Anti-PD-1 and Anti-vegfa bifunctional antibody, pharmaceutical composition thereof and use thereof

Abstract

To provide an anti-VEGFA / anti-PD-1 bifunctional antibody, a pharmaceutical composition thereof and a use thereof.SOLUTION: An anti-VEGFA / PD-1 bifunctional antibody comprises a first protein functional region targeting VEGFA and a second protein functional region targeting PD-1. The bifunctional antibody of the present invention can specifically bind to VEGFA and PD-1, specifically relieve immunosuppression of VEGFA and PD-1 in an organism, and inhibit tumor-induced angiogenesis, thus having good application prospect.SELECTED DRAWING: Figure 23

Description

[Technical field]

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

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

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

[0004] Vascular endothelial growth factor (VEGF) is a growth factor that can promote the division and proliferation of endothelial cells, promote the formation of new blood vessels, and improve vascular permeability, and it plays a role by binding to vascular endothelial growth factor receptors on the cell surface and activating tyrosine kinase signaling pathways. In tumor tissues, tumor cells, as well as macrophages and mast cells that infiltrate into tumors, secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine manner, promotes the proliferation and migration of endothelial cells, induces angiogenesis, promotes the continuous growth of tumors, enhances vascular permeability, causes fibrin deposition in the surrounding tissue, and promotes the infiltration of mononuclear cells, fibroblasts, and endothelial cells, which can facilitate the formation of tumor stroma and the invasion of tumor cells into new blood vessels, and also promote tumor metastasis. Therefore, inhibiting tumor angiogenesis is currently considered to be one of the most promising tumor treatment methods. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and neuropilin-1 (NRP-1). The first three receptors are similar in structure, belong to the tyrosine kinase superfamily, and consist of an extramembrane region, a transmembrane segment, and an intramembrane region, where the extramembrane region is composed of an immunoglobulin-like domain, and the intramembrane region is a tyrosine kinase region. VEGFR1 and VEGFR2 are located primarily on the surface of vascular endothelial cells, and VEGFR3 is located primarily on the surface of lymphatic endothelial cells.

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

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

[0007] VEGFR3 is one of the tyrosine kinase family members, mainly expressed in embryonic vascular endothelial cells and adult lymphatic endothelial cells, and VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis; NRP-1 is a non-tyrosine kinase transmembrane protein that cannot transmit biological signals independently and can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor (Ma Li, Chinese Journal of Birth Health and Heredity, Vol. 24(No. 5): pp. 146-148 (2016)).

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

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

[0010] Programmed cell death receptor-1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein membrane surface receptor that belongs to the CD28 immunoglobulin superfamily and is generally expressed on T cells, B cells and myeloid cells. PD-1 has two natural ligands, PD-L1 and PD-L2. Both PD-L1 and PD-L2 belong to the B7 superfamily and are constitutively or inducibly expressed on the membrane surface of various cells, including non-hematopoietic cells and various tumor cells. PD-L1 is mainly expressed on T cells, B cells, DCs and microvascular endothelial cells, as well as various tumor cells, while PD-L2 is expressed only on antigen-presenting cells, such as dendritic cells and macrophages. The interaction between PD-1 and its ligands can suppress lymphatic activation, T cell proliferation, and secretion of cytokines, such as IL-2 and IFN-γ.

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

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

[0013] Due to the broad spectrum of anti-tumor potential and remarkable efficacy of PD-1 antibodies, it is widely accepted in the industry that antibodies targeting the PD-1 pathway will provide a breakthrough in the treatment of various tumors: non-small cell lung cancer, renal cell carcinoma, ovarian cancer and melanoma (Homet MB, Parisi G. et al. Anti-PD-1 therapy in melanoma. Semin Oncol. 2015 June;42(3):466-473), as well as in lymphoma and anemia (Held SA, Heine A, et al. Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets 2013 September;13(7):768-74).

[0014] Bifunctional antibodies, also known as bispecific antibodies, are specific drugs that simultaneously target two different antigens and can be produced by immunoselective purification. In addition, bispecific antibodies can also be produced by genetic engineering, which has certain advantages due to the corresponding flexibility in terms of optimization of binding sites, consideration of synthesis type and yield. Currently, bispecific antibodies have been shown to exist in more than 45 types (Muller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 2010;24:89-98). Many bispecific antibodies have been developed in the IgG-ScFv format, i.e., the Morrison format (Coloma MJ, Morrison SL Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997;15:159-163), which has been demonstrated to be one of the ideal formats for bispecific antibodies due to its similarity to the naturally occurring IgG format and advantages in antibody engineering, expression and purification (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010;23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011;3:299-309). Summary of the Invention [Problem to be solved by the invention]

[0015] Currently, there is a need to develop bifunctional antibody drugs that target both PD-1 and VEGF (eg, VEGFA). [Means for solving the problem]

[0016] overview Through intensive research and inventive efforts, and based on the commercially available VEGFA monoclonal antibody Avastin (bevacizumab) and the previously obtained 14C12H1L1 (see China Patent Publication No. CN106977602A), the inventors obtained a humanized bifunctional antibody, named VP101, which can simultaneously bind to VEGFA and PD-1, and block the binding of VEGFA to VEGFR2 and the binding of PD-1 to PD-L1.

[0017] The present inventors have surprisingly found that VP101: Effectively binding to PD-1 on the surface of human immune cells, relieving the immunosuppression mediated by PD-L1 and PD-1, and promoting the secretion of IFN-γ and IL-2 by human immune cells; effectively inhibiting VEGFA-induced proliferation of vascular endothelial cells, thereby inhibiting tumor-induced angiogenesis; and / or It may be used in the preparation of medicines for preventing and treating malignant tumors, such as liver cancer, lung cancer, melanoma, renal tumor, ovarian cancer and lymphoma. It was found that this is possible.

[0018] The present invention is described in detail below.

[0019] One aspect of the present invention is a first protein functional region that targets VEGFA; A second protein functional domain that targets PD-1 A bispecific antibody comprising: Preferably, the first protein functional region is an anti-VEGFA antibody or an antigen-binding fragment thereof, a heavy chain variable region of the anti-VEGFA antibody comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15 to 17, respectively, and a light chain variable region of the anti-VEGFA antibody comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18 to 20, respectively; the second protein functional region is an anti-PD-1 antibody or an antigen-binding fragment thereof, a heavy chain variable region of the anti-PD-1 antibody comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 21 to 23, respectively, or a light chain variable region of the anti-PD-1 antibody comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 24 to 26, respectively; Concerning bispecific antibodies.

[0020] In some embodiments of the invention, the anti-VEGFA antibody or antigen-binding fragment thereof is selected from a Fab, a Fab', a F(ab')2, a Fd, a Fv, a dAb, a complementarity determining region fragment, a single chain antibody, a humanized antibody, a chimeric antibody, and a diabody; and / or the anti-PD-1 antibody or antigen-binding fragment thereof is selected from a Fab, a Fab', a F(ab')2, a Fd, a Fv, a dAb, a complementarity determining region fragment, a single chain antibody, a humanized antibody, a chimeric antibody, and a diabody; Bispecific antibodies are provided.

[0021] In some embodiments of the invention, the bispecific antibody is in the IgG-scFv format.

[0022] In some embodiments of the invention, the first protein functional region is an immunoglobulin, an immunoglobulin heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15 to 17, respectively, and an immunoglobulin light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18 to 20, respectively; the second protein functional region is a single chain antibody, a single chain antibody heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 21 to 23, respectively, and a single chain antibody light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 24 to 26, respectively; or The first protein functional region is a single chain antibody, a single chain antibody heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 21 to 23, respectively, and a single chain antibody light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 24 to 26, respectively; the second protein functional region is an immunoglobulin, an immunoglobulin heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15 to 17, respectively, and an immunoglobulin light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18 to 20, respectively.

[0023] In certain embodiments of the invention, a bispecific antibody is provided comprising a first protein functional domain that targets VEGFA and a second protein functional domain that targets PD-1; the first protein functional region is an immunoglobulin, an immunoglobulin heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15 to 17, respectively, and an immunoglobulin light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18 to 20, respectively; the second protein functional region is a single chain antibody, a single chain antibody heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 21 to 23, respectively, and a single chain antibody light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 24 to 26, respectively; or the first protein functional region is a single chain antibody, a single chain antibody heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 21 to 23, respectively, and a single chain antibody light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 24 to 26, respectively; and the second protein functional region is an immunoglobulin, an immunoglobulin heavy chain variable region comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 15 to 17, respectively, and an immunoglobulin light chain variable region comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18 to 20, respectively; Bispecific antibodies are provided.

[0024] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5, the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: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; or 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; the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:7. Bispecific antibodies are provided.

[0025] In some embodiments of the present invention, Bispecific antibodies are provided in which the immunoglobulin comprises a non-CDR region derived from a species other than mouse, for example a human antibody.

[0026] In some embodiments of the present invention, the immunoglobulin comprises a constant region derived from a human antibody; Preferably, the constant region of the immunoglobulin is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4. Bispecific antibodies are provided.

[0027] In some embodiments of the present invention, The heavy chain constant region of the immunoglobulin is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region is a human Ig kappa chain C region; Bispecific antibodies are provided.

[0028] In some embodiments of the invention, the immunoglobulin constant regions are humanized, for example, each heavy chain constant region is an Ig gamma-1 chain C region, ACCESSION:P01857, and each light chain constant region is an Ig kappa chain C region, ACCESSION:P01834.

[0029] In some embodiments of the present invention, the first protein functional domain and the second protein functional domain are linked directly or by a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, e.g., 1, 2, 3, 4, 5, or 6, and GGGGS (SEQ ID NO: 14) is a building block of the linker. Bispecific antibodies are provided.

[0030] In some embodiments of the invention, bispecific antibodies are provided, wherein the number of first protein functional regions and second protein functional regions is each independently one, two or more.

[0031] In some embodiments of the invention, bispecific antibodies are provided in which there is one immunoglobulin and two single chain antibodies, preferably two identical single chain antibodies.

[0032] In some embodiments of the invention there are provided bispecific antibodies, wherein the immunoglobulin is IgG, IgA, IgD, IgE or IgM, preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0033] In some embodiments of the present invention, bispecific antibodies are provided in which a single chain antibody is linked to the C-terminus of an immunoglobulin heavy chain. Since an immunoglobulin has two heavy chains, two single chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single chain antibody molecules are identical.

