Anti-pd-1-Anti-vegfa bispecific antibody, pharmaceutical composition and use thereof

Abstract

Provided are an anti-VEGFA-anti-PD-1 bispecific antibody and a use thereof. Specifically, the anti-VEGFA-anti-PD-1 bispecific antibody comprises: a PD-1-targeted first protein functional region and a VEGFA-targeted second protein functional region. According to an EU numbering system, mutation occurs at two positions of positions 234 and 235 of a heavy chain constant region of the immunoglobulin contained in the bispecific antibody, and after the mutation, an affinity constant of the bispecific antibody with FcγRI, FcγRIIa, FcγRIIIa, and / or C1q is decreased compared to that before the mutation. The bispecific antibody can specifically bind to VEGFA and PD-1, specifically relieve immunosuppression of VEGFA and PD-1 on an organism, and inhibit tumor-induced angiogenesis, and thus has good application prospects.

Description

Anti-pd-1-anti-vegfa bispecific antibodies, pharmaceutical compositions thereof, and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of tumor therapy and immunological biology, and relates to an anti-PD-1-anti-VEGFA bispecific antibody, a pharmaceutical composition thereof, and a use thereof. Specifically, the present application relates to an anti-human PD-1-anti-human VEGFA bispecific antibody, a pharmaceutical composition thereof, and a use thereof. BACKGROUND

[0002] Tumor, especially malignant tumor, is a disease that seriously endangers human health in the world today, and ranks second in the mortality caused by various diseases. Moreover, in recent years, its incidence has shown a significant upward trend. Malignant tumor has poor treatment effect, high rate of late metastasis, and poor prognosis. The conventional treatment methods currently used in clinical practice, such as radiotherapy, chemotherapy, and surgical treatment, although to a great extent, alleviate the pain, prolong the survival time, but these methods have great limitations, and their efficacy is difficult to further improve.

[0003] The growth of tumor has two distinct stages, i.e., from the slow growth period without blood vessels to the rapid proliferation period with blood vessels. The generation of blood vessels enables the tumor to obtain sufficient nutrition and complete the vascular switching period. If there is no generation of blood vessels, the growth of the primary tumor does not exceed 1-2 mm, and the metastasis cannot be realized.

[0004] Vascular endothelial growth factor (VEGF) is a growth factor that can promote the division and proliferation of endothelial cells, promote the formation of new blood vessels, and increase vascular permeability. It binds to the vascular endothelial growth factor receptor on the cell surface and exerts its function by activating the tyrosine kinase signal transduction pathway. In tumor tissue, tumor cells, tumor-infiltrating macrophages and mast cells can secrete high levels of VEGF to stimulate tumor vascular endothelial cells in a paracrine manner, promote endothelial cell proliferation and migration, induce angiogenesis, promote tumor sustained growth, increase vascular permeability, cause fibrin deposition in the surrounding tissue, promote monocyte, fibroblast endothelial cell invasion, and facilitate tumor matrix formation and tumor cell entry into new blood vessels, thus promoting tumor metastasis. Therefore, inhibiting tumor angiogenesis is considered to be one of the most promising methods for tumor treatment. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD and PIGF. The vascular endothelial growth factor receptor (VEGFR) includes VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4) and Neuropilin-1 (NRP-1). Among them, the first three receptors are similar in structure and belong to the tyrosine kinase superfamily, and are composed of three parts of extracellular region, transmembrane fragment and intracellular region. The extracellular region is composed of immunoglobulin-like domains, and the intracellular region belongs to the tyrosine kinase region. VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, and VEGFR3 is mainly located on the surface of lymphatic endothelial cells.

[0005] VEGF family molecules have different affinities for several receptors. VEGFA mainly binds to VEGFR1, VEGFR2 and NRP-1 to exert its function. VEGFR1 is the earliest discovered receptor. Under normal physiological conditions, the affinity of VEGFR1 to VEGFA is higher than that of VEGFR2 to VEGFA, but its intracellular tyrosine kinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5) (2016): 146-148).

[0006] VEGFR2 is a major regulator of angiogenesis and construction, and has a high tyrosine kinase activity compared with VEGFR1. VEGFR2 mediates the proliferation, differentiation and other behaviors of vascular endothelial cells, and the formation process and permeability of blood vessels after binding with ligand VEGFA (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3) (2007): 179-213.), and VEGFA mediates intracellular related protein gene transcription and expression through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway after binding with VEGFR2, and promotes the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13) (1999): 2221-2230.).

[0007] VEGFR3 belongs to one of the members of the tyrosine kinase family, and is mainly expressed in the vascular endothelial cells of the embryonic period and the lymphatic endothelial cells of the adult period. VEGFC and VEGFD bind with VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote the neogenesis of lymphatic vessels; NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transduce biological signals, but can mediate signal transduction after forming a complex with VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5) (2016): 146-148).

[0008] VEGFA and VEGFR2 are mainly involved in the regulation of angiogenesis, and before and after the binding of VEGFA and VEGFR2, a cascade reaction of numerous intermediate signals in the upstream and downstream pathways is triggered, and finally the physiological functions of endothelial cells are changed in different forms such as proliferation, survival, migration, increased permeability and infiltration into surrounding tissues (Dong Hongchao et al., Modern Oncology Medicine, Vol. 22, No. 9, September 2014, pp. 2231-3).

[0009] Currently, there are a variety of humanized monoclonal antibodies targeting human VEGF, especially VEGFA, such as Bevacizumab, which was approved by the U.S. Food and Drug Administration in 2004 for the treatment of non-small cell lung cancer, renal cell carcinoma, cervical cancer, metastatic colorectal cancer and other tumors.

[0010] Programmed cell death protein 1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein membrane surface receptor belonging to the CD28 immunoglobulin superfamily, which is widely expressed in T cells, B cells and myeloid cells. PD-1 has two natural ligands, PD-L1 and PD-L2. PD-L1 and PD-L2 belong to the B7 superfamily and are constitutively or inducibly expressed on the surface of various cell membranes, including non-hematopoietic system cells and various tumor cells. PD-L1 is mainly expressed in T cells, B cells, DCs and microvascular endothelial cells and various tumor cells. PD-L2 is only expressed in antigen-presenting cells such as dendritic cells and macrophages. The interaction of PD-1 with its ligand can inhibit the activation of lymphocytes, inhibit the proliferation of T cells and the secretion of cytokines such as IL-2 and IFN-γ.

[0011] A large number of studies have shown that the tumor microenvironment can protect tumor cells from destruction by immune cells, and the expression of PD-1 is up-regulated in infiltrating lymphocytes in the tumor microenvironment, and a variety of primary tumor tissues are positive for PD-L1 in immunohistochemical analysis, such as lung cancer, liver cancer, ovarian cancer, skin cancer, colon cancer, glioma, etc. At the same time, the expression of PD-L1 in tumors is significantly associated with poor prognosis in cancer patients. Blocking the interaction of PD-1 and its ligand can promote tumor-specific T cell immunity and improve the immune clearance efficiency of tumor cells. A large number of clinical trials have shown that antibodies targeting PD-1 or PD-L1 can promote CD8+ T cell infiltration into tumor tissue, up-regulate anti-tumor immune effectors such as IL-2, IFN-γ, granzyme B and perforin, thereby effectively inhibiting tumor growth.

[0012] In addition, antibodies against PD-1 can also be used to treat viral chronic infections. Viral chronic infections are often accompanied by loss of function and reduction in number of virus-specific effector T cells. By injecting PD-1 antibodies, the interaction of PD-1 and PD-L1 can be blocked, thereby effectively inhibiting the exhaustion of effector T cells in viral chronic infections.

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

[0014] Currently, the combination of anti-angiogenic therapy and immune checkpoint inhibitors has shown good efficacy in many tumors. The combination of anti-VEGF antibodies, such as Bevacizumab, and PD-1 / PD-L1 antibodies, such as Nivolumab, Pembrolizumab, Atezolizumab, etc., has shown good efficacy in ovarian cancer (Joyce F. Liu et al., JAMA Oncol. 2019; 5(12): 1731-1738.), non-small cell lung cancer (including non-small cell lung cancer with EGFR and / or ALK sensitive mutations) (Manegold C, et al., J Thorac Oncol. 2017 Feb; 12(2): 194-207.), renal cell carcinoma (Dudek AZ et al., J Clin Oncol. 2018; 36(suppl; abstr 4558).), hepatocellular carcinoma (Stein S et al., J Clin Oncol, 2018, 36(15_suppl):4074.; FDA approved Bevacizumab combined with Atezolizumab for the treatment of hepatocellular carcinoma 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). Presented at: American Society of Clinical Oncology; May 29-June 2, 2015; Chicago, IL. 2015; abstract 704; Hochster HS et al. Efficacy and safety of atezolizumab (atezo) and bevacizumab (bev) in a phase Ib study of microsatellite instability (MSI)-high metastatic colorectal cancer (mCRC).Presented at: American Society of Clinical Oncology Gastrointestinal Cancers Symposium; January 19-21, 2017; San Francisco, CA. 2017; abstract 673.), breast cancer (Yukinori Ozaki et al., A multicenter phase II study evaluating the efficacy of nivolumab plus paclitaxel plus bevacizumab triple-combination therapy as a first-line treatment in patients with HER2-negative metastatic breast cancer: WJOG9917B NEWBEAT trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020; 80(4 Suppl): Abstract nr PD1-03.), VEGFR2 antibody and PD-1 antibody drug combination also showed good anti-tumor effect in gastric and gastroesophageal junction adenocarcinoma (Herbst RS et al., Lancet Oncol. 2019; 20(8): 1109-1123.), PD-1 antibody (Pembrolizumab) and angiogenesis inhibitor (Lenvatinib) combination regimen showed good efficacy in the treatment of endometrial cancer, which was approved by FDA for the treatment of endometrial cancer in 2019. For melanoma (PD-1 antibodies Nivolumab and Pembrolizumab have been approved by FDA for the treatment of melanoma), cervical cancer (Krishnansu S. et al., N Engl J Med 2014; 370: 734-743.), glioma, prostate cancer (Antonarakis ES. et al., J Clin Oncol. 2020 Feb 10; 38(5): 395-405.), urothelial carcinoma (Joaquim Bellmunt. et al.N Engl J Med 2017; 376: 1015-1026; FDA approved Nivolumab for treating 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 the synergistic effect of the PD-1 pathway and the VEGF pathway in tumorigenesis, it can be expected that drugs that block both the PD-1 and VEGF pathways will have better anti-tumor effects.

[0015] Bispecific antibodies are also known as dual specific antibodies, which are specific drugs targeting two different antigens at the same time. They can be produced by immunosort purification. In addition, they can also be obtained by genetic engineering. Genetic engineering has corresponding flexibility in terms of optimization of binding sites, consideration of synthetic forms and yield, so it has certain advantages. At present, more than 45 forms of bispecific antibodies have been proved to exist (Muller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98). At present, a variety of bispecific antibodies have been developed in the form of IgG-ScFv, that is, Morrison mode (Coloma M.J., Morrison S.L. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997; 15: 159-163). Due to the advantages of this form similar to naturally occurring IgG in antibody engineering, expression and purification, it has been proved to be an ideal form of bispecific antibodies (Miller B.R., Demarest S.J., et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23: 549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011; 3: 299-309).

[0016] ADCC (antibody-dependent cell-mediated cytotoxicitye) is the antibody-dependent cell-mediated cytotoxicity, which refers to the Fab segment of the antibody combining with the antigen epitope of the virus infected cell or tumor cell, and the Fc segment combining with the Fc receptor (Fc Receptor, FcR) on the surface of the killer cell (NK cell, macrophage, etc.), which mediates the direct killing of the target cell by the killer cell.

[0017] CDC (complement dependent cytotoxicity) is the complement-dependent cytotoxicity, which refers to that when the antibody specifically binds to the corresponding antigen on the cell membrane surface, a complex is formed to activate the complement system, and then MAC is formed on the surface of the target cell, leading to the lysis of the target cell. Complement can cause the lysis of various bacterial and other pathogenic organism cells, and is an important defense mechanism of the body against pathogenic infection.

[0018] Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells, which are used to recognize the Fc region of antibodies to mediate immune responses. After the Fab region of the antibody recognizes the antigen, the Fc region of the antibody binds to the Fc receptor on the immune cells (such as killer cells), which initiates the response function of the immune cells, such as phagocytosis and ADCC.

