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

Abstract

An anti-VEGFA and anti-PD-1 bifunctional antibody, a pharmaceutical composition thereof and use thereof belong to the field of tumor therapy and molecular immunology. Specifically, the anti-VEGFA and anti-PD-1 bifunctional antibody comprises: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1. The bifunctional antibody can specifically bind to VEGFA and PD-1, specifically relieve immunosuppression of VEGFA and PD-1 on an organism, and inhibit tumor-induced angiogenesis, and thus has good application prospects.

Description

Bifunctional antibodies against PD-1 and VEGFA, their pharmaceutical compositions and their uses Technical Field

[0001] This invention belongs to the fields of tumor therapy and immunobiology, and relates to a bifunctional antibody against PD-1 and against VEGFA, its pharmaceutical composition, and its uses. Specifically, this invention relates to a bifunctional antibody against human PD-1 and against human VEGFA, its pharmaceutical composition, and its uses. Background Technology

[0002] Cancer, especially malignant cancer, is a serious threat to human health worldwide, ranking second among all causes of death. Moreover, its incidence has shown a significant upward trend in recent years. Malignant cancers have poor treatment outcomes, high rates of late-stage metastasis, and generally poor prognoses. While current clinical treatments such as radiotherapy, chemotherapy, and surgery have significantly alleviated pain and prolonged survival, these methods have considerable limitations, making further improvements in efficacy difficult.

[0003] Tumor growth has two distinct phases: a slow, avascular growth phase followed by a rapid, vascularized proliferation phase. Angiogenesis allows the tumor to obtain sufficient nutrients to complete the angiogenesis transition. Without angiogenesis, the primary tumor grows to no more than 1-2 mm, and metastasis cannot occur.

[0004] Vascular endothelial growth factor (VEGF) is a class of growth factors that promote endothelial cell proliferation, angiogenesis, and vascular permeability. It binds to VEGF receptors on the cell surface and functions by activating tyrosine kinase signaling pathways. In tumor tissues, tumor cells, invading macrophages, and mast cells secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine manner, promoting endothelial cell proliferation and migration, inducing angiogenesis, promoting sustained tumor growth, increasing vascular permeability, causing fibrin deposition in surrounding tissues, promoting the infiltration of monocytes, fibroblasts, and endothelial cells, facilitating tumor matrix formation and tumor cell entry into new blood vessels, and promoting tumor metastasis. Therefore, inhibiting tumor angiogenesis is considered one of the most promising cancer treatment methods. The VEGF family includes: VEGFA, VEGFB, VEGFC, VEGRD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and Neuropilin-1 (NRP-1). The first three receptors are structurally similar, all belonging to the tyrosine kinase superfamily, and each consists of an extramembrane domain, a transmembrane segment, and an intramembrane domain. The extramembrane domain is composed of an immunoglobulin-like domain, while the intramembrane domain is a tyrosine kinase domain. VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, while VEGFR3 is mainly located on the surface of lymphatic endothelial cells.

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

[0006] VEGFR2 is a major regulator of angiogenesis and angiogenesis, and VEGFR-2 has a high tyrosine kinase activity compared to VEGFR1. After VEGFR2 binds to its ligand VEGFA, it mediates the proliferation and differentiation of vascular endothelial cells, as well as the process of angiogenesis and vascular permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3): 179-213. (2007)). After VEGFA binds to VEGFR2, it mediates the transcriptional expression of intracellular related protein genes through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13): 2221-2230. (1999)).

[0007] VEGFR3 belongs to the tyrosine kinase family and is mainly expressed in vascular endothelial cells during embryonic development and lymphatic endothelial cells during adulthood. VEGFC and VEGGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphatic vessel regeneration. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transduce biological signals. It can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5): 146-148(2016)).

[0008] VEGFA and VEGFR2 are mainly involved in regulating angiogenesis. Before and after VEGFA and VEGFR2 bind, they trigger a series of intermediate signals in the upstream and downstream pathways to form a cascade reaction, which ultimately changes the physiological functions of endothelial cells in different forms such as proliferation, survival, migration, increased permeability and infiltration into surrounding tissues (Dong Hongchao et al., Modern Oncology Medicine, September 2014, Vol. 22, No. 9, pp. 2231-3).

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

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

[0011] Numerous studies have shown that the tumor microenvironment protects tumor cells from damage by immune cells. PD-1 expression is upregulated in lymphocytes infiltrating the tumor microenvironment, and various primary tumor tissues, such as lung cancer, liver cancer, ovarian cancer, skin cancer, colon cancer, and glioma, show PD-L1 positivity in immunohistochemical analysis. Furthermore, PD-L1 expression in tumors is significantly associated with poor prognosis in cancer patients. Blocking the interaction between PD-1 and its ligands can promote tumor-specific T-cell immunity and improve the efficiency of tumor cell immune clearance. Numerous clinical trials have demonstrated that antibodies targeting PD-1 or PD-L1 can promote CD8+ T-cell infiltration into tumor tissues and upregulate anti-tumor immune effector factors such as IL-2, IFN-γ, granzyme B, and perforin, thereby effectively inhibiting tumor growth.

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

[0013] Due to the broad-spectrum anti-tumor prospects and remarkable efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring about breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., Anti-PD-1 Therapy in Melanoma. Semin Oncol. 2015 Jun; 42(3):466-473), lymphoma, and anemia (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. 2013 Sep; 13(7):768-74).

[0014] Bispecific antibodies, also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens. They can be produced through immunosorting and purification. Alternatively, they can be obtained through genetic engineering. Genetic engineering offers advantages in terms of flexibility in optimizing binding sites, considering synthetic forms, and increasing yield. Currently, more than 45 forms have been identified (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24:89-98). Currently, many bispecific antibodies have been developed in the form of IgG-ScFv, also known as the Morrison model (Coloma MJ, Morrison SL: Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol., 1997; 15:159-163). Due to its similarity to naturally occurring IgG, this form has advantages in antibody engineering, expression, and purification, and has been proven to be an ideal form for bifunctional antibodies (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011; 3:299-309).

[0015] Currently, there is a need to develop a bifunctional antibody drug that simultaneously targets PD-1 and VEGF (e.g., VEGFA).

[0016] Summary of the Invention

[0017] Through in-depth research and creative work, the inventors have obtained a humanized bifunctional antibody, named VP101, based on the marketed VEGFA monoclonal antibody Avastin (Bevacizumab) and the previously obtained 14C12H1L1 (see Chinese Patent Publication CN106977602A). This antibody can simultaneously bind VEGFA and PD-1, and block the binding of VEGFA to VEGFR2 and the binding of PD-1 to PD-L1.

[0018] The inventors were surprised to discover that VP101 is capable of:

[0019] It effectively binds to PD-1 on the surface of human immune cells, relieves PD-L1 and PD-1-mediated immunosuppression, and promotes the secretion of IFN-γ and IL-2 by human immune cells;

[0020] Effectively inhibits VEGFA-induced proliferation of vascular endothelial cells, thereby suppressing tumor-induced angiogenesis; and / or

[0021] It has the potential to be used in the preparation of drugs for the prevention and treatment of malignant tumors such as liver cancer, lung cancer, melanoma, kidney tumors, ovarian cancer, and lymphoma.

[0022] This leads to the following invention:

[0023] One aspect of the present invention relates to a bispecific antibody comprising:

[0024] Targeting the first protein functional region of VEGFA, and

[0025] Targeting the second protein functional region of PD-1;

[0026] Preferably,

[0027] The first protein functional region is an anti-VEGFA antibody or its antigen-binding fragment, wherein the heavy chain variable region of the anti-VEGFA antibody contains amino acid sequences as shown in SEQ ID NOs:15-17 (HCDR1-HCDR3), and its light chain variable region contains amino acid sequences as shown in SEQ ID NOs:18-20 (LCDR1-LCDR3); and

[0028] The second protein functional region is an anti-PD-1 antibody or its antigen-binding fragment, wherein the heavy chain variable region of the anti-PD-1 antibody contains amino acid sequences as shown in SEQ ID NOs:21-23, namely HCDR1-HCDR3, and its light chain variable region contains amino acid sequences as shown in SEQ ID NOs:24-26, namely LCDR1-LCDR3.

[0029] In some embodiments of the present invention, the bispecific antibody, wherein,

[0030] The anti-VEGFA antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody;

[0031] And / or,

[0032] The anti-PD-1 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody.

[0033] In some embodiments of the present invention, the bispecific antibody is in IgG-scFv mode.

[0034] In some embodiments of the present invention, the first protein functional region is an immunoglobulin, the heavy chain variable region of which contains amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:15-17, and the light chain variable region of which contains amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:18-20; and the second protein functional region is a single-chain antibody, the heavy chain variable region of which contains amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:21-23, and the light chain variable region of which contains amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:24-26.

[0035] or,

[0036] The first protein functional region is a single-chain antibody, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:21-23 (HCDR1-HCDR3) and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:24-26 (LCDR1-LCDR3); and the second protein functional region is an immunoglobulin, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:15-17 (HCDR1-HCDR3) and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:18-20 (LCDR1-LCDR3).

[0037] In one specific embodiment of the present invention, the present invention relates to a bispecific antibody, comprising:

[0038] Targeting the first protein functional region of VEGFA, and

[0039] Targeting the second protein functional region of PD-1;

[0040] in,

[0041] The first protein functional region is an immunoglobulin, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:15-17 HCDR1-HCDR3, and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:18-20 LCDR1-LCDR3; and the second protein functional region is a single-chain antibody, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:21-23 HCDR1-HCDR3, and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:24-26 LCDR1-LCDR3;

[0042] or,

[0043] The first protein functional region is a single-chain antibody, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:21-23 (HCDR1-HCDR3), and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:24-26 (LCDR1-LCDR3); and the second protein functional region is an immunoglobulin, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:15-17 (HCDR1-HCDR3), and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:18-20 (LCDR1-LCDR3).

[0044] In some embodiments of the present invention, the bispecific antibody, wherein,

[0045] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5; the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:11.

