Pharmaceutical Composition Comprising Anti-CTLA4-Anti-PD-1 Bispecific Antibody and Chiauranib

Abstract

The present invention is in the field of tumor therapy and molecular immunology, and relates to a pharmaceutical composition comprising anti-CTLA4-anti-PD-1 bispecific antibody and chiauranib.Specifically, the pharmaceutical composition comprises chiauranib or its pharma-ceutically acceptable salt or its crystalline form, and at least one anti-CTLA4-anti-PD-1 bispecific antibody.The present invention also relates to the use of this pharmaceutical composition.

Description

[Technical field]

[0001] Technical Field The present invention is in the field of tumor therapy and molecular immunology, and relates to a pharmaceutical combination comprising an anti-CTLA4-anti-PD-1 bispecific antibody and chiauranib, and uses thereof. Specifically, the present invention relates to a pharmaceutical combination comprising an anti-CTLA4-anti-PD-1 bispecific antibody containing a mutation and chiauranib or a pharma- ceutical acceptable salt thereof or a crystalline form thereof, and uses thereof. [Background technology]

[0002] background The transmembrane receptor PD-1 (programmed cell death protein-1) is a member of the CD28 gene family and is expressed on activated T cells, B cells, and myeloid cells. The ligands of PD-1, PDL1 (programmed cell death 1 ligand 1, or PDL-1) and PDL2 (programmed cell death 1 ligand 2, or PDL-2), are members of the B7 superfamily. PDL1 is expressed in a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, and PDL2 is expressed exclusively in antigen-presenting cells, such as dendritic cells and macrophages.

[0003] The PD-1 / PDL1 signaling pathway plays an important role in controlling immune tolerance, microbial infection, and tumor immune evasion. PD-1 is mainly expressed in immune cells such as T cells, and PDL1, a ligand of PD-1, is highly expressed in several human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate inhibited T cells to attack cancer cells. Blocking PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, which play a role in killing tumor cells, and further inhibit local tumor growth (Julie R et al., 2012, N Engl J Med., 366:2455-2465). Furthermore, tumors with high expression of PDL1 are associated with cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA, 104:3360-5). An effective method is to inject anti-PD-1 antibodies into the body to regulate the expression of PD-1. Due to the broad anti-tumor effects and remarkable efficacy of PD-1 antibodies, it is widely accepted in the industry that antibodies targeting the PD-1 pathway will bring about breakthroughs in the treatment of various tumors, such as non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol., 42(3): 466-473), leukemia, and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets., 13(7): 768-74).

[0004] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and CD28 molecules are highly similar in terms of gene structure, chromosomal location, sequence homology, and gene expression. Both molecules are receptors for the costimulatory molecule B7 and are expressed primarily on the surface of activated T cells. CTLA4 binding to B7 inhibits T cell activation in mice and humans, playing a negative regulatory role in T cell activation.

[0005] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing the reactivity of T cells to various antigens. In this respect, in vivo and in vitro studies are essentially consistent. Currently, there are CTLA4 monoclonal antibodies in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colon cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun., 13:5).

[0006] Interleukin 2 (IL-2) is produced by T cells. It is a growth factor that regulates T cell subgroups and is an important factor in controlling immune responses. It promotes the proliferation of activated B cells and is involved in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors (including melanoma, renal tumors, etc.) and is currently undergoing clinical trials for the treatment of chronic viral infections (Chavez, AR, et al., 2009, Ann. NY Acad. Sci., 1182:p.14-27). CTLA4 and CTLA4 antibodies are important influencers of T cell function and interfere with the immune microenvironment in the body. In vitro and in vivo studies have demonstrated that CTLA4 antibodies can specifically alleviate the immunosuppression of CTLA4, activate T cells, and induce the production of IL-2, which is expected to have a wide range of applications in gene therapy for diseases such as tumors and parasitic infections.

[0007] CTLA4 antibodies exert specific therapeutic effects against diseases and show remarkable efficacy, and therefore may be used to complement traditional medicines and explore new avenues of gene therapy.

[0008] Bispecific antibodies, also called bifunctional antibodies, are specific drugs that simultaneously target two different antigens and can be purified and produced by immunomagnetic separation. Alternatively, they can be obtained by genetic engineering. Genetic engineering has certain advantages, since it allows for flexibility in terms of optimizing the binding site, considering the synthesis form, yield, etc. Currently, more than 45 forms of existence have been proven (Dafne Muller, Kontermann R E., 2010, BioDrugs, 24(2): 89-98). Most of the bispecific antibodies developed are in the form of IgG-scFv, i.e., the Morrison format (Coloma MJ, Morrison SL., 1997, Nat Biotechnol., 15: 159-163), which has proven to be one of the ideal forms of bifunctional antibodies due to its similarity to the naturally occurring IgG form and advantages in antibody engineering, expression, and purification (Miller BR, Demarest SJ, et al., 2010, Protein Eng Des Sel, 23: 549-57; Fitzgerald J, Lugovskoy A., 2011, MAbs, 3: 299-309).

[0009] ADCC (antibody-dependent cellular cytotoxicity) refers to the direct killing of target cells by killer cells (NK cells, macrophages, etc.) and is mediated by the binding of the Fab fragment of an antibody to an epitope on a virus-infected cell or tumor cell and the binding of the Fc fragment of the antibody to an Fc receptor (FcR) on the surface of the killer cell.

[0010] CDC (Complement-Dependent Cytotoxicity) refers to the specific binding of antibodies to corresponding antigens on the cell membrane surface, which leads to the formation of complexes and activation of the complement system, which then leads to the formation of MAC on the surface of the target cell, resulting in the lysis of the target cell. Complement can cause the lysis of various bacteria and other pathogens, and is an important defense mechanism against pathogen infection.