[0034] In some embodiments of the invention, bispecific antibodies are provided in which there are two single chain antibodies, one terminus of each single chain antibody linked to the C-terminus or N-terminus of one of the two heavy chains of the immunoglobulin.

[0035] In some embodiments of the invention, the disulfide bond is located at the V H and V L Methods for introducing a disulfide bond between the VH and VL of an antibody are well known in the art, and are described, for example, in U.S. Patent No. 5,747,654; Rajagopal et al., Prot. Engin. vol. 10 (1997) pp. 1453-1459; Reiter et al., Nat. Biotechnol. vol. 14 (1996) pp. 1239-1245; Reiter et al., Protein Engineering vol. 8 (1995) pp. 1323-1331; Webber et al., Molecular Immunology vol. 32 (1995) pp. 249-258; Reiter et al., Immunity vol. 2 (1995) pp. 281-287; Reiter et al., JBC vol. 269 (1994) pp. 18327-18331; Reiter et al., Inter. J. of Cancer, all of which are incorporated herein by reference. 58 (1994) pp. 142-149; or Reiter et al., Cancer Res. 54 (1994) pp. 2714-2718.

[0036] In some embodiments of the invention, the bispecific antibody is -5 Less than M, for example 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K less than or equal to M D binds to the VEGFA protein and / or the PD-1 protein at D is measured by a Fortebio molecular interaction instrument.

[0037] In some embodiments of the invention, the bispecific antibody has an EC 50 Binds to VEGFA protein in ECs; 50 was detected by indirect ELISA; and / or Bispecific antibodies with an EC of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.17 nM, less than 0.16 nM, or less than 0.15 nM 50 PD-1 protein in the EC 50 is detected by indirect ELISA, Bispecific antibodies are provided.

[0038] Another aspect of the invention pertains to an isolated nucleic acid molecule encoding a bispecific antibody according to any of the embodiments of the invention.

[0039] The present invention also relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0040] The present invention also relates to a host cell comprising an isolated nucleic acid molecule of the invention or comprising a vector of the invention.

[0041] Another aspect of the invention relates to a method for preparing a bispecific antibody according to any of the embodiments of the invention, comprising culturing a host cell of the invention under suitable conditions and isolating the bispecific antibody from the cell culture.

[0042] Another aspect of the invention relates to a conjugate comprising a bispecific antibody and a conjugation moiety, wherein the bispecific antibody is a bispecific antibody according to any of the embodiments of the invention and the conjugation moiety is a detectable label; preferably, the conjugation moiety is a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

[0043] Another aspect of the invention is a kit comprising a bispecific antibody according to any of the embodiments of the invention or comprising a complex of the invention, comprising: Preferably, the kit further comprises a secondary antibody capable of specifically binding to the bispecific antibody; optionally, the secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

[0044] Another aspect of the invention relates to the use of a bispecific antibody, as described in any of the embodiments of the invention, in the preparation of a kit for detecting the presence or level of VEGFA and / or PD-1 in a sample.

[0045] Another aspect of the invention relates to a pharmaceutical composition comprising a bispecific antibody according to any of the embodiments of the invention or comprising a conjugate of the invention; optionally, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0046] The bispecific antibody of the present invention or the pharmaceutical composition of the present invention can be formulated into any dosage form known in the pharmaceutical art, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, and sprays. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention shall be sterile and stable under the conditions of manufacture and storage. One of the preferred dosage forms is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: a required amount of the bispecific antibody of the present invention is added to a suitable solvent, and optionally other desired ingredients (including but not limited to pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) are added at the same time, followed by filtration and sterilization. Moreover, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or lyophilization) for convenient storage and use, which can be dispersed in a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0047] Furthermore, the bispecific antibodies of the invention can be present in a pharmaceutical composition in unit dose form to facilitate administration. In some embodiments, the unit dose is at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. When the pharmaceutical composition is in a liquid (e.g., injectable) dosage form, it can contain the bispecific antibodies of the invention in a concentration of at least 0.1 mg / mL, such as at least 0.25 mg / mL, at least 0.5 mg / mL, at least 1 mg / mL, at least 2.5 mg / mL, at least 5 mg / mL, at least 8 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, or at least 100 mg / mL.

[0048] The bispecific antibody or pharmaceutical composition of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intravesical, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal. However, for many therapeutic applications, the preferred route / mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal and intramuscular injection). Those skilled in the art will recognize that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibody or pharmaceutical composition of the invention is administered by intravenous infusion or injection.

[0049] The bispecific antibodies or pharmaceutical compositions provided herein can be used alone or in combination, or can be used in combination with additional pharma- ceutical effective agents (e.g., tumor chemotherapeutic agents). Such additional pharma- ceutical effective agents can be administered prior to, simultaneously with, or after administration of the bispecific antibodies of the invention or the pharmaceutical compositions of the invention.

[0050] In the present invention, the administration regimen can be adjusted to achieve the optimum desired response (e.g., a therapeutic or prophylactic response), for example, it can be a single administration or multiple administrations over a period of time, and can be characterized by a proportional reduction or increase in the dose depending on the exigencies of the treatment.

[0051] Another aspect of the invention relates to the use of a bispecific antibody or a complex of the invention according to any of the embodiments of the invention in the preparation of a medicament for the prevention and / or treatment of a malignant tumor, preferably wherein the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor and leukemia.

[0052] Another aspect of the present invention is a method for producing a (1) a medicine or agent for detecting the level of VEGFA in a sample; A medicine or drug for blocking the binding of VEGFA to VEGFR2; Medicines or agents for downregulating VEGFA activity or levels; A medicine or agent for reducing VEGFA stimulation of vascular endothelial cell proliferation; A medicine or drug for inhibiting vascular endothelial cell proliferation, or medicines or agents for blocking tumor angiogenesis; and / or (2) a medicine or agent for blocking the binding of PD-1 to PD-L1; Medicaments or agents for downregulating the activity or levels of PD-1; A medicine or drug for reducing immunosuppression of PD-1 in the body; A medicine or drug for promoting IFN-γ secretion in T lymphocytes, or Medicine or agent for promoting IL-2 secretion in T lymphocytes The present invention relates to the use of a bispecific antibody or a complex of the present invention according to any of the embodiments of the present invention in the preparation of a

[0053] In one embodiment of the invention, the use is non-therapeutic and / or non-diagnostic.

[0054] Another aspect of the invention is an in vivo or in vitro method comprising administering to a cell an effective amount of a bispecific antibody or a complex of the invention according to any embodiment of the invention, (1) a method for detecting the level of VEGFA in a sample; Methods for blocking the binding of VEGFA to VEGFR2 Methods for downregulating VEGFA activity or levels, Methods for reducing VEGFA stimulation of vascular endothelial cell proliferation, A method for inhibiting vascular endothelial cell proliferation, or Methods for blocking tumor angiogenesis; and / or (2) A method for blocking the binding of PD-1 to PD-L1, Methods for downregulating PD-1 activity or levels, Methods for reducing PD-1 immunosuppression in vivo A method for promoting IFN-γ secretion in T lymphocytes, or Method for promoting IL-2 secretion in T lymphocytes The present invention relates to a method selected from the group consisting of

[0055] In one embodiment of the present invention, the in vitro methods are non-therapeutic and / or non-diagnostic.

[0056] In the in vitro experiments of the present invention, both anti-VEGFA antibody and anti-VEGFA / anti-PD-1 bifunctional antibody can inhibit the proliferation of HUVEC cells, and both anti-PD-1 antibody and anti-VEGFA / anti-PD-1 bifunctional antibody can promote the secretion of IFN-γ and / or IL-2, and activate the immune response.

[0057] Another aspect of the invention relates to a method for preventing and / or treating a malignant tumor comprising administering to a subject in need thereof an effective amount of a bispecific antibody or a complex of the invention according to any of the embodiments of the invention, preferably wherein the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, e.g. non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor and leukemia.

[0058] A non-limiting typical range for a therapeutically or prophylactically effective amount of a bispecific antibody of the invention is 0.02-50 mg / kg, e.g., 0.1-50 mg / kg, 0.1-25 mg / kg, or 1-10 mg / kg. It should be noted that the dosage may vary depending on the type and severity of the condition being treated. Furthermore, for any particular patient, a particular dosing regimen will be adjusted over time according to the needs of the patient and the professional judgment of the physician; one of ordinary skill in the art will appreciate that the dosage ranges set forth herein are for illustrative purposes only and are not intended to limit the use and scope of the pharmaceutical compositions of the invention.

[0059] In the present invention, the subject may be a mammal, such as a human.

[0060] There is provided a bispecific antibody or conjugate according to any of the embodiments of the invention for use in the prevention and / or treatment of a malignant tumor, preferably wherein the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, e.g. non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor and leukemia.

[0061] (1) detecting the level of VEGFA in a sample; Blocking the binding of VEGFA to VEGFR2, Downregulation of VEGFA activity or levels, Attenuating VEGFA stimulation of vascular endothelial cell proliferation; Inhibition of vascular endothelial cell proliferation, or Blocking tumor angiogenesis; and / or (2) blocking the binding of PD-1 to PD-L1; Downregulation of PD-1 activity or levels, Relief of PD-1-induced immunosuppression in vivo Stimulation of IFN-γ secretion in T lymphocytes, or Stimulation of IL-2 secretion in T lymphocytes In accordance with one embodiment of the present invention, there is provided a bispecific antibody or conjugate according to any of the embodiments of the present invention for use in

[0062] Antibody drugs, especially monoclonal antibodies, have achieved good results in treating various diseases. The traditional experimental methods for obtaining these therapeutic antibodies are to immunize animals with antigens, obtain antibodies targeting the antigen in the immunized animals, or improve those antibodies with low affinity to the antigen by affinity maturation.

[0063] The light and heavy chain variable regions determine antigen binding; the variable region of each chain contains three hypervariable regions called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H chain) include HCDR1, HCDR2 and HCDR3, and the CDRs of the light chain (L chain) include LCDR1, LCDR2 and LCDR3, as named by Kabat et al., Bethesda Md, Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication (Vols. 1-3) 1991:91-3242).

[0064] Preferably, the CDRs can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool" in for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic Acids Research, Vol. 38, Suppl. 1 (2009): D301-D307.

