Combination medicine containing anti-PD-1-anti-VEGFA bispecific antibody and use thereof

Abstract

The present invention relates to cancer therapy and immunobiology, and provides a combination drug comprising an anti-PD-1-anti-VEGFA bispecific antibody, and its use. Specifically, the combination drug comprises at least one bispecific antibody and at least one PARP inhibitor, where the bispecific antibody comprises a first protein functional domain for targeting PD-1 and a second protein functional domain for targeting VEGFA; and according to the EU numbering system, the heavy chain constant region of the immunoglobulin contained in the bispecific antibody is mutated at two sites, i.e., positions 234 and 235, and after the mutation, the affinity constant of the bispecific antibody to FcγRI, FcγRIIa, FcγRIIIa and / or C1q is reduced compared with that of the bispecific antibody before the mutation. By combining PARPi with the bispecific antibody, the therapeutic effect against tumors is significantly improved compared with the case of using PARPi or the bispecific antibody alone, and the present invention has good applicability.

Description

[Technical field]

[0001] The present invention relates to a combination drug containing an anti-human PD-1-anti-VEGFA bispecific antibody and its use, in particular, to a combination drug containing an anti-human PD-1-anti-human VEGFA bispecific antibody and its use, in the field of tumor therapy and immunobiology. [Background technology]

[0002] Tumors, especially malignant tumors, are a serious threat to human health in today's world. Tumor growth has two distinct phases: from a slow, avascular growth phase to a vascular, rapid growth phase. Angiogenesis provides sufficient nutrients to the tumor to complete the vascular switch phase; without angiogenesis, primary tumors would not grow beyond 1-2 mm and metastasis would not occur.

[0003] Vascular endothelial growth factor (VEGF) is a growth factor that promotes the division and proliferation of endothelial cells, promotes the formation of new blood vessels, and enhances vascular permeability. It exerts its function by binding to vascular endothelial growth factor receptors on the cell surface and activating the tyrosine kinase signaling pathway. In tumor tissues, tumor cells, tumor-infiltrating macrophages, and mast cells secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine manner, promotes the proliferation and migration of endothelial cells, induces angiogenesis, promotes the sustained growth of tumors, and also enhances vascular permeability, causes fibrin deposition in surrounding tissues, promotes the infiltration of monocytes and fibroblast endothelial cells, promotes the formation of tumor matrix and the entry of tumor cells into new blood vessels, and promotes tumor metastasis. Therefore, inhibition of tumor angiogenesis is currently considered to be one of the most promising tumor treatment methods. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4) and Neuropilin-1 (NRP-1). The first three of these receptors are structurally similar, all belong to the tyrosine kinase superfamily, and all consist of three parts: an extramembrane region, a transmembrane segment, and an intramembrane region, of which the extramembrane region consists of an immunoglobulin-like domain, and the intramembrane region belongs to the tyrosine kinase region. VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, and VEGFR3 is mainly located on the surface of lymphatic endothelial cells.

[0004] VEGF family molecules have different affinities for several receptors. VEGFA mainly binds to VEGFR1, VEGFR2 and NRP-1 to exert its action. VEGFR1 is the earliest receptor discovered, and under normal physiological conditions, the affinity between VEGFR1 and VEGFA is higher than that between VEGFR2 and VEGFA, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5)(2016):146-148).

[0005] VEGFR2 is a major regulator of angiogenesis and construction, and VEGFR2 has a much higher tyrosine kinase activity than VEGFR1. VEGFR2 mediates the behavior of vascular endothelial cells, such as proliferation and differentiation, as well as the process of angiogenesis and vascular permeability after binding to the ligand VEGFA (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3)(2007):179-213.), and VEGFA mediates the transcriptional expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway after binding to VEGFR2, promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13)(1999):2221-2230.).

[0006] VEGFR3 is a member of the tyrosine kinase family and is mainly expressed in vascular endothelial cells during the embryonic stage and lymphatic endothelial cells during adulthood. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot transmit biological signals independently, but can mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5)(2016):146-148).

[0007] VEGFA and VEGFR2 are mainly involved in the control of angiogenesis, and before and after the binding of VEGFA with VEGFR2, multiple intermediate signals in the upstream and downstream pathways form cascade reactions, ultimately changing the physiological functions of endothelial cells in different forms, such as proliferation, survival, migration, increased permeability, and invasion into surrounding tissues (Dong Hongchao et al., Modern Oncology, Vol. 22, Issue 9, September 2014, pp. 2231-3).

[0008] Programmed cell death receptor-1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein surface receptor that belongs to the CD28 immunoglobulin superfamily and is ubiquitously expressed on T cells, B cells, and medullary cells. PD-1 has two natural ligands, PD-L1 and PD-L2. Both PD-L1 and PD-L2 belong to the B7 superfamily and are constitutively or inducibly expressed on the membrane surface of many types of cells, including non-hematopoietic cells and many types of tumor cells. PD-L1 is mainly expressed on T cells, B cells, DCs, and microvascular endothelial cells, and many types of tumor cells. PD-L2 is expressed exclusively on antigen-presenting cells, such as dendritic cells and macrophages. The interaction of PD-1 with its ligands inhibits lymphatic activation and inhibits T cell proliferation and secretion of cytokines such as IL-2 and IFN-γ.

[0009] Many studies have shown that the tumor microenvironment can protect tumor cells from the destruction of immune cells, while the expression of PD-1 on lymphocytes infiltrated into the tumor microenvironment is upregulated, and many types of primary tumor tissues appear as PD-L1 positive in immunohistochemical assays, such as lung cancer, liver cancer, ovarian cancer, skin cancer, colon cancer, and glioma. At the same time, the expression of PD-L1 in tumors is significantly correlated with poor prognosis of cancer patients. Blocking the interaction of PD-1 with its ligand can promote tumor-specific T cell immunity and enhance the efficiency of immune clearance of tumor cells. Numerous clinical trials have shown that antibodies targeting PD-1 or PD-L1 can promote the infiltration of CD8+ T cells into tumor tissues and upregulate anti-tumor immune effectors such as IL-2, IFN-γ, granzyme B, and perforin, thereby effectively inhibiting tumor growth.

[0010] Antibodies against the PD-1 pathway are generally considered in the industry to provide a breakthrough for a wide variety of tumor therapies, including those used to treat non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., Anti-PD-1 Therapy in Melanoma. Semin Oncol. Jun;42(3) (2015):466-473), lymphoma and anemia (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. Sep;13(7) (2013):768-74), microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) tumors, due to the broad anti-tumor promise and remarkable efficacy of PD-1 antibodies (a number of anti-PD-1 antibody drugs have been approved by the FDA and others for the treatment of tumors with MSI-H / dMMR characteristics).

[0011] Currently, the combination of antiangiogenic therapy and immune checkpoint inhibitors shows good therapeutic effects in many tumors, such as ovarian cancer (Joyce F. Liu et al., JAMA Oncol. 2019;5(12):1731-1738.), non-small cell lung cancer (non-small cell lung cancer with EGFR and / or ALK sensitivity mutations) (Manegold C, et al., J Thorac Oncol. 2017 Feb;12(2):194-207.), renal cell carcinoma (Dudek AZ et al., J Clin Oncol. 2018;36 (suppl; abstr 4558.), hepatocellular carcinoma (Stein S et al., J Clin Oncol, 2018, 36(15_suppl):4074.;FDA approved bevacizumab in combination with atezolizumab to treat hepatocellular carcinoma in 2020), colorectal cancer (including MSI-H / dMMR and non-MSI-H / dMMR types) (Bendell JC et al. American Society of Clinical Oncology; May 29-June 2, 2015; Chicago, IL. 2015; abstract 704;Hochster HS et al. American Society of Clinical Oncology Gastrointestinal Cancers Symposium; January 19-21, 2017; San Francisco, CA. 2017; abstract 673.), breast cancer (Yukinori Ozaki et al., AACR; Cancer Res 2020;80(4 Suppl):Abstract nr In the study, the combination of anti-VEGF antibodies such as bevacizumab with anti-PD-1 / PD-L1 antibodies such as nivolumab, pembrolizumab, and atezolizumab has shown good results.The combination of VEGFR2 antibody and PD-1 antibody drug also shows good antitumor effects in gastric and gastroesophageal junction adenocarcinoma (Herbst RS et al., Lancet Oncol. 2019;20(8):1109-1123.), and the combination of PD-1 antibody (pembrolizumab) and angiogenesis inhibitor (lenvatinib) has shown good therapeutic effects in the treatment of endometrial cancer and was approved by the FDA for the treatment of endometrial cancer in 2019. Melanoma (PD-1 antibodies nivolumab and pembrolizumab are approved by the FDA for the treatment of melanoma), cervical cancer (Krishnansu S. et al., N Engl J Med 2014; 370:734-743.), glioma, prostate cancer (Antonarakis ES. et al., J Clin Oncol. 2020 Feb 10;38(5):395-405.), urothelial carcinoma (Joaquim Bellmunt. et al., N Engl J Med 2017; 376:1015-1026; FDA approved nivolumab for the treatment of bladder cancer in 2017), esophageal cancer (Kato K et al., Lancet Oncol. 2019;20(11):1506-17.), mesothelioma (Scherpereel A et al., Lancet Oncol. 2019;20(2):239-253.), PD-1 / PDL-1 antibodies have shown good therapeutic effects, and considering the synergistic effect of the PD-1 and VEGF pathways in tumor development, drugs that simultaneously block both the PD-1 and VEGF pathways are expected to have better antitumor effects.

[0012] Poly ADP-ribose Polymerase (PARP) belongs to the PARP enzyme family, which includes PARP-1, PARP-2, PARP-3 and Vault-PARP. PARP plays an important role in DNA repair pathways. Upon DNA damage breaks, PARP is activated, which acts as a molecular receptor for DNA damage, recognizes and binds to the DNA break site, and further activates and catalyzes the poly ADP-ribosylation of receptor proteins, thus participating in the DNA repair process.

[0013] PARP inhibitors can exert antitumor effects by mediating synthetic lethality. In addition to PARP-mediated DNA repair, there are other DNA repair mechanisms. Homologous recombination refers to the recombination that occurs between or within DNA molecules that contain homologous sequences between non-sister chromatid monomers or on the same chromosome. DNA Homologous Recombination Repair (HRR) is an important repair method for DNA double-strand damage, and BRCA1 and BRCA2 are key proteins in this process. If a mutation occurs in the BRCA gene and the function of the BRCA1 and BRCA2 proteins is lost, it will cause HRR dysfunction, i.e., HRD (Homologous Recombination Deficiency). Abnormalities in other HRR-related genes, such as PALB2, ATM, BRCA1 / 2, CHEK2, BARD1, BRIP1, Mre11, CDK12, RAD50, NBS1, RAD51C, RAD51D and PALB2, or methylation of the BRCA1 gene promoter, and other yet unclear causes, all of which can cause HRD and lead to genomic instability (Lord CJ, Ashworth A. Science. 2017;355(6330):1152-1158.). Cancer and Tumor Genetic Profile (TCGA) study found that HRD (homologous recombination repair deficiency) exists in more than 50% of high-grade serous ovarian cancers. These include BRCA1 / 2 germline mutations (15%), BRCA1 / 2 somatic mutations (6%), epigenetic mutations BRCA1 promoter methylation (11%), EMSY amplification (8%), PTEN mutations (7%), RAD51C methylation (3%), ATM or ATR mutations (2%) and other HRD gene mutations (5%) (Turner NC. N Engl J Med. 2017;377(25):2490-2492).

[0014] Synthetic lethality refers to the phenomenon in which two non-lethal genes are simultaneously inactivated, causing cell death. PARP inhibitors bind to the PARP1 or PARP2 catalytic site, preventing the PARP protein from being removed from the DNA damage site, leading to stalled DNA replication forks and poor DNA replication. When PARP is inhibited and HRR-related proteins are deficient, synthetic lethality is caused.

[0015] For tumor cells with specific DNA repair defects (eg, BRCA1 or BRCA2 gene mutations), inhibition of PARP can block tumor cell DNA repair and promote tumor cell death by synthetic lethality.

[0016] PARP inhibitors (PARPi) have shown good therapeutic effects in clinical studies of cancers such as ovarian cancer, breast cancer, prostate cancer, and pancreatic cancer, and have been approved for use in the treatment of cancers such as ovarian cancer, fallopian tube cancer, peritoneal cancer, and breast cancer with homologous repair deficiency (J. Mateo et al. Ann Oncol. 2019 Sep 1;30(9):1437-1447.), and PARPi has also shown good efficacy in pancreatic cancer and prostate cancer. PARPi still shows antitumor effects against tumors without DNA repair deficiency (e.g., BRCA1 or BRCA2 gene mutations). In the NOVA clinical study, compared with placebo, niraparib significantly extended PFS in patients regardless of whether they had homologous recombination repair deficiency gene mutations (Mirza MR et al. N Engl J Med 2016; 375(22):2154-2164.), and other PARPi drugs, such as olaparib and rucaparib, also showed good efficacy in patients with tumors without homologous recombination repair deficiency gene mutations (J. Mateo et al. Ann Oncol. 2019 Sep 1;30(9):1437-1447.).

[0017] A total of four PARP inhibitors have already been approved for sale: Olaparib from AstraZeneca, Rucaparib from Clovis Oncology, Niraparib from Tesaro, and Talazoparib from Pfizer. Fluzoparib from China Hengrui Medicine has been approved in China for use in treating patients with platinum-sensitive recurrent ovarian, fallopian tube or primary peritoneal cancer with embryonic lineage BRCA mutations who have previously undergone double-stranded or higher chemotherapy.In addition, veliparib ER, ABT-472, ABT-767, stenoparib, AST-6828, AG-PD, ANG-2864, ANG-3038, ANG-3186, AZD-5305, AZ-0108, AZD-2461, AMXI-5001, AMXI-2001, AMXI-3001, AMXI-7001, AMXI-9001, pamiparib, ZYTP-1, CK-102, XZ-120312, YHP-743, iobenguane I 131, rucaparib cansylate, CVL-218, CPH-101, CPH-102, CBX-11, CBX-15, minocycline, DB-207, DPS-102, E-7016, iobenguane I 131, MK-2512, HCX-014, HWH-340, IDX-1197, IDX-1197, senaparib, IMP-04100, IMP-04111, IMP-041 49, IMP-04249, IMP-04307, IMP-04356, JPI-289, JPI-547, JPI-283, fluzoparib, GT-1620, Yobenguan I 131, DR-2313, MP-124, H-10, NT-125, BGP-15, NMSP-293, NMSP-293, NMSP-118, NMSP-648, NMS P-914, DB-207, NUV-1156, NUV-1176, JPI-289, Stenoparib, OX-401, NU-1025, NU-1085, PLX- 376, R-554, RBN-2397, RBN-012759, PJ-34, INO-1001, WW-46, BSI-401, iniparib, SOMCL-9112, SC-10914, HTMC-0435, SRX-3128, TSL-1502, PJ-34, CEP-8983, CK-102, THG-009, talazoparib SR, L-2286, mitoparib, and WB-1340 are currently under research and development.

