Anti-PD-1-anti-VEGFA bispecific antibodies, pharmaceutical compositions and uses thereof

Abstract

Anti-VEGFA-anti-PD-1 bispecific antibodies and uses thereof are provided. Specifically, the anti-VEGFA-anti-PD-1 bispecific antibodies comprise a first protein functional domain targeting PD-1 and a second protein functional domain targeting VEGFA. Mutations are made at positions 234 and 235 of the immunoglobulin heavy chain constant region contained in the bispecific antibodies according to the EU numbering system, and the affinity constants of the bispecific antibodies with FcγRI, FcγRIIa, FcγRIIIa, and / or C1q are reduced after the mutations compared to the affinity constants before the mutations. The bispecific antibodies specifically bind to VEGFA and PD-1, specifically alleviate immunosuppression by VEGFA and PD-1 in vivo, and can inhibit tumor-induced angiogenesis, thereby having good potential applications.

Description

[Technical field]

[0001] Technical Field The present invention belongs to the field of tumor therapy and immunobiology, and relates to an anti-PD-1-anti-VEGFA bispecific antibody, its pharmaceutical composition and its use. In particular, the present invention relates to an anti-human PD-1-anti-human VEGFA bispecific antibody, its pharmaceutical composition and its use. [Background technology]

[0002] background Tumors, especially malignant tumors, are serious health-threatening diseases in today's world and the second leading cause of death among various diseases. In recent years, their incidence has increased significantly. Malignant tumors are characterized by poor response to treatment, high rate of late metastasis, and poor prognosis. Although conventional treatment methods currently used clinically (e.g., radiation therapy, chemotherapy, and surgical treatment) can significantly relieve pain and extend survival time, these methods have significant limitations and it is difficult to further improve their effectiveness.

[0003] There are two distinct phases of tumor growth: a slow growth phase without the involvement of blood vessels and a rapid growth phase with the involvement of blood vessels. Angiogenesis allows the tumor to obtain sufficient nutrients to complete the vascular switch phase, and without angiogenesis, the primary tumor would be less than 1-2 mm, and therefore metastasis would not occur.

[0004] Vascular endothelial growth factor (VEGF) is a growth factor that can promote the division and proliferation of endothelial cells, promote the formation of new blood vessels, and improve vascular permeability, and it plays a role by binding to vascular endothelial growth factor receptors on the cell surface and activating tyrosine kinase signaling pathways. In tumor tissues, tumor cells, as well as macrophages and mast cells that infiltrate into tumors, 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 continuous growth of tumors, enhances vascular permeability, causes fibrin deposition in the surrounding tissue, and promotes the infiltration of mononuclear cells, fibroblasts, and endothelial cells, which can facilitate the formation of tumor stroma and the invasion of tumor cells into new blood vessels, and also promote tumor metastasis. Therefore, inhibiting 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 receptors are similar in structure, belong to the tyrosine kinase superfamily, and consist of an extramembrane region, a transmembrane segment, and an intramembrane region, where the extramembrane region is composed of an immunoglobulin-like domain, and the intramembrane region is a tyrosine kinase region. VEGFR1 and VEGFR2 are located primarily on the surface of vascular endothelial cells, and VEGFR3 is located primarily on the surface of lymphatic endothelial cells.

[0005] The molecules of the VEGF family have different affinities to these receptors. VEGFA mainly acts with VEGFR1, VEGFR2 and NRP-1. VEGFR1 is the earliest discovered receptor and has a higher affinity for pVEGFA than VEGFR2 under normal physiological conditions, but the tyrosinase activity in the intracellular segment is lower than that of VEGFR2 (Ma Li, Chinese Journal of Birth Health and Heredity, 2016, Vol. 24 (No. 5): pp. 146-148).

[0006] VEGFR2 is a major regulator of angiogenesis and vascular engineering, and has a much higher tyrosine kinase activity than VEGFR1. After binding to the ligand VEGFA, VEGFR2 mediates the proliferation, differentiation, etc. of vascular endothelial cells, as well as the process of blood vessel formation and blood vessel permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 2007, 62(3):179-213). After binding to VEGFR2, VEGFA mediates the transcriptional expression of related protein genes in cells via the downstream PLC-γ-PKC-Raf-MEK-MAPK signal pathway, thereby promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 1999, 18(13):2221-2230).

[0007] VEGFR3 is one of the tyrosine kinase family members, mainly expressed in embryonic vascular endothelial cells and adult lymphatic endothelial cells, and 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, and can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor (Ma Li, Chinese Journal of Birth Health and Heredity, 2016, Vol. 24(No. 5): pp. 146-148).

[0008] VEGFA and VEGFR2 are mainly involved in regulating angiogenesis before and after VEGFA binds to VEGFR2, forming a cascade reaction of multiple intermediate signals in the upstream and downstream pathways, and finally changing the physiological functions of endothelial cells, such as proliferation, survival, migration, increased permeability and invasion into peripheral tissues (Dong Hongchao et al., Journal of Modern Oncology, September 2014, Vol. 22(9):2231-3).

[0009] Currently, there are several humanized monoclonal antibodies targeting human VEGF, especially VEGFA, such as bevacizumab, which has been approved by the US Food and Drug Administration in 2004 for the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, cervical cancer and metastatic colorectal cancer.

[0010] Programmed cell death receptor-1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein membrane surface receptor that belongs to the CD28 immunoglobulin superfamily and is generally expressed on T cells, B cells and myeloid 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 various cells, including non-hematopoietic cells and various tumor cells. PD-L1 is mainly expressed on T cells, B cells, DCs and microvascular endothelial cells, as well as various tumor cells, while PD-L2 is expressed only on antigen-presenting cells, such as dendritic cells and macrophages. The interaction between PD-1 and its ligands can suppress lymphatic activation, T cell proliferation, and secretion of cytokines, such as IL-2 and IFN-γ.

[0011] Many studies have shown that the tumor microenvironment can protect tumor cells from damage by immune cells, that the expression of PD-1 is upregulated in lymphocytes infiltrating the tumor microenvironment, and that various primary tumor tissues, such as lung cancer, liver cancer, ovarian cancer, skin cancer, colon cancer and glioma, are PD-L1 positive in immunohistochemical analysis. At the same time, the expression of PD-L1 in tumors is significantly associated with poor prognosis of cancer patients. Blocking the interaction between PD-1 and its ligands can promote tumor-specific T cell immunity and enhance the efficiency of immune elimination of tumor cells. Numerous clinical trials have shown that antibodies targeting PD-1 or PD-L1 can inhibit the expression of CD8 + It has been shown that it can promote the infiltration of T cells into tumor tissues and upregulate anti-tumor immune effector factors, such as IL-2, IFN-γ, granzyme B and perforin, thereby effectively suppressing tumor growth.

[0012] In addition, anti-PD-1 antibodies can also be used in the treatment of chronic viral infections, which are often accompanied by the loss of function and a decrease in the number of virus-specific effector T cells. The interaction between PD-1 and PD-L1 can be blocked by injecting PD-1 antibodies, thereby effectively suppressing the exhaustion of effector T cells in chronic viral infections.

[0013] Due to the broad anti-tumor potential and remarkable efficacy of PD-1 antibodies, it is widely accepted in the industry that antibodies targeting the PD-1 pathway will bring about breakthroughs in the treatment of various tumors: non-small cell lung cancer, renal cell carcinoma, ovarian cancer, and melanoma (Homet MB, Parisi G. et al. Anti-PD-1 Therapy in Melanoma. Semin Oncol. 2015 June;42(3):466-473), as well as in lymphoma and anemia (Held SA, Heine A. et al. Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets 2013 September;13(7):768-74), microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) tumors (several anti-PD-1 antibody drugs have been approved by the FDA for the treatment of tumors with MSI-H / dMMR characteristics).

[0014] The current combination of antiangiogenic therapy and immune checkpoint inhibitors shows good efficacy in many tumors, such as ovarian cancer (Joyce F. Liu et al., JAMA Oncol., 2019;5(12):1731-1738), non-small cell lung cancer (NSCLC, including EGFR and / or ALK-sensitive mutations) (Manegold C, et al., J Thorac Oncol., 2017 February;12(2):194-207), renal cell carcinoma (Dudek AZ, et al., J Clin Oncol., 2018;36(Suppl.;Abstract, 4558)), hepatocellular carcinoma (Stein S, et al., J Clin Oncol, 2018, 36(15_Suppl):4074; bevacizumab in combination with atezolizumab approved by the FDA in 2020 for use in the treatment of hepatocellular carcinoma, colorectal cancer (including MSI-H / dMMR and non-MSI-H / dMMR subtypes) (Bendell JC et al., Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) and / or FOLFOX in patients (pts) with metastatic colorectal cancer (mCRC).American Society of Clinical Oncology;May 29-June 2, 2015;Chicago, IL. 2015;presented in Abstract 704;Hochster HS et al., Efficacy and safety of atezolizumab (atezo) and bevacizumab (bev) in a phase Ib study of microsatellite instability (MSI)-high metastatic colorectal cancer (mCRC).American Society of Clinical Oncology Gastrointestinal Cancers Symposium; January 19-21, 2017; San Francisco, CA.2017; Abstract p. 673), breast cancer (Yukinori Ozaki et al., A multicenter phase II study evaluating the efficacy of nivolumab plus paclitaxel plus bevacizumab triple-combination therapy as a first-line treatment in patients with HER2-negative metastatic breast cancer: WJOG9917B NEWBEAT trial [abstract]. In proceedings of the 2019 San Antonio Breast Cancer Symposium; December 10-14, 2019; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020; Vol. 80(4 Suppl): Abstract nr A combination of an anti-VEGF antibody (e.g., bevacizumab) and a PD-1 / PD-L1 antibody (e.g., nivolumab, pembrolizumab, and atezolizumab) for use in the treatment of endometrial cancer (PD1-03); a combination of a VEGFR2 antibody and a PD-1 antibody also showed good antitumor effects in adenocarcinoma of the stomach and esophagogastric junction (Herbst RS et al., Lancet Oncol. 2019;20(8):1109-1123); and a combination regimen of a PD-1 antibody (pembrolizumab) and an angiogenesis inhibitor (lenvatinib) showed good effects in the treatment of endometrial cancer and was approved by the FDA in 2019 for use in the treatment of endometrial cancer. Melanoma (PD-1 antibodies nivolumab and pembrolizumab are approved by the FDA for use in 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; nivolumab approved by FDA in 2017 for use in treating bladder cancer), 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), etc., PD-1 / PDL-1 antibodies have shown good efficacy, and considering that the PD-1 pathway has a synergistic effect with the VEGF pathway in tumor development, agents that block both the PD-1 and VEGF pathways can be expected to have good antitumor effects.

[0015] Bispecific antibodies, also known as bispecific antibodies, are specific drugs that simultaneously target two different antigens and can be produced by immunoselective purification. In addition, bispecific antibodies can also be produced by genetic engineering, which has certain advantages due to the corresponding flexibility in terms of optimization of binding sites, consideration of synthesis type and yield. Currently, bispecific antibodies have been shown to exist in more than 45 types (Muller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 2010;24:89-98). Many bispecific antibodies have been developed in the IgG-ScFv format, i.e., the Morrison format (Coloma MJ, Morrison SL Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997;15:159-163), which has been demonstrated to be one of the ideal formats of bispecific antibodies due to its similarity to the naturally occurring IgG format and advantages in antibody engineering, expression and purification (Miller BR, Demarest SJ et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010;23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011;3:299-309).