[0019] According to the different types of antibodies recognized by Fc receptors and the different cells expressing them, Fc receptors are mainly divided into three types of FcγR, FcαR and FcεR, and FcγR can be divided into four subtypes of FcγRI (also known as CD64), FcγRII (also known as CD32), FcγRIII (also known as CD16) and FcRn (also known as Neonatal Fc receptor). Among them, FcγRI, FcγRII and FcγRIII are closely related to ADCC effect. FcγRIII is the most important molecule for mediating ADCC, and has two highly homologous subtypes of FcγRIIIa and FcγRIIIb in different cell types. There are high-affinity FcγRIIIa subtypes caused by single nucleotide polymorphism (SNP) sites in the FcγRIIIa population, which are called FcγRIIIa_V158 and low-affinity FcγRIIIa_F158 subtypes, respectively. FcγRI has a high affinity for the Fc region of IgG and is involved in the ADCC process; FcγRII has three subtypes of FcγRIIa, FcγRIIb and FcγRIIc (also known as CD32a, CD32b and CD32c), among which FcγRIIa has ADCC activity; FcγRIIa has two subtypes in the human population due to single nucleotide mutation, which are called FcγRIIa_H131 and FcγRIIa_R131 (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4): 311-331).

[0020] The IgG family includes four members, IgG1, IgG2, IgG3 and IgG4, which differ in the fragment crystallizable (Fc) region of the heavy chain constant region by amino acids, resulting in different affinities to FcγRs. IgG1 is the most abundant subtype in human body and is also the most used subtype in monoclonal antibody drugs. IgG1 can bind to various FcγRs and can induce ADCC and CDC effects. IgG2 has the weakest affinity to FcγRs, but IgG2 can still induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 has the strongest binding capacity to FcγRs, can induce ADCC, and has stronger CDC effect than IgG1. IgG4 molecule has weaker binding to FcγRs other than FcγRI, and IgG4 molecule has lower possibility to induce CDC and NK cell-mediated ADCC.

[0021] At present, it is still necessary to develop new anti-PD-1-anti-VEGFA bispecific antibodies to reduce or eliminate the damage to immune cells caused by antibody-mediated ADCC and / or CDC activity of the anti-PD-1-anti-VEGFA bispecific antibodies, and to improve the efficacy of antibody drugs.

[0022] SUMMARY

[0023] The present inventors have made corresponding modifications to the Fc end of the anti-PD-1-anti-VEGFA antibody structure through in-depth research and creative labor, reduced the binding of the Fc region to the Fc receptor, thereby reducing the ADCC and CDC side effects on immune cells, and increasing the drug efficacy of the anti-PD-1-anti-VEGFA antibody drug.

[0024] Thus, the following invention is provided:

[0025] One aspect of the present invention relates to a bispecific antibody comprising:

[0026] a first protein functional region targeting PD-1, and

[0027] a second protein functional region targeting VEGFA;

[0028] wherein the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin.

[0029] wherein,

[0030] the immunoglobulin has a heavy chain variable region comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 34-36, respectively, and a light chain variable region comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 37-39, respectively; and the single chain antibody has a heavy chain variable region comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively, and a light chain variable region comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 31-33, respectively;

[0031] or,

[0032] the immunoglobulin has a heavy chain variable region comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 34-36, respectively, and a light chain variable region comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 37-39, respectively; and the single chain antibody has a heavy chain variable region comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively, and a light chain variable region comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 31-33, respectively;

[0033] the immunoglobulin is of human IgG1 subtype;

[0034] wherein the heavy chain constant region of the immunoglobulin has a mutation at any two or three of positions 234, 235 and 237 according to EU numbering system, and after the mutation, the affinity constant of the bispecific antibody to FcyRIIIa and / or Clq is reduced compared to that before the mutation; preferably, the affinity constant is measured by Fortebio Octet molecular interaction instrument.

[0035] In one or more embodiments of the present application, the bispecific antibody, wherein the heavy chain constant region of the immunoglobulin has a mutation as follows according to EU numbering system:

[0036] L234A and L235A; or

[0037] L234A and G237A; or

[0038] L235A and G237A;

[0039] or

[0040] L234A, L235A, G237A.

[0041] In the present application, if not otherwise specified, the letter before the site represents the amino acid before mutation, and the letter after the site represents the amino acid after mutation.

[0042] The present application also relates to a bispecific antibody comprising:

[0043] a first protein functional region targeting PD-1, and

[0044] a second protein functional region targeting VEGFA;

[0045] wherein the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin.

[0046] wherein,

[0047] the immunoglobulin has a heavy chain variable region comprising HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 28-30, respectively, and a light chain variable region comprising LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 31-33, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 34-36, respectively, and a light chain variable region comprising LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 37-39, respectively.

[0048] or,

[0049] the immunoglobulin has a heavy chain variable region comprising HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 34-36, respectively, and a light chain variable region comprising LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 37-39, respectively; and the single-chain antibody has a heavy chain variable region comprising HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 28-30, respectively, and a light chain variable region comprising LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 31-33, respectively.

[0050] The immunoglobulin is of human IgG1 subtype.

[0051] The present application also relates to a bispecific antibody comprising:

[0052] a first protein functional region targeting PD-1, and

[0053] a second protein functional region targeting VEGFA;

[0054] Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin.

[0055] in,

[0056] The immunoglobulin, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:31-33; and the single-chain antibody, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:37-39;

[0057] or,

[0058] The immunoglobulin, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:34-36 and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:37-39; and the single-chain antibody, wherein the heavy chain variable region comprises the amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:28-30 and the light chain variable region comprises the amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:31-33;

[0059] The immunoglobulin is human IgG1 subtype;

[0060] According to the EU numbering system, the heavy chain constant region of the immunoglobulin has one of the following combinations of mutations:

[0061] L234A and L235A; or

[0062] L234A and G237A; or

[0063] L235A and G237A; or

[0064] L234A, L235A, G237A.

[0065] In one or more embodiments of the present invention, the bispecific antibody, according to the EU numbering system, has or further has one or more mutations selected from the following:

[0066] 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.

[0067] In one or more embodiments of the present application, the bispecific antibody, wherein,

[0068] 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; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 17.

[0069] or,

[0070] the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 17; and, the amino acid sequence of the heavy chain variable region of the single chain antibody is 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.

[0071] In one or more embodiments of the present application, the bispecific antibody is selected from any one of (1)-(12) as follows:

[0072] (1)

[0073] 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; and, 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.

[0074] (2)

[0075] 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; and, 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;

[0076] (3)

[0077] 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; and, 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;

[0078] (4)

[0079] 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; and, 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;

[0080] (5)

[0081] 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; and, 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;

[0082] (6)

[0083] 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; and, 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;

[0084] (7)

[0085] 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; and, 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;

[0086] (8)

[0087] 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; and, 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;

[0088] (9)

[0089] 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; and, 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;

[0090] (10)

[0091] 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; and, 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;

[0092] (11)

[0093] 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; and, 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

[0094] (12)

[0095] 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; and, 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.

[0096] In one or more embodiments of the present application, the bispecific antibody is:

[0097] 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 thereof is set forth in SEQ ID NO: 26.

[0098] In one or more embodiments of the present application, the bispecific antibody is in the IgG-scFv format, i.e., the Morrison pattern.

[0099] In some embodiments of the present application, the bispecific antibody in one or more embodiments of the present application, wherein,

[0100] The immunoglobulin has a heavy chain constant region of human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and a light chain constant region of human Ig kappa chain C region.

[0101] In some embodiments of the present application, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834; or

[0102] The heavy chain constant region of the immunoglobulin adopts Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.

[0103] In one embodiment of the present application, the amino acid sequence of the heavy chain constant region Ig gamma-1 chain C region (ACCESSION: P01857) is as follows:

[0104]

[0105] In one embodiment of the application, the amino acid sequence of the heavy chain constant region Ig gamma-4 chain C region (ACCESSION: P01861.1) is as follows:

[0106]

[0107] In one embodiment of the application, the amino acid sequence of the light chain constant region Ig kappa chain C region (ACCESSION: P01834) is as follows:

[0108]

[0109] In some embodiments of the application, the bispecific antibody, wherein the single chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin. Since the immunoglobulin has two heavy chains, one immunoglobulin molecule has two single chain antibody molecules attached. Preferably, the two single chain antibody molecules are identical.

[0110] In some embodiments of the application, the bispecific antibody, wherein the single chain antibody is two, one end of each single chain antibody is attached to the C-terminus or N-terminus of the two heavy chains of the immunoglobulin, respectively.

[0111] In some embodiments of the application, the V H region of the single chain antibody has a disulfide bond between the V L region of the single chain antibody. Methods of introducing disulfide bonds between the V H region of the antibody and the V L region of the single chain antibody are well known in the art, see, e.g., U.S. Patent No. 5,747,654; Rajagopal et. al, Prot. Engin. 10 (1997) 1453-1459; Reiter et. al, Nat. Biotechnol. 14 (1996) 1239-1245; Reiter et. al, Protein Engineering 8 (1995) 1323-1331; Webber et. al, Molecular Immunology 32 (1995) 249-258; Reiter et. al, Immunity 2 (1995) 281-287; Reiter et. al, JBC 269 (1994) 18327-18331; Reiter et. al, Inter. J. of Cancer 58 (1994) 142-149; or Reiter et. al, Cancer Res. 54 (1994) 2714-2718; which are incorporated herein by reference.

[0112] In one or more embodiments of the present application, the bispecific antibody, wherein the first protein functional region and the second protein functional region are directly connected or connected through a connecting fragment; and / or the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are directly connected or connected through a connecting fragment.

[0113] In one or more embodiments of the present application, the bispecific antibody, wherein the connecting fragment is (GGGGS)n, n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

[0114] In one or more embodiments of the present application, the bispecific antibody, wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

[0115] In one or more embodiments of the present application, the bispecific antibody, wherein the single-chain antibody is connected at the C-terminus of the heavy chain of the immunoglobulin.

[0116] The present application also relates to a bispecific antibody, comprising:

[0117] a first protein functional region targeting PD-1, and

[0118] a second protein functional region targeting VEGFA;

[0119] the first protein functional region is 1, and the second protein functional region is 2;

[0120] wherein the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0121] the amino acid sequence of the heavy chain of the immunoglobulin is shown as SEQ ID NO: 24, and the amino acid sequence of the light chain of the immunoglobulin is shown as SEQ ID NO: 26;

[0122] the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown as SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown as SEQ ID NO: 17;

[0123] the single-chain antibody is connected at the C-terminus of the heavy chain of the immunoglobulin;

[0124] the first protein functional region and the second protein functional region are connected through a first connecting fragment; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected through a second connecting fragment; the first connecting fragment and the second connecting fragment are the same or different;

[0125] Preferably, the amino acid sequences of the first and second linker segments are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19;

[0126] Preferably, the amino acid sequences of the first and second linker segments are both as set forth in SEQ ID NO: 18.

[0127] In one or more embodiments of the application, the bispecific antibody, wherein the immunoglobulin or antigen binding fragment thereof binds FcyRI with an affinity constant of less than about 10 -6 M, for example, less than about 10 -7 M, 10 -8 M, or 10 -9 M or less; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.

[0128] In one or more embodiments of the application, the bispecific antibody, wherein the immunoglobulin or antigen binding fragment thereof binds C1q with an affinity constant of less than about 10 -9 M, for example, less than about 10 -7 M, 10 -8 M, or 10 -9 M or less; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.

[0129] In some embodiments of the application, the bispecific antibody, wherein the bispecific antibody binds VEGFA protein and / or PD-1 protein with a K -5 M, for example, less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less; preferably, the K D is measured by a Fortebio molecular interaction instrument. D

[0130] In some embodiments of the application, the bispecific antibody, wherein,

[0131] the bispecific antibody binds VEGFA protein with an EC 50 of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.15 nM, or less than 0.14 nM; preferably, the EC 50 is measured by an indirect ELISA method.

[0132] and / or,​

[0133] The bispecific antibody has an EC50 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 for binding to PD-1 protein; preferably, the EC50 is less than 0.1 nM, less than 0.05 nM, or less than 0.01 nM. 50 binds to PD-1 protein; preferably, the EC50 is 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 as measured by an indirect ELISA method.

[0134] In one or more embodiments of the present application, the bispecific antibody is a monoclonal antibody.

[0135] In one or more embodiments of the present application, the bispecific antibody is a humanized antibody.

[0136] In still another aspect of the present application, there is provided an isolated nucleic acid molecule encoding the bispecific antibody of any one of the present application.

[0137] In still another aspect of the present application, there is provided a vector comprising the isolated nucleic acid molecule of the present application.

[0138] In still another aspect of the present application, there is provided a host cell comprising the isolated nucleic acid molecule of the present application, or comprising the vector of the present application.

[0139] In still another aspect of the present application, there is provided a conjugate comprising an antibody or antigen binding fragment thereof and a conjugating moiety, wherein the immunoglobulin is the bispecific antibody of any one of the present application, and the conjugating moiety is a detectable label; preferably, the conjugating moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0140] In still another aspect of the present application, there is provided a kit comprising the bispecific antibody of any one of the present application, or comprising the conjugate of the present application.