[0018] Bifunctional antibodies, also called bispecific antibodies, are specific drugs that target two different antigens at the same time, and can be produced by immunoselection and purification. They can also be obtained by genetic engineering, which has certain advantages in terms of optimization of binding sites, consideration of synthesis form, and yield, and thus has flexibility. Currently, more than 45 kinds of bispecific antibodies have been proven to exist (Muller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24:89-98). Many bispecific antibodies currently developed are in the Morrison mode, an IgG-scFv format (Coloma MJ, Morrison SL Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997;15:159-163), which is similar to the naturally occurring IgG format and has proven to be an ideal format for bifunctional antibodies due to its advantages in antibody engineering, expression, and purification (Miller BR, Demarest SJ, et al., Protein Eng Des Sel 2010; 23:549-57; Fitzgerald J, Lugovskoy A. MAbs 2011; 3:299-309).

[0019] Preclinical studies have found that PARPi can trigger immune system recognition of tumor cells based on neo- and non-neo-antigens, suggesting that its combination with immune checkpoint inhibitors may achieve better therapeutic effects (Mateo J et al. Ann Oncol. 2019;30(9):1437-1447.). Inhibition of PARP increases the expression of PD-L1 on tumor cells, forming immune inhibition, thereby weakening the therapeutic effect of PARPi; therefore, inhibiting PARP and simultaneously blocking PD-(L)1 avoids the formation of immune inhibition, thereby allowing PARPi and PD-(L)1 inhibitors to exert stronger antitumor activity (Jiao S et al. Clin Cancer Res. 2017;23(14):3711-3720.). On the other hand, tumor cells have mutations in related genes that lead to their specific DNA repair deficiency, and the accumulation of DNA damage increases the difference between tumor cells and normal cells, making them more likely to be recognized by the immune system. The more mutations in a tumor, the more dependent its survival is on inhibition of the immune system. Therefore, the combination of PARPi and PD-1 drugs has a certain theoretical basis. In addition to PD-1, for example, anti-PD-1 / VEGFA bispecific antibodies can simultaneously block VEGF-A, which has a strong correlation with the expression of PD-1 in the tumor microenvironment, and inhibit tumor progression through mechanisms such as angiogenesis inhibition. Therefore, the development of more effective therapeutic methods or combination administration therapeutic modes has great clinical significance. Summary of the Invention

[0020] The present inventors have conducted intensive research and creative efforts, and have conducted research into the combination of anti-PD-1-anti-VEGFA antibodies and PARP inhibitors in tumor treatment, and have found that the combination of the two has a favorable effect on treating and / or preventing tumors, particularly malignant tumors. This provides the following invention.

[0021] One aspect of the invention relates to a pharmaceutical combination comprising at least one bispecific antibody and at least one PARP inhibitor, Among them, the bispecific antibody is a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; Among them, the immunoglobulin has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; Or, the immunoglobulin has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype, wherein the heavy chain constant region of said immunoglobulin is mutated at any two or three sites among positions 234, 235 and 237 according to the EU numbering system, and after the mutation, the bispecific antibody has a reduced affinity constant with FcγRIIIa and / or C1q compared to before the mutation, preferably, said affinity constant is measured by a Fortebio Octet molecular interaction device.

[0022] In one or more embodiments of the invention, in the pharmaceutical combination, the heavy chain constant region of said immunoglobulin has the following mutations according to the EU numbering system: L234A and L235A, or L234A and G237A, or L235A and G237A, or L234A, L235A, G237A.

[0023] In the present invention, unless otherwise specified, the alphabet before the site indicates the amino acid before mutation, and the alphabet after the site indicates the amino acid after mutation.

[0024] The present invention also relates to a pharmaceutical combination comprising at least one bispecific antibody and at least one PARP inhibitor, Among them, the bispecific antibody is a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; Among them, the immunoglobulin has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; Or, the immunoglobulin has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; The immunoglobulin is of the human IgG1 subtype.

[0025] The present invention also relates to a pharmaceutical combination comprising at least one bispecific antibody and at least one PARP inhibitor, Among them, the bispecific antibody is a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; Among them, the immunoglobulin has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; Or, the immunoglobulin has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype, Therein, according to the EU numbering system, the heavy chain constant region of said immunoglobulins has one of the following combinations of mutations: L234A and L235A, or L234A and G237A, or L235A and G237A, or L234A, L235A, G237A.

[0026] In one or more embodiments of the invention, in the pharmaceutical combination, the heavy chain constant region of the immunoglobulin is The present invention relates to a method for treating a pulmonary artery disease, the method comprising the steps of: (a) administering to a patient a pulmonary artery disease or the like a pulmonary artery disease, the method comprising the steps of ...

[0027] In one or more embodiments of the present invention, in the pharmaceutical combination, The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:17; Or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO:3.

[0028] In one or more embodiments of the present invention, the combination drug is any one selected from the group consisting of the following (1) to (12):

[0029] (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 7; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 11; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 17; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 7; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 11; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 17; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO:3; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:11, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO:3; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO:3; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 3; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 11, and the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 3, and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3.

[0030] In one or more embodiments of the invention, the pharmaceutical combination: The amino acid sequence of the heavy chain of said immunoglobulin is shown in SEQ ID NO:24, and the amino acid sequence of the light chain is shown in SEQ ID NO:26.

[0031] In one or more embodiments of the present invention, in the combination drug, the bispecific antibody is in the Morrison mode in the IgG-scFv format.

[0032] In some embodiments of the present invention, in the pharmaceutical combination, The heavy chain constant region of the immunoglobulin is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region of the immunoglobulin is selected from the light chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0033] In some embodiments of the present invention, in the pharmaceutical combination, The above immunoglobulin has a heavy chain constant region that is the human Ig gamma-1 chain C region or the human Ig gamma-4 chain C region, and a light chain constant region that is the human Ig kappa chain C region.

[0034] In some embodiments of the invention, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts the Ig gamma-1 chain C region, Accession No. P01857, the light chain constant region adopts the Ig kappa chain C region, Accession No. P01834, or The heavy chain constant region of the above immunoglobulin is Ig gamma-4 chain C region, accession number: P01861.1, and the light chain constant region is Ig kappa chain C region, accession number: P01834.

[0035] In one embodiment of the present invention, the amino acid sequence of the heavy chain constant region Ig gamma-1 chain C region (Accession Number: P01857) is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0036] In one embodiment of the present invention, the amino acid sequence of the heavy chain constant region Ig gamma-4 chain C region (Accession No.: P01861.1) is as follows: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41)

[0037] In one embodiment of the present invention, the amino acid sequence of the light chain constant region Ig kappa chain C region (Accession Number: P01834) is as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:42)

[0038] In some embodiments of the present invention, in the combination drug, the single chain antibody is connected to the C-terminus of the heavy chain of an immunoglobulin. Since an immunoglobulin consists of two heavy chains, two single chain antibody molecules are connected to one immunoglobulin molecule. Preferably, the two single chain antibody molecules are the same.

[0039] In some embodiments of the present invention, the combination drug contains two single-chain antibodies, one end of each single-chain antibody being connected to the C-terminus or N-terminus of each of the two heavy chains of the immunoglobulin, respectively.

[0040] In some embodiments of the invention, the V H and V L There is a disulfide bond between the V H and V LMethods for introducing disulfide bonds between the nucleotides are well known in the art and are described in, for example, U.S. Pat. No. 5,747,654; Rajagopal et al., Prot. Engin. 10 (1997) 1453-1459; Reiter et al., Nat. Biotechnol. 14 (1996) 1239-1245; Reiter et al., Protein Engineering 8 (1995) 1323-1331; Webber et al., Molecular Immunology 32 (1995) 249-258; Reiter et al., Immunity 2 (1995) 281-287; Reiter et al., JBC 269 (1994) 18327-18331; Reiter et al., Inter. J. of Cancer 58 (1994) 142-149, or Reiter et. al, Cancer Res. 54 (1994) 2714-2718, which are incorporated herein by reference.

[0041] In one or more embodiments of the invention, in the pharmaceutical combination, the first protein functional region is connected to the second protein functional region directly or via a connecting fragment, and / or the heavy chain variable region of the single chain antibody is connected to the light chain variable region of the single chain antibody directly or via a connecting fragment.

[0042] In one or more embodiments of the present invention, in the pharmaceutical combination, the connecting fragment is (GGGGS)n, n is a positive integer, and preferably n is 1, 2, 3, 4, 5 or 6.

[0043] In one or more embodiments of the present invention, in the pharmaceutical combination, the first protein functional region and the second protein functional region are independently one, two or more than two.

[0044] In one or more embodiments of the invention, in the pharmaceutical combination, the single chain antibody is attached to the C-terminus of the heavy chain of an immunoglobulin.

[0045] The present invention also relates to a pharmaceutical combination comprising at least one bispecific antibody and at least one PARP inhibitor, Among them, the bispecific antibody is a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; The first protein functional region is one, and the second protein functional region is two, wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; The amino acid sequence of the heavy chain of said immunoglobulin is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region of the single chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in SEQ ID NO: 17; The single chain antibody is linked to the C-terminus of an immunoglobulin heavy chain, The first protein functional region is connected to the second protein functional region via a first connecting fragment, and the heavy chain variable region of the single chain antibody is connected to the light chain variable region of the single chain antibody via a second connecting fragment, and the first connecting fragment and the second connecting fragment are homologous or different; Preferably, the amino acid sequences of the first connected fragment and the second connected fragment are independently selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19; Preferably, the amino acid sequences of the first connected fragment and the second connected fragment are both shown in SEQ ID NO:18.

[0046] In one or more embodiments of the invention, in the pharmaceutical combination, the bispecific antibody or antigen-binding fragment thereof is about 10 -6 Less than M, e.g., about 10 -7 M, 10 -8 M or 10 -9M or less, preferably said affinity constant is measured using a Fortebio Octet molecular interaction instrument.

[0047] In one or more embodiments of the invention, in the pharmaceutical combination, the bispecific antibody or antigen-binding fragment thereof is about 10 -9 Less than M, e.g., about 10 -7 M, 10 -8 M or 10 -9 It binds to C1q with an affinity constant of less than M or less, preferably said affinity constant being measured by a Fortebio Octet molecular interaction instrument.

[0048] In some embodiments of the present invention, in the pharmaceutical combination, the bispecific antibody or antigen-binding fragment thereof is -5 Less than M, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than M or less D and preferably binds to the VEGFA protein and / or the PD-1 protein at the above K D is measured by a Fortebio molecular interaction instrument.

[0049] In some embodiments of the present invention, in the pharmaceutical combination, The bispecific antibody or antigen-binding fragment thereof has an EC value of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.15 nM, or less than 0.14 nM. 50 and preferably binds to the VEGFA protein at the EC 50 was measured by indirect ELISA. and / or The bispecific antibody or antigen-binding fragment thereof has an EC value of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.17 nM, less than 0.16 nM, or less than 0.15 nM. 50 and preferably binds to the PD-1 protein at the EC 50is measured by indirect ELISA.

[0050] In one or more embodiments of the invention, the bispecific antibody is a monoclonal antibody.

[0051] In one or more embodiments of the invention, the bispecific antibody is a humanized antibody.

[0052] In some embodiments of the present invention, in the combination pharmaceutical, the PARP inhibitor is olaparib, rucaparib, niraparib, talazoparib, fluzoparib, veliparib ER, ABT-472, ABT-767, stenoparib, AST-6828, AG-PD, ANG-2864, ANG-3038, ANG-3186, AZD-5305, AZ-0108, AZD-2461, AMXI-5001, AMXI-2001, AMXI-3001, AMXI-7001, AMXI-9001, pamiparib, ZYTP-1, CK-102, XZ-120312, YHP-743, iobenguane I 131, rucaparib cansylate, CVL-218, CPH-101, CPH-102, CBX-11, CBX-15, minocycline, DB-207, DPS-102, E-7016, iobenguane I 131, MK-2512, HCX-014, HWH-340, IDX-1197, IDX-1197, senaparib, IMP-04100, IMP-04111, IMP-04149, IMP-04249, IMP-04307, IMP-04356, JPI-289, JPI-547, JPI-283, fluzoparib, GT-1620, iobenguane I 131, DR-2313, MP-124, H-10, NT-125, BGP-15, NMSP-293, NMSP-293, NMSP-118, NMSP-648, NMSP- 914, DB-207, NUV-1156, NUV-1176, JPI-289, Stenoparib, OX-401, NU-1025, NU-1085, PLX-376, R -554, RBN-2397, RBN-012759, PJ-34, INO-1001, WW-46, BSI-401, iniparib, SOMCL-9112, SC-10914, HTMC-0435, SRX-3128, TSL-1502, PJ-34, CEP-8983, CK-102, THG-009, talazoparib SR, L-2286, mitoparib and WB-1340.

[0053] In some embodiments of the invention, the pharmaceutical combination further comprises one or more tumor chemotherapeutic agents, Preferably, the tumor chemotherapeutic agent is selected from topoisomerase II (TOP2) inhibitors, preferably, the topoisomerase II (TOP2) inhibitor is etoposide; Preferably, the tumor chemotherapy drug is selected from paclitaxel-based drugs, and preferably, the paclitaxel-based drugs are selected from the group consisting of paclitaxel, albuminic paclitaxel, liposomal paclitaxel and docetaxel (taxotere); Preferably, the tumor chemotherapy drug is selected from platinum-based drugs, preferably, the platinum-based drugs are selected from the group consisting of cisplatin, carboplatin and oxaliplatin; Preferably, said tumor chemotherapeutic agent is one or more selected from the group consisting of gemcitabine, pemetrexed and capetabine.

[0054] In some embodiments of the present invention, the pharmaceutical combination is a fixed combination, for example in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or The above-mentioned combination medicine is a non-fixed combination, for example, the above-mentioned bispecific antibody, PARP inhibitor and tumor chemotherapeutic drug each take the form of a pharmaceutical composition. In some embodiments of the present invention, in the pharmaceutical combination, the pharmaceutical composition further comprises a pharma- ceutically acceptable vector and / or excipient.