[0016] ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) refers to the killing of target cells by killer cells (NK cells, macrophages, etc.) mediated by the binding of the Fab fragment of an antibody to an epitope on a virus-infected or tumor cell and the binding of the Fc fragment of the antibody to an Fc receptor (FcR) on the surface of the killer cell.

[0017] CDC (complement-dependent cytotoxicity) means that the specific binding of an antibody to an antigen on the corresponding cell membrane surface forms a complex and activates the complement system, which further forms MAC on the surface of the target cell, leading to the subsequent lysis of the target cell. Complement can cause the lysis of various bacteria and other pathogens, and is an important defense mechanism against pathogen infection.

[0018] Fc receptor belongs to the immunoglobulin family, which is expressed on the surface of specific immune cells to recognize the Fc region of the antibody and mediate immune response. After the Fab region recognizes the antigen, the Fc region of the antibody binds to the Fc receptor on the immune cell (e.g., killer cell) to initiate the response function of the immune cell, such as phagocytosis and ADCC.

[0019] According to the type of antibody recognized by the Fc receptor and the type of expressing cell, Fc receptors are mainly classified into three types, FcγR, FcαR and FcεR. FcγR can be further classified into four subtypes, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcRn (neonatal Fc receptor). Among these, FcγRI, FcγRII and FcγRIII are closely related to ADCC effect. FcγRIII is the most predominant molecule mediating ADCC and has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. In the FcγRIIIa population, there are two subtypes distinguished by the site of single nucleotide polymorphism (SNP), FcγRIIIa_V158 with high affinity and FcγRIIIa_F158 with low affinity. FcγRI has higher affinity for the Fc region of IgG and is involved in the ADCC process; FcγRII includes three subtypes, FcγRIIa, FcγRIIb and FcγRIIc (also called CD32a, CD32b and CD32c, respectively), among which FcγRIIa has ADCC activity; for FcγRIIa, there are two subtypes, FcγRIIa_H131 and FcγRIIa_R131, due to a single nucleotide mutation in humans (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, Vol. 11 (No. 4): pp. 311-331).

[0020] The IgG family includes four members, IgG1, IgG2, IgG3 and IgG4, which differ in amino acids in the fragment crystallizable (Fc) region of the heavy chain constant region, resulting in their different affinities for FcγR. IgG1 is the most abundant subtype in humans and is also the most common subtype used in monoclonal antibody medicine. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. IgG2 has the lowest affinity for FcγRs, but can further induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 is characterized by the highest binding ability to FcγRs and can induce ADCC and higher CDC effects than IgG1. IgG4 molecules bind weakly to FcγRs other than FcγRI, and IgG4 molecules have a lower possibility of causing CDC and NK cell-mediated ADCC. Summary of the Invention [Problem to be solved by the invention]

[0021] Currently, there remains a need to develop novel anti-PD-1-anti-VEGFA bispecific antibodies that reduce or eliminate the damage caused by antibody-mediated ADCC and / or CDC activity against immune cells to which the anti-PD-1-anti-VEGFA bispecific antibodies bind, and improve the efficacy of the antibody agents. [Means for solving the problem]

[0022] overview Through intensive research and inventive efforts, the present inventors accordingly modified the Fc fragment of the anti-PD-1-anti-VEGFA antibody structure to reduce the binding ability of the Fc region to Fc receptors, thereby reducing the toxicity and side effects of ADCC and CDC in immune cells and increasing the drug efficacy of the anti-PD-1-anti-VEGFA antibody drug.

[0023] The present invention is described in detail below. One aspect of the present invention is a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: 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; For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; or For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype; the heavy chain constant region of the immunoglobulin has mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and the affinity constant of the bispecific antibody to FcγRIIIa and / or C1q is reduced after the mutations compared to the affinity constant before the mutations; preferably, the affinity constant is measured by the Fortebio Octet system; Concerning bispecific antibodies.

[0024] In one or more embodiments of the invention, for bispecific antibodies, the immunoglobulin heavy chain constant region comprises the following mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A has.

[0025] In the present invention, unless otherwise specified, the letter before the position number represents the amino acid before the mutation, and the letter after the position number represents the amino acid after the mutation.

[0026] The present invention relates to a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: 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; For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; or For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype, Further related to bispecific antibodies.

[0027] The present invention relates to a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: 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; For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; or For immunoglobulins, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; for single-chain antibodies, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype; According to the EU numbering system, the immunoglobulin heavy chain constant region is: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A having Further related to bispecific antibodies.

[0028] In one or more embodiments of the invention, for the bispecific antibody, the heavy chain constant region of the immunoglobulin is, according to the EU numbering system, 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 The nucleic acid sequence of the present invention may have, or further have, one or more mutations selected from:

[0029] In one or more embodiments of the present invention, the amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:1, the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:3; the amino acid sequence of the single chain antibody heavy chain variable region is selected from SEQ ID NO:5 and SEQ ID NO:9, and the amino acid sequence of the single chain antibody light chain variable region is selected from SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:17; or the amino acid sequence of the immunoglobulin light chain variable region is selected from SEQ ID NO:5 and SEQ ID NO:9, and the amino acid sequence of the immunoglobulin light chain variable region is selected from SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:17; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:1, and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:3; Bispecific antibodies are provided.

[0030] In one or more embodiments of the present invention, the bispecific antibody has any one of the following structures (1) to (12): (1) the amino acid sequence of an immunoglobulin heavy chain variable region is set forth in SEQ ID NO:1, the amino acid sequence of an immunoglobulin light chain variable region is set forth in SEQ ID NO:3; the amino acid sequence of a single chain antibody heavy chain variable region is set forth in SEQ ID NO:5, and the amino acid sequence of a single chain antibody light chain variable region is set forth in SEQ ID NO:7; (2) the amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:3; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:5 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:11; (3) the amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:3; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:5 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:17; (4) the amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:3; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:9 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:7; (5) the amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:3; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:9 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:11; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:17; (7) The amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:5 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:7; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:3; (8) The amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:5 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:11; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:3; (9) The amino acid sequence of the immunoglobulin heavy chain variable region is set forth in SEQ ID NO:5 and the amino acid sequence of the immunoglobulin light chain variable region is set forth in SEQ ID NO:17; the amino acid sequence of the single chain antibody heavy chain variable region is set forth in SEQ ID NO:1 and the amino acid sequence of the single chain antibody light chain variable region is set forth in SEQ ID NO:3; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:11; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3. is selected from one of the following:

[0031] In one or more embodiments of the present invention, The amino acid sequence of the immunoglobulin heavy chain is set forth in SEQ ID NO: 24, and the amino acid sequence of the immunoglobulin light chain is set forth in SEQ ID NO: 26. Bispecific antibodies are provided.

[0032] In one or more embodiments of the invention, the bispecific antibody is in the form of an IgG-scFv, ie, the Morrison format.

[0033] In some embodiments of the invention, for a bispecific antibody in one or more embodiments of the invention: The heavy chain constant region of the immunoglobulin is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region of the immunoglobulin is a human Ig kappa chain C region.

[0034] In some embodiments of the invention, the immunoglobulin constant region is humanized, for example, the heavy chain constant region is an Ig gamma-1 chain C region, ACCESSION:P01857, and the light chain constant region is an Ig kappa chain C region, ACCESSION:P01834; or the heavy chain constant region of the immunoglobulin is an Ig gamma-4 chain C region, ACCESSION:P01861.1, and the light chain constant region of the immunoglobulin is an Ig kappa chain C region, ACCESSION:P01834.

[0035] In one embodiment of the invention, the amino acid sequence of the heavy chain constant region Ig gamma-1 chain C region (ACCESSION: P01857) is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40) In one embodiment of the invention, the amino acid sequence of the heavy chain constant region Ig gamma-4 chain C region (ACCESSION:P01861.1) is as follows: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41) In one embodiment of the present invention, the amino acid sequence of the light chain constant region Ig kappa chain C region (ACCESSION: P01834) is as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42) In some embodiments of the present invention, bispecific antibodies are provided in which a single chain antibody is linked to the C-terminus of an immunoglobulin heavy chain. Since an immunoglobulin has two heavy chains, two single chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single chain antibody molecules are identical.

[0036] In some embodiments of the invention, bispecific antibodies are provided in which there are two single chain antibodies, one terminus of each single chain antibody linked to the C-terminus or N-terminus of one of the two heavy chains of the immunoglobulin.

[0037] In some embodiments of the invention, the disulfide bond is located at the V H and V LMethods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, and are described, for example, in U.S. Pat. No. 5,747,654; Rajagopal et al., Prot. Engin., vol. 10 (1997) pp. 1453-1459; Reiter et al., Nat. Biotechnol., vol. 14 (1996) pp. 1239-1245; Reiter et al., Protein Engineering, vol. 8 (1995) pp. 1323-1331; Webber et al., Molecular Immunology, vol. 32 (1995) pp. 249-258; Reiter et al., Immunity, vol. 2 (1995) pp. 281-287; Reiter et al., JBC, vol. 269 (1994) pp. 18327-18331; Reiter et al., Inter. J. of Cancer, 58 (1994) pp. 142-149; or Reiter et al., Cancer Res., 54 (1994) pp. 2714-2718.

[0038] In one or more embodiments of the invention, bispecific antibodies are provided in which a first protein functional region is linked, either directly or by a linker fragment, to a second protein functional region; and / or a heavy chain variable region of a single chain antibody is linked, either directly or by a linker fragment, to a light chain variable region of a single chain antibody.

[0039] In one or more embodiments of the invention, for bispecific antibodies, the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5, or 6.

[0040] In one or more embodiments of the invention, for a bispecific antibody, the number of first protein functional regions and second protein functional regions is each independently one, two or more.

[0041] In one or more embodiments of the invention, bispecific antibodies are provided in which a single chain antibody is linked to the C-terminus of an immunoglobulin heavy chain.

[0042] The present invention relates to a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: the number of first protein functional regions is 1 and the number of second protein functional regions is 2; the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; the amino acid sequence of the immunoglobulin heavy chain is set forth in SEQ ID NO:24 and the amino acid sequence of the immunoglobulin light chain is set forth in SEQ ID NO:26; The amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:17; The single chain antibody is linked to the C-terminus of the immunoglobulin heavy chain; a first protein functional region is linked to a second protein functional region by a first linker fragment; a heavy chain variable region of the single chain antibody is linked to a light chain variable region of the single chain antibody by a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO: 18. Further related to bispecific antibodies.

[0043] In one or more embodiments of the invention, the immunoglobulin or antigen-binding fragment thereof is about 10 -6 Less than M, e.g., about 10 -7 M, 10 -8 Less than M or 10 -9 Bispecific antibodies are provided that bind to FcγRI with an affinity constant of less than or equal to M; preferably, the affinity constant is measured by the Fortebio Octet system.

[0044] In one or more embodiments of the invention, the immunoglobulin or antigen-binding fragment thereof is about 10 -9 Less than M, e.g., about 10 -7 M, 10 -8 Less than M or 10 -9 Bispecific antibodies are provided that bind to C1q with an affinity constant of less than or equal to M; preferably, the affinity constant is measured by the Fortebio Octet system.

[0045] In some embodiments of the invention, the bispecific antibody 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 or equal to M D binds to the VEGFA protein and / or the PD-1 protein at D is measured by the Fortebio system.

