Therapeutic combinations and their uses

Abstract

The present invention relates to the fields of tumor treatment and molecular immunology, and specifically to a therapeutic combination and its use. Specifically, the therapeutic combination comprises an anti-CTLA4-anti-PD-1 bispecific antibody and an anti-PD-1-anti-VEGFA bispecific antibody. The therapeutic combination of the present invention effectively treats or prevents tumors, and therefore has good application prospects.

Description

[Technical Field] 【0001】 The present invention relates to the fields of tumor therapy and molecular immunology, and specifically to a therapeutic combination and its use. Specifically, the therapeutic combination is a pharmaceutical composition or combination product. Specifically, the pharmaceutical composition or combination product comprises an anti-CTLA4-anti-PD-1 bispecific antibody or antigen-binding fragment thereof and an anti-PD-1-anti-VEGFA bispecific antibody or antigen-binding fragment thereof. [Background technology] 【0002】 The transmembrane receptor PD-1 (programmed cell death protein 1) is a member of the CD28 family and is expressed on activated T cells, B cells, and myeloid cells. Both PD-1 ligands, PDL1 (programmed cell death 1 ligand 1, or PD-L1) and PDL2 (programmed cell death 1 ligand 2, or PD-L2), are members of the B7 superfamily, where PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, and PDL2 is expressed exclusively on antigen-presenting cells such as dendritic cells and macrophages. 【0003】 The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune evasion. PD-1 is primarily expressed in immune cells such as T cells, and its ligand, PDL1, is highly expressed in several human tumor tissues. Blockade of the PD-1 / PDL1 signaling pathway can activate inhibited T cells, which then attack cancer cells. Blockade of PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, activate the tumor cell killing process, and further inhibit local tumor growth (Julie R et al., 2012, N Engl J Med., 366:2455-2465). In addition, tumors with high PDL1 expression are associated with cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA, 104:3360-5). An effective method is to administer anti-PD-1 antibodies to regulate the expression of PD-1. Due to the broad anti-tumor promise and surprising efficacy of PD-1 antibodies, it is widely recognized in the industry that antibodies targeting the PD-1 pathway will bring about a breakthrough in the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol., 42(3):466-473), leukemia, and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets., 13(7):768-74). 【0004】 Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and CD28 molecules are highly similar in gene structure, chromosomal location, sequence homology, and gene expression. Both molecules are receptors for the costimulatory molecule B7, which is expressed primarily on the surface of activated T cells. Binding of CTLA4 to B7 inhibits activation of mouse and human T cells and plays a negative regulatory role in T cell activation. 【0005】 CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing T cell reactivity to various antigens. In this regard, in vivo and in vitro studies are essentially consistent. Currently, CTLA4 monoclonal antibodies are in clinical trials for or have been approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun., 13:5). 【0006】 Interleukin-2 (IL-2) is produced by T cells. It is a growth factor that regulates T cell subgroups and is an important factor in regulating immune responses. It promotes the proliferation of activated B cells and participates in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for the treatment of malignancies, including melanoma and kidney tumors, and clinical trials for treating chronic viral infections are currently underway (Chavez, AR, et al., 2009, Ann. NY Acad. Sci., 1182:14-27). CTLA4 and CTLA4 antibodies are important influencers of T cell function and interfere with the immune microenvironment within the body. In vitro and in vivo studies have demonstrated that CTLA4 antibodies specifically alleviate CTLA4 immunosuppression, activate T cells, and induce IL-2 production, demonstrating promise for a wide range of gene therapy applications for diseases such as tumors and parasitic infections. 【0007】 CTLA4 antibodies produce specific therapeutic effects on diseases and show remarkable efficacy, and can be used to complement traditional medicines and to explore new avenues of gene therapy. 【0008】 Bispecific antibodies, also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens and can be produced by immunomagnetic separation. Alternatively, they can be obtained by genetic engineering. Genetic engineering offers flexibility in aspects such as optimization of binding sites, synthetic form, and yield, which offers certain advantages. Currently, more than 45 forms have been demonstrated (Dafne Muller, Kontermann R E., 2010, BioDrugs, 24(2):89-98). Many developed bispecific antibodies are in the form of IgG-scFv, i.e., the Morrison format (Coloma MJ, Morrison SL., 1997, Nat Biotechnol., 15:159-163), which has been demonstrated to be one of the ideal formats for bifunctional antibodies due to its similarity to the naturally occurring IgG form and advantages in antibody engineering, expression, and purification (Miller BR, Demarest SJ, et al., 2010, Protein Eng Des Sel, 23:549-57; Fitzgerald J, Lugovskoy A., 2011, MAbs, 3:299-309). 【0009】 Vascular endothelial growth factor (VEGF) is a growth factor that promotes endothelial cell division and proliferation, promotes the formation of new blood vessels, and improves vascular permeability. It acts by binding to vascular endothelial growth factor receptors on the cell surface and activating tyrosine kinase signaling pathways. In tumor tissue, tumor cells and tumor-infiltrating macrophages and mast cells secrete high levels of VEGF, which paracrinely stimulates tumor vascular endothelial cells, promotes endothelial cell proliferation and migration, induces angiogenesis, promotes tumor growth, improves vascular permeability, induces fibrin deposition in surrounding tissues, and promotes the infiltration of mononuclear cells, fibroblasts, and endothelial cells, facilitates the formation of tumor stroma and the invasion of tumor cells into new blood vessels, and promotes tumor metastasis. Therefore, inhibiting tumor angiogenesis is currently considered one of the most promising tumor treatments. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and neuropilin 1 (NRP-1). The first three receptors are members of the tyrosine kinase superfamily and share a similar structure consisting of an extramembrane region, a transmembrane segment, and an intramembrane region, where the extramembrane region is composed of an immunoglobulin-like domain and the intramembrane region is a tyrosine kinase domain. VEGFR1 and VEGFR2 are found primarily on the surface of vascular endothelial cells, and VEGFR3 is found primarily on the surface of lymphatic endothelial cells. 【0010】 Molecules in the VEGF family have different affinities for these receptors. VEGFA mainly acts together with VEGFR1, VEGFR2, and NRP-1. VEGFR1 was the first receptor identified and has a higher affinity for VEGFA than VEGFR2 under normal physiological conditions, but the tyrosinase activity of its intracellular segment is lower than that of VEGFR2 (Ma Li, Chinese Journal of Birth Health and Heredity, 24(5) (2016):146-148). 【0011】 VEGFR2 is a key regulator of angiogenesis and vascular engineering, possessing much higher tyrosine kinase activity than VEGFR1. After binding to its ligand VEGFA, VEGFR2 mediates endothelial cell proliferation, differentiation, and other processes, as well as angiogenesis and vascular permeability (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3) (2007):179-213). After binding to VEGFR2, VEGFA mediates gene transcription and expression of related intracellular proteins through the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway, thereby promoting endothelial cell proliferation (Takahashi T et al., Oncogene, 18(13) (1999):2221-2230). 【0012】 VEGFR3 is a member of the tyrosine kinase family and is mainly expressed in embryonic vascular endothelial cells and mature lymphatic endothelial cells. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot independently transmit biological signals and can only mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor (Ma Li, Chinese Journal of Birth Health and Heredity, 24(5) (2016):146-148). 【0013】 VEGFA and VEGFR2 are mainly involved in regulating angiogenesis before and after VEGFA binds to VEGFR2, forming a cascade of multiple intermediate signals in the upstream and downstream pathways, and finally altering the physiological functions of endothelial cells through proliferation, survival, migration, increased permeability, peripheral tissue invasion, and other patterns (Dong Hongchao et al., Sep. 2014, Journal of Modern Oncology, 22(9): 2231-3). 【0014】 For many cancer patients, the disease remains uncontrollable for a long time after surgery or chemotherapy, and the 5-year survival rate remains very low. In summary, the development of therapeutic approaches or combination dosing regimens with lower toxicity and higher efficacy is of great clinical significance. Summary of the Invention 【0015】 overview The inventors of the present invention have made intensive research and original efforts to combine an anti-CTLA4-anti-PD-1 bispecific antibody with an anti-PD-1-anti-VEGFA bispecific antibody, and surprisingly found that the combined antibody has a pharmacological effect of efficiently suppressing tumor growth, which is superior to that of either the anti-VEGFA-anti-PD-1 bispecific antibody or the anti-CTLA4-anti-PD-1 bispecific antibody alone. The present invention is described in detail below. 【0016】 One aspect of the invention relates to a therapeutic combination comprising an effective amount of a first bispecific antibody and an effective amount of a second bispecific antibody, where: (1) The first bispecific antibody is selected from the group consisting of: a first protein functional region that targets PD-1; and a second protein functional region that targets CTLA4; Includes; wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; where: the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; or the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; (2) the second bispecific antibody is one of the following: a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Includes; wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; where: the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 61 to 63, respectively; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; or The heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 58 to 60, respectively, and the light chain variable region thereof comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 61 to 63, respectively. 【0017】 In some embodiments of the present invention, for the therapeutic combination, in (1), the immunoglobulin is of the human IgG1 subtype; preferably, the heavy chain constant region of the immunoglobulin has the following combination of mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A, It has one of the following. 【0018】 In some embodiments of the present invention, for the therapeutic combination, in (2), the immunoglobulin is of the human IgG1 subtype; preferably, the heavy chain constant region of the immunoglobulin has the following combination of mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A It has one of the following. 【0019】 In some embodiments of the present invention, for the therapeutic combination, in (1), the immunoglobulin is of the human IgG1 subtype; preferably, the heavy chain constant region of the immunoglobulin has the following combination of mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A and In (2), the immunoglobulin is of the human IgG1 subtype; and wherein the heavy chain constant region of the immunoglobulin has the following combination of mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A It has one of the following. 