[0055] The pharmaceutical compositions of the present invention may be formulated into any pharma- ceutical known dosage form, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, tablets, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention must be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required amount of the bispecific antibody of the present invention in a suitable solvent, optionally incorporating other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Furthermore, for ease of storage and use, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or lyophilization). Such sterile, lyophilized powders can be dispersed in a suitable vector, such as sterile, pyrogen-free water, before use.

[0056] Furthermore, the bispecific antibody or PARP inhibitor of the present invention can be present in the pharmaceutical composition in the form of a unit dose for ease of administration. In certain embodiments, the unit dose is at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. When the pharmaceutical composition is in a liquid (e.g., injectable) dosage form, it may have a concentration of at least 0.1 mg / mL, such as at least 0.25 mg / mL, at least 0.5 mg / mL, at least 1 mg / mL, at least 2.5 mg / mL, at least 5 mg / mL, at least 8 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, or at least 100 mg / mL of the bispecific antibody of the present invention.

[0057] The bispecific antibody or pharmaceutical composition of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by the skilled artisan that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibody or pharmaceutical composition of the invention is administered by intravenous infusion or injection.

[0058] The bispecific antibodies or pharmaceutical compositions provided herein may be used alone or in combination with other pharmacologic active agents, such as tumor chemotherapeutic agents, which may be administered prior to, simultaneously with, or after administration of the bispecific antibodies or pharmaceutical compositions of the invention.

[0059] In the present invention, the mode of administration can be adjusted to obtain the optimum desired response (e.g., a therapeutic or prophylactic response), for example, a single administration, multiple administrations over a period of time, or the dose can be proportionally reduced or increased depending on the exigencies of the therapeutic situation.

[0060] The bispecific antibody of the present invention is a bispecific antibody according to any one of the aspects of the present invention, which is an anti-PD-1-anti-VEGFA bispecific antibody.

[0061] Another aspect of the present invention relates to a drug kit product comprising the pharmaceutical combination according to any one of the present invention and a product description.

[0062] A further aspect of the present invention relates to the use of a pharmaceutical combination according to any one of the present invention or a pharmaceutical kit product according to the present invention in the manufacture of a medicament for the treatment and / or prevention of malignant tumors, Preferably, the malignant tumor is selected from the group consisting of ovarian cancer, endometrial cancer, breast cancer, cervical cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, liver cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, brain cancer, esophageal cancer, such as esophageal squamous cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer, urothelial carcinoma, mesothelioma, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, myeloma, thyroid cancer, head and neck cancer, bone cancer, biliary tract cancer, and testicular cancer; Preferably, the tumor is a tumor with a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), Preferably, the tumor is a tumor in which there is no homologous recombination-mediated repair deficiency; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer, Preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR and / or ALK susceptibility mutations, Preferably, the non-small cell lung cancer is non-small cell lung cancer not associated with EGFR and / or ALK susceptibility mutations; Preferably, the liver cancer is hepatocellular carcinoma, Preferably, the renal tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer, Preferably, the urothelial cancer is bladder cancer, Preferably, the peritoneal cancer is a primary peritoneal cancer, Preferably, the ovarian cancer is an ovarian cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; Preferably, the fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the peritoneal cancer is a peritoneal cancer having a homologous recombination-mediated repair function deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the breast adenocarcinoma is a breast adenocarcinoma in which a homologous recombination-mediated repair function deficiency (eg, BRCA1 mutation and / or BRCA2 mutation) is present.

[0063] A further aspect of the present invention relates to the use of a pharmaceutical combination according to any one of the present invention or a pharmaceutical kit product according to the present invention in the manufacture of a medicament comprising: (1) an agent or reagent for detecting VEGFA levels in a sample; A drug or reagent that blocks the binding of VEGFA to VEGFR2, Agents or reagents that down-regulate VEGFA activity or levels; A drug or agent that relieves the stimulatory effect of VEGFA on the proliferation of vascular endothelial cells; A drug or reagent that inhibits the proliferation of vascular endothelial cells, or Agents or reagents that block tumor angiogenesis; and / or (2) A drug or reagent that blocks the binding of PD-1 to PD-L1; Agents or reagents that downregulate the activity or levels of PD-1; Drugs or reagents that release the inhibition of the body's immune system by PD-1; An agent or reagent that promotes IFN-γ secretion in T lymphocytes, or An agent or reagent that promotes IL-2 secretion in T lymphocytes. In the in vitro experiments of the present invention, both the anti-VEGFA antibody and the anti-VEGFA-anti-PD-1 bispecific antibody can inhibit the proliferation of HUVEC cells, and both the anti-PD-1 antibody and the anti-VEGFA-anti-PD-1 bispecific antibody can promote the secretion and activation of immune responses of IFNγ and / or IL-2.

[0064] A further aspect of the present invention relates to a method for treating and / or preventing malignant tumors, comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination according to any one of the present invention or the pharmaceutical kit product of the present invention, Preferably, the malignant tumor is selected from the group consisting of ovarian cancer, endometrial cancer, breast cancer, cervical cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, liver cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, brain cancer, esophageal cancer, such as esophageal squamous cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer, urothelial carcinoma, mesothelioma, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, myeloma, thyroid cancer, head and neck cancer, bone cancer, biliary tract cancer, and testicular cancer; Preferably, the tumor is a tumor with a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), Preferably, the tumor is a tumor in which there is no homologous recombination-mediated repair deficiency; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer, Preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR and / or ALK susceptibility mutations, Preferably, the non-small cell lung cancer is non-small cell lung cancer not associated with EGFR and / or ALK susceptibility mutations; Preferably, the liver cancer is hepatocellular carcinoma, Preferably, the renal tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer, Preferably, the urothelial cancer is bladder cancer, Preferably, the peritoneal cancer is a primary peritoneal cancer, Preferably, the ovarian cancer is an ovarian cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; Preferably, the fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the peritoneal cancer is a peritoneal cancer having a homologous recombination-mediated repair function deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the breast adenocarcinoma is a breast adenocarcinoma in which a homologous recombination-mediated repair function deficiency (eg, BRCA1 mutation and / or BRCA2 mutation) is present.

[0065] In some embodiments of the present invention, the methods for treating and / or preventing malignant tumors are administered before or after surgery and / or before or after radiation therapy.

[0066] In some embodiments of the present invention, in the method for treating and / or preventing a malignant tumor, the single dose of the bispecific antibody is 0.1 to 100 mg per kilogram of body weight, preferably 5 to 50 mg or 5 to 15 mg per kilogram of body weight; Administered once every 3, 4, 5, 6, or 10 days, 1, 2, or 3 weeks; and / or The administration route is intravenous infusion or injection.

[0067] In some embodiments of the present invention, in the method for treating and / or preventing a malignant tumor, the single dose of the PARP inhibitor is 0.1 to 100 mg per kilogram of body weight, preferably 5 to 50 mg or 5 to 15 mg per kilogram of body weight; Administered once every 3, 4, 5, 6, or 10 days, 1, 2, or 3 weeks; and / or The administration route is intravenous infusion or injection.

[0068] In some embodiments of the invention, in the method of treating and / or preventing malignant tumors, the bispecific antibody is administered simultaneously or not with a PARP inhibitor.

[0069] A typical non-limiting range of a therapeutically or prophylactically effective amount of the bispecific antibody and / or PARP inhibitor of the present invention is 0.02-100 mg / kg, e.g., 0.1-50 mg / kg, 0.1-25 mg / kg, or 1-10 mg / kg. It should be noted that dosages may vary depending on the type and severity of the condition requiring treatment. Furthermore, one of skill in the art should understand that for any particular patient, the particular dosing regimen should be adjusted over time based on the needs of the patient and the physician's professional assessment; dosage ranges described herein are for illustrative purposes only and are not intended to limit the use or scope of the pharmaceutical combination of the present invention.

[0070] In the present invention, the subject may be a mammal, such as a human.

[0071] Further aspects of the present invention relate to in vivo or in vitro methods selected from the group consisting of: (1) A method for detecting VEGFA levels in a sample, Method for blocking the binding of VEGFA to VEGFR2 Methods for downregulating VEGFA activity or levels Methods for reversing the stimulatory effect of VEGFA on the proliferation of vascular endothelial cells, A method for the preparation of a drug or reagent for inhibiting the proliferation of vascular endothelial cells; or Methods for blocking tumor angiogenesis, and / or (2) A method for blocking the binding of PD-1 and PD-L1, Methods for downregulating the activity or levels of PD-1; How to release PD-1 from the body's immune system A method for promoting IFN-γ secretion in T lymphocytes, or A method for promoting IL-2 secretion in T lymphocytes.

[0072] In one or more embodiments of the present invention, the combination medicine or drug kit product is used for treating and / or preventing malignant tumors, Preferably, the malignant tumor is selected from the group consisting of ovarian cancer, endometrial cancer, breast cancer, cervical cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, liver cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, brain cancer, esophageal cancer, such as esophageal squamous cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer, urothelial carcinoma, mesothelioma, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, myeloma, thyroid cancer, head and neck cancer, bone cancer, biliary tract cancer, and testicular cancer; Preferably, the tumor is a tumor with a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), Preferably, the tumor is a tumor in which there is no homologous recombination-mediated repair deficiency; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer, Preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR and / or ALK susceptibility mutations, Preferably, the non-small cell lung cancer is non-small cell lung cancer not associated with EGFR and / or ALK susceptibility mutations; Preferably, the liver cancer is hepatocellular carcinoma, Preferably, the renal tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer, Preferably, the urothelial cancer is bladder cancer, Preferably, the peritoneal cancer is a primary peritoneal cancer, Preferably, the ovarian cancer is an ovarian cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; Preferably, the fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the peritoneal cancer is a peritoneal cancer having a homologous recombination-mediated repair function deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the breast adenocarcinoma is a breast adenocarcinoma in which a homologous recombination-mediated repair function deficiency (eg, BRCA1 mutation and / or BRCA2 mutation) is present.

[0073] In one or more embodiments of the present invention, the combination pharmaceutical or drug kit product is used as follows: (1) Detecting VEGFA levels in the sample, Blocks the binding of VEGFA to VEGFR2, downregulating VEGFA activity or levels; It relieves the stimulatory effect of VEGFA on the proliferation of vascular endothelial cells, Inhibits the proliferation of vascular endothelial cells, or Blocking tumor angiogenesis, and / or (2) Blocks the binding of PD-1 and PD-L1 downregulating the activity or level of PD-1; Releases the inhibition of the body's immune system by PD-1, promotes IFN-γ secretion in T lymphocytes, or Promotes IL-2 secretion in T lymphocytes.

[0074] A further aspect of the invention relates to the use of a bispecific antibody in a pharmaceutical combination according to any one of the invention for the manufacture of a medicament for the treatment and / or prevention of a malignant tumor, wherein said malignant tumor is selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer and breast cancer, Preferably, the ovarian cancer is an ovarian cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; Preferably, the fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the peritoneal cancer is a peritoneal cancer having a homologous recombination-mediated repair function deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the breast adenocarcinoma is a breast adenocarcinoma in which a homologous recombination-mediated repair function deficiency (eg, BRCA1 mutation and / or BRCA2 mutation) is present.

[0075] A further aspect of the invention is a method for treating and / or preventing a malignant tumor, comprising administering to a subject in need thereof an effective amount of a bispecific antibody of the pharmaceutical combination according to any one of the invention, wherein said malignant tumor is selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer and breast cancer, Preferably, the ovarian cancer is an ovarian cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; Preferably, the fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the peritoneal cancer is a peritoneal cancer having a homologous recombination-mediated repair function deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the breast adenocarcinoma is a breast adenocarcinoma in which a homologous recombination-mediated repair function deficiency (eg, BRCA1 mutation and / or BRCA2 mutation) is present.

[0076] A further aspect of the invention relates to a bispecific antibody in the pharmaceutical combination according to any one of the invention for the treatment and / or prevention of a malignant tumor, wherein said malignant tumor is selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer and breast cancer, Preferably, the ovarian cancer is an ovarian cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; Preferably, the fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the peritoneal cancer is a peritoneal cancer having a homologous recombination-mediated repair function deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation), Preferably, the breast adenocarcinoma is a breast adenocarcinoma in which a homologous recombination-mediated repair function deficiency (eg, BRCA1 mutation and / or BRCA2 mutation) is present.

[0077] Antibody therapeutics, especially monoclonal antibodies, have already achieved good therapeutic effects in treating many diseases. The conventional experimental methods for obtaining these therapeutic antibodies are to immunize animals with an antigen, to obtain antibodies that target the antigen when the animal is immunized, or to improve antibodies with low affinity to the antigen by affinity maturation methods.

[0078] The variable regions of the light and heavy chains determine antigen binding, and each chain variable region contains three highly variable regions called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H Chain) contain HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L Chain) contain LCDR1, LCDR2, and LCDR3, which were named by Kabat et al., Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication (1-3) 1991:91-3242).

[0079] Preferably, the CDRs are defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38.suppl_1 (2009):D301-D307.

[0080] The amino acid sequences of the CDR regions of the monoclonal antibody sequences in the following items (1) to (13) were analyzed by technical means well known to those skilled in the art, for example, by the VBASE2 database, in particular according to the IMGT definition, and the results are as follows: (1) Bevacizumab The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3. The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GYTFTNYG (SEQ ID NO:28) HCDR2: INTYTGEP (SEQ ID NO:29) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO:30) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDISNY (SEQ ID NO:31) LCDR2: FTS (SEQ ID NO:32) LCDR3: QQYSTVPWT (SEQ ID NO:33) (2) 14C12, 14C12H1L1 or 14C12H1L1(M) The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO:34) HCDR2: ISGGGRYT (SEQ ID NO:35) HCDR3: ANRYGEAWFAY (SEQ ID NO:36) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDINTY (SEQ ID NO:37) LCDR2: RAN (SEQ ID NO:38) LCDR3: LQYDEFPLT (SEQ ID NO:39) (3) VP101(hG1WT) or VP101(hG1DM) The amino acid sequences of the nine CDR regions of the heavy chain are as follows: HCDR1: GYTFTNYG (SEQ ID NO:28) HCDR2: INTYTGEP (SEQ ID NO:29) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO:30) HCDR4: GFAFSSYD (SEQ ID NO:34) HCDR5: ISGGGRYT (SEQ ID NO:35) HCDR6: ANRYGEAWFAY (SEQ ID NO:36) HCDR7: QDINTY (SEQ ID NO:37) HCDR8: RAN (SEQ ID NO:38) HCDR9: LQYDEFPLT (SEQ ID NO:39) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDISNY (SEQ ID NO:31) LCDR2: FTS (SEQ ID NO:32) LCDR3: QQYSTVPWT (sequence number 33).