[0046] In some embodiments of the invention, the bispecific antibody has an EC 50 Binds to VEGFA protein in ECs; 50 was detected by indirect ELISA; and / or Bispecific antibodies with an EC 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 PD-1 protein in the EC 50 is detected by indirect ELISA, Bispecific antibodies are provided.

[0047] In one or more embodiments of the invention, the bispecific antibody is a monoclonal antibody.

[0048] In one or more embodiments of the invention, the bispecific antibody is a humanized antibody. Another aspect of the invention pertains to an isolated nucleic acid molecule encoding a bispecific antibody according to any of the embodiments of the invention.

[0049] Yet another aspect of the present invention pertains to vectors comprising the isolated nucleic acid molecules disclosed herein.

[0050] Yet another aspect of the present invention pertains to a host cell comprising an isolated nucleic acid molecule or vector described herein.

[0051] Another aspect of the invention relates to a conjugate comprising an antibody or antigen-binding fragment thereof and a conjugation moiety, wherein the immunoglobulin is a bispecific antibody according to any of the embodiments of the invention and the conjugation moiety is a detectable label; preferably, the conjugation moiety is a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

[0052] Another aspect of the invention is a kit comprising a bispecific antibody according to any of the embodiments of the invention or comprising a complex of the invention, comprising: Preferably, the kit further comprises a secondary antibody capable of specifically recognizing the immunoglobulin or an antigen-binding fragment thereof; optionally, the secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme. Regarding the kit.

[0053] Yet another aspect of the invention relates to the use of a bispecific antibody or conjugate according to any of the embodiments of the invention in the preparation of a kit for detecting the presence or level of PD-1 and / or VEGFA in a sample.

[0054] Another aspect of the invention relates to a pharmaceutical composition comprising a bispecific antibody or complex according to any of the embodiments of the invention; optionally, the pharmaceutical composition further comprises a pharma- ceutically acceptable vector and / or excipient.

[0055] The bispecific antibody of the present invention or the pharmaceutical composition of the present invention can be formulated into any dosage form known in the pharmaceutical art, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, and sprays. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention shall be sterile and stable under the conditions of manufacture and storage. One of the preferred dosage forms is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: a required amount of the bispecific antibody of the present invention is added to a suitable solvent, and optionally other desired ingredients (including but not limited to pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) are added at the same time, followed by filtration and sterilization. Furthermore, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or lyophilization) for convenient storage and use, which can be dispersed in an appropriate vector (e.g., sterile pyrogen-free water) prior to use.

[0056] Furthermore, the bispecific antibodies of the invention can be present in a pharmaceutical composition in unit dose form to facilitate administration. In some 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 can contain the bispecific antibodies of the invention in 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.

[0057] The bispecific antibody or pharmaceutical composition of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intravesical, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal. However, for many therapeutic applications, the preferred route / mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal and intramuscular injection). Those skilled in the art will recognize 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 can be used alone or in combination, or can be used in combination with additional pharma- ceutical effective agents (e.g., tumor chemotherapeutic agents). Such additional pharma- ceutical effective agents can be administered prior to, simultaneously with, or after administration of the bispecific antibodies of the invention or the pharmaceutical compositions of the invention.

[0059] In the present invention, the administration regimen can be adjusted to achieve the optimum desired response (e.g., a therapeutic or prophylactic response), for example, it can be a single administration or multiple administrations over a period of time, and can be characterized by a proportional reduction or increase in the dose depending on the exigencies of the treatment.

[0060] A still further aspect of the invention is the use of a bispecific antibody according to any of the embodiments of the invention or a conjugate according to the invention in the preparation of a medicament for treating and / or preventing a malignant tumor; preferably the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, the non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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. Regarding use.

[0061] Yet another aspect of the invention relates to the use of a bispecific antibody according to any of the embodiments of the invention or a conjugate according to the invention in the preparation of a medicament for: (1) a medicine or agent for detecting the level of VEGFA in a sample; A medicine or drug for blocking the binding of VEGFA to VEGFR2; Medicines or agents for downregulating VEGFA activity or levels; A medicine or agent for reducing VEGFA stimulation of vascular endothelial cell proliferation; A medicine or drug for inhibiting vascular endothelial cell proliferation, or medicines or agents for blocking tumor angiogenesis; and / or (2) a medicine or agent for blocking the binding of PD-1 to PD-L1; Medicaments or agents for downregulating the activity or levels of PD-1; A medicine or drug for reducing immunosuppression of PD-1 in the body; A medicine or drug for promoting IFN-γ secretion in T lymphocytes, or Medicine or agent for promoting IL-2 secretion in T lymphocytes The present invention relates to the use of a bispecific antibody or a complex of the present invention according to any of the embodiments of the present invention in the preparation of a

[0062] In the in vitro experiments of the present invention, both anti-VEGFA antibody and anti-VEGFA anti-PD-1 bispecific antibody can inhibit the proliferation of HUVEC cells, and both anti-PD-1 antibody and anti-VEGFA anti-PD-1 bispecific antibody can promote the secretion of IFN-γ and / or IL-2, and activate the immune response.

[0063] Yet another aspect of the invention is a method for treating and / or preventing malignant tumors, comprising administering an effective amount of a bispecific antibody according to any of the embodiments of the invention, or a conjugate according to the invention, to a subject in need thereof; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, the non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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. It concerns the method.

[0064] A non-limiting typical range for a therapeutically or prophylactically effective amount of a bispecific antibody of the invention is 0.02-50 mg / kg, e.g., 0.1-50 mg / kg, 0.1-25 mg / kg, or 1-10 mg / kg. It should be noted that the dosage may vary depending on the type and severity of the condition being treated. Furthermore, for any particular patient, a particular dosing regimen will be adjusted over time according to the needs of the patient and the professional judgment of the physician; one of ordinary skill in the art will appreciate that the dosage ranges set forth herein are for illustrative purposes only and are not intended to limit the use and scope of the pharmaceutical compositions of the invention.

[0065] In the present invention, the subject may be a mammal, such as a human. Yet another aspect of the present invention is a method for producing a medicament for use in a method for the preparation of a medicament (1) a method for detecting the level of VEGFA in a sample; Methods for blocking the binding of VEGFA to VEGFR2 Methods for downregulating VEGFA activity or levels, Methods for reducing VEGFA stimulation of vascular endothelial cell proliferation, A method for preparing a medicine or agent for inhibiting vascular endothelial cell proliferation, or Methods for blocking tumor angiogenesis; and / or (2) A method for blocking the binding of PD-1 to PD-L1, Methods for downregulating PD-1 activity or levels, Methods for reducing PD-1 immunosuppression in vivo A method for promoting IFN-γ secretion in T lymphocytes, or Method for promoting IL-2 secretion in T lymphocytes The present invention relates to an in vivo method or an in vitro method selected from the group consisting of:

[0066] In one or more embodiments of the invention, the bispecific antibody or conjugate is used in the treatment and / or prevention of malignant tumors; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, the non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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.

[0067] In one or more embodiments of the invention, the bispecific antibody or conjugate comprises: (1) detecting the level of VEGFA in a sample; Blocking the binding of VEGFA to VEGFR2, Downregulation of VEGFA activity or levels, Attenuating VEGFA stimulation of vascular endothelial cell proliferation; Inhibition of vascular endothelial cell proliferation, or Blocking tumor angiogenesis; and / or (2) blocking the binding of PD-1 to PD-L1; Downregulation of PD-1 activity or levels, Relief of PD-1-induced immunosuppression in vivo Stimulation of IFN-γ secretion in T lymphocytes, or Stimulation of IL-2 secretion in T lymphocytes Used for.

[0068] Antibody drugs, especially monoclonal antibodies, have achieved good results in treating various diseases. The traditional experimental methods for obtaining these therapeutic antibodies are to immunize animals with antigens, obtain antibodies targeting the antigen in the immunized animals, or improve those antibodies with low affinity to the antigen by affinity maturation.

[0069] The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions called complementarity determining regions (CDRs). The CDRs of the heavy chain (H chain) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L chain) include LCDR1, LCDR2, and LCDR3, as named by Kabat et al., Bethesda Md, Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 1991, (Vol. 1-3): 91-3242.

[0070] Preferably, the CDRs can also be 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 Vol. 38. Supplement 1 (2009): D301-D307.

[0071] The amino acid sequences of the CDR regions of the monoclonal antibody sequences (1) to (13) below were analyzed by technical means well known to those skilled in the art, for example, by the VBASE2 database and according to the IMGT regulations, and the results are as follows: (1) Bevacizumab The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:3.

[0072] 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 (sequence number 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 (SEQ ID NO: 33) For the antibody VP101(hG1DM) of the invention, amino acid mutations are introduced into the non-variable region of VP101(hG1WT). According to the EU numbering system, amino acid mutations are introduced at positions 234 and 235, and VP101(hG1DM) is obtained by introducing a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A) in the hinge region of the heavy chain.

[0073] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine methods that are widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0074] As used herein, when referring to the amino acid sequence of VEGFA protein (GenBank ID: NP_001165097.1), this includes the full-length VEGFA protein as well as fusion proteins of VEGFA, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse IgG 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 VEGFA protein can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "VEGFA protein" is intended to include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of VEGFA protein, this 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 as the underlined portion of SEQ ID NO: 33 (302 amino acids in total, without the last 6 His).

[0075] As used herein, when referring to the amino acid sequence of VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), this includes the full-length VEGFR2 protein, or the extracellular fragment of VEGFR2, VEGFR2-ECD, or a fragment containing VEGFR2-ECD, and further includes a fusion protein of VEGFR2-ECD, such as a fragment fused to Fc protein fragment (mFc or hFc) of mouse IgG 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 VEGFR2 protein can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "VEGFR2 protein" is intended to include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of VEGFR2 protein, this 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 set forth in SEQ ID NO: 34 (766 amino acids).

[0076] As used herein, unless otherwise specified, VEGFR is VEGFR1 and / or VEGFR2; its specific protein sequence is a sequence known in the prior art, and can refer to the sequence disclosed in existing literature or GenBank.For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0077] As used herein, when referring to the amino acid sequence of PD-1 protein (programmed cell death protein 1, NCBI GenBank: NM_005018), this includes the full-length PD-1 protein, or the extracellular fragment of PD-1, PD-1ECD, or a fragment containing PD-1ECD, which further includes fusion proteins of PD-1ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse IgG 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. Furthermore, when a sequence fragment of PD-1 protein is described, this also includes the corresponding sequence fragment of the natural or artificial variant.

[0078] As used herein, the term EC 50 means the concentration for 50% of the maximal effect, i.e. the concentration which produces 50% of the maximal effect.

