Use of antibodies in anti-tumor therapy

Abstract

The invention relates to application of an anti-CTLA4-anti-PD-1 bispecific antibody in anti-tumor treatment, the anti-CTLA4-anti-PD-1 bispecific antibody is used for treating tumors, the anti-CTLA4-anti-PD-1 bispecific antibody and an anti-VEGFR2 monoclonal antibody or an anti-VEGF monoclonal antibody are jointly applied, (1) the anti-CTLA4-anti-PD-1 bispecific antibody comprises a PD-1-targeting first protein functional area, a PD-1-targeting second protein functional area, a PD-1-targeting third protein functional area and a PD-1-targeting fourth protein functional area, and a second protein functional region targeting CTLA4; wherein the first protein functional area is immune globulin, and the second protein functional area is a single-chain antibody; or, the first protein functional area is a single-chain antibody, and the second protein functional area is immune globulin.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202210401367.9, with a Chinese filing date of April 14, 2022, and an invention title of "Use of Antibodies in Anti-Tumor Therapy". Technical Field

[0002] The present invention belongs to the field of molecular immunology, and specifically relates to the application of a class of monoclonal antibodies against vascular endothelial growth factor receptor VEGFR2 or monoclonal antibodies against VEGF in combination with anti-PD-1 / CTLA-4 bispecific antibodies in the treatment of tumors. Background Art

[0003] Tumor is a major disease that endangers human health, and there is an urgent need to develop effective treatment methods and drugs.

[0004] Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor for a growth factor that specifically acts on vascular endothelial cells, also known as KDR or FLK1. Currently, 5 types of VEGFRs have been discovered: VEGFR-1, VEGFR-2, VEGFR-3, NP-1, and NP-2. Among them, VEGFR-1 and VEGFR2 are mainly present in vascular endothelial cells, and VEGFR3 is mainly present in lymphatic endothelial cells. NP-1 and NP-2 are expressed not only in endothelial cells but also in some tumor cells. However, the biological activities of VEGF to promote the proliferation of vascular endothelial cells are mainly achieved by binding to VEGFR2.

[0005] The formation of new blood vessels plays an important role in various human diseases, such as retinopathy, arthritis, endometriosis, etc. The growth of tumors is usually accompanied by the formation of new blood vessels. As early as 1971, J. Folkman proposed that the growth and metastasis of tumors could be inhibited by blocking the formation of tumor blood vessels. In recent years, studies have found that the development, metastasis, and prognosis of more and more malignant tumors are related to vascular endothelial growth factor and its receptor family. Therefore, anti-tumor therapy targeting VEGF and its receptors has attracted attention again. Among the receptor family, the research targeting VEGFR-2 is the most extensive.

[0006] PD-1 (Programmed death-1) is a key immune checkpoint receptor expressed by activated T and B lymphocytes and mediates immunosuppression, and its ligands include at least PD-L1 and PD-L2. Anti-PD-1 antibodies specifically bind to programmed death-1 (PD-1) and block the inhibitory PD-1 / PD-L1 pathway (Topalian et al. (2012a) N Engl J Med 366:2443-54).

[0007] PD-L1 (Programmed death-ligand 1), also known as CD274 or B7-H1, is a 40 kDa type I transmembrane protein encoded by the CD274 gene and is a ligand of PD-1. PD-L1 is widely distributed not only on leukocytes and non-hematopoietic cells in lymphoid and non-lymphoid tissues but also on various cancer cells, highly expressed on the surface of a variety of tumor cells, and the malignancy and poor prognosis of tumors are closely related to the expression level of PD-L1. Both PD-L1 and PD-1 belong to the immunoglobulin superfamily and both have two extracellular Ig domains. The binding interfaces of PD-L1 with programmed death receptor-1 (PD-1) and B7-1 (CD80) are on the IgV-like domain (Lin et al. (2008) PNAS 105:3011-3016). PD-L1 contains a conserved short intracellular tail region (about 30 amino acids), and PD-1 contains two cytoplasmic tyrosine-based signaling motifs, namely immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). After T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif in its cytoplasmic tail, resulting in dephosphorylation of effector molecules (such as CD3ζ, PKCθ, and ZAP70) involved in the CD3+ T cell signaling cascade (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Clinical data indicate that high tumor expression of PD-L1 is associated with increased tumor invasiveness and poor prognosis.

[0008] PD-1 / PD-L1 is an important specific immune checkpoint. The formation of the PD-1 / PD-L1 complex transmits inhibitory signals and negatively regulates T cell immune responses; it inhibits TCR-mediated T cell activation, cytokine production, and T cell proliferation (Fife et al. (2011) Nature Immunology 10:1185-1193); induces exhaustion or anergy among antigen-specific T cells (Hofmeyer et al. (2011) Journal of Biomedicine and Biotechnology 2011:1-9); promotes the differentiation of Th1 cells into Foxp3+ regulatory T cells (Armanath et al. (2011) Science TransMed 3:1-13; Francisco et al. (2009) J.Exp.Med. 206:3015-3029); and induces apoptosis of effector T cells. Disruption of the PD-L1 gene results in upregulated T cell responses and the generation of autoreactive T cells (Latchman et al. (2004) PNAS 101:10691-10696). Blockade of antibodies against PD-1 or PD-L1 leads to increased anti-tumor immunity (Iwai et al. (2002) PNAS 99:12293-12297).

[0009] CTLA-4 is also one of the representative specific immune checkpoint proteins. CTLA-4 is the first target applied to clinical practice. Anti-CTLA-4 antibodies can promote T cell activation and improve its anti-tumor effect. Ipilimumab is the first CTLA-4 immune checkpoint inhibitor proven to improve the overall survival rate of treated metastatic melanoma patients and approved by the FDA for marketing. Current studies have shown that CTLA-4 inhibits T cell responses mainly through two pathways: one is by competitively binding to B7 with CD28 or recruiting phosphatases to the intracellular domain of CTLA-4, thereby reducing the signals of TCR (T cell receptor) and CD28. The other is to reduce the expression levels of CD80 and CD86 on antigen-presenting cells (APCs) or remove them from APCs through transendocytosis, thus reducing the participation of CD28 in T cell activation. In addition, CTLA-4 also mediates the binding of dendritic cells to CD80 / CD86 and induces the expression of the tryptophan-degrading enzyme IDO, resulting in the inhibition of TCR. CTLA-4 antibodies reduce Tregs by binding to CTLA-4 and activate TCR

[0010] The combined use of PD-1 antibodies and CTLA-4 (cytotoxic T lymphocyte-associated protein 4) antibody drugs has shown better efficacy than PD-1 antibody monotherapy in various tumors. However, the improvement in the efficacy of combined use is always accompanied by higher toxicity, which greatly limits its application.

[0011] Bevacizumab is an anti-angiogenic targeted drug that inhibits tumor metastasis by binding to vascular endothelial growth factor (VEGF) and blocking blood vessel growth. It is a broad-spectrum anti-cancer drug. Since its launch, bevacizumab has been mainly approved for the treatment of various types of cancers, such as brain cancer (e.g., invasive brain cancer, such as glioblastoma), kidney cancer, lung cancer (e.g., metastatic non-squamous non-small cell lung cancer), colon cancer, rectal cancer, cervical cancer (e.g., metastatic cervical cancer), endometrial cancer, ovarian cancer (e.g., advanced ovarian cancer) or fallopian tube cancer, kidney cancer, etc. Summary of the Invention

[0012] The inventors of the present invention have found that the combination of anti-CTLA4-anti-PD-1 bispecific antibodies with monoclonal antibodies against VEGFR2 or monoclonal antibodies against VEGF can effectively prevent and treat tumors with low toxic and side effects. Thus, the following invention is provided.

[0013] Specifically, the present invention relates to:

[0014] 1. An anti-CTLA4-anti-PD-1 bispecific antibody, which is preferably used for treating tumors. Preferably, the anti-CTLA4-anti-PD-1 bispecific antibody is administered in combination with component A, where component A is selected from a monoclonal antibody against VEGFR2 or its antigen-binding fragment, a monoclonal antibody against VEGF or its antigen-binding fragment, or a combination thereof.

[0015] Wherein

[0016] (1) The anti-CTLA4-anti-PD-1 bispecific antibody comprises:

[0017] A first protein functional region targeting PD-1, and

[0018] A second protein functional region targeting CTLA4;

[0019] 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;

[0020] Wherein,

[0021] The immunoglobulin described above comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 14 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 27-29 respectively), and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 16 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 30-32 respectively); and the single-chain antibody described above comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 2 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 33-35 respectively) and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 4 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 36-38 respectively);

[0022] Or,

[0023] The immunoglobulin described above comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 2 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 33-35 respectively) and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 4 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 36-38 respectively); and the single-chain antibody described above comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 14 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 27-29 respectively), and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 16 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 30-32 respectively);

[0024] (2) The anti-VEGFR2 monoclonal antibody comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 50 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 53-55 respectively) and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 52 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 56-58 respectively); or

[0025] (3) The anti-VEGF monoclonal antibody comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 60 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 63-65 respectively) and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 62 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 66-68 respectively).

[0026] 2. The anti-CTLA4-anti-PD-1 bispecific antibody according to item 1, wherein,

[0027] (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 14, SEQ ID NO: 18 or a variant thereof; 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 or a variant thereof; and, 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, SEQ ID NO: 43 or a variant thereof; and 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, SEQ ID NO: 44 or a variant thereof;

[0028] Or,

[0029] 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, SEQ ID NO: 43 or a variant thereof; and 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, SEQ ID NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 14, SEQ ID NO: 18 or a variant thereof; 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 or a variant thereof;

[0030] (2) The amino acid sequence of the heavy chain variable region of the anti-VEGFR2 monoclonal antibody is the sequence shown in SEQ ID NO: 50 or a variant thereof; and the amino acid sequence of the light chain variable region of the anti-VEGFR2 monoclonal antibody is the sequence shown in SEQ ID NO: 52 or a variant thereof; or

[0031] (3) The amino acid sequence of the heavy chain variable region of the anti-VEGF monoclonal antibody is the sequence shown in SEQ ID NO: 60 or a variant thereof; and the amino acid sequence of the light chain variable region of the anti-VEGF monoclonal antibody is the sequence shown in SEQ ID NO: 62 or a variant thereof.