[0011] Fc receptors belong to the immunoglobulin family and are expressed on the surface of certain immune cells, where they mediate immune responses by recognizing the Fc region of antibodies. When the Fab region of an antibody recognizes an antigen, the Fc region of the antibody binds to the Fc receptor on an immune cell (e.g., a killer cell) and initiates immune cell response functions such as phagocytosis and ADCC.

[0012] Depending on the type of antibody that the Fc receptor recognizes and the type of cell that expresses it, Fc receptors are mainly classified into three types: FcγR, FcαR, and FcεR. FcγR can be further classified into four subtypes: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcRn (neonatal Fc receptor). Of these, FcγRI, FcγRII, and FcγRIII are closely related to ADCC activity. FcγRIII is the most predominant molecule mediating ADCC and has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. The FcγRIIIa population has two subtypes, the high affinity FcγRIIIa_V158 and the low affinity FcγRIIIa_F158, which are distinguished by a single nucleotide polymorphism (SNP) site. FcγRI has high affinity for the Fc region of IgG and is involved in the ADCC process. FcγRII contains three subtypes, FcγRIIa, FcγRIIb, and FcγRIIc (also called CD32a, CD32b, and CD32c, respectively), of which FcγRIIa has ADCC activity, and for FcγRIIa, there are two subtypes in humans, FcγRIIa_H131 and FcγRIIa_R131, due to a single nucleotide mutation. FcγRIIb is an inhibitory receptor and is a typical inhibitory FcγR that inhibits the nearby ITAM pathway. For example, after immune complexes bind to the BCR, Fc fragments bind to FcγRIIb on the same cell, negatively regulating B cell activation and reducing antibody and cytokine secretion (Hogarth PM, Pietersz GA., 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331).

[0013] The IgG family includes four members, IgG1, IgG2, IgG3, and IgG4, which differ in amino acids in the fragment crystallizable (Fc) region of the heavy chain constant region and, as a result, in their affinity for FcγR. IgG1 is the most abundant subtype in humans and is also the most commonly used subtype in monoclonal antibody drugs. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. IgG2 has the lowest affinity for FcγRs, but it can still bind to FcγRIIa and induce monocyte-mediated ADCC. IgG3 has the highest binding ability to FcγRs, induces ADCC, and exerts a greater CDC effect than IgG1. IgG4 molecules bind weakly to FcγRs other than FcγRI, making them less likely to cause CDC or NK cell-mediated ADCC. However, antibodies of the IgG4 subtype can mediate ADCP effects through binding to FcγRI, and the ADCP effects present in antibody drugs targeting immune cells may damage immune cells and cause pharmacological side effects. Currently, there remains a need for the development of new anti-CTLA4-anti-PD-1 bispecific antibodies to reduce or eliminate the damage caused by antibody-mediated ADCC, ADCP, and / or CDC activity against immune cells to which the anti-CTLA4-anti-PD-1 bispecific antibodies bind, and to improve the efficacy of antibody pharmaceuticals.

[0014] Chemotherapeutic drugs are currently classified into nine main classes (He Jie, et al., Clinical Oncology, Beijing, People's Medical Publishing House, 2016: 230-237). The first class is a drug that directly binds to DNA and interferes with DNA replication, including various alkylating agents, mitomycin, bleomycin, dacarbazine, platinum-based drugs (e.g., cisplatin, carboplatin), camptothecin, and their derivatives. The second class is a drug that inhibits nucleic acid biosynthesis, which mainly acts on the enzyme system of tumor cells to block the synthesis of DNA and RNA precursors and inhibit the formation of DNA or RNA, mainly including methotrexate, fluorouracil, 6-mercaptopurine, hydroxyurea, cytarabine, etc. These drugs mainly act on cells in the S phase and are antimetabolite chemotherapy drugs and phase-specific anticancer drugs. The third class are chemotherapy drugs that affect transcription through a pharmacological mechanism in which they intercalate into the DNA double helix and form non-covalent bonds with it, preventing the DNA-dependent transcription of genetic information on DNA into mRNA, causing damage to template function and inhibition of transcription. The fourth class affects tubulin and mitosis, and mainly includes plant-based drugs such as vinca alkaloids, podophyllotoxins, and taxanes. The fifth class is drugs that affect ribosome function and inhibit protein synthesis. Representative of such drugs are harringtonine, which inhibit the initiation of protein synthesis and degrade ribosomes to release new peptide chains, but do not inhibit the binding of mRNA and tRNA to ribosomes. Such drugs cause a reduction in nuclear DNA and cytoplasmic RNA and depolymerization of polysomes, inhibiting mitosis. The sixth class is drugs that act on tumor cell membranes, such as concanavalin (Con-A) and phytohemagglutinin (PHA). These drugs bind to glycoprotein receptors on the cell membrane, thus affecting DNA synthesis in tumor cells and preventing them from dividing. The seventh class is apoptosis-inducing drugs, such as arsenic trioxide.The eighth class are hormones that regulate the endocrine system to treat tumors, including estrogens, antiestrogens, progestogens, androgens, antiandrogens, corticosteroids, and anticorticosteroids (including dichlorodiphenyldichloroethane and aminoglutethimide). The ninth class are targeted anticancer therapies, including monoclonal antibodies, epidermal growth factor signaling inhibitors (e.g., targeted agents against receptor tyrosine kinase pathways), ubiquitin proteasome inhibitors, and angiogenesis inhibitors.