[0081] The antibody VP101 (hG1DM) of the present invention introduces amino acid mutations in the non-variable region of VP101 (hG1WT). Amino acid mutations are introduced at positions 234 and 235 according to the EU numbering system: A point mutation from leucine to alanine (L234A) was introduced at position 234 in the heavy chain hinge region, and a point mutation from leucine to alanine (L235A) was introduced at position 235 to obtain VP101(hG1DM).

[0082] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratory procedures used herein are common procedures widely used in the art. At the same time, in order to better understand the present invention, the following provides definitions and explanations of relevant terms.

[0083] As used herein, when referring to the amino acid sequence of VEGFA protein, this includes, but is not limited to, the full length of VEGFA protein (GenBank ID: NP_001165097.1), and also includes fusion proteins of VEGFA, such as fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variants (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of VEGFA protein can be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "VEGFA protein" includes all such sequences, including natural or artificial variants thereof. And, when a sequence fragment of VEGFA protein is described, it also includes the corresponding sequence fragment of its natural or artificial variant. In one embodiment of the present invention, the amino acid sequence of VEGFA protein is shown in the underlined portion of SEQ ID NO: 33 (302 amino acids in total, excluding the last 6 His).

[0084] As used herein, the amino acid sequence of VEGFR2 protein (also called KDR) includes, but is not limited to, the full length of VEGFR2 protein (GenBank ID: NP_002244), or the extracellular fragment of VEGFR2, VEGFR2-ECD, or a fragment containing VEGFR2-ECD, and also includes fusion proteins of VEGFR2-ECD, such as fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variants (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of VEGFR2 protein can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "VEGFR2 protein" includes all such sequences, including natural or artificial variants thereof. And, when a sequence fragment of VEGFR2 protein is described, it also includes the corresponding sequence fragment of its natural or artificial variant. In one embodiment of the present invention, the amino acid sequence of the extracellular fragment of VEGFR2, VEGFR2-ECD, is shown in SEQ ID NO: 34 (766 amino acids).

[0085] As used herein, unless otherwise specified, the VEGFR is VEGFR1 and / or VEGFR2, and its specific protein sequence is a sequence known in the prior art, and can refer to the sequences disclosed in conventional literature or GenBank, for example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321), VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0086] As used herein, when referring to the amino acid sequence of PD-1 protein (Programmed cell death protein 1), this includes, but is not limited to, the full-length PD-1 protein (NCBI GenBank: NP_005009.2), or the extracellular fragment of PD-1, PD-1ECD, or a fragment containing PD-1ECD, and further includes a fusion protein of PD-1ECD, such as a fragment fused to an Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it will be understood by those skilled in the art that mutations or variants (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of PD-1 protein may be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "PD-1 protein" is intended to include all such sequences, including natural or artificial variants thereof. And, when a sequence fragment of PD-1 protein is described, it also includes the corresponding sequence fragment of the natural or artificial variant thereof.

[0087] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can produce 50% of the maximum effect.

[0088] As used herein, the term "antibody" refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. In the general sense, heavy chains are understood to mean the relatively large polypeptide chains in an antibody, and light chains are understood to mean the relatively small polypeptide chains in an antibody. Light chains can be classified as kappa and lambda light chains. Heavy chains are usually classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected through a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (VH ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L The light chain constant region consists of one domain, C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The regions can also be subdivided into regions of high variability (called complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). H and V L Each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. H and V L), each of which forms an antibody binding site. The assignment of amino acids to each region or domain is defined according to Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196(1987):901-917; Chothia et al. Nature 342(1989): 878-883, or the IMGT numbering system, with reference to the definitions in Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38.suppl_1 (2009): D301-D307. In particular, the heavy chain may comprise more than three CDRs, for example six, nine, or twelve. For example, in a bispecific antibody of the invention, the heavy chain may be an scFv in which the C-terminus of the heavy chain of an IgG antibody is connected to another antibody, in which case the heavy chain comprises nine CDRs. The term "antibody" is not limited by any particular method of producing the antibody. For example, this includes, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, for example IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0089] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, also referred to as an "antigen-binding portion." Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989). Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage methods of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides having at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. As used herein, the term "Fd fragment" refers to a V H and C H1 The term "Fv fragment" refers to an antibody fragment consisting of a single arm V domain of an antibody. L and V H The term "dAb fragment" refers to an antibody fragment consisting of the V domain. H The term "Fab fragment" refers to an antibody fragment consisting of the V domain (Ward et al., Nature 341 (1989):544-546). L , V H , C L and C H1 The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bridges in the hinge region.

[0090] In some cases, the antigen-binding fragment of an antibody is L and V HSingle chain antibodies (e.g., scFv) are monovalent molecules formed by pairing linkers that allow the domains to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242 (1988):423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 85 (1988):5879-5883). Such scFv molecules include the NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L The linker may have the general structure -COOH. A suitable prior art linker consists of a repeat of the amino acid sequence GGGGS or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers useful in the present invention are described by Alfthan et al., Protein Eng. 8 (1995):725-731, Choi et al., Eur. J. Immunol. 31 (2001):94-106, Hu et al., Cancer Res. 56 (1996):3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999):41-56 and Roovers et al., Cancer Immunol. (2001).

[0091] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a V H and V LA bivalent antibody is one in which the domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain to create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 (1993):6444-6448, and Poljak RJ et al., Structure 2 (1994):1121-1123).

[0092] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage) and can be specifically screened for antigen-binding fragments of the antibody in the same way as intact antibodies.

[0093] As used herein, reference to the term "antibody" includes not only complete antibodies but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

[0094] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or antibody fragment from a highly homogenous population of antibody molecules, i.e., a population of completely identical antibody molecules, except for natural mutations that may occur naturally. Monoclonal antibodies have high specificity for a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495,1975), but can also be obtained using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0095] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody which may be derived from a particular species or belong to a particular antibody class or subclass, and another portion of the light or / and heavy chain is derived from another antibody which may be derived from the same or a different species or belong to the same or a different antibody class or subclass, but which in any case retains binding activity for a target antigen (US Patent 4,816,567 Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 (1984):6851-6855).

[0096] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained after all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human antibody (e.g., mouse, rat or rabbit) with the desired specificity, affinity or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced with the corresponding amino acid residues of the non-human antibody or other antibodies to further improve or optimize the performance of the antibody. For further details on humanized antibodies, see, e.g., Jones et al., Nature, 321 (1986):522-525; Reichmann et al., Nature, (1988) 332:323 329; Presta, Curr. Op. Struct. Biol., 2 (1992):593-596; and Clark, Immunol. Today 21 (2000): 397-402.

[0097] As used herein, the term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. "Epitope" is also referred to in the art as "antigenic determinant". An epitope or antigenic determinant generally consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and generally has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope generally contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, and may be a "linear" epitope or a "conformational" epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all of the interaction sites between the protein and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein, whereas in a conformational epitope, the interaction sites occur across amino acid residues of the protein that are separated from each other.

[0098] As used herein, the term "isolate" or "isolated" refers to being obtained by hand from a natural state. If a particular "isolated" substance or component exists in nature, the natural environment in which it exists may have been altered or the substance may have been separated from its natural environment, or both. For example, a naturally occurring, non-isolated polynucleotide or polypeptide naturally occurs in the living body of an animal, and a highly purified version of the same polynucleotide or polypeptide, separated from such a natural state, is referred to as isolated. The term "isolate" or "isolated" does not exclude mixtures of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0099] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector is called an expression vector when it allows for the expression of a protein encoded by an inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction or transfection to allow the expression of the genetic material elements it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, Cox plasmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or artificial chromosomes such as P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papilloma baculoviruses (such as SV40). A vector may contain multiple expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication.

[0100] As used herein, the term "host cell" refers to cells useful for introducing a vector, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0101] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and an antigen against which it is directed. In one embodiment, an antibody that specifically binds to an antigen (or has specificity for an antigen) is one in which the antibody binds to the antigen at a concentration of about 10 -5Less than m, e.g., about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Affinity (K D In some embodiments of the present invention, the term "target" refers to specific binding to an antigen.

[0102] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies have an equilibrium dissociation constant of about 10, as determined, for example, using surface plasmon resonance (SPR) on a BIACORE instrument or a Fortebio molecular interaction instrument. -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less than the dissociation equilibrium constant (K D ) binds to the antigen.

[0103] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably, the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably, and the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are usually represented by abbreviations and abbreviation words well known in the art. For example, alanine can be represented by A or Ala.

[0104] As used herein, the term "pharmaceutical acceptable excipient" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancing agents. For example, pH adjusting agents include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween®-80, and ionic strength enhancing agents include, but are not limited to, sodium chloride.

[0105] As used herein, the term "adjuvant" refers to a non-specific immune enhancing agent that can enhance or change the type of immune response of the body to the antigen when delivered together with the antigen or in advance to the body together with the antigen.Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Brevis, lipopolysaccharide, cytokines, etc.Freund's adjuvant is the most commonly used adjuvant in current animal testing.Aluminum hydroxide adjuvant is more commonly used in clinical experiments.

[0106] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, it means an amount sufficient to prevent, inhibit, or delay the onset of a disease (e.g., a disease associated with PD-1 binding to PD-L1 or high VEGF expression, e.g., tumors), and an amount effective for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient suffering from the disease. Determination of such an effective amount is within the capabilities of a person skilled in the art. For example, an amount effective for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight, sex, etc., the mode of administration of the drug, and other treatments administered at the same time, etc. Effect of the Invention

[0107] The present invention achieves one or more of the following technical effects (1) to (6). (1) The Fc-terminal modification of the antibody of the present invention is C It completely eliminates the binding activity to the receptors FcγRI and FcγRIIIa_F158, and further completely eliminates the ADCC activity. (2) Modification of the Fc terminus of the antibody completely eliminates the binding activity of VP101(hG1WT) to complement C1q, and further completely eliminates CDC activity. (3) The bispecific antibody of the present invention can specifically bind to VEGFA, very effectively block the binding of VEGFA to VEGFR2, and specifically relieve the immune inhibition and angiogenesis promoting effects of VEGFA. (4) The bispecific antibody of the present invention can specifically bind to PD-1, very effectively block the binding of PD-1 to PD-L1, specifically remove the inhibition of the body's immune system by PD-1, and activate the immune response. (5) The combined administration of PARPi and the bispecific antibody of the present invention has a significantly superior therapeutic effect against tumors, particularly breast cancer or ovarian cancer, compared with the administration of PARPi or the bispecific antibody alone. (6) There is a synergistic effect between PARPi and the bispecific antibody of the present invention, resulting in a synergistic effect in the treatment or prevention of tumors. [Brief description of the drawings]

[0108] [Figure 1] 1 shows the results of determining the affinity constant between VP101 (hG1DM) and FcγRI. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Diagram 2] 1 is a graph showing the results of determining the affinity constant between bevacizumab and FcγRI. The antibody concentrations added in each pair of curves from the top to the bottom of the graph are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Diagram 3] This is a diagram showing the results of determining the affinity constant between nivolumab and FcγRI. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 4] This is a graph showing the results of determining the affinity constant between VP101 (hG1WT) and FcγRI. The antibody concentrations added in each pair of curves from the top to the bottom of the graph are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Diagram 5] This is a graph showing the results of determining the affinity constant between VP101 (hG4WT) and FcγRI. The antibody concentrations added in each pair of curves from the top to the bottom of the graph are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 6] 1 shows the results of determining the affinity constant between VP101 (hG1DM) and FcγRIIa_H131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 7] 1 shows the results of determining the affinity constant between bevacizumab and FcγRIIa_H131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 8] This is a diagram showing the results of determining the affinity constant between nivolumab and FcγRIIa_H131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 9] 1 shows the results of determining the affinity constant between VP101 (hG1WT) and FcγRIIa_H131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 10] 1 shows the results of determining the affinity constant between VP101 (hG4WT) and FcγRIIa_H131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 11] 1 shows the results of determining the affinity constant between VP101 (hG1DM) and FcγRIIa_R131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 12] 1 is a graph showing the results of determining the affinity constant between bevacizumab and FcγRIIa_R131. The antibody concentrations added in each pair of curves from the top to the bottom of the graph are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 13] This is a diagram showing the results of determining the affinity constant between nivolumab and FcγRIIa_R131. The antibody addition concentrations in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 14] 1 shows the results of determining the affinity constant between VP101 (hG1WT) and FcγRIIa_R131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 15]1 shows the results of determining the affinity constant between VP101 (hG4WT) and FcγRIIa_R131. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 16] 1 shows the results of determining the affinity constant between VP101 (hG1DM) and FcγRIIIa_V158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 17] 1 shows the results of determining the affinity constant between bevacizumab and FcγRIIIa_V158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 18] This is a diagram showing the results of determining the affinity constant between nivolumab and FcγRIIIa_V158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 19] 1 shows the results of determining the affinity constant between VP101 (hG1WT) and FcγRIIIa_V158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 20] 1 shows the results of determining the affinity constant between VP101 (hG4WT) and FcγRIIIa_V158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 21] This is a diagram showing the results of determining the affinity constant between VP101 (hG1DM) and FcγRIIIa_F158. The antigen concentrations in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 22]1 shows the results of determining the affinity constant between bevacizumab and FcγRIIIa_F158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Diagram 23] This is a diagram showing the results of determining the affinity constant between nivolumab and FcγRIIIa_F158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 24] 1 shows the results of determining the affinity constant between VP101 (hG1WT) and FcγRIIIa_F158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Diagram 25] 1 shows the results of determining the affinity constant between VP101 (hG4WT) and FcγRIIa_F158. The antibody concentrations added in each pair of curves from the top to the bottom of the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 26] This is a graph showing the results of determining the affinity constant between VP101 (hG1DM) and C1q. The antibody concentrations added in each pair of curves from the top to the bottom of the graph are 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. [Figure 27] 1 shows the results of affinity constant detection between bevacizumab and C1q. The antibody concentrations added in each pair of curves from top to bottom in the figure are 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. [Figure 28] This is a graph showing the results of determining the affinity constant between nivolumab and C1q. The antibody concentrations added in each pair of curves from top to bottom in the graph are 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. [Figure 29] This is a graph showing the results of determining the affinity constant between VP101 (hG1WT) and C1q. The antibody concentrations added in each pair of curves from the top to the bottom of the graph are 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. [Diagram 30] This is a graph showing the results of determining the affinity constant between VP101 (hG4WT) and C1q. The antigen concentrations in each pair of curves from top to bottom in the graph are 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. [Diagram 31] This shows the results of detecting the ADCC activity of VP101(hG1WT) and VP101(hG1DM) in the CHO-K1-PD1 target cell line expressing the PD-1 antigen. [Diagram 32] CDC activity of VP101(hG1WT) and VP101(hG1DM) was detected in CHO-K1-PD1 target cell line expressing PD-1 antigen. [Diagram 33] The effect of antibody VP101 (hG1DM) on the secretion of cytokine IFN-γ induced by mixed culture of PBMC and Raji-PDL1 cells is detected by ELISA. [Diagram 34] The effect of antibody VP101 (hG1DM) on the secretion of cytokine IL-2 induced by PBMC and Raji-PDL1 cell mixed culture is detected by ELISA method. [Diagram 35] This shows the results of detecting the ADCP activity of VP101 (hG1DM) in the CHO-K1-PD1 target cell line expressing the PD-1 antigen. [Diagram 36] The results show that VP101 (hG1DM) inhibits the proliferation of breast cancer tumor cells in a breast pad tumor xenograft model. [Figure 37] The results show that a combination of PARPi and VP101 (hG1DM) inhibits ovarian cancer cell migration. [Figure 38] This shows the effect of combined use of PARPi and VP101 (hG1DM) on the volume of subcutaneously implanted tumors in mice in a breast cancer subcutaneously implanted tumor model. [Figure 39] Effect of combined administration of PARPi and VP101 (hG1DM) on mouse body weight in a subcutaneously xenografted breast cancer tumor model. [Diagram 40] This shows the effect of combined use of PARPi and VP101 (hG1DM) on the volume of subcutaneously implanted tumors in mice in a subcutaneous ovarian cancer tumor model. [Diagram 41] Effect of combined administration of PARPi and VP101 (hG1DM) on mouse body weight in a subcutaneously xenografted ovarian cancer tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] Hereinafter, the embodiments of the present invention will be described in detail with reference to the examples. Those skilled in the art should understand that the following examples are merely for the purpose of illustrating the present invention and do not limit the scope of the present invention. If no specific techniques or conditions are described in the examples, they will be performed according to the techniques or conditions described in the literature in this field (for example, see "Molecular Cloning Experimental Guidelines" by J. Sambrook et al., translated by Huang Pedang et al., 3rd Edition, Science Press) or according to the product instructions. If the manufacturer of the reagents or equipment used is not specified, they may be commercially available general products.