[0079] As used herein, the term "antibody" refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. In a general sense, a heavy chain can be understood as the polypeptide chain having a larger molecular weight in an antibody, and a light chain refers to the polypeptide chain having a smaller molecular weight in an antibody. Light chains are classified as kappa and lambda light chains. Heavy chains are generally 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 linked by 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 (V H ) and the heavy chain constant region (CH 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 the light chain constant region (C L The light chain constant region consists of one domain, C L The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. H Area and V L The regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed between conserved regions, called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L) form the antibody binding site. The assignment of amino acids to regions or domains may be based on 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 rules, 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. In particular, the heavy chain may also 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 with the C-terminus of the heavy chain of an IgG antibody linked to another antibody, and in this case the heavy chain comprises nine CDRs. The term "antibody" is not limited by any particular method for producing the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, for example IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0080] As used herein, the term "antigen-binding fragment", also known as "antigen-binding portion", refers to a polypeptide comprising a fragment of a full-length antibody that specifically binds to the same antigen as the full-length antibody and / or retains the ability to compete with the full-length antibody for specific binding to an antigen. See generally, 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 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 antibody fragments (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0081] As used herein, the term "Fd fragment" refers to a V H Domain and C H1 "Fv fragment" refers to an antibody fragment consisting of the V domain of a single arm of an antibody; L Domains 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 Domain, V H Domain, C L Domain and C H1 the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0082] In some cases, the antigen-binding fragment of the antibody is L Domain and V HSingle chain antibodies (e.g., scFv) are antibodies in which the domains are paired to form a monovalent molecule via a linker that allows for the production of 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 have the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L It may have -COOH. Suitable linkers in the prior art consist of repeated GGGGS amino acid sequences or variants thereof. For example, linkers with the amino acid sequence (GGGGS)4 can be used, as well as variants thereof (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers useful in the present invention are disclosed 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).

[0083] In some cases, the antigen-binding fragment of the antibody is H Domain and V LDiabodies, or bivalent antibodies, are domains expressed in a single polypeptide chain.However, the linker used is too short to allow the two domains to pair on the same chain, so the domains pair with the complementary domains on another chain, generating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 (1993):6444-6448 and Poljak RJ et al., Structure 2 (1994):1121-1123).

[0084] Antigen-binding fragments of antibodies (such as the antibody fragments referred to 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 the antigen-binding fragments of antibodies can be screened for specificity in the same manner as intact antibodies.

[0085] As used herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0086] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or fragment thereof that is derived from a group of highly homogeneous antibodies, i.e., from a group of identical antibody molecules, except for natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained by hybridoma technology, first reported by Kohler et al. (Nature, 256:495, 1975), or by recombinant DNA technology (see, for example, U.S. Patent No. 4,816,567).

[0087] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light and / or heavy chain is derived from an antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of the light and / or heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but which in either case retains binding activity for the target antigen (see U.S. Patent No. 4,816,567 (Cabilly et al.); Morrison et al., Proc. Natl. Acad. Sci. USA, 81 (1984):6851-6855).

[0088] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (acceptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), which can be a non-human (e.g. mouse, rat or rabbit) antibody with the expected specificity, affinity or reactivity. Furthermore, some amino acid residues in the framework regions (FR) of the acceptor antibody can also be replaced with amino acid residues of the corresponding non-human antibody or with amino acid residues of 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, 1986, 321:522-525; Reichmann et al., Nature, 1998, 332:323-329; Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, and Clark, Immunol. Today 2000, 21:397-402.

[0089] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "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 carbohydrate or sugar side chain, and usually 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, which may be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by GE Morris (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are located linearly along the primary amino acid sequence of the protein. In a conformational epitope, the sites of interaction are located across amino acid residues of the protein that are separated from each other.

[0090] As used herein, the term "isolated" means to be obtained by artificial means from the natural state. A particular "isolated" substance or component may occur naturally, may be altered in its natural environment, or may be isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally occurs in a particular living animal, and the same polynucleotide or polypeptide of high purity isolated in such a natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0091] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector can express the protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material element carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages, such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used 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, and papova viruses (such as SV40). Vectors may contain various elements that regulate expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0092] As used herein, the term "host cell" means a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli or bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as an S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0093] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and an antigen that it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one in which the antibody binds to the antigen within about 10 -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Less than M or 10 -10 Affinity below M (K D ) to an antigen. In some embodiments of the invention, the term "target" refers to specific binding.

[0094] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. A smaller equilibrium dissociation constant indicates stronger antibody-antigen binding and a higher affinity between the antibody and the antigen. Generally, an antibody has a dissociation equilibrium constant of about 10, as determined, for example, by a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio system. -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Less than M or 10 -10 The dissociation equilibrium constant (K D ) binds to the antigen.

[0095] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Furthermore, herein, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0096] As used herein, the term "pharmaceutical acceptable auxiliary substance" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Gennaro AR, ed., 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic surfactants, anionic surfactants or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0097] As used herein, the term "adjuvant" refers to a non-specific immune stimulant that can enhance the immune response of the organism to an antigen or change the type of immune response when delivered to the organism together with or prior to the antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharides, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0098] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, a prophylactically effective amount (e.g., for a disease associated with the binding of PD-1 to PD-L1 or overexpression of VEGF, e.g., tumor) is an amount sufficient to prevent, inhibit or delay the onset of the disease (e.g., a disease associated with the binding of PD-1 to PD-L1 or overexpression of VEGF, e.g., tumor); a therapeutically effective amount is an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. It is undoubtedly within the scope of a person skilled in the art to determine such an effective amount. For example, an effective amount for therapeutic purposes 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, e.g., age, weight and sex, route of administration, and other treatments given at the same time, etc.

[0099] The term "MSI" refers to microsatellite instability. Microsatellites are short tandem repeats throughout the human genome, containing 10-50 repeats of one, two or more nucleotides. Microsatellites in certain abnormal cells, such as tumors, are altered in length by insertion or deletion of repeat units compared to normal cells. Such alterations are termed MSI. Based on the instability and extent, MSI can be classified as high frequency microsatellite instability (MSI-H), low frequency microsatellite instability (MSI-L), and microsatellite stable (MSS). The main cause of MSI is a deficiency in DNA mismatch repair (MMR). Human mismatch repair genes (MMR genes) can express the corresponding mismatch repair proteins by transcription and translation. The absence of any MMR protein can result in a deficiency in mismatch repair, and base pair mismatches accumulate in the process of DNA replication due to such deficiency, ultimately resulting in MSI. Approximately 15% of colorectal cancers are caused by the MSI pathway. It was first reported in colorectal cancer and can also occur in gastric cancer, esophageal cancer, adrenal cortical carcinoma, etc. (Baretti M et al., Pharmacol Ther., 2018;189:45-62). MSI-H / dMMR characteristics were also found in mesothelioma, sarcoma, adrenal cortical carcinoma, malignant melanoma, and ovarian germ cell neoplasms in subsequent studies.

[0100] MSI-H and dMMR represent the results of two different assays, which are biologically consistent and are called MSI-H / dMMR or MSI-high / dMMR, while MSI-L and MSS are phenotypes of competent mismatch repair (pMMR). The detection of dMMR is to carry out an immunohistochemical assay of protein expression for four mismatch genes, MSH2, MLH1, MSH6 and PMS2, based on tumor specimens (including surgical and aspirate specimens). The absence of any of the four proteins confirms dMMR, and a positive result for all four proteins indicates pMMR, i.e., complete mismatch repair function. The detection of MSI is to match and compare the length of repetitive DNA sequences (microsatellite sequences) in tumor cells and somatic cells. When five standard loci are detected using PCR based on the American NCI standard, mismatches at two or more loci indicate instability and are defined as MSI-H, one mismatched locus indicates MSI-L, and five matched loci indicate MSS. High-throughput sequencing (also called next-generation sequencing or NGS) can also be used as a method to detect microsatellite instability. When more microsatellite loci are selected for the PCR assay, for example more than five loci or additional microsatellite loci, mismatches at ≥ 30% loci are defined as MSI-H, matches at all loci are defined as MSS, and mismatches at 0-30% are defined as MSI-L.

[0101] Beneficial Effects The present invention achieves one or more of the following technical effects (1) to (4): (1) Modification of the Fc fragment of the antibody of the present invention completely eliminates the binding activity of VP101 (hG1WT) and the Fc receptors FcγRI and FcγRIIIa_F158, thereby completely eliminating ADCC activity.

[0102] (2) Modification of the Fc fragment of the antibody of the present invention completely eliminates the binding activity of VP101 (hG1WT) and complement C1q, thereby completely eliminating the CDC activity.

[0103] (3) The bispecific antibodies of the present invention can bind to VEGFA with sufficient specificity and effectively block the binding of VEGFA to VEGFA2, thereby specifically reducing the immunosuppressive effects of VEGFA and the promoting effects of VEGFA on angiogenesis in the body.

[0104] (4) The bispecific antibody of the present invention can bind to PD-1 with sufficient specificity and effectively block the binding of PD-1 to PD-L1, thereby specifically relieving the immunosuppression of PD-1 in the body and activating the immune response. [Brief description of the drawings]

[0105] [Figure 1] Affinity constant assay of VP101 (hG1DM) to FcγRI. Antibody concentrations for the top to bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively. [Diagram 2] Affinity constant assay of bevacizumab to FcγRI. Antibody concentrations for the top to bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively. [Diagram 3] Affinity constant assay of Nivolumab to FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively. [Figure 4] Affinity constant assay of VP101 (hG1WT) to FcγRI. Antibody concentrations for the top to bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively. [Diagram 5]Affinity constant assay of VP101 (hG4WT) to FcγRI. Antibody concentrations for the top to bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.12 nM, respectively. [Figure 6] Affinity constant assay of VP101 (hG1DM) to FcγRIIa_H131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 7] Affinity constant assay of bevacizumab to FcγRIIa_H131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 8] Affinity constant assay of Nivolumab to FcγRIIa_H131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 9] Affinity constant assay of VP101 (hG1WT) to FcγRIIa_H131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 10] Affinity constant assay of VP101 (hG4WT) to FcγRIIa_H131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 11] Affinity constant assay of VP101 (hG1DM) to FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 12] Affinity constant assay of bevacizumab to FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 13]Affinity constant assay of nivolumab to FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 14] Affinity constant assay of VP101 (hG1WT) for FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 15] Affinity constant assay of VP101 (hG4WT) for FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM, respectively. [Figure 16] Affinity constant assay of VP101 (hG1DM) to FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 17] Affinity constant assay of bevacizumab to FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 18] Affinity constant assay of Nivolumab to FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 19] Affinity constant assay of VP101 (hG1WT) for FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 20] Affinity constant assay of VP101 (hG4WT) to FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 21]Affinity constant assay of VP101 (hG1DM) to FcγRIIIa_F158. Antigen concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 22] Affinity constant assay of bevacizumab to FcγRIIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Diagram 23] Affinity constant assay of nivolumab to FcγRIIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 24] Affinity constant assay of VP101 (hG1WT) to FcγRIIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Diagram 25] Affinity constant assay of VP101 (hG4WT) to FcγRIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively. [Figure 26] Affinity constant assay of VP101 (hG1DM) to C1q. Antibody concentrations for the top to bottom curve pairs are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively. [Figure 27] Affinity constant assay of bevacizumab to C1q. Antibody concentrations for the top to bottom curve pairs are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively. [Figure 28] Affinity constant assay of Nivolumab to C1q. Antibody concentrations for the top to bottom curve pairs are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively. [Figure 29]Affinity constant assay of VP101 (hG1WT) to C1q. Antibody concentrations for the top to bottom curve pairs are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively. [Diagram 30] Affinity constant assay of VP101 (hG4WT) for C1q. The antigen concentrations for the top and bottom curve pairs are 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM, respectively. [Diagram 31] ADCC activity assay of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 target cell line expressing PD-1 antigen. [Diagram 32] CDC activity assay of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 target cell line expressing PD-1 antigen. [Diagram 33] Effect of antibody VP101 (hG1DM) on the secretion of the cytokine IFN-γ induced by mixed cultures of PBMC and Raji-PDL1 cells, detected by ELISA. [Diagram 34] Effect of antibody VP101 (hG1DM) on the secretion of cytokine IL-2 induced by mixed cultures of PBMC and Raji-PDL1 cells, detected by ELISA. [Diagram 35] ADCP activity assay of VP101(hG1DM) in CHO-K1-PD1 target cell line expressing PD-1 antigen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and are not interpreted as limiting the scope of the present invention. In the absence of clear description of techniques or conditions, they are carried out according to the techniques or conditions disclosed in the technical literature (see, for example, Guide to Molecular Cloning Experiments, J. Sambrook et al., and Huang Peitang et al., translated, 3rd edition, Science Press) or according to the product manual. The reagents or equipment used are all commercially available conventional products unless their manufacturers are specified.