[0032] 3. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 2, wherein the bispecific antibody is selected from any one of the following (1)-(20):

[0033] (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 4 or a variant thereof;

[0034] (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0035] (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 12 or a variant thereof;

[0036] (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 4 or a variant thereof;

[0037] (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0038] (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 12 or a variant thereof;

[0039] (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof;

[0040] (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof;

[0041] (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof;

[0042] (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof;

[0043] (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof;

[0044] (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof;

[0045] (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 42 or a variant thereof;

[0046] (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 44 or a variant thereof;

[0047] (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 42 or a variant thereof;

[0048] (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 44 or a variant thereof;

[0049] The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof;

[0050] (18)The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof;

[0051] (19)The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof;

[0052] And,

[0053] (20)The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof,

[0054] wherein the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

[0055] 4. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of Items 1 to 3, wherein,

[0056] the amino acid sequence of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 40 or a variant thereof, and the amino acid sequence of its light chain is the sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the variant sequence has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

[0057] 5. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of Items 1 to 4, wherein the first protein functional region is directly connected to the second protein functional region or connected by a linker fragment; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected by a linker fragment.

[0058] 6. The anti-CTLA4-anti-PD-1 bispecific antibody according to Item 5, wherein the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

[0059] 7. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of Items 1 to 6, wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

[0060] 8. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of Items 1 to 7, wherein the single-chain antibody (preferably the heavy chain variable region) is connected to the C-terminus of the heavy chain of the immunoglobulin.

[0061] 9. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of Items 1 to 8, wherein,

[0062] the immunoglobulin is of human IgG1 subtype;

[0063] wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has an A mutation, and the A mutation is selected from one of the following mutation combinations:

[0064] L234A and L235A; or

[0065] L234A and G237A; or

[0066] L235A and G237A; or

[0067] L234A, L235A, G237A; or

[0068] According to the EU numbering system, the heavy chain constant region of the immunoglobulin has a B mutation, and the B mutation is selected from one or more of the following mutations:

[0069] N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A;

[0070] Or, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has a combination of A mutation and B mutation;

[0071] Or

[0072] The heavy chain constant region of the anti-VEGFR2 monoclonal antibody or the anti-VEGF monoclonal antibody is Ig gamma-1 chain C region, ACCESSION P01857, and the light chain constant region is: Ig kappa chain C region, ACCESSION: P01834.

[0073] 10. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 9,

[0074] wherein the bispecific antibody comprises:

[0075] a first protein functional region targeting PD-1, and

[0076] a second protein functional region targeting CTLA4;

[0077] There is 1 of the first protein functional region and 2 of the second protein functional region;

[0078] wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody;

[0079] The amino acid sequence of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 40 or a variant thereof, and the amino acid sequence of its light chain is the sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence;

[0080] The amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 44 or a variant thereof, wherein the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence;

[0081] The single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin;

[0082] The first protein functional region is linked to the second protein functional region by a first linker fragment; and the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody by a second linker fragment; the first linker fragment and the second linker fragment are the same or different;

[0083] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO: 25 and SEQ ID NO: 26;

[0084] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are both as shown in SEQ ID NO: 26,

[0085] Preferably, the light chain sequence of the anti-PD-1 / CTLA-4 bispecific antibody is as shown in SEQ ID NO: 72, and the heavy chain sequence is as shown in SEQ ID NO: 70,

[0086] Preferably, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, complementarity-determining region (CDR) fragment, single-chain antibody (e.g., scFv), diabody or domain antibody.

[0087] 11. A pharmaceutical composition for treating tumors (especially malignant tumors), which comprises an effective amount of the anti-CTLA4-anti-PD-1 bispecific antibody described in any one of items 1-10 and component A defined in any one of items 1-10. Preferably, the pharmaceutical composition is in solid form or liquid form. Preferably, the mass ratio of the anti-CTLA4-anti-PD-1 bispecific antibody to component A is selected from (1:5)-(5:1), for example: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1; Optionally, the pharmaceutical composition further comprises one or more chemotherapeutic drugs (preferably the chemotherapeutic drug is an alkylating agent, an antimetabolite, an antibiotic, a plant drug and / or a hormonal drug, a platinum drug (such as cisplatin, carboplatin, oxaliplatin), an adriamycin, cyclophosphamide, paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, docetaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinblastine, tamoxifen, megestrol acetate, goserelin, asparaginase and / or a fluorouracil-based anti-tumor drug).

[0088] 12. Use of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1-10 in the preparation of a drug or kit for preventing and / or treating tumors (especially malignant tumors), preferably the drug or kit further comprises component A defined in any one of items 1-10, optionally, the drug or kit further comprises one or more drugs for treating tumors (preferably the drug is a chemotherapeutic agent or a growth inhibitor (such as an alkylating agent, anthracycline, antihormonal agent, aromatase inhibitor, antiandrogen, protein kinase inhibitor, lipid kinase inhibitor, antisense oligonucleotide, ribozyme, antimetabolite, topoisomerase inhibitor, cytotoxic agent or antitumor antibiotic, proteasome inhibitor, antimicrotubule agent, EGFR antagonist, VEGF antagonist, angiopoietin 2 antagonist, retinoid, tyrosine kinase inhibitor, histone deacetylase inhibitor and combinations thereof), targeted therapeutic agent (such as B-raf inhibitor, MEK inhibitor, K-ras inhibitor, c-Met inhibitor, Alk inhibitor, phosphatidylinositol 3-kinase inhibitor, Akt inhibitor, mTOR inhibitor, VEGF inhibitor, PARP inhibitor, dual phosphatidylinositol 3-kinase / mTOR inhibitor and combinations thereof), antibody-drug conjugate (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelator), T cell expressing a chimeric antigen receptor, antibody or antigen-binding fragment thereof, angiogenesis inhibitor, antitumor agent, cancer vaccine, adjuvant and combinations thereof, alkylating agent, antimetabolic drug, antibiotic, phytomedicine and / or hormonal drug, platinum drug (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, docetaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil antitumor drug).

[0089] 13. A method for preventing and / or treating tumors (especially malignant tumors), comprising administering to a subject a therapeutically effective amount of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1-10, in combination with component A as defined in any one of items 1-10, and more preferably, further in combination with one or more drugs for treating tumors (preferably the drugs are chemotherapeutic agents or growth inhibitors (such as alkylating agents, anthracyclines, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors and combinations thereof), targeted therapeutic agents (such as B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors and combinations thereof), antibody-drug conjugates (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and combinations thereof, antimetabolic drugs, antibiotics, plant drugs and / or hormonal drugs, platinum drugs (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel (such as albumin-bound paclitaxel, liposomal paclitaxel, docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil antitumor drugs), preferably, the anti-CTLA4-anti-PD-1 bispecific antibody, the component A and the tumor chemotherapy drug are administered simultaneously or sequentially.

[0090] 14. A kit for preventing and / or treating tumors (especially malignant tumors), comprising (a) a first pharmaceutical composition containing the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1-10 as an active ingredient; and (b) a second pharmaceutical composition containing component A defined in any one of items 1-10 as an active ingredient; and optionally (c) one or more drugs for treating tumors (preferably the drugs are chemotherapeutic agents or growth inhibitors (such as alkylating agents, anthracyclines, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors and their combinations), targeted therapeutic agents (such as B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors and their combinations), antibody-drug conjugates (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or their antigen-binding fragments, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and their combinations, alkylating agents, antimetabolic drugs, antibiotics, plant drugs and / or hormonal drugs, platinum drugs (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel (such as albumin-bound paclitaxel, liposomal paclitaxel, docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil antitumor drugs).

[0091] 15. A unit dosage form, preferably for treating tumors (especially malignant tumors), wherein the unit dosage form comprises: 1 to 1000 mg (preferably 10 - 1000 mg, preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg or 200 mg) of the anti - CTLA4 - anti - PD - 1 bispecific antibody according to any one of items 1 - 10, 1 to 1000 mg (preferably 10 - 1000 mg, preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg or 200 mg) of component A as defined in any one of items 1 - 10, and optionally one or more drugs for treating tumors (preferably the drug is a chemotherapeutic agent or a growth inhibitor (such as alkylating agent, anthracycline, anti - hormonal agent, aromatase inhibitor, anti - androgen, protein kinase inhibitor, lipid kinase inhibitor, antisense oligonucleotide, ribozyme, antimetabolite, topoisomerase inhibitor, cytotoxic agent or antitumor antibiotic, proteasome inhibitor, antimicrotubule agent, EGFR antagonist, VEGF antagonist, angiopoietin 2 antagonist, retinoid, tyrosine kinase inhibitor, histone deacetylase inhibitor and their combinations), targeted therapeutic agent (such as B - raf inhibitor, MEK inhibitor, K - ras inhibitor, c - Met inhibitor, Alk inhibitor, phosphatidylinositol 3 - kinase inhibitor, Akt inhibitor, mTOR inhibitor, VEGF inhibitor, PARP inhibitor, dual phosphatidylinositol 3 - kinase / mTOR inhibitor and their combinations), antibody - drug conjugate (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelator), T cell expressing chimeric antigen receptor, antibody or its antigen - binding fragment, angiogenesis inhibitor, antitumor agent, cancer vaccine, adjuvant and their combinations, alkylating agent, antimetabolic drug, antibiotic, phytomedicine and / or hormonal drug, platinum drug (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel (such as albumin - bound paclitaxel, liposomal paclitaxel, docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil - based antitumor drug); wherein the anti - CTLA4 - anti - PD - 1 bispecific antibody, component A and the chemotherapeutic drug are each separately packaged.