[0065] The amino acid sequences of the CDR regions of the monoclonal antibody sequences (1) to (13) below were analyzed by technical means well known to those skilled in the art, for example, by the VBASE2 database and according to the IMGT regulations, and the results are as follows: (1) Bevacizumab The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:7.

[0066] The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GYTFTNYG (SEQ ID NO: 15) HCDR2: INTYTGEP (SEQ ID NO: 16) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 17) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDISNY (SEQ ID NO: 18) LCDR2: FTS (SEQ ID NO: 19) LCDR3: QQYSTVPWT (SEQ ID NO: 20) (2) 14C12H1L1 The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:11.

[0067] The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO: 21) HCDR2: ISGGGRYT (SEQ ID NO: 22) HCDR3: ANRYGEAWFAY (SEQ ID NO: 23) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDINTY (sequence number 24) LCDR2: RAN (SEQ ID NO: 25) LCDR3: LQYDEFPLT (SEQ ID NO:26) (3) VP101 The amino acid sequences of the nine CDR regions of the heavy chain are as follows: HCDR1: GYTFTNYG (SEQ ID NO: 15) HCDR2: INTYTGEP (SEQ ID NO: 16) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 17) HCDR4: GFAFSSYD (SEQ ID NO: 21) HCDR5: ISGGGRYT (SEQ ID NO: 22) HCDR6: ANRYGEAWFAY (SEQ ID NO: 23) HCDR7: QDINTY (SEQ ID NO: 24) HCDR8: RAN (SEQ ID NO: 25) HCDR9: LQYDEFPLT (SEQ ID NO: 26) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDISNY (SEQ ID NO: 18) LCDR2: FTS (SEQ ID NO: 19) LCDR3: QQYSTVPWT (SEQ ID NO: 20) In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine methods that are widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0068] As used herein, when referring to the amino acid sequence of VEGFA protein (GenBank ID: NP_001165097.1), this includes the full-length VEGFA protein as well as fusion proteins of VEGFA, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse IgG or human IgG. However, it will be understood by those skilled in the art that mutations or variants (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of VEGFA protein can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "VEGFA protein" is intended to include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of VEGFA protein, this also includes the corresponding sequence fragment of its natural or artificial variant. In one embodiment of the present invention, the amino acid sequence of VEGFA protein is shown as the underlined portion of SEQ ID NO: 1 (302 amino acids in total, without the last 6 His).

[0069] As used herein, when referring to the amino acid sequence of VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), this includes the full-length VEGFR2 protein, or the extracellular fragment of VEGFR2, VEGFR2-ECD, or a fragment containing VEGFR2-ECD, and further includes a fusion protein of VEGFR2-ECD, such as a fragment fused to Fc protein fragment (mFc or hFc) of mouse IgG or human IgG. However, it will be understood by those skilled in the art that mutations or variants (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of VEGFR2 protein can be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "VEGFR2 protein" is intended to include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of VEGFR2 protein, this also includes the corresponding sequence fragment of its natural or artificial variant. In one embodiment of the invention, the amino acid sequence of the extracellular fragment of VEGFR2, VEGFR2-ECD, is shown as the wavy underlined portion of SEQ ID NO:4 (766 amino acids).

[0070] In the present invention, unless otherwise specified, VEGFR is VEGFR1 and / or VEGFR2; its specific protein sequence is a sequence known in the prior art, and can refer to the sequence disclosed in existing literature or GenBank.For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0071] As used herein, when referring to the amino acid sequence of PD-1 protein (programmed cell death protein 1, NCBI GenBank: NM_005018), this includes the full-length PD-1 protein, or the extracellular fragment of PD-1, PD-1ECD, or a fragment containing PD-1ECD, and also includes fusion proteins of PD-1ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse IgG or human IgG. However, it will be understood by those skilled in the art that mutations or variants (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of PD-1 protein may be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "PD-1 protein" is intended to include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of PD-1 protein, this also includes the corresponding sequence fragment of the natural or artificial variant.

[0072] As used herein, the term EC 50 means the concentration for 50% of the maximal effect, i.e. the concentration which produces 50% of the maximal effect.

[0073] As used herein, the term "antibody" refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. In a general sense, a heavy chain can be understood as the polypeptide chain having a larger molecular weight in an antibody, and a light chain refers to the polypeptide chain having a smaller molecular weight in an antibody. Light chains are classified as kappa and lambda light chains. Heavy chains are generally classified as μ, δ, γ, α or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (CH The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. H Area and V L The regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed between conserved regions, called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L), each of which forms an antibody binding site. The assignment of amino acids to regions or domains may be based on Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196 (1987):901-917; Chothia et al., Nature 342 (1989):878-883, or the IMGT numbering system definitions, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic Acids Research 38 Suppl_1 (2009):D301-D307. In particular, the heavy chain may also comprise more than three CDRs, for example six, nine, or twelve. For example, in a bispecific antibody of the invention, the heavy chain may be an ScFv with the C-terminus of the heavy chain of an IgG antibody linked to another antibody, and in this case the heavy chain comprises nine CDRs. The term "antibody" is not limited by any particular method for producing the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may be of different isotypes, for example IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0074] As used herein, the term "antigen-binding fragment", also known as "antigen-binding portion", refers to a polypeptide comprising a fragment of a full-length antibody that specifically binds to the same antigen as the full-length antibody and / or retains the ability to compete with the full-length antibody for specific binding to an antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibody fragments (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0075] As used herein, the term "Fd fragment" refers to a V H Domain and C H1 "Fv fragment" refers to an antibody fragment consisting of the V domain of a single arm of an antibody; L Domains and V H The term "dAb fragment" refers to an antibody fragment consisting of the V domain; H The term "Fab fragment" refers to an antibody fragment consisting of the V domain (Ward et al., Nature 341 (1989):544-546); L Domain, V H Domain, C L Domain and C H1 the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0076] In some cases, the antigen-binding fragment of the antibody is L Domain and V HSingle chain antibodies (e.g., scFv) are antibodies in which the domains are paired to form a monovalent molecule via a linker that allows for the production of a single polypeptide chain (see, e.g., Bird et al., Science 242 (1988):423-426, and Huston et al., Proc. Natl. Acad. Sci. USA 85 (1988):5879-5883). Such scFv molecules have the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L It may have -COOH. Suitable linkers in the prior art consist of a repeated GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, as well as variants thereof (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers useful in the present invention are disclosed by Alfthan et al., Protein Eng. 8 (1995): 725-731, Choi et al., Eur. J. Immunol. 31 (2001): 94-106, Hu et al., Cancer Res. 56 (1996): 3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999): 41-56, and Roovers et al., Cancer Immunol. (2001).

[0077] In some cases, the antigen-binding fragment of the antibody is H Domain and V LDiabodies, or bivalent antibodies, are domains expressed in a single polypeptide chain.However, the linker used is too short to allow the two domains to pair on the same chain, so the domains pair with the complementary domains on another chain, generating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90 (1993):6444-6448 and Poljak RJ et al., Structure 2 (1994):1121-1123).

[0078] Antigen-binding fragments of antibodies (such as the antibody fragments referred to above) can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as intact antibodies.

[0079] As used herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0080] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or fragment thereof that is derived from a group of highly homogeneous antibodies, i.e., from a group of identical antibody molecules, except for natural mutations that may occur naturally. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained by hybridoma technology, first reported by Kohler et al. (Nature, 256:495, 1975), or by recombinant DNA technology (see, for example, U.S. Patent No. 4,816,567).

[0081] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light and / or heavy chain is derived from an antibody (which may be from a particular species or belong to a particular antibody class or subclass) and another portion of the light and / or heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but which in either case retains binding activity for the target antigen (U.S. Patent No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 (1984):6851-6855).

[0082] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (acceptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), which can be a non-human (e.g. mouse, rat or rabbit) antibody with the expected specificity, affinity or reactivity. Furthermore, some amino acid residues in the framework regions (FR) of the acceptor antibody can also be replaced with amino acid residues of the corresponding non-human antibody or with amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further details regarding humanized antibodies, see, e.g., Jones et al., Nature 321 (1986):522-525; Reichmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2 (1992):593-596, and Clark, Immunol. Today 21 (2000):397-402.

[0083] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant". Epitopes or antigenic determinants generally consist of chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, epitopes generally contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which may be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by GE Morris (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, the sites of interaction span protein amino acid residues that are separated from one another.

[0084] As used herein, the term "isolated" means obtained by artificial means from a natural state. When a certain "isolated" substance or component occurs naturally, it may be that its natural environment is changed or that it is isolated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide exists in a certain living animal, and the same polynucleotide or polypeptide of high purity isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0085] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material element carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages, such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). Vectors can contain various elements that regulate expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0086] As used herein, the term "host cell" means a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli or bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as an S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0087] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and an antigen that it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one in which the antibody binds to the antigen within about 10 -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Less than M or 10 -10 Affinity below M (K D ) to an antigen. In some embodiments of the invention, the term "target" refers to specific binding.

[0088] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, an antibody has a dissociation equilibrium constant of about 10, as determined, for example, on a BIACORE instrument using surface plasmon resonance (SPR). -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Less than M or 10 -10 The dissociation equilibrium constant (K D ) binds to the antigen.

[0089] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "PcAb" have the same meaning and can be used interchangeably; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Furthermore, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0090] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or vehicle that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (e.g., see Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th Edition). See, Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0091] As used herein, the term "adjuvant" refers to a non-specific immune stimulatory agent that can enhance the immune response of the organism to an antigen or change the type of immune response when delivered to the organism together with the antigen or delivered to the organism in advance. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharides, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is frequently used in clinical trials.

