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

Abstract

To provide anti-VEGFA-anti-PD-1 bispecific antibodies, and to provide uses thereof.SOLUTION: An anti-VEGFA-anti-PD-1 bispecific antibody comprises 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 antibody according to the EU numbering system. After the mutations, the affinity constants of the bispecific antibody with FcγRI, FcγRIIa, FcγRIIIa, and / or C1q are reduced compared to the affinity constants before the mutations. The bispecific antibody specifically binds to VEGFA and PD-1, specifically alleviates VEGFA and PD-1 immunosuppression in vivo, and can inhibit tumor-induced angiogenesis, thereby exhibiting promising applications.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Technical Field The present invention relates to the fields of tumor therapy and immunobiology, and relates to an anti-PD-1-anti-VEGFA bispecific antibody, its pharmaceutical composition, and its use. More specifically, 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 threats in today's world and are the second leading cause of death among various diseases. Their incidence has increased significantly in recent years. Malignant tumors are characterized by poor response to treatment, a high rate of late-stage metastasis, and a poor prognosis. Conventional treatment methods currently used clinically (e.g., radiation therapy, chemotherapy, and surgery) can significantly alleviate pain and prolong survival, but these methods have significant limitations, and further improvement of their effectiveness is difficult.

[0003] Tumor growth occurs in two distinct phases: a slow, non-vascularized growth phase and a rapid, vascularized growth phase. Angiogenesis allows tumors to obtain sufficient nutrients to complete the vascular switch phase; without angiogenesis, primary tumors are less than 1–2 mm in size and therefore cannot metastasize.

[0004] Vascular endothelial growth factor (VEGF) is a growth factor that promotes the division and proliferation of endothelial cells, promotes the formation of new blood vessels, and improves vascular permeability. It acts by binding to vascular endothelial growth factor receptors on the cell surface and activating tyrosine kinase signaling pathways. In tumor tissue, tumor cells, as well as tumor-infiltrating macrophages and mast cells, secrete high levels of VEGF, which paracrinely stimulates tumor vascular endothelial cells, promotes endothelial cell proliferation and migration, induces angiogenesis, promotes continued tumor growth, enhances vascular permeability, causes fibrin deposition in surrounding tissues, and promotes the infiltration of mononuclear cells, fibroblasts, and endothelial cells, which facilitates the formation of tumor stroma and the invasion of new blood vessels by tumor cells, as well as tumor metastasis. Therefore, inhibiting tumor angiogenesis is currently considered one of the most promising tumor treatment strategies. 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 structurally similar and belong to the tyrosine kinase superfamily, consisting of an extramembrane region, a transmembrane segment, and an intramembrane region. The extramembrane region is composed of an immunoglobulin-like domain, and the intramembrane region is a tyrosine kinase domain. VEGFR1 and VEGFR2 are located primarily on the surface of vascular endothelial cells, while VEGFR3 is located primarily on the surface of lymphatic endothelial cells.

[0005] Molecules in the VEGF family have different affinities for these receptors. VEGFA mainly acts in conjunction with VEGFR1, VEGFR2, and NRP-1. VEGFR1, the earliest discovered receptor, has a higher affinity for pVEGFA than VEGFR2 under normal physiological conditions, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Birth Health and Heredity, 2016, Vol. 24(5): pp. 146-148).

[0006] VEGFR2 is a key regulator of angiogenesis and vascular engineering, possessing much higher tyrosine kinase activity than VEGFR1. After binding to its ligand VEGFA, VEGFR2 mediates endothelial cell proliferation, differentiation, and angiogenesis, as well as vascular 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 signaling pathway, thereby promoting endothelial cell proliferation (Takahashi T et al., Oncogene, 1999, 18(13):2221-2230).

[0007] VEGFR3 is a member of the tyrosine kinase family and is primarily expressed in embryonic vascular endothelial cells and adult lymphatic endothelial cells. VEGFC and VEGFD bind to VEGFR3 to stimulate lymphatic endothelial cell proliferation and migration, promoting lymphangiogenesis. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transmit biological signals 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(5): pp. 146-148).

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

[0009] Currently, there are several humanized monoclonal antibodies that target 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 death receptor-1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein surface receptor belonging to the CD28 immunoglobulin superfamily and is commonly 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 primarily expressed on T cells, B cells, DCs, microvascular endothelial cells, and various tumor cells, whereas PD-L2 is expressed exclusively on antigen-presenting cells, such as dendritic cells and macrophages. Interaction between PD-1 and its ligand can suppress lymphoid activation, T cell proliferation, and cytokine secretion, such as IL-2 and IFN-γ.

[0011] Numerous studies have shown that the tumor microenvironment can protect tumor cells from damage by immune cells, that PD-1 expression is upregulated on 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, PD-L1 expression in tumors is significantly associated with poor prognosis in 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 demonstrated that PD-1 Or antibodies targeting PD-L1 bind to 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] Furthermore, anti-PD-1 antibodies can also be used in the treatment of chronic viral infections, which are often accompanied by loss of function and a decrease in the number of virus-specific effector T cells. Blocking the interaction between PD-1 and PD-L1 by injecting PD-1 antibodies can effectively suppress the depletion of effector T cells in chronic viral infections.