[0107] In the following examples of the present invention, the commercial antibody bevacizumab (trade name Avastin®) for the same target was purchased from Roche as a control antibody or prepared according to Preparation Example 1.

[0108] In the following examples of the present invention, the commercial antibody Nivolumab (trade name Opdivo®) for the same target was purchased from BMS as a control antibody.

[0109] In the following examples of the present invention, the isotype control antibody used is human anti-hen egg white lysozyme IgG (anti-HEL, or human IgG abbreviated as hIgG), whose variable region sequences are derived from a study published by Acierno et al., entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol Biol., 2007;374(1):130-46). The hIgG1DM and hIgG4WT used in the examples are isotype control antibodies of anti-HEL with constant region sequences of hG1DM and hG4WT. The isotype control antibodies were prepared in the laboratory of Akeso Biopharma, Inc.

[0110] Preparation Example 1: Preparation of anti-VEGFA antibody bevacizumab China Patent Publication No. CN1259962A refers to the amino acid sequences of the heavy and light chain variable regions of the commercially available anti-VEGFA monoclonal antibody Avastin (bevacizumab). Genscript was commissioned to synthesize the nucleotide sequences encoding the heavy and light chain variable regions. Amino acid sequence of the heavy chain variable region of bevacizumab (bevacizumab-Hv): (123aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 1) Nucleotide sequence encoding the heavy chain variable region of bevacizumab: (369bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGA CTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC (SEQ ID NO: 2) Amino acid sequence of the light chain variable region of bevacizumab (Bevacizumab-Lv): (107aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 3) Nucleotide sequence encoding the light chain variable region of bevacizumab: (321bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCA CTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG (SEQ ID NO: 4) The heavy chain constant regions were all Ig gamma-1 chain C region, ACCESSION:P01857; the light chain constant regions were all Ig kappa chain C region, ACCESSION:P01834.

[0111] The heavy and light chain cDNA of bevacizumab were cloned into vector pcDNA3.1 to obtain the recombinant expression plasmid of antibody bevacizumab. The recombinant plasmid was transfected into 293F cells. The culture medium of 293F cells was purified and then detected.

[0112] The anti-VEGFA monoclonal antibody Avastin (bevacizumab) was thus obtained. Preparation Example 2: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as mutant 14C12H1L1(M) The amino acid sequences and encoding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication No. CN106967172A.

[0113] (1) 14C12 heavy and light chain variable region sequences Amino acid sequence of the heavy chain variable region of 14C12: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 5) Nucleotide sequence encoding the heavy chain variable region of 14C12: (354bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTAT CCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 6) Amino acid sequence of the light chain variable region of 14C12: (107aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 7) Nucleotide sequence encoding the light chain variable region of 14C12: (321 bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 8) (2) Heavy and light chain variable regions and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 Amino acid sequence of the heavy chain variable region of 14C12H1L1: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 9) Nucleotide sequence encoding the heavy chain variable region of 14C12H1L1: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 10) Amino acid sequence of the light chain variable region of 14C12H1L1: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 11) Nucleotide sequence encoding the light chain variable region of 14C12H1L1: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 12) Amino acid sequence of the heavy chain (14C12H1) of 14C12H1L1: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13) Nucleotide sequence encoding the heavy chain of 14C12H1L1 (14C12H1): (1344 bp) Amino acid sequence of the light chain of 14C12H1L1 (14C12L1): (214aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15) Nucleotide sequence encoding the light chain of 14C12H1L1 (14C12L1): (642 bp) (SEQ ID NO:16) (3) Heavy and light chain variable region sequences of 14C12H1L1(M) Individual amino acids in the framework regions (light chain) were mutated based on 14C12H1L1 to obtain 14C12H1L1(M).

[0114] The heavy chain variable region 14C12H1(M) of 14C12H1L1(M) is identical to the heavy chain variable region 14C12H1 of 14C12H1L1, i.e., the amino acid sequence is set forth in SEQ ID NO:9.

[0115] Light chain variable region 14C12L1(M): (108 aa, mutation positions are underlined in the amino acid sequence based on 14C12H1L1) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK R (SEQ ID NO:17) Preparation Example 3: Sequence design of bispecific antibodies 1. Sequence Design The structure of the bispecific antibody of the present invention is the Morrison type (IgG-scFv), i.e., the C-terminus of each of the two heavy chains of an IgG antibody is linked to an scFv fragment of a different antibody, and the main structural designs of the heavy and light chains are as shown in Table 1 below.

[0116] Based on bevacizumab, the VP101 antibody, in which the amino acid sequences of the heavy chain variable region and the light chain variable region of 14C12H1L1(M) are considered to be ScFv fragment portions, is referred to as VP101(M). Compared with 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves efficacy.

[0117] [Table 1]

[0118] In Table 1 above: (1) Those with a "V" designation in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. For those without a "V" designation, the corresponding heavy or light chain is full length, including the constant region. The corresponding sequences described in the preparation examples above refer to the amino acid sequences of these variable regions or full length, and the nucleotide sequences encoding them.

[0119] (2) The amino acid sequence of linker 1 is GGGGSGGGGSGGGGSGGGGS (sequence number 18). Optionally, the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 19) can be used as Linker 2, instead of Linker 1 described above.

[0120] (3) Bevacizumab-H used the Ig gamma-1 chain C region (ACCESSION: P01857) as the heavy chain constant region.

[0121] (4) Bevacizumab-G4H used the Ig gamma-4 chain C region (ACCESSION: P01861.1) as the heavy chain constant region.

[0122] 2. Expression and Purification of Antibody VP101(M) The heavy and light chain cDNA sequences of VP101 were each cloned into the vector pUC57simple (provided by Genscript) to obtain the plasmids pUC57simple-VP101H and pUC57simple-VP101L, respectively.

[0123] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were enzymatically digested (HindIII & EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned into vector pcDNA3.1, 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 loaded onto a HiTrap MabSelect SuRe column. The protein was further eluted in one step with elution buffer to isolate the target sample antibody VP101, and buffer exchanged into PBS.

[0124] 3. Detection of Antibody VP101(M) The purified samples were added to both reducing and non-reducing protein electrophoresis loading buffers and then boiled for SDS-PAGE electrophoresis detection.

[0125] To distinguish it from the mutated antibody of Preparation Example 4, VP101(M) is also referred to as VP101(hG1WT) in the present invention. VP101(M) described above is "wild type" and contains the Ig gamma-1 chain C region (ACCESSION: P01857) as the heavy chain constant region and the Ig kappa chain C region (ACCESSION: P01834) as the light chain constant region.

[0126] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (453aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20) Nucleotide sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (1359 bp) To distinguish it from the mutated antibody of Preparation Example 4, VP101(G4M) is also referred to as VP101(hG4WT) in the present invention. VP101(G4M) described above is "wild type" and contains the Ig gamma-4 chain C region (ACCESSION: P01861.1) as the heavy chain constant region and the Ig kappa chain C region (ACCESSION: P01834) as the light chain constant region.

[0127] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (450 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 22) Nucleotide sequence encoding the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (1350 bp) Preparation Example 4: Design of non-variable region amino acid mutations based on humanized bispecific antibody VP101 (hG1WT) Based on VP101(hG1WT) obtained in Preparation Example 3, VP101(hG1DM) was obtained by introducing a point mutation of leucine and alanine at position 234 (L234A) and a point mutation of leucine and alanine at position 235 (L235A) in the heavy chain.

[0128] Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1DM): (453 aa, mutation positions are underlined) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24) Nucleotide sequence encoding the heavy chain of the immunoglobulin moiety in VP101 (hG1DM): (1359 bp, mutation positions are underlined) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT(SEQ ID NO:25) The amino acid sequences and encoding nucleotide sequences of the light chains of the immunoglobulin portions of VP101(hG1DM), VP101(hG1WT) and VP101(hG4WT) are identical.

[0129] Amino acid sequence of the light chain of the immunoglobulin portion of VP101 (hG1DM): (214aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26) Nucleotide sequence encoding the light chain of the immunoglobulin portion in VP101 (hG1DM): (642 bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAGCGGACCGTGGCCGCTCCCAGTGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAATCCGGGACAGCCTCTGTGGTCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGAGTGGCAATTCACAGGAGAGCGTGACAGAACAGGACTCCAAAGATTCTACTTATAGTCTGTCAAGCACACTGACTCTGAGCAAGGCTGACTACGAAAAGCATAAAGTGTATGCATGTGAGGTCACCCACCAGGGGCTGAGCAGTCCAGTCACCAAGTCATTCAACAGAGGCGAGTGC (SEQ ID NO: 27)

Example

[0130] Example 1: Affinity constant assay of FcγrRI for VP101(hG1WT) and VP101(hG1DM) The Fc receptor FcγRI, also known as CD64, can bind to the Fc fragment of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to Fc receptors affects the safety and efficacy of the antibody.

[0131] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRI were determined using the Fortebio Octet system, and the ADCC activity of each antibody was evaluated.

[0132] The method for determining the affinity constant of an antibody to FcγRI using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 1 μg / mL of FcγRIa was immobilized on the HIS1K sensor for 50 s. The sensor was equilibrated in buffer for 60 s, and binding of CD64 immobilized on the sensor to antibodies at concentrations from 3.12 to 50 nM (serial two-fold dilutions) was determined for 120 s. The antibody was dissociated in buffer for 120 s. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 s each. The detection temperature was 30 °C and the frequency was 0.3 Hz. The data was analyzed by fitting with a 1:1 model, and the affinity constants were obtained.

[0133] The affinity constants of FcγRI for 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.

[0134] [Table 2]

[0135] N / A indicates that the antibody has no binding to the antigen or has a very weak binding signal, and therefore the result was not analyzed and no corresponding data was obtained.

[0136] 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; nivolumab can bind to FcγRI with an affinity constant of 6.20E-09M; VP101(hG1DM) has no binding to FcγRI or has a very weak binding signal, therefore the results were not analyzed and no corresponding data was obtained.

[0137] The results show that the affinities of the other antibodies to FcγRI are similar, except for the lack of binding of VP101(hG1DM) to FcγRI, whose binding activity was effectively eliminated.

[0138] Example 2: Affinity constant assay of FcγRIIa_H131 for VP101(hG1WT) and VP101(hG1DM) The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) can bind the Fc fragment of IgG antibodies and mediate the ADCC effect.

[0139] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIa_H131 were determined using the Fortebio Octet system, and the ADCC activity of each antibody was evaluated.