[0092] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, a prophylactically effective amount (e.g., for a disease associated with the binding of PD-1 to PD-L1 or overexpression of VEGF, e.g., tumor) is an amount sufficient to prevent, inhibit or delay the onset of the disease (e.g., a disease associated with the binding of PD-1 to PD-L1 or overexpression of VEGF, e.g., tumor); a therapeutically effective amount is an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. It is undoubtedly within the scope of a person skilled in the art to determine such an effective amount. For example, the effective amount in therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's immune system, the general condition of the patient, e.g., age, weight and sex, the form of drug administration, and other treatments performed at the same time, etc. Effect of the Invention

[0093] The bispecific antibody VP101 can effectively bind specifically to VEGFA, effectively block the binding of VEGFA to VEGFR2, and specifically alleviate the immunosuppressive effect of VEGFA and the promoting effect of VEGFA on angiogenesis in vivo; VP101 can effectively bind specifically to PD-1, effectively block the binding of PD-1 to PD-L1, and specifically alleviate the immunosuppressive effect of PD-1 in vivo and activate the immune response. [Brief description of the drawings]

[0094] [Figure 1] Figure 1 shows the results of SDS-PAGE detection of bifunctional antibody VP101. The four lanes from left to right show the samples and their respective loading amounts: antibody in loading buffer for non-reducing protein electrophoresis, 1 μg; antibody in loading buffer for reducing protein electrophoresis, 1 μg; marker, 5 μL; BSA, 1 μg. [Diagram 2] FIG. 2 shows the results of determining the kinetic profile parameters for the binding of antibody VP101 to PD-1. [Diagram 3] FIG. 3 shows the results of determining the kinetic profile parameters for the binding of antibody BsAbB7 to PD-1. [Figure 4] FIG. 4 shows the results of determining the kinetic profile parameters for the binding of antibody BsAbB8 to PD-1. [Diagram 5] FIG. 5 shows the results of determining the kinetic profile parameters for the binding of antibody 14C12H1L1 to PD-1. [Figure 6] FIG. 6 shows the results of detection of kinetic profile parameters for the binding of the antibody nivolumab to PD-1. [Figure 7] FIG. 7 shows the results of determining the kinetic profile parameters for the binding of antibody VP101 to VEGF. [Figure 8] FIG. 8 shows the results of determining the kinetic profile parameters for the binding of antibody BsAbB7 to VEGF. [Figure 9] FIG. 9 shows the results of determining the kinetic profile parameters for the binding of antibody BsAbB8 to VEGF. [Figure 10] FIG. 10 shows the results of determining the kinetic profile parameters for the binding of the antibody bevacizumab to VEGF. [Figure 11] FIG. 11 shows the binding activity of antibody VP101 to VEGFA as detected by indirect ELISA. [Figure 12] FIG. 12 shows the binding activity of antibodies VP101, BsAbB7, BsAbB8 and bevacizumab to VEGFA-his, respectively, as detected by indirect ELISA. [Figure 13] FIG. 13 shows the binding activity of antibody VP101 to PD-1 as detected by indirect ELISA. [Figure 14] FIG. 14 shows the binding activity of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab to PD-1, respectively, as detected by indirect ELISA. [Figure 15]FIG. 15 shows the activity of antibody VP101 in competing with VEGFR2 for binding to VEGFA as detected by competitive ELISA. [Figure 16] Figure 16 shows the activity of antibody VP101 in competing with PD-L1 for binding to PD-1, as detected by competitive ELISA. [Figure 17] FIG. 17 shows the binding EC50 of antibodies 14C12H1L1 and VP101 to 293T-PD-1 cell surface protein PD-1 as detected by FACS. [Figure 18] FIG. 18 shows the binding EC50 of antibodies VP101, BsAbB7 and BsAbB8 to the 293T-PD-1 cell surface protein PD-1 as detected by FACS. [Figure 19] Figure 19 shows the activity of antibodies VP101 and 14C12H1L1 in competing with PD-L1 for binding to the 293T-PD-1 cell surface protein PD-1, as detected by FACS. [Figure 20] Figure 20 shows the activity of antibodies 14C12H1L1, VP101, BsAbB7, BsAbB8, 14C12H1L and nivolumab in competing with PD-L1 for binding to the 293T-PD-1 cell surface protein PD-1 as detected by FACS. [Figure 21] FIG. 21 shows the neutralizing bioactivity of antibodies VP101, BsAbB7 and BsAbB8 in blocking VEGF to activate the NFAT signaling pathway. [Figure 22] FIG. 22 shows the effect of bevacizumab and VP101 on HUVEC cell proliferation. [Diagram 23] FIG. 23 shows the effect of VP101 on IFN-γ secretion in a mixed culture system of DC and PBMC cells. [Figure 24] FIG. 24 shows the effects of VP101, BsAbB7 and BsAbB8 on IFN-γ secretion in a mixed culture system of DC cells and PBMC cells. [Diagram 25]FIG. 25 shows the effects of VP101, BsAbB7 and BsAbB8 on IL-2 secretion in a mixed culture system of DC cells and PBMC cells. [Figure 26] Figure 26 shows the effect of antibodies 14C12H1L1 and VP101 on the secretion of cytokine IL-2 induced by co-culture of PBMC cells and Raji-PD-L1 cells, as detected by ELISA. [Figure 27] Figure 27 shows the effect of antibodies 14C12H1L1 and VP101 on the secretion of the cytokine IFN-γ induced by co-culture of PBMC cells and Raji-PD-L1 cells, as detected by ELISA. [Figure 28] Figure 28 shows the effect of antibodies 14C12H1L1, VP101, BsAbB7 and BsAbB8 on the secretion of cytokine IFN-γ induced by co-culture of PBMC cells and Raji-PD-L1 cells, as detected by ELISA. [Figure 29] Figure 29 shows the effect of antibodies 14C12H1L1, VP101, BsAbB7 and BsAbB8 on the secretion of cytokine IL-2 induced by co-culture of PBMC cells and Raji-PD-L1 cells, as detected by ELISA. [Diagram 30] FIG. 30 shows the inhibition of tumor growth by different concentrations of VP101. [Diagram 31] FIG. 31 shows the effect of different concentrations of VP101 on mouse body weight. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and are not interpreted as limiting the scope of the present invention. In the absence of clear description of techniques or conditions, they are carried out according to the techniques or conditions disclosed in the technical literature (see, for example, Guide to Molecular Cloning Experiments, J. Sambrook et al., and Huang Peitang et al., translated, 3rd edition, Science Press) or according to the product manual. The reagents or equipment used are all commercially available conventional products unless their manufacturers are specified.

[0096] In the following examples of the present invention, the commercial antibody bevacizumab (trade name Avastin®) for the same target was purchased from Roche as a control antibody or prepared according to Preparation Example 4.

[0097] In the following examples of the present invention, the commercial antibody Nivolumab (trade name Opdivo®) for the same target was purchased from BMS as a control antibody. In the following examples of the present invention, the amino acid sequences of the control antibodies BsAbB7 and BsAbB8 were identical to the amino acid sequences of BsAbB7 and BsAbB8 in China Patent Publication No. CN105175545A, respectively.

[0098] Preparation Example 1: Preparation of fusion proteins PD-1-mFc, PD-1-hFc, and PD-L1-hFc The preparation of fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc and SDS-PAGE electrophoresis detection were carried out by fully referring to Preparation Example 1 of China Patent Publication No. CN106632674A.

[0099] The amino acid sequences and coding nucleotide sequences of the fusion proteins PD-1-mFc, PD-1-hFc, and PD-L1-hFc in this Preparation Example are the same as those of PD-1-mFc, PD-1-hFc, and PDL-1-hFc, respectively, in Preparation Example 1 of Chinese Patent Publication No. CN106632674A.

[0100] The fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc were thus obtained.

[0101] Preparative Example 2: Expression and purification of the fusion protein VEGFA-His 1. Construction of Plasmid VEGFA-His PCR amplification was performed using VEGFA human cDNA (purchased from Origene) as a template, and the hVEGFA-His fragment was purified and isolated using a regular DNA product purification kit. The isolated hVEGFA-His fragment and expression vector pcDNA3.1 were enzymatically digested with XbaI and HindIII-HF, and the target gene fragment was isolated by gel extraction and ligated with the linear expression vector by T4 ligase. All ligation products were then transformed into DH5a chemically competent cells and coated on agar plates containing Amp. Well-isolated single colonies were selected for colony PCR identification, and PCR-positive clones were inoculated into LB culture medium for cultivation, and bacterial solutions were obtained and sent to Guangzhou Invitrogen Biotechnology for sequencing verification. Alignment of the sequencing results showed that the insertion sequences of the positive recon were completely accurate.

[0102] 2. Expression and purification of the fusion protein VEGFA-His The recombinant plasmid VEGFA-his was transfected into 293F cells (purchased from Invitrogen) for 7 days according to the manual of the Lipofectamine transfection kit (purchased from Invitrogen), and then the culture medium was subjected to high-speed centrifugation, the supernatant was concentrated and buffer-exchanged into binding buffer A, and then loaded onto a HisTrap column, and the protein was linearly eluted with elution buffer A. The primary purified sample was buffer-exchanged into binding buffer B of a HiTrap desalting column, loaded onto a HiTrap Q column, and the protein was linearly eluted with elution buffer B, and the target sample was isolated and buffer-exchanged into PBS. The purified sample was added to reducing protein electrophoresis loading buffer for SDS-PAGE electrophoresis detection.

[0103] The fusion protein VEGFA-His was thus obtained. The amino acid sequence of VEGFA-His is as follows (171 aa): APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCERPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCCRCDKPRRHHHHHH (SEQ ID NO:1) Here, the underlined portion is the amino acid sequence of VEGFA.

[0104] Nucleotide sequence encoding VEGFA-His (513 bp) GCACCCATGGCCGAGGGCGGCGGCCAGAACCACCACGAGGTGGTGAAGTTCATGGACGTGTACCAGAGAAGCTACTGCCACCCCATCGAGACCCTGGTGGACATCTTCCAGGAGTACCCCGACGAGATCG AGTACATCTTCAAGCCCAGCTGCGTGCCCTGATGAGATGCGGCGGCTGCTGCAACGACGAGGGCTGGAGTGCGTGCCCACCGAGGAGAGCAACATCACCATGCAGATCATGAGAATCAAGCCCCACCA GGGCCAGCACATCGGCGAGATGAGCTTCCTGCAGCACAACAAGTGCGAGTGCAGACCCAAGAAGGACAGAGCCAGACAGGAGAACCCCTGCGGCCCCTGCAGCGAGAGAAGAAAGCACCTGTCGTGCAG GACCCCAGACCTGCAAGTGCAGCTGCAAGAACACCGACAGCAGATGCAAGGCCAGACAGCTGGAGCTGAACGAGAGAACCTGCAGATGCGACAAGCCCAGAAGACATCATCACCATCACCAC (SEQ ID NO: 2) Preparative Example 3: Expression and purification of the fusion protein VEGFR2-hFc 1. Synthesis of gene VEGFR2-hFc: The amino acids corresponding to the extracellular fragment VEGFR2 ECD of the gene VEGFR2 (vascular endothelial growth factor receptor 2, NCBI GenBank: NP_002244) were fused with TEV and the Fc protein fragment of human IgG (hFc), respectively (SEQ ID NO: 3). The synthesis of the corresponding coding nucleotide sequence was commissioned to Genscript (SEQ ID NO: 4).