[0013] The broad anti-tumor potential and surprising efficacy of PD-1 antibodies have led to the development of antibodies targeting the PD-1 pathway 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 lymphoma and anemia (Held SA, Heine A. et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets 2013 Sep;13(7):768-74), and is widely accepted in the industry as a breakthrough in tumors with high microsatellite instability (MSI-H) or deficient mismatch repair (dMMR) (several anti-PD-1 antibody drugs have been approved by the FDA for the treatment of tumors with MSI-H / dMMR characteristics).

[0014] Current combinations of antiangiogenic therapy and immune checkpoint inhibitors have shown favorable efficacy in many tumors, including 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; 12(2):194-207), renal cell carcinoma (Dudek AZ et al., J Clin Oncol., 2018; 36(Suppl. Abstract, 4558)), and hepatocellular carcinoma (Stein S, et al., J Clin Oncol, 2018, 36(15_Suppl):4074; bevacizumab in combination with atezolizumab for use in the treatment of hepatocellular carcinoma, approved by the FDA in 2020, colorectal cancer (including MSI-H / dMMR and non-MSI-H / dMMR types) (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; Abstract p. 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 nivol umab 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 PD1-03. Combinations of anti-VEGF antibodies (e.g., bevacizumab) and PD-1 / PD-L1 antibodies (e.g., nivolumab, pembrolizumab, and atezolizumab) for use in the treatment of patients with HER2-negative metastatic breast cancer. Combinations of VEGFR2 antibodies and PD-1 antibodies also showed good antitumor effects in gastric and gastroesophageal junction adenocarcinoma (Herbst RS et al., Lancet Oncol. 2019;20(8):1109-1123; In addition, a combination regimen of a PD-1 antibody (pembrolizumab) and an angiogenesis inhibitor (lenvatinib) showed good efficacy in the treatment of endometrial cancer and was approved by the FDA for use in the treatment of endometrial cancer in 2019.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 was approved by the FDA for use in the treatment of bladder cancer in 2017), esophageal cancer (Kato K. et al., Lancet Oncol., 2019;20(11):1506-17), mesothelioma (Scherpereel A et al., Lancet Oncol., 2019;20(2):239-253), 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 anti-tumor effects.

[0015] Bispecific antibodies, also known as diabodies, are specific drugs that simultaneously target two different antigens and can be produced by immunoselective purification. Furthermore, bispecific antibodies can also be produced by genetic engineering, which has particular advantages due to the flexibility in terms of optimizing the binding site, considering the synthesis type, and yield. Currently, more than 45 types of bispecific antibodies have been reported (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). Due to its similarity to the naturally occurring IgG format and advantages in antibody engineering, expression, and purification, it has been demonstrated to be one of the ideal types of bispecific antibodies (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 therapeutic antibodies targets ting 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 the 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] Complement-dependent cytotoxicity (CDC) refers to the specific binding of antibodies to antigens on the corresponding cell membrane surface, forming complexes and activating the complement system, which further forms MAC on the surface of target cells, resulting in subsequent lysis of the target cells. Complement can cause the lysis of various bacteria and other pathogens, and is an important defense mechanism against pathogen infection.

[0018] Fc receptors belong to the immunoglobulin family and are expressed on the surface of specific immune cells to recognize the Fc region of antibodies and mediate immune responses. After the Fab region recognizes an antigen, the Fc region of the antibody binds to the Fc receptor on the immune cell (e.g., killer cell) to initiate immune cell response functions such as phagocytosis and ADCC.

[0019] Fc receptors are primarily classified into three types: FcγR, FcαR, and FcεR, depending on the type of antibody recognized by the Fc receptor and the type of cell on which they are expressed. 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 effects. FcγRIII is the primary molecule mediating ADCC and has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. Within the FcγRIIIa population, there are two subtypes distinguished by single nucleotide polymorphism (SNP): high-affinity FcγRIIIa_V158 and low-affinity FcγRIIIa_F158. FcγRI has a 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), of which FcγRIIa has ADCC activity; in humans, two subtypes of FcγRIIa exist, FcγRIIa_H131 and FcγRIIa_R131, due to a single nucleotide mutation (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, Vol. 11(Issue 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 pharmaceuticals. 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 a higher CDC effect than IgG1. IgG4 molecules bind weakly to FcγRs other than FcγRI, and IgG4 molecules have a lower potential to induce 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 creative 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 a Fortebio Octet system. This relates to bispecific antibodies.

[0024] In one or more embodiments of the invention, for bispecific antibodies, the heavy chain constant region of an 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 It has.

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

[0026] The present invention provides 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 provides 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 heavy chain constant region of an immunoglobulin may contain the following mutations: 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 bispecific antibodies, the heavy chain constant region of an immunoglobulin according to the EU numbering system 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 gene 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 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 light 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; Bispecific antibodies are provided.

[0030] In one or more embodiments of the present invention, the bispecific antibody has one of the following structures (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, 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: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. 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, 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: P01861.1. CESSION:P01834.

[0035] In one embodiment of the present invention, the amino acid sequence of the heavy chain constant region Ig gamma-1 chain C region (ACCESSION: P01857) is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40) In one embodiment of the present 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 present 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 of the single chain antibody. 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 present 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 single-chain antibody heavy chain variable region is linked, either directly or by a linker fragment, to a single-chain antibody light chain variable region.

