Use of antibodies in antitumor treatments

Abstract

The present invention relates to a bispecific antibody against CTLA4 and PD-1, which is used to treat tumors and is administered in combination with an anti-VEGFR2 monoclonal antibody or an anti-VEGF monoclonal antibody and comprises a first protein functional region that targets PD-1 and a second protein functional region that targets 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.

Description

[Technical field]

[0001] Technical Field The present invention relates to the field of molecular immunology, in particular to the use of anti-vascular endothelial growth factor receptor VEGFR2 monoclonal antibodies or anti-VEGF monoclonal antibodies in combination with anti-PD-1 / CTLA-4 bispecific antibodies in the treatment of tumors.

[0002] background Tumors are serious health threats and there is an urgent need to develop effective treatments and drugs.

[0003] Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor that specifically acts on the growth factor of vascular endothelial cells, and is also called KDR or FLK1. Currently, five main types of VEGFR have been discovered: VEGFR-1, VEGFR-2, VEGFR-3, NP-1 and NP-2. Here, 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, VEGF exerts its physiological activity, such as promoting the proliferation of vascular endothelial cells, mainly by binding to VEGFR2.

[0004] Angiogenesis plays an important role in various human diseases, such as retinopathy, arthritis, endometriosis, etc. Tumor growth is usually accompanied by angiogenesis. As early as 1971, J. Folkman proposed that tumor growth and metastasis can be inhibited by suppressing tumor angiogenesis. In recent years, more and more malignant tumors have been found to be associated with vascular endothelial growth factor and its receptor family in the development, metastasis, and prognosis. Therefore, antitumor therapy targeting VEGF and its receptor has attracted attention again, and among the receptor family, targeting VEGFR-2 has been most widely studied.

[0005] PD-1 is an important immune checkpoint receptor expressed by activated T and B lymphocytes and mediates immune suppression, and its ligands include at least PD-L1 and PD-L2. Anti-PD-1 antibodies specifically bind to programmed death receptor-1 (PD-1) and block the inhibitory PD-1 / PD-1 ligand pathway (Topalian et al., (2012a) N Engl J Med 366: 2443-54).

[0006] PD-L1 (programmed death ligand 1), also known as CD274 or B7-H1, is a 40 kDa single-pass transmembrane protein encoded by the CD274 gene and is a ligand for PD-1. PD-L1 is widely distributed in various cancer cells as well as in leukocytes and non-hematopoietic cells in lymphoid and non-lymphoid tissues, and is highly expressed on the surface of various 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 have two extracellular Ig domains. The binding interface of PD-L1 with programmed death receptor-1 (PD-1) and B7-1 (CD80) is in the IgV-like domain (Lin et al., (2008) PNAS 105: 3011-3016). PD-L1 contains a conserved short intracellular tail (approximately 30 amino acids), and PD-1 contains two cytoplasmic tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). After T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motifs in its cytoplasmic tail, resulting in the dephosphorylation of effector molecules (e.g., CD3zeta, PKCtheta, and ZAP70) involved in the signaling cascade of CD3+ T cells (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 suggest that high tumor expression of PD-L1 is associated with increased tumor invasiveness and poor prognosis.

[0007] 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. This 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 of cognate antigen-specific T cells (Hofmeyer et al., (2011) Journal of Biomedicine and Biotechnology 2011: 1-9), promotes 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 upregulates T cell responses and generates autoreactive T cells (Latchman et al., (2004) PNAS, 101:10691-10696). Blockade of PD-1 or PD-L1 with antibodies results in increased antitumor immunity (Iwai et al., (2002) PNAS, 99:12293-12297).

[0008] CTLA-4 is also one of the representative specific immune checkpoint proteins. CTLA-4 is the first target to be clinically applied. Anti-CTLA-4 antibodies can promote T cell activation and improve its antitumor effect. Ipilimumab is the first CTLA-4 immune checkpoint inhibitor that has been demonstrated to improve the overall survival of previously treated metastatic melanoma patients and has been approved by the FDA. Current research shows that CTLA-4 inhibits T cell responses mainly through two pathways. One is by reducing the signal of TCR (T cell receptor) and CD28 by competitive binding with CD28 to B7 or by recruiting phosphatase to the intracellular domain of CTLA-4. The other is by reducing the expression levels of CD80 and CD86 on antigen presenting cells (APCs) or by removing them from APCs by transendocytosis, thereby reducing the activation of T cells involving CD28. In addition, CTLA-4 mediates dendritic cell binding to CD80 / CD86, inducing expression of the tryptophan degrading enzyme IDO, which results in inhibition of the TCR. Antibodies to CTLA-4 deplete Tregs, which in turn activate the TCR by binding to CTLA-4.

[0009] The combination of PD-1 antibody and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody drug has shown better efficacy against several tumors than PD-1 antibody alone. However, the improved efficacy of this combination is always accompanied by high toxicity, which greatly limits its application.

[0010] Bevacizumab is an antiangiogenic targeted drug that binds to vascular endothelial growth factor (VEGF) to block blood vessel growth and inhibit tumor metastasis. Bevacizumab is a broad spectrum anticancer drug. Since its launch, bevacizumab has been approved primarily for the treatment of various cancers, such as brain cancer (e.g., invasive brain cancer, e.g., 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.

[0011] overview The present inventors have found that the combination of anti-CTLA4-anti-PD-1 bispecific antibody with anti-VEGFR2 monoclonal antibody or with anti-VEGF monoclonal antibody can effectively prevent and treat tumors with low toxicity and side effects. The present invention is described in detail below.

[0012] Specifically, the present invention relates to the following:

[0013] 1. 1. An anti-CTLA4-anti-PD-1 bispecific antibody, The anti-CTLA4-anti-PD-1 bispecific antibody is preferably used to treat tumors, preferably the anti-CTLA4-anti-PD-1 bispecific antibody is administered in combination with component A, wherein component A is selected from an anti-VEGFR2 monoclonal antibody or an antigen-binding fragment thereof, an anti-VEGF monoclonal antibody or an antigen-binding fragment thereof, or a combination thereof; Where: (1) Anti-CTLA4-anti-PD-1 bispecific antibody a first protein functional region that targets PD-1; and a second protein functional domain that targets 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 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 14 (preferably, HCDR1 to HCDR3 shown in SEQ ID NOs: 27 to 29, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 16 (preferably, LCDR1 to LCDR3 shown in SEQ ID NOs: 30 to 32, respectively), and the single-chain antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 2 (preferably, HCDR1 to HCDR3 shown in SEQ ID NOs: 33 to 35, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 4 (preferably, LCDR1 to LCDR3 shown in SEQ ID NOs: 36 to 38, respectively), or the immunoglobulin comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 2 (preferably, HCDR1 to HCDR3 shown in SEQ ID NOs: 33 to 35, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 4 (preferably, LCDR1 to LCDR3 shown in SEQ ID NOs: 36 to 38, respectively); the single-chain antibody comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 14 (preferably, HCDR1 to HCDR3 shown in SEQ ID NOs: 27 to 29, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 16 (preferably, LCDR1 to LCDR3 shown in SEQ ID NOs: 30 to 32, respectively); (2) The anti-VEGFR2 monoclonal antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 50 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 53 to 55, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 52 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 56 to 58, respectively), or (3) The anti-VEGF monoclonal antibody comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 60 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 63 to 65, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 62 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 66 to 68, respectively). Anti-CTLA4-anti-PD-1 bispecific antibody.

[0014] 2. (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, 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, 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; or the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:43 or a variant thereof, 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, 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; (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 mutant 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 mutant thereof; or (3) The anti-CTLA4-anti-PD-1 bispecific antibody according to item 1, wherein 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.

[0015] 3. The bispecific antibody is selected from the group consisting of the following (1) to (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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a mutant thereof, 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 mutant 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 mutant thereof; (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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a mutant thereof, 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 mutant 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 mutant thereof; is selected from one of 3. The anti-CTLA4-anti-PD-1 bispecific antibody of item 1 or 2, 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 to the corresponding sequence.