[0015] Chiauranib is a multi-target multi-pathway selective kinase inhibitor developed by Shenzhen Chipscreen Biosciences Co., Ltd. Chiauranib can selectively inhibit multiple kinase targets such as Aurora B, CSF1R, VEGFR / PDGFR / c-Kit, thereby inhibiting tumor cell proliferation, enhancing anti-tumor immunity, inhibiting tumor angiogenesis, and realizing the anti-tumor effect of multi-pathway mechanism. In previous clinical trials, monotherapy with chiauranib for small cell lung cancer (SCLC) patients who had failed multi-line treatment achieved very good efficacy compared with historical control data. A Chinese patent (publication number CN111728974A) discloses the structure of the chiauranib compound, which is N-(2-aminophenyl)-6-(7-methoxyquinoline-4-oxy)-1-naphthamide. The structural formula is as shown in Formula I. [ka] Summary of the Invention [Problem to be solved by the invention]

[0016] However, in various tumors, the disease cannot be controlled for a long time even after chemotherapy treatment, and the 5-year survival rate remains very low. Therefore, the development of less toxic and more effective therapeutic approaches or combination dosing regimens is of great clinical significance. [Means for solving the problem]

[0017] summary Through intensive research and creative efforts, the present inventors have appropriately and creatively combined anti-CTLA4 / anti-PD-1 antibody and chiauranib, and the resulting pharmaceutical combination has excellent anti-tumor effect. The present invention is described in detail below.

[0018] One aspect of the invention relates to a pharmaceutical combination comprising Chiauranib or a pharma- ceutical acceptable salt thereof (e.g., the hydrochloride salt) or a crystalline form thereof (e.g., non-solvated crystalline form A, B, or C) and at least one (e.g., one, two, or three) anti-CTLA4-anti-PD-1 bispecific antibody.

[0019] In some embodiments of the present invention, there is provided a pharmaceutical combination, wherein the mass ratio of chiauranib or a pharma- ceutical acceptable salt thereof or a crystalline form thereof to an anti-CTLA4-anti-PD-1 bispecific antibody is 1:(1-1000), preferably 1:(5-500), and more preferably 1:(10-100).

[0020] In some embodiments of the present invention, there is provided a pharmaceutical combination, in which the mass ratio of chiauranib or a pharma- ceutical acceptable salt thereof or a crystalline form thereof to an anti-CTLA4-anti-PD-1 bispecific antibody is 1:(5 to 1000), preferably 1:(10 to 500), and more preferably 1:(10 to 100), 1:(10 to 150), 1:(10 to 200), 1:(10 to 250), 1:(10 to 300), 1:(10 to 350), 1:(10 to 400), or 1:(10 to 450).

[0021] In some embodiments of the present invention, a pharmaceutical combination is provided, wherein the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 100 mg-1000 mg, 200 mg-800 mg, 200 mg-500 mg, 300 mg-600 mg, 400 mg-500 mg, or 450 mg.

[0022] In some embodiments of the present invention, pharmaceutical combinations are provided, wherein the unit dose of chiauranib or a pharma- ceutical acceptable salt thereof or a crystalline form thereof is a unit dose of 0.1 mg to 100 mg, 0.5 mg to 50 mg, 1 mg to 20 mg, 2 mg to 15 mg, 3 mg to 12 mg, 4 mg to 8 mg, or 5 mg.

[0023] In some embodiments of the present invention, a pharmaceutical combination is provided, wherein: The pharmaceutical combination is a fixed combination, e.g., a pharmaceutical composition, or the pharmaceutical combination is a non-fixed combination, e.g., Chiauranib or a pharma- ceutical acceptable salt or a crystalline form thereof, and the anti-CTLA4-anti-PD-1 bispecific antibodies in the non-fixed combination are each present in a separate pharmaceutical composition.

[0024] In some embodiments of the present invention, pharmaceutical compositions are provided, wherein said pharmaceutical compositions are independently a solid pharmaceutical composition or a liquid pharmaceutical composition.

[0025] In some embodiments of the invention, pharmaceutical combinations are provided, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable adjuvants, such as carriers and / or excipients.

[0026] In some embodiments of the present invention, a pharmaceutical combination is provided, wherein the pharmaceutical composition does not contain any active pharmaceutical ingredient other than chiauranib or a pharma- ceutical acceptable salt thereof or a crystalline form thereof, and the anti-CTLA4-anti-PD-1 bispecific antibody.

[0027] In some embodiments of the invention, a pharmaceutical combination is provided in which the anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional domain that targets PD-1; and A second protein functional domain that targets CTLA4 (where: the first protein functional region is an immunoglobulin and the second protein functional region is a single chain fragment variable region, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the single chain fragment variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; or the first protein functional region is a single chain fragment variable region and the second protein functional region is an immunoglobulin, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; the heavy chain variable region of the single chain fragment variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the heavy chain constant region of said immunoglobulin has mutations at any two or three of positions 234, 235, and 237 according to the EU numbering system, and the affinity constant of said bispecific antibody for FcγRIIIa and / or C1q is decreased after the mutations compared to before the mutations; preferably, said affinity constants are measured by a Fortebio Octet molecular interaction instrument.

[0028] In one or more embodiments of the invention, a pharmaceutical combination is provided having an anti-CTLA4-anti-PD-1 bispecific antibody, wherein after said mutations, the affinity constant of the bispecific antibody for FcγRIIIa, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb is decreased compared to before the mutations; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.

[0029] In some embodiments of the present invention, a pharmaceutical combination is provided comprising an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the heavy chain constant region of said immunoglobulin has the following mutations according to the EU numbering system: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, and G237A.

[0030] In the present invention, unless otherwise specified, the letter before the position number represents the amino acid before the mutation, and the letter after the position number represents the amino acid after the mutation.

[0031] In some embodiments of the invention, a pharmaceutical combination is provided, wherein the anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional domain that targets PD-1; and a second protein functional domain that targets CTLA4; where: the first protein functional region is an immunoglobulin and the second protein functional region is a single chain fragment variable region, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the single chain fragment variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; or

[0032] the first protein functional region is a single chain fragment variable region and the second protein functional region is an immunoglobulin, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36 to 38, respectively; the heavy chain variable region of the single chain fragment variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30 to 32, respectively;

[0033] the immunoglobulin is a human IgG1 subtype, According to the EU numbering system, said immunoglobulin comprises a heavy chain constant region having one of the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, and G237A.