[0046] or,

[0047] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9; the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:11; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:7.

[0048] In some embodiments of the present invention, the bispecific antibody, wherein,

[0049] The immunoglobulin includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0050] In some embodiments of the present invention, the bispecific antibody, wherein,

[0051] The immunoglobulin described herein has a constant region derived from human antibodies;

[0052] Preferably, the constant region of the immunoglobulin is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.

[0053] In some embodiments of the present invention, the bispecific antibody, wherein,

[0054] The immunoglobulin has a heavy chain constant region of human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and a light chain constant region of human Ig kappa chain C region.

[0055] In some embodiments of the present invention, the constant regions of the immunoglobulins are humanized. For example, the heavy chain constant regions are all Ig gamma-1 chain C region, ACCESSION: P01857; and the light chain constant regions are all Ig kappa chain C region, ACCESSION: P01834.

[0056] In some embodiments of the present invention, the bispecific antibody is wherein the first protein functional region and the second protein functional region are directly connected or connected through a linker fragment.

[0057] Preferably, the linker segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6. GGGGS (SEQ ID NO:14) is a component of the Linker.

[0058] In some embodiments of the present invention, the bispecific antibody wherein the first protein functional region and the second protein functional region are independently one, two, or more.

[0059] In some embodiments of the present invention, the bispecific antibody comprises one immunoglobulin and two single-chain antibodies, preferably two identical single-chain antibodies.

[0060] In some embodiments of the present invention, the bispecific antibody is wherein the immunoglobulin is IgG, IgA, IgD, IgE or IgM; preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0061] In some embodiments of the invention, the bispecific antibody is wherein the single-chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin. Since immunoglobulins consist of two heavy chains, one immunoglobulin molecule is linked to two single-chain antibody molecules. Preferably, the two single-chain antibody molecules are identical.

[0062] In some embodiments of the present invention, the bispecific antibody comprises two single-chain antibodies, one end of each single-chain antibody being attached to the C-terminus or N-terminus of the two heavy chains of the immunoglobulin.

[0063] In some embodiments of the present invention, the V of the single-chain antibody H With V L Disulfide bonds exist between them. In the antibody's V... H and V L Methods for introducing disulfide bonds between molecules are well known in the art, and for example, see US Patent 5,747,654; Rajagopal et al., Prot. Engin. 10 (1997) 1453-1459; Reiter et al., Nat. Biotechnol. 14 (1996) 1239-1245; Reiter et al., Protein Engineering 8 (1995) 1323-1331; Webber et al., Molecular Immunology 32 (1995) 249-258; Reiter et al., Immunity 2 (1995) 281-287; Reiter et al., JBC 269 (1994) 18327-18331; Reiter et al., Inter. J. of Cancer 58 (1994) 142-149; or Reiter et al., Cancer Res. 54 (1994) 2714-2718; which is incorporated herein by reference.

[0064] In some embodiments of the present invention, the bispecific antibody, wherein the bispecific antibody is less than 10 -5 M, for example, less than 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller KD Combining VEGFA protein and / or PD-1 protein; preferably, the K D Measured using a Fortebio molecular interaction analyzer.

[0065] In some embodiments of the present invention, the bispecific antibody, wherein,

[0066] The bispecific antibody is expressed in EC50 values ​​of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.15 nM, or less than 0.14 nM. 50 Binding to VEGFA protein; preferably, the EC 50 Measured by indirect ELISA method;

[0067] And / or,

[0068] The bispecific antibodies are expressed in EC5 concentrations 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 Binding to PD-1 protein; preferably, the EC 50 Measured by indirect ELISA method.

[0069] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes the bispecific antibody described in any one of the present invention.

[0070] The present invention also relates to a carrier comprising the isolated nucleic acid molecules of the present invention.

[0071] The present invention also relates to a host cell comprising the isolated nucleic acid molecules of the present invention, or comprising the vector of the present invention.

[0072] Another aspect of the invention relates to a method for using the bispecific antibody described in any one of the invention, comprising culturing the host cells of the invention under suitable conditions, and recovering the bispecific antibody from the cell culture.

[0073] Another aspect of the present invention relates to a conjugate comprising a bispecific antibody and a conjugation portion, wherein the bispecific antibody is any one of the bispecific antibodies described in the present invention, and the conjugation portion is a detectable label; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0074] Another aspect of the present invention relates to a kit comprising the bispecific antibody described in any one of the present invention, or comprising the conjugate described in the present invention;

[0075] Preferably, the kit further comprises a second antibody that specifically binds to the bispecific antibody; optionally, the second antibody further comprises a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0076] Another aspect of the invention relates to the use of any one of the bispecific antibodies described in the invention in the preparation of a kit for detecting the presence or level of VEGFA and / or PD-1 in a sample.

[0077] Another aspect of the invention relates to a pharmaceutical composition comprising the bispecific antibody described in any one of the invention or a conjugate comprising the invention; optionally, it further comprises pharmaceutically acceptable excipients.

[0078] The bispecific antibody or pharmaceutical composition of the present invention can be formulated into any dosage form known in the pharmaceutical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the bispecific antibody of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0079] Furthermore, the bispecific antibody of the present invention can be present in the pharmaceutical composition in unit dose form for ease of administration. In some embodiments, the unit dose is at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. When the pharmaceutical composition is a liquid (e.g., injectable) dosage form, it may contain the bispecific antibody of the present invention at a concentration of at least 0.1 mg / ml, such as at least 0.25 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml.

[0080] The bispecific antibody or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibody or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

[0081] The bispecific antibody or pharmaceutical composition provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., chemotherapy drugs for tumors). Such other pharmaceutically active agents can be administered before, simultaneously with, or after the administration of the bispecific antibody or pharmaceutical composition of this invention.

[0082] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0083] Another aspect of the invention relates to the use of any bispecific antibody or conjugate of the invention as described in any one of the invention in the preparation of a medicament for the prevention and / or treatment of malignant tumors; preferably, the malignant tumors are selected from colon cancer, rectal cancer, lung cancer such as non-small cell lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, and leukemia.

[0084] Another aspect of the present invention relates to the use of the bispecific antibody or conjugate of any one of the present invention in the preparation of the following drugs or reagents:

[0085] (1)

[0086] Drugs or reagents for detecting VEGFA levels in samples.

[0087] Drugs or reagents that block the binding of VEGFA to VEGFR2.

[0088] Drugs or reagents that downregulate the activity or level of VEGFA.

[0089] Drugs or reagents that can counteract the stimulatory effect of VEGFA on vascular endothelial cell proliferation.

[0090] Drugs or reagents that inhibit the proliferation of vascular endothelial cells, or

[0091] Drugs or reagents that block tumor angiogenesis;

[0092] and / or

[0093] (2)

[0094] Drugs or reagents that block the binding of PD-1 to PD-L1

[0095] Drugs or reagents that downregulate the activity or level of PD-1

[0096] Drugs or reagents that can counteract the immunosuppression caused by PD-1.

[0097] Drugs or reagents that promote IFN-γ secretion in T lymphocytes, or

[0098] Drugs or reagents that promote IL-2 secretion in T lymphocytes.

[0099] In one embodiment of the invention, the use is for non-therapeutic and / or non-diagnostic purposes.

[0100] Another aspect of the invention relates to an in vivo or in vitro method comprising the step of applying cells in an effective amount of the bispecific antibody or conjugate of the invention, said method being selected from the following:

[0101] (1)

[0102] Methods for detecting VEGFA levels in samples

[0103] Methods to block the binding of VEGFA and VEGFR2

[0104] Methods to downregulate the activity or level of VEGFA

[0105] Methods to relieve the stimulatory effect of VEGFA on vascular endothelial cell proliferation.

[0106] Methods to inhibit vascular endothelial cell proliferation, or

[0107] Methods to block tumor angiogenesis;

[0108] and / or

[0109] (2)

[0110] Methods to block the binding of PD-1 and PD-L1

[0111] Methods to downregulate PD-1 activity or its level

[0112] Methods to relieve the immunosuppression caused by PD-1

[0113] Methods to promote IFN-γ secretion in T lymphocytes, or

[0114] Methods to promote IL-2 secretion in T lymphocytes.

[0115] In one embodiment of the invention, the in vitro method is for non-therapeutic and / or non-diagnostic purposes.

[0116] In the in vitro experiments of this invention, both the anti-VEGFA antibody and the anti-VEGFA-anti-PD-1 bifunctional antibody can inhibit the proliferation of HUVEC cells, and both the anti-PD-1 antibody and the anti-VEGFA-anti-PD-1 bifunctional antibody can promote the secretion of IFNγ and / or IL-2 and activate the immune response.

[0117] Another aspect of the invention relates to a method for preventing and / or treating malignant tumors, comprising the step of administering to a subject in need an effective amount of any one of the bispecific antibodies or conjugates of the invention; preferably, 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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, and leukemia.

[0118] The typical non-limiting range of the therapeutic or preventative effective dose of the bispecific antibody of the present invention is 0.02–50 mg / kg, for example 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 symptoms requiring treatment. Furthermore, those skilled in the art will understand that for any given patient, a specific dosing regimen should be adjusted over time based on the patient's needs and the physician's professional evaluation; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0119] In this invention, the subject can be a mammal, such as a human.

[0120] The bispecific antibody or conjugate according to any one of the present invention is used for the prevention and / or treatment of malignant tumors; preferably, 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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, and leukemia.

[0121] The bispecific antibody or conjugate according to any one of the present invention is used for:

[0122] (1)

[0123] Detecting VEGFA levels in samples.

[0124] Blocking the binding of VEGFA to VEGFR2,

[0125] Downregulate the activity or level of VEGFA.

[0126] To relieve the stimulatory effect of VEGFA on vascular endothelial cell proliferation.

[0127] Inhibit vascular endothelial cell proliferation, or

[0128] Blocking tumor angiogenesis;

[0129] and / or

[0130] (2)

[0131] Blocking the binding of PD-1 to PD-L1

[0132] Downregulate the activity or level of PD-1.