[0105] VEGFR2, vascular endothelial growth factor receptor 2, NCBI GenBank NP_002244; hFc: Ig gamma-1 chain C region, ACCESSION: P01857,106-330; Amino acid sequence of fusion protein VEGFR2-hFc: (998aa)

[0106] [Table 1]

[0107] Here, the wavy underlined portion is the ECD portion of VEGFR2, the boxed portion is the TEV enzyme digestion site, and the solid line portion is the hFc portion.

[0108] Nucleotide sequence encoding the fusion protein VEGFR2-hFc: (2997bp)

[0109] [Table 2]

[0110] Here, the wavy underlined portion is the ECD portion of VEGFR2, the boxed portion is the TEV enzyme digestion site, and the solid line portion is the hFc portion.

[0111] 2. Construction of plasmid pUC57simple-VEGFR2-hFc: The VEGFR2-hFc coding gene synthesized by Genscript was cloned into the expression vector pUC57simple (provided by Genscript) to obtain the pUC57simple-VEGFR2-hFc plasmid.

[0112] 3. Construction of recombinant plasmid pcDNA3.1-VEGFR2-hFc: Plasmid pUC57simple-VEGFR2-hFc was enzymatically digested (Xba I and BamH I), and the fusion gene fragment VEGFR2-hFc isolated by electrophoresis was ligated with expression vector pcDNA3.1 (purchased from Invitrogen) to obtain pcDNA3.1-VEGFR2-hFc, which was transfected into competent E. coli cells DH5a (purchased from TIANGEN); transfection and cultivation were carried out according to the manual. The positive pcDNA3.1-VEGFR2-hFc colonies were screened, and E. coli was amplified according to conventional methods, and then the recombinant plasmid pcDNA3.1-VEGFR2-hFc was extracted using a kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., DP103-03) according to the kit's manual.

[0113] 4. Transfection of Recombinant Plasmid pcDNA3.1-VEGFR2-hFc into 293F Cells The recombinant plasmid pcDNA3.1-VEGFR2-hFc was transfected into 293F cells (purchased from Invitrogen) using a Lipofectamine transfection kit (purchased from Invitrogen).

[0114] 5. SDS-PAGE electrophoresis detection of VEGFR2-hFc protein After transfecting the recombinant plasmid pcDNA3.1-VEGFR2-hFc into 293F cells for 7 days, the culture medium was subjected to high-speed centrifugation, micropore membrane vacuum filtration, and Mabselect SuRe column purification to obtain VEGFR2-hFc fusion protein samples, and a portion of the sample was added to reducing protein electrophoresis loading buffer for SDS-PAGE electrophoresis detection.

[0115] The fusion protein VEGFR2-hFc was thus obtained.

[0116] Preparation Example 4: Preparation of anti-VEGFA antibody bevacizumab China Patent Publication No. CN1259962A refers to the amino acid sequences of the heavy and light chain variable regions of the commercially available VEGFA monoclonal antibody Avastin (bevacizumab). Genscript was commissioned to synthesize the nucleotide sequences encoding the heavy and light chain variable regions.

[0117] Amino acid sequence of the heavy chain variable region of bevacizumab: (123aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 5) Nucleotide sequence encoding the heavy chain variable region of bevacizumab: (369bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGA CTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC (SEQ ID NO: 6) Amino acid sequence of the light chain variable region of bevacizumab: (107aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 7) Nucleotide sequence encoding the light chain variable region of bevacizumab: (321bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG (SEQ ID NO: 8) Heavy chain constant regions were all Ig gamma-1 chain C region, ACCESSION: P01857; light chain constant regions were all Ig kappa chain C region, ACCESSION: P01834.

[0118] The heavy and light chain cDNA of bevacizumab were cloned into vector pcDNA3.1 to obtain the recombinant expression plasmid of antibody bevacizumab. The recombinant plasmid was transfected into 293F cells. The culture medium of 293F cells was purified and then detected.

[0119] The anti-VEGFA monoclonal antibody Avastin (bevacizumab) was thus obtained.

[0120] Preparation Example 5: Preparation and detection of anti-PD-1 humanized antibody 14C12H1L1 The preparation was carried out according to Examples 3-4 disclosed in Chinese Patent Publication No. CN106977602A.

[0121] The amino acid sequences of the heavy and light chain variable regions of humanized antibody 14C12H1L1, as well as the nucleotide sequences encoding them, are also the same as those disclosed in Examples 3-4 of Chinese Patent Publication No. CN106977602A, and are also provided herein below: Amino acid sequence of the heavy chain variable region of humanized antibody 14C12H1L1: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 9) Nucleotide sequence encoding the heavy chain variable region of humanized antibody 14C12H1L1: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 10) Amino acid sequence of the light chain variable region of humanized antibody 14C12H1L1: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 11) Nucleotide sequence encoding the light chain variable region of humanized antibody 14C12H1L1: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 12) The anti-PD-1 humanized antibody 14C12H1L1 was thus obtained.

[0122] Preparation Example 6: Preparation and identification of hIgG The sequence of human anti-hen egg white lysozyme IgG (anti-HEL, or human IgG abbreviated as hIgG) was reported in a study published by Acierno et al., entitled "Affinity The variable region sequence of Fab F10.6.6 from "Fv domain of anti-protein antibodies (Fv domain maturation increases the stability and plasticity of anti-protein antibodies)" (Acierno et al., J Mol Biol. 2007;374(1):130-46) was derived. The preparation method is as follows: Nanjing Genscript Biology was commissioned to carry out amino acid codon optimization and gene synthesis for the heavy and light chain (full sequence or variable region) genes of human IgG antibody by referring to the standard techniques introduced in "Guide to Molecular Cloning Experiments (3rd Edition)" and by using standard molecular cloning techniques, such as PCR, enzyme digestion, DNA gel extraction, ligation transformation, colony PCR or enzyme digestion identification, and the heavy and light chain genes were subcloned into antibody heavy chain expression vectors and antibody light chain expression vectors of mammalian expression system, respectively, and the heavy and light chain genes of those recombinant expression vectors were further sequenced and analyzed. After confirming that the sequences were accurate, endotoxin-free expression plasmids were prepared in large scale, and the heavy and light chain plasmids were transiently co-transfected into HEK293 cells expressing recombinant antibodies. After 7 days of culture, the cell culture medium was collected and affinity purified using an rProtein A column (GE), and the quality of the obtained antibody samples was determined using SDS-PAGE and SEC-HPLC standard analytical techniques.

[0123] hIgG was thus obtained and used in Examples 8-9 below.

[0124] Example 1: Sequence design, preparation and detection of the heavy and light chains of bifunctional antibody VP101 1. Sequence Design The structure of the bifunctional antibody VP101 of the present invention is the Morrison type (IgG-scFv), i.e., the C-terminus of the two heavy chains of an IgG antibody is each bound to an scFv fragment of a different antibody, and the main structural design of the heavy and light chains is as shown in Table 1 below.

[0125] [Table 3]

[0126] In Table 1 above: (1) Those with a "V" designation in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. For those without a "V" designation, the corresponding heavy or light chain is full length, including the constant region. The corresponding sequences described in the preparation examples above refer to the amino acid sequences of these variable regions or full length, and the nucleotide sequences encoding them. (2) The amino acid sequence of linker 1 is GGGGSGGGGSGG GGSGGGGS (sequence number 13).

[0127] 2. Expression and Purification of Antibody VP101 The heavy and light chain cDNA sequences of VP101 were each cloned into the vector pUC57simple (provided by Genscript) to obtain the plasmids pUC57simple-VP101H and pUC57simple-VP101L, respectively.

[0128] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were enzymatically digested (HindIII and EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned into vector pcDNA3.1, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was further eluted in one step with elution buffer to isolate the target sample antibody VP101 and buffer exchanged into PBS.

[0129] 2. Detection of Antibody VP101 The purified samples were added to both reducing and non-reducing protein electrophoresis loading buffers and then boiled for SDS-PAGE electrophoresis detection. The electropherogram of VP101 is shown in Figure 1. The target proteins of the reducing protein sample are at 75 kD and 25 kD, and the target protein of the non-reducing protein sample (single antibody) is at 200 kD.

[0130] Unless otherwise specified, the humanized antibody VP101 used in the following experiments was prepared by the methods of this example.

[0131] Example 2: Determination of kinetic parameters of humanized antibody VP101 1. Determination of kinetic parameters for binding of humanized antibody VP101 to PD-1-mFc The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 5 μg / mL of antibody was immobilized on the AHC sensor at a fixation height of approximately 0.4 nM. The sensor was equilibrated in the buffer for 60 s, and the antibody immobilized on the sensor was allowed to bind to PD-1-mFc at concentrations ranging from 0.62 to 50 nM (3-fold gradient dilution) for 120 s, and then the antigen and antibody were allowed to dissociate in the buffer for 300 s. The data were analyzed by fitting with a 1:1 model to obtain affinity constants. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0. The kinetic parameters for the binding of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and the control antibody nivolumab to PD-1-mFc are shown in Table 2, and the results of the detection of the kinetic characteristic parameters are shown in Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6, respectively.

[0132] [Table 4]

[0133] K D is the affinity constant; kon is the binding rate of the antigen and antibody; kdis is the dissociation rate of the antigen and antibody; K D =kdis / kon.

[0134] These results demonstrate that antibodies VP101 and BsAbB7 are comparable in terms of affinity for PD-1-mFc; the affinity constant of VP101 for PD-1-mFc is significantly smaller than that of BsAbB8, suggesting that VP101 has better binding activity; the constant dissociation rate for VP101 and PD-1-mFc is significantly smaller than that of BsAbB8 and 14C12H1L1, suggesting that VP101 binds more stably to antigen and has a slower dissociation rate than 14C12H1L1 and BsAbB8.

