5-fold serially diluted test samples / controls (test samples were target proteins #37, #38, #39, #40, #41, and #42; control was TTI-622 analog (Trillium, amino acid sequence as shown in SEQ ID NO:28, the same below)) were added, 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-human IgG secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance value at 450 nm was read on a microplate reader.

[0159] Results of enzyme-linked immunosorbent assay (ELISA) (Table 9) Figure 6 The results showed that, compared with the control TTI-622 analogue, target proteins #37, #38, and #39 all exhibited good binding ability.

[0160] Table 9: Binding affinity of SIRPα immunoglobulin fusion protein

[0161] Sample Name <![CDATA[OD 450 Maximum value <![CDATA[EC 50 (ng / ml)]]> TTI-622 analogues 0.611 111 #37 1.882 6.64 #38 1.197 35.01 #39 0.836 81.14 #40 0.542 208.3 #41 0.272 / #42 0.105 /

[0162] Note: " / " indicates that the corresponding EC was not obtained. 50 value.

[0163] Example 5: Blocking the binding of SIRPα and CD47

[0164] Human SIRPα antigen (purchased from ACRO, catalog number SIA-H52A8) was coated into microplates at a concentration of 2 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with Casein blocking buffer at room temperature for 1.5 hours, 200 μL / well. After washing with PBST, 5-fold serially diluted mixtures of the test samples / controls and His-tagged human CD47 protein (purchased from ACRO, catalog number CD7-H5227) were added (test samples were target proteins #37, #38, #39, #40, #41, and #42; control was a TTI-622 analog), 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-His-tagged secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0165] Results of enzyme-linked immunosorbent assay (ELISA) (Table 10) Figure 7 The results showed that, compared with the control group, target proteins #37, #38, and #39 all effectively blocked the binding of SIRPα and CD47. Specifically, the OD values ​​of #37 and #38 were significantly higher. 450 It has a smaller minimum value and IC50 value, and a stronger ability to block the binding of human CD47 to human SIRPα; #39 has a blocking ability comparable to that of TTI-622 analogues.

[0166] Table 10: Blocking the binding of human SIRPα and human CD47

[0167] Sample Name <![CDATA[OD 450 Minimum value IC50 (μg / ml) TTI-622 analogues 0.138 14.07 #37 0.049 0.2654 #38 0.068 5.939 #39 0.296 11.9 #40 0.501 / #41 0.965 / #42 1.126 /

[0168] Note: " / " indicates that the corresponding EC was not obtained. 50 value.

[0169] Example 6: THP-1 phagocytoses tumor cells

[0170] THP-1 cells (purchased from Procell) were cultured to the logarithmic growth phase, and then 150 nM PMA (purchased from Sigma, catalog number: P1585-1MG) was added and the cells were cultured in a 5% CO2 cell culture incubator for 24 hours. After washing twice with DPBS, the cells were cultured in RPMI medium for another 24 hours, and then 20 ng / ml IFN-γ (purchased from Sigma, catalog number: SRP3058-100UG) and 10 pg / ml IFN-γ were added. LPS (purchased from Sigma, catalog number: L2630-10MG) were cultured in a 5% CO2 cell culture incubator for 18 hours. After washing twice with DPBS, the cells were incubated at 4°C for 30 minutes with APC-labeled anti-human CD14 antibody (purchased from Invitrogen, catalog number: 367118) and PE-labeled anti-human CD80 antibody (purchased from BD, catalog number: 560925). After washing twice with 1% BSA, the fluorescence signal was read by flow cytometry (BD, model: Lyric) to evaluate the induction effect of THP-1.

[0171] Raji cells (purchased from ADCC, catalog number: CCL-86) were cultured to the logarithmic growth phase and incubated at 37°C for 20 minutes with CFSE (purchased from Invitrogen, catalog number: C34554A) at an 8000-fold dilution. APC-labeled anti-human CD14 antibody (purchased from Invitrogen, catalog number: 367118) at a 100-fold dilution was added to induced THP-1 cells and cultured at 4°C for 30 minutes. The two cell lines were then mixed at a 2:1 ratio, resulting in 1.2 x 103 CFSE-labeled Raji cells. 5 6 x 10 THP-1 cells labeled with anti-CD14 antibody per well after induction. 4 Add the test sample (#37, #38), antibody Fc fragment (Isotype control), and control TTI-622 analog to each well at a concentration of 10 nmol. After incubation at 37°C for 3 hours, the fluorescence signal is detected on a FACS Lyric flow cytometer.