[0133] Relieve the immunosuppression caused by PD-1.

[0134] Promotes IFN-γ secretion in T lymphocytes, or

[0135] Promotes IL-2 secretion in T lymphocytes.

[0136] Antibody therapies, especially monoclonal antibodies, have achieved good therapeutic effects in the treatment of a variety of diseases. Traditional experimental methods for obtaining these therapeutic antibodies involve immunizing animals with antigens to obtain antibodies targeting the antigens, or using affinity maturation methods to improve antibodies with low affinity for antigens.

[0137] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region 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; these were named by Kabat et al., see Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication (1-3) 1991: 91-3242.

[0138] Preferably, the CDR can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. “IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF.” Nucleic acids research 38. suppl_1(2009):D301-D307.

[0139] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of the monoclonal antibody sequences in items (1)-(13) below according to the IMGT definition using the VBASE2 database, the results are as follows:

[0140] (1) Bevacizumab

[0141] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0142] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0143] HCDR1:GYTFTNYG (SEQ ID NO:15)

[0144] HCDR2:INTYTGEP(SEQ ID NO:16)

[0145] HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:17)

[0146] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0147] LCDR1:QDISNY (SEQ ID NO:18)

[0148] LCDR2:FTS(SEQ ID NO:19)

[0149] LCDR3:QQYSTVPWT(SEQ ID NO:20)

[0150] (2)14C12H1L1

[0151] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11.

[0152] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0153] HCDR1:GFAFSSYD (SEQ ID NO:21)

[0154] HCDR2:ISGGGRYT(SEQ ID NO:22)

[0155] HCDR3:ANRYGEAWFAY(SEQ ID NO:23)

[0156] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0157] LCDR1:QDINTY(SEQ ID NO:24)

[0158] LCDR2:RAN(SEQ ID NO:25)

[0159] LCDR3:LQYDEFPLT(SEQ ID NO:26)

[0160] (3)VP101

[0161] The amino acid sequences of the nine CDR regions of its heavy chain are as follows:

[0162] HCDR1:GYTFTNYG (SEQ ID NO:15)

[0163] HCDR2:INTYTGEP(SEQ ID NO:16)

[0164] HCDR3:AKYPHYYGSSHWYFDV(SEQ ID NO:17)

[0165] HCDR4:GFAFSSYD (SEQ ID NO:21)

[0166] HCDR5:ISGGGRYT(SEQ ID NO:22)

[0167] HCDR6:ANRYGEAWFAY(SEQ ID NO:23)

[0168] HCDR7:QDINTY (SEQ ID NO:24)

[0169] HCDR8:RAN(SEQ ID NO:25)

[0170] HCDR9:LQYDEFPLT(SEQ ID NO:26)

[0171] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0172] LCDR1:QDISNY (SEQ ID NO:18)

[0173] LCDR2:FTS(SEQ ID NO:19)

[0174] LCDR3:QQYSTVPWT(SEQ ID NO:20)

[0175] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0176] As used herein, when referring to the amino acid sequence of the VEGFA protein (GenBank ID: NP_001165097.1), it includes the full-length VEGFA protein, as well as fusion proteins of VEGFA, such as fragments fused to the Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the VEGFA protein without affecting its biological function. Therefore, in this invention, the term "VEGFA protein" should include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of the VEGFA protein, it also includes the corresponding sequence fragment from its natural or artificial variants. In one embodiment of the invention, the amino acid sequence of the VEGFA protein is as shown in the underlined portion of SEQ ID NO:1 (excluding the last 6 His, totaling 302 amino acids).

[0177] As used herein, when referring to the amino acid sequence of the VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), it includes the full-length VEGFR2 protein, or the extracellular fragment VEGFR2-ECD of VEGFR2, or a fragment containing VEGFR2-ECD; it also includes fusion proteins of VEGFR2-ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the VEGFR2 protein without affecting its biological function. Therefore, in this invention, the term "VEGFR2 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the VEGFR2 protein, it also includes the corresponding sequence fragment from its natural or artificial variants. In one embodiment of the present invention, the amino acid sequence of the extracellular fragment VEGFR2-ECD of VEGFR2 is shown in the wavy line portion of SEQ ID NO:4 (766 amino acids).

[0178] As used herein, unless otherwise specified, VEGFR refers to VEGFR1 and / or VEGFR2; their specific protein sequences are known sequences in the prior art, and can be found in existing literature or sequences published in GenBank. For example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791).

[0179] As used herein, when referring to the amino acid sequence of PD-1 protein (Programmed cell death protein 1, NCBI GenBank: NM_005018), it includes the full-length PD-1 protein, or the extracellular fragment PD-1ECD, or a fragment containing PD-1ECD; it also includes fusion proteins of PD-1ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of PD-1 protein without affecting its biological function. Therefore, in this invention, the term "PD-1 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of PD-1 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0180] As used in this article, the term EC 50 The half-maximal concentration (50% of maximal effect) refers to the concentration that produces a 50% maximal effect.

[0181] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in an antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V LThe region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L These form antibody binding sites. The allocation of amino acids to each region or domain can follow the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions in the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 38. suppl_1 (2009): D301-D307. Specifically, the heavy chain may also contain more than three CDRs, such as six, nine, or twelve. For example, in the bifunctional antibody of the present invention, the heavy chain may be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains nine CDRs. The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0182] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also 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 generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0183] As used in this article, the term "Fd fragment" refers to a segment composed of V H and C H1 An antibody fragment composed of structural domains; the term "Fv fragment" refers to a fragment consisting of the V-shaped structure of a single arm of the antibody. L and V H Antibody fragments composed of structural domains; the term "dAb fragment" refers to an antibody fragment composed of V... H Antibody fragments composed of structural domains (Ward et al., Nature 341(1989):544-546); the term "Fab fragment" refers to a fragment composed of V... L V H C L and C H1 Antibody fragments composed of structural domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.

[0184] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where V L and V H The domain enables the formation of monovalent molecules by linker pairing with single polypeptide chains (see, for example, Bird et al., Science 242 (1988): 423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 85 (1988): 5879-5883). Such scFv molecules can have a general structure: NH2-V L -Connector-V H -COOH or NH2-V H -Connector-V L-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993): 6444-6448). Other linkers that can be used in this invention are described by Alfthan et al., Protein Eng. 8 (1995): 725-731, Choi et al., Eur. J. Immunol. 31 (2001): 94-106, Hu et al., Cancer Res. 56 (1996): 3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999): 41-56, and Roovers et al., Cancer Immunol. (2001).

[0185] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, where V... H and V L The domain is expressed on a single polypeptide chain, but the linker is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating 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).

[0186] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as those used for intact antibodies.

[0187] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0188] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see US Patent 4,816,567).

[0189] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), while the other portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (US Patent 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81(1984):6851-6855).

[0190] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, 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.

[0191] As used herein, the term “epitope” refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. “Epitope” is also referred to in the art as an “antigenic determinant.” Epitopes or antigenic determinants typically consist of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. For example, epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation, which can be “linear” or “conformal.” See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0192] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0193] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0194] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0195] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D The antigen is bound to the antigen. In some embodiments of the invention, the term "targeted" refers to specific binding.

[0196] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D ( ) Combined antigens, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR).

[0197] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0198] As used herein, the term "pharmaceuticalally acceptable excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is 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, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0199] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0200] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention (e.g., diseases associated with PD-1 binding to PD-L1 or VEGF overexpression, such as cancer) means an amount sufficient to prevent, halt, or delay the onset of a disease (e.g., diseases associated with PD-1 binding to PD-L1 or VEGF overexpression, such as cancer); an effective amount for disease treatment means an amount sufficient to cure or at least partially halt the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0201] Beneficial effects of the invention

[0202] The bispecific antibody VP101 of this invention can bind specifically to VEGFA and can effectively block the binding of VEGFA to VEGFR2, specifically relieving the immunosuppressive effect of VEGFA on the body and its promoting effect on angiogenesis; VP101 can bind specifically to PD-1 and can effectively block the binding of PD-1 to PD-L1, specifically relieving the immunosuppressive effect of PD-1 on the body and activating the immune response. Attached Figure Description

[0203] Figure 1: SDS-PAGE results of the bifunctional antibody VP101. The samples and their loading amounts in the four lanes from left to right are as follows: non-reducing protein electrophoresis loading buffer sample antibody, 1 μg; reduced protein electrophoresis loading buffer sample antibody, 1 μg; marker, 5 μl; BSA, 1 μg.

[0204] Figure 2: Detection results of kinetic characteristic parameters of antibody VP101 binding to PD-1.

[0205] Figure 3: Detection results of kinetic characteristic parameters of antibody BsAbB7 binding to PD-1.

[0206] Figure 4: Detection results of kinetic characteristic parameters of antibody BsAbB8 binding to PD-1.

[0207] Figure 5: Detection results of kinetic characteristic parameters of antibody 14C12H1L1 binding to PD-1.

[0208] Figure 6: Detection results of kinetic characteristic parameters of the binding of antibody Nivolumab to PD-1.

[0209] Figure 7: Detection results of kinetic characteristic parameters of antibody VP101 binding to VEGF.

[0210] Figure 8: Detection results of kinetic characteristic parameters of antibody BsAbB7 binding to VEGF.

[0211] Figure 9: Detection results of kinetic characteristic parameters of antibody BsAbB8 binding to VEGF.

[0212] Figure 10: Detection results of kinetic characteristic parameters of the binding of antibody Bevacizumab to VEGF.

[0213] Figure 11: The binding activity of antibody VP101 to VEGFA was detected by indirect ELISA.

[0214] Figure 12: The binding activity of antibodies VP101, BsAbB7, BsAbB8, Bevacizumab and VEGFA-his was detected by indirect ELISA.

[0215] Figure 13: Detection of the binding activity of antibody VP101 to PD-1 using an indirect ELISA method.

[0216] Figure 14: Binding activity of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1, and Nivolumab to PD-1 using the indirect ELISA method.