[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 present 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 provides 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 linked to a second protein functional region by a first linker fragment; a single-chain antibody heavy chain variable region linked to a single-chain antibody light chain variable region 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 M or less; 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, e.g. 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 VEGFA protein and / or PD-1 protein at K 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 EC 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 binds to PD-1 protein in 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 host cells containing the isolated nucleic acid molecules or vectors 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 embodiment of the invention or comprising a conjugate of the invention; 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. 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 conjugate according to any of the embodiments of the invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable vector and / or excipient.

[0055] The bispecific antibodies of the invention or pharmaceutical compositions of the 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 compositions of the invention are sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: a required amount of the bispecific antibody of the invention is added to an appropriate 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. Additionally, sterile injectable solutions may be prepared in the form of sterile lyophilized powders (e.g., vacuum-dried or freeze-dried) for convenient storage and use. Such sterile, lyophilized powder can be dispersed in a suitable vector (e.g., sterile, pyrogen-free water) before use.

[0056] Furthermore, bispecific antibodies of the invention can be present in a pharmaceutical composition in unit dose form for ease of 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 liquid (e.g., injectable) form, it can contain the bispecific antibodies of the invention at a concentration of at least 0.1 mg / mL, e.g., 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] A 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., powder, ointment, 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, a 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 pharmaceutically effective agents (e.g., tumor chemotherapeutic agents). Such additional pharmaceutically effective agents can be administered before, simultaneously with, or after administration of the bispecific antibodies of the invention or the pharmaceutical compositions of the invention.

[0059] In the present invention, dosage regimens can be adjusted to achieve the optimum desired response (e.g., a therapeutic or prophylactic response), e.g., it can be a single dose or multiple doses administered over a period of time, and can be characterized by proportionally reducing or increasing the dose as the therapeutic exigencies dictate.

[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 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. Regarding use.

[0061] Yet another aspect of the present invention relates to the use of a bispecific antibody according to any embodiment 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, medicines or drugs 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; medicines or agents for downregulating the activity or levels of PD-1; Medicines or drugs for reducing immunosuppression of PD-1 in vivo; A medicine or agent for promoting IFN-γ secretion in T lymphocytes, or Medicine or drug for promoting IL-2 secretion in T lymphocytes The present invention relates to the use of a bispecific antibody according to any of the embodiments of the invention or a conjugate of the invention in the preparation of a

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

[0063] Yet another aspect of the invention is a method for treating and / or preventing malignant tumors, comprising the step of administering an effective amount of a bispecific antibody according to any embodiment 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. Regarding 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 to 50 mg / kg, e.g., 0.1 to 50 mg / kg, 0.1 to 25 mg / kg, or 1 to 10 mg / kg. It should be noted that dosages may vary depending on the type and severity of the condition being treated. Furthermore, for any particular patient, specific dosing regimens will be adjusted over time according to the patient's needs and the physician's professional judgment; those skilled in the art will understand 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, for example a human. Yet another aspect of the present invention is a method for producing a semiconductor device comprising: (1) a method for detecting the level of VEGFA in a sample; A method for blocking the binding of VEGFA to VEGFR2, Methods for downregulating VEGFA activity or levels, A method 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, A method 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 VEGFA binding to VEGFR2 Downregulation of VEGFA activity or levels, Attenuation of VEGFA stimulation of vascular endothelial cell proliferation, Inhibition of vascular endothelial cell proliferation, or Blockade of tumor angiogenesis; and / or (2) blocking the binding of PD-1 to PD-L1; Downregulation of PD-1 activity or levels, Alleviating PD-1 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 the treatment of various diseases. The conventional experimental methods for obtaining these therapeutic antibodies are to immunize animals with an antigen, obtain antibodies targeting the antigen in the immunized animals, or improve those antibodies with low affinity for the antigen by affinity maturation.

[0069] The variable regions of the light and heavy chains determine antigen binding; each chain variable region contains three hypervariable regions called complementarity-determining regions (CDRs). The CDRs of the heavy chain (H chain) include HCDR1, HCDR2, and HCDR3, while the CDRs of the light chain (L chain) include LCDR1, L These include CDR2 and LCDR3, as designated by Kabat et al., Bethesda Md., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 1991, (Vols. 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, Suppl. 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 in accordance with the IMGT standard, 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 (SEQ ID NO: 37) LCDR2:RAN (SEQ ID NO: 38) LCDR3: LQYDEFPLT (SEQ ID NO: 39) (3) VP101(hG1WT) or VP101(hG1DM) The amino acid sequences of the nine CDR regions of the heavy chain are as follows: HCDR1: GYTFTNYG (SEQ ID NO: 28) HCDR2: INTYTGEP (SEQ ID NO: 29) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 30) HCDR4: GFAFSSYD (SEQ ID NO: 34) HCDR5: ISGGGRYT (SEQ ID NO: 35) HCDR6: ANRYGEAWFAY (SEQ ID NO: 36) HCDR7: QDINTY (SEQ ID NO: 37) HCDR8:RAN (SEQ ID NO: 38) HCDR9: LQYDEFPLT (SEQ ID NO: 39) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDISNY (SEQ ID NO: 31) LCDR2:FTS (SEQ ID NO: 32) LCDR3: QQYSTVPWT (SEQ ID NO: 33) For the antibody VP101(hG1DM) of the present 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. VP101(hG1DM) is obtained by introducing point mutations from leucine to alanine at position 234 (L234A) and from leucine to alanine at position 235 (L235A) in the hinge region of the heavy chain.