[0140] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIa_H131 using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor for an immobilization time of 60 seconds. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA (pH 7.4) during 600 s of blocking, and the binding of FcγRIIa_H131 immobilized on the sensor to antibodies at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine, pH 1.7 and 10 nM nickel sulfate. The detection temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by fitting with a 1:1 model, and the affinity constants were obtained.

[0141] The affinity constants of FcγRIIa_H131 for 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.

[0142] [Table 3]

[0143] N / A indicates that the antibody has no binding to the antigen or has a very weak binding signal, and therefore the result was not analyzed and no corresponding data was obtained.

[0144] 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; VP101 (hG1DM) can bind to FcγRIIa_H131 with an affinity constant of 3.57E-08M; but nivolumab has no binding to FcγRIIa_H131 or has a very weak binding signal, and therefore the results were not analyzed and no corresponding data was obtained.

[0145] The results show that except for no binding of nivolumab to FcγRIIa_H131, other antibodies bound to FcγRIIa_H131 with the following strong to weak affinities: VP101(hG1WT), VP101(hG4DM), VP101(hG4WT) and bevacizumab.

[0146] Example 3: Affinity constant assay of FcγRIIa_R131 for VP101(hG1WT) and VP101(hG1DM) The Fc receptor FcγRIIa_R131 (also known as CD32a_R131) binds the Fc fragment of IgG antibodies and can mediate the ADCC effect.

[0147] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIa_R131 were determined using the Fortebio Octet system, and the ADCC activity of each antibody was evaluated.

[0148] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_R131 was immobilized on the NTA sensor for an immobilization time of 60 seconds. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA (pH 7.4) during 600 s of blocking, and the binding of FcγRIIa_R131 immobilized on the sensor to antibodies at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine, pH 1.7 and 10 nM nickel sulfate. The detection temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by fitting with a 1:1 model, and the affinity constants were obtained.

[0149] The affinity constants of FcγRIIa_R131 for 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.

[0150] [Table 4]

[0151] N / A indicates that the antibody has no binding to the antigen or has a very weak binding signal, and therefore the result was not analyzed and no corresponding data was obtained.

[0152] The results show 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_H131 with an affinity constant of 3.35E-08M.

[0153] The results show that the antibodies bound to FcγRIIa_R131 with the following strong to weak affinities: VP101(hG1WT), VP101(hG1DM), VP101(hG4WT), bevacizumab and nivolumab.

[0154] Example 4: Affinity constant assay of FcγRIIb to VP101(hG1WT) and VP101(hG1DM) The Fc receptor FcγRIIb (also known as CD32b) binds the Fc fragment of IgG antibodies and can regulate immune cell function.

[0155] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIb were determined using a Fortebio Octet system to evaluate the binding ability of VP101(hG1WT) and VP101(hG1DM) to Fc receptors.

[0156] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIb using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of hFCGR2B-his was immobilized on the NTA sensor for an immobilization time of 60 seconds. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA (pH 7.4) during 600 s of blocking, and the binding of hFCGR2B-his immobilized on the sensor to antibodies at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was refreshed in 10 mM glycine, pH 1.7 and 10 nM nickel sulfate. The detection temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by fitting with a 1:1 model, and the affinity constants were obtained.

[0157] Example 5: Affinity constant assay of FcγRIIIa_V158 for VP101(hG1WT) and VP101(hG1DM) The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) binds the Fc fragment of IgG antibodies and can mediate the ADCC effect.

[0158] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIIa_V158 were determined using the Fortebio Octet system, and the ADCC activity of each antibody was evaluated.

[0159] The method for determining the affinity constant of antibodies to FcγRIIIa_V158 by the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in the buffer for 60 s, and the binding of hFcGR3A(V158)-his immobilized on the sensor to antibodies at concentrations from 31.25 to 500 nM (serial two-fold dilutions) was determined for 60 s. The antibody was dissociated in the buffer for 60 s. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 s each. The detection temperature was 30 °C, and the frequency was 0.3 Hz. The data was analyzed by fitting with a 1:1 model, and the affinity constants were obtained.

[0160] The affinity constants of FcγRIIIa_V158 for 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.

[0161] [Table 5]

[0162] N / A indicates that the antibody has no binding to the antigen or has a very weak binding signal, and therefore the result was not analyzed and no corresponding data was obtained.

[0163] The results show that VP101(hG1WT) can bind to FcγRIIIa_V158 with an affinity constant of 4.35E-08M; VP101(hG4DM) can bind to FcγRIIIa_V158 with an affinity constant of 1.34E-07M; bevacizumab can bind to FcγRIIIa_V158 with an affinity constant of 2.76E-08M; however, nivolumab and VP101(hG4WT) have no binding to FcγRIIIa_V158 or have very low binding signals, therefore the results were not analyzed and no corresponding data was obtained.

[0164] The results show that except for no binding of nivolumab and VP101(hG4WT) to FcγRIIIa_V158, other antibodies bound to FcγRIIIa_V158 with the following strong to weak affinities: bevacizumab, VP101(hG1WT) and VP101(hG1DM).

[0165] Example 6: Affinity constant assay of FcγRIIIa_F158 for VP101(hG1WT) and VP101(hG1DM) The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) binds the Fc fragment of IgG antibodies and can mediate the ADCC effect.

[0166] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIIa_F158 were determined using the Fortebio Octet system, and the ADCC activity of each antibody was evaluated.

[0167] The method for determining the affinity constants of VP101(hG1WT) and VP101(hG1DM) for FcγRIIIa_F158 using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in buffer for 60 s, and binding of hFcGR3A(F158)-his immobilized on the sensor to antibodies at concentrations from 31.25 to 500 nM (serial two-fold dilutions) was determined for 60 s. The antibody was dissociated in buffer for 60 s. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 s each. 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.

[0168] The affinity constants of FcγRIIIa_F158 for 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.

[0169] [Table 6]

[0170] N / A indicates that the antibody has no binding to the antigen or has a very weak binding signal, and therefore the result was not analyzed and no corresponding data was obtained.

[0171] The results show that VP101(hG1WT) can bind to FcγRIIIa_F158 with an affinity constant of 7.41E-08M; bevacizumab can bind to FcγRIIIa_F158 with an affinity constant of 9.32E-08M; however, nivolumab, VP101(hG4WT) and VP101(hG1DM) have no binding to FcγRIIIa_F158 or have very low binding signals, therefore the results were not analyzed and no corresponding data was obtained.

[0172] The results show that except for no binding of nivolumab, VP101(hG4WT) and VP101(hG1DM) to FcγRIIIa_F158, other antibodies bound to FcγRIIIa_F158 with the following strong to weak affinities: VP101(hG1WT) and bevacizumab.

[0173] Example 7: Affinity constant assay of C1q for VP101(hG1WT) and VP101(hG1DM) Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate the CDC effect. The ability of therapeutic monoclonal antibodies to bind C1q affects the safety and efficacy of the antibodies.

[0174] In this example, the affinity constants of VP101(hG1WT) and VP101(hG1DM) for C1q were determined using the Fortebio Octet system, and the CDC activity of each antibody was evaluated.

[0175] The method for determining the affinity constant of an antibody to C1q using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 50 μg / mL of antibody was immobilized on the FAB2G sensor with an immobilization height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s, and the binding of the antibody immobilized on the sensor to C1q at concentrations from 0.625 to 10 nM (serial two-fold dilutions) was determined for 60 s. The antigen and antibody were dissociated in the buffer for 60 s. The sensor was refreshed four times in 10 mM glycine, pH 1.7, for 5 s each. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 0.6 Hz. The data was analyzed by fitting with a 1:1 model to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 7.0 and the data analysis software was Fortebio Data Analysis 7.0.

[0176] The affinity constants of C1q for 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.

[0177] [Table 7]

[0178] N / A indicates that the antibody has no binding to the antigen or has a very weak binding signal, and therefore the result was not analyzed and no corresponding data was obtained.

[0179] The results show that VP101(hG1WT) can bind to C1q with an affinity constant of 9.76E-10M; bevacizumab can bind to C1q with an affinity constant of 1.14E-09M; however, nivolumab, VP101(hG4WT) and VP101(hG1DM) have no binding to C1q or have very low binding signals, therefore the results were not analyzed and no corresponding data was obtained.

[0180] Results: Nivolumab, VP101(hG4WT) and VP101(hG1DM) does not bind to C1q , VP101(hG1WT) and Bevacizumab bind to C1q with similar affinity .

[0181] Example 8: ADCC activity assay of VP101(hG1WT) and VP101(hG1DM) in 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 inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen and established a system of co-culture of normal human PBMCs and target cells to detect the ADCC activity of the antibodies at the cellular level.

[0182] The method for detecting the ADCC activity of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 cells expressing PD-1 antigen is as follows: In this experiment, human PD-1 overexpression vector pCDH-CMV-PD1FL-Puro (pCDH-CMV-Puro purchased from Youbio) was first constructed, the expression vector was packaged into a virus, and then infected into CHO-K1 cells, and the drug-resistant stable expressing membrane PD-1 protein CHO-K1-PD1 stable cell line was obtained after dosing and screening with puromycin (2 μg / mL). Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation device. The isolated PBMCs were resuspended in 1640 complete medium, stained with trypan blue, the cells were counted, and the cell viability was determined. The cells were incubated overnight at 37 °C and 5% CO2 in a saturated humidity incubator. The next day, CHO-K1-PD1 cells and PBMCs were harvested, centrifuged, the supernatant was removed, and then the cell pellet was resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as assay medium), centrifuged, and washed twice; the cells were counted, the cell viability was determined, and the concentration of the cells was adjusted to the proper range by using assay medium, and the CHO-K1-PD1 cell suspension (30,000 cells / well) was added to a 96-well plate according to the experimental design; 50 μL of antibody was added, mixed thoroughly, and pre-incubated at room temperature for 1 hour: after pre-incubation, the cells were added with PBMCs (900,000 / 50 μL / well), mixed thoroughly, and incubated in an incubator at 37° C. and 5% CO2 for 4 hours. After 4 hours, the 96-well plate was taken out and centrifuged at 250×g for 5 minutes, and 100 μL of cell supernatant was carefully transferred to a new 96-well flat-bottom microplate (without pipetting the cell pellet). 100 μL of freshly prepared reaction solution was added to each well according to the instructions of the cytotoxicity detection kit. The cells were incubated in the dark at room temperature for 30 minutes. The OD values ​​at 490 nm and 650 nm were measured, where the OD value of each group = OD 490nm -OD 650nm The ADCC activity was calculated for each group according to the formula: ADCC (%)=(treated group−negative control group) / (maximum LDH release in target cells−spontaneous LDH release in target cells)×100%.

[0183] The results of detection of the ADCC activity of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 cells expressing the PD-1 antigen are expressed as ADCC%, and the results are shown in FIG.

[0184] The results show that the positive control 14C12H1L1(G1WT) has significant ADCC activity in the mixed culture system of PBMC and CHO-K1-PD1, and the ADCC system is normal. Compared with the isotype control antibody hIgG1DM, VP101(hG1WT) showed significant ADCC activity and was dose-dependent, while VP101(G1DM) showed no ADCC activity. The results show that VP101(hG1DM) produced by mutation based on VP101(hG1WT) has no ADCC activity at the cytological level, and the ADCC effect is eliminated.

[0185] Example 9: CDC activity assay of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 cells expressing PD-1 antigen To detect the CDC effect of antibodies VP101(hG1WT) and VP101(hG1DM) at the cellular level, the inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen (see Example 8 for the construction method), and established a system of co-culture of target cells and normal human complement serum to detect the CDC activity of the antibodies at the cellular level.