[0016] 4. 4. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 3, wherein the amino acid sequence of the immunoglobulin heavy chain is the sequence shown in SEQ ID NO: 40 or a variant thereof, and the amino acid sequence of the immunoglobulin 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 to the corresponding sequence.

[0017] 5. The anti-CTLA4-anti-PD-1 bispecific antibody of any one of items 1 to 4, wherein a first protein functional region is linked to a second protein functional region either directly or via a linker fragment and / or a heavy chain variable region of the single chain antibody is linked to a light chain variable region of the single chain antibody either directly or via a linker fragment.

[0018] 6. The linker fragment is (GGGGS) nand n is a positive integer, preferably n is 1, 2, 3, 4, 5 or 6.

[0019] 7. 7. The anti-CTLA4-anti-PD-1 bispecific antibody of any one of items 1 to 6, wherein the number of first and second protein functional regions is each independently 1, 2 or more.

[0020] 8. 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 linked to the C-terminus of the heavy chain of the immunoglobulin.

[0021] 9. the immunoglobulin is of the human IgG1 subtype, wherein, according to the EU numbering system, the immunoglobulin comprises the following mutation: L234A and L235A or L234A and G237A or L235A and G237A or L234A, L235A and G237A and a heavy chain constant region having an A mutation selected from According to the EU numbering system, the immunoglobulin has 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 the immunoglobulin comprises a heavy chain constant region having a combination of A and B mutations according to the EU numbering system, or 9. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 8, wherein the heavy chain constant region of the anti-VEGFR2 monoclonal antibody or the anti-VEGF monoclonal antibody is the Ig gamma-1 chain C region, accession P01857, and the light chain constant region thereof is the Ig kappa chain C region, accession P01834.

[0022] 10. The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional domain that targets CTLA4; the number of first protein functional regions is 1 and the number of second protein functional regions is 2; wherein the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; the amino acid sequence of the heavy chain of said immunoglobulin is as set forth in SEQ ID NO:40 or a variant thereof and the amino acid sequence of the light chain of said immunoglobulin is as set forth in SEQ ID NO:24 or a variant thereof, wherein 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 to the corresponding sequence; 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% identity to the corresponding sequence; the single chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; a first protein functional region is linked to a second protein functional region via 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 via a second linker fragment, the first linker fragment and the second linker fragment being the same or different; Preferably, the first linker fragment and the second linker fragment each have an amino acid sequence independently selected from SEQ ID NO: 25 and SEQ ID NO: 26; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are as shown in SEQ ID NO: 26; Preferably, the light chain of the anti-PD-1 / CTLA-4 bispecific antibody has the sequence shown in SEQ ID NO:72 and the heavy chain of the anti-PD-1 / CTLA-4 bispecific antibody has the sequence shown in SEQ ID NO:70; Preferably, the antigen-binding fragment is Fab, Fab', F(ab') 2 10. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 9, which is selected from a fragment of a polypeptide, a Fd, an Fv, a dAb, a Fab / c, a complementarity determining region (CDR) fragment, a single chain antibody (e.g., scFv), a bivalent antibody, or a domain antibody.

[0023] 11. A pharmaceutical composition for treating a tumor (particularly a malignant tumor), comprising: A pharmaceutical composition comprising an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10 and component A as defined in any one of items 1 to 10, wherein the pharmaceutical composition is preferably in solid or liquid form, preferably the mass ratio of the anti-CTLA4-anti-PD-1 bispecific antibody to component A is selected from (1:5) to (5:1), for example 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1, and optionally the pharmaceutical composition further comprises one or more chemotherapeutic agents (preferably the chemotherapeutic agents are alkylating agents, antimetabolites, antibiotics, further comprising plant-derived and / or hormonal drugs, platinum-based drugs (e.g., cisplatin, carboplatin, and oxaliplatin), adriamycin, cyclophosphamide, paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, docetaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol, goserelin, asparaginase, and / or fluorouracil antineoplastic agents; Pharmaceutical compositions.

[0024] 12. Use of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10 in the manufacture of a medicament or kit for preventing and / or treating a tumor (particularly a malignant tumor), The medicament or kit preferably further comprises a component A as defined in any one of items 1 to 10, and optionally the medicament or kit further comprises one or more agents for treating a tumor (preferably the agents are chemotherapeutic agents or growth inhibitors (e.g. alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxins or antitumor antibiotics). , proteasome inhibitors, anti-microtubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof), targeted therapeutic agents (e.g., B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylserine-3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, dual phosphatidylserine-3-kinase inhibitors, phospho-ositol-3-kinase / mTOR inhibitors and combinations thereof), antibody-drug conjugates (e.g., maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antineoplastic agents, cancer vaccines, adjuvants, and combinations thereof, alkylating agents, antimetabolites, antibiotics, plant-derived and / or hormonal agents, platinum-based agents (e.g., cisplatin, carboplatin, and oxaliplatin), adriamycin, cyclophosphamide, paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, docetaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol, goserelin, asparaginase, and / or fluorouracil antineoplastic agents). use.

[0025] 13. A method for preventing and / or treating a tumor (particularly a malignant tumor), comprising: A therapeutically effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody according to any one of paragraphs 1 to 10 in combination with component A as defined in any one of paragraphs 1 to 10, more preferably with one or more agents for treating tumors (preferably the agents are chemotherapeutic agents or growth inhibitors (e.g. alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxins or are antitumor antibiotics, proteasome inhibitors, anti-microtubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof), targeted therapeutic agents (e.g., B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylserine-3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, VEGF inhibitors, PARP inhibitors, dual phosphatidylserine-3-kinase inhibitors, oxitol-3-kinase / mTOR inhibitors and combinations thereof), antibody-drug conjugates (e.g., maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antineoplastic agents, cancer vaccines, adjuvants, and combinations thereof, alkylating agents, antimetabolites, antibiotics, plant-derived and / or hormonal agents, platinum-based agents (e.g., cisplatin, carbamazepine, cyclosporine ... rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol, goserelin, asparaginase, and / or fluorouracil; wherein the anti-CTLA4-anti-PD-1 bispecific antibody, component A and the antitumor chemotherapeutic agent are preferably administered simultaneously or sequentially; method.

[0026] 14. A kit for preventing and / or treating a tumor (particularly a malignant tumor), comprising: (a) a first pharmaceutical composition comprising an anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10 as an active ingredient, (b) a second pharmaceutical composition comprising component A as defined in any one of items 1 to 10 as an active ingredient, and optionally (c) one or more drugs for treating a tumor (preferably the drugs are chemotherapeutic agents or growth inhibitory agents (e.g. alkylating agents, anthracyclines, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides , ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, anti-microtubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors and combinations thereof), targeted therapeutic agents (e.g., B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylserine-3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, V EGF inhibitors, PARP inhibitors, dual phosphatidylcholine-phosphate / mTOR inhibitors and combinations thereof), antibody-drug conjugates (e.g., maytansine, monomethyl auristatin E, calicheamicin, esperamicin and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antineoplastic agents, cancer vaccines, adjuvants and combinations thereof, alkylating agents, antimetabolites, antibiotics, plant-derived drugs and / or hormones. and / or fluorouracil (anti-cancer drugs), including but not limited to cisplatin, platinum-based agents (e.g., cisplatin, carboplatin, and oxaliplatin), adriamycin, cyclophosphamide, paclitaxel (e.g., albumin-bound paclitaxel, liposomal paclitaxel, and docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol, goserelin, asparaginase, and / or fluorouracil (anti-cancer drugs). kit.