[0034] In some embodiments of the invention, a pharmaceutical combination is provided having an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the heavy chain constant region of the immunoglobulin further comprises the following amino acids according to the EU numbering system: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, The gene has one or more mutations selected from P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A.

[0035] In one or more embodiments of the invention, a pharmaceutical combination is provided in which the anti-CTLA4-anti-PD-1 bispecific antibody is in the form of an IgG-scFv, i.e., the Morrison format.

[0036] In some embodiments of the invention, pharmaceutical combinations with anti-CTLA4-anti-PD-1 bispecific antibodies are provided, wherein the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:14 and SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:16 and SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of the single chain fragment variable part is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41, and SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single chain fragment variable part is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42, and SEQ ID NO:44;

[0037] Or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41, and SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42, and SEQ ID NO:44; the amino acid sequence of the heavy chain variable region of the single chain fragment variable part is selected from SEQ ID NO:14 and SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single chain fragment variable part is selected from SEQ ID NO:16 and SEQ ID NO:20.

[0038] In some embodiments of the present invention, a pharmaceutical combination is provided in which the anti-CTLA4-anti-PD-1 bispecific antibody is selected from any one of the following (1) to (20):

[0039] (1) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 4;

[0040] (2) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 8;

[0041] (3) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 12;

[0042] (4) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 4;

[0043] (5) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 8;

[0044] (6) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is set forth in SEQ ID NO: 12;

[0045] (7) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:16;

[0046] (8) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:20;

[0047] (9) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:16;

[0048] (10) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO:20;

[0049] (11) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 16;

[0050] (12) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 20;

[0051] (13) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 42;

[0052] (14) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 44;

[0053] (15) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 42;

[0054] (16) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 44;

[0055] (17) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 16;

[0056] (18) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 16;

[0057] (19) the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 20;

[0058] (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 20.

[0059] In some embodiments of the invention, a pharmaceutical combination is provided having an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO: 40 and the amino acid sequence of the light chain is set forth in SEQ ID NO: 24.

[0060] In some embodiments of the invention, pharmaceutical combinations comprising an anti-CTLA4-anti-PD-1 bispecific antibody are provided, wherein the immunoglobulin or antigen-binding fragment thereof binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb at a concentration of about 10 -7 Affinity constants greater than M, e.g., about 10 -6 M, 10 -5 M, 10 -4 M or 10 -3 binds with an affinity constant that is greater than or equal to M, preferably the affinity constant is measured by a Fortebio Octet molecular interaction instrument; Preferably, the immunoglobulin or antigen-binding fragment thereof has no binding signal or a binding signal of less than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb; preferably, the binding signal refers to a response value measured by a Fortebio Octet molecular interaction device.

[0061] In some embodiments of the invention, a pharmaceutical combination is provided having an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the immunoglobulin or antigen-binding fragment thereof binds C1q at a concentration of about 10 -9 Affinity constants greater than M, e.g., about 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 M or greater. Preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument. Preferably, the immunoglobulin or antigen-binding fragment thereof has no or a binding signal of less than 0.1 nm to C1q. Preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction device.

[0062] In some embodiments of the invention, a pharmaceutical combination is provided comprising an anti-CTLA4-anti-PD-1 bispecific antibody, wherein a first protein functional domain is linked to a second protein functional domain directly or via a linker fragment, and / or a heavy chain variable domain of a single chain fragment variable domain is linked to a light chain variable domain of a single chain fragment variable domain directly or via a linker fragment.

[0063] In some embodiments of the invention, a pharmaceutical combination is provided comprising an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the linker fragment is (GGGGS)n, where n is a positive integer, preferably n is 1, 2, 3, 4, 5, or 6.

[0064] In some embodiments of the invention, a pharmaceutical combination is provided comprising an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the number of first protein functional domains and second protein functional domains is each independently one, two or more.

[0065] In one or more embodiments of the invention, a pharmaceutical combination is provided with an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the number of first protein functional domains is one and the number of second protein functional domains is two.

[0066] In one or more embodiments of the present invention, a pharmaceutical combination is provided with an anti-CTLA4-anti-PD-1 bispecific antibody, in which the single-chain fragment variable region is linked to the C-terminus of an immunoglobulin heavy chain. Since an immunoglobulin has two heavy chains, two single-chain fragment variable region molecules are linked to one immunoglobulin molecule. Preferably, the two single-chain fragment variable region molecules are identical. Preferably, the single-chain fragment variable region is linked to the C-terminus of an immunoglobulin heavy chain by forming an amide bond via the above-mentioned linker fragment.

[0067] In one or more embodiments of the invention, the constant regions of the immunoglobulins are humanized, for example, the heavy chain constant region is the Ig gamma-1 chain C region, accession number P01857, and the light chain constant region is the Ig kappa chain C region, accession number P01834.

[0068] In one or more embodiments of the invention, a pharmaceutical combination is provided, wherein the anti-CTLA4-anti-PD-1 bispecific antibody binds to the CTLA4 protein and / or the PD-1 protein at a concentration of about 10 -5 K under M D , for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K below M D Combine with.

[0069] In one or more embodiments of the invention, a pharmaceutical combination is provided, wherein the anti-CTLA4-anti-PD-1 bispecific antibody is a monoclonal antibody. In one or more embodiments of the invention, a pharmaceutical combination is provided, wherein the anti-CTLA4-anti-PD-1 bispecific antibody is a humanized antibody.

[0070] In some embodiments of the invention, pharmaceutical combinations are provided that comprise an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the single chain fragment variable region is linked to the C-terminus of the immunoglobulin heavy chain.