[0217] Figure 15: The competitive ELISA method was used to detect the activity of antibody VP101 in competitively binding to VEGFA with VEGFR2.

[0218] Figure 16: The competitive ELISA method was used to detect the activity of antibody VP101 in competitively binding to PD-1 with PD-L1.

[0219] Figure 17: FACS detection of the binding of antibodies 14C12H1L1 and VP101 to 293T-PD-1 cell surface protein PD-1 in EC 50 .

[0220] Figure 18: FACS detection of the binding of VP101, BsAbB7, BsAbB8 to PD-1, a protein on the surface of 293T-PD-1 cells. 50 .

[0221] Figure 19: FACS assay to detect the activity of antibodies 14C12H1L1, VP101 and PD-L1 in competitively binding to the 293T-PD-1 cell surface protein PD-1.

[0222] Figure 20: FACS assay to detect the activity of antibodies 14C12H1L1, VP101, BsAbB7, BsAbB8, 14C12H1L, and Nivolumab in competitively binding to the 293T-PD-1 cell surface protein PD-1 with PD-L1.

[0223] Figure 21: Detection of the neutralizing bioactivity of antibodies VP101, BsAbB7, and BsAbB in blocking the VEGF-activated NFAT signaling pathway.

[0224] Figure 22: Effects of Bevacizumab and VP101 on HUVEC cell proliferation.

[0225] Figure 23: Effect of VP101 on IFN-γ secretion in a mixed culture system of DC and PBMC.

[0226] Figure 24: Effects of VP101, BsAbB7, and BsAbB8 on IFN-γ secretion in a mixed culture system of DC and PBMC.

[0227] Figure 25: Effects of VP101, BsAbB7, and BsAbB8 on IL-2 secretion in a mixed culture system of DC and PBMC.

[0228] Figure 26: Effects of antibodies 14C12H1L1 and VP101 on the secretion of IL-2 induced by co-culture of PBMC and Raji-PD-L1 cells, as detected by ELISA.

[0229] Figure 27: Effects of antibodies 14C12H1L1 and VP101 on the secretion of cytokine IFN-γ induced by co-culture of PBMC and Raji-PD-L1 cells, as detected by ELISA.

[0230] Figure 28: Effects of antibodies 14C12H1L1, VP101, BsAbB7, and BsAbB8 on the secretion of cytokine IFN-γ induced by co-culture of PBMC and Raji-PD-L1 cells, as detected by ELISA.

[0231] Figure 29: Effects of antibodies 14C12H1L1, VP101, BsAbB7, and BsAbB8 on the secretion of IL-2 induced by co-culture of PBMC and Raji-PD-L1 cells, as detected by ELISA.

[0232] Figure 30: Inhibitory effect of different concentrations of VP101 on tumor growth.

[0233] Figure 31: Effect of different concentrations of VP101 on mouse body weight. Detailed Implementation

[0234] 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 for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.

[0235] In the following embodiments of the present invention, the marketed drug antibody Bevacizumab (trade name) targeting the same target is used. As a control antibody, it can be purchased from Roche, Inc., or it can be prepared according to Preparation Example 4.

[0236] In the following embodiments of the present invention, the marketed drug antibody Nivolumab (trade name) targeting the same target is used. The control antibody was purchased from BMS, Bristol Myers Squibb.

[0237] In the following embodiments of the present invention, the amino acid sequences of the control antibodies BsAbB7 and BsAbB8 are identical to those of BsAbB7 and BsAbB8 in Chinese Patent Publication CN105175545A.

[0238] Preparation Example 1: Preparation of fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc

[0239] The preparation of fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc and their SDS-PAGE electrophoresis detection were carried out entirely according to Preparation Example 1 of Chinese Patent Publication CN106632674A.

[0240] In this preparation example, the amino acid sequences and their encoding nucleic acid sequences of the fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc are the same as those of PD-1-mFc, PD-1-hFc and PDL-1-hFc in Preparation Example 1 of Chinese Patent Publication CN106632674A.

[0241] The fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc were prepared.

[0242] Preparation Example 2: Expression and purification of the fusion protein VEGFA-His

[0243] 1. Constructing the VEGFA-His plasmid

[0244] VEGFA human cDNA (purchased from Origene) was used as a template for PCR amplification, and the hVEGFA-His fragment was purified and recovered using a standard DNA product purification kit. The recovered hVEGFA-His fragment was digested with the expression vector pcDNA3.1 using XbaI & HindIII-HF enzymes. The target gene fragment and linear expression vector were recovered by gel electrophoresis and ligated using T4 ligase. All ligation products were transformed into DH5α chemocompetent cells, plated on Agar plates containing ampicillin, and well-isolated single colonies were selected for colony PCR identification. Clones with positive PCR results were inoculated into LB medium and cultured, and the bacterial culture was sent to Guangzhou Yingjun Biotechnology Co., Ltd. for sequencing verification. Sequencing results showed that the inserted sequence in the positive recombinants was completely correct.

[0245] 2. Expression and purification of the fusion protein VEGFA-His

[0246] Following the Lipofectamin transfection kit (Invitrogen), recombinant plasmid VEGFA-his was transfected into 293F cells (Invitrogen) for 7 days. The culture medium was centrifuged at high speed, the supernatant was concentrated, and the medium was replaced with Binding Buffer A before loading onto a HisTrap column. Proteins were linearly eluted with Elution Buffer A. The purified sample was then transferred to a HiTrap Desalting column, the medium was replaced with Binding Buffer B, and the sample was loaded onto a HiTrap Q column. Proteins were linearly eluted with Elution Buffer B, the target sample was recovered, and the medium was replaced with PBS. The purified sample was then added to reduced protein electrophoresis loading buffer for SDS-PAGE analysis.

[0247] The fusion protein VEGFA-His was successfully synthesized.

[0248] The amino acid sequence of VEGFA-His is as follows (171aa):

[0249]

[0250] The underlined portion represents the amino acid sequence of VEGFA.

[0251] Nucleic acid sequence encoding VEGFA-His (513 bp)

[0252]

[0253]

[0254] Preparation Example 3: Expression and purification of the fusion protein VEGFR2-hFc

[0255] 1. Synthesis of the gene VEGFR2-hFc:

[0256] The amino acids corresponding to the extracellular fragment VEGFR2ECD of the gene VEGFR2 (Vascular Endothelial Growth Factor Receptor 2, NCBI GenBank: NP_002244) were fused with the Fc protein fragment (hFc) of TEV and human IgG, respectively (SEQ ID NO: 3). The corresponding encoding nucleic acid sequences were synthesized by GenScript (SEQ ID NO: 4).

[0257] VEGFR2:Vascular Endothelial Growth Factor Receptor 2,NCBI GenBank:NP_002244;

[0258] hFc:Ig gamma-1 chain C region, ACCESSION: P01857, 106-330;

[0259] The amino acid sequence of the fusion protein VEGFR2-hFc: (998aa)

[0260]

[0261]

[0262] The wavy underlined portion represents the ECD portion of VEGFR2, the bordered portion represents the TEV restriction site, and the solid underlined portion represents the hFc portion.

[0263] The nucleic acid sequence encoding the fusion protein VEGFR2-hFc is as follows: (2997 bp)

[0264]

[0265]

[0266]

[0267] The wavy underlined part represents the ECD portion of VEGFR2, the bordered part represents the TEV restriction site, and the solid underlined part represents the hFc portion.

[0268] 2. Construction of pUC57simple-VEGFR2-hFc plasmid:

[0269] The VEGFR2-hFc encoding gene synthesized by GenScript was cloned into the pUC57simple expression vector (provided by GenScript) to obtain the pUC57simple-VEGFR2-hFc plasmid.

[0270] 3. Construction of the pcDNA3.1-VEGFR2-hFc recombinant plasmid:

[0271] The plasmid pUC57simple-VEGFR2-hFc was digested with enzymes (Xba I and BamHI), and the resulting fusion gene fragment VEGFR2-hFc was recovered by electrophoresis and ligated into the pcDNA3.1 expression vector (purchased from Invitrogen) to obtain pcDNA3.1-VEGFR2-hFc. This pcDNA3.1-VEGFR2-hFc was then transfected into competent E. coli cells DH5α (purchased from TIANGEN), and transfection and culture were performed according to the manufacturer's instructions. Positive pcDNA3.1-VEGFR2-hFc clones were screened, and E. coli were amplified using standard methods. The pcDNA3.1-VEGFR2-hFc recombinant plasmid was then extracted using a kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., DP103-03) according to the kit's instructions.

[0272] 4. Recombinant plasmid pcDNA3.1-VEGFR2-hFc was transfected into 293F cells.

[0273] The recombinant plasmid pcDNA3.1-VEGFR2-hFc was transfected into 293F cells (purchased from Invitrogen) according to the method of the lipofectamin transfection kit (purchased from Invitrogen).

[0274] 5. SDS-PAGE electrophoresis detection of VEGFR2-hFc protein

[0275] Seven days after transfecting 293F cells with the recombinant plasmid pcDNA3.1-VEGFR2-hFc, the culture medium was purified by high-speed centrifugation, vacuum filtration through a microporous membrane, and Mabselect SuRe column to obtain the VEGFR2-hFc fusion protein sample. A portion of the sample was added to the reduced protein electrophoresis loading buffer and detected by SDS-PAGE electrophoresis.

[0276] The fusion protein VEGFR2-hFc was successfully synthesized.

[0277] Preparation Example 4: Preparation of Bevacizumab, an anti-VEGFA antibody

[0278] The amino acid sequences of the heavy chain variable region and light chain variable region of the marketed VEGFA monoclonal antibody Avastin (Bevacizumab) are based on Chinese Patent Publication CN1259962A. The nucleic acid sequences encoding the heavy chain variable region and light chain variable region were synthesized by Genscript Biotech.