[0172] like Figure 8 The experimental results showed that, under the condition of antibody / sample concentration of 10 nM, the proportions of THP-1 cells phagocytosing Raji cells mediated by TTI-622 analog, #37 and #38 were 81.58%, 89.29% and 86.73%, respectively, all higher than the Isotype control (50.91%), and the phagocytosis proportions of #37 and #38 were higher than those of TTI-622 analog.

[0173] Example 7 Construction of bispecific antibodies

[0174] The SIRPαV2 D1 domain mutant provided by this invention was used to construct bispecific antibodies by combining a rituximab fragment or a nivolumab fragment with a constant region as shown in SEQ ID NO:59 or SEQ ID NO:60.

[0175] SIRPα-anti-CD20 bispecific antibody (i.e., SIRPαV2 D1 domain mutant-rituximab fragment-constant region bispecific antibody (#46, #47, #48, #49, #50, #51) as shown in SEQ ID NO:59): Specifically, the C-terminus of the SIRPαV2 D1 domain mutant sequence corresponding to the above-mentioned #37, #38, #39, #40, #41, #42 fusion proteins is linked to a linker peptide sequence (GGGGSGGGGS), which is then linked to the N-terminus of the rituximab heavy chain variable region. This, along with the constant region sequence as shown in SEQ ID NO:59, constructs a recombinant heavy chain sequence. The heavy chain sequence from N-terminus to C-terminus is: SIRPαV2 D1 domain mutant, linker peptide (GGGGSGGGGS), rituximab heavy chain variable region, as shown in SEQ ID NO:59. NO:59 indicates the constant region; the light chain is the light chain of anti-CD20 rituximab (see Table 11 for structural information).

[0176] SIRPα-anti-PD-1 bispecific antibody (i.e., SIRPαV2 D1 domain mutant-nivolumab fragment-constant region bispecific antibody (#52, #53, #54, #55) as shown in SEQ ID NO:60): Specifically, the C-terminus of the SIRPαV2 D1 domain mutant sequence corresponding to the above-mentioned #37 and #38 fusion proteins is linked to a linker peptide (GGGGSGGGGSGGGGS), which is then linked to the N-terminus of the variable region of the nivolumab heavy chain. This is combined with the constant region sequence as shown in SEQ ID NO:60 to construct a recombinant heavy chain sequence. From the N-terminus to the C-terminus, the sequence is as follows: SIRPαV2 D1 domain mutant, linker peptide (GGGGSGGGGSGGGGS), nivolumab heavy chain variable region, constant region as shown in SEQ ID NO:60; the light chain is the nivolumab light chain, forming molecules #52 and #54 respectively. The N-terminus of the SIRPαV2D1 domain mutant sequence corresponding to the above-mentioned #37 and #38 fusion proteins was linked to the C-terminus of the constant region as shown in SEQ ID NO:60 via a linker peptide sequence (GGGGSGGGGSGGGGS), constructing a recombinant heavy chain sequence. From the N-terminus to the C-terminus, the sequence is as follows: nivolumab heavy chain variable region, constant region as shown in SEQ ID NO:60, linker peptide (GGGGSGGGGSGGGGSGGGS), SIRPαV2 D1 domain mutant, and light chain is the light chain of nivolumab, forming molecules #53 and #55 respectively (see Table 11 for structural information).