[0092] 16. A method for preventing or treating cancer or tumor, wherein one or more unit dosage forms according to item 15 are administered to a subject in need thereof, preferably, the anti - CTLA4 - anti - PD - 1 bispecific antibody, component A and the chemotherapeutic drug in the unit dosage form are each administered separately.

[0093] 17. A single-dose pharmaceutical unit, preferably for the treatment of tumors (especially malignant tumors), comprising 0.1 - 10000 mg (preferably 1 - 1000 mg, preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg, 200 mg or 100 mg) of the anti-CTLA4 anti-PD-1 bispecific antibody according to any one of items 1 - 10, and 0.1 - 10000 mg (preferably 1 - 1000 mg, preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg, 200 mg or 100 mg) of component A as defined in any one of items 1 - 10.

[0094] 18. The anti-CTLA4 - anti-PD-1 bispecific antibody according to any one of items 1 - 10, the pharmaceutical composition according to item 11, the application according to item 12, the method according to item 13, the kit according to item 14, the unit preparation according to item 15, or the method according to item 16, wherein

[0095] the tumor is selected from one or more of the following:

[0096] cervical cancer (such as metastatic cervical cancer), lung cancer such as non-small cell lung cancer (e.g., squamous non-small cell lung cancer or non-squamous non-small cell lung cancer), esophageal cancer, esophageal squamous cell carcinoma, ileocecal adenocarcinoma, ampullary adenocarcinoma, small cell lung cancer, gastric cancer (e.g., advanced gastric cancer, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma), renal cancer, renal endometrial cancer, adrenocortical carcinoma, prostate cancer, thyroid cancer, peritoneal cancer, adenocarcinoma, pancreatic cancer, glioma, head and neck cancer, bone cancer, testicular cancer, leukemia, myeloma, lymphoma, sarcoma, mesothelioma, hepatocellular carcinoma, colon cancer, cholangiocarcinoma, bile duct cancer, rectal cancer, colon cancer, endometrial cancer, ovarian cancer (such as advanced ovarian cancer) or fallopian tube cancer, large cell neuroendocrine carcinoma, urothelial cancer (e.g., upper urothelial cancer or bladder cancer), breast cancer, triple-negative breast cancer, peripheral T-cell lymphoma, melanoma, nasopharyngeal cancer, and highly microsatellite unstable (MSI-H) or mismatch repair defective (dMMR) solid tumors, brain cancer (such as invasive brain cancer, e.g., glioblastoma), ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenoma, mucinous or serous cystadenocarcinoma, leiomyosarcoma, rhabdomyosarcoma, choriocarcinoma, malignant mole, malignant Sertoli-Leydig cell tumor, malignant granulosa cell tumor, dysgerminoma.

[0097] 19. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of Items 1-10, the pharmaceutical composition according to Item 11, the application according to Item 12, the method according to Item 13, the kit according to Item 14, the unit dosage form according to Item 15, or the method according to Item 16, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, Component A and / or the chemotherapeutic drug are in a form suitable for intravenous injection or intravenous drip, preferably in a liquid form.

[0098] 20. According to the method of Item 13 or 16, wherein the step of administering an effective amount of the anti-CTLA4-anti-PD-1 bispecific antibody to the subject is before or after surgical treatment, and / or before or after radiotherapy.

[0099] 21. According to the method of Item 13 or 16, wherein

[0100] the single-dose administration dose of the anti-CTLA4 anti-PD-1 bispecific antibody is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single-dose administration dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg;

[0101] the single-dose administration dose of Component A is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single-dose administration dose of Component A is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg,

[0102] preferably, the administration is twice a day to about once every other day, or once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;

[0103] preferably, the administration method is intravenous drip or intravenous injection.

[0104] In some embodiments, the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody is administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg body weight.

[0105] In some embodiments, the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody is administered in one or more uniform doses effective to treat the cancer. In some specific embodiments, the uniform dose ranges from about 1 mg to about 1000 mg. In some specific embodiments, the uniform dose is selected from about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg.

[0106] In some embodiments, the administration treatment of the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody is carried out in a cycle of 2 weeks (14 days) or 3 weeks (21 days), and preferably the anti-PD-1 and CTLA4 bispecific antibody is intravenously administered on the first day (D1) of each cycle. That is, the anti-PD-1 and CTLA4 bispecific antibody is administered at a frequency of once every two weeks (q2w) or once every three weeks (q3w).

[0107] The present invention also provides the use of an anti-CTLA4-anti-PD-1 bispecific antibody, an anti-VEGFR2 monoclonal antibody, and an anti-VEGF monoclonal antibody in the preparation of a product for treating cancer, the product comprising a container containing a fixed dose of the anti-CTLA4-anti-PD-1 bispecific antibody and the anti-VEGFR2 monoclonal antibody (or the anti-VEGF monoclonal antibody). In some specific embodiments, the container is an ampoule. The fixed dose is selected from about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, or about 1000 mg. In some specific embodiments, the product further comprises a package insert or a drug instruction manual for guiding the user to administer the fixed dose of the anti-CTLA4-anti-PD-1 bispecific antibody, the anti-VEGFR2 monoclonal antibody, or the anti-VEGF monoclonal antibody to a cancer patient. In some specific embodiments, the product comprises one or more ampoules, and the ampoules contain about 50 mg, 100 mg, 200 mg, 300 mg, 350 mg, 400 mg, 500 mg, or 600 mg of the anti-VEGFR2 monoclonal antibody or the anti-VEGF monoclonal antibody. In some specific embodiments, the product is packaged in the form of a solution of the anti-PD-1 and CTLA4 bispecific antibody, the anti-VEGFR2 monoclonal antibody, or the anti-VEGF monoclonal antibody at 50 mg / 5 mL / vial, 100 mg / 10 mL / vial, 200 mg / 10 mL / vial, or 350 mg / 35 mL / vial.

[0108] The dosage of the chemotherapeutic agent can be determined by those of ordinary skill in the art. For example, for platinum-based chemotherapeutic agents, taking cisplatin as an example, the single-dose administration dose of cisplatin is calculated according to the Calvert formula:

[0109] Cisplatin dose (mg) = the area under the curve (AUC) (mg / ml / min) × [creatinine clearance rate (ml / min) + 25], where the value of AUC is 4-7, preferably 5;

[0110] It is administered once every 2-6 weeks, preferably once every 3 weeks or once every 4 weeks;

[0111] and / or

[0112] The administration method is intravenous drip or intravenous injection.

[0113] In one or more embodiments of the present invention, the chemotherapeutic agent or growth inhibitor is selected from alkylating agents, anthracyclines, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0114] In one or more embodiments of the present invention, the targeted therapeutic agent is selected from B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors, and combinations thereof.

[0115] In one or more embodiments of the present invention, the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and radioisotope chelators.

[0116] In the present invention, some expressions have the following meanings:

[0117] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease, the regression of which is demonstrated by a decrease in the severity of the disease symptoms, an increase in the frequency and duration of the disease-free symptom stage, or the prevention of damage or disability caused by the affliction of the disease. The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0118] A "preventively effective amount" of a drug is an amount of any drug that, when administered alone or in combination with an antitumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject at risk of cancer recurrence, inhibits the occurrence or recurrence of cancer. In certain embodiments, the preventively effective amount completely prevents the occurrence or recurrence of cancer. "Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of the occurrence or recurrence of cancer, or completely preventing the occurrence or recurrence of cancer.

[0119] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include but are not limited to sodium chloride.

[0120] "Recurrent" cancer is cancer that regenerates at the original site or at a distant site after responding to initial treatment (e.g., surgery). "Locally recurrent" cancer is cancer that appears at the same location as the previously treated cancer after treatment.

[0121] "Metastatic" cancer refers to cancer that spreads from one part of the body (e.g., the lungs) to another part of the body.

[0122] The variable regions of the light and heavy chains determine antigen binding: each variable region of the chain contains three hypervariable regions, called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) contain HCDR1, HCDR2, HCDR3, and the CDRs of the light chain (L) contain LCDR1, LCDR2, LCDR3; they were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md). Given the sequences of the variable regions of the heavy and light chains of a known antibody, there are currently several methods for determining the CDR regions of the antibody, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of each definition of the CDRs of an antibody or its variants will be within the scope of the terms defined and used herein. Given the amino acid sequence of the variable region of a given antibody, a person skilled in the art can generally determine the specific CDRs without relying on any experimental data outside of the sequence itself.

[0123] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease, as evidenced by a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of the disease-free phase, or the prevention of damage or disability caused by the affliction of the disease. The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0124] A "prophylactically effective amount" of a drug refers to any amount of the drug that, when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject at risk of cancer recurrence, inhibits the occurrence or recurrence of cancer. In certain embodiments, the prophylactically effective amount completely prevents the occurrence or recurrence of cancer. "Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of the occurrence or recurrence of cancer, or completely preventing the occurrence or recurrence of cancer.

[0125] "Recurrent" cancer is cancer that regenerates at the original site or at a distant site after responding to initial treatment (e.g., surgery). "Locally recurrent" cancer is cancer that appears at the same location as the previously treated cancer after treatment.

[0126] "Metastatic" cancer refers to cancer that has spread from one part of the body (e.g., the lungs) to another part of the body.

[0127] The term "single drug dose unit" refers to a single drug dosage form, such as an injection, e.g., placed in an ampoule, containing the anti-CTLA4 anti-PD-1 bispecific antibody, anti-VEGFR2 antibody, or anti-VEGF antibody described in the present invention to be administered to a subject at the time of the dosing regimen (preferably per kg body weight of the subject). In a specific embodiment of the present invention, the dosing regimen includes, for example, administering the single drug dose unit according to a dosing cycle of twice a day to about once 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.