[0073] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the laboratory procedures used herein for cell culture, molecular genetics, nucleic acid chemistry and immunology are routine methods widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of relevant 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 mouse IgG or human IgG Fc protein fragments (mFc or hFc). However, those skilled in the art will understand 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 occurring or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "VEGFA protein" is intended to include all such sequences, including their natural or artificial variants. Furthermore, when a sequence fragment of VEGFA protein is described, this also includes the corresponding sequence fragment of the 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, excluding the last six His residues).

[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, the extracellular fragment of VEGFR2, VEGFR2-ECD, or a fragment containing VEGFR2-ECD. This also includes fusion proteins of VEGFR2-ECD, such as fragments fused to the Fc protein fragments (mFc or hFc) of mouse IgG or human IgG. However, those skilled in the art will understand 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 occurring or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "VEGFR2 protein" is intended to include all such sequences, including natural or artificial variants thereof. Furthermore, when a sequence fragment of a VEGFR2 protein is described, this also includes the corresponding sequence fragment of the 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 refers to VEGFR1 and / or VEGFR2; its specific protein sequence is a known sequence in the prior art, and can refer to the sequences 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, the extracellular fragment of PD-1, PD-1ECD, or fragments containing PD-1ECD, and 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, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of PD-1 protein can be naturally occurring or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" is intended to include all such sequences, including naturally occurring or artificially engineered variants thereof. Furthermore, when a sequence fragment of PD-1 protein is described, this also includes the corresponding sequence fragment of that naturally occurring or artificially engineered variant.

[0078] As used herein, the term EC 50 means the concentration for 50% of the maximum effect, ie the concentration that causes 50% of the maximum effect.

[0079] As used herein, the term "antibody" refers to an immunoglobulin molecule generally consisting 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 connected by a "J" region of about 12 or more amino acids, and 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 region 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. Amino acid assignments to regions or domains can be found in the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or in Chothia & Lesk J. Mol. Biol. 196(1987):901-917; Chothia et al. Nature 342 (1989): pp. 878-883, or the IMGT numbering system definitions, 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, Suppl. 1 (2009): pp. D301-D307. In particular, the heavy chain may also comprise more than three CDRs, for example, six, nine, or twelve CDRs. For example, in the bispecific antibodies of the present invention, the heavy chain may be an ScFv having 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 intended to be limited by any particular method for producing the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of a different isotype, for example, IgG (e.g., subtype 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, Chapter 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'), 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 on the polypeptide.

[0081] As used herein, the term "Fd fragment" refers to a V H Domain and C H1 The term "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 an antibody is V L Domains and V HSingle-chain antibodies (e.g., scFvs) are monovalent molecules in which the domains are paired together via a linker that allows for the creation 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 The linker may have -COOH. Suitable linkers in the prior art consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, and variants thereof can also be used (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 an antibody is V H Domains and V L Diabodies, or bivalent antibodies, are antibodies in which the domains are expressed on a single polypeptide chain. However, the linker used is too short to allow pairing between the two domains on the same chain, so the domains pair with complementary domains on another chain, creating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 2004, 103:111-114, 2004). d. Sci. USA 90 (1993):6444-6448, and Poljak RJ et al., Structure 2 (1994):1121-1123).

[0084] Antigen-binding fragments of antibodies (e.g., the antibody fragments referred to above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage), and antigen-binding fragments of antibodies can be screened for specificity in the same manner as are 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 derived from a highly homogeneous group of antibodies, i.e., derived from a group of identical antibody molecules, excluding spontaneous mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen, as opposed to a monoclonal antibody. Monoclonal antibodies can generally be obtained by hybridoma technology, first reported by Kohler et al. (Nature, Vol. 256:495, 1975), or by recombinant DNA technology (see, e.g., U.S. Pat. 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 derived 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 derived 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 regarding 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." Epitopes or antigenic determinants generally consist of chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, epitopes generally contain a unique space. A conformational protein may contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a conformational protein, which may be "linear" or "conformational." See, e.g., Epitope Mapping Protocols See Methods in Molecular Biology, Vol. 66, edited by G. E. Morris (1996). In a linear epitope, all of the interaction sites between the protein and the interacting molecule (e.g., an antibody) are located linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites are located across amino acid residues of the protein that are separated from one another.

[0090] As used herein, the term "isolated" means to be obtained by artificial means from natural state.A certain "isolated" substance or component may occur in nature, or its natural environment may be changed, or it may be isolated from the natural environment, or both.For example, a certain non-isolated polynucleotide or polypeptide naturally occurs in a certain living animal, and the same polynucleotide or polypeptide with high purity isolated from such natural state is called 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 elements 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 phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., 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 a replication origin.

[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 S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK293 cell, or 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 its target antigen. 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 at a rate of 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 (K D ) means to bind to an antigen. In some embodiments of the invention, the term "target" means to specifically bind.

[0094] As used herein, the term "K D " refers to the dissociation equilibrium of a specific antibody-antigen interaction. The equilibrium dissociation constant (Eq. 10) is used to describe the binding affinity between an antibody and an antigen. A smaller equilibrium dissociation constant indicates stronger antibody-antigen binding and higher affinity between the antibody and the antigen. Generally, antibodies have an equilibrium dissociation 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 "pharmaceutically acceptable auxiliary substance" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 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; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0097] As used herein, the term "adjuvant" refers to a nonspecific immunostimulatory agent that can enhance the immune response of a living body to an antigen or, when delivered to the living body together with or prior to an antigen, can alter the type of immune response. 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 achieve, or at least partially achieve, 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., a 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., a 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. Determining such an effective amount is undoubtedly within the skill of one in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, e.g., age, weight, and sex, the route of administration, and other concurrently administered treatments.