[0177] Table 11: Examples of molecular structures of bispecific antibodies

[0178] Serial Number Heavy chain Light chain #46 #37 SIRPαV2 D1 domain mutant - (GGGGS)2- rituximab heavy chain variable region - constant region shown in SEQ ID NO:59 rituximab light chain #47 #38 SIRPαV2 D1 domain mutant - (GGGGS)2- rituximab heavy chain variable region - constant region shown in SEQ ID NO:59 rituximab light chain #48 #39 SIRPαV2 D1 domain mutant - (GGGGS)2- rituximab heavy chain variable region - constant region shown in SEQ ID NO:59 rituximab light chain #49 40# SIRPαV2 D1 domain mutant - (GGGGS)2-rituximab heavy chain variable region - constant region shown in SEQ ID NO:59 rituximab light chain #50 #41 SIRPαV2 D1 domain mutant - (GGGGS)2- rituximab heavy chain variable region - constant region shown in SEQ ID NO:59 rituximab light chain #51 #42 SIRPαV2 D1 domain mutant - (GGGGS)2- rituximab heavy chain variable region - constant region shown in SEQ ID NO:59 rituximab light chain #52 #37 SIRPαV2 D1 domain mutant - (GGGGS)3-nivolumab heavy chain variable region - constant region shown in SEQ ID NO:60 Nivolumab light chain #53 The SIRPαV2 D1 domain mutant in the nivolumab heavy chain variable region - constant region shown in SEQ ID NO:60 - (GGGGS)4-#37 Nivolumab light chain #54 #38 SIRPαV2 D1 domain mutant - (GGGGS)3-nivolumab heavy chain variable region - constant region shown in SEQ ID NO:60 Nivolumab light chain #55 The SIRPαV2 D1 domain mutant in the nivolumab heavy chain variable region - constant region shown in SEQ ID NO:60 - (GGGGS)4-#38 Nivolumab light chain

[0179] The DNA fragments corresponding to the above sequences were ligated to the target vectors pcDNA3.1 or pcDNA3.4 to construct recombinant expression vectors. These vectors were then transformed into TOP10 competent cells. After colonies grew the following day, single clones were picked and sent for sequencing. Nucleotide sequences completely identical to the target sequence were selected for plasmid extraction for cell transfection. For cell transfection, the recombinant heavy chain plasmid and light chain plasmid were mixed and incubated with ExpiFectamine™ 293 transfection reagent at a ratio of 1:2.7. After standing for 20 minutes, the mixture was added to 293SQ cells in logarithmic growth phase and cultured in a cell culture shaker at 37°C, 80% humidity, 8% CO2, and 220 rpm. Enhancers were added the following day, and the cell culture supernatant was collected after 4 days. The target protein was obtained by affinity chromatography using Protein A magnetic beads, as shown in Table 12.

[0180] Table 12: Molecular sequences of bispecific antibodies

[0181]

[0182]

[0183]

[0184] Example 8 Blood Agglutination

[0185] Human erythrocytes were diluted to a 4% concentration using PBS, 25 μL / well, and added to a 96-well plate. The test samples / controls (as shown in Table 13) were added, and the plates were incubated at 37°C for 3 hours before taking pictures.

[0186] Table 13: Blood Agglutination Test Samples / References

[0187] Group Sample to be tested Reference Group E #46、#47、#48、#49、#50、#51 TTI-622 analogues, Hu5F9 analogues Group F #53 Nivolumab analogues, Hu5F9 analogues Group G #54、#55 TTI-622 analogues, Hu5F9 analogues

[0188] The results of the coagulation test are as follows Figure 9 , Figure 10 and Figure 11 As shown. Wherein: Figure 9 The results showed that the positive control Hu5F9 analog produced a hemagglutination reaction, while the TTI-622 analog and #46, #47, #48, #49, #50 and #51 did not produce a hemagglutination reaction. Figure 10 The results showed that the positive control Hu5F9 analog produced a hemagglutination reaction, while the nivolumab analog and #53 did not produce a hemagglutination reaction. Figure 11 The results showed that the positive control Hu5F9 analog produced a blood agglutination reaction, while the TTI-622 analog and #55 did not produce a blood agglutination reaction, and #54 produced a weak blood agglutination reaction.

[0189] Example 9: Binding with red blood cells

[0190] Add red blood cells diluted with PBS to 0.25% red blood cells / well to 96-well plates, add serially diluted test samples / controls (as shown in Table 14), 100 μL / well, and incubate at 4°C for 1 hour. After washing twice with 1% BSA, add fluorescently labeled goat anti-human IgG Fc antibody, 100 μL / well, and incubate at 4°C for 40 minutes. After washing twice with 1% BSA, detect the fluorescence signal on a FACS Lyric flow cytometer.