[0186] The method for detecting the CDC activity of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 cells expressing PD-1 antigen is as follows: On the day of detection, CHO-K1-PD1 cells were harvested by trypsinization and centrifuged at 170×g for 5 min; the cell pellet was resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as assay medium), centrifuged repeatedly, and washed twice; the cells were counted, the cell viability was determined, and the cell concentration was adjusted to the proper range by using assay medium, and the CHO-K1-PD1 cell suspension (30,000 cells / well) was added to a 96-well plate according to the experimental design; 50 μL of antibody was added, mixed thoroughly, and pre-incubated at room temperature for 10 min: after pre-incubation, 50 μL / well of normal human complement serum (final concentration: 2%) was added to the cells, mixed thoroughly, and incubated in an incubator at 37° C. and 5% CO2 for 4 h. After 4 hours, the cells were centrifuged at 250×g for 5 minutes; 100 μL of cell supernatant was carefully transferred to a new 96-well flat-bottom plate (without pipetting the cell pellet). 100 μL of freshly prepared reaction solution was added to each well according to the instructions of the cytotoxicity detection kit. The cells were incubated in the dark at room temperature for 30 minutes. The OD values ​​at 490 nm and 650 nm were measured, where the OD value of each group = OD 490nm -OD 650nm CDC activity was calculated for each group according to the formula: CDC (%)=(treated group-negative control group) / (maximum LDH release in target cells-spontaneous LDH release in target cells)×100%.

[0187] The detection results of the CDC activity of VP101(hG1WT) and VP101(hG1DM) in CHO-K1-PD1 cells expressing the PD-1 antigen were expressed as CDC%, and the results are shown in FIG.

[0188] The results show that the CDC% of the positive control antibody 14C12H1L1(G1WT) had a significant difference from that of the isotype control antibody hIgG1DM group in the mixed culture system of normal human complement serum and CHO-K1-PD1, indicating that the CDC detection system is normal. At the equivalent dose level, there was no significant difference in the CDC% for both VP101(hG1WT) and VP101(hG1DM) compared with the isotype control.

[0189] Example 10: Pharmacodynamic activity of VP101 (hG1DM) in a mixed culture system (MLR) of peripheral blood mononuclear cells and Raji-PDL1 cells In this experiment, human PD-L1 overexpression vector plenti6.3-PD-L1-BSD (plenti6.3-BSD purchased from invitrogen) was first constructed, the expression vector was packaged into a virus and then infected into Raji cells, and the Raji-PDL1 stable cell line for stable expression of membrane PD-L1 protein was obtained after dosing and screening with BSD (10μg / mL). Normal human PBMCs were isolated according to Ficoll peripheral blood mononuclear cell isolation device, and the isolated PBMCs were resuspended in 1640 complete medium, counted, and frozen. The PBMCs were recovered, added with SEB (Staphylococcus aureus enterotoxin B), stimulated, and cultured for 2 days. After 2 days, log phase Raji-PDL1 cells were harvested, mitomycin C (Sigma, working concentration was 25 μg / mL) was added, incubated in an incubator for 60 min, mitomycin C-treated Raji-PDL1 cells were centrifuged and washed; PBMC stimulated with SEB for 2 days were harvested and washed; these cells were mixed and cultured according to a 1:1 cell number ratio in the presence or absence of antibodies. After 3 days, cell supernatants were harvested by centrifugation, and IL-2 and IFN-γ concentrations in the supernatants were detected by ELISA.

[0190] The results of IFN-γ secretion are shown in Figure 33. The results show that VP101(hG1DM) can effectively promote the secretion of IFN-γ, and its activity is significantly superior to that of nivolumab.

[0191] The results of IL-2 secretion are shown in Figure 34. The results show that VP101(hG1DM) can effectively promote IL-2 secretion in a dose-dependent manner, and its activity is significantly superior to that of nivolumab.

[0192] Example 11: No antibody-dependent phagocytic activity of VP101 (hG1DM) in PD-1 positive cells Antibody-dependent cellular phagocytosis (ADCP) refers to the binding of Fc fragments of antibodies bound to cell surface antigens to Fc receptors of phagocytically active cells (such as macrophages), thus mediating the phagocytosis of antibody-bound cells. For immune checkpoint inhibitor antibodies such as PD-1 antibodies, the presence of ADCP activity causes damage to PD-1-expressing immune cells that exert anti-tumor killing effects, thereby affecting their anti-tumor activity.

[0193] In this experiment, mouse macrophages were used as effector cells, and PD-1 overexpressed CHO-K1-PD1 cell line (see Example 8 for construction method) was used as target cells. The ADCP effect mediated by the cells was detected. In this experiment, flow cytometry was applied to detect the ADCP activity of VP101(hG1DM) in cells expressing PD-1, and the results show that VP101(hG1DM) had no ADCP activity, but Nivolumab, a commercial antibody against the same target, had significant ADCP activity. The method is detailed as follows: Bone marrow from femurs of C57 mice (purchased from Guangdong Medical Laboratory Animal Center) was first collected aseptically and lysed by red blood cell lysis buffer for 5 min on ice. Lysis was terminated with DMEM complete medium (containing 10% FBS), and the lysate was centrifuged at 1000 rpm and washed twice. The cell pellet was resuspended in 10 mL of DMEM complete medium, and M-CSF was added at a working concentration of 100 ng / mL. The cells were cultured for 7 days at 37°C and 5% CO2 in a cell culture chamber for induction. Half of the medium was replaced and M-CSF was added on days 3 and 5. The induction of the cells was terminated on day 7. The cells were digested with 0.25% trypsin. Macrophages were collected and centrifuged at 750×g for 5 min. The supernatant was removed, and the cells were resuspended in DMEM complete medium (containing 10% FBS) and counted. Cells were adjusted to the appropriate density and plated into 96-well conical flat-bottom plates for further use.

[0194] CHO-K1-PD1 cells were harvested by conventional methods, centrifuged at 170×g for 5 min, resuspended, counted, and viability determined. Cells were washed once with PBS. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM in PBS, and cells were resuspended in the appropriate amount of diluted CFSE (staining density: 10,000,000 cells / mL) and incubated in an incubator for 20 min. Staining was stopped by adding 6 mL of DMEM complete medium (containing 10% FBS). Cells were centrifuged at 170×g for 5 min, the supernatant was removed; 1 mL of DMEM complete medium was added. Cells were incubated in an incubator for 10 min. Antibodies were diluted in DMEM complete medium to 20 μg / mL, 2 μg / mL and 0.2 μg / mL (working concentrations were 10 μg / mL, 1 μg / mL and 0.1 μg / mL) and isotype control antibodies hIgG1DM and hIgG4 were designed. Freshly induced mature macrophages were harvested, centrifuged at 750×g for 5 min and the supernatant was removed; cells were counted and transferred to a 96-well conical flat-bottom plate, centrifuged at 1000×g for 5 min and the supernatant was removed; the cell density of CHO-K1-PD1-CFSE was adjusted; the diluted antibodies and target cells were mixed according to the ratio of 50 μL:50 μL into the 96-well conical flat-bottom plate containing macrophages according to the experimental design. Cells were resuspended, mixed thoroughly and incubated at 37° C. for 2 hours in an incubator. 150 μL of 1% PBSA at normal temperature was added to each well, centrifuged at 1000×g for 5 min, and the supernatant was removed; cells were washed once with 200 μL of PBSA; APC anti-mouse / human CD11b antibody (diluted 1:500 in PBSA) was added to the corresponding samples at 100 μL / sample, mixed thoroughly, and incubated on ice for 40 min. 150 μL of 1% PBSA was added to each well, centrifuged at 1000×g for 5 min, and the supernatant was removed; each well was washed once with 200 μL of PBSA. 200 μL of 1% PBSA was added to each well for resuspension, followed by analysis using a Beckman flow cytometer.

[0195] Macrophages in the system are APCs+ Macrophages that were positive and involved in phagocytosis were APC and CFSE double positive. The phagocytosis rate was determined as the ratio of the number of double positive cells to the number of APC positive cells to evaluate antibody-dependent ADCP activity. The ADCP activity of each group, expressed as P%, was calculated according to the following formula:

[0196]

number

[0197] The results are shown in Figure 35. The results show that nivolumab had a significant ADCP effect in macrophage + CHO-K1-PD1 system; the phagocytosis rate of VP101 (hG1DM) was equivalent to that of the isotype control antibody, indicating that VP101 (hG1DM) had no ADCP effect. The results indicate that VP101 (hG1DM) may have a good antitumor effect.