[0128] In the present invention, unless otherwise specified, the "first" (e.g., the first protein functional region, the first linker fragment) and "second" (e.g., the second protein functional region, the second linker fragment) are for the purpose of distinguishing in reference or for clarity of expression, and do not have the typical meaning of order. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1. Detection of the bioactivity of the anti-PD-1 / CTLA-4 bispecific antibody Cadonilimab in combination with the anti-VEGFR2 antibody HpL3 in promoting INF-γ secretion by mixed lymphocyte reaction (MLR).

[0130] Figure 2 . Detection of the bioactivity of Cadonilimab in combination with bevacizumab in promoting the activation and secretion of IL-2 by PBMC by mixed lymphocyte reaction (MLR). Detailed implementation manners

[0131] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not indicated by the manufacturer can be obtained through commercial purchase as conventional products.

[0132] In the following embodiments of the present invention, the isotype control antibody anti-HEL is an antibody targeting human anti-hen egg lysozyme (HEL). The variable region sequences of these antibodies are from Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-46. The heavy chain variable region sequence is shown in SEQ ID No: 45, and the light chain variable region sequence is shown in SEQ ID NO: 47). The constant region fragment of hIgG1WT (i.e., anti-HEL) uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region (SEQ ID NO: 46), and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region (SEQ ID NO: 48); anti-HEL was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0133] The bevacizumab (anti-VEGF monoclonal antibody) used in the examples is a commercial drug produced by Roche Pharmaceuticals.

[0134] Preparation Example 1: Sequence Design of Anti-CTLA4 Antibody

[0135] The amino acid sequences of the heavy and light chains of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3, as well as the encoding nucleic acid sequences, are the same as those of 4G10, 4G10H1L1, and 4G10H3L3 in Chinese Patent Publication CN 106967172A.

[0136] (1) The heavy chain variable region sequence and light chain variable region sequence of 4G10

[0137] Nucleic acid sequence of the heavy chain variable region: (372 bp, SEQ ID NO: 1)

[0138] The amino acid sequence it encodes: (124 aa, SEQ ID NO: 2)

[0139] Nucleic acid sequence of the light chain variable region: (378 bp, SEQ ID NO: 3)

[0140] The amino acid sequence it encodes: (126 aa, SEQ ID NO: 4)

[0141] (2) The heavy chain variable region sequence and light chain variable region sequence of the humanized monoclonal antibody 4G10H1L1

[0142] Nucleic acid sequence of the heavy chain variable region (4G10H1V): (345 bp, SEQ ID NO: 5)

[0143] The amino acid sequence it encodes: (115 aa, SEQ ID NO: 6)

[0144] Nucleic acid sequence of the light chain variable region (4G10L1V): (327 bp, SEQ ID NO: 7)

[0145] The amino acid sequence it encodes: (109 aa, SEQ ID NO: 8)

[0146] (3) The heavy chain variable region sequence and light chain variable region sequence of the humanized monoclonal antibody 4G10H3L3

[0147] Nucleic acid sequence of the heavy chain variable region (4G10H3V): (345bp, SEQ ID NO:9)

[0148] The amino acid sequence of the encoded heavy chain variable region (4G10H3V): (115 aa, SEQ ID NO: 10)

[0149] Nucleic acid sequence of the light chain variable region (4G10L3V): (327 bp, SEQ ID NO: 11)

[0150] The encoded amino acid sequence (4G10L3V): (109 aa, SEQ ID NO: 12)

[0151] Preparation Example 2: Sequence Design of Anti-PD-1 Antibody 14C12 and Its Humanized Antibody 14C12H1L1

[0152] The amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the encoding nucleic acid sequences, are the same as those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A respectively.

[0153] (1) The heavy chain variable region sequence and light chain variable region sequence of 14C12

[0154] The nucleic acid sequence of the heavy chain variable region: (354 bp, SEQ ID NO: 13)

[0155] The encoded amino acid sequence: (118 aa, SEQ ID NO: 14)

[0156] The nucleic acid sequence of the light chain variable region: (321 bp, SEQ ID NO: 15)

[0157] The encoded amino acid sequence: (107 aa, SEQ ID NO: 16)

[0158] (2) The heavy chain variable region sequence, light chain variable region sequence, heavy chain sequence and light chain sequence of the humanized monoclonal antibody 14C12H1L1

[0159] The nucleic acid sequence of the heavy chain variable region: (354 bp, SEQ ID NO: 17)

[0160] The encoded amino acid sequence: (118 aa, SEQ ID NO: 18)

[0161] The nucleic acid sequence of the light chain variable region: (321 bp, SEQ ID NO: 19)

[0162] The encoded amino acid sequence: (107 aa, SEQ ID NO: 20)

[0163] The DNA sequence of the heavy chain of 14C12H1L1 (14C12H1): (1344 bp,

[0164] SEQ ID NO: 21)

[0165] The encoded amino acid sequence: (448 aa, SEQ ID NO: 22)

[0166] DNA sequence of the 14C12H1L1 light chain (14C12L1): (642 bp, SEQ ID NO: 23)

[0167] The amino acid sequence encoded by it: (214 aa, SEQ ID NO: 24)

[0168] Preparation Example 3: Sequence Design of Bifunctional Antibodies CP001(M), CP002(M), CP003(M) and CP004(M)

[0169] The structural patterns of the bispecific antibodies CP001(M), CPb002(M), CP003(M) and CP004(M) belong to the Morrison pattern (IgG-scFv), that is, the scFv fragments of another antibody are connected to the C-termini of the two heavy chains of an IgG antibody through linker fragments, and the design compositions of their heavy and light chains are as shown in Table A below.

[0170] Table A: Sequence design of CP001(M), CP002(M), CP003(M) and CP004(M)

[0171]

[0172] In the above Table A:

[0173] The amino acid sequence of Linker1 is (GGGGS)3 (SEQ ID NO: 25)

[0174] The amino acid sequence of Linker2 is (GGGGS)4 (SEQ ID NO: 26)

[0175] In addition, in the above Table A, for the scFv fragments of the antibodies CP001(M), CP002(M), CP003(M) and CP004(M), 4G10H1V(M), 4G10L1V(M), 4G10H3V(M), 4G10L3V(M) are mutations of individual amino acids in their framework regions on the basis of 4G10H1V, 4G10L1V, 4G10H3V, 4G10L3V respectively, effectively optimizing the structure of the antibody and improving its effectiveness.

[0176] (1) 4G10H1V(M): (115 aa, the amino acid sequence mutation sites made on the basis of 4G10H1V are underlined, SEQ ID NO: 41)

[0177] (2) 4G10L1V(M): (110 aa, the amino acid sequence mutation sites made on the basis of 4G10L1V are underlined, SEQ ID NO: 42)

[0178] (3)4G10H3V(M): (115 aa, the amino acid sequence mutation sites based on 4G10H3V are underlined, SEQ ID NO: 43)

[0179] (4)4G10L3V(M): (110 aa, the amino acid sequence mutation sites based on 4G10L3V are underlined, SEQ ID NO: 44)

[0180] In order to distinguish it from the following mutated antibodies, in the embodiments of the present invention, CP004(M) is also referred to as CP004(hG1WT). As the "wild type", the above CP004(M) uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region, and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0181] Preparation Example 4: Non-Variable Region Amino Acid Mutation Design Based on Humanized Bifunctional Antibody CP004

[0182] Based on CP004(hG1WT) obtained in Preparation Example 3, the present inventors introduced a point mutation from leucine to alanine (L234A) at the 234th site of its heavy chain, a point mutation from leucine to alanine (L235A) at the 235th site, and a point mutation from glycine to alanine (G237A) at the 237th site, and obtained CP004(hG1TM).

[0183] The DNA sequence of the heavy chain of the immunoglobulin part in CP004(hG1TM): (1344 bp, the mutation sites are underlined, SEQ ID NO: 39)

[0184] The amino acid sequence of the heavy chain of the immunoglobulin part in CP004(hG1TM): (448 aa, the mutation sites are underlined, SEQ ID NO: 40)

[0185] The DNA sequences of the light chains of CP004(hG1TM) and CP004(hG1WT) are the same, and the encoded amino acid sequences are also the same. For the specific sequences, see Preparation Example 3.

[0186] Preparation Example 5: Preparation of Anti-PD-1 / CTLA-4 Bispecific Antibody Cadonilimab

[0187] Anti-PD-1 / CTLA-4The structural pattern of the bispecific antibody Cadonilimab (i.e., CP004(hG1TM)) belongs to the Morrison pattern (IgG-scFv), that is, the C-terminals of the two heavy chains of an immunoglobulin part (IgG) antibody are each connected to the VH end of the scFv fragment of another antibody through a linker fragment. Its immunoglobulin part is based on a PD-1 antibody, and the scFv fragment is based on an anti-CTLA-4 antibody, which are linked by a linker fragment in the middle.

[0188] The light chain sequence (SEQ ID NO:72) and heavy chain sequence (SEQ ID NO:70) of the anti-PD-1 / CTLA-4 bispecific antibody Cadonilimab (i.e., CP004(hG1TM)) described in this example are derived from WHO Drug Information, Proposed INN: List 124 (WHO Drug Information, 2020; 34(4): 947-949), and the preparation method is as follows.

[0189] The DNA sequences of the heavy chain-encoding gene of the candidate monoclonal antibody, including the constant region and variable region, and the DNA sequence of the light chain-encoding gene, including the constant region and variable region, are respectively cloned into the pUC57simple vector (provided by GenScript Corporation) to obtain the pUC57simple-H and pUC57simple-L plasmids.

[0190] The above plasmids are respectively digested with enzymes (HindIII&EcoRI), and the recovered heavy and light chains obtained by electrophoresis are subcloned into the expression vector pcDNA3.1 (purchased from Invitrogen Corporation). The recombinant plasmids are extracted and co-transfected into 293F cells according to the combination of pcDNA3.1-H + pcDNA3.1-L. After culturing the cells for 7 days, the culture medium is centrifuged at high speed, concentrated in the supernatant and loaded onto a HiTrap MabSelect SuRe column, and purified using a protein purification liquid chromatography system (AKTA Purifier 10, GE).