[0141] Preferably, the kit further comprises a second antibody that specifically recognizes the immunoglobulin or antigen binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0142] In still another aspect of the present application, there is provided the use of the bispecific antibody of any one of the present application, or the conjugate of the present application, in the manufacture of a kit for detecting the presence or level of PD-1 and / or VEGFA in a sample.

[0143] In still another aspect of the present application, there is provided a pharmaceutical composition comprising the bispecific antibody of any one of the present application, or the conjugate of the present application; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0144] The bispecific antibody of the present application or the pharmaceutical composition of the present application can be formulated into any dosage form known in the pharmaceutical art, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form will depend on the intended means of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the bispecific antibody of the present application in the required amount in an appropriate solvent with one or more of the other ingredients enumerated above, as desired (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ion strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtered sterilization. Furthermore, sterile injectable solutions can be prepared as sterile freeze-dried powders (e.g., by vacuum drying or freeze-drying) to allow reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0145] In addition, the bispecific antibody of the present application can be present in unit dosage form in the pharmaceutical composition to facilitate administration. In certain embodiments, the unit dosage 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. In the case where the pharmaceutical composition is in a liquid (e.g., injectable) dosage form, it can comprise the bispecific antibody of the present application at a concentration of at least 0.1 mg / ml, such as at least 0.25 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml.

[0146] The bispecific antibody or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibody or pharmaceutical composition of the present application is administered by intravenous infusion or injection.

[0147] The bispecific antibody or the pharmaceutical composition provided by the present application can be used alone or in combination, and can also be used in combination with another pharmaceutically active agent (such as a tumor chemotherapeutic drug). Such another pharmaceutically active agent can be administered before, simultaneously with, or after the administration of the bispecific antibody of the present application or the pharmaceutical composition of the present application.

[0148] In the present application, the administration regimen can be adjusted to obtain the best intended response (such as a therapeutic or prophylactic response). For example, a single administration can be made, multiple administrations can be made over a period of time, or the dose can be proportionally reduced or increased according to the urgency of the treatment situation.

[0149] Another aspect of the present application relates to the use of the bispecific antibody of any one of the present application or the conjugate of the present application in the preparation of a medicament for the treatment and / or prevention of a malignant tumor; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia;

[0150] Preferably, the lung cancer is non-small cell lung cancer, small cell lung cancer; preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR and / or ALK sensitive mutation;

[0151] Preferably, the liver cancer is hepatocellular carcinoma;

[0152] Preferably, the renal tumor is renal cell carcinoma;

[0153] Preferably, the breast cancer is triple-negative breast cancer;

[0154] Preferably, the urothelial cancer is bladder cancer.

[0155] Another aspect of the present application relates to the use of the bispecific antibody of any one of the present application or the conjugate of the present application in the preparation of a medicament for the treatment and / or prevention of a malignant tumor; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia;

[0156] (1)

[0157] a medicament or reagent for detecting the level of VEGFA in a sample,

[0158] a medicament or reagent for blocking the binding of VEGFA to VEGFR2,

[0159] a medicament or reagent for down-regulating the activity or level of VEGFA,

[0160] a medicament or reagent for relieving the stimulation of vascular endothelial cell proliferation by VEGFA,

[0161] a drug or agent that inhibits the proliferation of vascular endothelial cells, or

[0162] a drug or agent that blocks tumor angiogenesis;

[0163] and / or

[0164] (2)

[0165] a drug or agent that blocks the binding of PD-1 to PD-L1,

[0166] a drug or agent that down-regulates the activity or level of PD-1,

[0167] a drug or agent that releases the body from the immunosuppression of PD-1,

[0168] a drug or agent that promotes the secretion of IFN-γ in T lymphocytes, or

[0169] a drug or agent that promotes the secretion of IL-2 in T lymphocytes.

[0170] In the in vitro experiments of the present application, the anti-VEGFA antibody and the anti-VEGFA-anti-PD-1 bispecific antibody can both inhibit the proliferation of HUVEC cells, and the anti-PD-1 antibody and the anti-VEGFA-anti-PD-1 bispecific antibody can both promote the secretion of IFNγ and / or IL-2 to activate the immune response.

[0171] Yet another aspect of the present application relates to a method for treating and / or preventing a malignant tumor, comprising the step of administering to a subject in need thereof an effective amount of the bispecific antibody of any one of the present application or the conjugate of the present application; 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 cancer, cervical cancer, esophageal cancer, microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia;

[0172] Preferably, the lung cancer is non-small cell lung cancer, small cell lung cancer; preferably, the non-small cell lung cancer is EGFR and / or ALK sensitive mutation of non-small cell lung cancer;

[0173] Preferably, the liver cancer is hepatocellular carcinoma;

[0174] Preferably, the renal tumor is renal cell carcinoma;

[0175] Preferably, the breast cancer is triple-negative breast cancer;

[0176] Preferably, the urothelial cancer is bladder cancer.

[0177] A typical non-limiting range for a therapeutically or prophylactically effective amount of the bispecific antibody of the present application is 0.02-50 mg / kg, such as 0.1-50 mg / kg, 0.1-25 mg / kg, or 1-10 mg / kg. It is noted that the dosage will vary depending on the type and severity of the symptoms to be treated. Furthermore, those of skill in the art will appreciate that the particular dosage regimen, including the amount, timing, and route of administration, will also depend on the particular patient's needs and on the physician's professional judgment; the dosage ranges given here are intended for illustrative purposes only and do not limit the scope of use or practice of the pharmaceutical compositions of the present application.

[0178] In the present application, the subject can be a mammal, such as a human.

[0179] Yet another aspect of the present application relates to a method in or in vitro, selected from:

[0180] (1)

[0181] a method of detecting the level of VEGFA in a sample,

[0182] a method of blocking the binding of VEGFA to VEGFR2,

[0183] a method of down-regulating the activity or level of VEGFA,

[0184] a method of relieving the stimulation of vascular endothelial cell proliferation by VEGFA,

[0185] a method of inhibiting the proliferation of vascular endothelial cells by a drug or agent, or

[0186] a method of blocking tumor angiogenesis;

[0187] and / or

[0188] (2)

[0189] a method of blocking the binding of PD-1 to PD-L1,

[0190] a method of down-regulating the activity or level of PD-1,

[0191] a method of relieving the immunosuppression of the body by PD-1,

[0192] a method of promoting the secretion of IFN-γ in T lymphocytes, or

[0193] a method of promoting the secretion of IL-2 in T lymphocytes.

[0194] In one or more embodiments of the present application, the bispecific antibody or conjugate is used for treating and / or preventing a malignant tumor; 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 cancer, cervical cancer, esophageal cancer, microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) cancer, urothelial cancer, mesothelioma, endometrial cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and leukemia;

[0195] Preferably, the lung cancer is non-small cell lung cancer, small cell lung cancer; preferably, the non-small cell lung cancer is EGFR and / or ALK sensitive mutation non-small cell lung cancer;

[0196] Preferably, the liver cancer is hepatocellular carcinoma;

[0197] Preferably, the renal tumor is renal cell carcinoma;

[0198] Preferably, the breast cancer is triple-negative breast cancer;

[0199] Preferably, the urothelial cancer is bladder cancer.

[0200] In one or more embodiments of the present application, the bispecific antibody or conjugate is used for:

[0201] (1)

[0202] detecting the level of VEGFA in a sample,

[0203] blocking the binding of VEGFA to VEGFR2,

[0204] down-regulating the activity or level of VEGFA,

[0205] relieving the stimulation of vascular endothelial cell proliferation by VEGFA,

[0206] inhibiting the proliferation of vascular endothelial cells, or

[0207] blocking tumor angiogenesis;

[0208] and / or

[0209] (2)

[0210] blocking the binding of PD-1 to PD-L1,

[0211] down-regulating the activity or level of PD-1,

[0212] relieving the immune suppression of the body by PD-1,

[0213] promoting the secretion of IFN-γ in T lymphocytes, or

[0214] promoting IL-2 secretion in T lymphocytes.

[0215] Antibody therapeutics, particularly monoclonal antibodies, have shown great efficacy in the treatment of a variety of diseases. The traditional experimental method to obtain these therapeutic antibodies is to immunize animals with antigens, and to obtain antibodies targeting the antigens in the immunized animals, or to improve those antibodies with low affinity to antigens by affinity maturation methods.

[0216] The variable regions of light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions of about 10-15 amino acids each, referred to as Complementarity Determining Regions (CDRs) (the CDRs of the H Chain include HCDR1, HCDR2, HCDR3, and the CDRs of the L Chain include LCDR1, LCDR2, LCDR3; they are named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication (1-3) 1991:91-3242.

[0217] Preferably, the CDRs can also be defined by the IMGT numbering system, please refer to Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc.“IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF.” Nucleic acids research 38.suppl_1 (2009): D301-D307.

[0218] By means of techniques well known to those skilled in the art, for example, by analyzing the amino acid sequences of the CDR regions of the monoclonal antibodies in items (1)-(13) below according to IMGT definition through the VBASE2 database, the results are as follows:

[0219] (1) Bevacizumab

[0220] The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 3.

[0221] The amino acid sequences of the 3 CDR regions of the heavy chain variable region are as follows:

[0222] HCDR1: GYTFTNYG (SEQ ID NO: 28)

[0223] HCDR2: INTYTGEP (SEQ ID NO: 29)

[0224] HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 30)

[0225] The amino acid sequences of the 3 CDR regions of the light chain variable region thereof are as follows:

[0226] LCDR1: QDISNY (SEQ ID NO: 31)

[0227] LCDR2: FTS (SEQ ID NO: 32)

[0228] LCDR3: QQYSTVPWT (SEQ ID NO: 33)

[0229] (2) 14C12, 14C12H1L1, or 14C12H1L1(M)

[0230] The amino acid sequences of the 3 CDR regions of the heavy chain variable region thereof are as follows:

[0231] HCDR1: GFAFSSYD (SEQ ID NO: 34)

[0232] HCDR2: ISGGGRYT (SEQ ID NO: 35)

[0233] HCDR3: ANRYGEAWFAY (SEQ ID NO: 36)

[0234] The amino acid sequences of the 3 CDR regions of the light chain variable region thereof are as follows:

[0235] LCDR1: QDINTY (SEQ ID NO: 37)

[0236] LCDR2: RAN (SEQ ID NO: 38)

[0237] LCDR3: LQYDEFPLT (SEQ ID NO: 39)

[0238] (3) VP101 (hGlWT) or VP101 (hGlDM)

[0239] The amino acid sequences of the 9 CDR regions of the heavy chain thereof are as follows:

[0240] HCDR1: GYTFTNYG (SEQ ID NO: 28)

[0241] HCDR2: INTYTGEP (SEQ ID NO: 29)

[0242] HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 30)

[0243] HCDR4: GFAFSSYD (SEQ ID NO: 34)

[0244] HCDR5: ISGGGRYT (SEQ ID NO: 35)

[0245] HCDR6: ANRYGEAWFAY (SEQ ID NO: 36)

[0246] HCDR7: QDINTY (SEQ ID NO: 37)

[0247] HCDR8: RAN (SEQ ID NO: 38)

[0248] HCDR9: LQYDEFPLT (SEQ ID NO: 39)

[0249] The amino acid sequences of the 3 CDR regions of the light chain variable region of the antibody VP101 (hGlDM) are as follows:

[0250] LCDR1: QDISNY (SEQ ID NO: 31)

[0251] LCDR2: FTS (SEQ ID NO: 32)

[0252] LCDR3: QQYSTVPWT (SEQ ID NO: 33).

[0253] The antibody VP101 (hGlDM) of the present application introduces amino acid mutations in the non-variable region of VP101 (hGlWT). The amino acid mutations are introduced at positions 234, 235 according to the EU numbering system:

[0254] The VP101 (hGlDM) is obtained by introducing a point mutation of leucine to alanine at position 234 (L234A) and a point mutation of leucine to alanine at position 235 (L235A) in the hinge region of its heavy chain.

[0255] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are in accordance with conventional techniques in the respective fields. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0256] As used herein, when referring to the amino acid sequence of VEGFA protein (GenBank ID: NP_001165097.1), it includes the full length of VEGFA protein, also includes fusion proteins of VEGFA, such as fragments fused with Fc protein fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of VEGFA protein without affecting its biological function. Therefore, in the present application, the term "VEGFA protein" should include all such sequences, including its natural or artificial variants. And when describing sequence fragments of VEGFA protein, it also includes the corresponding sequence fragments in its natural or artificial variants. In an embodiment of the present application, the amino acid sequence of VEGFA protein is shown in the underlined part of SEQ ID NO: 33 (without the last 6 His, a total of 302 amino acids).