[0027] 15. A unit dosage form preferably used for treating a tumor (particularly a malignant tumor), comprising: The unit formulation contains 1 to 10,000 mg (preferably 10 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg) of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10, and 1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg) of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10.Component A as defined in any one of items 1 to 10 and optionally one or more agents for treating tumors (preferably the agents are chemotherapeutic agents or growth inhibitors, such as alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, metabolic antagonists, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, agonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof), targeted therapeutics (e.g., 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, OR inhibitors and combinations thereof), antibody-drug conjugates (e.g., maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and radioisotope chelators), T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antineoplastic agents, cancer vaccines, adjuvants, and combinations thereof, alkylating agents, antimetabolites, antibiotics, plant-derived and / or hormonal agents, platinum-based agents (e.g., cisplatin, , carboplatin and oxaliplatin), adriamycin, cyclophosphamide, paclitaxel (e.g., albumin-bound paclitaxel, liposomal paclitaxel and docetaxel), etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloids, tamoxifen, megestrol, goserelin, asparaginase and / or fluorouracil antineoplastic agents); wherein the anti-CTLA4-anti-PD-1 bispecific antibody, component A, and the chemotherapeutic agent are packaged separately; Unit dosage form.

[0028] 16. 1. A method for preventing or treating cancer or tumors, comprising: 16. The method of claim 15, further comprising administering to a subject in need of prevention or treatment of cancer or tumor one or more of the unit preparations according to claim 15, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, component A and chemotherapeutic agent in the unit preparation are each administered separately. method.

[0029] 17. It is preferably used to treat tumors (especially malignant tumors), 0.1 to 10000 mg (preferably 1 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of the anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10, and 0.1 to 10000 mg (preferably 1 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of component A defined in any one of items 1 to 10, Single dose unit.

[0030] 18. The tumor is: Cervical cancer (e.g., metastatic cervical cancer), lung cancer, e.g., 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, adrenal cortical cancer, 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, biliary tract cancer, bile duct cancer, rectal cancer, colon cancer, endometrial cancer, ovarian cancer (e.g., advanced ovarian cancer) or fallopian tube cancer, large cell neuroendocrine carcinoma, urothelial cancer (e.g., upper tract urothelial carcinoma or bladder cancer), breast cancer, triple negative breast cancer, peripheral T cell lymphoma, melanoma, nasopharyngeal carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, brain cancer (e.g., aggressive brain cancer, e.g., glioblastoma), ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenoma, mucinous or serous cystadenocarcinoma, leiomyosarcoma, rhabdomyosarcoma, choriocarcinoma, malignant hydatidiform mole, malignant Sertoli cell-stromal cell tumor, malignant granulocytoma, and dysgerminoma. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10, the pharmaceutical composition according to item 11, the use according to item 12, the method according to item 13, the kit according to item 14, the unit formulation according to item 15, or the method according to item 16, which is selected from one or more of the following:

[0031] 19. The anti-CTLA4-anti-PD-1 bispecific antibody according to any one of items 1 to 10, the pharmaceutical composition according to item 11, the use according to item 12, the method according to item 13, the kit according to item 14, the unit formulation 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 agent are in a form suitable for intravenous injection or intravenous infusion, preferably in liquid form.

[0032] 20. Item 17. The method according to item 13 or 16, wherein the step of administering an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody to the subject is before or after a surgical procedure and / or before or after radiation therapy.

[0033] twenty one. the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 0.1-100 mg, preferably 1-10 mg, per kg body weight; alternatively, the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg, or 100 mg, for each subject; the unit dose of component A is 0.1 to 100 mg, preferably 1 to 10 mg, per kg of body weight; alternatively, the unit dose of component A is 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg, for each subject; Preferably, the dose is administered twice a day to about once every other day, or once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, or 3 weeks; Item 17. The method according to item 13 or 16, wherein the administration route is preferably intravenous infusion or intravenous injection.

[0034] 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 body weight, 2 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 6 mg / kg body weight, 9 mg / kg body weight, 10 mg / kg body weight, 11 mg / kg body weight, 12 mg / kg body weight, 13 mg / kg body weight, 14 mg / kg body weight, 15 mg / kg body weight, 16 mg / kg body weight, 17 mg / kg body weight, 18 mg / kg body weight, 19 mg / kg body weight, 20 mg / kg body weight, 21 mg / kg body weight, 22 mg / kg body weight, 23 mg / kg body weight, 24 mg / kg body weight or 25 mg / kg body weight.

[0035] 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 flat doses capable of effectively treating cancer. In some specific embodiments, the flat dose ranges from about 1 mg to about 1000 mg. In some specific embodiments, the flat 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.

[0036] In some embodiments, administration of the anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody is in two-week (14 day) or three-week (21 day) cycles, and preferably the anti-PD-1-anti-CTLA4 bispecific antibody is administered intravenously on the first day (D1) of each cycle, i.e., the anti-PD-1-anti-CTLA4 bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).

[0037] 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 manufacture of a product for treating cancer, comprising a container housing a fixed dose of an anti-CTLA4-anti-PD-1 bispecific antibody or an anti-VEGFR2 monoclonal antibody (or an 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 instructions instructing the user to administer a fixed dose of anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody to a cancer patient. In some specific embodiments, the product comprises one or more ampoules containing about 50 mg, 100 mg, 200 mg, 300 mg, 350 mg, 400 mg, 500 mg or 600 mg of anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody. In some specific embodiments, the product is packaged as a 50 mg / 5 mL, 100 mg / 10 mL, 200 mg / 10 mL or 350 mg / 35 mL solution of anti-PD-1-anti-CTLA4 bispecific antibody, anti-VEGFR2 monoclonal antibody or anti-VEGF monoclonal antibody per vial.

[0038] The amount of chemotherapeutic agent can be determined by one of skill in the art. For platinum-based chemotherapeutic agents, in the case of cisplatin, the unit dose of cisplatin is calculated according to the Calvert formula: Cisplatin dose (mg) = area under the curve, i.e., AUC (mg / mL / min) × [creatinine clearance rate (mL / min) + 25] where AUC is 4-7, preferably 5. The dose is administered once every 2-6 weeks, preferably once every 3 weeks or once every 4 weeks, and / or the route of administration is intravenous infusion or intravenous injection.

[0039] In one or more embodiments of the invention, the chemotherapeutic agent or growth inhibitory agent is selected from alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, metabolic antagonists, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, anti-microtubule inhibitors, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0040] In one or more embodiments of the invention, the targeted therapeutic agent is selected from a B-raf inhibitor, a MEK inhibitor, a K-ras inhibitor, a c-Met inhibitor, an Alk inhibitor, a phosphatidylcholinesterase-3-kinase inhibitor, an Akt inhibitor, an mTOR inhibitor, a VEGF inhibitor, a PARP inhibitor, a dual phosphatidylcholinesterase-3-kinase / mTOR inhibitor, and combinations thereof.

[0041] In one or more embodiments of the invention, the antibody-drug conjugate comprises an agent selected from the group consisting of maytansine, monomethylauristatin E, calicheamicin, esperamicin, and a radioisotope chelator.

[0042] In the present invention, some expressions have the following meanings:

[0043] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability caused by illness of the disease. The ability of a therapeutic agent to promote regression of a disease can be assessed, for example, in human subjects in clinical trials, in animal model systems predictive of efficacy in humans using a variety of methods known to those of skill in the art, or by determining the activity of the agent in in vitro assays.

[0044] A "prophylactically effective amount" of an agent refers to any amount of agent that, when administered alone or in combination with an anti-neoplastic agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or at risk of cancer recurrence, inhibits the onset or recurrence of cancer. In some embodiments, a prophylactically effective amount prevents the onset or recurrence of cancer entirely. "Inhibiting" the onset or recurrence of cancer means reducing the likelihood of the onset or recurrence of cancer or preventing the onset or recurrence of cancer entirely.

[0045] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient. Such carriers and / or excipients are well known in the art (e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 1999). thEd., Pennsylvania, Mack Publishing Company, 1995), 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 non-ionic surfactants, e.g., Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride.

[0046] A "recurrent" cancer is one that grows back at the original site or at a distant site after previous treatment (e.g., surgery). A "locally recurrent" cancer is one that grows back at the same site after treatment as the previously treated cancer.

[0047] "Metastatic" cancer refers to cancer that has spread from one part of the body (eg, the lungs) to another part.