[0191] The purified sample, that is, the antibody Cadonilimab, is added with a reducing protein electrophoresis loading buffer and a non-reducing protein electrophoresis loading buffer, and after boiling, SDS-PAGE electrophoresis detection is carried out. The target protein of the reducing protein sample is at 70kD and 30KD, and the target protein of the non-reducing protein sample (single antibody) is at 200kD. It conforms to the theoretical size, and thus the purified antibody is obtained.

[0192] Preparation Example 6: Preparation of anti-VEGFR2 antibody HpL3

[0193] The antibody light chain variable region and heavy chain variable region sequences of the anti-VEGFR2 antibody HpL3 described in this example, as well as the preparation method, are all cited from the patent "A Class of Monoclonal Antibodies Against Vascular Endothelial Growth Factor Receptor VEGFR2, Their Encoding Genes and Applications" with the application number CN201610573836.X.

[0194] The amino acid sequence of the heavy chain variable region of HpL3 is shown as SEQ ID NO: 50, the DNA sequence of its encoding gene is shown as SEQ ID NO: 49, and the sequences of heavy chain variable region CDR1-3 are shown as SEQ ID NO: 53-55 respectively.

[0195] The amino acid sequence of the light chain variable region of HpL3 is shown as SEQ ID NO: 52, the DNA sequence of its encoding gene is shown as SEQ ID NO: 51, and the sequences of light chain variable region CDR1-3 are shown as SEQ ID NO: 56-58 respectively.

[0196] HpL3 uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region, and Igkappa chain C region, ACCESSION: P01834 as the light chain constant region

[0197] The DNA sequences of the heavy chain encoding gene of the candidate monoclonal antibody, including the constant region and variable region, and the DNA sequence of the light chain encoding gene, including the constant region and variable region, are respectively cloned into the pUC57simple vector (provided by GenScript Corporation) to obtain pUC57simple-PCABH and pUC57simple-L3 plasmids respectively.

[0198] The above plasmids are respectively digested (HindIII&EcoRI), and the heavy chain and light chain obtained by electrophoresis recovery are respectively subcloned into the expression vector pcDNA3.1 (purchased from Invitrogen Corporation). The recombinant plasmids are extracted and co-transfected into 293F cells according to the combination of pcDNA3.1-PCABH + pcDNA3.1-L3. After culturing the cells for 7 days, the culture medium is centrifuged at high speed, concentrated by the supernatant, and loaded onto a HiTrap MabSelect SuRe column, and purified using a protein purification liquid chromatography system (AKTA Purifier 10, GE).

[0199] The purified sample, namely the antibody HpL3, was added to the reducing protein electrophoresis loading buffer and the non-reducing protein electrophoresis loading buffer. After boiling, SDS-PAGE electrophoresis was performed for detection. The target protein of the reducing protein sample was at 45 kD and 30 kD, and the target protein of the non-reducing protein sample (single antibody) was at 150 kD. This was in line with the theoretical size, and thus the purified monoclonal antibody was obtained.

[0200] Example 1. Mixed lymphocyte reaction MLR to detect the bioactivity of promoting INF-γ secretion by the combination of the anti-PD-1 / CTLA-4 bispecific antibody Cadonilimab and the anti-VEGFR2 antibody HpL3

[0201] Preparation of Raji-PDL1 cells:

[0202] The human PD-L1 overexpression lentiviral vector plenti6.3 / V5-PDL1FL-BSD was packaged with virus and used to infect Raji cells. After drug screening with 10 μg / ml BSD (Blasticidin), the drug-resistant cell line Raji-PDL1 was obtained. plenti6.3 / V5-PDL1FL-BSD was synthesized by the supplier GenScript Nanjing with PDL1FL (PDL1, Genebank ID: NP_054862.1), and then obtained by digestion and ligation to plenti6.3 / V5-BSD (Invitrogen catalog number k531520).

[0203] Two days before the experiment, PBMCs (isolated from normal human peripheral blood) were resuscitated and cultured in a complete medium in an incubator at 37 °C and 5% carbon dioxide. After 2 h, when the PBMCs recovered, SEB (final concentration 0.5 μg / mL) (Toxin technology, catalog number: BT202) was added and the cells were stimulated for two days. On the day of the experiment, Raji-PDL1 cells were collected, centrifuged, resuspended and counted (resuspended with 1640 + 10% FBS), and the cell density was adjusted to 2×10 6 / mL, and MMC (Mito-mycin C, final concentration 2 μg / mL, Mitomycin C, manufacturer: Stressmarq, catalog number: SIH-246) was added and the cells were treated in an incubator at 37 °C and 5% carbon dioxide for 1 h. PBMCs stimulated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with 1640 basal medium, resuspended in a complete medium and counted. According to PBMC 1×10 5 / well, Raji-PDL1 1×10 5Inoculate into 96-well U-bottom plates (3799) at 50 μL / well; add drugs according to the experimental design, and set up blank controls and negative controls, and culture for 3 days (the final volume of the system is 200 μL); after 3 days, centrifuge at 250 x g for 5 min (Beckman centrifuge), collect the cell supernatant, and detect the IFN-γ content using a Becton Dickinson kit.

[0204] As Figure 1 shown, in the co-culture system of PBMC and Raji-PDL1 cells, the addition of Cadonilimab and HpL3 antibody can significantly induce PBMC to further secrete IFN-γ, showing a significant dose-dependent relationship. In terms of the activity level of promoting IFN-γ secretion, compared with Cadonilimab and HpL3 antibody monotherapy, the combination of Cadonilimab and HpL3 has better potential for promoting IFN-γ secretion.

[0205] Example 2. Mixed lymphocyte reaction (MLR) to detect the biological activity of Cadonilimab combined with bevacizumab in promoting PBMC activation and IL-2 secretion

[0206] Two days before the experiment, resuscitate PBMC (isolated from peripheral blood voluntarily donated by healthy volunteers after informed consent), and culture in complete medium in a 37 °C, 5% carbon dioxide incubator; after 2 h, wait for the PBMC to recover, and add SEB (final concentration 0.5 μg / mL) (Toxin technology, catalog number: BT202) to stimulate for two days; on the day of the experiment, collect Raji-PDL1 (constructed by Akeso) cells, centrifuge, resuspend and count (resuspend with 1640 + 10% FBS), adjust the cell density to 2×10 6 / mL, add MMC (Mito-mycin C, final concentration 2 μg / mL) (mitomycin C, manufacturer: Stressmarq, catalog number: SIH-246) and incubate in a 37 °C, 5% carbon dioxide incubator for 1 hour; collect PBMC after being stimulated with SEB for two days and Raji-PDL1 cells after being treated with MMC, wash twice with 1640 basal medium, resuspend the cells with analysis medium and count, and inoculate at 1×10 5 / well for PBMC and 1×10 5 / well for Raji-PDL1 (40 μL / well each) into 96-well U-bottom plates (3799); routinely digest and collect Hela cells (purchased from the Chinese Academy of Sciences), and inoculate at 2×10 4 / well (40 μL / well) into 96-well U-bottom plates (3799); add drugs according to the experimental design, and set up negative controls and isotype controls, and culture for 3 days (the final volume of the system is 200 μL); after 3 days, centrifuge at 250 x g for 5 min (Beckman centrifuge), collect the cell supernatant, and detect the IL-2 content using a Becton Dickinson kit.

[0207] As shown Figure 2 in the co-culture system of PBMC, Raji-PDL1, Hela (cervical cancer) cells and VEGF (batch number: 20180126-1, produced by Akeso), the addition of Cadonilimab and bevacizumab can significantly induce PBMC to further secrete IL-2, showing a significant dose-dependent relationship. In terms of the activity level of promoting IL-2 secretion, compared with Cadonilimab and bevacizumab monotherapy, the combination of Cadonilimab and bevacizumab has better potential for promoting IL-2 secretion.

[0208] The amino acid sequence of the heavy chain variable region of bevacizumab is as shown in SEQ ID NO:60, the DNA sequence of its encoding gene is as shown in SEQ ID NO:59, and the sequences of heavy chain variable region CDR1-3 are as shown in SEQ ID NO:63-65 respectively. The amino acid sequence of the light chain variable region of bevacizumab is as shown in SEQ ID NO: 62, the DNA sequence of its encoding gene is as shown in SEQ ID NO:61, and the sequences of light chain variable region CDR1-3 are as shown in SEQ ID NO:66-68 respectively.