[0279] The amino acid sequence of the variable region of the Bevacizumab heavy chain is: (123aa)

[0280]

[0281] Nucleic acid sequence encoding the variable region of the Bevacizumab heavy chain: (369 bp)

[0282]

[0283] The amino acid sequence of the variable region of the Bevacizumab light chain is: (107aa)

[0284]

[0285] Nucleic acid sequence encoding the variable region of the Bevacizumab light chain: (321 bp)

[0286]

[0287] The constant region of the heavy chain was the Ig gamma-1 chain C region, ACCESSION: P01857; the constant region of the light chain was the Ig kappa chain C region, ACCESSION: P01834.

[0288] The heavy chain cDNA and light chain cDNA of Bevacizumab were cloned into the pcDNA3.1 vector to obtain the recombinant expression plasmid of the antibody Bevacizumab. The recombinant plasmid was transfected into 293F cells. The 293F cell culture medium was purified and then analyzed.

[0289] The anti-VEGFA monoclonal antibody Avastin (Bevacizumab) was successfully prepared.

[0290] Preparation Example 5: Preparation and Detection of Humanized Anti-PD-1 Antibody 14C12H1L1

[0291] The procedure was carried out entirely in accordance with the descriptions of Examples 3-4 in Chinese Patent Publication CN106977602A.

[0292] The amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized antibody 14C12H1L1, as well as their encoding nucleic acid sequences, are the same as those described in Examples 3-4 of Chinese Patent Publication CN106977602A, and are also provided here:

[0293] The amino acid sequence of the variable region of the heavy chain of the humanized antibody 14C12H1L1: (118aa)

[0294]

[0295] Nucleic acid sequence encoding the variable region of the heavy chain of the humanized antibody 14C12H1L1: (354 bp)

[0296]

[0297]

[0298] The amino acid sequence of the variable region of the light chain of the humanized antibody 14C12H1L1: (107 aa)

[0299]

[0300] Nucleic acid sequence encoding the variable region of the light chain of humanized antibody 14C12H1L1: (321 bp)

[0301]

[0302] A humanized antibody against PD-1, 14C12H1L1, was prepared.

[0303] Preparation Example 6: Preparation and Identification of Human Anti-Egg Lysosomal Antibody hIgG

[0304] Human anti-Hen Egg Lysozyme IgG (anti-HEL, i.e., human IgG, abbreviated as hIgG) has a sequence derived from the variable region of the Fab F10.6.6 sequence in Acierno et al.'s study on Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The preparation method is as follows:

[0305] Nanjing GenScript Biotechnology was commissioned to optimize the amino acid codons and synthesize the heavy and light chain (full sequence or variable region) genes of human IgG antibodies. Following standard techniques described in *Molecular Cloning: A Laboratory Manual (3rd Edition)*, PCR, enzyme digestion, DNA gel recovery, ligation transformation, colony PCR, or enzyme digestion identification were used to subclone the heavy and light chain genes into antibody heavy chain and antibody light chain expression vectors in mammalian expression systems. Further sequencing analysis of the heavy and light chain genes in the recombinant expression vectors was performed. After sequencing verification, endotoxin-free expression plasmids were prepared in large quantities and transiently co-transfected into HEK293 cells for recombinant antibody expression. After 7 days of culture, cell culture medium was collected and purified using rProtein A column (GE). The harvested antibody samples were quality-assessed using standard SDS-PAGE and SEC-HPLC analytical techniques.

[0306] hIgG was prepared and used in Examples 8-9 below.

[0307] Example 1: Sequence design, preparation, and detection of the heavy and light chains of the bifunctional antibody VP101

[0308] 1. Sequence Design

[0309] The bifunctional antibody VP101 in this invention has a structural pattern belonging to the Morrison pattern (IgG-scFv), that is, the C-terminus of both heavy chains of an IgG antibody is linked to the scFv fragment of another antibody. The main components of its heavy and light chains are designed as shown in Table 1 below.

[0310] Table 1: Composition Design of Heavy and Light Chains in VP101

[0311]

[0312] In Table 1 above:

[0313] (1) Those marked with "V" in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Those without "V" are the full-length heavy or light chains containing the constant region. The amino acid sequences of these variable regions or full-length chains and their encoding nucleic acid sequences are all based on the corresponding sequences described in the preparation examples above.

[0314] (2) The amino acid sequence of Linker 1 is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:13)

[0315] 2. Expression and purification of antibody VP101

[0316] The heavy chain cDNA sequence and light chain cDNA sequence of VP101 were cloned into the pUC57simple vector (provided by GenScript) to obtain the pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.

[0317] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested with HindIII and EcoRI, respectively. The heavy and light chains obtained by electrophoresis were subcloned into the pcDNA3.1 vector, 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, the supernatant was concentrated, and then loaded onto a HiTrap MabSelect SuRe column. Protein was eluted with Elution Buffer in one step, and the target sample antibody VP101 was recovered. The medium was then replaced with PBS.

[0318] 2. Detection of antibody VP101

[0319] The purified samples were added to reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer, respectively, and then boiled before SDS-PAGE electrophoresis detection. The electrophoresis pattern of VP101 is shown in Figure 1. The target protein in the reduced protein sample was located at 75kD and 25kD, while the target protein in the non-reduced protein sample (single antibody) was located at 200kD.

[0320] Unless otherwise specified, the humanized antibody VP101 used in the following experiments was prepared according to the method described in this embodiment.

[0321] Example 2: Determination of kinetic parameters of humanized antibody VP101

[0322] 1. Determination of kinetic parameters of binding between humanized antibody VP101 and PD-1-mFc

[0323] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL antibody was immobilized on the AHC sensor at a height of approximately 0.4 nm. The sensor was equilibrated in the buffer for 60 s. The antibody immobilized on the sensor then bound to PD-1-mFc at a concentration of 0.62–50 nM (three-fold serial dilution) for 120 s. The antigen and antibody dissociated in the buffer for 300 s. Data were analyzed using a 1:1 model to obtain the affinity constant. Data acquisition software was Fortebio Data Acquisition 7.0, and data analysis software was Fortebio Data Analysis 7.0. The kinetic parameters of the binding of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1, and the control antibody Nivolumab to PD-1-mFc are shown in Table 2. The results of the kinetic characteristic parameter detection are shown in Figures 2, 3, 4, 5, and 6, respectively.

[0324] Table 2: Kinetic parameters of binding of humanized antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and control antibody Nivolumab to PD-1-mFc

[0325] Sample IDK D (M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)VP1011.68E-103.22E+051.44E+045.40E-05 3.16E-050.14-0.28BsAbB71.62E-103.27E+052.60E+045.30E-056.24E-050.01-0.11BsAbB84.0 6E-103.39E+052.04E+041.37E-044.61E-050.01-0.1314C12H1L11.64E-104.55E+051.61E+047. 47E-052.98E-050.24-0.28Nivolumab2.32E-105.85E+052.03E+041.36E-043.47E-050.02-0.14

[0326] K D K is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; K D =kdis / kon.

[0327] The results showed that antibodies VP101 and BsAbB7 had comparable affinity to PD-1-mFc; the affinity constant of VP101 for PD-1-mFc was significantly smaller than that of BsAbB8, suggesting that VP101 had better binding activity; the dissociation rate constant of VP101 for PD-1-mFc was significantly smaller than that of BsAbB8 and 14C12H1L1, suggesting that VP101 was more stable in binding to the antigen and had a slower dissociation rate than 14C12H1L1 and BsAbB8.

[0328] 2. Determination of kinetic parameters of binding between humanized antibody VP101 and VEGF-His

[0329] 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 s. The sensor equilibrated in buffer for 60 s. VEGF immobilized on the sensor bound to antibody at concentrations ranging from 12.34 to 1000 nM (three-fold serial dilution) for 120 s. Antigen-antibody dissociation occurred in buffer for 300 s. Data were analyzed using a 1:1 model to obtain affinity constants. Data acquisition software was Fortebio Data Acquisition 7.0, and data analysis software was Fortebio Data Analysis 7.0.

[0330] The kinetic parameters of the binding of antibodies VP101, BsAbB7, BsAbB8 and control antibody Bevacizumab to VEGF-His are shown in Table 3, and the detection results of the kinetic characteristic parameters are shown in Figures 7, 8, 9 and 10, respectively.

[0331] Table 3: Kinetic parameters of VEGF-His binding to antibodies VP101, BsAbB7, BsAbB8 and control antibody Bevacizumab

[0332] Sample IDK D(M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)VP1015.21E-101.55E+059. 67E+038.05E-054.66E-050.39-0.60BsAbB75.14E-101.57E+059.67E+038.05E- 054.83E-050.36-0.53BsAbB86.33E-101.71E+051.07E+041.08E-044.64E-050. 39-0.56Bevacizumab7.24E-101.23E+057.09E+038.90E-054.53E-050.29-0.41

[0333] The results showed that antibodies VP101 and BsAbB7 had comparable affinity to the antigen, and their affinity constants were significantly smaller than those of BsAbB8 and the control antibody Bevacizumab, suggesting that VP101 had better binding activity. The dissociation rate constant of VP101 with VEGF-His was significantly smaller than that of BsAbB8, suggesting that VP101 was more stable in binding to the antigen and had a slower dissociation rate than BsAbB8.

[0334] Example 3: ELISA method for detecting the binding activity of antibody VP101 to antigen

[0335] 1. Indirect ELISA method for determining the binding activity of antibody VP101 to antigen VEGFA-his

[0336] The specific method is as follows:

[0337] The microplate was coated with VEGFA-His and incubated at 37°C for 2 hours. After washing, it was blocked with 1% BSA for 2 hours. After washing, serially diluted antibodies were added and incubated at 37°C for 30 minutes. After washing, enzyme-labeled goat anti-human IgG secondary antibody working solution was added and incubated at 37°C for 30 minutes. After washing, TMB chromogenic solution was added and the reaction was developed in the dark for 5 minutes. The reaction was stopped by adding stop solution. The microplate was immediately placed in a microplate reader, and the OD values ​​of each well were read at 450 nm. The data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0338] The results of detecting the binding of antibody VP101 to antigen VEGFA-his are shown in Figure 11. The absorbance intensities at each dose are shown in Table 4. A curve was fitted with antibody concentration on the x-axis and absorbance value on the y-axis to calculate the antibody binding EC50. 50 The results are shown in Table 4 below.