[0048] The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain contains three hypervariable regions, or complementarity determining regions (CDRs) (heavy (H) CDRs contain HCDR1, HCDR2, and HCDR3, and light (L) CDRs contain LCDR1, LCDR2, and LCDR3, as defined by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md). There are several ways to determine the CDR regions of an antibody when the sequences of the heavy and light chain variable regions of the antibody are known, including the Kabat, IMGT, Chothia, and ABM numbering systems. However, the application of all definitions of CDR to an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. If the amino acid sequence of the variable region of an antibody is known, one of skill in the art can generally determine the particular CDRs without reliance on any experimental data other than the sequence itself.

[0049] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability caused by illness of the disease. The ability of a therapeutic agent to promote regression of a disease can be assessed, for example, in human subjects in clinical trials, in animal model systems predictive of efficacy in humans using a variety of methods known to those of skill in the art, or by determining the activity of the agent in in vitro assays.

[0050] A "prophylactically effective amount" of an agent refers to any amount of agent that, when administered alone or in combination with an anti-neoplastic agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or at risk of cancer recurrence, inhibits the onset or recurrence of cancer. In some embodiments, a prophylactically effective amount prevents the onset or recurrence of cancer entirely. "Inhibiting" the onset or recurrence of cancer means reducing the likelihood of the onset or recurrence of cancer or preventing the onset or recurrence of cancer entirely.

[0051] A "recurrent" cancer is one that grows back at the original site or at a distant site after previous treatment (e.g., surgery). A "locally recurrent" cancer is one that grows back at the same site after treatment as the previously treated cancer.

[0052] "Metastatic" cancer refers to cancer that has spread from one part of the body (eg, the lungs) to another part.

[0053] The term "single dosage unit" refers to a single pharmaceutical dosage form, e.g., an injection in an ampoule, comprising an anti-CTLA4-anti-PD-1 bispecific antibody, anti-VEGFR2 antibody or anti-VEGF antibody of the invention, administered to a subject at the time of the regimen, preferably per kg of body weight of the subject. In specific embodiments of the invention, the regimen includes administering a single dosage unit, for example, twice daily to about every other day, or according to a dosing cycle once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

[0054] In the present invention, the terms "first" (e.g., a first protein functional domain or a first linker fragment) and "second" (e.g., a second protein functional domain or a second linker fragment) are used for expressive distinction or clarity and do not have a typical sequential meaning, unless otherwise specified. [Brief description of the drawings]

[0055] [Figure 1] Figure 1. Assay results for the bioactivity of cadrinimab, an anti-PD-1 / CTLA-4 bispecific antibody, in combination with the anti-VEGFR2 antibody HpL3 on promoting INF-γ secretion as measured by mixed lymphocyte reaction (MLR). [Diagram 2] Figure 2. Assay results for the bioactivity of cadrinimab in combination with bevacizumab on PBMC activation and promotion of IL-2 secretion as measured by mixed lymphocyte reaction (MLR).

[0056] Detailed Description Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. Those skilled in the art will understand that the following examples are merely for illustrating the present invention and should not be construed as limiting the scope of the present invention. Experimental procedures for which conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used are all commercially available conventional products unless their manufacturers are specified.

[0057] In the following examples of the present invention, the isotype control antibody used, i.e., anti-HEL, is an antibody targeting human anti-hen egg lysozyme (HEL), and the variable region sequences of these antibodies are from the study reported by Acierno et al. entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol biol., 2007; 374(1): 130-46), where the sequence of the heavy chain variable region is shown in SEQ ID NO: 45 and the sequence of the light chain variable region is shown in SEQ ID NO: 47. In the constant region fragment of hIgG1WT (i.e., anti-HEL), the heavy chain constant region (SEQ ID NO: 46) is Ig gamma-1 chain C region, Accession: P01857, and the light chain constant region (SEQ ID NO: 48) is Ig kappa chain C region, Accession: P01834. Anti-HEL was prepared by Akeso Biopharma, Inc.

[0058] Bevacizumab (an anti-VEGF monoclonal antibody) used in the examples is a commercially available drug manufactured by Roche.

[0059] Preparation Example 1: Sequence design of anti-CTLA4 antibody The amino acid sequences of the heavy and light chains of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 and the nucleic acid sequences encoding them are identical to those of 4G10, 4G10H1L1 and 4G10H3L3, respectively, in Chinese Patent Application Publication No. CN106967172A.

[0060] (1) Sequences of the heavy and light chain variable regions of 4G10 Nucleic acid sequence of the heavy chain variable region: (372 bp, SEQ ID NO:1) The encoded amino acid sequence: (124 aa, SEQ ID NO:2) Nucleic acid sequence of the light chain variable region: (378 bp, SEQ ID NO:3) The encoded amino acid sequence: (126 aa, SEQ ID NO: 4) (2) Sequences of the heavy and light chain variable regions of humanized monoclonal antibody 4G10H1L1 Nucleic acid sequence of the heavy chain variable region (4G10H1V): (345 bp, SEQ ID NO:5) The encoded amino acid sequence: (115 aa, SEQ ID NO:6) Nucleic acid sequence of the light chain variable region (4G10L1V): (327 bp, SEQ ID NO:7) The encoded amino acid sequence: (109 aa, SEQ ID NO:8) (3) Sequences of the heavy and light chain variable regions of humanized monoclonal antibody 4G10H3L3 Nucleic acid sequence of the heavy chain variable region (4G10H3V): (345 bp, SEQ ID NO:9) The encoded amino acid sequence of the heavy chain variable region (4G10H3V): (115aa, SEQ ID NO:10) Nucleic acid sequence of the light chain variable region (4G10L3V): (327 bp, SEQ ID NO:11) The encoded amino acid sequence (4G10L3V): (109aa, SEQ ID NO:12)

[0061] Preparation Example 2: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 The amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 and the nucleic acid sequences encoding them are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication No. CN106967172A.

[0062] (1) Sequences of the heavy and light chain variable regions of 14C12 Nucleic acid sequence of the heavy chain variable region: (354 bp, SEQ ID NO: 13) The encoded amino acid sequence: (118aa, SEQ ID NO:14) Nucleic acid sequence of the light chain variable region: (321 bp, SEQ ID NO: 15) The encoded amino acid sequence: (107aa, SEQ ID NO:16) (2) Heavy and light chain variable regions and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 Nucleic acid sequence of the heavy chain variable region: (354 bp, SEQ ID NO: 17) The encoded amino acid sequence: (118aa, SEQ ID NO:18) Nucleic acid sequence of the light chain variable region: (321 bp, SEQ ID NO: 19) The encoded amino acid sequence: (107aa, SEQ ID NO:20) DNA sequence of 14C12H1L1 heavy chain (14C12H1): (1344 bp, SEQ ID NO:21) The encoded amino acid sequence: (448aa, SEQ ID NO:22) DNA sequence of 14C12H1L1 light chain (14C12L1): (642 bp, SEQ ID NO:23) The encoded amino acid sequence: (214 aa, SEQ ID NO: 24)

[0063] Preparation Example 3: Sequence design of bifunctional antibodies CP001(M), CP002(M), CP003(M) and CP004(M) The structural pattern of the bifunctional antibodies CP001(M), CP002(M), CP003(M) and CP004(M) is the Morrison format (IgG-scFv), i.e., the C-terminus of the two heavy chains of one IgG antibody is separately linked to the scFv fragment of another antibody via a linker fragment. The components for the design of the heavy and light chains are shown in Table A below.

[0064] [Table 1]

[0065] In Table A above, The amino acid sequence of linker 1 is (GGGGS) 3 (SEQ ID NO: 25). The amino acid sequence of linker 2 is (GGGGS) 4 (Sequence number: 26).

[0066] In Table A above, the scFv fragments 4G10H1V(M), 4G10L1V(M), 4G10H3V(M) and 4G10L3V(M) of the CP001(M), CP002(M), CP003(M) and CP004(M) antibodies were based on 4G10H1V, 4G10L1V, 4G10H3V and 4G10L3V, respectively, and contained mutations at individual amino acids in the framework regions, which effectively optimized the structure of the antibodies and improved their efficacy.