[0209] Sequence Listing:

[0210] SEQ ID No.1: Nucleic acid sequence of the heavy chain variable region of 4G10

[0211] caggtcaagctgcaggagtctggacctgagctggtgaagcctggagcttcaatgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagaaccttgaatggattggacttattaatccttacaataatattactaactacaaccagaagttcatgggcaaggccacatttactgtagacaagtcatccagcacagcctacatggaactcctcagactgacatctgaagactctggagtctatttctgtgcaagactcgactataggtcttattggggccaagggactctggtcactgtctctgcagccaaaacgacacccccatctgtct at

[0212] SEQ ID No.2: Amino acid sequence of the heavy chain variable region of 4G10

[0213] QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY

[0214] SEQ ID No.3: Nucleic acid sequence of the variable region of the 4G10 light chain

[0215] caggctgttgtgactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcacttgtcgctcaagtactggggctgttacaactagtaactttgccaactgggtccaagaaaaaccagatcatttattcactagtctaataggtggtaccaacaaccgagctccaggtgttcctgccagattctcaggctccctgattggagacaaggctgccctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaaccattgggtgttcggtggaggaaccaaactgactgtcctaggccagcccaagtcttcgccatcagtcaccctgtttcaagggcaattctgc

[0216] SEQ ID No.4: Amino acid sequence of the variable region of the 4G10 light chain

[0217] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC

[0218] SEQ ID No.5: Nucleic acid sequence of the variable region of the 4G10H1L1 heavy chain

[0219] caggtgcagctggtggagtctggggccgagctggtgaagcccggcgcctccatgaagatctcttgcaaggccagcggatacagtttcactggctataccatgaactgggtcaaacaggctccaggacagggactggagtggatcgggctgattaatccttacaacaacatcaccaactacaaccagaagttcatgggaaaagcaacctttacagtggacaagagcatttccacagcctacatggaactgagccggctgacttcagacgatagcggggtctatttttgtgcaaggctggattatcgctcttactgggggcagggaactctggtcactgtctccgct

[0220] SEQ ID No.6: Amino acid sequence of the variable region of the heavy chain of 4G10H1L1

[0221] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA

[0222] SEQ ID No.7: Nucleic acid sequence of the variable region of the light chain of 4G10H1L1

[0223] caggctgtcgtcactcaggaaccttcactgactgtgagcccaggaggaactgtcaccctgacatgcggaagctccaccggagcagtgaccacatccaacttcgccaattgggtccaggaaaagccaggccaggcatttcgatccctgatcggaggcacaaacaatcgggcttcttgggtgcccgcaagattctcaggaagcctgctggggggaaaagccgctctgaccattagtggcgctcagcctgaggacgaagccgagtacttctgcgctctgtggtatagcaaccactgggtgtttggcgggggaacaaagctgactgtgctg

[0224] SEQ ID No.8: Amino acid sequence of the variable region of the light chain of 4G10H1L1

[0225] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL

[0226] SEQ ID No.9: Nucleic acid sequence of the variable region of the heavy chain of 4G10H3L3

[0227] caggtgcagctggtcgagtctggggccgaagtgaagaaacccggcgcctcagtgaaggtcagctgcaaggccagcgggtacagtttcactggatataccatgaactgggtccgacaggcccctggccaggggctggagtggatcggcctgattaacccttacaacaacatcactaactacgcacagaagttccaggggagagtgacctttacagtggacaccagcatttccacagcctac atggaactgtcccggctgagatctgacgatacaggcgtgtacttctgcgctaggctggattaccgcagctattggggacagggcacactggtgactgtcagcgca

[0228] SEQ ID No.10: Amino acid sequence of the variable region of the heavy chain of 4G10H3L3

[0229] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA

[0230] SEQ ID No.11: Nucleic acid sequence of the variable region of the light chain of 4G10H3L3

[0231] caggctgtcgtcactcaggaaccttcactgaccgtgtctcctggcgggactgtcaccctgacatgcggcagctccacaggggccgtgaccacaagtaacttcccaaattgggtccagcagaagccaggacaggctccccggagtctgatcggaggcaccaacaacaaggccagctggacacccgcacggttcagcggcagcctgctgggcggcaaggccgctctgacaattagcggagcccagcctgaggacgaagccgagtactattgcgctctgtggtactccaaccactgggtgttcggcggcggcaccaagctgactgtgctg

[0232] SEQ ID No.12: Amino acid sequence of the variable region of the light chain of 4G10H3L3

[0233] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL

[0234] SEQ ID No.13: Nucleic acid sequence of the variable region of the heavy chain of 14C12

[0235] gaggtcaaactggtggagagcggcggcgggctggtgaagcccggcgggtcactgaaactgagctgcgccgcttccggcttcgcctttagctcctacgacatgtcatgggtgaggcagacccctgagaagcgcctggaatgggtcgctactatcagcggaggcgggcgatacacctactatcctgactctgtcaaagggagattcacaattagtcgggataacgccagaaatactctgtatctgcagatgtctagtctgcggtccgaggatacagctctgtactattgtgcaaaccggtacggcgaagcatggtttgcctattggggacagggcaccctggtgacagtctctgcc

[0236] SEQ ID No.14: Amino acid sequence of the heavy chain variable region of 14C12

[0237] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA

[0238] SEQ ID No.15: Nucleic acid sequence of the light chain variable region of 14C12

[0239] gacattaagatgacacagtccccttcctcaatgtacgctagcctgggcgagcgagtgaccttcacatgcaaagcatcccaggacatcaacacatacctgtcttggtttcagcagaagccaggcaaaagccccaagaccctgatctaccgggccaatagactggtggacggggtccccagcagattctccggatctggcagtgggcaggattactccctgaccatcagctccctggagtatgaagacatgggcatctactattgcctgcagtatgatgagttccctctgacctttggagcaggcacaaaactggaactgaag

[0240] SEQ ID No.16: Amino acid sequence of the light chain variable region of 14C12

[0241] DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK

[0242] SEQ ID No.17: Nucleic acid sequence of the heavy chain variable region of 14C12H1L1

[0243] gaagtgcagctggtcgagtctgggggagggctggtgcagcccggcgggtcactgcgactgagctgcgcagcttccggattcgcctttagctcctacgacatgtcctgggtgcgacaggcaccaggaaagggactggattgggtcgctactatctcaggaggcgggagatacacctactatcctgacagcgtcaagggccggttcacaatctctagagataacagtaagaacaatctgtatctgcagatgaacagcctgagggctgaggacaccgcactgtactattgtgccaaccgctacggggaagcatggtttgcctattgggggcagggaaccctggtgacagtctctagt

[0244] SEQ ID No.18: Amino acid sequence of the heavy chain variable region of 14C12H1L1

[0245] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS

[0246] SEQ ID No.19: Nucleic acid sequence of the light chain variable region of 14C12H1L1

[0247] gacattcagatgactcagagcccctcctccatgtccgcctctgtgggcgacagggtcaccttcacatgccgcgctagtcaggatatcaacacctacctgagctggtttcagcagaagccagggaaaagccccaagacactgatctaccgggctaatagactggtgtctggagtcccaagtcggttcagtggctcagggagcggacaggactacactctgaccatcagctccctgcagcctgaggacatggcaacctactattgcctgcagtatgatgagttcccactgacctttggcgccgggacaaaactggagctgaag

[0248] SEQ ID No.20: Amino acid sequence of the variable region of the light chain of 14C12H1L1

[0249] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK

[0250] SEQ ID No.21: DNA sequence (1344 bp) of the heavy chain (14C12H1) of 14C12H1L1

[0251]

[0252] SEQ ID No.22: Amino acid sequence (448 aa) encoded by the heavy chain of 14C12H1L1 (14C12H1)

[0253] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0254] SEQ ID No.23: DNA sequence (642 bp) of the light chain of 14C12H1L1 (14C12L1)

[0255] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT

[0256] SEQ ID No.24: Amino acid sequence (214 aa) encoded by the light chain of 14C12H1L1 (14C12L1)

[0257] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0258] SEQ ID No.25: Amino acid sequence of Linker1

[0259] GGGGSGGGGSGGGGS

[0260] SEQ ID No.26: Amino acid sequence of Linker2

[0261] GGGGSGGGGSGGGGSGGGGS

[0262] SEQ ID No.27: HCDR1 of 14C12: GFAFSSYD

[0263] SEQ ID No.28: HCDR2 of 14C12: ISGGGRYT

[0264] SEQ ID No.29: HCDR3 of 14C12: ANRYGEAWFAY

[0265] SEQ ID No.30: LCDR1 of 14C12: QDINTY

[0266] SEQ ID No.31: LCDR2 of 14C12: RAN

[0267] SEQ ID No.32: LCDR3 of 14C12: LQYDEFPLT

[0268] SEQ ID No.33: HCDR1 of 4G10: GYSFTGYT

[0269] SEQ ID No.34: HCDR2 of 4G10: INPYNNIT

[0270] SEQ ID No.35: HCDR3 of 4G10: ARLDYRSY

[0271] SEQ ID No.36: LCDR1 of 4G10: TGAVTTSNF

[0272] SEQ ID No.37: LCDR2 of 4G10: GTN

[0273] SEQ ID No.38: LCDR3 of 4G10: ALWYSNHWV

[0274] SEQ ID No.39: DNA sequence of the heavy chain of the immunoglobulin portion of CP004 (hG1TM), with mutation sites underlined

[0275] GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGA GCCCCTAGCGTCTTCCTCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGACCATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCAGGTGGCAGCAGGGAAACGTGTTCTCCTGCAGCGTGATGCACGAAGCCCTCCACAACCACTACACCCAGAAAAGCCTGTCCCTGAGCCCCGGCAAA

[0276] SEQ ID No.40: Amino acid sequence of the heavy chain of the immunoglobulin portion in CP004 (hG1TM), with the mutation sites underlined

[0277] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA G APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0278] SEQ ID No.41: Amino acid sequence of 4G10H1V(M), with the mutation sites underlined

[0279] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ C LEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA

[0280] SEQ ID No.42: Amino acid sequence of 4G10L1V(M), with the mutation sites underlined

[0281] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFG C GTKLTVL R

[0282] SEQ ID No.43: Amino acid sequence of 4G10H3V(M), with the mutation sites underlined

[0283] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ C LEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA

[0284] SEQ ID No.44: Amino acid sequence of 4G10L3V(M), with the mutation sites underlined

[0285] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFG C GTKLTVL R

[0286] SEQ ID No: 45: Heavy chain variable region sequence of hIgG

[0287] EVQLEQSGAELMKPGASVKISCKATGYTFTTYWIEWIKQRPGHSLEWIGEILPGSDSTYYNEKVKGKVTFTADASSNTAYMQLSSLTSEDSAVYYCARGDGFYVYWGQGTTLTVSS

[0288] SEQ ID No: 46 Heavy chain constant region sequence of hIgG

[0289] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0290] SEQ ID No: 47 Light chain variable region sequence of hIgG

[0291] DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYTSQSMSGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSGSWPRTFGGGTKLDIK

[0292] SEQ ID No: 48 Light chain constant region sequence of hIgG

[0293] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0294] SEQ ID No: 49 Nucleic acid sequence of the variable region of the heavy chain of HpL3 (VEGFR2)

[0295] GAAGTGCAGCTGGTGCAGAGCGGCGGGGGACTGGTGAAGCCCGGCGGGTCCCTGCGACTGTCTTGCGCCGCTAGT GGCTTCACCTTCAGCTCCTACTCT ATGAACTGGGTGAGACAGGCCCCTGGAAAAGGCCTGGAGTGGGTCTCTAGT ATCTCAAGCTCCTCTAGTTACATC TACTATGCCGACAGCGTGAAGGGGCGGTTCACCATCTCAAGAGATAACGCTAAAAATAGTCTGTATCTGCAGATGAACAGCCTGAGGGCAGAAGACACTGCCGTGTACTATTGT GCTCGCGT CACCGACGCATTTGATATT TGGGGGCAGGGAACCATGGTGACAGTCTCAAGC (Note: The underlined sequences represent CDR sequences)

[0296] SEQ ID No: 50 Amino acid sequence of the variable region of the heavy chain of HpL3 EVQLVQSGGGLVKPGGSLRLSCAAS GFT FSSYS MNWVRQAPGKGLEWVSS ISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARVTDAFD I WGQGTMVTVSS (Note: The underlined sequences represent CDR sequences)

[0297] SEQ ID No: 51 Nucleic acid sequence of the variable region of the light chain of HpL3

[0298] GACATTCAGATGACTCAGAGCCCTTCTTCAGTGTCCGCCTCTATCGGCGACCGGGTCACCATTACATGCAGAGCTTCC CAGGGCCTCGATAACTGG CTGGGGTGGTACCAGCAGAAGCCTGGGAAAGCCCCAAAGCTGCTGATCTAC GACGCTTCCAATCTGGATACCGGAGTGCCATCTCGATTCAGTGGCTCAGGGAGCGGAACATACTTTACTCTGACCATCAGCTCCCTGCAGGCTGAGGACTTCGCAGTGTATTTTTGC CAGCAGGCAAAGGCCTTCCCTCCCACC TTTGGCGGGGGAACAAAAGTGGACATCAAG (Note: The underlined sequences represent CDR sequences)

[0299] SEQ ID No: 52 Amino acid sequence of the variable region of the HpL3 light chain

[0300] DIQMTQSPSSVSASIGDRVTITCRAS QGLDNW LGWYQQKPGKAPKLLIY DAS NLDTGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFC QQAKAFPPT FGGGTKVDIK (Note: The underlined sequences represent CDR sequences)

[0301] HCDR1-HCDR3 of the variable region of the HpL3 heavy chain

[0302] HCDR1: GFTFSSYS (SEQ ID NO: 53)

[0303] HCDR2: ISSSSSYI (SEQ ID NO: 54)

[0304] HCDR3: ARVTDAFDI (SEQ ID NO: 55)

[0305] LCDR1-LCDR3 of the variable region of the HpL3 light chain

[0306] LCDR1: QGLDNW (SEQ ID NO: 56)

[0307] LCDR2: DAS (SEQ ID NO: 57)

[0308] LCDR3: QQAKAFPPT (SEQ ID NO: 58)

[0309] SEQ ID No: 59 Nucleic acid sequence of the variable region of the Bevacizumab heavy chain (VEGF)

[0310] GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC

[0311] SEQ ID No: 60 Amino acid sequence of the heavy chain variable region of Bevacizumab

[0312] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS

[0313] SEQ ID No: 61 Nucleic acid sequence of the light chain variable region of Bevacizumab

[0314] GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG

[0315] Amino acid sequence of the variable region of the light chain of SEQ ID No: 62 Bevacizumab

[0316] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK

[0317] The amino acid sequences of the three CDR regions of the variable region of the heavy chain of Bevacizumab are as follows:

[0318] HCDR1: GYTFTNYG (SEQ ID NO: 63)

[0319] HCDR2: INTYTGEP (SEQ ID NO: 64)

[0320] HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO: 65)

[0321] The amino acid sequences of the three CDR regions of the variable region of the light chain of Bevacizumab are as follows:

[0322] LCDR1: QDISNY (SEQ ID NO: 66)

[0323] LCDR2: FTS (SEQ ID NO: 67)

[0324] LCDR3: QQYSTVPWT (SEQ ID NO:68)

[0325] Nucleic acid sequence of the heavy chain of SEQ ID No.69 CP004(hG1TM)

[0326]

[0327] Amino acid sequence (full length) of the heavy chain of SEQ ID No.70 CP004 (hG1TM)

[0328] EVQLVESGGGLVQPGGSLRLSCAAS GFAFSSYD MSWVRQAPGKGLDWVAT ISGGGRYT YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC ANRYGEAWFAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQVQLVESGAEVKKPGASVKVSCKAS GYSFTGYT MNWVRQAPGQCLEWIGL INPYNN IT NYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFC ARLDYRSY WGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSS TGAVTTSNF PNWVQQKPGQAPRSLIG GTN NKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYC ALWYSNHWV FGCGTKLTVLR (Note: The underlined sequences indicate CDR sequences)

[0329] Nucleic acid sequence of the light chain of SEQ ID No.71 CP004(hG1TM) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT

[0330] Amino acid sequence (full length) of the light chain of SEQ ID No.72 CP004(hG1TM)

[0331] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A pharmaceutical composition comprising an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody and a component, wherein Component A is an anti-VEGF monoclonal antibody or an antigen-binding fragment thereof, wherein (1) The anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional region targeting PD-1, and a second protein functional region targeting CTLA4; 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; wherein, the immunoglobulin comprises HCDR1-HCDR3 in the heavy-chain variable region shown in SEQ ID NO:14 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 27-29 respectively), and LCDR1-LCDR3 in the light-chain variable region shown in SEQ ID NO:16 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 30-32 respectively); and the single-chain antibody comprises HCDR1-HCDR3 in the heavy-chain variable region shown in SEQ ID NO:2 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 33-35 respectively) and LCDR1-LCDR3 in the light-chain variable region shown in SEQ ID NO:4 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 36-38 respectively); or, the immunoglobulin comprises HCDR1-HCDR3 in the heavy-chain variable region shown in SEQ ID NO:2 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 33-35 respectively) and LCDR1-LCDR3 in the light-chain variable region shown in SEQ ID NO:4 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 36-38 respectively); and the single-chain antibody comprises HCDR1-HCDR3 in the heavy-chain variable region shown in SEQ ID NO:14 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 27-29 respectively), and LCDR1-LCDR3 in the light-chain variable region shown in SEQ ID NO:16 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 30-32 respectively); (2) The anti-VEGF monoclonal antibody comprises HCDR1-HCDR3 in the heavy-chain variable region shown in SEQ ID NO:60 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 63-65 respectively) and LCDR1-LCDR3 in the light-chain variable region shown in SEQ ID NO:62 (preferably LCDR1-LCDR3 shown in SEQ ID NOs: 66-68 respectively).

2. The pharmaceutical composition according to claim 1, wherein, (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the sequences shown in SEQ ID NO: 14, SEQ ID NO: 18 or variants thereof; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the sequences shown in SEQ ID NO: 16, SEQ ID NO: 20 or variants thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from the sequences shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 43 or variants thereof; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from the sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42, SEQ ID NO: 44 or variants thereof; Or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the sequences shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 43 or variants thereof; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42, SEQ ID NO: 44 or variants thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from the sequences shown in SEQ ID NO: 14, SEQ ID NO: 18 or variants thereof; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from the sequences shown in SEQ ID NO: 16, SEQ ID NO: 20 or variants thereof; Or (2) The amino acid sequence of the heavy chain variable region of the anti-VEGF monoclonal antibody is the sequence shown in SEQ ID NO: 60 or a variant thereof; and the amino acid sequence of the light chain variable region of the anti-VEGF monoclonal antibody is the sequence shown in SEQ ID NO: 62 or a variant thereof, wherein the sequence of the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein The bispecific antibody is selected from any one of the following (1)-(20): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 4 or a variant thereof; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 8 or a variant thereof; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 12 or a variant thereof; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 4 or a variant thereof; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 8 or a variant thereof; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 12 or a variant thereof; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 6 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof; (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof; (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 42 or a variant thereof; (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 44 or a variant thereof; (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 42 or a variant thereof; (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 44 or a variant thereof; (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof; (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 14 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 16 or a variant thereof; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof; And, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 43 or a variant thereof, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a variant thereof; and, the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 18 or a variant thereof, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20 or a variant thereof, The sequence of said variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the amino acid sequence of the heavy chain of said immunoglobulin is the sequence shown in SEQ ID NO: 40 or a variant thereof, and the amino acid sequence of its light chain is the sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the sequence of said variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein Said first protein functional region is directly connected to said second protein functional region or connected through a linking fragment; and / or the heavy chain variable region of said single-chain antibody is directly connected to the light chain variable region of said single-chain antibody or connected through a linking fragment.

6. The pharmaceutical composition according to claim 5, wherein, Said linking fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein, Said first protein functional region and second protein functional region are independently 1, 2 or more than 2.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein, Said single-chain antibody (preferably the heavy chain variable region) is linked to the heavy chain of the immunoglobulin, preferably linked to the C-terminus of the heavy chain of the immunoglobulin.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein said immunoglobulin is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of said immunoglobulin has an A mutation, and the A mutation is selected from one of the following mutation combinations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, G237A; or According to the EU numbering system, the heavy chain constant region of said immunoglobulin has a B mutation, and the B mutation is selected from one or more of the following mutations: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A; or Or, according to the EU numbering system, the heavy chain constant region of said immunoglobulin has a combination of A mutation and B mutation; The heavy chain constant region of the anti-VEGFR2 monoclonal antibody or the anti-VEGF monoclonal antibody is Ig gamma-1 chain C region, ACCESSION P01857, and the light chain constant region is: Ig kappa chain C region, ACCESSION: P01834.