[0110] In the following examples of the present invention, the same targeting commercially available antibody bevacizumab (trade name Avastin (registered trademark)) is used as a control antibody, which can be purchased from Roche or prepared with reference to Preparation Example 1.

[0111] In the following examples of the present invention, the same targeting commercial antibody Nivolumab (trade name Opdivo®) used as a control antibody was purchased from Bristol-Myers Squibb (BMS).

[0112] In the following examples of the present invention, the PARP inhibitor Olaparib used was purchased from selleckchem.

[0113] In the following examples of the present invention, the isotype control antibody used is human anti-hen egg lysozyme IgG (anti-HEL, i.e. human IgG, abbreviated as hIgG), whose variable region sequence is derived from Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The hIgG1DM and hIgG4WT used in the examples are isotype control antibodies, i.e. anti-HEL with hG1DM and hG4WT constant region sequences, which were produced in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0114] In the following examples of the present invention, the breast cancer cells MDA-MB-231, the ovarian cancer cells SNU-251 and the SK-OV-3 cells used all have BRCA1 and BRCA2 mutations.

[0115] Production Example 1: Production of anti-VEGFA antibody bevacizumab The amino acid sequences of the heavy and light chain variable regions of the commercially available anti-VEGFA monoclonal Avastin (bevacizumab) can be found in Chinese Patent Publication CN1259962A. The nucleic acid sequences encoding the heavy and light chain variable regions were synthesized by GenScript. Amino acid sequence of bevacizumab heavy chain variable region (bevacizumab-Hv): (123 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 1)

[0116] Nucleic acid sequence encoding the bevacizumab heavy chain variable region: (369 bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGA CTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC (SEQ ID NO: 2)

[0117] Amino acid sequence of bevacizumab light chain variable region (bevacizumab-Lv): (107 aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 3)

[0118] Nucleic acid sequence encoding the bevacizumab light chain variable region: (321 bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCA CTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG (SEQ ID NO: 4)

[0119] The heavy chain constant regions used in all cases were the Ig gamma-1 chain C region, accession number: P01857, and the light chain constant regions used in all cases were the Ig kappa chain C region, accession number: P01834.

[0120] The heavy and light chain cDNAs of bevacizumab were cloned into the pcDNA3.1 vector to obtain recombinant expression plasmids of the antibody bevacizumab. The recombinant plasmids were transfected into 293F cells. Detection was performed after purifying the 293F cell culture medium.

[0121] The anti-VEGFA monoclonal antibody Avastin (bevacizumab) was produced.

[0122] Preparation Example 2: Sequence design of anti-PD-1 antibody 14C12, its humanized antibody 14C12H1L1, and mutant 14C12H1L1(M) The amino acid sequences of the heavy and light chains, and the encoding nucleic acid sequences of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are the same as those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication CN106967172A. (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12 Amino acid sequence of the heavy chain variable region of 14C12: (118 aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 5) Nucleic acid sequence encoding the heavy chain variable region of 14C12: (354 bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTAT CCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 6)

[0123] Amino acid sequence of the light chain variable region of 14C12: (107 aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 7)

[0124] Nucleic acid sequence encoding the light chain variable region of 14C12: (321 bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 8)

[0125] (2) Heavy chain variable region sequence and light chain variable region sequence, heavy chain sequence and light chain sequence of humanized monoclonal antibody 14C12H1L1 14C12H1L1 Heavy chain variable region amino acid sequence: (118 aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 9) 14C12H1L1 Nucleic acid sequence encoding the heavy chain variable region: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTAC TATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (Sequence number 10)

[0126] 14C12H1L1 Amino acid sequence of the light chain variable region: (107 aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 11)

[0127] 14C12H1L1 Nucleic acid sequence encoding the light chain variable region: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 12)

[0128] 14C12H1L1 heavy chain (14C12H1) amino acid sequence: (448 aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

[0129] Nucleic acid sequence encoding 14C12H1L1 heavy chain (14C12H1): (1344 bp)

[0130] Amino acid sequence of 14C12H1L1 light chain (14C12L1): (214 aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15)

[0131] Nucleic acid sequence encoding 14C12H1L1 light chain (14C12L1): (642 bp) (SEQ ID NO:16)

[0132] (3) Heavy chain variable region sequence and light chain variable region sequence of 14C12H1L1(M) Based on 14C12H1L1, individual amino acids in the framework region (light chain) were mutated to obtain 14C12H1L1(M). Heavy chain variable region 14C12H1L1(M): The heavy chain variable region of 14C12H1L1 is the same as 14C12H1, i.e., the amino acid sequence is shown in SEQ ID NO:9.

[0133] Light chain variable region 14C12L1(M) of 14C12H1L1(M): (108 aa, mutation sites in the amino acid sequence based on 14C12H1L1 are underlined) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK R (SEQ ID NO:17)

[0134] Production Example 3: Sequence design of bispecific antibodies 1. Sequence Design The structural mode of the bispecific antibody of the present invention belongs to the Morrison mode (IgG-scFv), i.e., an scFv fragment of one IgG antibody is connected to the C-terminus of the two heavy chains of the other antibody, and the main compositional design of the heavy and light chains is as shown in Table 1 below. In addition to the above-mentioned bevacizumab, the VP101 antibody, which has the amino acid sequences of the heavy chain variable region and the light chain variable region of the above-mentioned 14C12H1L1(M) as the scFv fragment portion, is called VP101(M). Compared to 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves its efficacy. [Table 1]

[0135] In Table 1 above: (1) When "V" is written in the lower right corner, it refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. When "V" is not written, the corresponding heavy chain or light chain is the full length including the constant region. For the amino acid sequences of these variable regions or full length and their encoding nucleic acid sequences, please refer to the corresponding sequences described in the above production examples.

[0136] (2) Amino acid sequence of linker 1: GGGGSGGGGSGGGGSG GGGS (SEQ ID NO: 18) Alternatively, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19) can be used as linker 2 in place of linker 1 described above. (3) Bevacizumab-H used Ig gamma-1 chain C region, accession number: P01857, as the heavy chain constant region. (4) Bevacizumab-G4H used Ig gamma-4 chain C region, accession number: P01861.1, as the heavy chain constant region.

[0137] 2. Expression and purification of antibody VP101(M) The heavy chain cDNA sequence and the light chain cDNA sequence of VP101(M) were each cloned into a pUC57simple (provided by GenScript) vector to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively. Plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested with enzymes (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, and the supernatant was concentrated and then applied to a HiTrap MabSelect SuRe column. The protein was eluted in one step using Elution Buffer to recover the target antibody VP101, and the solution was replaced with PBS.

[0138] 3. Detection of antibody VP101(M) The purified samples were mixed with reducing protein electrophoresis test buffer and non-reducing protein electrophoresis test buffer, respectively, and then boiled, followed by SDS-PAGE electrophoresis detection.

[0139] In the present invention, VP101(M) is also referred to as VP101(hG1WT) to distinguish it from the antibody after mutation in Production Example 4. The above-mentioned VP101(M) is the "wild type", and uses Ig gamma-1 chain C region, accession number: P01857, as the heavy chain constant region, and Ig kappa chain C region, accession number: P01834, as the light chain constant region.

[0140] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (453aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20)

[0141] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (1359 bp)

[0142] In the present invention, VP101(G4M) is also referred to as VP101(hG4WT) to distinguish it from the antibody after mutation in Production Example 4. The above-mentioned VP101(G4M) is the "wild type", and Ig gamma-4 chain C region, accession number: P01861.1, was used as the heavy chain constant region, and Ig kappa chain C region, accession number: P01834, was used as the light chain constant region.

[0143] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (450aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 22)

[0144] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (1350bp)

[0145] Production Example 4: Design of amino acid mutations in the non-variable region based on humanized bispecific antibody VP101 (hG1WT) The inventors obtained VP101(hG1DM) by introducing a point mutation (L234A) from leucine to alanine at position 234 of the heavy chain of VP101(hG1WT) obtained in Preparation Example 3, and a point mutation (L235A) from leucine to alanine at position 235.

[0146] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (453 aa, mutation sites are underlined) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24)

[0147] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (1359 bp, mutation sites are underlined) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT(SEQ ID NO:25)

[0148] The light chain amino acid sequences of the immunoglobulin portions of VP101(hG1DM), VP101(hG1WT) and VP101(hG4WT) are the same, and the encoding nucleic acid sequences are also the same.

[0149] Amino acid sequence of the light chain of the immunoglobulin portion of VP101(hG1DM): (214 aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26) Nucleic acid sequence encoding the light chain of the immunoglobulin portion of VP101 (hG1DM): (642 bp) (SEQ ID NO:27) EXAMPLES

[0150] Example 1: Measurement of affinity constants between FcγRI and VP101 (hG1WT) and VP101 (hG1DM) The Fc receptor FcγRI (also known as CD64) binds to the Fc terminus of IgG antibodies and can participate in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic monoclonal antibody to bind to an Fc receptor impacts the safety and efficacy of the antibody.

[0151] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with FcγRI were measured using a Fortebio Octet molecular interaction device, and the ADCC activity of each antibody was evaluated. The experimental method for detecting the affinity constant between the corresponding antibody and FcγRI by the Fortebio Octet molecular interaction device is briefly described as follows: the sample dilution buffer was PBS, 0.02% Tween®-20, 0.1% BSA, pH 7.4. 1 μg / mL FcγRIa was immobilized on the HIS1K sensor, the time was 50 s, the sensor was equilibrated in the buffer for 60 s, CD64 immobilized on the sensor bound to each antibody, the antibody concentration was 3.12-50 nM (2-fold dilution), the time was 120 s, the antibody was dissociated in the buffer, the time was 120 s. The sensor was regenerated with 10 mM glycine at pH 1.5, the time was 5 s, and repeated four times. The detection temperature was 30°C, and the frequency was 0.3 Hz. The data was fitted with a 1:1 model and analyzed to obtain the affinity constant.

[0152] The affinity constant measurement results of FcγRI to VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as the control antibodies nivolumab and bevacizumab, are shown in Table 1 and Figures 1 to 5. [Table 2]

[0153] N / A indicates that the antibody and antigen were not bound or the binding signal was very low, and the results were not analyzed, so no corresponding data was available.

[0154] The results show that VP101(hG1WT) can bind to FcγRI with an affinity constant of 3.95E-09M, VP101(hG4WT) can bind to FcγRI with an affinity constant of 8.52E-09M, bevacizumab can bind to FcγRI with an affinity constant of 3.68E-09M, and nivolumab can bind to FcγRI with an affinity constant of 6.20E-09M, but VP101(hG1DM) does not bind to FcγRI or the binding signal is extremely low, and the results were not analyzed, so no corresponding data was obtained.

[0155] The results show that, except for VP101(hG1DM) which did not bind to FcγRI, the affinities of the other antibodies to FcγRI were similar, and the binding activity of VP101(hG1DM) was effectively eliminated.

[0156] Example 2: Measurement of affinity constants between FcγRIIa_H131 and VP101 (hG1WT) and VP101 (hG1DM) The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) binds to the Fc terminus of IgG antibodies and can mediate ADCC.

[0157] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with FcγRIIa_H131 were measured using a Fortebio Octet molecular interaction device, and the ADCC activity of each antibody was evaluated.

[0158] The experimental method for detecting the affinity constants of VP101 (hG1WT) and VP101 (hG1DM) with FcγRIIa_H131 using the Fortebio Octet molecular interaction device is briefly described as follows: the immobilization diluent was PBS, 0.02% Tween (registered trademark)-20, 0.1% BSA, pH 7.4, and the analyte dilution buffer was PBS, 0.02% Tween (registered trademark)-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor, the immobilization time was 60 s, the sensor was equilibrated in PBS, 0.02% Tween (registered trademark)-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer for 600 s to block the sensor, FcγRIIa_H131 immobilized on the sensor bound to the antibody, the antibody concentration was 12.5-200 nM (2-fold gradient dilution), the time was 60 s, and the antibody was dissociated in the buffer, the time was 60 s. The sensor was regenerated using 10 mM Glycine, pH 1.7 and 10 mM nickel sulfate. The detection temperature was 30°C and the frequency was 0.6 Hz. The data was fitted with a 1:1 model and analyzed to obtain the affinity constant.