[0191] Table 14: Blood Agglutination Test Samples / References

[0192] Group Sample to be tested Reference Group H #46、#47、#48、#49、#50、#51 TTI-622 analogues, Hu5F9 analogues Group I #53 Nivolumab analogues, Hu5F9 analogues Group J #54、#55 TTI-622 analogues, Hu5F9 analogues

[0193] Table 15: Maximum fluorescence value when bound to erythrocytes

[0194]

[0195] like Figure 12 As shown in Table 15, Figure 12 The results show that Hu5F9 analogs bind strongly to erythrocytes, while #47, #48, #49, #50, and #51, as well as TTI-622 analogs, do not bind to erythrocytes, and #46 binds weakly to erythrocytes, with a significantly lower binding strength than Hu5F9 analogs. Table 15 shows that Hu5F9 analogs bind strongly to erythrocytes, while nivolumab analogs, TTI-622 analogs, #53, #54, and #55 do not bind to erythrocytes.

[0196] Example 10 Binding affinity of bispecific antibodies

[0197] (1) Affinity of SIRPα-anti-CD20 bispecific antibody to bind to CD47

[0198] Human CD47 antigen (purchased from Sino Biological, catalog number 12283-HCCH) was coated onto microplates at a concentration of 0.5 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with 2% BSA blocking buffer at room temperature for 1 hour, 200 μL / well. After washing with PBST, 5-fold serially diluted test samples / controls were added, with test samples being target proteins #46, #47, #48, #49, #50, and #51, and the control being a TTI-622 analog, 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-human IgG secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0199] Results of enzyme-linked immunosorbent assay (ELISA) (Table 16) Figure 13 The results show that #46, #47, and #48 all exhibit good bonding strength.

[0200] Table 16: Affinity of SIRPα Immunoglobulin Fusion Protein to CD47

[0201] Sample Name <![CDATA[OD 450 Maximum value <![CDATA[EC 50 (μg / ml)]]> TTI-622 analogues 1.64 0.060 #46 2.41 0.004 #47 2.38 0.066 #48 2.37 0.309 #49 1.20 2.033 #50 0.35 5.508 #51 0.09 >10

[0202] (2) Affinity of SIRPα-anti-PD-1 bispecific antibody to bind to CD47

[0203] Human CD47 antigen (purchased from Sino Biological, catalog number 12283-HCCH) was coated onto microplates at a concentration of 0.5 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with 2% BSA blocking buffer at room temperature for 1 hour, 200 μL / well. After washing with PBST, 5-fold serially diluted test samples / controls were added (Group A: test samples were target proteins #52 and #53, and control was target protein #37; Group B: test samples were target proteins #54 and #55, and control was target protein #38), 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-human IgG secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0204] The ELISA results of the target proteins #52, #53, #54, and #55 binding to human CD47 are shown in Table 17. According to EC... 50 Based on the values ​​and curves, #52, #53, #54, and #55 all showed good affinity for binding CD47.

[0205] Table 17: Affinity of SIRPα immunoglobulin fusion protein to CD47

[0206]

[0207] (3) Affinity of SIRPα-anti-PD-1 bispecific antibody to bind to PD-1

[0208] Human PD-1 antigen (purchased from ACRO, catalog number PD1-H5221) was coated onto microplates at a concentration of 1 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with 2% BSA blocking buffer at room temperature for 1.5 hours, 200 μL / well. After washing with PBST, 5-fold serially diluted test samples / controls (Group C: test samples were #52 and #53 target proteins, and the control was nivolumab analog; Group D: test samples were #54 and #55 target proteins, and the control was nivolumab analog), 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-human IgG secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0209] The ELISA results of the target proteins #52, #53, #54, and #55 binding to human PD-1 are shown in Table 18. According to EC... 50 Based on the values ​​and curves, #52, #53, #54, and #55 all showed good affinity for binding to PD-1.

[0210] Table 18: Affinity of SIRPα immunoglobulin fusion protein to PD-1

[0211]

[0212] Example 11: Blocking binding affinity of bispecific antibodies

[0213] (1) SIRPα-anti-CD20 bispecific antibody blocks the binding of SIRPα to CD47.

[0214] Human SIRPα antigen (purchased from ACRO, catalog number SIA-H52A8) was coated into microplates at a concentration of 2 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with Casein blocking buffer at room temperature for 1.5 hours, 200 μL / well. After washing with PBST, 5-fold serially diluted mixtures of the test samples / controls and His-tagged human CD47 protein (purchased from ACRO, catalog number CD7-H5227) were added (the test samples were #46, #47, #48, #49, #50, and #51 target proteins, and the control was a TTI-622 analog), 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-His-tagged secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0215] Results of enzyme-linked immunosorbent assay (ELISA) (Table 19) Figure 14 The results show that, compared to TTI-622 analogues, the OD values ​​of #46 and #47 are... 450 Both the minimum value and IC50 value are smaller; #48 has a blocking ability comparable to that of the TTI-622 analog; while #49, #50, and #51 have weaker abilities to block human CD47 and human SIRPα. This indicates that SIRPα-anti-CD20 bispecific antibodies #46 and #47 have a stronger ability to block the binding of human CD47 and human SIRPα.