[0067] (1) 4G10H1V(M): (115 aa, mutation positions are underlined in the amino acid sequence based on 4G10H1V, SEQ ID NO: 41) (2) 4G10L1V(M): (110 aa, mutation positions are underlined in the amino acid sequence based on 4G10L1V, SEQ ID NO: 42) (3) 4G10H3V(M): (115 aa, mutation positions are underlined in the amino acid sequence based on 4G10H3V, SEQ ID NO: 43) (4) 4G10L3V(M): (110 aa, mutation positions are underlined in the amino acid sequence based on 4G10L3V, SEQ ID NO: 44)

[0068] In the following, in order to distinguish it from the mutant antibody, in the examples of the present invention, CP004(M) is also referred to as CP004(hG1WT). The above-mentioned CP004(M) is a "wild type" that contains an Ig gamma-1 chain C region (Accession: P01857) as a heavy chain constant region and an Ig kappa chain C region (Accession: P01834) as a light chain constant region.

[0069] Preparation Example 4: Amino acid mutation design of the non-variable region based on the humanized bifunctional antibody CP004 Based on CP004 (hG1WT) obtained in Preparation Example 3, CP004 (hG1TM) was obtained by introducing a leucine-alanine point mutation (L234A) at position 234 in the heavy chain, a leucine-alanine point mutation (L235A) at position 235, and a glycine-alanine point mutation (G237A) at position 237. DNA sequence of the heavy chain of the immunoglobulin portion of CP004 (hG1TM): (1344 bp, mutation positions are underlined, SEQ ID NO:39) Amino acid sequence of the heavy chain of the immunoglobulin portion of CP004 (hG1TM): (448 aa, mutation positions are underlined, SEQ ID NO: 40)

[0070] CP004(hG1TM) and CP004(hG1WT) share the same DNA sequence and the same encoded amino acid sequence for the light chain. The specific sequence is shown in Preparation 3.

[0071] Preparation Example 5: Preparation of the anti-PD-1 / CTLA-4 bispecific antibody, kadonilimab The structural pattern of the anti-PD-1 / CTLA-4 bispecific antibody kadonilimab (i.e., CP004 (hG1TM)) is the Morrison format (IgG-scFv), i.e., the C-terminus of the two heavy chains of one immunoglobulin moiety (IgG) antibody is linked via a linker fragment to the VH terminus of the scFv fragment of another antibody, where the immunoglobulin moiety is based on the PD-1 antibody and the scFv fragment is based on the anti-CTLA-4 antibody, linked by a linker fragment in between.

[0072] 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 cadnilimab antibody (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:

[0073] The DNA sequence of the heavy chain-encoding gene containing the constant and variable regions of the candidate monoclonal antibody and the DNA sequence of the light chain-encoding gene containing the constant and variable regions of the candidate monoclonal antibody were separately cloned into a pUC57simple (provided by GenScript) vector to obtain plasmids pUC57simple-H and pUC57simple-L.

[0074] The above plasmids were enzymatically digested (HindIII and EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned separately into the expression vector pcDNA3.1 (purchased from Invitrogen). The recombinant plasmids were extracted, and the combination of pcDNA3.1-H+pcDNA3.1-L was co-transfected into 293F cells. After 7 days of cell culture, the culture was centrifuged at high speed. The supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column and purified using a protein purification liquid chromatography system (AKTA Purifier 10, GE).

[0075] The purified sample, i.e., antibody kadonilimab, was added to both reduced and non-reduced protein electrophoresis loading buffers and then boiled for SDS-PAGE electrophoresis. The target proteins in the reduced protein sample were 70 kD and 30 kD, and the target protein in the non-reduced protein sample (single antibody) was 200 kD. The proteins fit the theoretical size, and thus a purified antibody was obtained.

[0076] Preparation Example 6: Preparation of anti-VEGFR2 antibody HpL3 The sequences and preparation methods of the antibody light chain variable region and heavy chain variable region of the anti-VEGFR2 antibody HpL3 described in this Example were cited from Patent Application No. CN2016105736.X, "AN ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR VEGFR2 MONOCLONAL ANTIBODY AND AN ENCODING GENE AND USE THEREOF."

[0077] The amino acid sequence of the heavy chain variable region of HpL3 is shown in SEQ ID NO: 50, the DNA sequence of its encoding gene is shown in SEQ ID NO: 49, and the sequences of CDR1 to 3 of the heavy chain variable region are shown in SEQ ID NO: 53 to 55, respectively.

[0078] The amino acid sequence of the light chain variable region of HpL3 is shown in SEQ ID NO: 52, the DNA sequence of its encoding gene is shown in SEQ ID NO: 51, and the sequences of CDR1 to 3 of the light chain variable region are shown in SEQ ID NO: 56 to 58, respectively.

[0079] HpL3 contains an Ig gamma-1 chain C region (accession: P01857) as a heavy chain constant region, and an Ig kappa chain C region (accession: P01834) as a light chain constant region.

[0080] The DNA sequence of the heavy chain-encoding gene containing the constant and variable regions of the candidate monoclonal antibody and the DNA sequence of the light chain-encoding gene containing the constant and variable regions of the candidate monoclonal antibody were separately cloned into a pUC57simple (provided by GenScript) vector to obtain plasmids pUC57simple-PCABH and pUC57simple-L3.

[0081] The above plasmids were enzymatically digested (HindIII and EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned separately into the expression vector pcDNA3.1 (purchased from Invitrogen). The recombinant plasmids were extracted, and the combination of pcDNA3.1-PCABH+pcDNA3.1-L3 was co-transfected into 293F cells. After 7 days of cell culture, the culture was centrifuged at high speed. The supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column and purified using a protein purification liquid chromatography system (AKTA Purifier 10, GE).

[0082] The purified sample, i.e., antibody HpL3, was added to both reduced and non-reduced protein electrophoresis loading buffers and then boiled for SDS-PAGE electrophoresis. The target proteins in the reduced protein sample were 45 kD and 30 kD, and the target protein in the non-reduced protein sample (single antibody) was 150 kD. The proteins fit the theoretical size, and thus a purified monoclonal antibody was obtained.

[0083] Example 1: Bioactivity of the anti-PD-1 / CTLA-4 bispecific antibody kadonilimab in combination with the anti-VEGFR2 antibody HpL3 in promoting INF-γ secretion as measured by mixed lymphocyte reaction (MLR) Preparation of Raji-PDL1 cells The human PD-L1 overexpressing lentiviral vector plenti6.3 / V5-PDL1FL-BSD was infected into Raji cells after virus packaging, and the drug-resistant cell line Raji-PDL1 was obtained after BSD (blasticidin, 10ug / mL) drug screening. plenti6.3 / V5-PDL1FL-BSD was obtained by synthesizing the gene PDL1FL (PDL1, Genebank ID: NP_054862.1) from the supplier Nanjing Genscript, and then ligating it into plenti6.3 / V5-BSD (Invitrogen Cat. No: k531520) by enzymatic digestion.

[0084] Two days before the experiment, PBMCs (isolated from normal human peripheral blood) were thawed and cultured in complete medium at 37°C in a 5% carbon dioxide incubator. After 2 hours, when the PBMCs had recovered, they were stimulated with SEB (final concentration 0.5 μg / mL) (Toxin technology, Cat. No: BT202) for 2 days. On the day of the experiment, Raji-PDL1 cells were harvested, centrifuged, resuspended (in 1640+10% FBS), counted, and 2 × 10 6 The density of the cells was adjusted to 1 × 10 cells / mL, MMC (mitomycin C, final concentration 2 μg / mL, manufacturer: Stressmarq, Cat. No: SIH-246) was added, and the mixture was treated at 37°C for 1 hour in a 5% carbon dioxide incubator. PBMCs 2 days after SEB stimulation and Raji-PDL1 cells after MMC treatment were collected and washed twice with 1640 basal medium, the cells were resuspended in complete medium, counted, and the cells were diluted to 1 × 10 5 1 x 10 PBMCs / well 5 Cells were seeded into a 96-well U-shaped plate (3799) according to Raji-PDL1 (50 μL / well each). Drugs were added according to the experimental design, blank and negative controls were set up, and cells were cultured for 3 days (the final volume of the system was 200 μL). After 3 days, cells were centrifuged at 250×g for 5 minutes (Beckman centrifuge), cell supernatant was collected, and IFN-γ content was detected using the Dakewe kit.