[0257] As used herein, when referring to the amino acid sequence of VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), it includes the full length of VEGFR2 protein, or the extracellular fragment VEGFR2-ECD of VEGFR2 or fragments containing VEGFR2-ECD; also includes fusion proteins of VEGFR2-ECD, such as fragments fused with Fc protein fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of VEGFR2 protein without affecting its biological function. Therefore, in the present application, the term "VEGFR2 protein" should include all such sequences, including its natural or artificial variants. And when describing sequence fragments of VEGFR2 protein, it also includes the corresponding sequence fragments in its natural or artificial variants. In an embodiment of the present application, the amino acid sequence of the extracellular fragment VEGFR2-ECD of VEGFR2 is shown in SEQ ID NO: 34 (766 amino acids).

[0258] As used herein, if not specified, the VEGFR is VEGFR1 and / or VEGFR2; its specific protein sequence is known in the prior art, which can be referred to the sequences disclosed in the prior art or GenBank. For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0259] As used herein, when referring to the amino acid sequence of PD-1 protein (Programmed cell death protein 1, NCBI GenBank: NM_005018), it includes the full length of PD-1 protein, or the extracellular fragment of PD-1, PD-1 ECD, or a fragment comprising PD-1 ECD; also includes fusion proteins of PD-1 ECD, such as fragments fused with Fc protein fragment of mouse or human IgG (mFc or hFc). However, one skilled in the art understands that mutations or variations (including but not limited to substitutions, deletions and / or additions) can be naturally produced or artificially introduced in the amino acid sequence of PD-1 protein without affecting its biological function. Therefore, in the present application, the term "PD-1 protein" shall include all such sequences, including natural or artificial variants thereof. And when describing sequence fragments of PD-1 protein, it also includes the corresponding sequence fragments in natural or artificial variants thereof.

[0260] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, refers to the concentration that can cause 50% of maximal effect.

[0261] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). In general terms, a heavy chain can be understood as a polypeptide chain of larger molecular weight in an antibody, and a light chain refers to a polypeptide chain of smaller molecular weight in an antibody. Light chains can be classified as kappa and lambda light chains. Heavy chains can be generally classified as mu, delta, gamma, alpha, or epsilon, and define the isotype of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, a variable region and a constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region is composed of three domains (C H1 , C H2 , and C H3 ). Each light chain is composed of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region is composed of one domain C L . The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V LRegions can also be subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V H and V L consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy chain / light chain pair (V H and V L ) form the antibody binding site, respectively. The assignment of amino acids to each region or domain can follow the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196 (1987): 901-917; Chothia et al. Nature 342 (1989): 878-883 or the IMGT numbering system definition, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38.suppl_1 (2009): D301-D307. In particular, a heavy chain can contain more than 3 CDRs, e.g., 6, 9, or 12. For example, in a bispecific antibody of the application, a heavy chain can be the C-terminal end of a heavy chain of an IgG antibody linked to a ScFv of another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0262] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989). Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides having at least a portion of an antibody that is sufficient to confer specific antigen-binding

[0263] As used herein, the term "Fd fragment" means an antibody fragment consisting of the V H and C H1 domains; the term "Fv fragment" means an antibody fragment consisting of the V L and V H domains of a single arm of an antibody; the term "dAb fragment" means an antibody fragment consisting of a V H domain (Ward et al., Nature 341 (1989): 544-546); the term "Fab fragment" means an antibody fragment consisting of the V L , V H , C L , and C H1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.

[0264] In some cases, an antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the V L and V H domains are paired by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242 (1988): 423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 85 (1988): 5879-5883). Such scFv molecules can have the general structure: NH2-V L - linker - V H -COOH or NH2-V H - linker - V L-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4may be used, but variants thereof can also be used (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers useful in the present application are described by Alfthan et al., Protein Eng. 8 (1995): 725-731, Choi et al., Eur. J. Immunol. 31 (2001): 94-106, Hu et al., Cancer Res. 56 (1996): 3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999): 41-56, and Roovers et al., Cancer Immunol. (2001).

[0265] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody comprising V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448, and Poljak R.J. et al., Structure 2 (1994): 1121-1123).

[0266] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as is used for whole antibodies.

[0267] In this document, unless the context clearly indicates otherwise, the term "antibody" includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0268] As used herein, the terms "monoclonal antibody" and "monoclonal" refer to a population of antibody molecules that are identical in their amino acid sequences, except for naturally occurring mutations that can arise during production. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Polyclonal antibodies, in contrast, are typically made in animals containing at least two or more different antibodies, which typically recognize different epitopes on the antigen. Monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256: 495, 1975, but can also be made by recombinant DNA methods (see, e.g., U.S. Patent 4,816,567).

[0269] As used herein, the term "chimeric antibody" refers to an antibody in which some portion of the light or / and heavy chains are derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), while some portion of the light or / and heavy chains are derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which still retains the binding activity for the target antigen (U.S. Patent 4,816,567 Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 (1984): 6851-6855).

[0270] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment in which all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by CDR regions of a non-human antibody (donor antibody), which can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework regions (FRs) of the recipient antibody can be replaced by corresponding amino acid residues of the non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize the properties of the antibody. For more details on humanized antibodies, see, e.g., Jones et al., Nature, 321 (1986): 522-525; Reichmann et al., Nature, (1988) 332: 323 329; Presta, Curr. Op. Struct. Biol., 2 (1992): 593-596; and Clark, Immunol. Today 21 (2000): 397-402.

[0271] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. An "epitope" is also referred to in the art as an "antigenic determinant". An epitope or antigenic determinant typically consists of chemically active surface groupings of molecules such as amino acids or carbohydrate and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all of the points of interaction between the protein and the interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across amino acid residues of the protein that are separated from one another.

[0272] As used herein, the term "isolated" or "isolated" refers to an artificially created means from the natural state. If a certain "isolated" substance or component appears in nature, it is possible that the natural environment of the substance has been changed, or the substance has been separated from the natural environment, or both. For example, a certain polynucleotide or polypeptide naturally exists in a certain living animal body without being isolated, and a high-purity same polynucleotide or polypeptide isolated from such a natural state is called isolated. The term "isolated" or "isolated" does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0273] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements carried in the genetic material of the vector 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 (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages 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 (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0274] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0275] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen refers to an antibody that binds to the antigen with an affinity of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less. In some embodiments of the application, the term "targeting" refers to specific binding. D ) to the antigen. In some embodiments of the application, the term "targeting" refers to specific binding.

[0276] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, an antibody binds to an antigen with an affinity of less than about 10 -5M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to an antigen, for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument or a Fortebio molecular interaction instrument.

[0277] As used herein, the terms "monoclonal antibody" and "monoclonal" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by their commonly known single and three letter codes. For example, alanine can be represented by A or Ala.

[0278] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0279] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that, when delivered into the body together with or prior to an antigen, can enhance the immune response of the body to the antigen or change the type of immune response. There are many kinds of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments at present. Aluminum hydroxide adjuvant is more commonly used in clinical experiments.

[0280] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an amount effective to prevent a disease (e.g., a disease associated with PD-1 binding to PD-L1 or overexpression of VEGF such as a tumor) is an amount that is sufficient to prevent, arrest, or delay the onset of the disease (e.g., a disease associated with PD-1 binding to PD-L1 or overexpression of VEGF such as a tumor); an amount effective to treat a disease is an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of a drug, and other therapies being administered concurrently, and the like.

[0281] The term "MSI (microsatellite instability)" is microsatellite instability. Microsatellite is a short tandem repeat sequence widely distributed in the human genome, with single nucleotide, double nucleotide or high nucleotide repeats, with a repeat number of 10-50 times. Compared with normal cells, the microsatellite in some abnormal tissue cells such as tumors changes in length due to insertion or deletion of repeat units, which is called MSI. According to the instability and degree of MSI, it can be divided into microsatellite high instability type (MSI-H), microsatellite low instability type (MSI-L) and microsatellite stable type (MSS). The main cause of MSI is the defect of DNA mismatch repair (MMR) function. After transcription and translation of human mismatch repair genes (MMR genes), the corresponding mismatch repair proteins can be expressed. If the expression of any MMR protein is missing, the mismatch repair function of the cell will be defective, the repair function of base mismatch during DNA replication will be lost, and the accumulation will cause the occurrence of microsatellite instability (MSI). About 15% of colorectal cancer is caused by MSI pathway. It was first discovered in colorectal cancer and can also occur in gastric cancer, endometrial cancer, adrenal cortex tumor, etc. (Baretti M et al. Pharmacol Ther. 2018; 189: 45-62.), and subsequent studies have also found MSI-H / dMMR characteristics in mesothelioma, sarcoma, adrenal cortex cancer, malignant melanoma and ovarian germ cell tumor.

[0282] MSI-H and dMMR represent the results generated by two different detection methods, dMMR is consistent with MSI-H in biology, referred to as MSI-H / dMMR or MSI-high / dMMR, MSI-L and MSS are the phenotypes of MMR normal (proficient mismatch repair, pMMR). The dMMR detection method is the immunohistochemical protein detection of four mismatch genes MSH2, MLH1, MSH6 and PMS2 based on tumor specimens (including surgical specimens, puncture specimens), if any of the four proteins is absent, it is dMMR; if all four proteins are positively expressed, the tumor is pMMR, that is, the mismatch repair function is complete. The detection of MSI is the one-to-one matching detection of the length of tumor cells and somatic cell repetitive DNA sequences (microsatellite sequences), and the length is compared. When using the PCR method and based on the NCI standard to detect 5 standard sites, if two or more sites are inconsistent, it is unstable, defined as MSI-H, if one site is inconsistent, it is called MSI-L (microsatellite low instability), and if 5 sites are consistent, it is MSS. High-throughput sequencing (or next-generation sequencing, NGS) can also be used as a method for detecting microsatellite instability. When more microsatellite sites, such as more than 5 or other microsatellite sites, are detected by PCR, usually ≥30% of the sites are inconsistent, which is called MSI-H, all sites are consistent, which is defined as MSS, and between 0 and 30% is MSI-L.

[0283] Advantages of the application

[0284] The application achieves any one or more of the technical effects described in (1) to (4) below:

[0285] (1) The modification of the antibody Fc end of the application completely eliminates the binding activity of VP101 (hG1WT) to FcγRI and FcγRIIIa_F158, and further completely eliminates the ADCC activity. C (2) The modification of the antibody Fc end of the application completely eliminates the binding activity of VP101 (hG1WT) to complement C1q, and further completely eliminates the CDC activity.

[0286] (2) The modification of the antibody Fc end of the application completely eliminates the binding activity of VP101 (hG1WT) to complement C1q, and further completely eliminates the CDC activity.

[0287] (3) The bispecific antibody of the application can specifically bind to VEGFA and effectively block the binding of VEGFA to VEGFR2, and specifically relieve the promotion of VEGFA on immune suppression and angiogenesis of the body.

[0288] (4) The bispecific antibody of the present application can specifically bind to PD-1 and effectively block the binding of PD-1 to PD-L1, thereby specifically releasing the immune suppression of PD-1 and activating the immune response. BRIEF DESCRIPTION OF DRAWINGS

[0289] Figure 1: The result graph of the affinity constant detection of VP101 (hGlDM) and FcyRI. The concentration of the added antibody in each pair of curves from top to bottom is 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0290] Figure 2: The result graph of the affinity constant detection of Bevacizumab and FcyRI. The concentration of the added antibody in each pair of curves from top to bottom is 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0291] Figure 3: The result graph of the affinity constant detection of Nivolumab and FcyRI. The concentration of the added antibody in each pair of curves from top to bottom is 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0292] Figure 4: The result graph of the affinity constant detection of VP101 (hGlWT) and FcyRI. The concentration of the added antibody in each pair of curves from top to bottom is 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0293] Figure 5: The result graph of the affinity constant detection of VP101 (hG4WT) and FcyRI. The concentration of the added antibody in each pair of curves from top to bottom is 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0294] Figure 6: The result graph of the affinity constant detection of VP101 (hGlDM) and FcyRIIa_H131. The concentration of the added antibody in each pair of curves from top to bottom is 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0295] Figure 7: The result graph of the affinity constant detection of Bevacizumab and FcyRIIa_H131. The concentration of the added antibody in each pair of curves from top to bottom is 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0296] Figure 8: Nivolumab affinity constant determination results plot with FcyRIIa_H131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0297] Figure 9: VP101 (hG1 WT) affinity constant determination results plot with FcyRIIa_H131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0298] Figure 10: VP101 (hG4 WT) affinity constant determination results plot with FcyRIIa_H131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0299] Figure 11: VP101 (hG1 DM) affinity constant determination results plot with FcyRIIa_R131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0300] Figure 12: Bevacizumab affinity constant determination results plot with FcyRIIa_R131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0301] Figure 13: Nivolumab affinity constant determination results plot with FcyRIIa_R131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0302] Figure 14: VP101 (hG1 WT) affinity constant determination results plot with FcyRIIa_R131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0303] Figure 15: VP101 (hG4 WT) affinity constant determination results plot with FcyRIIa_R131. The concentration of antibody added in each pair of curves from top to bottom in the figure is 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, respectively.