[0159] The affinity constant measurement results of FcγRIIa_H131 with VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as the control antibodies nivolumab and bevacizumab, are shown in Table 2 and Figures 6 to 10. [Table 3]

[0160] N / A indicates that the antibody and antigen were not bound or the binding signal was very low, and the results were not analyzed, so no corresponding data was available.

[0161] The results show that VP101(hG1WT) can bind to FcγRIIa_H131 with an affinity constant of 2.28E-08M, VP101(hG4WT) can bind to FcγRIIa_H131 with an affinity constant of 3.68E-08M, bevacizumab can bind to FcγRIIa_H131 with an affinity constant of 6.44E-08M, and VP101(hG1DM) can bind to FcγRIIa_H131 with an affinity constant of 3.57E-08M, but nivolumab does not bind to FcγRIIa_H131 or the binding signal is extremely low, and the results were not analyzed, so no corresponding data was obtained.

[0162] The results reveal that, except for nivolumab, which did not bind to FcγRIIa_H131, all other antibodies bound to FcγRIIa_H131, with the strongest affinities being VP101(hG1WT), VP101(hG1DM), VP101(hG4WT), and bevacizumab.

[0163] Example 3: Measurement of affinity constants between FcγRIIa_R131 and VP101 (hG1WT) and VP101 (hG1DM) The Fc receptor FcγRIIa_R131 (also known as CD32a _R131) binds to the Fc terminus of IgG antibodies and can mediate ADCC.

[0164] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with FcγRIIa_R131 were measured using a Fortebio Octet molecular interaction device, and the ADCC activity of each antibody was evaluated.

[0165] The experimental method for detecting the affinity constant between VP101(hG1WT) and VP101(hG1DM) using the Fortebio Octet molecular interaction device is briefly described as follows: the immobilization diluent was PBS, 0.02% Tween(registered trademark)-20, 0.1% BSA, pH 7.4, and the analyte dilution buffer was PBS, 0.02% Tween(registered trademark)-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_R131 was immobilized on the NTA sensor, the immobilization time was 60 s, the sensor was equilibrated in PBS, 0.02% Tween (registered trademark)-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer for 600 s to block the sensor, FcγRIIa_R131 immobilized on the sensor bound to the antibody, the antibody concentration was 12.5 to 200 nM (2-fold gradient dilution), the time was 60 s, and the antibody was dissociated in the buffer, the time was 60 s. The sensor was regenerated using 10 mM Glycine, pH 1.7 and 10 mM nickel sulfate. The detection temperature was 30°C and the frequency was 0.6 Hz. The data was fitted with a 1:1 model and analyzed to obtain the affinity constant.

[0166] The affinity constant measurement results of FcγRIIa_R131 with VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as the control antibodies nivolumab and bevacizumab, are shown in Table 3 and Figures 11 to 15.

[0167] [Table 4]

[0168] N / A indicates that the antibody and antigen were not bound or the binding signal was very low, and the results were not analyzed, so no corresponding data was available.

[0169] The results showed that VP101(hG1WT) can bind to FcγRIIa_R131 with an affinity constant of 2.42E-08M, VP101(hG4WT) can bind to FcγRIIa_R131 with an affinity constant of 3.57E-08M, bevacizumab can bind to FcγRIIa_R131 with an affinity constant of 5.16E-08M, nivolumab can bind to FcγRIIa_R131 with an affinity constant of 6.93E-08M, and VP101(hG1DM) can bind to FcγRIIa_R131 with an affinity constant of 3.35E-08M.

[0170] The results reveal that all antibodies bind to FcγRIIa_R131, with the strongest affinities being VP101(hG1WT), VP101(hG1DM), VP101(hG4WT), bevacizumab, and nivolumab.

[0171] Example 4: Measurement of affinity constants between FcγRIIb and VP101 (hG1WT) and VP101 (hG1DM) The Fc receptor FcγRIIb (also known as CD32b) binds to the Fc terminus of IgG antibodies and may be involved in regulating immune cell function.

[0172] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with FcγRIIb were measured using a Fortebio Octet molecular interaction device, and the binding ability of VP101(hG1WT) and VP101(hG1DM) with Fc receptors was evaluated. The experimental method for detecting the affinity constants of VP101 (hG1WT) and VP101 (hG1DM) with FcγRIIb using the Fortebio Octet molecular interaction device is briefly described as follows: the immobilization diluent was PBS, 0.02% Tween (registered trademark)-20, 0.1% BSA, pH 7.4, and the analyte dilution buffer was PBS, 0.02% Tween (registered trademark)-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL hFCGR2B-his was immobilized on the NTA sensor, the immobilization time was 60 s, the sensor was equilibrated in PBS, 0.02% Tween (registered trademark)-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer for 600 s to block the sensor, the hFCGR2B-his immobilized on the sensor bound to the antibody, the antibody concentration was 12.5-200 nM (2-fold gradient dilution), the time was 60 s, and the antibody was dissociated in the buffer, the time was 60 s. The sensor was regenerated using 10 mM Glycine, pH 1.7 and 10 mM nickel sulfate. The detection temperature was 30 °C and the frequency was 0.6 Hz. The data was fitted with a 1:1 model and analyzed to obtain the affinity constant.

[0173] Example 5: Measurement of affinity constants between FcγRIIIa_V158 and VP101 (hG1WT) and VP101 (hG1DM) The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) binds to the Fc terminus of IgG antibodies and can mediate the ADCC effect.

[0174] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with FcγRIIIa_V158 were measured using a Fortebio Octet molecular interaction device, and the ADCC activity of each antibody was evaluated.

[0175] The experimental method for detecting the affinity constant between the corresponding antibody and FcγRIIIa_V158 by the Fortebio Octet molecular interaction device is briefly described as follows: the sample dilution buffer was PBS, 0.02% Tween (registered trademark)-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_V158 was immobilized on the HIS1K sensor, the time was 120 s, the sensor was equilibrated in the buffer for 60 s, the hFcGR3A (V158)-his immobilized on the sensor bound to each antibody, the antibody concentration was 31.25-500 nM (2-fold dilution), the time was 60 s, the antibody was dissociated in the buffer, the time was 60 s. The sensor was regenerated with 10 mM glycine at pH 1.5, the time was 5 s, and repeated four times. The detection temperature was 30 °C, and the frequency was 0.3 Hz. The data were analyzed by fitting with a 1:1 model to obtain affinity constants.

[0176] The affinity constant measurement results of FcγRIIIa_V158 with VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as the control antibodies nivolumab and bevacizumab, are shown in Table 4 and Figures 16 to 20.

[0177] [Table 5]

[0178] N / A indicates that the antibody and antigen were not bound or the binding signal was very low, and the results were not analyzed, so no corresponding data was available.

[0179] The results showed that VP101 (hG1WT) could bind to FcγRIIIa_V158 with an affinity constant of 4.35E-08M, VP101 (hG1DM) could bind to FcγRIIIa_V158 with an affinity constant of 1.34E-07M, bevacizumab could bind to FcγRIIIa_V158 with an affinity constant of 2.76E-08M, and nivolumab, VP101 (hG4WT) could not bind to FcγRIIIa_V158 or the binding signal was extremely low, and the results were not analyzed, so no corresponding data was obtained.

[0180] The results show that, except for nivolumab and VP101(hG4WT), which did not bind to FcγRIIIa_V158, all other antibodies bound to FcγRIIIa_V158, with the strongest affinity being bevacizumab, VP101(hG1WT), and VP101(hG1DM).

[0181] Example 6: Measurement of affinity constants between FcγRIIIa_F158 and VP101 (hG1WT) and VP101 (hG1DM) The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) binds to the Fc terminus of IgG antibodies and can mediate ADCC.

[0182] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with FcγRIIIa_F158 were measured using a Fortebio Octet molecular interaction device, and the ADCC activity of each antibody was evaluated.

[0183] The experimental method for detecting the affinity constants of VP101 (hG1WT) and VP101 (hG1DM) with FcγRIIIa_F158 using the Fortebio Octet molecular interaction device is briefly described as follows: the sample dilution buffer was PBS, 0.02% Tween (registered trademark)-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_F158 was immobilized on the HIS1K sensor, the time was 120 s, the sensor was equilibrated in the buffer for 60 s, the hFcGR3A (F158)-his immobilized on the sensor bound to each antibody, the antibody concentration was 31.25 to 500 nM (2-fold dilution), the time was 60 s, the antibody was dissociated in the buffer, the time was 60 s. The sensor was regenerated with 10 mM glycine at pH 1.5, the time was 5 s, and the regeneration was repeated four times. The detection temperature was 30° C. and the frequency was 0.3 Hz. The data were analyzed by fitting with a 1:1 model to obtain the affinity constants.

[0184] The affinity constant measurement results of FcγRIIIa_F158 with VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as the control antibodies nivolumab and bevacizumab, are shown in Table 5 and Figures 21 to 25.

[0185] [Table 6]

[0186] N / A indicates that the antibody and antigen were not bound or the binding signal was very low, and the results were not analyzed, so no corresponding data was available.

[0187] The results show that VP101(hG1WT) can bind to FcγRIIIa_F158 with an affinity constant of 7.41E-08M, and bevacizumab can bind to FcγRIIIa_F158 with an affinity constant of 9.32E-08M, but nivolumab, VP101(hG4WT) and VP101(hG1DM) do not bind to FcγRIIIa_F158 or the binding signals are extremely low, and the results were not analyzed, so no corresponding data was obtained.

[0188] The results show that, except for nivolumab, VP101(hG4WT) and VP101(hG1DM) which did not bind to FcγRIIIa_F158, all other antibodies bound to FcγRIIIa_F158, with the strongest affinity being VP101(hG1WT) followed by bevacizumab.

[0189] Example 7: Measurement of affinity constants between C1q and VP101 (hG1WT) and VP101 (hG1DM) Serum complement C1q can bind to the Fc terminus of IgG antibodies and mediate the CDC effect. The ability of a therapeutic monoclonal antibody to bind C1q impacts the safety and efficacy of the antibody.

[0190] In this experiment, the affinity constants of VP101(hG1WT) and VP101(hG1DM) with C1q were measured using a Fortebio Octet molecular interaction device, and the CDC activity of each antibody was evaluated.

[0191] The experimental method for detecting the affinity constant between the corresponding antibody and C1q by the Fortebio Octet molecular interaction device is briefly described as follows: the sample dilution buffer was PBS, 0.02% Tween (registered trademark)-20, 0.1% BSA, pH 7.4. 50 μg / mL of antibody was immobilized on the FAB2G sensor, the immobilization height was about 2.0 nm, the sensor was equilibrated in the buffer for 60 s, the antibody immobilized on the sensor bound to C1q, the C1q concentration was 0.625 nM to 10 nM (2-fold dilution), the time was 60 s, and the antigen-antibody dissociated in the buffer, the time was 60 s. The sensor was regenerated with 10 mM glycine at pH 1.7, the time was 5 s, and repeated four times. The sample plate vibration speed was 1000 rpm, the detection temperature was 30 degrees, and the detection frequency was 0.6 Hz. The data was fitted and analyzed with a 1:1 model to obtain the affinity constant. The data collection software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0192] The affinity constant measurement results of C1q with VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as the control antibodies nivolumab and bevacizumab, are shown in Table 6 and Figures 26 to 30.

[0193] [Table 7] N / A indicates that the antibody and antigen were not bound or the binding signal was very low, and the results were not analyzed, so no corresponding data was available. The results show that VP101(hG1WT) can bind to C1q with an affinity constant of 9.76E-10M, and bevacizumab can bind to C1q with an affinity constant of 1.14E-09M, but nivolumab, VP101(hG4WT) and VP101(hG1DM) did not bind to C1q or the binding signals were very low, and the results were not analyzed, so no corresponding data was obtained. The results show that, except for nivolumab, VP101(hG4WT) and VP101(hG1DM) which did not bind to FcγRIIIa_F158, all other antibodies bound to C1q and the affinity of VP101(hG1WT) was similar to that of bevacizumab.

[0194] Example 8: Detection of ADCC activity of VP101(hG1WT) and VP101(hG1DM) against CHO-K1-PD1 cells expressing PD-1 antigen To detect the ADCC effect of antibodies VP101(hG1WT) and VP101(hG1DM) at the cellular level, the present inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen, and established a co-culture system of normal human PBMCs and target cells to detect the ADCC activity of the antibodies at the cellular level.

[0195] The specific method for detecting the ADCC activity of VP101(hG1WT) and VP101(hG1DM) against CHO-K1-PD1 cells expressing PD-1 antigen is as follows: The experiment first constructed the human PD-1 overexpression vector pCDH-CMV-PD1FL-Puro (pCDH-CMV-Puro was purchased from Youbao Biology), packaged the expression vector into a virus, and then infected CHO-K1 cells. After screening by administering puromycin (2 μg / mL), a drug-resistant CHO-K1-PD1 stable cell line stably expressing membrane PD-1 protein was obtained. Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell separation solution operating instructions, the isolated PBMCs were resuspended in 1640 complete medium, and the cell count and activity were measured by trypan blue staining, and incubated overnight in an incubator at 37℃, 5% CO2, and saturated humidity. The next day, CHO-K1-PD1 cells and PBMCs were collected respectively, and the supernatant was removed by centrifugation, and the cell precipitate was resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as analysis medium), and the cells were washed twice by centrifugation, and the cell count and activity rate were measured. The cell concentration was adjusted to an appropriate range using analysis medium. According to the test design, the CHO-K1-PD1 cell suspension was added to a 96-well plate at 30000 / well, 50 μL of antibody was added, mixed evenly, and pre-incubated at room temperature for 1 h. After pre-incubation, PBMCs were added at 900,000 / well, mixed thoroughly, and incubated in an incubator at 37℃, 5% CO2 for 4hrs. After 4 hours, the 96-well plate was removed and centrifuged at 250×g for 5 min. 100 μL of cell supernatant (be careful not to aspirate the cells at the bottom of the plate) was transferred to a new 96-well flat-bottom microplate, and 100 μL of the prepared reaction solution was added per well according to the Cytotoxicity Detection Kit instructions, and incubated at room temperature for 30 min in the dark. The OD values ​​were measured at 490 nm and 650 nm, respectively, and the OD value of each group = OD 490 nm -OD 650 nm The ADCC activity of each group was calculated as follows: ADCC (%) = (experimental group - negative control group) / (maximum LDH release from target cells - spontaneous LDH release from target cells) x 100%.