[0135] 2. Determination of kinetic parameters for binding of humanized antibody VP101 to VEGF-His The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 1 μg / mL VEGF-His was immobilized on the HIS1K sensor for 20 seconds, then the sensor was equilibrated in buffer for 60 seconds, and the VEGF immobilized on the sensor was bound to the antibody at a concentration of 12.34-1000 nM (3-fold gradient dilution) for 120 seconds, and then the antigen and antibody were dissociated in buffer for 300 seconds. The data were analyzed by fitting with a 1:1 model to obtain the affinity constants. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0136] The kinetic parameters for the binding of antibodies VP101, BsAbB7, BsAbB8 and the control antibody bevacizumab to VEGF-His are shown in Table 3, and the results of the determination of the kinetic profile parameters are shown in Figures 7, 8, 9 and 10, respectively.

[0137] [Table 5]

[0138] These results reveal that the antibodies VP101 and BsAbB7 are comparable in terms of affinity to the antigen, and the affinity constant of VP101 is significantly smaller than that of BsAbB8 and the control antibody bevacizumab, suggesting that VP101 has better binding activity; the dissociation rate constant of VP101 for VEGF-His is significantly smaller than that of BsAbB8, suggesting that VP101 binds more stably to the antigen and has a slower dissociation rate than BsAbB8.

[0139] Example 3: Detection of the binding activity of antibody VP101 to antigen by ELISA 1. Detection of the binding activity of antibody VP101 to the antigen VEGFA-his by indirect ELISA The method is as follows: A microplate was coated with VEGFA-His and incubated at 37°C for 2 hours. After washing, the microplate was blocked with 1% BSA for 2 hours. After washing, gradient diluted antibodies were added to the microplate and incubated at 37°C for 30 minutes. After washing, enzyme-labeled goat anti-human IgG secondary antibody working solution was added to the microplate and incubated at 37°C for 30 minutes. After washing, TMB color development solution was added to the microplate for 5 minutes in the absence of light, and then stop solution was added to stop the color reaction. The microplate was then immediately placed in a microplate reader and the OD value of each well of the microplate was recorded at 450 nm. Data was analyzed and processed using SoftMax Pro 6.2.1.

[0140] The results of detection of the binding of antibody VP101 to the antigen VEGFA-His are shown in Figure 11. The absorbance at each dose is shown in Table 4. Binding EC of the antibody 50 was calculated by curve fitting using antibody concentration as the abscissa and absorbance as the ordinate, and the results are shown in Table 4 below.

[0141] [Table 6]

[0142] These results demonstrate that antibody VP101 can efficiently bind to VEGFA protein, that the binding efficiency is dose-dependent, and that the two antibodies are comparable in terms of binding activity to human VEGFA.

[0143] 2. Detection of the binding activity of antibodies VP101, BsAbB7 and BsAbB8 to the antigen VEGFA-His by indirect ELISA The method is as follows: A microplate was coated with VEGFA-His and incubated overnight at 4°C. After washing, the microplate was blocked with 1% BSA (dissolved in PBS) for 2 hours. After washing, gradient dilutions of antibodies were added to the microplate and incubated at 37°C for 30 minutes. After washing, horseradish peroxidase-labeled goat anti-human IgG Fc (Jackson, 109-035-098) working solution was added to the microplate and incubated at 37°C for 30 minutes. After washing, TMB (Neogen, 308177) for color development was added to the microplate for 5 minutes in the absence of light, and then stop solution was added to stop the color reaction. The microplate was then immediately placed in a microplate reader and the OD value of each well in the microplate was recorded at 450 nm. The data was analyzed and processed using SoftMax Pro 6.2.1.

[0144] The results of the binding of antibody VP101 to the antigen VEGFA-His are shown in Figure 12. The absorbance of each dose is shown in Table 5. Binding EC of the antibody 50 was calculated by curve fitting using the antibody concentration as the abscissa and the absorbance value as the ordinate, and the results are shown in Table 5 below.

[0145] [Table 7]

[0146] These results reveal that antibodies VP101, BsAbB7, BsAbB8 and bevacizumab can all efficiently bind to VEGF protein, their binding efficiencies are dose-dependent, and antibody VP101 has higher binding activity to human VEGF than BsAbB7 and BsAbB8.

[0147] 3. Detection of the binding activity of antibody VP101 to antigen PD-1 by indirect ELISA The method is as follows: The microplate was coated with human PD-1-mFc and incubated overnight at 4°C. After blocking with 1% BSA at 37°C for 2 hours, the antibody was added to the microplate and then incubated at 37°C for 30 minutes. After washing the microplate and tapping it dry, HRP-labeled goat anti-human IgG (H+L) secondary antibody (Jackson, 109-035-088) was added and the microplate was incubated at 37°C for 30 minutes. After washing the microplate and tapping it dry, TMB (Neogen, 308177) was added for 5 minutes for color development, and then stop solution was added to stop the color development. The microplate was then immediately placed in a microplate reader and the OD value of each well in the microplate was recorded at 450 nm. SoftMax Pro Data were analyzed and processed using 6.2.1.

[0148] The detection results of the binding of antibody VP101 to the antigen PD-1 are shown in Figure 13. The absorbance of each dose is shown in Table 6. By quantitative analysis of the bound antibody VP101, curve simulation was performed to obtain the binding efficiency EC 50 was obtained and is shown in Table 6 below.

[0149] [Table 8]

[0150] These results demonstrate that the antibody VP101 can efficiently bind to the PD-1 protein, and that the binding efficiency is dose-dependent.

[0151] 4. Detection of the binding activity of antibodies VP101, BsAbB7 and BsAbB8 to the antigen PD-1 by indirect ELISA The method is as follows: The microplate was coated with human PD-1-mFc and incubated overnight at 4°C. After blocking with 1% BSA at 37°C for 2 hours, the antibody was added to the microplate and then incubated at 37°C for 30 minutes. After washing the microplate and tapping dry, horseradish peroxidase-labeled goat anti-human IgG Fc (Jackson, 109-035-098) was added and the microplate was incubated at 37°C for 30 minutes. After washing the microplate and tapping dry, TMB (Neogen, 308177) was added for color development for 5 minutes, and then stop solution was added to stop the color development. The microplate was then immediately placed into a microplate reader and the OD value of each well in the microplate was recorded at 450 nm. The data was analyzed and processed using SoftMax Pro 6.2.1.

[0152] The detection results of the binding of antibody VP101 to antigen PD-1 are shown in Figure 14. The absorbance of each dose is shown in Table 7. By quantitative analysis of the bound antibody VP101, curve simulation was performed to obtain the binding efficiency EC 50 was obtained and is shown in Table 7 below.

[0153] [Table 9]

[0154] These results demonstrate that antibody VP101 can efficiently bind to PD-1 protein, that the binding efficiency is dose-dependent, and that antibody VP101 has higher binding activity to human PD-1 than BsAbB7 and BsAbB8.

[0155] 5. Detection of the activity of antibody VP101 in competing with VEGFR2 for binding to the antigen VEGFA by competitive ELISA The method is detailed below: The microplate was coated with VEGF-His and incubated at 37°C for 2 hours. After washing, the microplate was blocked with 1% BSA at 37°C for 1 hour. After washing, gradient diluted antibodies and human VEGFR2 ECD-mFc-bio (final concentration: 0.02 μg / mL) were added to the microplate and incubated at room temperature for 2 hours. After washing, HRP-labeled streptavidin SA-HRP (1:4000) working solution was added to the microplate and incubated at 37°C for 30 minutes. After washing, the microplate was added TMB color development solution for color development in the absence of light for 5 minutes, and then stop solution was added to stop the color development reaction. The microplate was then immediately placed in a microplate reader and the OD value of each well in the microplate was recorded at 450 nm. The data was analyzed and processed using SoftMax Pro 6.2.1.

[0156] The detection results are shown in Figure 15. The absorbance of each dose is shown in Table 8. Through quantitative analysis of the absorbance of the bound antibody, curve simulation was performed to determine the binding efficiency EC 50 The results were obtained (Table 8).

[0157] [Table 10]

[0158] The results demonstrate that antibody VP101 can effectively bind to the antigen VEGFA and inhibit the binding of VEGFR2 to VEGFA, and its efficiency in inhibiting the binding of VEGFR2 to VEGFA is dose-dependent.

[0159] 6. Detection of antibody VP101 in competition with PD-L1 for binding to antigen PD-1 by competitive ELISA The method is detailed below: A microplate was coated with PD-1-hFc and incubated overnight at 4℃. After blocking the microplate with 1% BSA for 2 hours, different concentrations of antibodies were mixed with PD-L1-hFc for 10 minutes each (see Table 10 for dilution concentrations). After incubation at 37℃ for 30 minutes, the microplate was washed and tapped dry. Then, enzyme-labeled secondary antibody was added and the microplate was incubated at 37℃ for 30 minutes. After washing the microplate and tapping dry, TMB was added for color development for 5 minutes, and then stop solution was added to stop the color development. The microplate was then immediately placed into a microplate reader and the OD value of each well in the microplate was recorded at 450nm (see Table 10). The data was analyzed and processed using SoftMax Pro 6.2.1.

[0160] The detection results are shown in Figure 16. The absorbance of each dose is shown in Table 9. By quantitative analysis of the bound antibody VP101, curve simulation was performed to obtain the binding efficiency EC 50 The results were obtained (Table 9).

[0161] [Table 11]

[0162] These results demonstrate that the antibody VP101 can effectively bind to the antigen PD-1 and inhibit the binding of the ligand PD-L1 to PD-1, and its efficiency in inhibiting the binding of PD-L1 to PD-1 is dose-dependent.

[0163] Example 4: Binding of antibody VP101 to cell membrane surface antigens First, 293T cells expressing PD-1 antigen were constructed, and then the specific binding ability of the antibody to the cell membrane surface antigen was analyzed and confirmed by flow cytometry.

[0164] 1. Construction of 293T cells expressing PD-1 antigen The PD-1 vector pLenti6.3-PD-1 (vector pLenti6.3 was purchased from Invitrogen) was transfected into 293T cells, and a clone group of 293T-PD-1 cells stably expressing PD-1 was obtained by screening.