【0020】 In some embodiments of the present invention, for the therapeutic combination, in (1) above: the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:14 and SEQ ID NO:18; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:16 and SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41 and SEQ ID NO:43; the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:44; or The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41 and SEQ ID NO:43; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:44; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:14 and SEQ ID NO:18; and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:16 and SEQ ID NO:20. 【0021】 In some embodiments of the present invention, for the therapeutic combination, the characteristic (1) is selected from any of the following 1) to 20): 1) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 4; 2) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 8; 3) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 12; 4) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 4; 5) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 8; 6) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 12; 7) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:2, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:4; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:16; 8) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:2, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:4; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:20; 9) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:6, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:8; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:16; 10) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:6, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:8; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO:20; 11) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 12; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 16; 12) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 12; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 20; 13) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 41, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 42; 14) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 43, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 44; 15) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 41, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 42; 16) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 43, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 44; 17) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 41, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 42; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 16; 18) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 43, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 44; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 16; 19) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 41, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 42; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 20; and 20) The heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 43, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 44; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 20. 【0022】 In some embodiments of the present invention, for the therapeutic combination, in (1) above: The immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:40 and a light chain having the amino acid sequence set forth in SEQ ID NO:24. 【0023】 In some embodiments of the present invention, for the therapeutic combination, in (1) above: The first bispecific antibody may comprise: a first protein functional region that targets PD-1; and a second protein functional region that targets CTLA4; Includes; the number of said first protein functional regions is one and the number of said second protein functional regions is two; wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; The immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 40 and a light chain having the amino acid sequence set forth in SEQ ID NO: 24; The single chain antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 44; The single chain antibody is linked to the C-terminus or N-terminus of an immunoglobulin heavy chain; the first protein functional region is linked to a second protein functional region via a first linker fragment; the single chain antibody heavy chain variable region is linked to the single chain antibody light chain variable region via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment have amino acid sequences independently selected from SEQ ID NO: 25 and SEQ ID NO: 26, respectively; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:26. 【0024】 In some embodiments of the present invention, for the therapeutic combination, in (2) above: the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:45 and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:47; the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:14 and SEQ ID NO:18 and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:49; or The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 14 and SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 49; the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 45, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 47. 【0025】 In some embodiments of the present invention, the characteristic (2) of the therapeutic combination is selected from any one of the following 1) to 12): 1) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 47; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 16; 2) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 47; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 20; 3) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 47; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 49; 4) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 47; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 16; 5) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 47; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 20; 6) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 47; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 49; 7) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 47; 8) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 47; 9) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 49; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 47; 10) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 16; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 47; 11) the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 47; and 12) The heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 49; the heavy chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region of the single chain antibody has the amino acid sequence set forth in SEQ ID NO: 47. 【0026】 In some embodiments of the present invention, for the therapeutic combination, in (2) above: The immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:54 and a light chain having the amino acid sequence set forth in SEQ ID NO:56. 【0027】 In some embodiments of the present invention, for the therapeutic combination, in (2) above: The second bispecific antibody may comprise: a first protein functional region that targets PD-1; and a second protein functional region that targets VEGFA; Includes; the number of said first protein functional regions is one and the number of said second protein functional regions is two; wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; The immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 54 and a light chain having the amino acid sequence set forth in SEQ ID NO: 56; The single chain antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 49; The single chain antibody is linked to the C-terminus or N-terminus of an immunoglobulin heavy chain; the first protein functional region is linked to a second protein functional region via a first linker fragment; the single chain antibody heavy chain variable region is linked to the single chain antibody light chain variable region via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment have amino acid sequences independently selected from SEQ ID NO: 25 and SEQ ID NO: 26, respectively; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:26. 【0028】 In some embodiments of the present invention, for the therapeutic combination, in (1) and / or (2) above: The first protein functional region is linked, either directly or via a linker fragment, to the second protein functional region; and / or the heavy chain variable region of the single chain antibody is linked, either directly or via a linker fragment, to the light chain variable region of the single chain antibody. 【0029】 In some embodiments of the present invention, for the therapeutic combination, in (1) and / or (2) above: The linker fragment is (GGGGS)n, where n is a positive integer, preferably n is 1, 2, 3, 4, 5 or 6. 【0030】 In some embodiments of the present invention, for the therapeutic combination, in (1) and / or (2) above: The number of first protein functional regions and second protein functional regions can each independently be one, two or more. 【0031】 In some embodiments of the present invention, for the therapeutic combination, in (1) and / or (2) above: Single chain antibodies are linked to the C-terminus or N-terminus of an immunoglobulin heavy chain. 【0032】 In some embodiments of the invention, for a therapeutic combination, the first bispecific antibody and the second bispecific antibody are mixed together and the therapeutic combination is a pharmaceutical composition. 【0033】 In some embodiments of the invention, for a therapeutic combination, the first bispecific antibody and the second bispecific antibody are present in separate packages, and the therapeutic combination is a combination product. 【0034】 In some embodiments of the invention, for therapeutic combinations, the mass ratio of the first bispecific antibody to the second bispecific antibody is (10:1) to (1:5), preferably (5:1) to (1:3) or (3:1) to (1:2), and more preferably 2:1. 【0035】 In some embodiments of the present invention, for a therapeutic combination, the molar ratio of the first bispecific antibody to the second bispecific antibody is (10:1) to (1:10), preferably (5:1) to (1:5), (3:1) to (1:3), or (2:1) to (1:2), and more preferably 1:1. 【0036】 In some embodiments of the invention, for a therapeutic combination, the unit dose of the first bispecific antibody and second bispecific antibody is independently 100 mg to 2500 mg, 100 mg to 2000 mg, 100 mg to 1500 mg, 100 mg to 1200 mg, 100 mg to 1000 mg, 200 mg to 800 mg, 200 mg to 500 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg based on the mass of the first bispecific antibody or second bispecific antibody. 【0037】 In some embodiments of the present invention, the therapeutic combination further comprises an effective amount of an anti-tumor chemotherapeutic agent, such as an alkylating agent, antimetabolite, anti-tumor antibiotic, plant-based anti-cancer agent, hormone, or immunological agent. 【0038】 In some embodiments of the invention, the first bispecific antibody and the second bispecific antibody are active pharmaceutical ingredients (APIs) or active ingredients. In some embodiments of the invention, the therapeutic combination consists of a first bispecific antibody, a second bispecific antibody, and a pharmaceutically acceptable adjuvant. 【0039】 In some embodiments of the present invention, the first bispecific antibody, the second bispecific antibody, and the anti-tumor chemotherapeutic agent are active pharmaceutical ingredients (APIs) or active ingredients. In some embodiments of the present invention, the therapeutic combination consists of a first bispecific antibody, a second bispecific antibody, an anti-tumor chemotherapeutic agent, and a pharmaceutically acceptable adjuvant. In some embodiments of the present invention, the therapeutic combination further comprises one or more pharmaceutically acceptable adjuvants; preferably, further comprises a package insert. 