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

[0100] MSI-H and dMMR represent the results of two different assays, which are biologically consistent and are referred to as MSI-H / dMMR or MSI-high / dMMR, while MSI-L and MSS are phenotypes of pMMR. dMMR detection involves performing an immunohistochemical assay of protein expression for four mismatch genes, MSH2, MLH1, MSH6, and PMS2, on tumor specimens (including surgical and aspirate specimens). Absence of any of the four proteins confirms dMMR, while positive results for all four proteins indicate pMMR, i.e., complete mismatch repair function. MSI detection involves matching and comparing the lengths 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 known as next-generation sequencing or NGS) can also be used as a method to detect microsatellite instability. When more microsatellite loci are selected for PCR assays, e.g., more than five loci or additional microsatellite loci, mismatches at ≥30% of 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 to 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 attenuating the immunosuppressive and angiogenic promoting effects of VEGFA in vivo.

[0104] (4) The bispecific antibodies 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 alleviating the immunosuppression induced by PD-1 in vivo and activating the immune response. [Brief explanation of the drawings]

[0105] [Figure 1] Affinity constant assay of VP101 (hG1DM) 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 2] Affinity constant assay of bevacizumab 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 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 and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 5]Affinity constant assay of VP101 (hG4WT) for 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 6] Affinity constant assay of VP101 (hG1DM) to FcγRIIa_H131. Antibody concentrations for the top and 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 and 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 and 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 and 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) 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 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 and 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 and 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 and 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) for FcγRIIIa_V158. Antibody concentrations for the top and 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. The antibody concentrations for the top and 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 and 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) for FcγRIIIa_V158. The antibody concentrations for the top and 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) for FcγRIIIa_F158. The 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 and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 23] Affinity constant assay of nivolumab to FcγRIIIa_F158. Antibody concentrations for the top and 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) for FcγRIIIa_F158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 25] Affinity constant assay of VP101 (hG4WT) for FcγRIIa_F158. Antibody concentrations for the top and 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) for C1q. Antibody concentrations for the top and 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 and 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 for C1q. Antibody concentrations for the top and 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) for C1q. Antibody concentrations for the top and bottom curve pairs are 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. [Figure 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. [Figure 31] ADCC activity assay of VP101(hG1WT) and VP101(hG1DM) in the CHO-K1-PD1 target cell line expressing the PD-1 antigen. [Figure 32] CDC activity assay of VP101(hG1WT) and VP101(hG1DM) in the CHO-K1-PD1 target cell line expressing the PD-1 antigen. [Figure 33] Effect of antibody VP101 (hG1DM) on the secretion of cytokine IFN-γ induced by mixed culture of PBMC cells and Raji-PDL1 cells, detected by ELISA. [Figure 34] Effect of antibody VP101 (hG1DM) on the secretion of cytokine IL-2 induced by mixed culture of PBMC cells and Raji-PDL1 cells, detected by ELISA. [Figure 35] ADCP activity assay of VP101(hG1DM) in the CHO-K1-PD1 target cell line expressing the PD-1 antigen. DETAILED DESCRIPTION OF THE INVENTION

[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 to be construed as limiting the scope of the present invention. If there is no explicit 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. If the reagents or equipment used are not specified by their manufacturers, they are all conventional products that are commercially available.

[0107] In the following examples of the present invention, the commercially available 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 commercially available 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 sequence is derived from a study published by Acierno et al., titled "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 the 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 Chinese Patent Publication No. CN1259962A discloses 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: (369 bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGA CTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC (SEQ ID NO: 2) Amino acid sequence of the light chain variable region of bevacizumab (bevacizumab-Lv): (107aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 3) Nucleotide sequence encoding the light chain variable region of bevacizumab: (321 bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCA CTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG (SEQ ID NO: 4) The heavy chain constant regions were all Ig gamma-1 chain C regions, ACCESSION: P01857; the light chain constant regions were all Ig kappa chain C regions, ACCESSION: P01834.

[0111] The heavy and light chain cDNAs of bevacizumab were cloned into the vector pcDNA3.1 to obtain a recombinant expression plasmid for the 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 obtained in this way. Preparative Example 2: Sequence Design of Anti-PD-1 Antibody 14C12 and Its Humanized Antibody 14C12H1L1, and 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: (118 aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 5) Nucleotide sequence encoding the heavy chain variable region of 14C12: (354 bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTAT CCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 6) Amino acid sequence of the light chain variable region of 14C12: (107 aa) 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: (118 aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 9) Nucleotide sequence encoding the heavy chain variable region of 14C12H1L1: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAG GCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 10) Amino acid sequence of the light chain variable region of 14C12H1L1: (107 aa) 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: (448 aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13) Nucleotide sequence encoding the heavy chain (14C12H1) of 14C12H1L1: (1344 bp) Amino acid sequence of the light chain (14C12L1) of 14C12H1L1: (214 aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15) Nucleotide sequence encoding the light chain of 14C12H1L1 (14C12L1): (642 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCAGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCT GAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAG GAGAGTGTGACTGAACAGGACTCAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 16) (3) Heavy and light chain variable region sequences of 14C12H1L1(M) Individual amino acids in the framework region (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 bispecific antibody of the present invention has a Morrison type (IgG-scFv) structure, i.e., the C-termini of the two heavy chains of an IgG antibody are each linked to the 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 light chain variable region of 14C12H1L1(M) are considered to be the ScFv fragment portion, is referred to as VP101(M). Compared to 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves its efficacy.