[0165] 2. Detection of antibody binding to cell surface antigens The antigen-expressing 293T-PD-1 cells obtained in the previous step were digested with pancreatin by the conventional pancreatin digestion method, and the number of cells in each collection tube was 2 × 10 5 The antibody dilution solutions with concentrations gradiently diluted with PBSA (1% BSA) were incubated with 293T-PD-1 cells on ice for 2 hours, and then 100 μL of FITC goat anti-human IgG (1:500) was added to each tube and incubated on ice for 1 hour. Then, PBS was used for washing, and 300 μL of PBSA was used to resuspend the cells, and the fluorescent signal (MFI) was detected in the FITC channel of the flow cytometer.

[0166] The results are shown in Figure 17, and the MFI values ​​for each concentration are shown in Table 10. Fluorescence quantification analysis and curve fitting of the bound 14C12H1L1 antibody revealed that the binding EC 50 was calculated to be 3.5 nM.

[0167] [Table 12]

[0168] These results clearly show that the VP101 antibody can effectively bind to the PD-1 antigen on the surface of 293T-PD-1 host cells, the binding efficiency is dose-dependent, bevacizumab has no binding activity to 293T-PD-1, and suggest that the binding of VP101 to 293T-PD-1 is specific.

[0169] 3. Binding of antibodies VP101, BsAbB7 and BsAbB8 to cell surface antigens was detected by referring to the experimental procedure described in step 2 of this example.

[0170] The results are shown in Figure 18, and the MFI values ​​for each concentration are shown in Table 11. Fluorescence quantification analysis and curve fitting of the bound antibodies revealed that the binding EC 50 The values ​​were calculated to be 7.853 nM, 3.607 nM, 7.896 nM, 9.943 nM and 10.610 nM, respectively.

[0171] [Table 13]

[0172] These results demonstrate that the VP101 antibody can bind to PD-1 on the membrane surface of 293T-PD-1 in a dose-dependent manner. Bevacizumab has no binding activity to 293T-PD-1, suggesting that the binding of VP101 to 293T-PD-1 is specific.

[0173] Example 5: Competitive binding of antibody VP101 to cell membrane surface antigens 1. Competitive flow cytometry was used to measure the EC of VP101 in competing with PD-L1 for binding to the cell membrane surface antigen PD-1. 50 This method is employed to detect 293T-PD-1 cells were digested in a conventional manner and divided into several samples, each containing 300,000 cells, which were then subjected to centrifugation and washing. Then, 100μL of the corresponding gradient diluted antibody was added to each tube and incubated on ice for 30 minutes; then, 100μL of PD-L1-mFc was added to each tube, the mixture was mixed thoroughly to reach a final concentration of 20nM, and then incubated on ice for 1 hour. Then, 500μL of 1% PBSA was added, the mixture was centrifuged at 5600rpm for 5 minutes, and the supernatant was removed. Then, 100μL of FITC-coated anti-mouse antibody diluted at a ratio of 1:500 was added to each tube, the mixture was mixed thoroughly, and then incubated on ice for 40 minutes in the absence of light. Then, the mixture was centrifuged, washed, resuspended, and then transferred to a loading tube for testing.

[0174] The results are shown in Figure 19, and the MFI values ​​for each concentration are shown in Table 12. Fluorescence quantification analysis and curve fitting determined the binding EC 50 The values ​​were calculated to be 8.33 nM and 4.37 nM, respectively.

[0175] [Table 14]

[0176] The results demonstrate that the VP101 antibody can effectively block the binding of PDL-1 to PD-1 on the surface of 293T-PD-1 host cells in a dose-dependent manner.

[0177] 2. EC of VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab in competing with PD-L1 for binding to the cell membrane surface antigen PD-1 50 The values ​​were detected by using competitive flow cytometry and by referring to the experimental procedure described in step 1 of this example.

[0178] The results are shown in Figure 20, and the MFI values ​​for each concentration are shown in Table 13. By fluorescence quantification analysis and curve fitting, the competitive binding EC50 values ​​of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and Nivolumab were calculated to be 15.04 nM, 22.25 nM, 19.25 nM, 9.21 nM and 9.72 nM, respectively.

[0179] [Table 15]

[0180] The results demonstrate that the activity of antibody 14C12H1L1 is comparable to that of the commercial antibody nivolumab targeting PD-1, and is superior to that of the bifunctional antibody VP101, which is superior to that of BsAbB7 and BsAbB8.

[0181] Example 6: Detection of the neutralizing biological activity of antibodies VP101, BsAbB7 and BsAbB8 in blocking VEGF to activate the NFAT signal pathway 1. Construction of 293T-NFAT-(opv)KDR(C7) cells The KDR(VEGFR2) vector pCDH-KDRFL(OPV)-GFP-Puro (vector pCDH-GFP-Puro was purchased from Youbio) and the NFAT vector pNFAT-luc-hygro (vector pGL4-luc2P-hygro was purchased from Promega) were transfected into 293T cells, and a clonal group of 293T-NFAT-(opv)KDR(C7) cells stably expressing the KDR and NFAT luciferase reporter genes was obtained by screening.

[0182] 2.293T-NFAT-(opv)KDR(C7) cells were collected and centrifuged for 5 minutes, and the supernatant was removed; DMEM+10% FBS medium was used to resuspend the cells, count the cell number, and detect the cell viability; Then, the cell concentration was adjusted to be in the appropriate range, and 50000 cells / 50μL of the cell suspension was added into each well of a black 96-well plate; Corresponding antibodies (final concentrations of 300, 100, 10, 2, 0.2, 0.02, 0.002 nM) and VEGF (final concentration of 30 ng / mL) were diluted according to the experimental design, and the antibodies targeting VEGF were pre-incubated with VEGF at room temperature for 1 h before being added to the cells. Blank and isotype controls (final volume of each well was 100 μL) were designed and incubated in a carbon dioxide incubator at 37°C with 5% CO2 for 4 h; 50 μL of luciferase assay system was added to each well, and the relative fluorescence units (RLU) were detected by a multi-label microplate tester within 5 min.

[0183] The experimental results are shown in FIG. 50 The values ​​are shown in Table 14.

[0184] [Table 16]

[0185] From these results, the EC 50 The EC of BsAbB7 was 1.240 nM. 50 The EC of BsAbB8 was 1.217 nM. 50 The EC value of bevacizumab was 1.728 nM. 50 is 0.773 nM, and the experimental results show that the activity of VP101 and BsAbB7 in blocking VEGF to activate the NFAT signal pathway is better than that of BsAbB8.

[0186] Example 7: Experiments with VP101 antibody inhibiting VEGFA-induced HUVEC cell proliferation HUVEC cells (purchased from Allcell) in good growth condition were cultured at a cell concentration of 1.5 × 10 4After adjusting the concentration to 100 μL / mL, the cells were seeded in a 96-well plate at 200 μL / well, and then incubated in an incubator at 37° C. and 5% CO2 for 24 hours. After confirming that the cells had attached to the wells, the culture medium was discarded. Then, 20 nM VEGFA prepared by using 2% FBS containing 1640 was added to the 96-well plate at 200 μL / well, and antibodies at different concentrations were added, followed by incubation for 72 hours. After 72 hours, the culture medium was discarded, and MTT was added. After 4 hours, MTT was discarded, DMSO was added, and the OD value was measured at 490 nm using a microplate reader.

[0187] The results are shown in Figure 22. The results reveal that both humanized antibody VP101 and bevacizumab can effectively inhibit VEGFA-induced HUVEC cell proliferation in a dose-dependent manner, and the pharmacological activity of VP101 in inhibiting VEGFA-induced HUVEC cell proliferation is higher than that of bevacizumab at the same dose.

[0188] Example 8: Promotion of secretion of cytokines IFN-γ and IL-2 in mixed lymphocyte reaction 1. VP101, 14C12H1L1 and nivolumab promote IFN-γ secretion in a mixed culture system of DC and PBMC cells PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare) and added with IL-4 (Peprotech 200-04, 1000 U / mL) and GM-CSF (Peprotech 300-03, 1000 U / mL) for induction for 6 days, and then TNF-α (Peprotech 300-01A, 200 U / mL) was further added for induction for 3 days to obtain mature DC cells.

[0189] On the day of co-culture, fresh PBMCs were isolated from peripheral blood of another donor, and the resulting mature DC cells were mixed with freshly isolated PBMCs of another donor at a ratio of 1:10, while different concentrations of antibodies (hIgG as a control) were added at the same time. After co-culture for 5-6 days, cell supernatants were collected and assayed for IFN-γ content using an ELISA kit (purchased from Dakewe).

[0190] The effect of VP101 on IFN-γ secretion in a mixed culture system of DC cells and PBMC cells is shown in Figure 23. As can be seen from Figure 23, VP101 can effectively promote IFN-γ secretion in a dose-dependent manner. Furthermore, at doses of 30 nM and 300 nM, VP101 has greater activity in promoting IFN-γ secretion than the same 14C12H1L1, and at the dose level of 30 nM, it has greater activity in promoting IFN-γ secretion than the same nivolumab.

[0191] 2. Promotion of IL-2 and IFN-γ secretion by VP101, BsAbB7, and BsAbB8b in a mixed culture system of DC and PBMC cells Refer to step 1 of this example for the experimental method, i.e., PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare), and added with IL-4 (Peprotech 200-04, 1000U / mL) and GM-CSF (Peprotech 300-03, 1000U / mL) for induction for 6 days, and then TNF-α (Peprotech 300-01A, 200U / mL) was further added for induction for 3 days to obtain mature DC cells.

[0192] On the day of co-culture, fresh PBMCs were isolated from peripheral blood of another donor, and the resulting mature DC cells were mixed with freshly isolated PBMCs of another donor at a ratio of 1:10, while different concentrations of antibodies (hIgG as control) were added at the same time; after co-culture for 5-6 days, the cell supernatants were collected and assayed for IL-2 and IFN-γ content using ELISA kits (purchased from Dakewe).

[0193] The effect of VP101 on IFN-γ secretion in a mixed culture system of DC cells and PBMC cells is shown in Figure 24. As can be seen from Figure 24, VP101 can effectively promote IFN-γ secretion in a dose-dependent manner. The pharmacological activity of VP101 in promoting the secretion of IFN-γ is significantly better than that of BsAbB7 and BsAbB8.