[0071] In some embodiments of the invention, a pharmaceutical combination is provided in which the anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional domain 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 said first protein functional domain is an immunoglobulin and said second protein functional domain is a single chain fragment variable domain; The amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO: 40, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 24; The amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is set forth in SEQ ID NO: 44; the single chain fragment variable region is linked to the C-terminus of the heavy chain of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment; the heavy chain variable region of the single chain fragment variable region is linked to the light chain variable region of the single chain fragment variable region via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment each have an amino acid sequence independently selected from SEQ ID NO: 25 and SEQ ID NO: 26; Preferably, the amino acid sequences of said first linker fragment and said second linker fragment are shown in SEQ ID NO:26).

[0072] Another aspect of the invention relates to a kit product comprising a pharmaceutical combination according to any of the embodiments of the invention and a package insert.

[0073] In some embodiments of the invention, the kit product comprises a first product and a second product in separate packages, Where: The first article of manufacture comprises an anti-CTLA4-anti-PD-1 bispecific antibody according to any embodiment of the invention; the second product comprises Chiauranib or a pharma- ceutically acceptable salt thereof (e.g., the hydrochloride salt) or a crystalline form thereof (e.g., unsolvated crystalline form A, B, or C); Preferably, the first product and the second product further independently comprise one or more pharma- ceutically acceptable adjuvants; Preferably, the kit product further comprises a package insert.

[0074] In one or more embodiments of the invention, a kit product is provided, wherein the unit dose of the first product is 100 mg to 1000 mg, 200 mg to 800 mg, 200 mg to 500 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg based on the mass of the anti-CTLA4-anti-PD-1 bispecific antibody.

[0075] In one or more embodiments of the invention, a kit product is provided, wherein the unit dose of the second product is 0.1 mg to 100 mg, 0.5 mg to 50 mg, 0.5 mg to 10 mg, 1 mg to 10 mg, 2 mg to 8 mg, or 1 mg to 5 mg, or 5 mg, based on the weight of chiauranib or a pharma- ceutically acceptable salt thereof or a crystalline form thereof.

[0076] In one or more embodiments of the invention, a kit product is provided, wherein the unit dose of the second product is 1 mg to 20 mg, 2 mg to 15 mg, 3 mg to 12 mg, 4 mg to 8 mg, or 5 mg, based on the mass of chiauranib or a pharma- ceutically acceptable salt thereof or a crystalline form thereof.

[0077] Yet another aspect of the invention relates to the use of a pharmaceutical combination according to any of the embodiments of the invention for the preparation of a medicament for the treatment or prevention of tumors; Preferably, the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, and nasopharyngeal cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of tumors of MSI-H / dMMR phenotype (colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors).

[0078] MSI refers to microsatellite instability. Microsatellites are short tandem repeats that span the human genome and contain 10-50 repeats of one, two, or more nucleotides. Microsatellites in certain abnormal cells, such as tumors, are altered in length compared to normal cells by insertion or deletion of repeat units. Such alterations are called MSI. Based on the instability and degree, MSI can be classified as microsatellite high instability (MSI-H), microsatellite low instability (MSI-L), and microsatellite stable (MSS). The main cause of MSI is a defect in DNA mismatch repair (MMR). Human mismatch repair genes (MMR genes) can express the corresponding mismatch repair proteins through transcription and translation. Lack of expression of MMR proteins leads to mismatch repair deficiency, which leads to the accumulation of base pair mismatches during DNA replication, ultimately causing MSI. Approximately 15% of colorectal cancers are attributed to the MSI pathway. This was first reported in colorectal cancer, but can also occur in gastric cancer, endometrial cancer, and adrenal cortical carcinoma (Baretti M et al., Pharmacol Ther., 2018; 189: 45-62). Subsequent studies have found MSI-H / dMMR signatures in mesothelioma, sarcoma, adrenal cortical carcinoma, melanoma, and ovarian germ cell tumors.

[0079] MSI-H and dMMR are the results of two different assays, which are biologically consistent and are called MSI-H / dMMR or MSI-high / dMMR, while MSI-L and MSS are phenotypes of mismatch repair proficient (pMMR). dMMR detection is to perform immunohistochemical assays of protein expression of four mismatch genes, MSH2, MLH1, MSH6, and PMS2, based on tumor specimens (including surgical and aspirate specimens). Absence of expression of any of the four proteins establishes dMMR; positive results of all four proteins indicate pMMR, i.e., complete mismatch repair function. MSI detection is to match and compare the lengths of repetitive DNA sequences (microsatellite sequences) in tumor cells and somatic cells. When five standard loci are detected using PCR based on the US NCI criteria, mismatches at two or more loci indicate instability defined as MSI-H, one mismatched locus indicates MSI-L, and five matched loci indicate MSS. High-throughput sequencing (also called next-generation sequencing, NGS) can also be used as a method to detect microsatellite instability. When more microsatellite loci are selected in a PCR assay, such as more than five loci or additional microsatellite loci, mismatches at 30% or more of the loci are defined as MSI-H, matches at all loci are defined as MSS, and mismatches between 0% and 30% are defined as MSI-L.

[0080] A pharmaceutical combination according to any embodiment of the invention for use in the treatment or prevention of a tumor; Preferably, the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, and nasopharyngeal cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of tumors of MSI-H / dMMR phenotype (colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors).

[0081] Yet another aspect of the present invention relates to a method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of a pharmaceutical combination according to any of the embodiments of the present invention. Preferably, the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer and nasopharyngeal cancer. Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer. Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma. Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of tumors of MSI-H / dMMR phenotype (colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors).

[0082] In one or more embodiments of the present invention, a method of treating or preventing a tumor is provided, comprising administering to a subject in need thereof an effective amount of an anti-CTLA4-anti-PD-1 bispecific antibody before or after surgical treatment and / or before or after radiation therapy.