【0040】 Yet another aspect of the invention relates to the use of a therapeutic combination according to any of the aspects of the invention for the preparation of a therapeutic drug for the treatment or prevention of tumors; Preferably, the tumor is selected from one or more of pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical tumor, plasma cell carcinoma, head and neck cancer, brain cancer, throat cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, lung cancer, breast cancer, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is a tumor of the following MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm, The compound is selected from one or more of the following: 【0041】 A therapeutic combination according to any of the aspects of the invention for use in the treatment or prevention of tumors: Preferably, the tumor is selected from one or more of pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical tumor, plasma cell carcinoma, head and neck cancer, brain cancer, throat cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, lung cancer, breast cancer, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is a tumor of the following MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm, The compound is selected from one or more of the following: 【0042】 Yet another aspect of the present invention relates to a method of treating or preventing tumors, comprising administering to a subject in need thereof an effective amount of a therapeutic combination according to any of the aspects of the present invention; wherein: Preferably, the tumor is selected from one or more of pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical tumor, plasma cell carcinoma, head and neck cancer, brain cancer, throat cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, lung cancer, breast cancer, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is a tumor of the following MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell neoplasm, The compound is selected from one or more of the following: 【0043】 Yet another aspect of the present invention is preferably used to treat tumors and comprises 1 to 10,000 mg (preferably 10 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg) of an anti-CTLA4-anti-PD-1 bispecific antibody according to any of the aspects of the present invention and 1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg) of an anti-CTLA4-anti-PD-1 bispecific antibody according to any of the aspects of the present invention. and optionally one or more chemotherapeutic agents according to the present invention (such as a platinum-based drug and / or a fluorouracil antineoplastic agent); wherein the anti-CTLA4-anti-PD-1 bispecific antibody, anti-PD-1-anti-VEGFA bispecific antibody, and the chemotherapeutic agents are packaged separately. 【0044】 The present invention relates to a method for preventing or treating cancer or tumor, wherein the method comprises administering to a subject in need thereof one or more unit dosage forms according to the present invention, preferably wherein the anti-PD-1-anti-VEGFA bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapeutic agent are each administered separately. 【0045】 Yet another aspect of the present invention relates to a single dosage unit that is preferably used to treat a tumor and comprises 0.1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of an anti-CTLA4-anti-PD-1 bispecific antibody according to any of the aspects of the present invention and 0.1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of an anti-PD-1-anti-VEGFA bispecific antibody according to any of the aspects of the present invention. 【0046】 In one or more embodiments of the invention, the anti-CTLA4-anti-PD-1 bispecific antibody, VEGFA-anti-PD-1 bispecific antibody and / or chemotherapeutic agent are preferably present in a liquid form suitable for intravenous injection or infusion. 【0047】 In one or more embodiments of the invention, the step of administering to the subject an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody according to any aspect of the invention and / or an anti-PD-1-anti-VEGFA bispecific antibody according to any aspect of the invention occurs before or after surgical therapy and / or before or after radiation therapy. 【0048】 In one or more embodiments of the invention, the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody according to any of the aspects of the invention and / or the anti-PD-1-anti-VEGFA bispecific antibody according to any of the aspects of the invention is 0.1 to 100 mg per kg of body weight, preferably 1 to 10 mg per kg of body weight; or the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody according to any of the aspects of the invention and / or the anti-PD-1-anti-VEGFA bispecific antibody according to any of the aspects of the invention is 10 to 1000 mg per kg of body weight, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg per kg of body weight for each subject; Preferably, the dose is administered twice daily to about every other day, or once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks; Preferably, the route of administration is intravenous drip infusion, intravenous injection, subcutaneous injection, or intraperitoneal injection. 【0049】 MSI refers to microsatellite instability. Microsatellites are short tandem repeats in the human genome, containing 10 to 50 repeats of one, two, or more nucleotides. Microsatellites in certain abnormal cells, such as tumors, are altered in length by the insertion or deletion of repeat units compared to normal cells. This alteration is also called MSI. Based on the level of instability and its severity, MSI can be classified as microsatellite instability-high (MSI-H), microsatellite instability-low (MSI-L), and microsatellite stable (MSS). The primary cause of MSI is DNA mismatch repair (MMR) deficiency. Human mismatch repair genes (MMR genes) can express the corresponding mismatch repair proteins through transcription and translation. The absence of any MMR protein can lead to mismatch repair deficiency, and base pair mismatches accumulate during the DNA replication process due to such deletions, ultimately resulting in MSI. Approximately 15% of colorectal cancers are thought to be the result of the MSI pathway. This was first reported in colorectal cancer, and can also occur in gastric cancer, endometrial cancer, adrenocortical carcinoma, etc. (Baretti M et al., Pharmacol Ther., 2018; 189:45-62). MSI-H / dMMR signatures have also been found in subsequent studies in mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell neoplasms. 【0050】 MSI-H and dMMR represent two distinct assay results and are biologically consistent and are referred to as MSI-H / dMMR or MSI-high / dMMR, whereas MSI-L and MSS represent pMMR phenotypes. dMMR detection involves immunohistochemical analysis of the protein expression of four mismatch repair genes, MSH2, MLH1, MSH6, and PMS2, in tumor specimens (including surgical and aspirate specimens). Absence of any of the four proteins confirms dMMR; positive results for all four proteins suggest pMMR, i.e., intact mismatch repair function. MSI detection involves matching and comparing the lengths of repetitive DNA sequences (microsatellite sequences) in tumor cells and somatic cells. When five standard loci are detected using PCR based on the National Cancer Institute (NCI) standards, mismatches at two or more loci indicate instability, defined as MSI-H, mismatches at one locus indicate MSI-L, and matched loci indicate MSS. High-throughput sequencing (also known as next-generation sequencing, or NGS) can also be used to detect microsatellite instability. When more microsatellite loci are selected for PCR assays, such as more than five loci or additional microsatellite loci, mismatches at ≥30% of loci are defined as MSI-H, matches at all loci are defined as MSS, and mismatches at 0-30% are defined as MSI-L. 【0051】 In one or more embodiments of the invention, the bispecific antibody is in the form of an IgG-scFv, i.e., Morrison format. In one or more embodiments of the present invention, for bispecific antibodies, the single-chain antibody is linked to the C-terminus of the immunoglobulin heavy chain. Since an immunoglobulin has two heavy chains, two single-chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single-chain antibody molecules are identical. Preferably, the single-chain antibody is linked to the C-terminus of the immunoglobulin heavy chain by forming an amide bond via the linker fragment described above. 【0052】 In one or more embodiments of the present invention, the immunoglobulin constant regions are humanized, for example, the heavy chain constant region is the Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is the Ig kappa chain C region, ACCESSION: P01834. In one or more embodiments of the invention, the bispecific antibody is a monoclonal antibody. In one or more embodiments of the invention, the bispecific antibody is a humanized antibody. 【0053】 Antibody drugs, especially monoclonal antibodies (mAbs), have achieved good efficacy in treating various diseases. Traditional methods for obtaining these therapeutic antibodies include immunizing animals with an antigen to obtain antibodies that target the antigen in the immunized animals, or modifying those antibodies to have lower affinity for the antigen by affinity maturation. 【0054】 The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions, i.e., complementarity-determining regions (CDRs) (heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include LCDR1, LCDR2, and LCDR3; see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda, Md.). The amino acid sequences of the CDRs of the following monoclonal antibodies (1) to (7) were analyzed by technical means well known to those skilled in the art, and the results are as follows: 【0055】 (1) 14C12 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:14, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:16. The three CDRs of the heavy chain variable region have the following amino acid sequences: HCDR1:GFAFSSYD (SEQ ID NO:27) HCDR2: ISGGGRYT (SEQ ID NO: 28) HCDR3: ANRYGEAWFAY (SEQ ID NO: 29) The three CDRs of the light chain variable region have the following amino acid sequences: LCDR1:QDINTY (SEQ ID NO: 30) LCDR2:RAN (SEQ ID NO: 31) LCDR3:LQYDEFPLT (SEQ ID NO: 32) 【0056】 (2) 14C12H1L1 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:18, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:20. The amino acid sequences of the three CDRs of the heavy chain variable region are identical to those of 14C12. The amino acid sequences of the three CDRs of the light chain variable region are identical to those of 14C12. 【0057】 (3) 14C12H1L1(M) The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:18, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:49. The amino acid sequences of the three CDRs of the heavy chain variable region are identical to those of 14C12. The amino acid sequences of the three CDRs of the light chain variable region are identical to those of 14C12. 【0058】 (4) 4G10 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:2, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:4. The three CDRs of the heavy chain variable region have the following amino acid sequences: HCDR1: GYSFTGYT (SEQ ID NO: 33) HCDR2: INPYNNIT (SEQ ID NO: 34) HCDR3: ARLDYRSY (SEQ ID NO: 35) The three CDRs of the light chain variable region have the following amino acid sequences: LCDR1:TGAVTTSNF (SEQ ID NO: 36) LCDR2:GTN (SEQ ID NO: 37) LCDR3:ALWYSNHWV (SEQ ID NO: 38) 【0059】 (5) 4G10H1L1 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:6, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:8. The amino acid sequences of the three CDRs of the heavy chain variable region are identical to those of 4G10. The amino acid sequences of the three CDRs of the light chain variable region are identical to those of 4G10. 【0060】 (6) 4G10H3L3 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:12. The amino acid sequences of the three CDRs of the heavy chain variable region are identical to those of 4G10. The amino acid sequences of the three CDRs of the light chain variable region are identical to those of 4G10. 【0061】 (7) Bevacizumab The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:45, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:47. The three CDRs of the heavy chain variable region have the following amino acid sequences: HCDR1: GYTFTNYG (SEQ ID NO: 58) HCDR2: INTYTGEP (SEQ ID NO: 59) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 60) The three CDRs of the heavy chain variable region have the following amino acid sequences: LCDR1:QDISNY (SEQ ID NO: 61) LCDR2:FTS (SEQ ID NO: 62) LCDR3:QQYSTVPWT (SEQ ID NO: 63) 【0062】 In the present invention, unless otherwise defined, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the experimental procedures of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, definitions and explanations for relevant terms are provided below. 