[0196] The results of detecting the ADCC activity of VP101(hG1WT) and VP101(hG1DM) against CHO-K1-PD1 cells expressing the PD-1 antigen are shown in ADCC%. The results are shown in FIG. The results show that in the mixed culture system of PBMC and CHO-K1-PD1, the positive control 14C12H1L1(G1WT) has significant ADCC activity, indicating that the ADCC system is normal. Compared with the isotype control antibody hIgG1DM, VP101(hG1WT) exhibits significant ADCC activity and shows dose-dependency, while VP101(G1DM) shows no ADCC activity. The results show that VP101(hG1DM), which is a mutation based on VP101(hG1WT), has no ADCC activity at the cytological level, and the ADCC effect is eliminated.

[0197] Example 9: Detection of CDC activity of VP101(hG1WT) and VP101(hG1DM) against CHO-K1-PD1 cells expressing PD-1 antigen In order to detect the CDC effect of antibodies VP101(hG1WT) and VP101(hG1DM) at the cellular level, the present inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen (see Example 8 for the construction method), and constructed a system in which target cells were co-cultured with normal human complement serum to detect the CDC activity of the antibodies at the cellular level.

[0198] The specific method for detecting the CDC activity of VP101(hG1WT) and VP101(hG1DM) against CHO-K1-PD1 cells expressing PD-1 antigen is as follows: On the day of detection, CHO-K1-PD1 cells were collected by pancreatic enzyme digestion, centrifuged at 170×g for 5 min, and then resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as analysis medium), and washed twice by centrifugation. The cell number and viability were measured, and the cell concentration was adjusted to an appropriate range using analysis medium. Based on the test design, the CHO-K1-PD1 cell suspension was added to a 96-well plate at 30,000 / well, 50 μL of antibody was added, mixed evenly, and pre-incubated at room temperature for 10 min. After pre-incubation, normal human complement serum (final concentration 2%) was added at 50 μL / well, mixed thoroughly, and incubated in a 37℃, 5% CO2 incubator for 4 hours. After 4 hours, the plates were centrifuged at 250×g for 5 min, and 100 μL of the cell supernatant (be careful not to aspirate the cells at the bottom of the plate) was carefully aspirated and transferred to a new 96-well flat-bottom microplate. 100 μL of the prepared reaction solution was added per well according to the Cytotoxicity Detection Kit instructions, and incubated at room temperature for 30 min in the dark. The OD values ​​were measured at 490 nm and 650 nm, respectively, and the OD value of each group = OD 490 nm -OD 650 nm The CDC activity of each group was calculated as follows: CDC (%) = (experimental group - negative control group) / (maximum LDH release from target cells - spontaneous LDH release from target cells) x 100%.

[0199] The results of detecting the CDC activity of VP101(hG1WT) and VP101(hG1DM) against CHO-K1-PD1 cells expressing the PD-1 antigen are shown in CDC%. The results are shown in FIG.

[0200] The results showed that in the mixed culture system of normal human complement serum and CHO-K1-PD1, the positive control antibody 14C12H1L1(G1WT) had a significant difference in CDC% compared with the isotype control antibody hIgG1DM group, indicating that this CDC detection system is normal. Meanwhile, at the same level, the CDC% of VP101(hG1WT) and VP101(hG1DM) was not significantly different from the isotype control.

[0201] Example 10: Detection of pharmacological activity of VP101 (hG1DM) in a mixed culture system (MLR) of peripheral blood mononuclear cells and Raji-PDL1 cells The experiment first constructed a human PD-L1 overexpression vector plenti6.3-PD-L1-BSD (plenti6.3-BSD was purchased from Invitrogen), packaged the expression vector into viruses, infected Raji cells, and screened by administering BSD (10 μg / mL), and obtained a Raji-PDL1 stable cell line that stably expressed membrane PD-L1 protein. Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation medium operating instructions, and the isolated PBMCs were resuspended in 1640 complete medium, counted, and frozen for storage. The PBMCs were resuscitated and stimulated with SEB (Staphylococcus aureus enterotoxin antigen) for 2 days. After 2 days, log-phase Raji-PDL1 cells were harvested and treated with mitomycin C (Sigma, working concentration 25 μg / mL) in an incubator for 60 min. The Raji-PDL1 cells after mitomycin C treatment were washed by centrifugation. At the same time, PBMCs were harvested and washed 2 days after SEB stimulation, and mixed cultured at a cell number ratio of 1:1 with or without antibody. After 3 days, the supernatant was harvested by centrifugation, and the IL-2 and IFN-γ concentrations in the supernatant were detected by ELISA.

[0202] The IFN-γ secretion results are shown in Figure 33. The results show that VP101(hG1DM) can effectively promote the secretion of IFN-γ, and the activity is significantly superior to that of Nivolumab.

[0203] The IL-2 secretion results are shown in Figure 34. The results show that VP101(hG1DM) can effectively promote the secretion of IL-2, and exhibits a dose-dependent effect, with activity significantly superior to that of nivolumab.

[0204] Example 11: VP101 (hG1DM) does not have antibody-mediated phagocytic activity against PD-1-expressing positive cells Antibody-mediated cellular phagoxytosis (ADCP) is a phenomenon in which the Fc portion of an antibody that binds to a cell surface antigen binds to the Fc receptor of a cell with phagocytic activity, such as a macrophage, and then mediates the phagocyte's phagocytosis of the antibody-bound cell. In the case of immune checkpoint inhibitor antibodies, such as PD-1 antibodies, the presence of ADCP activity causes damage to immune cells expressing PD-1, which exerts anti-tumor killer effects, and affects their anti-tumor activity.

[0205] In this experiment, mouse-derived macrophages were used as effector cells, and CHO-K1-PD1 cell line (see Example 8 for construction method) overexpressing PD-1 was used as target cells to detect its mediated ADCP effect. In this experiment, flow cytometry was used to detect the ADCP activity of VP101 (hG1DM) on PD-1 expressing cells, and the results showed that it had no ADCP activity, but had obvious ADCP activity with the commercially available PD-1 antibody nivolumab as a target. The specific method is as follows:

[0206] First, femoral bone marrow from C57 mice (purchased from Guangdong Medical Laboratory Animal Center) was collected under sterile conditions, lysed on ice with red blood cell lysate for 5 min, lysed with DMEM complete medium (containing 10% FBS) to stop lysis, and centrifuged at 1000 rpm twice. The cell mass was resuspended in 10 mL DMEM complete medium, M-CSF was added to a working concentration of 100 ng / mL, and induced in a cell incubator at 37°C and 5% CO2 for 7 days, during which half the liquid was replaced and M-CSF was replenished on the 3rd and 5th days. On the 7th day, cell induction was completed, digested with 0.25% pancreatic enzymes, macrophages were collected, centrifuged at 750×g for 5 min, the supernatant was removed, suspended in DMEM complete medium (containing 10% FBS), counted, adjusted the cell density, and dispensed into a 96-well cone-bottom plate for storage.

[0207] CHO-K1-PD1 cells were harvested in the usual way, centrifuged at 170xg for 5 min, resuspended, counted, and viability was measured, and washed once with PBS. CFSE was diluted to 2.5 μM in PBS, and an appropriate amount of CFSE-resuspended cells (staining density: 10 million cells / mL) was taken and placed in an incubator for 20 min. Staining was terminated by adding 6 mL of DMEM complete medium (containing 10% FBS), centrifuged at 170xg for 5 min, the supernatant was discarded, 1 mL of DMEM complete medium was added, and the cells were placed in an incubator for 10 min. The antibodies were diluted in DMEM complete medium to 20 μg / mL, 2 μg / mL, 0.2 μg / mL (working concentration 10 μg / mL, 1 μg / mL, 0.1 μg / mL), and the isotype control antibodies hIgG1DM and hIgG4 were designed. Fresh and induced mature macrophages were collected, centrifuged at 750xg for 5 min, the supernatant was discarded, counted, transferred to a 96-well cone bottom plate, centrifuged at 1000xg for 5 min, the supernatant was discarded, the CHO-K1-PD1-CFSE cell density was adjusted, and the antibodies diluted according to the experimental design were added to the 96-well cone bottom plate containing the target cells and the corresponding macrophages in 50 μL:50 μL, respectively, resuspended and mixed, and placed in a 37 °C incubator to incubate for 2 h. Add 150 μL of room temperature 1% PBSA per well, centrifuge at 1000xg for 5 min, discard the supernatant, wash once with 200 μL of PBSA, add 100 μL / sample of APC anti-mouse / human CD11b antibody (diluted 500-fold with PBSA) to the corresponding sample, mix evenly, and incubate on ice for 40 min. Add 150 μL of 1% PBSA per well, centrifuge at 1000xg for 5 min, discard the supernatant, wash once with 200 μL of PBSA per well. Add 200 μL of 1% PBSA per well to resuspend and load into Beckman rheometer.

[0208] Macrophages in the input system were APC+ positive, and macrophages that had undergone phagocytosis were double positive for APC and CFSE. The ratio of the number of double positive cells to the number of APC positive cells was defined as the phagocytosis rate, and antibody-mediated ADCP activity was evaluated. The ADCP activity of each group was calculated using the following formula and expressed as P%:

number

[0209] The results are shown in Figure 35. The results showed that nivolumab had obvious ADCP effect on macrophage + CHO-K1-PD1 system, and the phagocytosis rate of VP101(hG1DM) was comparable to that of the same type control antibody, indicating that VP101(hG1DM) has no ADCP effect. These results indicate that VP101(hG1DM) is likely to have a superior antitumor effect.

[0210] Example 12: Animal experiments of VP101 (hG1DM) monotherapy for the treatment of breast cancer In this experiment, the antitumor activity of the in vivo proliferation of transplanted breast adenocarcinoma cells VP101 (hG1DM) was investigated. The study design is shown in Table 7 below.

[0211] The severely immunodeficient NCG mice used in the study were female, 5 weeks old, and purchased from Jiangsu Jisu Yaokang Biotechnology Co., Ltd. The isotype control antibody hIgG4 used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and the construction method was the same. PBMCs were isolated and activated from Zhongshan Kangfang Biopharmaceutical Co., Ltd., and the method was the same as before.

[0212] The collected breast adenocarcinoma cell line MDA-MB-231 cells were inoculated into a total of 12 mice, 2 million / 50 μL / mouse, breast pad inoculation. After 30 days of growth in the body, the mice were equally divided into two groups according to the tumor volume, a negative control group, and a VP101 (hG1DM) group. On the day of inoculation, 1 million PBMC / 50 μL / mouse were subcutaneously inoculated, and the day of PBMC inoculation was set as day 0, and the administration was performed on the day of inoculation (day 0), 7th day, 14th day, and 21st day. The tumor volume was measured with a caliper, and after classification, the tumor size was measured twice a week using a caliper, and the calculation formula TV = 0.5 × ab2 Tumor volume was calculated according to the formula: where a is the long axis of the tumor, b is the short axis of the tumor, and TV is the volume of the tumor.

[0213] [Table 8] The experimental results are shown in Figure 36. The results show that VP101 (hG1DM) can effectively inhibit the volumetric growth of breast adenocarcinoma cell line MDA-MB-231 cells compared with the isotype control antibody, showing good anti-tumor, especially anti-breast adenocarcinoma, effects.

[0214] Example 13: Study of inhibition of ovarian cancer cells by combined administration of PARPi and VP101 (hG1DM) SNU-251 ovarian cancer cells in the logarithmic growth phase (Shenzhen Haodi Huatuo Biotechnology Co., Ltd., product number: HTX2700C) were taken, and a single-cell suspension was prepared by pancreatic enzyme digestion, counted, and the cell density was adjusted to 2 × 10 5 The cells were inoculated at 1000μg / well into a 6-well plate and cultured at 37°C for 24 h in RPMI 1640 medium (Gibco, product number: 22400-089).

[0215] After the cells had grown to 60%-70%, they were divided into groups for administration, and a control group and drug group were set up and cultured in a 37℃, 5% CO2 incubator for 24 hours. Place the Transwell chamber into a 24-well plate, add 100 μL of RPMI 1640 medium to the upper chamber, place in an incubator to equilibrate for 1 h, digest the cells in the inhibited 6-well plate with pancreatic enzymes, prepare a single-cell suspension in serum-free medium, count, and confirm the cell density at 4 × 10 4 The concentration was adjusted to 100 cells / 100 μL and inoculated into the upper chamber of the chamber, and 600 μL of RPMI 1640 medium containing 10% fetal bovine serum was added to the lower chamber, followed by incubation in a 37°C, 5% CO2 incubator for 24 hours. After the culture was completed, the Transwell chamber was removed, the medium was discarded, and the lower chamber cells were fixed with PBS for 2 min, then the lower chamber cells were fixed with 4% paraformaldehyde for 20 min, and the chamber was washed twice with PBS. The cells in the lower chamber were stained with 500 μL 0.1% crystal violet staining solution for 15 min, and the remaining staining solution was washed with PBS. The cells in the lower chamber were carefully wiped with a cotton swab, and the chamber was placed on a slide to observe the cells that had migrated to the lower chamber in three fields under a microscope and take pictures. The number of cells was calculated using ImageJ software, and the migration degree of the cells was calculated according to the formula:

[0216] The experimental results are shown in Figure 37. The results show that the effect of inhibiting ovarian cancer cell migration was significantly enhanced when single-agent Olaparib or single-agent VP101(hG1DM) was used at the same concentration, and when PARP inhibitor Olaparib was used in combination with VP101(hG1DM).

[0217] Example 14: Animal experiments of combined administration of PARPi and VP101 (hG1DM) for the treatment of breast cancer To measure the in vivo tumor inhibitory activity of the combination of the PARP inhibitor Olaparib and the anti-PD-1-anti-VEGFA dual-function antibody VP101 (hG1DM), MDA-MB-231 cells (human breast cancer cells, purchased from ATCC) were first used to subcutaneously inoculate 5-7 week-old NCG mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.), and on the 23rd day after inoculation, the mice were randomly divided into 4 groups according to the tumor volume, with 6 mice in each group. The day of grouping was designated as D0, and the administration of Olaparib was started on the day of grouping, D0. On D2, 4 million activated PBMCs were intraperitoneally injected, and the administration of VP101 (hG1DM) was started. The administration method for the combination administration group was to prepare the drugs separately and administer them before and after (there is no specific requirement for the administration order and interval time, and one drug is administered after another drug). The model creation and specific administration method are shown in Table 8. After administration, the length and width of the tumor in each group were measured, and the tumor volume was calculated.