[0198] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand.Various modifications and substitutions can be made in the details according to all the teachings disclosed, and all these modifications are within the protection scope of the present invention.The full scope of the present invention is provided by the appended claims and any equivalents thereof. The present disclosure provides, for example: [Section 1] a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; or the first protein functional domain is a single chain antibody and the second protein functional domain is an immunoglobulin; With respect to the immunoglobulin, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; with respect to the single-chain antibody, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; or With respect to the immunoglobulin, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; with respect to the single-chain antibody, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype; the heavy chain constant region of said immunoglobulin has mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and the affinity constant of said bispecific antibody to FcγRIIIa and / or C1q is reduced after the mutations compared to the affinity constant before the mutations; preferably, said affinity constant is measured by a Fortebio Octet system. Bispecific antibodies. [Section 2] According to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutation: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A The bispecific antibody according to item 1, comprising: [Section 3] a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; or the first protein functional domain is a single chain antibody and the second protein functional domain is an immunoglobulin; With respect to the immunoglobulin, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; with respect to the single-chain antibody, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; or With respect to the immunoglobulin, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 34 to 36, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 37 to 39, respectively; with respect to the single-chain antibody, the heavy chain variable region comprises HCDR1 to HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 28 to 30, respectively, and the light chain variable region comprises LCDR1 to LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 31 to 33, respectively; the immunoglobulin is of the human IgG1 subtype; 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 L234A, L235A and G237A having Bispecific antibodies. [Section 4] According to the EU numbering system, the heavy chain constant region of the immunoglobulin is 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 The bispecific antibody according to any one of items 1 to 3, which has one or more mutations selected from the following: [Section 5] the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from 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 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 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 SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3. The bispecific antibody according to any one of items 1 to 4. [Section 6] The bispecific antibody is selected from the group consisting of the following (1) to (12): (1) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:7; (2) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:11; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1. wherein the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:17; (4) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:7; (5) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:11; (6) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:17; (7) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; (8) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:11; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; (9) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:9 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:11; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:1 and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:3; and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 3. The bispecific antibody according to any one of items 1 to 5, wherein the bispecific antibody is selected from any one of the following: [Section 7] 7. The bispecific antibody according to any one of items 1 to 6, wherein the amino acid sequence of the heavy chain of the immunoglobulin is set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain of the immunoglobulin is set forth in SEQ ID NO: 26. [Section 8] The immunoglobulin or antigen-binding fragment thereof is -6 Less than M, e.g., about 10 -7 M, 10 -8 Less than M or 10 -9 binds to FcγRI with an affinity constant of M or less; preferably, said affinity constant is measured by the Fortebio Octet system. The bispecific antibody according to any one of items 1 to 7. [Section 9] The immunoglobulin or the antigen-binding fragment thereof is -9 Less than M, e.g., about 10 -7 M, 10 -8 Less than M or 10 -9 binds to C1q with an affinity constant of M or less; preferably, said affinity constant is measured by the Fortebio Octet system; The bispecific antibody according to any one of items 1 to 8. [Section 10] Item 10. The bispecific antibody according to any one of Items 1 to 9, wherein the first protein functional region is linked to the second protein functional region, either directly or via a linker fragment; and / or the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody, either directly or via a linker fragment. [Section 11] 11. The bispecific antibody of clause 10, wherein the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5, or 6. [Section 12] Item 12. The bispecific antibody according to any one of items 1 to 11, wherein the number of the first protein functional domain and the second protein functional domain is each independently 1, 2 or more. [Section 13] Item 13. The bispecific antibody according to any one of Items 1 to 12, wherein the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin. [Section 14] a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA A bispecific antibody comprising: the number of said first protein functional domains is 1 and the number of said second protein functional domains is 2; the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; the amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO:24 and the amino acid sequence of the light chain of said immunoglobulin is set forth in SEQ ID NO:26; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:17; the single chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; the first protein functional region is linked to the second protein functional region by a first linker fragment; the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody by a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO: 18. Bispecific antibodies. [Section 15] 15. An isolated nucleic acid molecule encoding the bispecific antibody according to any one of items 1 to 14. [Section 16] A vector comprising the isolated nucleic acid molecule of claim 15. [Section 17] 17. A host cell comprising the isolated nucleic acid molecule of paragraph 15 or the vector of paragraph 16. [Section 18] A conjugate comprising an antibody or an antigen-binding fragment thereof and a conjugation moiety, wherein the immunoglobulin is the bispecific antibody according to any one of items 1 to 14, and the conjugation moiety is a detectable label; preferably, the conjugation moiety is a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance, or an enzyme. [Section 19] A kit comprising the bispecific antibody according to any one of items 1 to 14 or the complex according to item 18; Preferably, the kit further comprises a secondary antibody capable of specifically recognizing the immunoglobulin or antigen-binding fragment thereof; optionally, the secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme. [Section 20] 20. Use of the bispecific antibody according to any one of items 1 to 14, or the conjugate according to item 18, in the preparation of a kit for detecting the presence or level of PD-1 and / or VEGFA in a sample. [Section 21] A pharmaceutical composition comprising the bispecific antibody according to any one of items 1 to 14, or the conjugate according to item 18, optionally further comprising a pharma- ceutically acceptable vector and / or excipient. [Section 22] Use of the bispecific antibody according to any one of items 1 to 14 or the conjugate according to item 18 in the preparation of a medicament for treating and / or preventing a malignant tumor; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; Preferably, said lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, said non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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. use. [Section 23] A method for treating and / or preventing a malignant tumor, comprising a step of administering an effective amount of the bispecific antibody according to any one of items 1 to 14, or the conjugate according to item 18, to a subject in need thereof; preferably, the malignant tumor is colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, digestive cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, frequent myeloma, or the like. Selected from crosatellite instability-high (MSI-H) and mismatch repair deficient (dMMR) cancers, urothelial carcinoma, mesothelioma, endometrial carcinoma, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia; Preferably, said lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, said non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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. method. [Section 24] 15. The bispecific antibody according to any one of items 1 to 14 for use in the treatment and / or prevention of a malignant tumor; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; Preferably, said lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, said non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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. Bispecific antibodies. [Section 25] 19. The conjugate according to item 18 for use in the treatment and / or prevention of malignant tumors; preferably, the malignant tumors are selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma and leukemia; Preferably, said lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, said non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; 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. Complex.

Claims

1. A bispecific antibody targeting VEGFA and PD-1, (a) a human IgG1 immunoglobulin that binds to VEGFA, wherein said human IgG1 immunoglobulin comprises two pairs of polypeptide chains, wherein each pair of polypeptide chains comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain of said human IgG1 immunoglobulin is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain of said human IgG1 immunoglobulin is set forth in SEQ ID NO:26; and (b) two single chain antibodies that bind to PD-1, wherein the amino acid sequence of the VH region of each single chain antibody is set forth in SEQ ID NO:9, and the amino acid sequence of the VL region of each single chain antibody is set forth in SEQ ID NO:17; Including, wherein one end of each single chain antibody is linked to the C-terminus of each heavy chain of said human IgG1 immunoglobulin by a first linker fragment, wherein said VH region and said VL region of each single chain antibody are linked by a second linker fragment, wherein said first linker fragment and said second linker fragment are identical or different, preferably wherein the amino acid sequences of said first linker fragment and second linker fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19, and preferably wherein the amino acid sequences of said first linker fragment and second linker fragment are set forth in SEQ ID NO:

18. Bispecific antibodies.

2. The bispecific antibody described in claim 1, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

18.

3. The bispecific antibody described in claim 1, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

19.

4. A bispecific antibody as described in claim 1, wherein the amino acid sequence of the first linker fragment is set forth in SEQ ID NO: 18 and the amino acid sequence of the second linker fragment is set forth in SEQ ID NO:

19.

5. A bispecific antibody as described in claim 1, wherein the amino acid sequence of the first linker fragment is set forth in SEQ ID NO: 19 and the amino acid sequence of the second linker fragment is set forth in SEQ ID NO:

18.

6. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 5.

7. An isolated nucleic acid molecule described in claim 6, comprising DNA.

8. A vector comprising the isolated nucleic acid molecule of claim 6 or 7.

9. The vector described in claim 8, which is an expression vector.

10. A vector described in claim 8 or 9, selected from a plasmid, a phagemid, a cosmid and an artificial chromosome.

11. The vector of claim 10, wherein the artificial chromosome is a yeast artificial chromosome, a bacterial artificial chromosome, or a P1-derived artificial chromosome.

12. The vector described in claim 8 or 9, wherein the vector is a bacteriophage selected from a lambda phage and an M13 phage.

13. The vector described in claim 8 or 9, wherein the vector is an animal virus.

14. The vector of claim 13, wherein the vector is selected from a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a pox virus, a baculovirus, a papilloma virus, and a papovavirus.

15. The vector of claim 8, wherein the vector comprises a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene, a replication initiation site, or any combination thereof.

16. A host cell comprising the isolated nucleic acid molecule of claim 6 or 7 or the vector of any one of claims 8 to 15.

17. A method comprising transforming, transducing or transfecting a host cell with a vector according to any one of claims 8 to 15.

18. A method comprising transforming, transducing or transfecting a host cell with a first vector and a second vector, wherein said first vector encodes a heavy chain polypeptide of a bispecific antibody and said second vector encodes a light chain polypeptide of said bispecific antibody, (i) the heavy chain polypeptide of the bispecific antibody comprises an amino acid sequence set forth in SEQ ID NO:24 and a single chain antibody, wherein one end of the single chain antibody is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO:24 by a first linker fragment, the amino acid sequence of the VH region of the single chain antibody is set forth in SEQ ID NO:9, the amino acid sequence of the VL region of the single chain antibody is set forth in SEQ ID NO:17, the VH region and the VL region of the single chain antibody are linked by a second linker fragment, and the first linker fragment and the second linker fragment are the same or different; and (ii) the amino acid sequence of the light chain polypeptide of the bispecific antibody is set forth in SEQ ID NO: 26; Preferably, wherein the amino acid sequences of said first linker fragment and said second linker fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19, preferably wherein the amino acid sequences of said first linker fragment and said second linker fragment are set forth in SEQ ID NO:

18. method.

19. The method described in claim 18, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

18.

20. The method described in claim 18, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

19.

21. The method described in claim 18, wherein the amino acid sequence of the first linker fragment is set forth in SEQ ID NO: 18 and the amino acid sequence of the second linker fragment is set forth in SEQ ID NO:

19.

22. The method described in claim 18, wherein the amino acid sequence of the first linker fragment is set forth in SEQ ID NO: 19 and the amino acid sequence of the second linker fragment is set forth in SEQ ID NO:

18.

23. The host cell of claim 16, wherein the host cell is selected from a prokaryotic cell, a fungal cell, an insect cell and an animal cell.

24. The host cell of claim 23, wherein the host cell is selected from Escherichia coli, Bacillus subtilis, yeast cells, Aspergillus, S2 Drosophila cells, Sf9 cells, fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells and isolated human cells.

25. The host cell described in claim 24, wherein the host cell is a CHO cell.

26. The method of any one of claims 17 to 22, wherein the host cell is selected from a prokaryotic cell, a fungal cell, an insect cell and an animal cell.

27. The method of claim 26, wherein the host cell is selected from Escherichia coli, Bacillus subtilis, yeast cells, Aspergillus, S2 Drosophila cells, Sf9 cells, fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells and isolated human cells.

28. The method of claim 27, wherein the host cell is a CHO cell.

29. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 5, further comprising a pharma- ceutical acceptable vector and / or excipient.

30. Use of a bispecific antibody according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 29 in the preparation of a medicament for treating and / or preventing malignant tumors.

31. The malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia; Preferably, said lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, said non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the renal tumor is a renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer.

31. The use according to claim 30.

32. The use described in claim 31, wherein the lung cancer is non-small cell lung cancer.

33. The use of claim 31, wherein the lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer.

34. (1) A medicine or drug for blocking the binding of VEGFA to VEGFR2. Medicaments or agents for downregulating VEGFA activity or levels; A medicine or agent for reducing VEGFA stimulation of vascular endothelial cell proliferation; A medicine or drug for inhibiting vascular endothelial cell proliferation, or medicines or agents for blocking tumor angiogenesis; and / or (2) A drug or agent for blocking the binding of PD-1 to PD-L1; Medicaments or agents for downregulating the activity or levels of PD-1; A medicine or drug for reducing immunosuppression of PD-1 in a living body; A medicine or agent for promoting IFN-γ secretion in T lymphocytes, or Medicine or agent for promoting IL-2 secretion in T lymphocytes 6. Use of a bispecific antibody according to any one of claims 1 to 5 in the preparation of a

35. A bispecific antibody according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 29 for use in the treatment and / or prevention of malignant tumors.

36. The malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor, cervical cancer, esophageal cancer, microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia; Preferably, said lung cancer is non-small cell lung cancer or small cell lung cancer; preferably, said non-small cell lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the renal tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; 36. The bispecific antibody or pharmaceutical composition of claim 35, wherein preferably said urothelial cancer is bladder cancer.

37. The bispecific antibody or pharmaceutical composition of claim 36, wherein the lung cancer is non-small cell lung cancer.

38. The bispecific antibody or pharmaceutical composition of claim 36, wherein the lung cancer is EGFR and / or ALK sensitive mutant non-small cell lung cancer.

39. (1) Blocking the binding of VEGFA to VEGFR2, downregulating VEGFA activity or levels; Attenuating VEGFA stimulation of vascular endothelial cell proliferation; Inhibition of vascular endothelial cell proliferation, or Blocking tumor angiogenesis; and / or (2) blocking the binding of PD-1 to PD-L1; downregulating PD-1 activity or levels; Relief of PD-1 immunosuppression in vivo, Stimulation of IFN-γ secretion in T lymphocytes, or Stimulation of IL-2 secretion in T lymphocytes; A bispecific antibody according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 29 for use in 40. A method for preparing a bispecific antibody according to any one of claims 1 to 5, comprising culturing a host cell according to claim 16 under suitable conditions, and isolating the bispecific antibody from the cell culture.

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