【0063】 As used herein, when referring to the amino acid sequence of CTLA4 protein (cytotoxic T-lymphocyte-associated antigen 4), it includes the full-length CTLA4 protein or the extracellular fragment of CTLA4, the CTLA4 ECD, or a fragment containing the CTLA4 ECD; it also includes fusion proteins of CTLA4 ECD, 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 occur naturally or be artificially introduced into the amino acid sequence of the CTLA4 protein without affecting its biological function. Therefore, in the present invention, the term "CTLA4 protein" should include all such sequences, including natural or artificial variants thereof. In addition, when a sequence fragment of the CTLA4 protein is described, it also includes the corresponding sequence fragment of the natural or artificial variant. 【0064】 As used herein, reference to the amino acid sequence of PD-1 protein (NCBI GenBank: NM_005018) includes the full-length PD-1 protein, the extracellular fragment of PD-1, PD-1ECD, or fragments containing PD-1ECD; and also includes fusion proteins of PD-1ECD, such as fragments fused to 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 occur naturally or be artificially introduced into the amino acid sequence of PD-1 protein without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" should include all such sequences, including natural or artificial variants thereof. In addition, when a sequence fragment of PD-1 protein is described, it also includes the corresponding sequence fragment of such a natural or artificial variant. 【0065】 As used herein, unless otherwise specified, B7 refers to B7-1 and / or B7-2, the specific sequences of which are known in the art and may refer to sequences disclosed in existing literature or GenBank, e.g., B7-1 (CD80, NCBI Gene ID: 941); B7-2 (NCBI Gene ID: CD86, 942). 【0066】 As used herein, reference to the amino acid sequence of VEGFA protein (GenBank ID: NP_001165097.1) includes the full-length VEGFA protein as well as fusion proteins of VEGFA, such as fragments fused to mouse or human IgG Fc protein fragments (mFc or hFc). However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can occur naturally or be artificially introduced into the amino acid sequence of VEGFA protein without affecting its biological function. Therefore, in the present invention, the term "VEGFA protein" should include all such sequences, including natural or artificial variants thereof. In addition, when a sequence fragment of VEGFA protein is described, it also includes the corresponding sequence fragment of a natural or artificial variant thereof. 【0067】 As used herein, when referring to the amino acid sequence of VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), it includes the full-length VEGFR2 protein, the extracellular fragment of VEGFR2, VEGFR2-ECD, or a fragment containing VEGFR2-ECD. It also includes fusion proteins of VEGFR2-ECD, such as fragments fused to 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 occur naturally or be artificially introduced into the amino acid sequence of the VEGFR2 protein without affecting its biological function. Therefore, in the present invention, the term "VEGFR2 protein" should include all such sequences, including natural or artificial variants thereof. In addition, when a sequence fragment of a VEGFR2 protein is described, it also includes the corresponding sequence fragment of a natural or artificial variant thereof. 【0068】 As used herein, unless otherwise specified, VEGFR refers to VEGFR1 and / or VEGFR2, the specific sequences of which are known in the art and may refer to sequences disclosed in existing literature or GenBank, for example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI Gene ID: 3791). 【0069】 As used herein, EC 50 The term refers to the concentration for 50% of the maximal effect, ie the concentration capable of producing 50% of the maximal effect. 【0070】 As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. In a general sense, a heavy chain can be understood as the polypeptide chain having a larger molecular weight in an antibody, and a light chain refers to the polypeptide chain having a smaller molecular weight in an antibody. Light chains are classified as kappa and lambda light chains. Heavy chains are generally classified as μ, δ, γ, α, and ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L The light chain constant region consists of one domain, C L The antibody constant region mediates the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. H Area and V L The region can be further subdivided into hypervariable regions (called complementarity determining regions, or CDRs) dispersed between conserved regions called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L) form the antibody binding site. The amino acid assignment for each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD. (1987 and 1991)), Chothia & Lesk, (1987) J. Mol. Biol., 196:901-917, or Chothia et al. (1989) Nature, 342:878-883. In particular, the heavy chain may further comprise 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 an scFv having the C-terminus of the heavy chain of an IgG antibody linked to another antibody, and in this case, the heavy chain comprises nine CDRs. The term "antibody" is not intended to be limited by any particular method for producing the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies can be of different isotypes, such as IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM. 【0071】 Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., DNA recombination, or enzymatic or chemical cleavage), and the antigen-binding fragments can be screened for specificity in the same way as intact antibodies. 【0072】 As used herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to it includes not only complete antibodies but also antigen-binding fragments of antibodies. 【0073】 As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody fragment derived from a population of highly homologous antibodies, i.e., derived from a population of identical antibody molecules, excluding natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies, in contrast to a monoclonal antibody, generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology, first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using DNA recombination (e.g., U.S. Patent No. 4,816,567). 【0074】 As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDRs of a human immunoglobulin (acceptor antibody) are replaced by the CDRs of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework regions (FRs) of the acceptor antibody can also be replaced by corresponding amino acid residues of a non-human antibody or by amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further details regarding humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000). 【0075】 As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant." Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules, such as amino acids, carbohydrates, or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope generally includes at least 34, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" or "conformational." See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). 【0076】 In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are located linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites are located across amino acid residues of the protein that are separated from each other. 【0077】 As used herein, the term "isolated" refers to being obtained by artificial means from a natural state. A particular "isolated" substance or component may occur naturally either in its natural environment or it may be isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide may occur naturally in a particular living animal, and a highly purified version of the same polynucleotide or polypeptide isolated from such a natural environment would be referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance. 【0078】 As used herein, the term "E. coli expression system" refers to an expression system consisting of an E. coli (strain) and a vector, where the E. coli (strain) is derived from commercially available strains such as, but not limited to, GI698, ER2566, BL21(DE3), B834(DE3), and BLR(DE3). 【0079】 As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (such as SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may further contain a replication origin. 【0080】 As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or an Aspergillus cell, an insect cell such as an S2 Drosophila cell or an Sf9 cell, or an animal cell such as a fibroblast, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell. 【0081】 As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen that it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody binds to an antigen within, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less, about 10 -5 Affinity lower than M (K D ) means to bind to an antigen. In some embodiments of the present invention, the term "targeting" refers to specific binding. 【0082】 As used herein, "K D The term "antibody-antigen binding affinity" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the stronger the antibody-antigen binding and indicates a higher affinity between the antibody and the antigen. Generally, antibodies bind to antigens at a dissociation equilibrium constant of about 10, as measured, for example, by a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio Octet system. -5 Lower than M, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10The dissociation equilibrium constant (K D ) binds to the antigen. 【0083】 As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably; and the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Moreover, as used herein, amino acids are generally represented by the one-letter and three-letter abbreviations known in the art. For example, alanine is represented by A or Ala. 【0084】 As used herein, the term "pharmaceutically acceptable adjuvant" or "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. Such carriers and / or excipients are well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th Ed., Pennsylvania, Mack Publishing Company, 1995) and include, but are 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 sodium chloride. 【0085】 As used herein, the term "adjuvant" refers to a nonspecific immune-enhancing agent that, when delivered together with or prior to an antigen, enhances or alters the immune response of the organism to the antigen. Various adjuvants exist, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, and cytokines. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more frequently used in clinical trials. 【0086】 As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactically effective amount (e.g., for a disease associated with CTLA4 binding to B7 or CTLA4 overactivity, e.g., a tumor) is an amount sufficient to prevent, terminate, or delay the onset of the disease (e.g., a disease associated with CTLA4 binding to B7 or CTLA4 overactivity, e.g., a tumor); a therapeutically effective amount is an amount sufficient to cure or at least partially terminate the disease and its complications in a patient suffering from the disease. Determining such an effective amount is undoubtedly within the capabilities of one skilled in the art. For example, an amount effective for therapeutic purposes depends on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the route of administration, and other concurrently administered treatments. 【0087】 A "recurrent" cancer is one that recurs in the original or distant site after responding to prior treatment (e.g., surgery). A "locally recurrent" cancer is one that occurs after treatment in the same site as the previously treated cancer. 【0088】 "Metastatic" cancer refers to cancer that spreads from one part of the body (eg, the lungs) to another. 【0089】 As used herein, the term "completely eliminated" refers to the absence of a binding signal or a very weak binding signal detected by existing instrumentation (e.g., a Fortebio Octet system). In one embodiment of the present invention, the absence of a binding signal or a very weak binding signal refers to a binding signal (i.e., response) of less than 0.1 nm. 