[0339] Table 4: Binding of bifunctional antibodies to VEGFA-his (indirect ELISA)

[0340]

[0341] The results showed that antibody VP101 could effectively bind to VEGFA protein, and its binding efficiency was dose-dependent, both of which were comparable to the binding activity of human VEGFA.

[0342] 2. Indirect ELISA method was used to determine the binding activity of antibodies VP101, BsAbB7, and BsAbB8 to antigen VEGFA-his.

[0343] The specific method is as follows:

[0344] The microplate was coated with VEGFA-His and incubated overnight at 4°C. After washing, it was blocked with 1% BSA (dissolved in PBS) for 2 hours. After washing, serially diluted antibody was added 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 and incubated at 37°C for 30 minutes. After washing, TMB (Neogen, 308177) was added for color development in the dark for 5 minutes, and the reaction was stopped by adding stop solution. The microplate was immediately placed in a microplate reader, and the OD values ​​of each well were read at 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software.

[0345] The binding results of antibody VP101 and antigen VEGFA-his are shown in Figure 12. The absorbance intensities at each dose are shown in Table 5. A curve was fitted with antibody concentration on the x-axis and absorbance value on the y-axis to calculate the antibody binding EC50. 50 The results are shown in Table 5 below.

[0346] Table 5: Binding activities of VP101, BsAbB7, BsAbB8, and Bevacizumab with VEGFA-his (indirect ELISA)

[0347]

[0348] The results showed that antibodies VP101, BsAbB7, BsAbB8, and Bevacizumab could all effectively bind to VEGF protein, and their binding efficiency was dose-dependent. The binding activity of antibody VP101 to human VEGF was stronger than that of BsAbB7 and BsAbB8.

[0349] 3. The binding activity of antibody VP101 to antigen PD-1 was determined by indirect ELISA.

[0350] The specific method is as follows:

[0351] Human PD-1-mFc was coated onto an ELISA plate and incubated overnight at 4°C. After blocking with 1% BSA at 37°C for 2 hours, antibodies were added, and the plates were incubated at 37°C for 30 minutes. The plates were washed and dried, and HRP-labeled goat anti-human IgG (H+L) secondary antibody (Jackson, 109-035-088) was added, followed by incubation at 37°C for 30 minutes. The plates were washed and dried again, and TMB (Neogen, 308177) was added for color development for 5 minutes. The color development was stopped by adding stop solution. The plates were immediately placed in an ELISA reader, and the OD values ​​of each well were read at 450 nm. Data were analyzed using SoftMax Pro 6.2.1 software.

[0352] The results of detecting the binding of antibody VP101 to antigen PD-1 are shown in Figure 13. The light absorption intensity at each dose is shown in Table 6. Quantitative analysis of the bound antibody VP101 was performed, and the EC50 curve simulated the antibody binding efficiency. 50 As shown in Table 6 below.

[0353] Table 6: Binding of bifunctional antibodies to PD-1 (indirect ELISA)

[0354]

[0355]

[0356] The results showed that antibody VP101 could effectively bind to PD-1 protein, and its binding efficiency was dose-dependent.

[0357] 4. Indirect ELISA method to determine the binding activity of antibodies VP101, BsAbB7, and BsAbB8 to antigen PD-1.

[0358] The specific method is as follows:

[0359] Human PD-1-mFc was coated onto an ELISA plate and incubated overnight at 4°C. After blocking with 1% BSA (dissolved in PBS) at 37°C for 2 hours, antibodies were added, and the plate was incubated at 37°C for 30 minutes. The plate was washed and dried, and horseradish peroxidase-labeled goat anti-human IgG,Fc (Jackson, 109-035-098) was added and incubated at 37°C for 30 minutes. The plate was washed and dried, and TMB (Neogen, 308177) was added for color development for 5 minutes. The color development was stopped by adding stop solution. The plate was immediately placed in an ELISA reader, and the OD values ​​of each well were read at 450 nm. Data were analyzed using SoftMax Pro 6.2.1 software.

[0360] The results of detecting the binding of antibody VP101 to antigen PD-1 are shown in Figure 14. The absorbance intensities at different doses are shown in Table 7. Quantitative analysis of the bound antibody VP101 was performed, and the EC50 curve simulated the antibody binding efficiency. 50 As shown in Table 7 below.

[0361] Table 7: Binding activities of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1, and Nivolumab with PD-1 (indirect ELISA)

[0362]

[0363]

[0364] The results showed that antibody VP101 could effectively bind to PD-1 protein, and its binding efficiency was dose-dependent. The binding activity of antibody VP101 to human PD-1 was significantly stronger than that of BsAbB7 and BsAbB8.

[0365] 5. Competitive ELISA method was used to determine the activity of antibody VP101 and VEGFR2 in competitively binding to antigen VEGFA.

[0366] The specific method is as follows:

[0367] The ELISA plate was coated with VEGF-His and incubated at 37°C for 2 hours. After washing, it was blocked with 1% BSA at 37°C for 1 hour. After washing, serially diluted antibody and human VEGFR2ECD-mFc-bio (final concentration 0.02 μg / ml) were added, and the plate was incubated at room temperature for 2 hours. After washing, HRP-labeled streptavidin SA-HRP (1:4000) working solution was added, and the plate was incubated at 37°C for 30 minutes. After washing, TMB chromogenic solution was added, and the plate was developed in the dark for 5 minutes. The chromogenic reaction was terminated by adding stop solution. The ELISA plate was immediately placed in a microplate reader, and the OD values ​​of each well were read at 450 nm. The data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0368] The detection results are shown in Figure 15. The absorbance intensities for each dose are shown in Table 8. The binding EC was obtained by quantitatively analyzing the absorbance intensity of the bound antibody and using curves to simulate the antibody binding efficiency. 50 (Table 8)

[0369] Table 8: Competitive ELISA detection antibodies against VEGFR2-mFc antigen VEGFA-His

[0370]

[0371]

[0372] The results showed that antibody VP101 could effectively bind to antigen VEGFA and inhibit VEGFR2 binding to VEGFA, and its efficiency in inhibiting VEGFR2 binding to VEGFA was dose-dependent.

[0373] 6. Competitive ELISA method to determine the competitive binding of antibody VP101 and PD-L1 to antigen PD-1.

[0374] The specific method is as follows:

[0375] The microplate was coated with PD-1-hFc and incubated overnight at 4°C. After blocking with 1% BSA for 2 hours, different concentrations of antibody were mixed with PD-L1-hFc for 10 minutes (dilution concentrations are shown in Table 10). The plates were incubated at 37°C for 30 minutes, washed, and blotted dry. Secondary antibody was added, and the plates were incubated at 37°C for 30 minutes. The plates were washed and blotted dry, and TMB was added for color development for 5 minutes. The color development was stopped by adding stop solution. The microplate was immediately placed in a microplate reader, and the OD values ​​of each well were read at 450 nm (see Table 10). The data were analyzed using SoftMax Pro 6.2.1 software.

[0376] The detection results are shown in Figure 16. The absorbance intensities for each dose are shown in Table 9. The binding EC was obtained by quantitatively analyzing the absorbance of the bound antibody VP101 and obtaining the binding efficiency by simulating the antibody binding curve. 50 (Table 9)

[0377] Table 9: Competitive ELISA Detection of Bifunctional Antibody Competitive Binding to PD-L1

[0378]

[0379] The results showed that antibody VP101 could effectively bind to antigen PD-1 and inhibit the binding of ligand PD-L1 to PD-1, and its efficiency in inhibiting PD-L1 binding to PD-1 was dose-dependent.

[0380] Example 4: Binding of antibody VP101 to cell membrane surface antigens

[0381] First, a 293T antibody expressing the PD-1 antigen was constructed, and then flow cytometry was used to analyze and verify the antibody's specific binding ability to the cell membrane surface antigen.

[0382] 1. Constructing 293T cells expressing PD-1 antigen

[0383] The PD-1 vector pLenti6.3-PD-1 (pLenti6.3 was purchased from Invitrogen) was transfected into 293T cells, and after screening, a stable clonal population of 293T-PD-1 cells expressing PD-1 was obtained.

[0384] 2. Detecting the binding of antibodies to cell surface antigens.

[0385] The 293T-PD-1 cells expressing the antigen were obtained using the above steps via conventional trypsin digestion, with each collection tube containing 2 × 10⁻⁶ cells. 5 Antibody concentration gradient dilutions were prepared using PBSA (1% BSA) and incubated with 293T-PD-1 cells on ice for 2 hours. 100 μL of FITC goat anti-human IgG (1:500) was added to each tube and incubated on ice for 1 hour. The cells were washed with PBS, resuspended in 300 μL of PBSA, and the fluorescence signal (MFI) was detected using the FITC channel on a flow cytometer.

[0386] The results are shown in Figure 17, and the MFI values ​​for each concentration are shown in Table 10. The binding EC50 of the VP101 antibody was calculated by quantitative fluorescence analysis and curve fitting of the bound 14C12H1L1 antibody. 50 It is 3.5 nM.

[0387] Table 10: Fluorescence intensity analysis of VP101 binding to 293T-PD-1 surface antigen by FACS

[0388] Antibody (nM) 0.14 0.41 1.23 3.70 11.11 33.33 100 EC 50 Bevacizumab3.22.22.02.32.73.85.7-Nivolumab33.374.9171.9357.9481.9498.3478.42.114C1 2H1L148.199.7201.5409.0600.2655.4670.82.9VP10130.861.8135.7286.9487.7534.0528.63.5

[0389] The results showed that the VP101 antibody could effectively bind to the PD-1 antigen on the surface of 293T-PD-1 host cells, and its binding efficiency was dose-dependent. Bevacizumab had no binding activity against 293T-PD-1, indicating that the binding of VP101 to 293T-PD-1 is specific.

[0390] 3. Detect the binding of antibodies VP101, BsAbB7, and BsAbB8 to cell surface antigens, following the experimental steps described in step 2 of this embodiment.