[0083] In one or more embodiments of the invention, a method for treating or preventing a tumor is provided, wherein the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 0.1 to 100 mg, preferably 1 to 10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg) per kg of body weight, or the unit dose of the anti-CTLA4-anti-PD-1 bispecific antibody is 0.1 to 100 mg, preferably 1 to 10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg) per kg of body weight. The amount is 10 to 1000 mg (e.g., 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) for each subject, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg; Preferably, the dose is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the route of administration is intravenous infusion or intravenous injection.

[0084] In some embodiments, administration of the anti-CTLA4-anti-PD-1 bispecific antibody is in two-week (14 day) or three-week (21 day) cycles, and preferably, the anti-CTLA4-anti-PD-1 bispecific antibody is administered intravenously on the first day (D1) of each cycle. For example, the anti-CTLA4-anti-PD-1 bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).

[0085] In one or more embodiments of the invention, a method for treating or preventing tumors is provided, in which an anti-CTLA4-anti-PD-1 bispecific antibody and chiauranib are administered simultaneously or sequentially.

[0086] Antibody drugs, especially monoclonal antibodies (mAbs), have achieved good efficacy in treating various diseases. Traditional methods for obtaining these therapeutic antibodies include immunizing animals with an antigen to obtain antibodies that target the antigen in the immunized animal, or modifying antibodies with low affinity to the antigen by affinity maturation.

[0087] The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain is composed of three hypervariable regions, or complementarity determining regions (CDRs), (heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include 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.).

[0088] The amino acid sequences of the CDR regions of the monoclonal antibody sequences (1) to (13) below were analyzed by technical means known to those skilled in the art, for example, the VBASE2 database, and the results are as follows:

[0089] (1)14C12 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16. The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO: 27) HCDR2: ISGGGRYT (SEQ ID NO: 28) HCDR3: ANRYGEAWFAY (sequence number 29). The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDINTY (sequence number 30) LCDR2: RAN (SEQ ID NO: 31) LCDR3: LQYDEFPLT (sequence number 32).

[0090] (2)14C12H1L1 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20. The amino acid sequences of the three CDR regions of the heavy chain variable region are identical to the amino acid sequence of 14C12. The amino acid sequences of the three CDR regions of the light chain variable region are identical to the amino acid sequence of 14C12.

[0091] (3) 4G10 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GYSFTGYT (SEQ ID NO: 33) HCDR2: INPYNNIT (SEQ ID NO: 34) HCDR3: ARLDYRSY (sequence number 35). The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: TGAVTTSNF (SEQ ID NO: 36) LCDR2: GTN (SEQ ID NO: 37) LCDR3:ALWYSNHWV (sequence number 38).

[0092] (4) 4G10H1L1 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8. The amino acid sequences of the three CDR regions of the heavy chain variable region are identical to the amino acid sequence of 4G10. The amino acid sequences of the three CDR regions of the light chain variable region are identical to the amino acid sequence of 4G10.

[0093] (5)4G10H3L3 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12. The amino acid sequences of the three CDR regions of the heavy chain variable region are identical to the amino acid sequence of 4G10. The amino acid sequences of the three CDR regions of the light chain variable region are identical to the amino acid sequence of 4G10.

[0094] (6) BiAb001(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO: 27) HCDR2: ISGGGRYT (SEQ ID NO: 28) HCDR3: ANRYGEAWFAY (SEQ ID NO: 29) HCDR4: GYSFTGYT (SEQ ID NO: 33) HCDR5: INPYNNIT (SEQ ID NO: 34) HCDR6: ARLDYRSY (SEQ ID NO: 35) HCDR7: TGAVTTSNF (SEQ ID NO: 36) HCDR8: GTN (SEQ ID NO: 37) HCDR9: ALWYSNHWV (sequence number 38). The amino acid sequences of the three CDR regions associated with the light chain variable region are as follows: LCDR1: QDINTY (sequence number 30) LCDR2: RAN (SEQ ID NO: 31) LCDR3: LQYDEFPLT (sequence number 32).

[0095] (7) BiAb002(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are identical to the amino acid sequence of BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are identical to those of BiAb001(M).

[0096] (8)BiAb003(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are identical to the amino acid sequence of BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are identical to those of BiAb001(M).

[0097] (9)BiAb004(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are identical to the amino acid sequence of BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are identical to those of BiAb001(M).

[0098] In the antibody BiAb004(hG1TM) of the present invention, amino acid mutations have been introduced into the non-variable region of BiAb004(M). According to the EU numbering system, the amino acid mutations have been introduced at positions 234, 235, and 237: BiAb004 (hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the hinge region of the heavy chain.

[0099] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.In addition, the experimental procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures widely used in the corresponding fields.Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0100] In the present specification, when the amino acid sequence of CTLA4 protein (cytotoxic T-lymphocyte-associated antigen 4) is mentioned, this includes, but is not limited to, the full length of CTLA4 protein, or the extracellular fragment of CTLA4, CTLA4ECD, or a fragment containing CTLA4ECD. Also included are fusion proteins of CTLA4ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, the skilled artisan will understand that mutations or variants (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of CTLA4 protein may be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "CTLA4 protein" includes all such sequences, including natural or artificial variants thereof. Furthermore, when a sequence fragment of CTLA4 protein is described, it also includes the corresponding sequence fragment in the natural or artificial variants thereof.

[0101] In the present specification, when the amino acid sequence of PD-1 protein is referred to, this includes, but is not limited to, the full-length PD-1 protein (NCBI GenBank: NM_005018), or the extracellular fragment of PD-1, PD-1ECD, or a fragment containing PD-1ECD. Also included are fusion proteins of PD-1ECD, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variants (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of PD-1 protein may be naturally generated or artificially introduced without affecting its biological function. Thus, in the present invention, the term "PD-1 protein" is intended to include all such sequences, including natural or artificial variants thereof. Furthermore, when a sequence fragment of PD-1 protein is described, it also includes the corresponding sequence fragment in a natural or artificial variant thereof.