【0090】 In the present invention, the terms "first" (e.g., first bispecific antibody, first protein functional region, or first linker fragment) and "second" (e.g., second bispecific antibody, second protein functional region, or second linker fragment) are used to distinguish or for clarity of presentation and do not have a typical sequential meaning, unless otherwise specified. 【0091】 In the present invention, "about" or "approximately" means that the defined value or physical quantity varies within a range of 10%, 20%, or 30%, unless otherwise specified. For example, it may be about 100 minutes or approximately 100 minutes, 90 to 110 minutes, 80 to 120 minutes, or 70 to 130 minutes. 【0092】 The earlier applications, Chinese Patent Publication Nos. CN112300286A and CN112830972A, are hereby incorporated by reference in their entirety. 【0093】 Beneficial Effects of the Invention The present invention achieves one or more of the following technical effects (1) to (2): (1) The therapeutic combination of the present invention effectively prevents or treats tumors. (2) The combination of the anti-PD-1-anti-VEGFA bispecific antibody and the anti-CTLA4-anti-PD-1 bispecific antibody has a pharmacological effect of effectively inhibiting tumor growth (better than that of the anti-VEGFA-anti-PD-1 bispecific antibody or the anti-CTLA4-anti-PD-1 bispecific antibody alone), thereby achieving a synergistic technical effect. [Brief explanation of the drawings] 【0094】 【Figure 1】 FIG. 1 shows the effect of anti-CTLA4-anti-PD-1 bispecific antibody in combination with anti-PD-1-anti-VEGFA bispecific antibody on cytokine IL-2 secretion induced by stimulated PBMC and Raji-PDL1 cell cocultures. 【Figure 2】 FIG. 2 shows the detection of the promoting effect of anti-CTLA4-anti-PD-1 bispecific antibody in combination with anti-PD-1-anti-VEGFA bispecific antibody on the secretion of the cytokine IFN-γ by mixed lymphocyte reaction. DETAILED DESCRIPTION OF THE INVENTION 【0095】 Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be understood as limiting the scope of the present invention. If no unique techniques or conditions are specified, the examples will be carried out according to the techniques or conditions described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the product manual. If the manufacturer of the reagents or equipment used is not specified, they are conventional products available on the market. 【0096】 In the following examples relating to the present invention: Human peripheral blood mononuclear cells were isolated and prepared at Akeso Biopharma, Inc. with the donor's informed consent. Raji-PDL1 is a human PD-L1-expressing cell line constructed by Akeso Biopharma, Inc. via transfection based on the human B cell line Raji. 【0097】 Ficoll-Paque™ PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare, Cat. No. 17-1440-02. Human IL-2 ELISA kit was purchased from Dakewe Biotech Co., Ltd., Cat. No. 1110202. RPMI 1640 medium, DMEM medium, trypsin-EDTA (0.25%) phenol red, and blastidin were all purchased from Gibco. 【0098】 Staphylococcus aureus enterotoxin B (SEB) was purchased from Dianotech, Cat. No. BT202. FBS was purchased from Excell bio, Cat. No. FSP500. Mitomycin C (MMC) was purchased from Stressmarq, Cat. No. SIH-246-10MG, Batch No. SM286474. 【0099】 The sequence of the isotype control, human anti-hen egg lysozyme IgG (anti-HEL antibody, or human IgG abbreviated as hIgG), was derived from the variable region sequence of Fab F10.6.6 in a study by Acierno et al. entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol Biol., 2007; 374(1):130-146). The hIgG1DM used in the examples is an isotype control antibody prepared by Akeso Biopharma, Inc., using anti-HEL with the hG1DM constant region sequence, Batch No. 20190520. 【0100】 Preparation Example 1: Sequence design of anti-CTLA4 antibody The amino acid sequences and coding nucleotide sequences of the heavy and light chains of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to those of 4G10, 4G10H1L1, and 4G10H3L3, respectively, in Chinese Patent Publication No. CN106967172A. (1) 4G10 heavy and light chain variable region sequences Nucleotide sequence of the heavy chain variable region: (372 bp) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAG AAGTTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (Sequence number 1) The amino acid sequence it encodes: (124 aa) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO: 2) 【0101】 Nucleotide sequence of the light chain variable region: (378bp) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGA TTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC (Sequence number 3) The amino acid sequence it encodes: (126 aa) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO: 4) 【0102】 (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 4G10H1L1 Nucleotide sequence of heavy chain variable region (4G10H1V): (345bp) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCA CCAACTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT (Sequence number 5) The amino acid sequence it encodes: (115 aa) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 6) 【0103】 Nucleotide sequence of the light chain variable region (4G10L1V): (327 bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACA ATCGGGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG (Sequence number 7) The amino acid sequence it encodes: (109 aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 8) 【0104】 (3) Heavy and light chain variable region sequences of humanized monoclonal antibody 4G10H3L3 Nucleotide sequence of heavy chain variable region (4G10H3V): (345 bp) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCA CTAACTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA (Sequence number 9) The amino acid sequence it encodes (4G10H3V): (115aa) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 10) 【0105】 Nucleotide sequence of the light chain variable region (4G10L3V): (327 bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACA ACAAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (Sequence number 11) Encoded amino acid sequence (4G10L3V): (109aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 12) 【0106】 Preparation Example 2: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 The amino acid sequences and coding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication No. CN106967172A. 【0107】 (1) Heavy and light chain variable region sequences of 14C12 Nucleotide sequence of the heavy chain variable region: (354 bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTAC TATCCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (Sequence number 13) The amino acid sequence it encodes: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 14) 【0108】 Nucleotide sequence of the light chain variable region: (321 bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGAC TGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (Sequence number 15) The amino acid sequence it encodes: (107 aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 16) 【0109】 (2) Heavy and light chain variable regions and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 Nucleotide sequence of the heavy chain variable region: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTAC TATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (Sequence number 17) The amino acid sequence it encodes: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 18) 【0110】 Nucleotide sequence of the light chain variable region: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGAC TGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (Sequence number 19) The amino acid sequence it encodes: (107 aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 20) 【0111】 DNA sequence of the heavy chain (14C12H1) of 14C12H1L1: (1344 bp) The amino acid sequence it encodes: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) 【0112】 DNA sequence of the light chain of 14C12H1L1 (14C12L1): (642 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 23) The amino acid sequence encoded thereby: (214 aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24) 【0113】 Preparation Example 3: Sequence design of bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) The structural pattern of the bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) is the Morrison format (IgG-scFv), i.e., the C-terminus of the two heavy chains of one IgG antibody is separately linked to the scFv fragment of another antibody via a linker fragment. The components for the heavy and light chain design are shown in Table A below. 【0114】 Table A: Sequence design of BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) [Table 1] 【0115】 In Table A above: The amino acid sequence of linker 1 is (GGGGS)3 (SEQ ID NO: 25). The amino acid sequence of linker 2 is (GGGGS)4 (SEQ ID NO: 26). 【0116】 In Table A above, the scFv fragments 4G10H1V(M), 4G10L1V(M), 4G10H3V(M) and 4G10L3V(M) of the BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) antibodies contain mutations in individual amino acids of the framework regions based on 4G10H1V, 4G10L1V, 4G10H3V and 4G10L3V, respectively, which effectively optimized the structure of the antibodies and improved their efficacy. 【0117】 (1) 4G10H1V(M): (115 aa, the positions of mutations in the amino acid sequence based on 4G10H1V are underlined) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ CLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 41) (2) 4G10L1V(M): (110 aa, the positions of mutations in the amino acid sequence based on 4G10L1V are underlined) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 42) (3) 4G10H3V(M): (115 aa, the positions of mutations in the amino acid sequence based on 4G10H3V are underlined) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ C LEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 43) (4) 4G10L3V(M): (110 aa, the positions of mutations in the amino acid sequence based on 4G10L3V are underlined) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 44) 【0118】 To distinguish it from mutated antibodies, BiAb004(M) will hereafter be referred to as BiAb004(hG1WT) in the examples of the present invention. The previously described BiAb004(M) is a "wild-type" antibody that contains the Ig γ-1 chain C region (ACCESSION: P01857) as the heavy chain constant region and the Ig κ chain C region (ACCESSION: P01834) as the light chain constant region. 【0119】 Preparation Example 4: Design of non-variable region amino acid mutations based on humanized bifunctional antibody BiAb004 Based on BiAb004(hG1WT) obtained in Preparation Example 3, a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) were introduced into the heavy chain to obtain BiAb004(hG1TM). 【0120】 DNA sequence of the heavy chain of the immunoglobulin portion in BiAb004(hG1™): (1344 bp, positions of mutations are underlined) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGA GCCCCTACGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCA AGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAA CCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCC CTCCTGTCCTCGACTCCGACGGCTCCTTCTTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA (Sequence number 39) 【0121】 Amino acid sequence of the heavy chain of the immunoglobulin portion in BiAb004(hG1™): (448 aa, mutation positions are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40) 【0122】 BiAb004(hG1TM) and BiAb004(hG1WT) share the same DNA sequence and the same encoded amino acid sequence for the light chain. The specific sequence is shown in Preparation Example 3. 【0123】 Preparation Example 5: Preparation of anti-VEGFA antibody bevacizumab The amino acid sequences of the heavy and light chain variable regions of the commercially available anti-VEGFA monoclonal antibody Avastin (bevacizumab) are described in Chinese Patent Publication No. CN1259962A. The synthesis of the nucleotide sequences encoding the heavy and light chain variable regions was commissioned to Genscript. 【0124】 Amino acid sequence of bevacizumab heavy chain variable region (bevacizumab-Hv): (123aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 45) Nucleotide sequence encoding the bevacizumab heavy chain variable region: (369 bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAG CCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC (Sequence number 46) 【0125】 Amino acid sequence of bevacizumab light chain variable region (bevacizumab-Lv): (107aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 47) Nucleotide sequence encoding the bevacizumab light chain variable region: (321 bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCC TGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG (Sequence number 48) 【0126】 All heavy chain constant regions were Ig γ-1 chain C region, ACCESSION: P01857; all light chain constant regions were Ig κ chain C region, ACCESSION: P01834. 