[0117] [Table 1]

[0118] In Table 1 above: (1) Those with a "V" in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. 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 sequences and the nucleotide sequences encoding them.

[0119] (2) The amino acid sequence of linker 1 is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18). Optionally, the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 19) is added to the aforementioned Linker 1 Alternatively, it can be used as linker 2.

[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] The plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested with HindIII and EcoRI, and the heavy and light chains isolated by electrophoresis were subcloned into the vector pcDNA3.1. 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 using elution buffer, and the target sample antibody VP101 was isolated 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. The 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): (453 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20) Nucleotide sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG1WT): (1359 bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCA ACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAACTCCTCGGAGGCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGC CATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCT CTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCC GAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTTCCTGTACAGCAAACTGACCGTCGATAAATCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTCCGTGATGCATGAAGCACTGCACAACCATTATACCCAGAAGTCTCTGAGCCTGTCCCCCGGCAAG (SEQ ID NO: 21) 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 in VP101 (hG4WT): (1350 bp) Preparation Example 4: Non-variable region amino acid mutation design 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 moiety in 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 portion in VP101 (hG1DM): (1359 bp, mutation positions underlined) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCTGGAGGCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAA ACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGC CAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACC CCTCCTGTCCTCGACTCCGACGGCTCCTTCTTTCCTGTACAGCAAACTGACCGTCGATAAATCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTCCGTGATGCATGAAGCACTGCACAACCATTATACCCAGAAGTCTCTGAGCCTGTCCCCCGGCAAG (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 moiety in VP101 (hG1DM): (214 aa) 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) [Examples]

[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 participates in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic monoclonal antibody 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 for FcγRI using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 1 μg / mL FcγRIa was immobilized on a HIS1K sensor for 50 seconds. The sensor was equilibrated in buffer for 60 seconds, and binding of CD64 immobilized on the sensor to the antibody at concentrations ranging from 3.12 to 50 nM (serial two-fold dilutions) was determined for 120 seconds. The antibody was allowed to dissociate in buffer for 120 seconds. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 seconds 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 the affinity constant.

[0133] The affinity constants of FcγRI for VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as for 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; 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; and VP101(hG1DM) has no binding to FcγRI or an extremely 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 that VP101(hG1DM) does not bind to FcγRI, and the binding activity of VP101(hG1DM) is 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) binds to the Fc fragment of IgG antibodies and can 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 a 60-second immobilization time. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA (pH 7.4) for 600 seconds, followed by blocking. The binding of FcγRIIa_H131 to the immobilized antibody at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in the buffer for 60 seconds. 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 were analyzed by fitting with a 1:1 model to obtain the affinity constant.

[0141] The affinity constants of FcγRIIa_H131 for VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as for 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 Therefore, the results were not analyzed and no corresponding data were 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; however, nivolumab had no binding to FcγRIIa_H131 or an extremely 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 to 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 a 60-second immobilization time. The sensor was equilibrated in a buffer containing PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA (pH 7.4) for 600 seconds, followed by blocking. The binding of FcγRIIa_R131 to the immobilized antibody at concentrations ranging from 12.5 to 200 nM (serial two-fold dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in the buffer for 60 seconds. 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 were analyzed by fitting with a 1:1 model to obtain the affinity constant.

[0149] The affinity constants of FcγRIIa_R131 for VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as for 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; 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 for 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 the 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 hFCGR2B-his was immobilized on the NTA sensor for a 60-second immobilization time. The sensor was equilibrated in a buffer of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA (pH 7.4) for 600 seconds of blocking, and 12.5–20 μg / mL of the immobilized hFCGR2B-his was added to the NTA sensor. Binding to the antibody at a concentration of 0 nM (serial two-fold dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in buffer for 60 seconds. 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 were analyzed by fitting with a 1:1 model to obtain the affinity constant.

[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 to 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 an antibody for FcγRIIIa_V158 using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on a HIS1K sensor for 120 seconds. The sensor was equilibrated in buffer for 60 seconds, and binding of the immobilized hFcGR3A(V158)-his antibody at concentrations ranging from 31.25 to 500 nM (serial two-fold dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in buffer for 60 seconds. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 seconds 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 the affinity constant.

[0160] The affinity constants of FcγRIIIa_V158 for VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as for 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; therefore, the result was not analyzed and no corresponding data was obtained.

[0163] The results showed that VP101(hG1WT) could bind to FcγRIIIa_V158 with an affinity constant of 4.35E-08M; VP101(hG4DM) could 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; whereas nivolumab and VP101(hG4WT) have no or very low binding signals to FcγRIIIa_V158, and 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, the 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 to 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 PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_F158 was immobilized on a HIS1K sensor for 120 seconds. The sensor was equilibrated in buffer for 60 seconds, and binding of the immobilized hFcGR3A(F158)-his antibody at concentrations ranging from 31.25 to 500 nM (serial two-fold dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in buffer for 60 seconds. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 seconds 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 for 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; 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 or very low binding signals to FcγRIIIa_F158, and therefore the results were not analyzed and corresponding data were not 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 antibody.