[0102] In this specification, unless otherwise specified, B7 refers to B7-1 and / or B7-2, the specific protein sequences of which are known in the art and can be referenced to existing literature or sequences disclosed in GenBank, e.g., B7-1 (CD80, NCBI gene ID: 941), B7-2 (CD86, NCBI gene ID: 942).

[0103] As used herein, EC 50 The term refers to the concentration of 50% of the maximal effect, ie the concentration capable of producing 50% of the maximal effect.

[0104] The term "antibody" as used herein generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. In a general sense, heavy chains can be understood as the larger polypeptide chain in an antibody, and light chains refer to the smaller polypeptide chain in an antibody. Light chains can be classified as kappa and lambda light chains. Heavy chains are generally classified as μ, δ, γ, α, and ε, and antibodies are defined as IgM, IgD, IgG, IgA, and IgE isotypes, respectively. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 , and C H3 Each light chain is composed of a light chain variable region (V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). H Area and V LThe regions can be further subdivided into hypervariable regions (called complementarity determining regions, or CDRs), with conserved regions called framework regions (FRs) distributed between the CDRs. H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L ) form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. In particular, the heavy chain can further comprise more than three CDRs, such as six, nine, or twelve. For example, in the bifunctional antibody of the present invention, the heavy chain is an scFv in which the C-terminus of the heavy chain of an IgG antibody is linked to another antibody, in which case the heavy chain comprises nine CDRs. The term "antibody" is not limited by the particular method for producing the antibody. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies, among others. The antibodies may be of different isotypes, for example IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0105] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., DNA recombination, or enzymatic or chemical cleavage) and the antigen-binding fragments of antibodies are screened for specificity in the same manner as are intact antibodies.

[0106] Unless otherwise clearly defined by the context, when the term "antibody" is referred to herein, this includes not only complete antibodies but also antigen-binding fragments of antibodies.

[0107] The terms "mAb" and "monoclonal antibody" as used herein refer to an antibody or antibody fragment obtained from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules, excluding natural mutations that may occur spontaneously. Monoclonal antibodies are highly specific to a single epitope on an antigen. In comparison with monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that globally recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using DNA recombination (see, e.g., U.S. Patent No. 4,816,567).

[0108] The term "humanized antibody" as used herein refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g. mouse, rat, or rabbit) antibody with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced with the corresponding amino acid residues of the non-human antibody or amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For details on humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today, 21: 397-402 (2000).

[0109] The term "epitope" as used herein refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant". Epitopes or antigenic determinants are generally composed of chemically active surface groups of molecules, such as amino acids, carbohydrates, or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial structure and can be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are located linearly along the primary amino acid sequence of the protein. In a conformational epitope, the sites of interaction are located across amino acid residues of the protein that are separated from each other.

[0110] The term "isolated" as used herein refers to being obtained by artificial means from a natural state. If a particular "isolated" substance or component exists in nature, it may be that its natural environment has been altered or that it has been isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally exists in a particular living animal, and the same polynucleotide or polypeptide of high purity isolated from such a natural state is called an isolated polynucleotide or isolated polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0111] The term "E. coli expression system" as used herein refers to an expression system consisting of an E. coli (strain) and a vector, where the E. coli (strain) is derived from commercially available strains such as, but not limited to, GI698, ER2566, BL21(DE3), B834(DE3), and BLR(DE3).

[0112] The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), phages (e.g., lambda phage or M13 phage), and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). The vector may contain various elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may further contain a replication origin.

[0113] The term "host cell" as used herein refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0114] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen that it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) refers to an antibody that binds to an antigen with a specific binding affinity of about 10 -5 Affinity (K D ), e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity below M (K D In some embodiments of the present invention, the term "targeted" refers to specific binding.

[0115] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the stronger the binding between the antibody and the antigen and the higher the affinity between the antibody and the antigen. In general, antibodies have a dissociation equilibrium constant of about 10 -5 The dissociation equilibrium constant (K D ), e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to an antigen, which is measured, for example, by a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio Octet molecular interaction instrument.

[0116] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably. The terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably. The terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, as used herein, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0117] The term "pharmaceutical acceptable carrier and / or excipient" as used herein refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. Such carriers and / or excipients are known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th Ed., Pennsylvania, Mack Publishing Company, 1995) and include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride.

[0118] The term "adjuvant" as used herein refers to a non-specific immune enhancing agent that, when delivered to an organism together with or prior to an antigen, can enhance or change the type of immune response of the organism to the antigen. There are various types of adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments. In clinical trials, aluminum hydroxide adjuvant is more frequently used.

[0119] The term "effective amount" as used herein refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, a prophylactically effective amount (e.g., in the case of a disease associated with CTLA4 binding to B7 or CTLA4 overactivity, e.g., tumors) is an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., in the case of a disease associated with CTLA4 binding to B7 or CTLA4 overactivity, e.g., tumors), and a therapeutically effective amount is an amount sufficient to cure or at least partially stop the disease and its complications in a patient suffering from the disease. It is certainly within the capabilities of a person skilled in the art to determine such an effective amount. For example, an amount effective for therapeutic purposes depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and sex, the route of administration, and other treatments performed at the same time, etc.

[0120] A "recurrent" cancer is one that regrows at the original site or at a site distant from the original site after responding to a previous treatment (e.g. surgery). A "locally recurrent" cancer is one that occurs after treatment in the same location as the previously treated cancer.

[0121] "Metastatic" cancer refers to cancer that has spread from one part of the body (such as the lungs) to another part.

[0122] As used herein, the term "completely eliminated" means that there is no binding signal or the binding signal is very weak as detected by existing instruments (e.g., Fortebio Octet molecular interaction instrument). In one embodiment of the present invention, there is no binding signal or the binding signal is very weak means that the binding signal (i.e., the response value or response) is less than 0.1 nm.