[0216] Table 19: Blocking the binding of human SIRPα and human CD47

[0217] Sample Name <![CDATA[OD 450 Minimum value IC50 (μg / ml) TTI-622 analogues 0.136 21.72 #46 0.05 0.104 #47 0.087 7.88 #48 0.333 14.85 #49 0.639 / #50 1.083 / #51 1.181 /

[0218] Note: " / " indicates that the corresponding EC was not obtained. 50 value.

[0219] (2) SIRPα-anti-PD-1 bispecific antibody blocks the binding of SIRPα to CD47.

[0220] Human SIRPα antigen (purchased from ACRO, catalog number SIA-H52A8) was coated onto microplates at a concentration of 2 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with Casein blocking buffer at room temperature for 1.5 hours, 200 μL / well. After washing with PBST, 5-fold serially diluted mixtures of the test sample / control and His-tagged human CD47 antigen (purchased from ACRO, catalog number CD7-H5227) were added (test sample: target protein #53, control: target protein #37), 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-His secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0221] The results are shown in Table 20. The results show that the target protein #53 exhibits excellent ability to block the binding of CD47 to SIRPα.

[0222] Table 20: Blocking the binding of human SIRPα and human CD47

[0223] Sample Name <![CDATA[OD 450 Minimum value IC50 (nM) #53 0.030 1.986 #37 0.038 7.085

[0224] (3) SIRPα-anti-PD-1 bispecific antibody blocks the binding of PD-1 to PD-L1.

[0225] Human PD-1 antigen (purchased from ACRO, catalog number PD1-H5257) was coated into microplates at a concentration of 1 μg / ml, 100 μL / well, and incubated overnight at 4°C. The next day, after washing with PBST, the plates were blocked with 2% BSA blocking buffer at room temperature for 1.5 hours, 200 μL / well. After washing with PBST, 5-fold serially diluted mixtures of the test sample / control and His-tagged human PD-L1 antigen (purchased from SinoBiologocal, catalog number 10084-H08H) were added (the test sample was #53 target protein, and the control was a nivolumab analog), 100 μL / well, and incubated at room temperature for 2 hours. After washing with PBST, HRP-labeled anti-His secondary antibody was added, and the plates were incubated at room temperature for 1 hour. After TMB reaction solution was added for color development, the absorbance was read at 450 nm using a microplate reader.

[0226] The results are shown in Table 21. The results show that the target protein #53 exhibits excellent ability to block the binding of PD-1 and PD-L1.

[0227] Table 21: Blocking the binding of human SIRPα and human CD47

[0228] Sample Name <![CDATA[OD 450 Minimum value IC50 (nM) #53 0.075 1.662 nivolumab analogues 0.126 1.77

[0229] Example 12 Animal Model Experiment

[0230] Raji cells in the logarithmic growth phase were collected, washed with PBS, and prepared into a single-cell suspension. This suspension was then subcutaneously inoculated into tumors in 6-8 week old NOD-SCID mice until the tumor volume reached approximately 100 mm². 3 Mice were randomly divided into seven groups: experimental group #46, experimental group #47, control group #37 + rituximab analogue, control group #38 + rituximab analogue, control group TTI-622 analogue + rituximab analogue, control group rituximab analogue, and PBS control group. The mice were administered drugs according to the experimental protocol in Table 22. Tumor volume and mouse weight were measured twice weekly to evaluate drug efficacy.

[0231] Table 22: Dosing Regimen

[0232]

[0233] The results are as follows Figure 15 As shown in Table 23, compared with the PBS control group, all other groups effectively inhibited the growth of Raji cells. The tumor growth inhibition rate (TGI) results for each group are shown in Table 23. The tumor volume and body weight of each mouse in each group are shown in Table 24. Figure 16 and Figure 17 As shown, the results indicate that #37 and #38 in combination with rituximab analogues and #46 and #47 bispecific antibodies can effectively inhibit the growth of tumor cells, with little impact on mouse body weight, demonstrating high safety.