[0304] Figure 16: Plot of the affinity constant determination of VP101 (hGl DM) to FcyRIIIa_V158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0305] Figure 17: Plot of the affinity constant determination of Bevacizumab to FcyRIIIa_V158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0306] Figure 18: Plot of the affinity constant determination of Nivolumab to FcyRIIIa_V158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0307] Figure 19: Plot of the affinity constant determination of VP101 (hGl WT) to FcyRIIIa_V158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0308] Figure 20: Plot of the affinity constant determination of VP101 (hG4 WT) to FcyRIIIa_V158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0309] Figure 21: Plot of the affinity constant determination of VP101 (hGl DM) to FcyRIIIa_F158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0310] Figure 22: Plot of the affinity constant determination of Bevacizumab to FcyRIIIa_F158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0311] Figure 23: Plot of the affinity constant determination of Nivolumab to FcyRIIIa_F158. The concentration of antibody added in each pair of curves from top to bottom is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0312] Figure 24: VP101 (hGl WT) affinity constant determination results plot with FcyRIIIa_F158. The concentration of antibody added in each pair of curves from top to bottom in the figure is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0313] Figure 25: VP101 (hG4 WT) affinity constant determination results plot with FcyRIIa_F158. The concentration of antibody added in each pair of curves from top to bottom in the figure is 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, respectively.

[0314] Figure 26: VP101 (hGl DM) affinity constant determination results plot with Clq. The concentration of antibody added in each pair of curves from top to bottom in the figure is 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, respectively.

[0315] Figure 27: Bevacizumab affinity constant determination results plot with Clq. The concentration of antibody added in each pair of curves from top to bottom in the figure is 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, respectively.

[0316] Figure 28: Nivolumab affinity constant determination results plot with Clq. The concentration of antibody added in each pair of curves from top to bottom in the figure is 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, respectively.

[0317] Figure 29: VP101 (hGl WT) affinity constant determination results plot with Clq. The concentration of antibody added in each pair of curves from top to bottom in the figure is 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, respectively.

[0318] Figure 30: VP101 (hG4 WT) affinity constant determination results plot with Clq. The concentration of antibody added in each pair of curves from top to bottom in the figure is 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, respectively.

[0319] Figure 31: VP101 (hGl WT) and VP101 (hGl DM) ADCC activity detection results in CHO-K1-PD1 target cell system expressing PD-1 antigen.

[0320] Figure 32: VP101 (hGl WT) and VP101 (hGl DM) CDC activity detection results in CHO-K1-PD1 target cell system expressing PD-1 antigen.

[0321] Figure 33: Effect of antibody VP101 (hGlDM) on the secretion of the cytokine IFN-γ induced by the mixed culture of PBMC, Raji-PDL1 cells, detected by ELISA.

[0322] Figure 34: Effect of antibody VP101 (hGlDM) on the secretion of the cytokine IL-2 induced by the mixed culture of PBMC, Raji-PDL1 cells, detected by ELISA.

[0323] Figure 35: Detection results of the ADCP activity of VP101 (hGlDM) in the CHO-K1-PD1 target cell system expressing PD-1 antigen. DETAILED DESCRIPTION

[0324] The embodiments of the present application will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art (for example, refer to J. Sambrook et al., Huang Peitang et al. Translated, Molecular Cloning Experiment Guide, Third Edition, Science Press) or according to the product manual are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0325] In the following examples of the present application, the same target drug antibody Bevacizumab (trade name Avastin®, Genentech, Inc.) is used as a control antibody, which is purchased from Roche or can also be prepared according to Preparation Example 1.

[0326] In the following examples of the present application, the same target drug antibody Nivolumab (trade name Opdivo®, Bristol-Myers Squibb) is used as a control antibody, which is purchased from BMS.

[0327] ​​In the following examples of the present application, the isotype control antibody used is human anti-Hen Egg Lysozyme (human IgG, hIgG for short) with variable region sequence from Acierno et al. 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 hIgG1 DM and hIgG4 WT used in the examples are isotype control antibodies of anti-HEL with hG1 DM and hG4 WT constant region sequences, which are prepared in the laboratory of Kangfang Biomedicals Co., Ltd.

[0328] Preparation Example 1: Preparation of Bevacizumab, an antibody against VEGFA

[0329] The amino acid sequences of the heavy chain variable region and the light chain variable region of the marketed anti-VEGFA monoclonal antibody Avastin (Bevacizumab) are referred to in Chinese Patent Publication CN1259962A. The nucleic acid sequences encoding the heavy chain variable region and the light chain variable region were synthesized by GenScript.

[0330] Amino acid sequence of the heavy chain variable region of Bevacizumab (Bevacizumab-Hv): (123 aa)

[0331]

[0332] Nucleic acid sequence encoding the heavy chain variable region of Bevacizumab: (369 bp)

[0333]

[0334]

[0335] Amino acid sequence of the light chain variable region of Bevacizumab (Bevacizumab-Lv): (107 aa)

[0336]

[0337] Nucleic acid sequence encoding the light chain variable region of Bevacizumab: (321 bp)

[0338]

[0339] The heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857; and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.

[0340] The heavy chain cDNA and the light chain cDNA of Bevacizumab were cloned into pcDNA3.1 vector respectively to obtain the recombinant expression plasmid of the antibody Bevacizumab. The recombinant plasmid was transfected into 293F cells. The 293F cell culture solution was purified and then detected.

[0341] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) was prepared.

[0342] Preparation Example 2: Sequence design of the antibody 14C12 against PD-1, the humanized antibody 14C12H1L1 thereof, and the mutant 14C12H1L1 (M)

[0343] The amino acid sequences and the encoding nucleic acid sequences of the heavy chain and the light chain of the antibody 14C12 against PD-1 and the humanized antibody 14C12H1L1 thereof are the same as those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A respectively.

[0344] (1) The heavy chain variable region sequence and the light chain variable region sequence of 14C12

[0345] The amino acid sequence of the heavy chain variable region of 14C12: (118 aa)

[0346]

[0347] The nucleic acid sequence encoding the heavy chain variable region of 14C12: (354 bp)

[0348]

[0349] The amino acid sequence of the light chain variable region of 14C12: (107 aa)

[0350]

[0351] The nucleic acid sequence encoding the light chain variable region of 14C12: (321 bp)

[0352]

[0353] (2) The heavy chain variable region sequence and the light chain variable region sequence, the heavy chain sequence and the light chain sequence of the humanized monoclonal antibody 14C12H1L1

[0354] 14C12H1L1 heavy chain variable region amino acid sequence: (118 aa)

[0355]

[0356] 14C12H1L1 heavy chain variable region nucleic acid sequence: (354 bp)

[0357]

[0358] 14C12H1L1 light chain variable region amino acid sequence: (107 aa)

[0359]

[0360] 14C12H1L1 light chain variable region nucleic acid sequence: (321 bp)

[0361]

[0362] 14C12H1L1 heavy chain (14C12H1) amino acid sequence: (448 aa)

[0363]

[0364] 14C12H1L1 heavy chain (14C12H1) nucleic acid sequence: (1344 bp)

[0365]

[0366]

[0367] 14C12H1L1 light chain (14C12L1) amino acid sequence: (214 aa)

[0368]

[0369] 14C12H1L1 light chain (14C12L1) nucleic acid sequence: (642 bp)

[0370]

[0371] (3) 14C12H1L1 (M) heavy chain variable region sequence and light chain variable region sequence

[0372] 14C12H1L1 (M) was obtained by mutating individual amino acids in the framework region (light chain) of 14C12H1L1.

[0373] 14C12H1L1 (M) heavy chain variable region 14C12H1 (M):

[0374] The heavy chain variable region 14C12H1 of 14C12H1L1 is identical to that of 14C12H1L1, i.e. the amino acid sequence is shown in SEQ ID NO: 9.

[0375] The light chain variable region 14C12L1(M) of 14C12H1L1(M):

[0376]

[0377] Preparation Example 3: Sequence design of bispecific antibody

[0378] 1. Sequence design

[0379] The structural pattern of the bispecific antibody in the present application belongs to the Morrison pattern (IgG-scFv), i.e. the scFv fragment of another antibody is connected at the C-terminus of both heavy chains of one IgG antibody, and the main composition of the heavy chain and the light chain is designed as shown in Table 1 below.

[0380] On the basis of the above-mentioned Bevacizumab, the VP101 antibody with the amino acid sequences of the heavy chain variable region and the light chain variable region of 14C12H1L1(M) as the ScFv fragment part is called VP101(M). Compared with 14C12H1L1, 14C12H1L1(M) effectively optimizes the structure of the bispecific antibody and improves its effectiveness.

[0381] Table 1: Composition design of heavy chain and light chain of VP101(M) and VP101(G4M)

[0382]

[0383] In the above Table 1:

[0384] (1) The right lower corner marked with "V" refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Without the mark "V", the corresponding heavy chain or light chain is full-length containing the constant region. The amino acid sequences and the encoding nucleic acid sequences of these variable regions or full-lengths are all referred to the corresponding sequences described in the above preparation examples.

[0385] (2) The amino acid sequence of Linker 1 is: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18)

[0386] Alternatively, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19) can be used as Linker 2 to replace the aforementioned Linker 1.

[0387] (3) Bevacizumab-H which adopts Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region.

[0388] (4) Bevacizumab-G4H which adopts Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region.

[0389] 2. Expression and purification of antibody VP101 (M)

[0390] The heavy chain cDNA sequence and the light chain cDNA sequence of VP101 (M) were cloned into pUC57simple (provided by Geneart) vector respectively, obtaining pUC57simple-VP101H and pUC57simple-VP101L plasmids respectively.

[0391] The plasmids pUC57simple-VP101H and pUC57simple-VP101L were respectively subjected to enzyme digestion (HindIII & EcoRI), and the recovered heavy chain and light chain were respectively subcloned into pcDNA3.1 vector, and the recombinant plasmid was extracted and co-transfected into 293F cells. After the cells were cultured for 7 days, the culture solution was subjected to high-speed centrifugation, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column, and the protein was eluted with Elution Buffer to recover the target sample antibody VP101 (M), and then the solution was changed to PBS.

[0392] 3. Detection of antibody VP101 (M)

[0393] The purified sample was added to the reduced protein electrophoresis loading buffer and the non-reduced protein electrophoresis loading buffer respectively, boiled, and then subjected to SDS-PAGE electrophoresis detection.

[0394] In order to distinguish from the mutated antibody in Preparation Example 4, VP101 (M) is also referred to as VP101 (hG1WT) in the present application. The above-mentioned VP101 (M) is referred to as a "wild type", which adopts Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region, and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0395] The amino acid sequence of the heavy chain of the immunoglobulin part in VP101 (hG1WT): (453 aa)

[0396]

[0397] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG1 WT): (1359 bp)

[0398]

[0399]

[0400] For the sake of distinguishing from the mutated antibody in Preparation Example 4, VP101 (G4M) is also referred to as VP101 (hG4 WT) in the present application. The above-mentioned VP101 (G4M) is referred to as "wild type", which adopts Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region, and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0401] Amino acid sequence of the heavy chain of the immunoglobulin portion in VP101 (hG4 WT): (450 aa)

[0402]

[0403] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG4 WT): (1350 bp)

[0404]

[0405]

[0406] Preparation Example 4: Design of non-variable region amino acid mutation based on humanized bispecific antibody VP101 (hG1 WT)

[0407] On the basis of VP101 (hG1 WT) obtained in Preparation Example 3, the present inventors introduced a point mutation of leucine to alanine at position No. 234 (L234A) and a point mutation of leucine to alanine at position No. 235 (L235A) in the heavy chain, thereby obtaining VP101 (hG1 DM).

[0408] Amino acid sequence of the heavy chain of the immunoglobulin portion in VP101 (hG1 DM): (453 aa, the mutation site is underlined)

[0409]

[0410] Nucleic acid sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG1 DM): (1359 bp, the mutation site is underlined)

[0411]

[0412]

[0413] The light chain amino acid sequences of the immunoglobulin portions of VP101 (hG1DM), VP101 (hG1WT), and VP101 (hG4WT) are identical, as are their encoding nucleic acid sequences.