10. The pharmaceutical composition according to any one of claims 1 to 9, Among them, The bispecific antibody comprises: a first protein functional region targeting PD-1, and a second protein functional region targeting CTLA4; There is 1 first protein functional region and 2 second protein functional regions; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is as shown in SEQ ID NO: 40 or a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, and the amino acid sequence of its light chain is as shown in SEQ ID NO: 24 or a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto; The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 43 or a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 44 or a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology thereto; The single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; The first protein functional region is linked to the second protein functional region through a first linker fragment; and the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody through a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO:25 and SEQ ID NO: 26; Preferably, the amino acid sequences of the first and second linking fragments are both as shown in SEQ ID NO:

26. Preferably, the light chain sequence of the anti-PD-1 / CTLA-4 bispecific antibody is as shown in SEQ ID NO: 72, and the heavy chain sequence is as shown in SEQ ID NO:

70. Preferably, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, complementarity-determining region (CDR) fragment, single-chain antibody (e.g., scFv), diabody or domain antibody.

11. The pharmaceutical composition according to any one of claims 1-10, wherein the mass ratio of the anti-CTLA4-anti-PD-1 bispecific antibody to component A is selected from (1:5)-(5:1), such as: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1; preferably, the pharmaceutical composition is in solid form or liquid form, optionally, the pharmaceutical composition further comprises one or more chemotherapeutic drugs (preferably the chemotherapeutic drug is an alkylating agent, antimetabolite, antibiotic, plant drug and / or hormonal drug, platinum drugs such as cisplatin, carboplatin, oxaliplatin, doxorubicin, cyclophosphamide, paclitaxel (such as albumin-bound paclitaxel, liposomal paclitaxel, docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil anti-tumor drugs).

12. Use of the anti-CTLA4-anti-PD-1 bispecific antibody and Component A as defined in any one of claims 1-10 in the preparation of a medicament or a kit for preventing and / or treating tumors (especially malignant tumors), optionally, the medicament or the kit further comprises one or more drugs for treating tumors (preferably the drug is a chemotherapeutic agent or a growth inhibitor (such as an alkylating agent, anthracycline, antihormonal agent, aromatase inhibitor, antiandrogen, protein kinase inhibitor, lipid kinase inhibitor, antisense oligonucleotide, ribozyme, antimetabolite, topoisomerase inhibitor, cytotoxic agent or antitumor antibiotic, proteasome inhibitor, antimicrotubule agent, EGFR antagonist, VEGF antagonist, angiopoietin 2 antagonist, retinoid, tyrosine kinase inhibitor, histone deacetylase inhibitor and combinations thereof), targeted therapeutic agent (such as B-raf inhibitor, MEK inhibitor, K-ras inhibitor, c-Met inhibitor, Alk inhibitor, phosphatidylinositol 3-kinase inhibitor, Akt inhibitor, mTOR inhibitor, VEGF inhibitor, PARP inhibitor, dual phosphatidylinositol 3-kinase / mTOR inhibitor and combinations thereof), antibody-drug conjugate (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelator), T cell expressing a chimeric antigen receptor, antibody or antigen-binding fragment thereof, angiogenesis inhibitor, antitumor agent, cancer vaccine, adjuvant and combinations thereof, alkylating agent, antimetabolic drug, antibiotic, plant drug and / or hormonal drug, platinum drug (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, docetaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil antitumor drug).

13. A kit for preventing and / or treating tumors (especially malignant tumors), comprising (a) a first pharmaceutical composition containing an anti-CTLA4-anti-PD-1 bispecific antibody defined in any one of claims 1-10 as an active ingredient; and (b) a second pharmaceutical composition containing component A defined in any one of claims 1-10 as an active ingredient; and optionally (c) one or more drugs for treating tumors (preferably the drugs are chemotherapeutic agents or growth inhibitors (such as alkylating agents, anthracyclines, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors and their combinations), targeted therapeutic agents (such as B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors and their combinations), antibody-drug conjugates (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or their antigen-binding fragments, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and their combinations, alkylating agents, antimetabolic drugs, antibiotics, plant drugs and / or hormonal drugs, platinum drugs (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel (such as albumin-bound paclitaxel, liposomal paclitaxel, docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil antitumor drugs).

14. Unit preparation, preferably for treating tumors (especially malignant tumors), wherein, The unit dosage form comprises: 1 to 10,000 mg (preferably 10 - 1,000 mg, more preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg or 200 mg) of the anti-CTLA4 - anti-PD-1 bispecific antibody defined in any one of claims 1 - 10; 1 to 10,000 mg (preferably 1 - 1,000 mg, more preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg, 200 mg or 100 mg) of component A defined in any one of claims 1 - 10; and optionally one or more anti-tumor drugs (preferably the drug is a chemotherapeutic agent or a growth inhibitor (such as an alkylating agent, anthracycline, antihormonal agent, aromatase inhibitor, antiandrogen, protein kinase inhibitor, lipid kinase inhibitor, antisense oligonucleotide, ribozyme, antimetabolite, topoisomerase inhibitor, cytotoxic agent or antitumor antibiotic, proteasome inhibitor, antimicrotubule agent, EGFR antagonist, VEGF antagonist, angiopoietin 2 antagonist, retinoid, tyrosine kinase inhibitor, histone deacetylase inhibitor and combinations thereof), targeted therapeutic agent (such as B-raf inhibitor, MEK inhibitor, K-ras inhibitor, c-Met inhibitor, Alk inhibitor, phosphatidylinositol 3-kinase inhibitor, Akt inhibitor, mTOR inhibitor, VEGF inhibitor, PARP inhibitor, dual phosphatidylinositol 3-kinase / mTOR inhibitor and combinations thereof), antibody-drug conjugate (such as maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelator), T cell expressing a chimeric antigen receptor, antibody or antigen-binding fragment thereof, angiogenesis inhibitor, antitumor agent, cancer vaccine, adjuvant and combinations thereof, antimetabolic drug, antibiotic, phytomedicine and / or hormonal drug, platinum drug (such as cisplatin, carboplatin, oxaliplatin), doxorubicin, cyclophosphamide, paclitaxel (such as albumin-bound paclitaxel, liposomal paclitaxel, docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol acetate, goserelin, asparaginase and / or fluorouracil antitumor drug); wherein the anti-CTLA4 - anti-PD-1 bispecific antibody, component A and the chemotherapeutic drug are separately packaged.

15. A single-dose pharmaceutical unit, preferably for the treatment of tumors (especially malignant tumors), comprising 0.1 - 10000 mg (preferably 1 - 1000 mg, preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg, 200 mg or 100 mg) of the anti-CTLA4 anti-PD-1 bispecific antibody as defined in any one of claims 1 - 10, and 0.1 - 10000 mg (preferably 1 - 1000 mg, preferably 50 - 500 mg, 100 - 400 mg, 150 - 300 mg, 150 - 250 mg, 200 mg or 100 mg) of component A as defined in any one of claims 1 - 10.

16. The pharmaceutical composition according to any one of claims 1 - 11, the use according to claim 12, the kit according to claim 13, the unit dosage form according to claim 14, or the single-dose pharmaceutical unit according to claim 15, wherein the tumor is selected from one or more of the following: cervical cancer (such as metastatic cervical cancer), lung cancer such as non-small cell lung cancer (e.g., squamous non-small cell lung cancer or non-squamous non-small cell lung cancer), esophageal cancer, esophageal squamous cell carcinoma, ileocecal adenocarcinoma, ampullary adenocarcinoma, small cell lung cancer, gastric cancer (e.g., advanced gastric cancer, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma), kidney cancer, endometrial cancer of the kidney, adrenocortical carcinoma, prostate cancer, thyroid cancer, peritoneal cancer, adenocarcinoma, pancreatic cancer, glioma, head and neck cancer, bone cancer, testicular cancer, leukemia, myeloma, lymphoma, sarcoma, mesothelioma, hepatocellular carcinoma, colon cancer, cholangiocarcinoma, bile duct cancer, rectal cancer, colon cancer, endometrial cancer, ovarian cancer (such as advanced ovarian cancer) or fallopian tube cancer, large cell neuroendocrine carcinoma, urothelial cancer (e.g., upper urothelial cancer or bladder cancer), breast cancer, triple-negative breast cancer, peripheral T-cell lymphoma, melanoma, nasopharyngeal carcinoma, and highly microsatellite-instable (MSI-H) or mismatch repair-deficient (dMMR) solid tumors, brain cancer (such as invasive brain cancer, e.g., glioblastoma), ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenoma, mucinous or serous cystadenocarcinoma, leiomyosarcoma, rhabdomyosarcoma, choriocarcinoma, malignant mole, malignant Sertoli-Leydig cell tumor, malignant granulosa cell tumor, dysgerminoma.

17. The pharmaceutical composition according to any one of claims 1 - 11, the use according to claim 12, the kit according to claim 13, the unit dosage form according to claim 14, or the single-dose pharmaceutical unit according to claim 15, wherein the anti-CTLA4 - anti-PD-1 bispecific antibody, component A and / or the chemotherapeutic agent are in a form suitable for intravenous injection or intravenous drip, preferably in a liquid form.

18. Use of the anti-CTLA4 anti-PD-1 bispecific antibody as defined in any one of claims 1 - 10 and component A as defined in any one of claims 1 - 10 in the preparation of a kit for the prevention and / or treatment of tumors, wherein, The kit contains an anti-CTLA4 anti-PD-1 bispecific antibody with a single-dose of 0.1-100 mg per kg body weight (preferably 1-10 mg) or 10-1000 mg per subject (preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg); Component A with a single-dose of 0.1-100 mg per kg body weight (preferably 1-10 mg) or 10-1000 mg per subject (preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg), Preferably, it is administered twice a day to about once 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 administration method is intravenous drip or intravenous injection.

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