[0085] As shown in Figure 1, in a mixed culture system of PBMC and Raji-PDL1 cells, the addition of kadonilimab and HpL3 antibody can significantly induce IFN-γ secretion in PBMC in a significant dose-dependent manner. With regard to the level of IFN-γ secretion-promoting activity, the combination of kadonilimab and HpL3 antibody has a better IFN-γ secretion-promoting ability than kadonilimab and HpL3 antibody alone.

[0086] Example 2: Bioactivity of Kadonilimab in Combination with Bevacizumab on PBMC Activation and Promotion of IL-2 Secretion Measured by Mixed Lymphocyte Reaction (MLR) Two days before the experiment, PBMCs (isolated from voluntary peripheral blood donations from healthy volunteers after informed consent) were thawed and cultured in complete medium at 37°C in a 5% carbon dioxide incubator. After 2 hours, when the PBMCs had recovered, they were stimulated with SEB (final concentration 0.5 μg / mL) (Toxin technology, Cat. No: BT202) for 2 days. On the day of the experiment, Raji-PDL1 cells (Akeso Biopharma, Inc.) were harvested, centrifuged, resuspended (in 1640+10% FBS), counted, and 2 × 10 6 The cells were adjusted to a density of 1 × 10 cells / mL, MMC (mitomycin C, final concentration 2 μg / mL) (mitomycin C, manufacturer: Stressmarq, Cat. No: SIH-246) was added, and the mixture was treated at 37°C for 1 hour in a 5% carbon dioxide incubator. PBMCs 2 days after SEB stimulation and Raji-PDL1 cells after MMC treatment were collected and washed twice with 1640 basal medium, the cells were resuspended in analysis medium, counted, and the cells were diluted to 1 × 10 5 1 x 10 PBMCs / well 5 Hela cells (purchased from the Chinese academy of sciences) were conventionally digested and 2 × 10 4The cells were seeded into a 96-well U-shaped plate (3799) according to the number of cells / well (40 μL / well). Drugs were added according to the experimental design, negative and isotype controls were set up, and the cells were cultured for 3 days (the final volume of the system was 200 μL). After 3 days, the cells were centrifuged at 250×g for 5 minutes (Beckman centrifuge), the cell supernatant was collected, and the IL-2 content was detected using the Dakewe kit.

[0087] As shown in Figure 2, in a mixed culture system of PBMC, Raji-PDL1 and Hela (cervical cancer) cells, and VEGF (batch number: 20180126-1, manufactured by Akeso Biopharma Inc.), the addition of kadonilimab and bevacizumab can significantly induce IL-2 secretion in PBMC, both in a significant dose-dependent manner. With regard to the level of IL-2 secretion-promoting activity, the combination of kadonilimab and bevacizumab has better IL-2 secretion-promoting ability than kadonilimab and bevacizumab alone.

[0088] The amino acid sequence of the heavy chain variable region of bevacizumab is shown in SEQ ID NO: 60, the DNA sequence of its encoding gene is shown in SEQ ID NO: 59, and the sequences of CDR1 to 3 of the heavy chain variable region are shown in SEQ ID NO: 63 to 65, respectively. The amino acid sequence of the light chain variable region of bevacizumab is shown in SEQ ID NO: 62, the DNA sequence of its encoding gene is shown in SEQ ID NO: 61, and the sequences of CDR1 to 3 of the light chain variable region are shown in SEQ ID NO: 66 to 68, respectively.

[0089] Sequence Listing SEQ ID NO:1: Nucleic acid sequence of the heavy chain variable region of 4G10 [ka]

[0090] SEQ ID NO:2: Amino acid sequence of the heavy chain variable region of 4G10 [ka]

[0091] SEQ ID NO:3: Nucleic acid sequence of the light chain variable region of 4G10 [ka]

[0092] SEQ ID NO: 4: Amino acid sequence of the light chain variable region of 4G10 [ka]

[0093] SEQ ID NO:5: Nucleic acid sequence of the heavy chain variable region of 4G10H1L1 [ka]

[0094] SEQ ID NO:6: Amino acid sequence of the heavy chain variable region of 4G10H1L1 [ka]

[0095] SEQ ID NO:7: Nucleic acid sequence of the light chain variable region of 4G10H1L1 [ka]

[0096] SEQ ID NO:8: Amino acid sequence of the light chain variable region of 4G10H1L1 [ka]

[0097] SEQ ID NO:9: Nucleic acid sequence of the heavy chain variable region of 4G10H3L3 [ka]

[0098] SEQ ID NO:10: Amino acid sequence of the heavy chain variable region of 4G10H3L3 [ka]

[0099] SEQ ID NO:11: Nucleic acid sequence of the light chain variable region of 4G10H3L3 [ka]

[0100] SEQ ID NO:12: Amino acid sequence of the light chain variable region of 4G10H3L3 [ka]

[0101] SEQ ID NO: 13: Nucleic acid sequence of the heavy chain variable region of 14C12 [ka]

[0102] SEQ ID NO: 14: Amino acid sequence of the heavy chain variable region of 14C12 [ka]

[0103] SEQ ID NO: 15: Nucleic acid sequence of the light chain variable region of 14C12 [ka]

[0104] SEQ ID NO: 16: Amino acid sequence of the light chain variable region of 14C12 [ka]

[0105] SEQ ID NO: 17: Nucleic acid sequence of the heavy chain variable region of 14C12H1L1 [ka]

[0106] SEQ ID NO: 18: Amino acid sequence of the heavy chain variable region of 14C12H1L1 [ka]

[0107] SEQ ID NO: 19: Nucleic acid sequence of the light chain variable region of 14C12H1L1 [ka]

[0108] SEQ ID NO: 20: Amino acid sequence of the light chain variable region of 14C12H1L1 [ka]

[0109] SEQ ID NO:21: DNA sequence of 14C12H1L1 heavy chain (14C12H1) (1344 bp) [ka] TIFF2024515618000023.tif187161

[0110] SEQ ID NO: 22: Encoded amino acid sequence (448aa) of 14C12H1L1 heavy chain (14C12H1) [ka]

[0111] SEQ ID NO: 23: DNA sequence of 14C12H1L1 light chain (14C12L1) (642 bp) [ka]

[0112] SEQ ID NO: 24: Encoded amino acid sequence of 14C12H1L1 light chain (14C12L1) (214aa) [ka]

[0113] SEQ ID NO:25: Amino acid sequence of linker 1 [ka]

[0114] SEQ ID NO:26: Amino acid sequence of linker 2 [ka]

[0115] HCDR1 of SEQ ID NO: 27:14C12 [ka]

[0116] HCDR2 of SEQ ID NO:28:14C12 [ka]

[0117] HCDR3 of SEQ ID NO:29:14C12 [ka]

[0118] LCDR1 of SEQ ID NO: 30:14C12 [ka]

[0119] LCDR2 of SEQ ID NO:31:14C12 [ka]

[0120] LCDR3 of SEQ ID NO:32:14C12 [ka]

[0121] HCDR1 of SEQ ID NO:33:4G10 [ka]

[0122] HCDR2 of SEQ ID NO:34:4G10 [ka]

[0123] SEQ ID NO: 35: HCDR3 of 4G10 [ka]

[0124] LCDR1 of SEQ ID NO: 36:4G10 [ka]

[0125] LCDR2 of SEQ ID NO: 37:4G10 [ka]

[0126] LCDR3 of SEQ ID NO: 38:4G10 [ka]

[0127] SEQ ID NO: 39: DNA sequence of the heavy chain of the immunoglobulin portion of CP004 (hG1TM), mutation positions are underlined [ka] TIFF2024515618000042.tif60161

[0128] SEQ ID NO: 40: Amino acid sequence of the heavy chain of the immunoglobulin portion of CP004 (hG1TM), mutation positions are underlined [ka]

[0129] SEQ ID NO: 41: Amino acid sequence of 4G10H1V(M), mutation positions are underlined [ka]

[0130] SEQ ID NO: 42: Amino acid sequence of 4G10L1V(M), mutation positions are underlined [ka]

[0131] SEQ ID NO: 43: Amino acid sequence of 4G10H3V(M), mutation positions are underlined [ka]

[0132] SEQ ID NO: 44: Amino acid sequence of 4G10L3V(M), mutation positions are underlined [ka]

[0133] SEQ ID NO: 45: Sequence of the heavy chain variable region of hIgG [ka]

[0134] SEQ ID NO: 46 Sequence of the heavy chain constant region of hIgG [ka]

[0135] SEQ ID NO: 47 Sequence of the light chain variable region of hIgG [ka]

[0136] SEQ ID NO: 48 Sequence of the light chain constant region of hIgG [ka]

[0137] SEQ ID NO: 49 Nucleic acid sequence of the heavy chain variable region of HpL3 (VEGFR2) [ka] (Note: the underlined parts represent the CDR sequences.)