[0414] Amino acid sequence of the light chain of the immunoglobulin portion in VP101 (hG1DM): (214 aa)

[0415]

[0416] Nucleic acid sequence encoding the light chain of the immunoglobulin portion in VP101 (hG1DM): (642 bp)

[0417]

[0418]

[0419] Example 1: Determination of affinity constant of FcyRI to VP101 (hG1WT) and VP101 (hG1DM)

[0420] Fc receptor FcyRI, also known as CD64, binds to the Fc end of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of a therapeutic monoclonal antibody to bind to Fc receptors affects the safety and effectiveness of the antibody.

[0421] This experiment used a Fortebio Octet molecular interaction instrument to detect the affinity constant of VP101 (hG1WT) and VP101 (hG1DM) to FcyRI, in order to evaluate the ADCC activity of each antibody.

[0422] The experimental method for detecting the affinity constant of the corresponding antibody to FcyRI by Fortebio Octet molecular interaction instrument is briefly described as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 1 μg / mL of FcyRIa is immobilized on a HIS1K sensor for 50 s, and the sensor is balanced in buffer for 60 s. The CD64 immobilized on the sensor binds to each antibody, and the antibody concentration is 3.12-50 nM (two-fold dilution), for 120 s. The antibody is dissociated in buffer for 120 s. The sensor is regenerated with 10 mM glycine, pH 1.5, for 5 s, repeated 4 times. The detection temperature is 30°C, and the frequency is 0.3 Hz. The data is analyzed by fitting a 1:1 model to obtain the affinity constant.

[0423] The affinity constant determination results of FcyRI to VP101 (hG1WT), VP101 (hG4WT) and VP101 (hG1DM) and control antibodies Nivolumab, Bevacizumab are shown in Table 1 and Figures 1-5.

[0424] Table 1: Kinetic parameters of VP101 (hG1WT) and VP101 (hG1DM) antibodies and their subtypes binding to FcyRI

[0425] Antibody K D (M) kon (1 / Ms) SE (kon) kdis (1 / s) SE (kdis) Rmax (nm)

[0426] VP101 (hG1DM) N / A N / A N / A N / A N / A N / A A Bevacizumab 3.68E-09 6.61E+05 2.07E+04 2.44E-03 9.02E-05 0.46-0.49 Nivolumab 6.20E-09 6.98E+05 2.22E+04 4.32E-03 9.88E-05 0.48-0.53 VP101 (hG1WT) 3.95E-09 5.67E+05 1.82E+04 2.24E-03 9.02E-05 0.68-0.80 VP101 (hG4WT) 8.52E-09 6.28E+05 2.12E+04 5.35E-03 1.07E-04 0.61-0.65

[0427] N / A means that the antibody has no binding or very low binding signal to the antigen, and the results are not analyzed, so the corresponding data cannot be obtained.

[0428] The results show that VP101 (hG1WT) can bind to FcyRI with an affinity constant of 3.95E-09 M; VP101 (hG4WT) can bind to FcyRI with an affinity constant of 8.52E-09 M; Bevacizumab can bind to FcyRI with an affinity constant of 3.68E-09 M; Nivolumab can bind to FcyRI with an affinity constant of 6.20E-09 M; and VP101 (hG1DM) has no binding or very low binding signal to FcyRI, and the results are not analyzed, so the corresponding data cannot be obtained.

[0429] The results show that the affinities of other antibodies to FcyRI are similar, except that VP101 (hG1DM) has no binding to FcyRI. The binding activity of VP101 (hG1DM) is effectively eliminated.

[0430] Example 2: FcγRIIa_H131 affinity constant determination of VP101 (hG1WT) and VP101 (hG1DM)

[0431] Fc receptor FcγRIIa_H131, also known as CD32a_H131, can bind to the Fc end of IgG antibodies and mediate ADCC.

[0432] In this experiment, the Fortebio Octet molecular interaction instrument was used to detect the affinity constant of VP101 (hG1WT) and VP101 (hG1DM) to FcγRIIa_H131 to evaluate the ADCC activity of each antibody.

[0433] The experimental method for detecting the affinity constant of VP101 (hG1WT) and VP101 (hG1DM) to FcγRIIa_H131 by Fortebio Octet molecular interaction instrument is briefly described as follows: the solidification diluent is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4, and the analyte dilution buffer is PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor, the immobilization time was 60 s, and the sensor was equilibrated in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer for 600 s to block the sensor. The FcγRIIa_H131 immobilized on the sensor was combined with the antibody, the antibody concentration was 12.5-200 nM (two-fold gradient dilution), the time was 60 s, the antibody was dissociated in the buffer, and the time was 60 s. The sensor was regenerated with 10 mM Glycine, pH 1.7 and 10 mM nickel sulfate. The detection temperature was 30°C, and the frequency was 0.6 Hz. The data was analyzed by fitting with a 1:1 model to obtain the affinity constant.

[0434] The results of the affinity constant determination of FcγRIIa_H131 to VP101 (hG1WT), VP101 (hG4WT) and VP101 (hG1DM) and control antibodies Nivolumab, Bevacizumab are shown in Table 2 and Figures 6-10.

[0435] Table 2: Kinetic parameters of VP101 (hG1WT) and VP101 (hG1DM) antibodies and their subtypes binding to FcγRIIa_H131

[0436] Antibody K D(M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)VP101(hG1DM)3.57E-081.15E+051.01E+044.11E-033.27E-040.41-0.53Bevacizumab6.44E-082.10E+051.78E+041.36E-025.14E-040.49-0.68NivolumabN / AN / AN / AN / AN / AN / AVP101(hG1WT)2.28E-082.41E+051.48E+045.50E-033.49E-041.40-1.52VP101(hG4WT)3.68E-082.13E+052.43E+047.83E-036.65E-040.41-0.57

[0437] N / A means that the antibody has no or very low binding signal with the antigen, and the result is not analyzed, so the corresponding data cannot be obtained.

[0438] The results show that VP101(hG1WT) can bind to FcγRIIa_H131 with an affinity constant of 2.28E-08 M; VP101(hG4WT) can bind to FcγRIIa_H131 with an affinity constant of 3.68E-08 M; Bevacizumab can bind to FcγRIIa_H131 with an affinity constant of 6.44E-08 M; VP101(hG1DM) can bind to FcγRIIa_H131 with an affinity constant of 3.57E-08 M; and Nivolumab has no or very low binding signal with FcγRIIa_H131, and the result is not analyzed, so the corresponding data cannot be obtained.

[0439] The results show that, except for Nivolumab which has no binding with FcγRIIa_H131, the other antibodies all have binding with FcγRIIa_H131, and the affinity from strong to weak is: VP101(hG1WT), VP101(hG1DM), VP101(hG4WT), Bevacizumab.

[0440] Example 3: Affinity constant determination of FcγRIIa_R131 with VP101(hG1WT) and VP101(hG1DM)

[0441] Fc receptor FcγRIIa_R131 (also known as CD32a_R131) can bind to the Fc end of IgG antibodies and mediate ADCC.

[0442] The affinity constant of VP101 (hGlWT) and VP101 (hGlDM) to FcyRIIa_R131 was measured by Fortebio Octet molecular interaction instrument to evaluate the ADCC activity of each antibody.

[0443] The experimental method for measuring the affinity constant of VP101 (hGlWT) and VP101 (hGlDM) by Fortebio Octet molecular interaction instrument is briefly described as follows: the solidification diluent is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4, and the analyte dilution buffer is PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of FcyRIIa_R131 is immobilized on the NTA sensor for 60 s, and the sensor is equilibrated in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer for 600 s to block the sensor. The FcyRIIa_R131 immobilized on the sensor is combined with the antibody, and the antibody concentration is 12.5-200 nM (two-fold gradient dilution), the time is 60 s, and the antibody is dissociated in the buffer for 60 s. The sensor is regenerated using 10 mM Glycine, pH 1.7 and 10 mM nickel sulfate. The detection temperature is 30°C, and the frequency is 0.6 Hz. The data is analyzed by fitting the 1:1 model to obtain the affinity constant.

[0444] The results of the determination of the affinity constant of FcyRIIa_R131 to VP101 (hGlWT), VP101 (hGlDM), and control antibodies Nivolumab, Bevacizumab are shown in Table 3 and Figures 11-15.

[0445] Table 3: Kinetic parameters of VP101 (hGlWT) and VP101 (hGlDM) antibodies and their subtypes binding to FcyRIIa_R131

[0446] Antibody KD(M) kon(1 / Ms) SE(kon) kdis(1 / s) SE(kdis) Rmax(nm) VP101(hG1DM) 3.35E-08 1.20E+05 9.72E+03 4.03E-03 3.08E-04 0.57 -0.69 Bevacizumab 5.16E-08 2.59E+05 1.72E+04 1.33E-02 4.52E-04 0.42 -0.69 Nivolumab 6.93E-08 4.78E+05 1.09E+05 3.31E-02 2.54E-03 0.08 -0.16 VP101(hG1WT) 2.42E-08 2.14E+05 1.30E+04 5.17E-03 3.28E-04 1.75 -1.92 VP101(hG4WT) 3.57E-08 1.99E+05 1.23E+04 7.09E-03 3.40E-04 0.81 -1.05

[0447] N / A means that the antibody has no or very low binding signal with the antigen, and the result is not analyzed, so the corresponding data cannot be obtained.

[0448] The results show that VP101(hG1WT) can bind to FcγRIIa_R131, and the affinity constant is 2.42E-08M; VP101(hG4WT) can bind to FcγRIIa_R131, and the affinity constant is 3.57E-08M; Bevacizumab can bind to FcγRIIa_R131, and the affinity constant is 5.16E-08M; Nivolumab can bind to FcγRIIa_R131, and the affinity constant is 6.93E-08M; VP101(hG1DM) can bind to FcγRIIa_R131, and the affinity constant is 3.35E-08M.

[0449] The results show that the antibodies all bind to FcγRIIa_R131, and the affinity from strong to weak is: VP101(hG1WT), VP101(hG1DM), VP101(hG4WT), Bevacizumab, Nivolumab.

[0450] Example 4: Affinity constant determination of FcγRIIb to VP101(hG1WT) and VP101(hG1DM)

[0451] Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc end of IgG antibody and participate in the regulation of the function of immune cells.

[0452] The Fortebio Octet molecular interaction instrument was used to detect the affinity constant of VP101 (hG1 WT) and VP101 (hG1 DM) to FcγRIIb, so as to evaluate the binding ability of VP101 (hG1 WT) and VP101 (hG1 DM) to Fc receptors.

[0453] The experimental method of Fortebio Octet molecular interaction instrument for detecting the affinity constant of VP101 (hG1 WT) and VP101 (hG1 DM) to FcγRIIb is as follows: the solidification diluent is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4, and the analyte dilution buffer is PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4. 5 μg / mL of hFCGR2B-his is immobilized on the NTA sensor, the immobilization time is 60 s, and the sensor is equilibrated in PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 buffer for 600 s to block the sensor. The hFCGR2B-his immobilized on the sensor is combined with the antibody, the antibody concentration is 12.5-200 nM (two-fold gradient dilution), the time is 60 s, the antibody is dissociated in the buffer, and the time is 60 s. The sensor is regenerated with 10 mM Glycine, pH 1.7 and 10 mM nickel sulfate. The detection temperature is 30°C, and the frequency is 0.6 Hz. The data is analyzed by fitting with 1:1 model to obtain the affinity constant.

[0454] Example 5: Determination of the affinity constant of FcγRIIIa_V158 to VP101 (hG1 WT) and VP101 (hG1 DM)

[0455] Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc end of IgG antibody and mediate ADCC effect.

[0456] In the experiment, the Fortebio Octet molecular interaction instrument was used to detect the affinity constant of VP101 (hG1 WT) and VP101 (hG1 DM) to FcγRIIIa_V158, so as to evaluate the ADCC activity of each antibody.

[0457] The experimental method for detecting the affinity constant of the corresponding antibody to FcyRIIIa_V158 by Fortebio Octet molecular interaction instrument is briefly described as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcyRIIIa_V158 is immobilized on a HIS1K sensor for 120 s, and the sensor is balanced in the buffer for 60 s. The hFcGR3A (V158)-his immobilized on the sensor is combined with each antibody, and the antibody concentration is 31.25-500 nM (two-fold dilution), for 60 s. The antibody is dissociated in the buffer for 60 s. The sensor is regenerated with 10 mM glycine, pH 1.5, for 5 s, repeated 4 times. The detection temperature is 30°C, and the frequency is 0.3 Hz. The data are analyzed by fitting with a 1:1 model to obtain the affinity constant.

[0458] The results of the affinity constant determination of FcyRIIIa_V158 to VP101 (hG1WT), VP101 (hG4WT) and VP101 (hG1DM) and control antibodies Nivolumab, Bevacizumab are shown in Table 4 and FIGS. 16-20.