[0218] [Table 9]

[0219] The results are shown in Figure 38. The results showed that compared with the Olaparib monotherapy group and the VP101 (hG1DM) monotherapy group, the combination of VP101 (hG1DM) and the PARP inhibitor Olaparib showed combined antitumor efficacy in a mouse mammary gland cancer model, and the inhibition of tumors by the combination was superior to that of each test drug monotherapy group. Furthermore, as shown in Figure 39, the test drug VP101 (hG1DM) and the PARP inhibitor Olaparib were well tolerated when used alone or in combination, and each group had no effect on the body weight of tumor-bearing mice.

[0220] Example 15: Animal experiments of combined administration of PARPi and VP101 (hG1DM) for the treatment of ovarian cancer To measure the in vivo tumor inhibitory activity of the combination of PARP inhibitor Olaparib and anti-PD-1-anti-VEGFA dual-function antibody VP101 (hG1DM), SK-OV-3 human ovarian cancer cells (purchased from ATCC) were first used to subcutaneously inoculate 5-7 week-old NCG mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.), and 3 million activated PBMCs were injected intraperitoneally on the 39th day after inoculation. The mice were randomly divided into 4 groups according to tumor volume, with 6 mice in each group. The day of grouping was designated as D0, and administration began on the day of grouping, D0. The administration method for the combination administration group was to prepare the drugs separately and administer them before and after (there is no specific requirement for the administration order and interval time, and one drug is administered after another drug). The model creation and specific administration method are shown in Table 9. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0221] [Table 10]

[0222] The results are shown in Figure 40. The results show that compared with the Olaparib monotherapy group and the VP101 (hG1DM) monotherapy group, the combination of VP101 (hG1DM) and the PARP inhibitor Olaparib showed combined antitumor efficacy in a mouse ovarian cancer model, and the inhibition of tumors by the combination was superior to that of each test drug monotherapy group. Furthermore, as shown in FIG. 41, the test drug VP101 (hG1DM) and the PARP inhibitor olaparib were well tolerated when used alone or in combination, and each group did not affect the body weight of tumor-bearing mice.

[0223] Although the specific embodiments of the present invention are described in detail, it should be understood by those skilled in the art. According to all the teachings disclosed, modifications and substitutions to these details are possible, and these modifications are within the scope of protection of the present invention. The full scope of the present invention is defined by the appended claims and any equivalents thereof.

Claims

1. A pharmaceutical composition for the treatment and / or prevention of a malignant tumor in a subject, comprising at least one bispecific antibody administered in combination with at least one PARP inhibitor, The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin comprises a heavy chain constant region having mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and wherein after the mutations the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced compared to before the mutations.

2. A pharmaceutical product comprising an effective amount of a bispecific antibody for the treatment and / or prevention of a malignant tumor in a subject who has previously been administered an effective amount of a PAPR inhibitor, The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin comprises a heavy chain constant region having mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and wherein after the mutations the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced compared to before the mutations.

3. A pharmaceutical product comprising an effective amount of a PARP inhibitor for the treatment and / or prevention of malignant tumors in a subject who has been administered an effective amount of a bispecific antibody, The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin comprises a heavy chain constant region having mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and wherein after the mutations the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced compared to before the mutations.

4. The heavy chain constant region of said immunoglobulin comprises the following mutations according to the EU numbering system: L234A and L235A, or L234A and G237A, or L235A and G237A, or The pharmaceutical agent according to any one of claims 1 to 3, having L234A, L235A, and G237A.

5. A pharmaceutical composition for the treatment and / or prevention of a malignant tumor in a subject, comprising at least one bispecific antibody administered in combination with at least one PARP inhibitor, The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin has the following mutations according to the EU numbering system: L234A and L235A, or L234A and G237A, or L235A and G237A, or a heavy chain constant region having L234A, L235A and G237A, Pharmaceuticals.

6. A pharmaceutical product comprising an effective amount of a bispecific antibody for the treatment and / or prevention of a malignant tumor in a subject who has been administered an effective amount of a PAPR inhibitor, The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin has the following mutations according to the EU numbering system: L234A and L235A, or L234A and G237A, or L235A and G237A, or a heavy chain constant region having L234A, L235A and G237A, Pharmaceuticals.

7. A pharmaceutical product comprising an effective amount of a PARP inhibitor for the treatment and / or prevention of malignant tumors in a subject who has been administered an effective amount of a bispecific antibody, The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin has the following mutations according to the EU numbering system: L234A and L235A, or L234A and G237A, or L235A and G237A, or a heavy chain constant region having L234A, L235A and G237A, Pharmaceuticals.

8. The heavy chain constant region of the immunoglobulin is The pharmaceutical agent according to any one of claims 1 to 7, having one or more mutations selected from the group consisting of N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A.

9. The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region of the single chain antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9, and the light chain variable region of the single chain antibody has an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:17, Or, The heavy chain variable region of the immunoglobulin has an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9, and the light chain variable region of the immunoglobulin has an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:17, and the heavy chain variable region of the single chain antibody has an amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the single chain antibody has an amino acid sequence shown in SEQ ID NO:

3. A pharmaceutical product according to any one of claims 1 to 8. (1) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:7, (2) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 11, (3) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 17, (4) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:9, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:7; (5) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 11, (6) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 17, (7) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:3; (8) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:11, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:3; (9) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO:17, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO:3; (10) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 3; (11) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 11, and the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 3, or (12) The pharmaceutical product according to any one of claims 1 to 9, wherein the heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 9 and the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 17, and the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO:

3.

11. 11. The pharmaceutical product of any one of claims 1 to 10, wherein the immunoglobulin comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 24 and a light chain having the amino acid sequence shown in SEQ ID NO:

26.

12. The pharmaceutical product of any one of claims 1 to 10, wherein the immunoglobulin comprises a heavy chain constant region having the mutations L234A and L235A.

13. The bispecific antibody comprising: A heavy chain comprising HCDR1-HCDR9 having the amino acid sequences set forth in SEQ ID NOs: 28, 29, 30, 34, 35, 36, 37, 38 and 39, respectively; and a light chain comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; The pharmaceutical product according to any one of claims 1 to 12, comprising:

14. 14. The pharmaceutical product of any one of claims 1 to 13, wherein the first protein functional region is connected to the second protein functional region either directly or via a connecting fragment, and / or the heavy chain variable region of the single chain antibody is connected to the light chain variable region of the single chain antibody either directly or via a connecting fragment.

15. 15. The method of claim 14, wherein the connecting fragment is (GGGGS)n, where n is a positive integer, preferably where n is 1, 2, 3, 4, 5 or 6.

16. The pharmaceutical product according to any one of claims 1 to 15, wherein the number of the first protein functional region and the second protein functional region is independently two or more than one.

17. The single chain antibody is attached to the C-terminus of an immunoglobulin heavy chain. A pharmaceutical product according to any one of claims 1 to 16.

18. A pharmaceutical product described in any one of claims 1 to 10 and 12 to 17, wherein the immunoglobulin comprises a light chain constant region including a human Ig κ chain C region, preferably, the light chain constant region of the immunoglobulin is a human Ig κ chain C region, accession number: P01834.

19. The number of the first protein functional regions is two, and the number of the second protein functional regions is one, wherein the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; the immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:24 and a light chain having the amino acid sequence set forth in SEQ ID NO:26; the single chain antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:9 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:17; the single chain antibody is attached to the C-terminus of an immunoglobulin heavy chain; The first protein functional region is connected to the second protein functional region via a first connecting fragment, and the heavy chain variable region of the single chain antibody is connected to the light chain variable region of the single chain antibody via a second connecting fragment, and the first connecting fragment and the second connecting fragment are homologous or different. A pharmaceutical product according to any one of claims 1 to 18.

20. The pharmaceutical described in claim 19, wherein the first connected fragment and the second connected fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19, and preferably, the amino acid sequences of the first connected fragment and the second connected fragment are shown in SEQ ID NO:

18.

21. The PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, fluzoparib, veliparib ER, ABT-472, ABT-767, stenoparib, AST-6828, AG-PD, ANG-2864, ANG-3038, ANG-3186, AZD-5305, AZ-0108, AZD-2461, AMXI-5001, AMXI-2001, AMXI-3001, AMXI-7001, AMXI-9001, pamiparib, ZYTP-1, CK-102, XZ-120312, YHP-743, and iobenguane I. 131, rucaparib cansylate, CVL-218, CPH-101, CPH-102, CBX-11, CBX-15, minocycline, DB-207, DPS-102, E-7016, iobenguane I 131, MK-2512, HCX-014, HWH-340, IDX-1197, IDX-1197, senaparib, IMP-04100, IMP-04111, IMP-04149, IMP-04249, IMP-04307, IMP-04356, JPI-289, JPI-547, JPI-283, fluzoparib, GT-1620, iobenguane I 131, DR-2313, MP-124, H-10, NT-125, BGP-15, NMSP-293, NMSP-118, NMSP-648, NMSP-914, DB-207, NUV-1156, NUV-1176, JPI-289, Stenoparib, OX-401, NU-1025, NU-1085, PLX-376, one or more selected from the group consisting of R-554, RBN-2397, RBN-012759, PJ-34, INO-1001, WW-46, BSI-401, iniparib, SOMCL-9112, SC-10914, HTMC-0435, SRX-3128, TSL-1502, PJ-34, CEP-8983, CK-102, THG-009, talazoparib SR, L-2286, mitoparib and WB-1340; A pharmaceutical product according to any one of claims 1 to 20.

22. The pharmaceutical product further comprises one or more tumor chemotherapeutic agents, and / or the subject has previously been administered one or more tumor chemotherapeutic agents; Preferably, the tumor chemotherapeutic agent is selected from topoisomerase II (TOP2) inhibitors, preferably, the topoisomerase II (TOP2) inhibitor is etoposide; Preferably, the tumor chemotherapy drug is selected from paclitaxel-based drugs, and preferably, the paclitaxel-based drugs are selected from the group consisting of paclitaxel, albuminic paclitaxel, liposomal paclitaxel and docetaxel; Preferably, the tumor chemotherapy drug is selected from platinum-based drugs, preferably, the platinum-based drugs are selected from the group consisting of cisplatin, carboplatin and oxaliplatin; Preferably, the tumor chemotherapeutic agent is one or more selected from the group consisting of gemcitabine, pemetrexed and capetabine; A pharmaceutical product according to any one of claims 1 to 21.

23. The pharmaceutical product is a fixed combination, for example in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition, or The pharmaceutical product is a non-fixed combination, for example, the bispecific antibody and the PARP inhibitor are each in the form of a pharmaceutical composition. A pharmaceutical product according to any one of claims 1 to 22.

24. 24. The pharmaceutical product of claim 23, wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable vector and / or excipient.

25. A kit comprising a pharmaceutical agent according to any one of claims 1 to 24 and product instructions.

26. The pharmaceutical product according to any one of claims 1 to 24 or the kit according to claim 25, wherein the malignant tumor is selected from the group consisting of ovarian cancer, endometrial cancer, breast cancer, cervical cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, liver cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, brain cancer, esophageal cancer, for example, esophageal squamous cell carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer, urothelial carcinoma, mesothelioma, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, leukemia, myeloma, thyroid cancer, head and neck cancer, bone cancer, biliary tract cancer, and testicular cancer.

27. ​​The malignant tumor is a tumor having a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), The malignant tumor is a tumor in which there is no homologous recombination-mediated repair function deficiency, The lung cancer is non-small cell lung cancer or small cell lung cancer, The non-small cell lung cancer is non-small cell lung cancer with EGFR and / or ALK susceptibility mutation, the non-small cell lung cancer is non-small cell lung cancer not associated with EGFR and / or ALK susceptibility mutations; the liver cancer is hepatocellular carcinoma, the renal tumor is renal cell carcinoma; the breast adenocarcinoma is triple-negative breast adenocarcinoma; the urothelial cancer is bladder cancer; the peritoneal cancer is primary peritoneal cancer, The ovarian cancer is an ovarian cancer having a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), The fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), the peritoneal cancer is a peritoneal cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; or The breast cancer is a breast cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation).

27. The pharmaceutical or kit of claim 26.

28. The pharmaceutical agent or method according to any one of claims 1 to 24, 26 and 27, wherein the pharmaceutical agent is administered to a subject before or after surgery and / or before or after radiation therapy. The kit according to any one of claims 25 to 27.

29. The bispecific antibody, for administration in a single dose of 0.1 to 100 mg per kilogram of body weight, preferably 5 to 50 mg or 5 to 15 mg per kilogram of body weight; for administration once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks; and / or A pharmaceutical product according to any one of claims 1 to 24 and 26 to 28 or a kit according to any one of claims 25 to 28, for intravenous infusion or injection.

30. The PARP inhibitor, for administration in a single dose of 0.1 to 100 mg per kilogram of body weight, preferably 5 to 50 mg or 5 to 15 mg per kilogram of body weight; for administration once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks; and / or A pharmaceutical product according to any one of claims 1 to 24 and 26 to 29 or a kit according to any one of claims 25 to 29, for intravenous infusion or injection.

31. Use of a bispecific antibody in the manufacture of a pharmaceutical for the treatment and / or prevention of a malignant tumor selected from the group consisting of ovarian cancer, fallopian tube cancer, peritoneal cancer and breast cancer, comprising: The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Where: the first protein functional region is an immunoglobulin, the immunoglobulin comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; and the second protein functional region is a single chain antibody, the single chain antibody comprising a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; Or, the first protein functional region is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 34 to 36, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 37 to 39, respectively; the second protein functional region is an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively, and a light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the immunoglobulin comprises a heavy chain constant region having mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and wherein after the mutations, the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q is reduced compared to before the mutations.

32. The ovarian cancer is an ovarian cancer having a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), The fallopian tube cancer is a fallopian tube cancer having a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation), the peritoneal cancer is a peritoneal cancer in which a homologous recombination-mediated repair deficiency (e.g., a BRCA1 mutation and / or a BRCA2 mutation) is present; or The breast cancer is a breast cancer having a homologous recombination-mediated repair deficiency (e.g., BRCA1 mutation and / or BRCA2 mutation).

32. The use according to claim 31.

Images