[0391] The results are shown in Figure 18, and the MFI values ​​for each concentration are shown in Table 11. The binding EC50 values ​​of Nivolumab, 14C12H1L1, VP101, BsAbB7, and BsAbB8 antibodies were calculated by quantitative fluorescence analysis and curve fitting. 50 The values ​​are 7.853 nM, 3.607 nM, 7.896 nM, 9.943 nM, and 10.610 nM, respectively.

[0392] Table 11: Fluorescence intensity analysis of VP101, BsAbB7, and BsAbB8 bound to 293T-PD-1 surface antigen by FACS

[0393]

[0394] The results showed that the VP101 antibody could bind to PD-1 on the 293T-PD1 membrane surface in a dose-dependent manner. Bevacizumab showed no binding activity to 293T-PD-1, indicating that the binding of VP101 to 293T-PD-1 is specific.

[0395] Example 5: Competitive binding of antibody VP101 to cell membrane surface antigens

[0396] 1. The EC50 of VP101 competing with PD-L1 for binding to the cell membrane antigen PD-1 was determined by competitive flow cytometry. 50 The specific method is as follows:

[0397] 293T-PD-1 cells were routinely digested, with 300,000 cells per sample, and centrifuged and washed. 100 μL of serially diluted antibody was added to each tube, and the cells were incubated on ice for 30 min. 100 μL of PD-L1-mFc was added to each tube, mixed to a final concentration of 20 nM, and incubated on ice for 1 hour. 500 μL of 1% PBSA was added, and the cells were centrifuged at 5600 rpm for 5 min, and the supernatant was discarded. 100 μL of 1:500 diluted FITC Goat anti-mouse antibody was added to each tube, mixed, and incubated on ice in the dark for 40 min. The cells were centrifuged, washed, resuspended, and transferred to flow cytometry tubes for analysis.

[0398] The results are shown in Figure 19, and the MFI values ​​for each concentration are shown in Table 12. The competitive binding EC50 values ​​of antibodies VP101 and 14C12H1L1 were calculated using quantitative fluorescence analysis and curve fitting. 50 The values ​​are 8.33 nM and 4.37 nM, respectively.

[0399] Table 12: Fluorescence intensity analysis of 14C12H1L1 and VP101 competitively binding to 293T-PD-1 surface antigen by FACS

[0400]

[0401] The results showed that the VP101 antibody effectively blocked the binding of PDL-1 to PD-1 on the surface of 293T-PD-1 host cells in a dose-dependent manner.

[0402] 2. Competitive flow cytometry was used to determine the EC50 of VP101, BsAbB7, BsAbB8, 14C12H1L1, Nivolumab, and PD-L1 in competitive binding to the cell membrane surface antigen PD-1. 50 The method refers to the experimental steps described in 1 of this embodiment.

[0403] The results are shown in Figure 20, and the MFI values ​​for each concentration are shown in Table 13. The competitive binding EC50 values ​​of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1, and Nivolumab were calculated using quantitative fluorescence analysis and curve fitting. 50 The values ​​were 15.04 nM, 22.25 nM, 19.25 nM, 9.21 nM, and 9.72 nM, respectively.

[0404] Table 13: Fluorescence intensity analysis of VP101, BsAbB7, BsAbB8, 14C12H1L1, and Nivolumab competitively binding to 293T-PD-1 surface antigen by FACS.

[0405] Antibody (nM) 0.14 0.41 1.23 3.71 1.11 33.33 100 300 EC 50 R 2 VP1011441.941380.621368.151288.34982.69112.908.498.2115.040.9971BsAbB71412.621377.2 71339.601341.271094.35417.709.239.1822.250.9985BsAbB81578.361521.501427.201429.8511 37.74359.699.739.6819.250.996214C12H1L11384.081551.051462.851296.64580.4512.9313.37 14.999.210.9950Nivolumab1539.581552.371483.841300.81713.5670.9260.7756.929.720.9969

[0406] The results showed that the activity of antibody 14C12H1L1 was comparable to that of the commercially available PD-1 single-target antibody Nivolumab, and superior to that of the bifunctional antibody VP101. The activity of antibody VP101 was superior to that of BsAbB7 and BsAbB8.

[0407] Example 6: Detection of the neutralizing bioactivity of antibodies VP101, BsAbB7, and BsAbB8 in blocking the VEGF-activated NFAT signaling pathway

[0408] 1. Construction of 293T-NFAT-(opv)KDR(C7) cells

[0409] KDR(VEGFR2) vector pCDH-KDRFL(OPV)-GFP-Puro (pCDH-GFP-Puro was purchased from Ubisoft Biotech) and NFAT vector pNFAT-luc-hygro (pGL4-luc2P-hygro was purchased from Promega) were transfected into 293T cells. After screening, a clonal population of 293T-NFAT-(opv)KDR(C7) cells stably expressing KDR and NFAT luciferase reporter genes was obtained.

[0410] 2. Collect 293T-NFAT-(opv)KDR(C7) cells, centrifuge for 5 min to remove supernatant, resuspend cells in DMEM + 10% FBS medium, count cell number and viability, and adjust cell concentration to an appropriate range. Add 50,000 cells / 50 μL of cell suspension to each well of a black 96-well plate.

[0411] Dilute the corresponding antibodies (final concentrations 300, 100, 10, 2, 0.2, 0.02, 0.002 nM) and VEGF (final concentration 30 ng / mL) according to the experimental design. The antibody targeting VEGF was pre-incubated with VEGF at room temperature for 1 h before being added to the cells. Blank and isotype controls (final volume 100 μL per well) were designed and incubated at 37°C with 5% CO2 for 4 h. Add 50 μL of Luciferase Assay System to each well, and detect relative fluorescence units (RLUs) using a multi-label microplate reader within 5 min.

[0412] The experimental results are shown in Figure 21. The EC values ​​of each antibody are... 50 See Table 14.

[0413] Table 14: Reporter assay results of the neutralizing bioactivity of VP101, BsAbB7, and BsAbB8 in blocking the VEGF-activated NFAT signaling pathway.

[0414] SampleVP101BsAbB7BsAbB8BevacizumabEC 50 (nM) 1.2400 1.2170 1.7280 0.7730

[0415] The results show that VP101's EC 50 EC with 1.240 nM, BsAbB7 50 EC50 of 1.217 nM, BsAbB8 50 The EC50 of Bevacizumab is 1.728 nM. 50 The value was 0.773 nM, indicating that VP101 and BsAbB7 were more effective than BsAbB8 in blocking the VEGF-activated NFAT signaling pathway.

[0416] Example 7: Experiment on the inhibition of VEGFA-induced HUVEC cell proliferation by VP101 antibody

[0417] Harvesting healthy HUVEC cells (purchased from Allcell) and adjusting the cell concentration to 1.5 × 10⁻⁶ cells. 4 / ml, seeded into 96-well plates, 200μL / well, and cultured in a 37℃, 5% CO2 incubator for 24h. After observing good cell adhesion, the culture medium was discarded, and 20nM VEGFA was prepared with 2% FBS 1640 and added to the 96-well plates, 200μl / well. Different concentrations of antibody were added, and the plates were cultured for another 72h. After 72h, the culture medium was discarded, MTT was added, and after 4h, MTT was discarded and DMSO was added. The OD value at 490nm was measured using a microplate reader.

[0418] The results are shown in Figure 22. The results show that both humanized antibodies VP101 and Bevacizumab can effectively inhibit VEGFA-induced HUVEC cell proliferation in a dose-dependent manner; and at the same dose, VP101 has higher pharmacological activity than Bevacizumab in inhibiting VEGFA-induced HUVEC cell proliferation.

[0419] Example 8: Promoting the secretion of cytokines IFN-γ and IL-2 in mixed lymphocyte responses

[0420] 1. VP101, 14C12H1L1, and Nivolumab promote IFN-γ secretion in a mixed culture system of DC and PBMCs.

[0421] PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare). The isolated PBMCs were induced for 6 days with IL-4 (Peprotech 200-04, 1000 U / ml) and GM-CSF (Peprotech 300-03, 1000 U / ml), and then TNF-α (Peprotech 300-01A, 200 U / ml) was added for an additional 3 days to obtain mature DC cells.

[0422] On the day of co-culture, peripheral blood from another donor was collected to isolate fresh PBMCs. The obtained mature DCs were mixed with freshly isolated PBMCs from another donor at a ratio of 1:10 and cultured. At the same time, different concentrations of antibodies (hIgG as a control) were added. After co-culturing for 5-6 days, the cell supernatant was collected and the content of IFN-γ (purchased from Dakota) was detected using an ELISA kit.

[0423] The effect of VP101 on IFN-γ secretion in a co-culture system of DC cells and PBMCs is shown in Figure 23. As can be seen from Figure 23, VP101 effectively promotes IFN-γ secretion in a dose-dependent manner. Furthermore, at doses of 30 nM and 300 nM, VP101 exhibits higher IFN-γ secretion-promoting activity than an equivalent dose of 14C12H1L1, and at the 30 nM dose level, its IFN-γ secretion-promoting activity is higher than that of an equivalent dose of Nivolumab.

[0424] 2. VP101, BsAbB7, and BsAbB8 promote the secretion of IL-2 and IFN-γ in a mixed culture system of DC and PBMC.

[0425] The experimental method is the same as step 1 in this embodiment, that is, PBMCs are isolated using Ficoll-Paque Plus (GE Healthcare), and the isolated PBMCs are induced with IL-4 (Peprotech 200-04, 1000U / ml) and GM-CSF (Peprotech 300-03, 1000U / ml) for 6 days. Then, TNF-α (Peprotech 300-01A, 200U / ml) is added for an additional 3 days of induction to obtain mature DC cells.

[0426] On the day of co-culture, peripheral blood from another donor was collected to isolate fresh PBMCs. The obtained mature DCs were mixed with freshly isolated PBMCs from another donor at a ratio of 1:10 and cultured. At the same time, different concentrations of antibodies (hIgG as a control) were added. After co-culturing for 5-6 days, the cell supernatant was collected and the levels of IL-2 and IFN-γ (purchased from Dakota) were detected using an ELISA kit.