[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 for 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 a FAB2G sensor at an immobilization height of approximately 2.0 nm. The sensor was equilibrated in buffer for 60 seconds, and the binding of the antibody immobilized on the sensor to C1q at concentrations ranging from 0.625 to 10 nM (serial two-fold dilutions) was determined for 60 seconds. The antigen and antibody were allowed to dissociate in buffer for 60 seconds. The sensor was refreshed four times in 10 mM glycine, pH 1.7, for 5 seconds each. The sample plate shaking speed was 1000 rpm, the temperature was 30 °C, and the frequency was 0.6 Hz. Data were analyzed using a 1:1 model. The data were collected using Fortebio Data Acquisition 7.0, and the data were analyzed using Fortebio Data Analysis 7.0.

[0176] The affinity constants of C1q for VP101(hG1WT), VP101(hG4WT), and VP101(hG1DM), as well as for 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; 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 or very low binding signals to C1q, and therefore the results were not analyzed and no corresponding data were obtained.

[0180] The results show that nivolumab, VP101(hG4WT) and VP101(hG1DM) do not bind to C1q, while 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 co-culture system 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 the PD-1 antigen is as follows: In this experiment, we first constructed the human PD-1 overexpression vector pCDH-CMV-PD1FL-Puro (pCDH-CMV-Puro purchased from Youbio), packaged the expression vector into a virus, and then infected CHO-K1 cells to generate stable drug-resistant PD-1 cells. CHO-K1-PD1 stable cell lines expressing membrane PD-1 protein were obtained after treatment and screening with puromycin (2 μg / mL). Normal human PBMCs were isolated using a Ficoll peripheral blood mononuclear cell isolation system. The isolated PBMCs were resuspended in 1640 complete medium, stained with trypan blue, and counted to determine cell viability. The cells were incubated overnight at 37°C and 5% CO2 in a humidified incubator. The next day, CHO-K1-PD1 cells and PBMCs were collected and centrifuged, the supernatant was removed, and the cell pellet was then 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 cell concentration was adjusted to an appropriate 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, PBMCs (900,000 / 50 μL / well) were added to the cells, mixed thoroughly, and incubated in an incubator at 37°C and 5% CO2 for 4 hours. After 4 hours, the 96-well plate was removed 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) x 100%.

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

[0184] The results show that the positive control 14C12H1L1(G1WT) had significant ADCC activity in a mixed culture system of PBMC and CHO-K1-PD1, indicating that the ADCC system was normal. Compared to the isotype control antibody hIgG1DM, VP101(hG1WT) exhibited significant ADCC activity and demonstrated dose-dependence, whereas VP101(G1DM) did not exhibit ADCC activity. The results also show that VP101(hG1DM), produced by mutations based on VP101(hG1WT), did not have ADCC activity at the cytological level, indicating that the ADCC effect was 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 co-culture system 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 the 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 minutes; the cell pellet was resuspended in RPMI-1640 (containing 1% BSA) (hereinafter referred to as assay medium), repeatedly centrifuged, and washed twice; the cells were counted, the cell viability was determined, and the cell concentration was adjusted to an appropriate 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, The cells were mixed thoroughly and pre-incubated at room temperature for 10 minutes. 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 hours. After 4 hours, the cells were centrifuged at 250 × g for 5 minutes; 100 μL of the cell supernatant was carefully transferred to a new 96-well flat-bottom plate (do not pipette 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 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) x 100%.