[0459] Table 4: Kinetic parameters of VP101 (hG1WT) and VP101 (hG1DM) antibodies and their subtypes binding to FcyRIIIa_V158

[0460] Antibody K D (M) kon (1 / Ms) SE (kon) kdis (1 / s) SE (kdis) Rmax (nm) VP101 (hG1DM) 1.34E-07 6.05E+05 2.36E+05 8.11E-02 7.42E-03 0.07 -0.21 Bevacizumab 2.76E-08 5.06E+05 1.14E+05 1.39E-02 1.41E-03 0.13 -0.51 Nivolumab N / A N / A N / A N / A N / A N / A N / A VP101 (hG1WT) 4.35E-08 2.39E+05 3.14E+04 1.04E-02 8.73E-04 0.80 -1.22 VP101 (hG4WT) N / A N / A N / A N / A N / A N / A N / A

[0461] N / A means that the antibody has no binding or very low binding signal to the antigen, and the result is not analyzed, so the corresponding data cannot be obtained.

[0462] The results show that VP101 (hG1 WT) can bind to FcyRIIIa_V158 with an affinity constant of 4.35E-08 M; VP101 (hG1 DM) can bind to FcyRIIIa_V158 with an affinity constant of 1.34E-07 M; Bevacizumab can bind to FcyRIIIa_V158 with an affinity constant of 2.76E-08 M; and Nivolumab and VP101 (hG4 WT) cannot bind to FcyRIIIa_V158 or have very low binding signals, so the results are not analyzed, and therefore no corresponding data can be obtained.

[0463] The results show that, in addition to Nivolumab and VP101 (hG4 WT) not binding to FcyRIIIa_V158, other antibodies bind to FcyRIIIa_V158, and the affinity from strong to weak is: Bevacizumab, VP101 (hG1 WT), VP101 (hG1 DM).

[0464] Example 6: Determination of the affinity constant of FcyRIIIa_F158 to VP101 (hG1 WT) and VP101 (hG1 DM)

[0465] Fc receptor FcyRIIIa_F158 (also known as CD16a_F158) can bind to the Fc end of IgG antibodies and mediate ADCC.

[0466] In this experiment, the Fortebio Octet molecular interaction instrument was used to detect the affinity constant of VP101 (hG1 WT) and VP101 (hG1 DM) to FcyRIIIa_F158, in order to evaluate the ADCC activity of each antibody.

[0467] The experimental method for detecting the affinity constant of VP101 (hG1 WT) and VP101 (hG1 DM) to FcyRIIIa_F158 by Fortebio Octet molecular interaction instrument is briefly described as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcyRIIIa_F158 is immobilized on HIS1K sensor for 120 s, and the sensor is balanced in buffer for 60 s. hFcGR3A (F158)-his immobilized on the sensor binds to each antibody, and the antibody concentration is 31.25-500 nM (two-fold dilution), for 60 s. The antibody is dissociated in buffer for 60 s. The sensor is regenerated with 10 mM glycine, pH 1.5, for 5 s, repeated 4 times. The detection temperature is 30°C, and the frequency is 0.3 Hz. The data is analyzed by 1:1 model fitting to obtain the affinity constant.

[0468] The affinity constant determination results of FcyRIIIa_F158 to VP101 (hG1WT), VP101 (hG4WT) and VP101 (hG1DM) and control antibodies Nivolumab, Bevacizumab are shown in Table 5 and Figures 21-25.

[0469] Table 5: Kinetic parameters of VP101 (hG1WT) and VP101 (hG1DM) antibodies and their subtypes binding to FcyRIIIa_F158

[0470] Antibody K D (M) kon (1 / Ms) SE (kon) kdis (1 / s) SE (kdis) Rmax (nm) VP101 (hG1DM) N / A N / A N / A N / A N / A N / A Bevacizumab 9.32E-08 2.64E+05 7.16E+04 2.46E-02 2.09E-03 0.08-0.20 Nivolumab N / A N / A N / A N / A N / A N / A VP101 (hG1WT) 7.41E-08 2.47E+05 5.20E+04 1.83E-02 1.55E-03 0.15-0.48 VP101 (hG4WT) N / A N / A N / A N / A N / A N / A

[0471] N / A means that the antibody has no or very low binding signal with the antigen, and the results are not analyzed, so the corresponding data cannot be obtained.

[0472] The results show that VP101 (hG1WT) can bind to FcyRIIIa_F158 with an affinity constant of 7.41E-08 M; Bevacizumab can bind to FcyRIIIa_F158 with an affinity constant of 9.32E-08 M; and Nivolumab, VP101 (hG4WT) and VP101 (hG1DM) have no or very low binding signal with FcyRIIIa_F158, and the results are not analyzed, so the corresponding data cannot be obtained.

[0473] The results show that, in addition to Nivolumab, VP101 (hG4WT) and VP101 (hG1DM) not binding to FcyRIIIa_F158, other antibodies bind to FcyRIIIa_F158, and the affinity from strong to weak is: VP101 (hG1WT), Bevacizumab.

[0474] Example 7: Affinity constant determination of C1q to VP101 (hG1WT) and VP101 (hG1DM)

[0475] Serum complement Clq can bind to the Fc end of IgG antibodies, mediating the CDC effect. The ability of a therapeutic monoclonal antibody to bind Clq affects the safety and effectiveness of the antibody.

[0476] In this experiment, the affinity constant of VP101 (hGlWT) and VP101 (hGlDM) to Clq was detected by Fortebio Octet molecular interaction instrument to evaluate the CDC activity of each antibody.

[0477] The experimental method for detecting the affinity constant of the corresponding antibody to Clq by Fortebio Octet molecular interaction instrument is briefly described as follows: the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 50 μg / mL of antibody is immobilized on FAB2G sensor, and the immobilization height is about 2.0 nm. The sensor is equilibrated in the buffer for 60 s. The antibody immobilized on the sensor is combined with Clq, and the Clq concentration is 0.625 nM-10 nM (two-fold dilution). The time is 60 s. The antigen-antibody is dissociated in the buffer, and the time is 60 s. The sensor is regenerated with 10 mM glycine, pH 1.7, and the time is 5 s, repeated 4 times. The sample plate shaking rate is 1000 rpm, the detection temperature is 30 degrees, and the detection frequency is 0.6 Hz. The data is analyzed by fitting the 1:1 model to obtain the affinity constant. The data acquisition software is Fortebio Data Acquisition 7.0, and the data analysis software is Fortebio Data Analysis 7.0.

[0478] The results of the determination of the affinity constant of Clq to VP101 (hGlWT), VP101 (hG4WT) and VP101 (hGlDM) and control antibodies Nivolumab, Bevacizumab are shown in Table 6 and Figures 26-30.

[0479] Table 6: Kinetic parameters of VP101 (hGlWT) and VP101 (hGlDM) antibodies and their subtypes binding to Clq

[0480] Antibody K D(M) kon (1 / Ms) SE (kon) kdis (1 / s) SE (kdis) Rmax (nm) VP101 (hG1 DM) N / A N / A N / A N / A N / A N / A N / A Bevacizumab 1.14E-09 6.52E+06 5.64E+05 7.40E-03 5.58E-04 0.51 -0.63 Nivolumab N / A N / A N / A N / A N / A N / A N / A VP101 (hG1 WT) 9.76E-10 5.73E+06 5.49E+05 5.59E-03 6.12E-04 0.32 -0.51

[0481] VP101 (hG4 WT) N / A N / A N / A N / A N / A N / A

[0482] N / A means that the antibody has no or very low binding signal with the antigen, and the result is not analyzed, so the corresponding data cannot be obtained.

[0483] The results show that VP101 (hG1 WT) can bind to C1q with an affinity constant of 9.76E-10 M; Bevacizumab can bind to C1q with an affinity constant of 1.14E-09 M; while Nivolumab, VP101 (hG4 WT) and VP101 (hG1 DM) have no or very low binding signal with C1q, and the results are not analyzed, so the corresponding data cannot be obtained.

[0484] The results show that, except for Nivolumab, VP101 (hG4 WT) and VP101 (hG1 DM) have no binding with FcγRIIIa_F158, other antibodies have binding with C1q, and the affinity of VP101 (hG1 WT) is similar to that of Bevacizumab.

[0485] Example 8: Detection of ADCC activity of VP101 (hG1 WT) and VP101 (hG1 DM) on CHO-K1-PD1 cells expressing PD-1 antigen

[0486] In order to detect the ADCC effect of antibodies VP101 (hG1 WT) and VP101 (hG1 DM) at the cellular level, the inventors constructed CHO-K1-PD1 cells expressing PD-1 antigen, and established a system of co-culturing normal human PBMC with target cells for detecting the ADCC activity of antibodies at the cellular level.

[0487] The detection of ADCC activity of VP101 (hG1 WT) and VP101 (hG1 DM) on CHO-K1-PD1 cells expressing PD-1 antigen is as follows:

[0488] The experiment first constructed a human PD-1 overexpression vector pCDH-CMV-PD1FL-Puro (pCDH-CMV-Puro was purchased from Ubio Biotechnology), and the expression vector was packaged into virus to infect CHO-K1 cells. After adding Puromycin (2 μg / mL) for screening, a CHO-K1-PD1 stable cell line stably expressing membrane PD-1 protein was obtained. According to the operation instruction of Ficoll peripheral blood mononuclear cell separation liquid, normal human PBMC was obtained by separation. The separated PBMC was resuspended with 1640 complete culture medium, and the cell number and viability were counted by trypan blue staining. The cells were incubated overnight in a 37°C, 5% CO2, saturated humidity incubator. The next day, CHO-K1-PD1 cells and PBMC were collected, centrifuged to remove the supernatant, and the cell pellet was resuspended with RPMI-1640 (containing 1% BSA) (hereinafter referred to as analysis medium), and the cells were washed twice by centrifugation. The cell number and viability were counted, and the cell concentration was adjusted to the appropriate range with analysis medium. According to the test design, 30000 cells / well of CHO-K1-PD1 cell suspension were added to a 96-well plate; 50 μl of antibody was added, and the mixture was incubated at room temperature for 1 h; after pre-incubation, 900,000 cells / 50 μl / well of PBMC were added, and the mixture was mixed well; the mixture was incubated at 37°C in a 5% CO2 incubator for 4 hrs. After 4 hrs, the 96-well plate was removed, centrifuged at 250 x g for 5 min; 100 μl of cell supernatant (note that do not suck to the plate bottom cells) was carefully transferred to a new 96-well flat-bottom microplate, and 100 μl of freshly prepared reaction solution was added to each well according to the Cytotoxicity Detection Kit instruction, and the mixture was incubated at room temperature for 30 min. The OD values were measured at 490 nm and 650 nm, respectively, and the OD values of each group = OD 490nm -OD 650nm According to ADCC (%) = (experimental group-negative control group) / (target cell LDH maximum release-target cell LDH spontaneous release) x 100%, the ADCC activity of each group was calculated.

[0489] The results of ADCC activity detection of VP101 (hG1 WT) and VP101 (hG1 DM) on CHO-K1-PD1 cells expressing PD-1 antigen are expressed by ADCC%. The results are shown in Figure 31.

[0490] The results show that the positive control 14C12H1L1 (G1WT) has significant ADCC activity in the PBMC and CHO-K1-PD1 mixed culture system, indicating that the ADCC system is normal. Compared with the isotype control antibody hIgG1DM, VP101 (hG1WT) shows significant ADCC activity and presents a dose-dependent relationship, while VP101 (G1DM) shows no ADCC activity. The results show that VP101 (hG1DM) generated by mutation on the basis of VP101 (hG1WT) has no ADCC activity at the cellular level, and the ADCC effect has been eliminated.

[0491] Example 9: Detection of CDC activity of VP101 (hG1WT) and VP101 (hG1DM) on CHO-K1-PD1 cells expressing PD-1 antigen

[0492] 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 construction method), and established a system of co-culturing target cells with normal human complement serum to detect the CDC activity of antibodies at the cellular level.

[0493] The detection of CDC activity of VP101 (hG1WT) and VP101 (hG1DM) on CHO-K1-PD1 cells expressing PD-1 antigen is as follows:

[0494] On the day of detection, trypsinize and collect CHO-K1-PD1 cells, centrifuge at 170 x g for 5 min, then resuspend the cell pellet with RPMI-1640 (containing 1% BSA) (hereinafter referred to as analysis medium), repeat the centrifugal washing twice; count the cells and the viability, adjust the cell concentration to the appropriate range with analysis medium, according to the test design, add CHO-K1-PD1 cell suspension to a 96-well plate, 30000 / well; add 50 μl of antibody, mix well, and pre-incubate at room temperature for 10 min; after pre-incubation, add normal human complement serum (final concentration 2%), 50 μL / well, mix well, and incubate in a 37°C, 5% CO2 incubator for 4 hours. After 4 hours, centrifuge at 250 x g for 5 min; carefully aspirate 100 μl of cell supernatant (note not to aspirate the cells at the bottom of the plate) and transfer it to a new flat-bottom 96-well plate, according to the Cytotoxicity Detection Kit instructions, add 100 μl of freshly prepared reaction solution to each well, incubate at room temperature for 30 min in the dark. Measure the OD values at 490 nm and 650 nm, respectively, and the OD values of each group = OD 490nm -OD 650nmCDC activity of each group was calculated according to CDC (%) = (experimental group - negative control group) / (maximum release of target cell LDH - spontaneous release of target cell LDH) x 100%.