[0234] Table 23: Tumor production inhibition rate in each group

[0235] Group Dosage Tumor growth inhibition rate (TGI) 1 Vehicle (PBS) 0 2 rituximab analogues 81% 3 #46 98% 4 #47 99% 5 TTI-622 analogue + rituximab analogue 91% 6 #37+rituximab analogues 99.6% 7 #38+rituximab analogues 96%

Claims

1. A polypeptide, characterized in that, The polypeptide comprises a SIRPαV2 D1 domain mutant, which comprises: (1) a Q37H mutation and an N80Q mutation, or (2) an I31L mutation, a Q37H mutation and an N80Q mutation, relative to a wild-type SIRPαV2 D1 domain comprising an amino acid sequence set forth in SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein, The SIRPαV2 D1 domain mutant comprises an amino acid sequence set forth in SEQ ID NO: 3 or 4.

3. Use of the polypeptide of claim 1 or 2 in the preparation of a fusion protein, a monospecific antibody, a bispecific antibody or a multispecific antibody.

4. A SIRPa immunoglobulin fusion protein, characterized in that, The SIRPα immunoglobulin fusion protein comprises the following moieties linked directly or indirectly, the indirect linkage comprising linkage via a linker, a flexible sequence or a hinge region sequence: (1) the polypeptide of claim 1 or 2; and (2) an immunoglobulin constant domain or a fragment thereof.

5. The SIRPa immunoglobulin fusion protein of claim 4, wherein, The SIRPα immunoglobulin fusion protein satisfies at least one of the following conditions: (i) the polypeptide moiety is located at the N-terminus of the immunoglobulin constant domain or a fragment thereof; or (ii) the immunoglobulin is a human immunoglobulin or a mutant thereof; or (iii) the immunoglobulin constant domain or a fragment thereof comprises an immunoglobulin hinge region domain and an immunoglobulin Fc domain; or (iv) the SIRPα immunoglobulin fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO:

10. The human immunoglobulin is IgG1, IgG2, IgG3 or IgG4.

6. The SIRPa immunoglobulin fusion protein of claim 5, wherein, The amino acid sequence of the immunoglobulin hinge region domain is set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and / or the amino acid sequence of the immunoglobulin Fc domain is set forth in SEQ ID NO: 25 or SEQ ID NO:

26.

7. The SIRPa immunoglobulin fusion protein of claim 5, wherein The bispecific antibody comprises:

8. A bispecific antibody, characterized in that, a first binding domain comprising: the polypeptide of claim 1 or 2, or the SIRPα immunoglobulin fusion protein of any one of claims 4 to 7, and a second binding domain comprising: an antibody or an antigen-binding fragment thereof targeting a second antigen; and the first binding domain is linked directly or via a linker at the C-terminus, the N-terminus or the hinge region of the second binding domain. The linker is (GGGGS)n, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8.

9. The bispecific antibody of claim 8, wherein The bispecific antibody satisfies at least one of the following conditions:

10. The bispecific antibody of claim 9, wherein (1) the second antigen is a tumor cell antigen; or (2) the antibody or the antigen-binding fragment thereof targeting the second antigen is selected from an anti-CD20 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof; or ​ (3) the first binding domain of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 3, and the second binding domain comprises: i) CDR-H1 of SEQ ID NO: 43, CDR-H2 of SEQ ID NO: 44, and CDR-H3 of SEQ ID NO: 45, and ii) CDR-L1 of SEQ ID NO: 46, CDR-L2 of SEQ ID NO: 47, and CDR-L3 of SEQ ID NO: 48; or (4) the first binding domain of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 4, and the second binding domain comprises: i) CDR-H1 of SEQ ID NO: 43, CDR-H2 of SEQ ID NO: 44, and CDR-H3 of SEQ ID NO: 45, and ii) CDR-L1 of SEQ ID NO: 46, CDR-L2 of SEQ ID NO: 47, and CDR-L3 of SEQ ID NO: 48; or (5) the first binding domain of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 3, and the second binding domain comprises: i) CDR-H1 of SEQ ID NO: 49, CDR-H2 of SEQ ID NO: 50, and CDR-H3 of SEQ ID NO: 51, and ii) CDR-L1 of SEQ ID NO: 52, CDR-L2 of SEQ ID NO: 53, and CDR-L3 of SEQ ID NO: 54; or (6) the first binding domain of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 4, and the second binding domain comprises: i) CDR-H1 of SEQ ID NO: 49, CDR-H2 of SEQ ID NO: 50, and CDR-H3 of SEQ ID NO: 51, and ii) CDR-L1 of SEQ ID NO: 52, CDR-L2 of SEQ ID NO: 53, and CDR-L3 of SEQ ID NO: 54; or (7) the first binding domain of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 3, and the second binding domain comprises: i) the amino acid sequence of a heavy chain variable domain VH of SEQ ID NO: 55, and ii) the amino acid sequence of a light chain variable domain VL of SEQ ID NO: 56; or (8) the first binding domain of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 4, and the second binding domain comprises: i) the amino acid sequence of a heavy chain variable domain VH of SEQ ID NO: 55, and ii) the amino acid sequence of a light chain variable domain VL of SEQ ID NO: 56; or (9) the first binding domain comprises the amino acid sequence of SEQ ID NO: 3, and the second binding domain comprises i) the amino acid sequence of the heavy chain variable domain VH of SEQ ID NO: 57, and ii) the amino acid sequence of the light chain variable domain VL of SEQ ID NO: 58; or (10) the first binding domain comprises the amino acid sequence of SEQ ID NO: 4, and the second binding domain comprises i) the amino acid sequence of the heavy chain variable domain VH of SEQ ID NO: 57, and ii) the amino acid sequence of the light chain variable domain VL of SEQ ID NO: 58; or (11) the bispecific antibody further comprises a constant region domain, and the constant region domain comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 60; or (12) the bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 29, and the light chain amino acid sequence of SEQ ID NO: 30; or (13) the bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 31, and the light chain amino acid sequence of SEQ ID NO: 30; or (14) the bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 36, and the light chain amino acid sequence of SEQ ID NO: 37; or (15) the bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 38, and the light chain amino acid sequence of SEQ ID NO: 37; or (16) the bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 39, and the light chain amino acid sequence of SEQ ID NO: 37; or (17) the bispecific antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 40, and the light chain amino acid sequence of SEQ ID NO:

37.

11. The bispecific antibody of claim 10, wherein The tumor cell antigen is selected from the group consisting of 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-a, GCC, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Nectin-4, Notch 2, Notch 1, PD-1, PD-L1, PD-L2, PDGFR-a, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, or CD138.

12. A pharmaceutical composition for treating a disease, characterized by, The pharmaceutical composition comprises the polypeptide of claim 1 or 2, the fusion protein of any one of claims 4-7, or the bispecific antibody of any one of claims 8-11, and a pharmaceutically acceptable carrier thereof.

13. The pharmaceutical composition of claim 12, wherein, The pharmaceutical composition further comprises at least one additional agent.

14. The pharmaceutical composition of claim 13, wherein, The additional agent is an antibody or a non-antibody therapeutic agent.

15. A nucleic acid encoding the polypeptide of claim 1 or 2, the fusion protein of any one of claims 4-7, or the bispecific antibody of any one of claims 8-11.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15 or the vector of claim 16.

18. A SIRPa immunoglobulin fusion protein, monospecific antibody, bispecific antibody, or multispecific antibody, characterized in that, which comprises a CD47 binding domain comprising the amino acid sequence of the polypeptide of claim 1 or 2.

19. Use of the polypeptide of claim 1 or 2, the fusion protein of any one of claims 4-7, the bispecific antibody of any one of claims 8-11, the pharmaceutical composition of any one of claims 12-14, the nucleic acid of claim 15, the vector of claim 16, the host cell of claim 17, or the fusion protein, monospecific antibody, bispecific antibody, or multispecific antibody of claim 18, in the manufacture of a medicament for treating a disease, wherein the disease is a cancer, an autoimmune disease, or an inflammatory disease.

20. A method of treating a disease comprising administering to a subject in need thereof an effective amount of the polypeptide of claim 1 or 2, the fusion protein of any one of claims 4-7, the bispecific antibody of any one of claims 8-11, the pharmaceutical composition of any one of claims 12-14, the nucleic acid of claim 15, the vector of claim 16, the host cell of claim 17, or the fusion protein, monospecific antibody, bispecific antibody, or multispecific antibody of claim 18, wherein the disease is a cancer, an autoimmune disease, or an inflammatory disease.

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