[0138] SEQ ID NO: 50 Amino acid sequence of the heavy chain variable region of HpL3 [ka] (Note: the underlined parts represent the CDR sequences.)

[0139] SEQ ID NO: 51 Nucleic acid sequence of the light chain variable region of HpL3 [ka] (Note: the underlined parts represent the CDR sequences.)

[0140] SEQ ID NO: 52 Amino acid sequence of the light chain variable region of HpL3 [ka] (Note: the underlined parts represent the CDR sequences.) HCDR1 to HCDR3 of the heavy chain variable region of HpL3 HCDR1: GFTFSSYS (SEQ ID NO: 53) HCDR2: ISSSSSYI (SEQ ID NO: 54) HCDR3: ARVTDAFDI (SEQ ID NO: 55) LCDR1 to LCDR3 of the light chain variable region of HpL3 LCDR1: QGLDNW (SEQ ID NO: 56) LCDR2: DAS (SEQ ID NO: 57) LCDR3: QQAKAFPPT (SEQ ID NO: 58)

[0141] SEQ ID NO: 59 Nucleic acid sequence of the heavy chain variable region of bevacizumab (VEGF) [ka]

[0142] SEQ ID NO: 60 Amino acid sequence of the heavy chain variable region of bevacizumab [ka]

[0143] SEQ ID NO: 61 Nucleic acid sequence of the light chain variable region of bevacizumab [ka]

[0144] SEQ ID NO: 62 Amino acid sequence of the light chain variable region of bevacizumab [ka]

[0145] The amino acid sequences of the three CDRs of the heavy chain variable region of bevacizumab are as follows: [ka]

[0146] The amino acid sequences of the three CDRs of the light chain variable region of bevacizumab are as follows: [ka]

[0147] SEQ ID NO:69 Nucleic acid sequence of CP004 (hG1TM) heavy chain [ka] TIFF2024515618000063.tif169161

[0148] SEQ ID NO: 70 Amino acid sequence of CP004 (hG1TM) heavy chain (full length) [ka] (Note: the underlined parts represent the CDR sequences.)

[0149] SEQ ID NO:71 Nucleic acid sequence of CP004 (hG1TM) light chain [ka]

[0150] SEQ ID NO: 72 Amino acid sequence of CP004 (hG1TM) light chain (full length) [ka]

Claims

1. A pharmaceutical composition comprising an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody and an effective amount of component A, wherein component A is selected from an anti-VEGFR2 monoclonal antibody or an antigen-binding fragment thereof, an anti-VEGF monoclonal antibody or an antigen-binding fragment thereof, or a combination thereof; Where: (1) The anti-CTLA4-anti-PD-1 bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional domain that targets 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 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 14 and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 16, and the single chain antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 2 and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 4, or The immunoglobulin comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 2 and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 4, and the single-chain antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 14 and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 16, (2) The anti-VEGFR2 monoclonal antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 50 and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 52; or (3) The anti-VEGF monoclonal antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 60 and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 62; Pharmaceutical compositions.

2. The immunoglobulin comprises HCDR1 to HCDR3 as shown in SEQ ID NOs: 27 to 29, respectively, and LCDR1 to LCDR3 as shown in SEQ ID NOs: 30 to 32, respectively, and the single chain antibody comprises HCDR1 to HCDR3 as shown in SEQ ID NOs: 33 to 35, respectively, and LCDR1 to LCDR3 as shown in SEQ ID NOs: 36 to 38, respectively; or The immunoglobulin comprises HCDR1 to HCDR3 as set forth in SEQ ID NOs: 33 to 35, respectively, and LCDR1 to LCDR3 as set forth in SEQ ID NOs: 36 to 38, respectively, and the single chain antibody comprises HCDR1 to HCDR3 as set forth in SEQ ID NOs: 27 to 29, respectively, and LCDR1 to LCDR3 as set forth in SEQ ID NOs: 30 to 32, respectively; or The anti-VEGFR2 monoclonal antibody comprises HCDR1 to HCDR3 as set forth in SEQ ID NOs: 53 to 55, respectively, and LCDR1 to LCDR3 as set forth in SEQ ID NOs: 56 to 58, respectively; or The anti-VEGF monoclonal antibody comprises HCDR1 to HCDR3 as shown in SEQ ID NOs: 63 to 65, respectively, and LCDR1 to LCDR3 as shown in SEQ ID NOs: 66 to 68, respectively; The pharmaceutical composition according to claim 1.

3. (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the sequence shown in SEQ ID NO: 14 or SEQ ID NO: 18 or a mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the sequence shown in SEQ ID NO: 16 or SEQ ID NO: 20 or a mutant thereof, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 41 or SEQ ID NO: 43 or a mutant thereof, and the amino acid sequence of the light chain variable region of the single chain antibody is selected from the sequence shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 42 or SEQ ID NO: 44 or a mutant thereof, or the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the sequence shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41 or SEQ ID NO:43 or a variant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the sequence shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42 or SEQ ID NO:44 or a variant thereof, the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the sequence shown in SEQ ID NO:14 or 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 the sequence shown in SEQ ID NO:16 or SEQ ID NO:20 or a variant thereof, (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 mutant 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 mutant thereof; or (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 mutant 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 mutant thereof; 2. The pharmaceutical composition of claim 1, wherein the sequence of said variant has at least 90% homology with the corresponding sequence.

4. The bispecific antibody is selected from the group consisting of the following (1) to (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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42 or a mutant thereof, 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 mutant 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 mutant 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 mutant thereof, the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44 or a mutant thereof, 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 mutant 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 mutant thereof. is selected from one of 3. The pharmaceutical composition of claim 1 or 2, wherein the sequence of the variant has at least 90% homology with the corresponding sequence.

5. 3. The pharmaceutical composition according to claim 1 or 2, wherein 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 the light chain of the immunoglobulin is the sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the variant sequence has at least 90% homology with the corresponding sequence.

6. 3. The pharmaceutical composition of claim 1 or 2, wherein a first protein functional domain is linked to a second protein functional domain either directly or via a linker fragment and / or the heavy chain variable domain of said single chain antibody is linked to a light chain variable domain of said single chain antibody either directly or via a linker fragment.

7. The linker fragment is (GGGGS) ｎ 7. The pharmaceutical composition of claim 6, wherein n is a positive integer.

8. The pharmaceutical composition of claim 7, wherein n is 1, 2, 3, 4, 5 or 6.

9. 3. The pharmaceutical composition of claim 1 or 2, wherein the number of first and second protein functional domains is, independently, one, two or more.

10. 3. The pharmaceutical composition of claim 1 or 2, wherein said single chain antibody is linked to a heavy chain of said immunoglobulin.