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

[0188] The results showed that the CDC% of the positive control antibody 14C12H1L1(G1WT) was significantly different from that of the isotype control antibody hIgG1DM group in the mixed culture system of normal human complement serum and CHO-K1-PD1, indicating the normality of the CDC detection system.At equivalent dose levels, there was no significant difference in CDC% for both VP101(hG1WT) and VP101(hG1DM) compared to 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, the 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 used to infect Raji cells. Raji-PDL1 stable cell lines expressing stable membrane PD-L1 protein were obtained after treatment and screening with BSD (10 μg / mL). Normal human PBMCs were isolated using a Ficoll peripheral blood mononuclear cell isolation system. The isolated PBMCs were resuspended in 1640 complete medium, counted, and frozen. The PBMCs were recovered, stimulated with SEB (Staphylococcus aureus enterotoxin B), and cultured for 2 days. After two days, log-phase Raji-PDL1 cells were harvested and treated with mitomycin C (Sigma, working concentration 25 μg / mL) for 60 minutes in an incubator. The mitomycin C-treated Raji-PDL1 cells were centrifuged and washed; PBMCs stimulated with SEB for two days were harvested and washed; these cells were mixed and cultured at a 1:1 cell number ratio in the presence or absence of antibodies. After three days, the cell supernatants were harvested by centrifugation, and the 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 IFN-γ secretion, 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) is a process in which the Fc fragment of an antibody bound to a cell surface antigen is phagocytosed. This means that they bind to Fc receptors of active cells (such as macrophages) and thus mediate 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 overexpressing 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. The results show that VP101 (hG1DM) did not have ADCP activity, but nivolumab, a commercially available antibody against the same target, had significant ADCP activity. The method is detailed as follows: Bone marrow from femurs of C57 mice (purchased from the Guangdong Medical Laboratory Animal Center) was first collected aseptically and lysed with red blood cell lysis buffer on ice for 5 minutes. Lysis was completed 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. For induction, the cells were cultured in a cell culture chamber at 37°C and 5% CO2 for 7 days. Half of the medium was replaced, and M-CSF was added on days 3 and 5. Cell induction was completed on day 7. The cells were digested with 0.25% trypsin. Macrophages were collected and centrifuged at 750 × g for 5 minutes. The supernatant was removed, and the cells were resuspended in DMEM complete medium (containing 10% FBS) and counted. The cells were adjusted to the appropriate density and filled 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 minutes, 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 minutes. Staining was stopped by adding 6 mL of DMEM complete medium (containing 10% FBS). Cells were centrifuged at 170 × g for 5 minutes, the supernatant was removed, and 1 mL of DMEM complete medium was added. Cells were incubated in an incubator for 10 minutes. The 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 the isotype control antibodies hIgG1DM and hIgG4 were designed. Freshly induced mature macrophages were harvested and centrifuged at 750 × g for 5 minutes, and the supernatant was removed. The cells were counted, transferred to a 96-well conical flat-bottom plate, centrifuged at 1000 × g for 5 minutes, and the supernatant was removed. The cell density of CHO-K1-PD1-CFSE was adjusted. The diluted antibodies and target cells were mixed in a 50 μL:50 μL ratio into the 96-well conical flat-bottom plate containing the macrophages according to the experimental design. The cells were resuspended, mixed thoroughly, and incubated in an incubator at 37 °C for 2 hours. 150 μL of 1% PBSA at room temperature was added to each well, centrifuged at 1000 × g for 5 minutes, and the supernatant was removed; cells were washed once with 200 μL of PBSA; 100 μL of APC anti-mouse / human CD11b antibody (diluted 1:500 in PBSA) was added to the corresponding samples, mixed thoroughly, and incubated on ice for 40 minutes. 150 μL of 1% PBSA was added to each well, centrifuged at 1000 × g for 5 minutes, 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 the macrophage + CHO-K1-PD1 system; the phagocytosis rate of VP101(hG1DM) was comparable to that of the isotype control antibody, indicating that VP101(hG1DM) had no ADCP effect. The results suggest that VP101(hG1DM) may have a favorable antitumor effect.

[0198] Although 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 of these modifications are within the scope of protection of the present invention. The full scope of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. a first protein functional region that targets PD-1; Second protein functional domain that targets VEGFA 1. 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; For the 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 the 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 the 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 the 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 a Fortebio Octet system. Bispecific antibodies.

2. According to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A 2. The bispecific antibody of claim 1, having the following structure:

3. a first protein functional region that targets PD-1; Second protein functional domain that targets VEGFA 1. 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; For the 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 the 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 the 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 the 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 heavy chain constant region of said immunoglobulin comprises the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A having Bispecific antibodies.

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 of any one of claims 1 to 3, having one or more mutations selected from:

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 claims 1 to 4.

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 claims 1 to 5, wherein the bispecific antibody is selected from any one of the following:

7. 7. The bispecific antibody of any one of claims 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.

8. The immunoglobulin or antigen-binding fragment thereof is －６ Less than M, for example about 10 －７ M, 10 －８ Less than M or 10 －９ binds to FcγRI with an affinity constant of M or less; preferably, the affinity constant is measured by the Fortebio Octet system. The bispecific antibody according to any one of claims 1 to 7.

9. The immunoglobulin or the antigen-binding fragment thereof is about 10 －９ Less than M, for example 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 claims 1 to 8.

10. 10. The bispecific antibody of any one of claims 1 to 9, wherein the first protein functional region is linked, either directly or by a linker fragment, to the second protein functional region; and / or the heavy chain variable region of the single chain antibody is linked, either directly or by a linker fragment, to the light chain variable region of the single chain antibody.

11. 11. The bispecific antibody of claim 10, wherein the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5, or 6.

12. 12. The bispecific antibody of claim 1, wherein the number of first protein functional regions and second protein functional regions is each independently 1, 2 or more.

13. 13. The bispecific antibody of claim 1, wherein the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

14. a first protein functional region that targets PD-1; A second protein functional domain that targets VEGFA 1. 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.

15. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 14.

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

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

18. 15. A conjugate comprising an antibody or antigen-binding fragment thereof and a conjugation moiety, wherein the immunoglobulin is the bispecific antibody of any one of claims 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.

19. A kit comprising the bispecific antibody of any one of claims 1 to 14 or the conjugate of claim 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, for example, a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

20. 19. Use of the bispecific antibody of any one of claims 1 to 14, or the conjugate of claim 18, in the preparation of a kit for detecting the presence or level of PD-1 and / or VEGFA in a sample.

21. 19. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 14 or the conjugate of claim 18, optionally wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vector and / or excipient.

22. 19. Use of the bispecific antibody of any one of claims 1 to 14 or the conjugate of claim 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, 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. use.

23. A method for treating and / or preventing malignant tumors, comprising the step of administering an effective amount of the bispecific antibody of any one of claims 1 to 14 or the conjugate of claim 18 to a subject in need thereof; preferably, the malignant tumor is selected from the group consisting of 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, frequent myeloid leukemia, and leukemia. selected from crosatellite instability-high (MSI-H) and mismatch repair deficient (dMMR) cancers, urothelial carcinoma, 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. method.

24. 15. The bispecific antibody of any one of claims 1 to 14 for use 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. Bispecific antibodies.

25. 19. The conjugate of claim 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) cancers, 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. Complex.

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