【0127】 The heavy and light chain cDNAs of bevacizumab were cloned into the vector pcDNA3.1 to obtain recombinant expression plasmids for the antibody bevacizumab. The recombinant plasmids were used to transfect 293F cells. The 293F cell cultures were purified and then detected. This resulted in the anti-VEGFA monoclonal antibody Avastin (bevacizumab). 【0128】 Preparation Example 6: Sequence design of mutant 14C12H1L1(M) of anti-PD-1 humanized antibody 14C12H1L1 14C12H1L1(M) was obtained by mutation of amino acids within the framework region (light chain) of 14C12H1L1. Heavy chain variable region 14C12H1L1(M): Its sequence is identical to that of the heavy chain variable region 14C12H1 of 14C12H1L1, i.e., the amino acid sequence is set forth in SEQ ID NO:18. 【0129】 Light chain variable region 14C12L1(M): (108 aa; mutation positions are underlined in the amino acid sequence based on 14C12H1L1) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK R (SEQ ID NO: 49) 【0130】 Preparation Example 7: Sequence design of bispecific antibodies 1. Sequence design The structure of the bispecific antibody described herein is the Morrison type (IgG-scFv), i.e., the C-terminus of each of the two heavy chains of an IgG antibody is linked to the scFv fragment of another antibody, and the main structural designs of the heavy and light chains are as shown in Table B below. 【0131】 Based on the previously described bevacizumab, the VP101 antibody contains the amino acid sequences of the heavy and light chain variable regions of 14C12H1L1(M) as an scFv fragment, and is therefore also referred to as VP101(M). Compared to 14C12H1L1, 14C12H1L1(M) demonstrated an effective optimization of the bispecific antibody structure and improved efficacy. 【0132】 Table B: Heavy and light chain design of VP101(M) and VP101(G4M) [Table 2] 【0133】 In Table B above: (1) The "V" label in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Where there is no "V" label, the corresponding heavy or light chain is full-length, including the constant region. The corresponding sequences in the preparation examples above refer to the amino acid sequences of these variable regions or full-length sequences and the nucleotide sequences encoding them. 【0134】 (2) The amino acid sequence of linker 2 is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 26). Optionally, the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 25) may be used as Linker 1 instead of Linker 2 described above. (3) Bevacizumab-H contains the Igγ-1 chain C region (ACCESSION: P01857) as the heavy chain constant region. (4) Bevacizumab-G4H contains the Igγ-4 chain C region (ACCESSION: P01861.1) as the heavy chain constant region. 【0135】 2. Expression and purification of antibody VP101(M) The heavy and light chain cDNA sequences of VP101(M) were cloned into the vector pUC57simple (provided by Genscript) to obtain the plasmids pUC57simple-VP101H and pUC57simple-VP101L, respectively. Plasmids pUC57simple-VP101H and pUC57simple-VP101L were digested (HindIII and EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned separately into the pcDNA3.1 vector. The recombinant plasmids were then extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, and the supernatant was concentrated and applied to a HiTrap MabSelect SuRe column. The protein was further eluted in one step with elution buffer, and the target sample antibody VP101 was isolated and transferred to PBS by buffer exchange. 【0136】 3. Detection of antibody VP101(M) The purified samples were added to both reducing and non-reducing protein electrophoresis loading buffer, boiled, and subjected to SDS-PAGE electrophoresis analysis. 【0137】 VP101(M) is also referred to as VP101(hG1WT) in the present invention to distinguish it from the mutant antibody of Preparation Example 8. The previously described VP101(M) is a "wild-type" antibody and contains the Ig γ-1 chain C region (ACCESSION: P01857) as the heavy chain constant region and the Ig κ chain C region (ACCESSION: P01834) as the light chain constant region. 【0138】 Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG1WT): (453 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50) 【0139】 Nucleotide sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG1WT): (1359 bp) 【0140】 VP101(G4M) is also referred to as VP101(hG4WT) in the present invention to distinguish it from the mutant antibody of Preparation Example 8. The previously described VP101(G4M) is a "wild-type" antibody and contains the Ig γ-4 chain C region (ACCESSION: P01861.1) as the heavy chain constant region and the Ig κ chain C region (ACCESSION: P01834) as the light chain constant region. 【0141】 Amino acid sequence of the heavy chain of the immunoglobulin portion of VP101 (hG4WT): (450 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 52) 【0142】 Nucleotide sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG4WT): (1350 bp) 【0143】 Preparation Example 8: Non-variable region amino acid mutation design based on humanized bispecific antibody VP101 (hG1WT) Based on VP101(hG1WT) obtained in Preparation Example 7, VP101(hG1DM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A) in the heavy chain. 【0144】 Amino acid sequence of the heavy chain of the immunoglobulin moiety in VP101 (hG1DM): (453 aa, positions of mutations are underlined) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54) 【0145】 Nucleotide sequence encoding the heavy chain of the immunoglobulin portion in VP101 (hG1DM): (1359 bp, positions of mutations underlined) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGCGCAAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCTGGAGGCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAA ACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGC CAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACC CCTCCTGTCCTCGACTCCGACGGCTCCTTCTTTCCTGTACAGCAAACTGACCGTCGATAAATCTAGGTGGCAGCAGGGCAACGTGTTCTCTTGTTCCGTGATGCATGAAGCACTGCACAACCATTATACCCAGAAGTCTCTGAGCCTGTCCCCCGGCAAG (Sequence number 55) 【0146】 The amino acid sequences of the light chains of the immunoglobulin portions of VP101(hG1DM), VP101(hG1WT) and VP101(hG4WT) are identical, and the encoding nucleotide sequences are also identical. 【0147】 Amino acid sequence of the light chain of the immunoglobulin moiety in VP101 (hG1DM): (214 aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56) Nucleotide sequence encoding the light chain of the immunoglobulin portion in VP101 (hG1DM): (642 bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAGCGGACCGTGGCCGCTCCCAGTGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAATCCGGGACAGCCTCTGTGGTCTGTCTGCTGAACAACTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGAGTGGCAATTCACAGGAGAGCGTGACAGAACAGGACTCCAAAGATTCTACTTATAGTCTGTCAAGCACACTGACTCTGAGCAAGGCTGACTACGAAAAGCATAAAGTGTATGCATGTGAGGTCACCCACCAGGGGCTGAGCAGTCCAGTCACCAAGTCATTCAACAGAGGCGAGTGC (SEQ ID NO: 57) 【Example】 【0148】 Example 1: Detection of the promoting effect of an anti-CTLA4-anti-PD-1 bispecific antibody in combination with an anti-PD-1-anti-VEGFA bispecific antibody on the secretion of cytokine IL-2 by mixed lymphocyte reaction Two days before the experiment, PBMCs (from healthy donors) were thawed and cultured in complete medium at 37°C in a 5% carbon dioxide incubator. After 2 hours, when the PBMCs returned to normal, SEB (final concentration: 0.5 μg / mL) was added for 2 days of stimulation. On the day of the experiment, Raji-PDL1 cells were collected, centrifuged, resuspended (in 1640 + 10% FBS), counted, and adjusted to a density of 200 W / mL. MMC (mitomycin C; final concentration: 2 μg / mL) was added, and the mixture was treated at 37°C for 1 hour in a 5% carbon dioxide incubator. PBMCs after 2 days of SEB stimulation and Raji-PDL1 cells after MMC treatment were collected and washed twice with 1640 basal medium. The cells were resuspended in assay medium and counted. The cells were then used to treat PBMCs at 100,000 / well and Raji-PDL1 cells at 100,000 / well. The cells were inoculated into a 96-well U-shaped plate (Corning, Model: 3799) at 100,000 cells / well (40 μL / well each); drugs were added according to the experimental design, blank controls (Raji cell group alone and PBMC group alone) and negative controls (Raji cell + PBMC group and Raji + PBMC + VEGF group) were set up, and the cells were cultured for 3 days (final volume of the system was 200 μL); the final concentrations of the first bispecific antibody and the second bispecific antibody when combined were 3, 30, or 300 nM; and VEGF-his (Akeso Biopharma, Inc., Batch No. 20180126-1) was added to a final concentration of 500 ng / mL when VEGF was added (VEGF in the experiment was VEGFA). After 3 days, the cells were centrifuged at 250 xg for 5 minutes (Beckman centrifuge), and the cell supernatant was collected and the IL-2 content was detected using the Dakewe kit. 【0149】 The results are shown in Figure 1. The results indicate that in VEGF-containing and VEGF-free mixed culture systems, the anti-CTLA4-anti-PD-1 bispecific antibody BiAb004 (hG1™) and the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM), alone or in combination, could significantly induce PBMCs to secrete IL-2, and the effect of the combination was superior to that of the bispecific antibody alone at the same dose when the final antibody concentrations were the same. 【0150】 Example 2: Detection of the stimulatory effect of anti-CTLA4-anti-PD-1 bispecific antibody in combination with anti-PD-1-anti-VEGFA bispecific antibody on cytokine secretion by mixed lymphocyte reaction Normal human PBMCs were obtained by isolation according to the manufacturer's instructions using Ficoll-Paque™ Plus (GE, Cat. No. 17-1440-02). One day before the experiment, PBMCs were thawed and cultured in complete medium at 37°C in a 5% carbon dioxide incubator; on the day of the experiment, PBMCs were collected, centrifuged, resuspended, counted, adjusted for cell density, and diluted to 1 x 10 5 96-well U-shaped plates (Corning, 3799) were inoculated according to the experimental design, in which VEGF (Akeso Biopharma, Inc., Batch No. 20210508) was incubated with the antibody for 30 minutes at room temperature (VEGF in the experiment was VEGFA). At the same time, a negative control (PBMC + SEB + A549 cells + VEGF group, i.e., no antibody group) and an isotype control (PBMC + SEB + A549 cells + VEGF + hIgG1DM group, i.e., hIgG1DM group in the figure) were set up and incubated with PBMC for 30 minutes at 37°C in a 5% carbon dioxide incubator. A549 lung cancer cells (purchased from Chinese Academy of Sciences, Cat. No. SCSP-503) were routinely collected, centrifuged, resuspended, counted, adjusted for cell density, and then cultured at 1 × 10 4The cells were inoculated into a 96-well U-shaped plate according to the above procedure, and SEB (final concentration: 0.1 μg / mL) (Toxin Technology, Cat. No. BT202) was added at the same time, followed by co-culture for 3 days (final system volume: 200 μL). After 3 days, the cells were centrifuged at 250 × g for 5 minutes (Beckman centrifuge), and the cell supernatant was collected. The IFN-γ content was detected using a Dakewe kit (Dakewe, Cat. No. 1110002). 【0151】 The results are shown in Figure 2. Compared with isotype control antibodies, the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) and the anti-CTLA4-anti-PD-1 bispecific antibody BiAb004 (hG1™) effectively promoted the secretion of the cytokine IFN-γ in a mixed lymphocyte system, and the combination of BiAb004 (hG1™) + VP101 (hG1DM) showed mixed anti-tumor efficacy. The combination group was superior to the monotherapy groups at the same dose in promoting the secretion of the cytokine IFN-γ, and the combined activity of 150 nM BiAb004 (hG1™) + VP101 (hG1DM) was superior to that of 300 nM VP101 (hG1DM) monotherapy. 【0152】 Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to these details according to all the teachings disclosed, and all of these changes are within the scope of protection of the present invention. The full scope of the present invention is provided by the appended claims and any equivalents thereof.