11. The pharmaceutical composition of claim 10, wherein the heavy chain variable region of the single chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

12. the immunoglobulin is of the human IgG1 subtype, wherein, according to the EU numbering system, the immunoglobulin comprises the following mutations: L234A and L235A or L234A and G237A or L235A and G237A or L234A, L235A and G237A and a heavy chain constant region having an A mutation selected from According to the EU numbering system, the immunoglobulin contains 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 the immunoglobulin comprises a heavy chain constant region having a combination of A and B mutations according to the EU numbering system, The pharmaceutical composition according to claim 1 or 2, wherein the heavy chain constant region of the anti-VEGFR2 monoclonal antibody or the anti-VEGF monoclonal antibody is an Ig gamma-1 chain C region, accession number P01857, and the light chain constant region is an Ig kappa chain C region, accession number P01834.

13. The bispecific antibody is a first protein functional region that targets PD-1; and a second protein functional domain that targets CTLA4; the number of first protein functional regions is 1 and the number of second protein functional regions is 2; wherein the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; The heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 40 or a sequence having at least 90% homology thereto, and the light chain has the amino acid sequence shown in SEQ ID NO: 24 or a sequence having at least 90% homology thereto, the heavy chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 90% homology thereto, and the light chain variable region of the single chain antibody has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 90% homology thereto; the single chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin; 3. The pharmaceutical composition of claim 1 or 2, wherein a first protein functional domain is linked to a second protein functional domain via a first linker fragment, and the heavy chain variable domain of the single chain antibody is linked to the light chain variable domain of the single chain antibody via a second linker fragment, and the first linker fragment and the second linker fragment are the same or different.

14. The pharmaceutical composition described in claim 13, wherein the first linker fragment and the second linker fragment each have an amino acid sequence independently selected from SEQ ID NO:25 and SEQ ID NO:

26.

15. The anti-PD-1 / CTLA-4 bispecific antibody light chain has the sequence set forth in SEQ ID NO:72 and the anti-PD-1 / CTLA-4 bispecific antibody heavy chain has the sequence set forth in SEQ ID NO:70; or 15. The pharmaceutical composition of claim 14, wherein the antigen-binding fragment is selected from a Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, a single chain antibody, a bivalent antibody, or a domain antibody.

16. The pharmaceutical composition according to claim 1 or 2, wherein the mass ratio of the anti-CTLA4-anti-PD-1 bispecific antibody to component A is selected from the range of (1:5) to (5:1).

17. The pharmaceutical composition of claim 16, wherein the mass ratio of the anti-CTLA4-anti-PD-1 bispecific antibody to component A is 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:

1.

18. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition is in solid or liquid form, or wherein the pharmaceutical composition further comprises one or more chemotherapeutic agents.

19. The pharmaceutical composition of claim 18, wherein the chemotherapeutic agent is an alkylating agent, antimetabolite, antibiotic, plant-derived agent and / or hormonal agent, platinum-based agent, adriamycin, cyclophosphamide, paclitaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, vinca alkaloid, tamoxifen, megestrol, goserelin, asparaginase and / or fluorouracil-based antitumor agent.

20. The pharmaceutical composition of claim 19, wherein the platinum-based drug is cisplatin, carboplatin or oxaliplatin, or the paclitaxel is albumin-bound paclitaxel, liposomal paclitaxel or docetaxel.

21. Use of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15 and component A as defined in any one of claims 1 to 15 in the manufacture of a medicament or a kit for preventing and / or treating a tumor.

22. The use of claim 21, wherein the medicament or kit further comprises one or more agents for treating a tumor.

23. The use according to claim 22, wherein the drug is a chemotherapeutic agent or growth inhibitor, a targeted therapeutic agent, an antibody-drug conjugate, a T cell expressing a chimeric antigen receptor, an antibody or an antigen-binding fragment thereof, an angiogenesis inhibitor, an antitumor agent, a cancer vaccine, an adjuvant and combinations thereof, an alkylating agent, antimetabolite, an antibiotic, a plant-derived drug and / or a hormonal drug, a platinum-based drug, adriamycin, cyclophosphamide, paclitaxel, etoposide, gemcitabine, pemetrexed, capecitabine, olaparib, rucaparib, niraparib, talazoparib, fluzoparib, a vinca alkaloid, tamoxifen, megestrol, goserelin, asparaginase and / or a fluorouracil-based antitumor agent.

24. The proliferation inhibitor is an alkylating agent, an anthracycline, an antihormonal agent, an aromatase inhibitor, an antiandrogenic agent, a protein kinase inhibitor, a lipid kinase inhibitor, an antisense oligonucleotide, a ribozyme, an antimetabolite, a topoisomerase inhibitor, a cytotoxic or antitumor antibiotic, a proteasome inhibitor, an antimicrotubule agent, an EGFR antagonist, a VEGF antagonist, angiopoietin 2 antagonist, a retinoid, a tyrosine kinase inhibitor, a histone deacetylase inhibitor, or a combination thereof, or the platinum-based drug is cisplatin, carboplatin, or oxaliplatin, or paclitaxel.

24. The use of claim 23, wherein the antibody-drug conjugate is maytansine, monomethylauristatin E, calicheamicin, esperamicin, or a radioisotope chelator, or the targeted therapeutic agent is a B-raf inhibitor, a MEK inhibitor, a K-ras inhibitor, a c-Met inhibitor, an Alk inhibitor, a phosphatidylinositol 3-kinase inhibitor, an Akt inhibitor, an mTOR inhibitor, a VEGF inhibitor, a PARP inhibitor, a dual phosphatidylinositol 3-kinase / mTOR inhibitor, or a combination thereof.

25. A kit for preventing and / or treating a tumor, comprising:

19. A kit comprising: (a) a first pharmaceutical composition comprising an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15 as an active ingredient; and (b) a second pharmaceutical composition comprising component A as defined in any one of claims 1 to 15 as an active ingredient.

26. The kit of claim 25, further comprising (c) one or more agents for treating a tumor.

27. The kit of claim 26, wherein the agent is defined in claim 23 or 24.

28. A unit dosage form comprising 1 to 10,000 mg of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15, and 1 to 10,000 mg of component A as defined in any one of claims 1 to 15.

29. The unit dosage form of claim 28, further comprising one or more pharmaceutical agents as defined in claim 23 or 24.

30. The unit dosage form of claim 28, comprising 10 to 1000 mg of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15, or 1 to 1000 mg of component A as defined in any one of claims 1 to 15.

31. The unit dosage form of claim 28, comprising 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15, or component A as defined in any one of claims 1 to 15.

32. A single dose unit comprising 0.1 to 10,000 mg of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15, and 0.1 to 10,000 mg of component A as defined in any one of claims 1 to 15.

33. A single dosage unit according to claim 32, comprising 1 to 1000 mg of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15, or 1 to 1000 mg of component A as defined in any one of claims 1 to 15.

34. A single-dose unit according to claim 32, comprising 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg of an anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 15 or component A as defined in any one of claims 1 to 15.

35. The tumor is: Cervical cancer, lung cancer, esophageal cancer, esophageal squamous cell carcinoma, ileocecal adenocarcinoma, ampullary adenocarcinoma, small cell lung cancer, stomach cancer, kidney cancer, endometrial cancer, adrenal cortical 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, biliary tract cancer, bile duct cancer, rectal cancer, colon cancer, endometrial cancer, ovarian or fallopian tube cancer, large cell neuroendocrine cancer Cancer, urothelial carcinoma, breast cancer, triple-negative breast cancer, peripheral T-cell lymphoma, melanoma, nasopharyngeal carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, brain cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenoma, mucinous or serous cystadenocarcinoma, leiomyosarcoma, rhabdomyosarcoma, chorioepithelial carcinoma, malignant hydatidiform mole, malignant sustentacular cell-stromal cell tumor, malignant granular cell tumor and dysgerminoma 22. The use according to claim 21 , selected from one or more of:

36. The use of claim 35, wherein the tumor is metastatic cervical cancer, squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, advanced gastric cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, advanced ovarian cancer, upper tract urothelial carcinoma, bladder cancer or invasive brain cancer.

37. The use of claim 35, wherein the tumor is glioblastoma or non-small cell lung cancer.

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