[0194] The effect of VP101 on IL-2 secretion in a mixed culture system of DC cells and PBMC cells is shown in Figure 25. As can be seen from Figure 25, VP101 can effectively promote the secretion of IL-2 in a dose-dependent manner, and the pharmacological activity of VP101 in promoting the secretion of IL-2 is better than that of BsAbB7 and BsAbB8.

[0195] 3. VP101, 14C12H1L1 and nivolumab promote secretion of IL-2 and IFN-γ in a mixed culture system of PBMC cells and Raji-PD-L1 cells PD-L1 was stably transfected into Raji cells via lentiviral infection, and Raji-PD-L1 cells stably expressing PD-L1 were obtained after treatment and screening; PBMCs were cultured with mitomycin C-treated Raji-PD-L1 after stimulation with SEB for 2 days.

[0196] The results are shown in Figures 26 and 27. The results reveal that VP101 can effectively promote the secretion of IL-2 and IFN-γ, and at a dose level of 300 nM, the activity of VP101 in promoting the secretion of IL-2 is significantly better than that of equivalent amounts of 14C12H1L1 and nivolumab.

[0197] The isotype control antibody examined was human anti-chicken lysozyme (anti-HEL, or human IgG, abbreviated as hIgG), which was prepared as described in Preparative Example 6 above.

[0198] 4. The promotion of IL-2 and IFN-γ secretion by VP101, BsAbB7 and BsAbB8 in a mixed culture system of PBMC cells and Raji-PD-L1 cells was investigated by referring to the experimental method described in step 3 of this Example.

[0199] The results of IFN-γ secretion are shown in Figure 28. The results show that VP101 can effectively promote the secretion of IFN-γ in a dose-dependent manner. At the same time, VP101 is significantly better than BsAbB7 at the same dose, while the pharmacological activity of VP101 is significantly better than that of BsAbB8 at the dose levels of 3 nM and 30 nM.

[0200] The results of IL-2 secretion are shown in Figure 29. The results show that VP101 can effectively promote the secretion of IL-2 in a dose-dependent manner. At the same time, the pharmacological activity of VP101 is significantly better than that of BsAbB7 at doses of 3nM and 300nM, while VP101 is comparable to that of BsAbB8 at the same dose.

[0201] Example 9: In vivo tumor growth inhibition experiment by VP101 To detect the tumor suppression activity of VP101 in vivo, U87MG cells (human glioma cells purchased from ATCC) were first subcutaneously inoculated into 5-7 week old female Scid Beige mice (purchased from Vital River), and the modeling and specific modes of administration are shown in Table 15. After administration, the length and width of each group of tumors were measured, and the tumor volume was calculated.

[0202] [Table 17]

[0203] The results are shown in Figure 30. The results show that, compared with the isotype control antibody hIgG (preparation method is the same as that in Preparation Example 6), different doses of bevacizumab and VP101 can effectively inhibit the growth of mouse tumors, and high doses of VP101 are better at inhibiting tumors than low doses of VP101.

[0204] Furthermore, as can be seen in FIG. 31, VP101 does not affect the body weight of tumor-bearing mice.

[0205] The contents of the present invention provide a complete and clear description of the disclosed embodiments, but it is not limited thereto. For those skilled in the art, modifications and replacements to the present invention are possible under the guidance of these descriptions, and such modifications and replacements are included within the scope of the present invention. The full scope of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. a first protein functional region that targets VEGFA; A second protein functional domain that targets PD-1 1. A bispecific antibody comprising: the first protein functional region is an anti-VEGFA antibody or antigen-binding fragment thereof, a heavy chain variable region of the anti-VEGFA antibody comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:15-17, respectively, and a light chain variable region of the anti-VEGFA antibody comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:18-20, respectively; the second protein functional region is an anti-PD-1 antibody or an antigen-binding fragment thereof, a heavy chain variable region of the anti-PD-1 antibody comprising HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs:21 to 23, respectively, or a light chain variable region of the anti-PD-1 antibody comprising LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs:24 to 26, respectively; Bispecific antibodies.

2. The anti-VEGFA antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody, and diabody; and / or The anti-PD-1 antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody and diabody; The bispecific antibody of claim 1.

3. the first protein functional region is an immunoglobulin, a heavy chain variable region of said immunoglobulin comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:15-17, respectively, and a light chain variable region of said immunoglobulin comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:18-20, respectively; the second protein functional region is a single chain antibody, a heavy chain variable region of said single chain antibody comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:21-23, respectively, and a light chain variable region of said single chain antibody comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:24-26, respectively; or the first protein functional region is a single chain antibody, a heavy chain variable region of said single chain antibody comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:21-23, respectively, and a light chain variable region of said single chain antibody comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:24-26, respectively; the second protein functional region is an immunoglobulin, a heavy chain variable region of said immunoglobulin comprising HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:15-17, respectively, and a light chain variable region of said immunoglobulin comprising LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:18-20, respectively; The bispecific antibody of claim 1.

4. the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: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; or, 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; the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:5 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:

7. The bispecific antibody of claim 3.

5. 5. The bispecific antibody of claim 3 or 4, wherein the immunoglobulin is IgG, IgA, IgD, IgE or IgM; preferably, the immunoglobulin is IgG.

6. 5. A bispecific antibody according to claim 3 or 4, in which there are two single chain antibodies, one end of each single chain antibody being linked to the C-terminus or N-terminus of one of the two heavy chains of the immunoglobulin.

7. 5. The bispecific antibody of claim 3 or 4, wherein the immunoglobulin comprises non-CDR regions derived from a species other than mouse, e.g. derived from a human antibody.

8. the immunoglobulin comprises a constant region derived from a human antibody; Preferably, the constant region of the immunoglobulin is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4. A bispecific antibody according to claim 3 or 4.

9. the heavy chain constant region of the immunoglobulin is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region is a human Ig kappa chain C region; A bispecific antibody according to claim 3 or 4.

10. the first protein functional domain and the second protein functional domain are linked directly or by a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, e.g., 1, 2, 3, 4, 5, or 6; A bispecific antibody according to claim 1 or 2.

11. 3. The bispecific antibody of claim 1 or 2, wherein the number of said first protein functional domains and said second protein functional domains is each independently one, two or more.

12. The bispecific antibody has an EC ５０ Binds to VEGFA protein at the EC ５０ is detected by indirect ELISA; and / or The bispecific antibody has an EC ５０ PD-1 protein is bound to the EC ５０ is detected by indirect ELISA; A bispecific antibody according to claim 1 or 2.

13. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 12.

14. A vector comprising the isolated nucleic acid molecule of claim 13.

15. 15. A host cell comprising the isolated nucleic acid molecule of claim 13 or the vector of claim 14.

16. 13. A method for preparing the bispecific antibody of any one of claims 1 to 12, comprising culturing the host cell of claim 15 under suitable conditions and isolating the bispecific antibody from the cell culture.

17. A conjugate comprising a bispecific antibody and a conjugation moiety, wherein the bispecific antibody is the bispecific antibody of any one of claims 1 to 12, and the conjugation moiety is a detectable label; preferably, the conjugation moiety is a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

18. A kit comprising the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17; Preferably, the kit further comprises a secondary antibody capable of specifically binding to the bispecific antibody; optionally, the secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

19. Use of the bispecific antibody according to any one of claims 1 to 12 in the preparation of a kit for detecting the presence or level of VEGFA and / or PD-1 in a sample.

20. 18. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17, and optionally a pharma- ceutically acceptable excipient.

21. 18. Use of the bispecific antibody according to any one of claims 1 to 12, or the conjugate according to claim 17, in the preparation of a medicament for preventing and / or treating a malignant tumor, preferably wherein said malignant tumor is selected from colon cancer, rectal cancer, lung cancer, such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor and leukemia.

22. (1) a pharmaceutical agent or drug for detecting the level of VEGFA in a sample; A medicine or agent for blocking the binding of VEGFA to VEGFR2; Medicaments or agents for downregulating VEGFA activity or levels; A medicine or agent for reducing VEGFA stimulation of vascular endothelial cell proliferation; A medicine or drug for inhibiting vascular endothelial cell proliferation, or medicines or agents for blocking tumor angiogenesis; and / or (2) A drug or agent for blocking the binding of PD-1 to PD-L1; Medicaments or agents for downregulating the activity or levels of PD-1; A medicine or drug for reducing immunosuppression of PD-1 in a living body; A medicine or agent for promoting IFN-γ secretion in T lymphocytes, or Medicine or agent for promoting IL-2 secretion in T lymphocytes 20. Use of the bispecific antibody of any one of claims 1 to 12, or the conjugate of claim 17, in the preparation of

23. An in vivo or in vitro method comprising administering to a cell an effective amount of the bispecific antibody according to any one of claims 1 to 12 or the complex according to claim 17, (1) A method for detecting the level of VEGFA in a sample; Methods for blocking the binding of VEGFA to VEGFR2; Methods for downregulating VEGFA activity or levels; A method for reducing VEGFA stimulation of vascular endothelial cell proliferation; A method for inhibiting vascular endothelial cell proliferation, or Methods for blocking tumor angiogenesis; and / or (2) A method for blocking the binding of PD-1 to PD-L1; Methods for downregulating PD-1 activity or levels; A method for reducing immunosuppression of PD-1 in vivo; A method for promoting IFN-γ secretion in T lymphocytes, or A method for promoting IL-2 secretion in T lymphocytes; A method selected from the group consisting of

24. 18. A method for preventing and / or treating a malignant tumor comprising administering to a subject in need thereof an effective amount of the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17, preferably wherein the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor and leukemia.

25. The bispecific antibody according to any one of claims 1 to 12 or the conjugate according to claim 17 for use in the prevention and / or treatment of a malignant tumor, preferably wherein the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain tumor and leukemia.

26. (1) detecting the level of VEGFA in a sample; Blocking the binding of VEGFA to VEGFR2; downregulating VEGFA activity or levels; Attenuating VEGFA stimulation of vascular endothelial cell proliferation; Inhibition of vascular endothelial cell proliferation, or Blocking tumor angiogenesis; and / or (2) blocking the binding of PD-1 to PD-L1; downregulating PD-1 activity or levels; Relief of PD-1 immunosuppression in vivo, Stimulation of IFN-γ secretion in T lymphocytes, or Stimulation of IL-2 secretion in T lymphocytes The bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17 for use in

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