Claims

[Claim 1] A therapeutic combination for use in the treatment of tumors, comprising an effective amount of anti-PD-1 / anti-CTLA4 bispecific antibody and an effective amount of anti-PD-1 / anti-VEGFA bispecific antibody, Here: (1) The anti-PD-1 / anti-CTLA4 bispecific antibody is as follows: (i) A first protein functional region that targets PD-1 and includes a first heavy chain variable region comprising HCDR1 to HCDR3 containing amino acid sequences defined in SEQ ID NOs. 27 to 29, and a first light chain variable region comprising LCDR1 to LCDR3 containing amino acid sequences defined in SEQ ID NOs. 30 to 32, and (ii) A second protein functional region that targets CTLA4 and includes a second heavy chain variable region containing HCDR1 to HCDR3 containing amino acid sequences defined in SEQ ID NOs. 33 to 35, and a second light chain variable region containing LCDR1 to LCDR3 containing amino acid sequences defined in SEQ ID NOs. 36 to 38, Including; Here, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin; and (2) The anti-PD-1 / anti-VEGFA bispecific antibody is as follows: (iii) A third protein functional region that targets VEGFA and includes a third heavy chain variable region containing HCDR1 to HCDR3 containing amino acid sequences defined in SEQ ID NOs. 58 to 60, and a third light chain variable region containing LCDR1 to LCDR3 containing amino acid sequences defined in SEQ ID NOs. 61 to 63, and (iv) A fourth protein functional region that targets PD-1 and includes a fourth heavy chain variable region containing HCDR1 to HCDR3, each containing the amino acid sequences defined in SEQ ID NOs. 27 to 29, and a fourth light chain variable region containing LCDR1 to LCDR3, each containing the amino acid sequences defined in SEQ ID NOs. 30 to 32, Including; Here, the third protein functional region is an immunoglobulin and the fourth protein functional region is a single-chain antibody; or the third protein functional region is a single-chain antibody and the fourth protein functional region is an immunoglobulin. The aforementioned therapeutic combination. [Claim 2] The therapeutic combination for use according to Claim 1, wherein each immunoglobulin is an IgG1 isotype; and comprises a heavy chain constant region comprising one of the following combinations of mutations according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A. [Claim 3] In (1) above: (i) The amino acid sequence of the first heavy chain variable region is selected from SEQ ID NO: 14 and SEQ ID NO: 18, and the amino acid sequence of the first light chain variable region is selected from SEQ ID NO: 16 and SEQ ID NO: 20; and (ii) The amino acid sequence of the second heavy chain variable region is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41 and SEQ ID NO: 43, and the amino acid sequence of the second light chain variable region is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 and SEQ ID NO:

44. A therapeutic combination for use as described in claim 1. [Claim 4] The therapeutic combination for use according to Claim 1, wherein the first and second heavy chain and light chain variable regions are selected from any of the following 1) to 10): 1) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 2, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 4; 2) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 6, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 8; 3) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 10, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 12; 4) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 2, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 4; 5) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 6, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 8; 6) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 10, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 12; 7) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 41, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 42; 8) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 43, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 44; 9) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 41, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO: 42; 10) The first heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the first light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; the second heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 43, and the second light chain variable region includes the amino acid sequence specified in SEQ ID NO:

44. [Claim 5] The first protein functional region of the anti-PD-1 / anti-CTLA4 bispecific antibody is an immunoglobulin, and the second protein functional region of the anti-PD-1 / anti-CTLA4 bispecific antibody is a single-chain antibody. The therapeutic combination for use according to claim 1, wherein the immunoglobulin comprises a heavy chain having the amino acid sequence defined in SEQ ID NO: 40 and a light chain having the amino acid sequence defined in SEQ ID NO:

24. [Claim 6] The first protein functional region of the anti-PD-1 / anti-CTLA4 bispecific antibody is an immunoglobulin, and the second protein functional region of the anti-PD-1 / anti-CTLA4 bispecific antibody is a single-chain antibody; The immunoglobulin comprises a heavy chain having the amino acid sequence specified in SEQ ID NO: 40 and a light chain having the amino acid sequence specified in SEQ ID NO: 24; The single-chain antibody comprises a heavy chain variable region having the amino acid sequence specified in SEQ ID NO: 43, and a light chain variable region having the amino acid sequence specified in SEQ ID NO: 44; The single-chain antibody is linked to the C-terminus or N-terminus of the heavy chain of immunoglobulin via a first linker; and The heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody via a second linker. A therapeutic combination for use as described in claim 1. [Claim 7] The therapeutic combination for use according to claim 6, wherein the first linker and the second linker each comprise an amino acid sequence independently selected from SEQ ID NO: 25 and SEQ ID NO:

26. [Claim 8] The therapeutic combination for use according to claim 6, wherein the amino acid sequences of the first linker and the second linker comprise SEQ ID NO:

26. [Claim 9] (i) The amino acid sequence of the third heavy chain variable region is defined in SEQ ID NO: 45, and the amino acid sequence of the third light chain variable region is defined in SEQ ID NO: 47; and (ii) The amino acid sequence of the fourth heavy chain variable region is selected from SEQ ID NO: 14 and SEQ ID NO: 18, and the amino acid sequence of the fourth light chain variable region is selected from SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO:

49. A therapeutic combination for use as described in claim 1. [Claim 10] The therapeutic combination for use according to claim 1, wherein the third and fourth heavy chain and light chain variable regions are selected from any of the following 1) to 6): 1) The third heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 45, and the third light chain variable region includes the amino acid sequence specified in SEQ ID NO: 47; the fourth heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the fourth light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; 2) The third heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 45, and the third light chain variable region includes the amino acid sequence specified in SEQ ID NO: 47; the fourth heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the fourth light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; 3) The third heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 45, and the third light chain variable region includes the amino acid sequence specified in SEQ ID NO: 47; the fourth heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 14, and the fourth light chain variable region includes the amino acid sequence specified in SEQ ID NO: 49; 4) The third heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 45, and the third light chain variable region includes the amino acid sequence specified in SEQ ID NO: 47; the fourth heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the fourth light chain variable region includes the amino acid sequence specified in SEQ ID NO: 16; 5) The third heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 45, and the third light chain variable region includes the amino acid sequence specified in SEQ ID NO: 47; the fourth heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the fourth light chain variable region includes the amino acid sequence specified in SEQ ID NO: 20; 6) The third heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 45, and the third light chain variable region includes the amino acid sequence specified in SEQ ID NO: 47; the fourth heavy chain variable region includes the amino acid sequence specified in SEQ ID NO: 18, and the fourth light chain variable region includes the amino acid sequence specified in SEQ ID NO:

49. [Claim 11] The therapeutic combination for use according to Claim 1, wherein the third protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is an immunoglobulin, and the fourth protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is a single-chain antibody, wherein the immunoglobulin comprises a heavy chain containing the amino acid sequence defined in SEQ ID NO: 54 and a light chain containing the amino acid sequence defined in SEQ ID NO:

56. [Claim 12] The fourth protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is a single-chain antibody, and the third protein functional region of the anti-PD-1 / anti-VEGFA bispecific antibody is an immunoglobulin; Here, the immunoglobulin comprises a heavy chain containing the amino acid sequence specified in SEQ ID NO: 54 and a light chain containing the amino acid sequence specified in SEQ ID NO: 56; Here, the single-chain antibody comprises a heavy chain variable region containing the amino acid sequence specified in SEQ ID NO: 18, and a light chain variable region containing the amino acid sequence specified in SEQ ID NO: 49; Here, the single-chain antibody is linked to the C-terminus or N-terminus of the heavy chain of immunoglobulin via a first linker; and, Here, the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody via a second linker. A therapeutic combination for use as described in claim 1. [Claim 13] The therapeutic combination for use according to claim 12, wherein the first linker and the second linker each comprise an amino acid sequence independently selected from SEQ ID NO: 25 and SEQ ID NO:

26. [Claim 14] The therapeutic combination for use according to claim 12, wherein the amino acid sequences of the first linker and the second linker comprise SEQ ID NO:

26. [Claim 15] The therapeutic combination for use according to claim 1, wherein a first protein functional region is linked to a second protein functional region directly or via a linker, and a third protein functional region is linked to a fourth protein functional region directly or via a linker. [Claim 16] The therapeutic combination for use according to claim 15, wherein the linker fragment is (GGGGS)n {wherein n is 1, 2, 3, 4, 5, or 6}. [Claim 17] The therapeutic combination for use according to claim 1, wherein an anti-PD-1 / anti-CTLA4 bispecific antibody and an anti-PD-1 / anti-VEGFA bispecific antibody are administered in the same composition. [Claim 18] The therapeutic combination for use according to claim 1, wherein an anti-PD-1 / anti-CTLA4 bispecific antibody and an anti-PD-1 / anti-VEGFA bispecific antibody are administered in separate compositions. [Claim 19] The therapeutic combination for use according to claim 1, wherein the mass ratio of the anti-PD-1 / anti-CTLA4 bispecific antibody to the anti-PD-1 / anti-VEGFA bispecific antibody is (10:1) to (1:5). [Claim 20] The therapeutic combination for use according to Claim 1, wherein the effective amounts of the anti-PD-1 / anti-CTLA4 bispecific antibody and the anti-PD-1 / anti-VEGFA bispecific antibody are independently 100 mg to 2500 mg, 100 mg to 2000 mg, 100 mg to 1500 mg, 100 mg to 1200 mg, 100 mg to 1000 mg, 200 mg to 800 mg, 200 mg to 500 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg, based on the mass of the anti-PD-1 / anti-CTLA4 bispecific antibody and / or the anti-PD-1 / anti-VEGFA bispecific antibody. [Claim 21] The therapeutic combination for use according to Claim 1, wherein the effective amount of anti-PD-1 / anti-CTLA4 bispecific antibody and the effective amount of anti-PD-1 / anti-VEGFA bispecific antibody is 0.1 mg / kg to 100 mg / kg. [Claim 22] The therapeutic combination for use according to claim 21, wherein an effective amount of anti-PD-1 / anti-CTLA4 bispecific antibody and an effective amount of anti-PD-1 / anti-VEGFA bispecific antibody are administered every two weeks, three weeks, or six weeks. [Claim 23] The therapeutic combination for use according to claim 1, further comprising administering an effective amount of an antitumor chemotherapeutic agent selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, plant-based anticancer agents, hormones, and immunoassayants. [Claim 24] The therapeutic combination for use according to any one of claims 1 to 23, wherein the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical tumor, plasma cell carcinoma, head and neck cancer, brain cancer, throat cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, lung cancer, breast cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma. [Claim 25] A therapeutic combination for use according to claim 24, (a) Lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; (b) The gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; (c) The lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma; or (d) The tumor is a solid tumor with an MSI-H / dMMR phenotype; wherein the tumor is selected from one or more of the following tumors with an MSI-H / dMMR phenotype: colorectal cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell neoplasms. The aforementioned therapeutic combination. [Claim 26] A therapeutic combination for use in the treatment of tumors, comprising administering an effective amount of anti-PD-1 / anti-CTLA4 bispecific antibody and an effective amount of anti-PD-1 / anti-VEGFA bispecific antibody to a subject in need thereof, wherein: (1) The anti-PD-1 / anti-CTLA4 bispecific antibody is as follows: (i) A first protein functional region that targets PD-1 and includes a first heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18 and a first light chain variable region containing the amino acid sequence of SEQ ID NO: 20; and (ii) A second protein functional region that targets CTLA4 and includes a second heavy chain variable region containing the amino acid sequence of SEQ ID NO: 43 and a second light chain variable region containing the amino acid sequence of SEQ ID NO: 44; This comprises; where the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; and (2) The anti-PD-1 / anti-VEGFA bispecific antibody is as follows: (i) A third protein functional region that targets VEGFA and includes a third heavy chain variable region containing the amino acid sequence of SEQ ID NO: 45 and a third light chain variable region containing the amino acid sequence of SEQ ID NO: 47; (ii) A fourth protein functional region that targets PD-1 and includes a fourth heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18 and a fourth light chain variable region containing the amino acid sequence of SEQ ID NO: 49; This includes; where the third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The aforementioned therapeutic combination. [Claim 27] ​​(i) The immunoglobulin of the anti-PD-1 / anti-CTLA4 bispecific antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 40 and a light chain containing the amino acid sequence of SEQ ID NO: 24; and (ii) The immunoglobulin of the anti-PD-1 / anti-VEGFA bispecific antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 54 and a light chain containing the amino acid sequence of SEQ ID NO:

56. A therapeutic combination for use according to claim 26.

Images