[0495] The results of the CDC activity detection of VP101 (hG1 WT) and VP101 (hG1 DM) on CHO-K1-PD1 cells expressing PD-1 antigen are shown in CDC %, as shown in Figure 32.

[0496] The results show that, in the normal human complement serum and CHO-K1-PD1 mixed culture system, the positive control antibody 14C12H1L1 (G1 WT) has a significant difference in CDC % compared with the isotype control antibody hIgG1 DM group, indicating that the CDC detection system is normal. At the same dose level, compared with the isotype control, the CDC % of VP101 (hG1 WT) and VP101 (hG1 DM) has no significant difference.

[0497] Example 10: Detection of the pharmacodynamic activity of VP101 (hG1 DM) in the mixed culture system (MLR) of peripheral blood mononuclear cells and Raji-PDL1 cells

[0498] The experiment first constructed a human PD-L1 overexpression vector plenti6.3-PD-L1-BSD (plenti6.3-BSD was purchased from invitrogen), and after the expression vector was packaged into virus and infected Raji cells, the Raji-PDL1 stable cell line stably expressing membrane PD-L1 protein was obtained after being screened with BSD (10 μg / mL). According to the operation instruction of Ficoll peripheral blood mononuclear cell separation liquid, normal human PBMC was obtained, and after being resuspended, counted and frozen in 1640 complete culture medium, the PBMC was recovered, SEB (Staphylococcus aureus enterotoxin antigen) was added for stimulation and culture for two days. After two days, the logarithmic phase Raji-PDL1 cells were collected, and mitomycin C (Sigma, working concentration 25 μg / mL) was added for treatment in the incubator for 60 min, and the Raji-PDL1 cells treated with mitomycin C were centrifuged and washed. At the same time, the PBMC stimulated for two days was collected and washed, and the mixed culture was carried out according to the ratio of 1:1 of cell number under the condition of with or without antibody. After 3 days, the supernatant was collected by centrifugation, and the concentrations of IL-2 and IFN-γ in the supernatant were detected by ELISA.

[0499] The results of IFN-γ secretion are shown in Figure 33. The results show that VP101 (hG1 DM) can effectively promote the secretion of IFN-γ, and the activity is significantly better than Nivolumab.

[0500] The IL-2 secretion results are shown in Figure 34. The results show that VP101 (hG1DM) can effectively promote the secretion of IL-2 in a dose-dependent manner, and the activity is significantly better than Nivolumab.

[0501] Example 11: VP101 (hG1DM) does not have antibody-mediated cellular phagocytosis activity on PD-1 expression positive cells

[0502] Antibody-dependent cellular phagocytosis (ADCP) refers to the binding of the Fc fragment of an antibody bound to the surface antigen of a cell to the Fc receptor of a phagocytic cell such as a macrophage, which in turn mediates the phagocytosis of the antibody-bound cell by the phagocytic cell. For immune checkpoint inhibitor antibodies, such as PD-1 antibodies, the presence of ADCP activity will cause damage to immune cells expressing PD-1 that play a role in killing tumors, thereby affecting their anti-tumor activity.

[0503] In this experiment, mouse macrophages were used as effector cells, and a CHO-K1-PD1 cell line overexpressing PD-1 (construction method see Example 8) was used as target cells to detect its mediation of ADCP effect. In this experiment, flow cytometry was used to detect the ADCP activity of VP101 (hG1DM) on cells expressing PD-1, and the results showed that it did not have ADCP activity, while the same target PD-1 antibody Nivolumab had obvious ADCP activity. The specific method is as follows:

[0504] First, the femur bone marrow of C57 mice (purchased from Guangdong Medical Laboratory Animal Center) was taken under sterile conditions, and lysed with red blood cell lysis solution on ice for 5 min, and terminated with DMEM complete culture medium (containing 10% FBS), and washed twice at 1000 rpm. The cell mass was resuspended with 10 mL of DMEM complete culture medium, and M-CSF was added to a working concentration of 100 ng / mL, and induced to culture at 37°C, 5% CO2 in a cell incubator for 7 days, wherein the medium was half-changed and M-CSF was supplemented on the 3rd and 5th day. The cells were induced for 7 days, and then digested with 0.25% trypsin, and the macrophages were collected, centrifuged at 750xg for 5 min, the supernatant was discarded, and the cells were resuspended with DMEM complete culture medium (containing 10% FBS) and counted, and the cell density was adjusted and aliquoted into 96-well conical bottom plates for use.

[0505] CHO-K1-PD1 cells were collected by routine method, centrifuged at 170xg for 5 min, counted and determined the viability after resuspension, and washed once with PBS. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM with PBS, and an appropriate amount of diluted CFSE was used to resuspend the cells (staining density: 10 million cells / mL), and incubated in an incubator for 20 min. 6 mL of DMEM complete medium (containing 10% FBS) was added to terminate the staining, centrifuged at 170xg for 5 min, and the supernatant was discarded; 1 mL of DMEM complete medium was added, and incubated in an incubator for 10 min. The antibody was diluted to 20 μg / mL, 2 μg / mL, and 0.2 μg / mL (working concentration 10 μg / mL, 1 μg / mL, and 0.1 μg / mL) with DMEM complete medium, and a homologous control antibody hIgG1 DM and hIgG4 were designed. Freshly induced mature macrophages were collected, centrifuged at 750xg for 5 min, counted, and transferred to a 96-well conical bottom plate, centrifuged at 1000xg for 5 min, and the supernatant was discarded; the density of CHO-K1-PD1-CFSE cells was adjusted; according to the experimental design, the diluted antibody was added to the target cells at a ratio of 50 μL:50 μL in the corresponding 96-well conical bottom plate containing macrophages, resuspended and mixed, and incubated in a 37°C incubator for 2 h. 150 μL of 1% PBSA at room temperature was added to each well, centrifuged at 1000xg for 5 min, and the supernatant was discarded; washed once with 200 μL of PBSA; 100 μL / sample of APC anti-mouse / human CD11b antibody (diluted 500 times with PBSA) was added to the corresponding sample, mixed, and incubated on ice for 40 min. 150 μL of 1% PBSA was added to each well, centrifuged at 1000xg for 5 min, and the supernatant was discarded; each well was washed once with 200 μL of PBSA. Each well was resuspended with 200 μL of 1% PBSA, and the Beckman flow cytometer was used.

[0506] The macrophages in the system were APC+ positive, and the phagocytosed macrophages were APC and CFSE double positive. The ratio of double positive cells to APC positive cells was used as the phagocytosis rate, and the antibody-mediated ADCP activity was evaluated. The ADCP activity of each group was calculated according to the following formula, and P% was used to represent it:

[0507]

[0508] The results are shown in FIG. 35.

[0509] The results show that Nivolumab has obvious ADCP effect in the macrophage + CHO-K1-PD1 system; the phagocytosis rate of VP101 (hG1DM) is equivalent to that of the isotype control antibody, indicating that VP101 (hG1DM) does not have ADCP effect. The results show that VP101 (hG1DM) is likely to have better anti-tumor effect.

[0510] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. The foregoing is intended to cover all modifications and alternatives within the scope of the present application. It is to be understood that the scope of the application fully encompasses other applications, methods, and systems that could be developed by a person with skill in the art having the benefit of the benefit of this disclosure. Therefore, the specific embodiments discussed are illustrative, and not restrictive, of the scope of the application.

Claims

1. A bispecific antibody, include: Targeting the first protein domain of PD-1, and Targeting the second protein domain of VEGFA; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; in, The immunoglobulin, whose heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs:28-30, and whose light chain variable region comprises LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody, whose heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs:34-36, and whose light chain variable region comprises LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs:37-39, respectively; or, The immunoglobulin, whose heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and whose light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively; and the single-chain antibody, whose heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and whose light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; The immunoglobulin is of human IgG1 subtype; Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin is mutated at any two or three of positions 234, 235 and 237, and after mutation, the affinity constant of the bispecific antibody to FcγRIIIa and / or C1q is reduced compared with before mutation; preferably, the affinity constant is measured by Fortebio Octet molecular interaction instrument.

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

3. A bispecific antibody, include: Targeting the first protein domain of PD-1, and Targeting the second protein domain of VEGFA; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; in, The immunoglobulin, whose heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs:28-30, and whose light chain variable region comprises LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs:31-33, respectively; and the single-chain antibody, whose heavy chain variable region comprises HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs:34-36, and whose light chain variable region comprises LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs:37-39, respectively; or, The immunoglobulin, whose heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:34-36, respectively, and whose light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:37-39, respectively; and the single-chain antibody, whose heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs:28-30, respectively, and whose light chain variable region comprises LCDR1-LCDR3 as shown in SEQ ID NOs:31-33, respectively; The immunoglobulin is of human IgG1 subtype; Wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has one of the following mutation combinations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, G237A.

4. The bispecific antibody according to any one of claims 1 to 3, in, According to the EU numbering system, the heavy chain constant region of the immunoglobulin further has one or more mutations selected from the following: 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.

5. The bispecific antibody according to any one of claims 1 to 4, in, The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from 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; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:1; and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:

3.

6. The bispecific antibody according to any one of claims 1 to 5, which is selected from any one of the following (1) to (12): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 7; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 11; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 3; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:7; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:11; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:7; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:11; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:3; and (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:17; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

3.

7. The bispecific antibody according to any one of claims 1 to 6: The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:24, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO:

26.

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

9. The bispecific antibody according to any one of claims 1 to 8, in, The immunoglobulin or antigen-binding fragment thereof is less than about 10 -9 M, for example, less than about 10 -7 M, 10 -8 M or 10 -9 The affinity constant is 2 M or less and binds to C1q; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.

10. The bispecific antibody according to any one of claims 1 to 9, in, The first protein functional region is directly connected to the second protein functional region or connected through a connecting fragment; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a connecting fragment.

11. The bispecific antibody according to claim 10, in, The connecting fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

12. The bispecific antibody according to any one of claims 1 to 11, in, The first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

13. The bispecific antibody according to any one of claims 1 to 12, in, The single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

14. A bispecific antibody, include: Targeting the first protein domain of PD-1, and Targeting the second protein domain of VEGFA; The number of the first protein functional region is 1, and the number of the second protein functional region is 2; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 17; The single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; The first protein functional region is connected to the second protein functional region via a first connecting fragment; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a second connecting fragment; the first connecting fragment and the second connecting fragment are the same or different; Preferably, the amino acid sequences of the first connecting fragment and the second connecting fragment are independently selected from SEQ ID NO: 18 and SEQ ID NO: 19; Preferably, the amino acid sequences of the first connecting fragment and the second connecting fragment are both as shown in SEQ ID NO:

18.

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

18. A conjugate comprising an antibody or an antigen-binding fragment thereof and a conjugated moiety, in, The immunoglobulin is the bispecific antibody according to any one of claims 1 to 14, and the coupling moiety is a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

19. A kit comprising the bispecific antibody according to any one of claims 1 to 14, or the conjugate according to claim 18; Preferably, the kit further comprises a second antibody that specifically recognizes the immunoglobulin or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

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

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

22. 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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) cancer, 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 non-small cell lung cancer with EGFR and / or ALK sensitive mutations; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer.

23. A method for treating and / or preventing a malignant tumor, comprising the step of administering to a subject in need thereof an effective amount of the bispecific antibody of any one of claims 1 to 14 or the conjugate of claim 18; preferably, the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, lymphoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) cancer, 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 non-small cell lung cancer with EGFR and / or ALK sensitive mutations; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer.

24. The bispecific antibody according to any one of claims 1 to 14, for use in the treatment and / or prevention of a malignant tumor; 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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, cervical cancer, esophageal cancer, microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) cancer, 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 non-small cell lung cancer with EGFR and / or ALK sensitive mutations; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer.

25. The conjugate according to claim 18, for use in 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 cancer, cervical cancer, esophageal cancer, microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) cancer, 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 non-small cell lung cancer with EGFR and / or ALK sensitive mutations; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the kidney tumor is renal cell carcinoma; Preferably, the breast cancer is triple-negative breast cancer; Preferably, the urothelial cancer is bladder cancer.