[0427] The effect of VP101 on IFN-γ secretion in a co-culture system of DC cells and PBMCs is shown in Figure 24. As can be seen from Figure 24, VP101 effectively promotes IFN-γ secretion in a dose-dependent manner. The pharmacological activity of VP101 in promoting IFN-γ secretion is significantly superior to that of BsAbB7 and BsAbB8.

[0428] The effect of VP101 on IL-2 secretion in a mixed culture system of DC cells and PBMCs is shown in Figure 25. As can be seen from Figure 25, VP101 can effectively promote IL-2 secretion in a dose-dependent manner, and the pharmacological activity of VP101 in promoting IL-2 secretion is superior to that of BsAbB7 and BsAbB8.

[0429] 3. VP101, 14C12H1L1, and Nivolumab promoted the secretion of IL-2 and IFN-γ in a mixed culture system of PBMC and Raji-PD-L1.

[0430] Raji cells were stably transfected with PD-L1 by lentiviral infection, and Raji-PD-L1 cells stably expressing PD-L1 were obtained by drug screening. PBMCs were stimulated with SEB for two days and then co-cultured with Raji-PD-L1 cells treated with mitomycin C.

[0431] The results are shown in Figures 26 and 27. The results show that VP101 can effectively promote the secretion of IL-2 and IFN-γ. At a dose level of 300 nM, the IL-2 secretion-promoting activity of VP101 is significantly better than that of equal doses of 14C12H1L1 and Nivolumab.

[0432] The isotype control antibody used in the study was human anti-Hen Egg Lysozyme IgG (anti-HEL, i.e., human IgG, abbreviated as hIgG), which was prepared according to the previous preparation example 6.

[0433] 4. VP101, BsAbB7, and BsAbB8 promoted the secretion of IL-2 and IFN-γ in a mixed culture system of PBMC and Raji-PD-L1, following the experimental method described in step 3 of this embodiment.

[0434] The IFN-γ secretion results are shown in Figure 28. The results indicate that VP101 effectively promotes IFN-γ secretion in a dose-dependent manner. Furthermore, at the same dose, VP101 is significantly superior to BsAbB7, and at dose levels of 3 nM and 30 nM, the pharmacological activity of VP101 is significantly superior to that of BsAbB8.

[0435] The IL-2 secretion results are shown in Figure 29. The results indicate that VP101 effectively promotes IL-2 secretion in a dose-dependent manner. Furthermore, at doses of 3 nM and 300 nM, the pharmacological activity of VP101 is significantly superior to that of BsAbB7, while at the same dose, VP101 is comparable to BsAbB8.

[0436] Example 9: Experiment on the in vivo inhibition of tumor growth by VP101

[0437] To detect the in vivo tumor-suppressive activity of VP101, 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). The modeling and specific administration methods are shown in Table 15. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0438] Table 15: Dosing regimens for VP101 treatment in U87MG xenograft Scid Beige mouse model

[0439]

[0440]

[0441] The results are shown in Figure 30. The results indicate that, compared with the isotype control antibody hIgG (prepared in the same way as in Example 6 above), Bevacizumab and different doses of VP101 can effectively inhibit the growth of mouse tumors, and the high dose of VP101 is better at inhibiting tumors than the low dose of VP101.

[0442] Furthermore, as shown in Figure 31, VP101 had no effect on the body weight of tumor-bearing mice.

[0443] Although the present invention has been fully and clearly described with respect to the specific embodiments disclosed herein, it is not limited thereto. Modifications and substitutions to the invention may be made by those skilled in the art based on the guidance of these descriptions, and such modifications and substitutions are included within the scope of protection of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A bispecific antibody comprising: Targeting the first protein functional region of VEGFA, and Targeting the second protein functional region of PD-1; in: The first protein functional region is an anti-VEGFA antibody or its antigen-binding fragment, wherein the heavy chain variable region of the anti-VEGFA antibody contains amino acid sequences as shown in SEQ ID NOs:15-17 (HCDR1-HCDR3), and its light chain variable region contains amino acid sequences as shown in SEQ ID NOs:18-20 (LCDR1-LCDR3); and The second protein functional region is an anti-PD-1 antibody or its antigen-binding fragment, wherein the heavy chain variable region of the anti-PD-1 antibody contains amino acid sequences as shown in SEQ ID NOs:21-23, namely HCDR1-HCDR3, and its light chain variable region contains amino acid sequences as shown in SEQ ID NOs:24-26, namely LCDR1-LCDR3.

2. The bispecific antibody according to claim 1, wherein, The anti-VEGFA antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody; And / or, The anti-PD-1 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody.

3. The bispecific antibody according to claim 1, wherein, The first protein functional region is an immunoglobulin, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:15-17 HCDR1-HCDR3, and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:18-20 LCDR1-LCDR3; and the second protein functional region is a single-chain antibody, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs:21-23 HCDR1-HCDR3, and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs:24-26 LCDR1-LCDR3; or, The first protein functional region is a single-chain antibody, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs: 21-23 (HCDR1-HCDR3) and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs: 24-26 (LCDR1-LCDR3); and the second protein functional region is an immunoglobulin, whose heavy chain variable region contains amino acid sequences as shown in SEQ ID NOs: 15-17 (HCDR1-HCDR3) and whose light chain variable region contains amino acid sequences as shown in SEQ ID NOs: 18-20 (LCDR1-LCDR3).

4. The bispecific antibody according to claim 3, wherein, The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:5; the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:7; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:9; and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

11. or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:9; the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:11; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:

7.

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

6. The bispecific antibody according to claim 3 or 4, wherein, The single-chain antibody consists of two strands, with one end of each strand attached to either the C-terminus or the N-terminus of one of the two heavy chains of the immunoglobulin.

7. The bispecific antibody according to claim 3 or 4, wherein, The immunoglobulin includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

8. The bispecific antibody according to claim 3 or 4, wherein, The immunoglobulin described herein has a constant region derived from human antibodies; Preferably, the constant region of the immunoglobulin is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.

9. The bispecific antibody according to claim 3 or 4, wherein, The immunoglobulin has a heavy chain constant region of human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and a light chain constant region of human Ig kappa chain C region.

10. The bispecific antibody according to claim 1 or 2, wherein, The first and second protein functional regions are directly connected or connected through a linker fragment; Preferably, the connection segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6.

11. The bispecific antibody according to claim 1 or 2, wherein, The first protein functional region and the second protein functional region are independently one, two, or more.

12. The bispecific antibody according to claim 1 or 2, wherein, The bispecific antibody is expressed in EC50 values ​​of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.15 nM, or less than 0.14 nM. 50 Binding to VEGFA protein; preferably, the EC 50 Measured by indirect ELISA method; And / or, The bispecific antibodies are expressed in EC5 concentrations 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 Binding to PD-1 protein; preferably, the EC 50 Measured by indirect ELISA method.

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

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

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

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

17. A conjugate comprising a bispecific antibody and a conjugated moiety, wherein, The bispecific antibody is the bispecific antibody according to any one of claims 1 to 12, and the conjugated portion is a detectable label; preferably, the conjugated portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

18. A kit comprising the bispecific antibody of any one of claims 1 to 12, or comprising the conjugate of claim 17; Preferably, the kit further comprises a second antibody that specifically binds to the bispecific antibody; optionally, the second antibody further comprises a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored 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. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17; optionally, it further comprising pharmaceutically acceptable excipients.

21. Use of the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17 in the preparation of a medicament for the prevention and / or treatment of malignant tumors; preferably, 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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, and leukemia.

22. 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 the following drugs or reagents: (1) Drugs or reagents for detecting VEGFA levels in samples. Drugs or reagents that block the binding of VEGFA to VEGFR2. Drugs or reagents that downregulate the activity or level of VEGFA. Drugs or reagents that can counteract the stimulatory effect of VEGFA on vascular endothelial cell proliferation. Drugs or reagents that inhibit the proliferation of vascular endothelial cells, or Drugs or reagents that block tumor angiogenesis; and / or (2) Drugs or reagents that block the binding of PD-1 to PD-L1 Drugs or reagents that downregulate the activity or level of PD-1 Drugs or reagents that can counteract the immunosuppression caused by PD-1. Drugs or reagents that promote IFN-γ secretion in T lymphocytes, or Drugs or reagents that promote IL-2 secretion in T lymphocytes.

23. A method, in vivo or in vitro, comprising the step of applying cells in an effective amount of the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17, said method being selected from the following: (1) Methods for detecting VEGFA levels in samples Methods to block the binding of VEGFA and VEGFR2 Methods to downregulate the activity or level of VEGFA Methods to relieve the stimulatory effect of VEGFA on vascular endothelial cell proliferation. Methods to inhibit vascular endothelial cell proliferation, or Methods to block tumor angiogenesis; and / or (2) Methods to block the binding of PD-1 and PD-L1 Methods to downregulate PD-1 activity or its level Methods to relieve the immunosuppression caused by PD-1 Methods to promote IFN-γ secretion in T lymphocytes, or Methods to promote IL-2 secretion in T lymphocytes.

24. A method for preventing and / or treating malignant tumors, comprising administering to a subject in need an effective amount of the bispecific antibody of any one of claims 1 to 12 or the conjugate of claim 17; preferably, 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, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer, and leukemia.

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

26. The bispecific antibody according to any one of claims 1 to 12 or the conjugate according to claim 17, wherein it is used for: (1) Detecting VEGFA levels in samples, Blocking the binding of VEGFA to VEGFR2, Downregulate the activity or level of VEGFA. To relieve the stimulatory effect of VEGFA on vascular endothelial cell proliferation. Inhibit vascular endothelial cell proliferation, or Blocking tumor angiogenesis; and / or (2) Blocking the binding of PD-1 to PD-L1 Downregulate the activity or level of PD-1. Relieve the immunosuppression caused by PD-1. Promotes IFN-γ secretion in T lymphocytes, or Promotes IL-2 secretion in T lymphocytes.

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