[0123] In the present invention, the terms "first" (e.g., a first protein functional domain, a first linker fragment, or a first product) and "second" (e.g., a second protein functional domain, a second linker fragment, or a second product) are used for expressive distinction or clarification, unless specifically stated otherwise, and do not have a typical sequential meaning.

[0124] In the present invention, "about" or "approximately" means that the defined value or physical quantity varies within a range of 10%, 20%, or 30%, unless otherwise specified. For example, about 100 minutes or approximately 100 minutes can be 90 minutes to 110 minutes, 80 minutes to 120 minutes, or 70 minutes to 130 minutes.

[0125] The crystalline forms of Chiauranib include, but are not limited to, nonsolvated crystalline form A, nonsolvated crystalline form B, and nonsolvated crystalline form C. Reference is made to the description in the Chinese granted patent CN107868044B, in which: The X-ray powder diffraction pattern of the unsolvated crystalline form A has characteristic peaks at reflection angles 2θ of 6.88°, 9.26°, 12.74°, 13.82°, 18.58°, 20.86°, and 25.72°; The X-ray powder diffraction pattern of the unsolvated crystalline form B has characteristic peaks at reflection angles 2θ of 4.88°, 9.68°, 12.74°, 14.52°, 17.72°, 24.30°, and 25.26°; The X-ray powder diffraction pattern of the unsolvated crystalline form C has characteristic peaks at reflection angles 2θ of 4.84°, 9.68°, 12.92°, 14.60°, 16.46°, 17.44°, 22.00°, and 25.28°.

[0126] Beneficial Effects of the Invention The present invention achieves one or more of the following technical effects (1) to (2):

[0127] (1) The pharmaceutical combination of the present invention has excellent effects in treating or preventing tumors. (2) In the pharmaceutical combination of the present invention, the anti-CTLA4-anti-PD-1 bispecific antibody and chiauranib have a synergistic effect, and thus a synergistic effect is achieved in the treatment or prevention of tumors, where the tumors include, but are not limited to, melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, or nasopharyngeal cancer.Preferably, the lung cancer is non-small cell lung cancer, small cell lung cancer, or squamous cell lung cancer, and preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma. [Brief description of the drawings]

[0128] [Figure 1] Figure 1: Affinity constant assay of BiAb004 (hG1TM) for FcγRI. Antibody concentrations for the curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM from top to bottom, respectively. [Diagram 2] Figure 2: Affinity constant assay of BiAb004 (hG1WT) for FcγRI. Antibody concentrations for the curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM from top to bottom, respectively. [Diagram 3] Figure 3: Affinity constant assay of BiAb004 (hG1TM) for FcγRIIIa_V158. Antibody concentrations for the curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM from top to bottom, respectively. [Figure 4] Figure 4: Affinity constant assay of BiAb004 (hG1WT) for FcγRIIIa_V158. Antibody concentrations for the curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM from top to bottom, respectively. [Diagram 5] Figure 5: Affinity constant assay of BiAb004 (hG1TM) for FcγRIIIa_F158. Antibody concentrations for the curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM from top to bottom, respectively. [Figure 6] Figure 6: Affinity constant assay of BiAb004 (hG1WT) for FcγRIIIa_F158. Antibody concentrations for the curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM from top to bottom, respectively. [Figure 7] Figure 7: Affinity constant assay of BiAb004 (hG1TM) for FcγRIIa_H131. Antibody concentrations for the curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM from top to bottom, respectively. [Figure 8] Figure 8: Affinity constant assay of BiAb004 (hG1WT) for FcγRIIa_H131. Antibody concentrations for the curve pair from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 9] Figure 9: Affinity constant assay of BiAb004 (hG1TM) for FcγRIIa_R131. Antibody concentrations for the curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, from top to bottom, respectively. [Figure 10] Figure 10: Affinity constant assay of BiAb004 (hG1WT) for FcγRIIa_R131. Antibody concentrations for the curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, from top to bottom, respectively. [Figure 11] Figure 11: Affinity constant assay of BiAb004 (hG1TM) for FcγRIIb. Antibody concentrations for the curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM from top to bottom, respectively. [Figure 12] Figure 12: Affinity constant assay of BiAb004 (hG1WT) for FcγRIIb. Antibody concentrations for the curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, from top to bottom, respectively. [Figure 13]Figure 13: Affinity constant assay of BiAb004 (hG1TM) for C1q. Antibody concentrations for the curve pairs from top to bottom are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 14] Figure 14: Affinity constant assay of BiAb004 (hG1WT) for C1q. Antibody concentrations for the curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, from top to bottom, respectively. [Figure 15] Figure 15: Affinity constant assay of 5C10H2L2-IgG1mt for C1q. Antibody concentrations for the curve pair are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM from top to bottom, respectively. [Figure 16] FIG. 16: ADCC activity assay of BiAb004(hG1WT) and BiAb004(hG1TM) against 293T-CTLA4-PD1 cells expressing CTLA-4 and PD-1 antigens. [Figure 17] FIG. 17: IFN-γ secretion by mixed lymphocytes induced by BiAb004(hG1TM) in combination with Chiauranib. [Figure 18] FIG. 18: IL-2 secretion by mixed lymphocytes induced by BiAb004(hG1TM) in combination with Chiauranib. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0129] Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrating the present invention, and do not limit the scope of the present invention. The examples in which no specific technique or condition is specified are carried out according to the techniques or conditions described in the publications in the field (see, for example, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the attached instructions. The reagents or equipment used are commercially available conventional products unless the manufacturer is specified.

[0130] In the following examples of the present invention: BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center. Human peripheral blood mononuclear cells were isolated and prepared at Akeso Biopharma Inc. with informed consent of the donors. Raji-PDL1 is a cell line expressing human PD-L1 that was constructed by transfection based on the human B cell line Raji by Akeso Biopharma Inc. Ficoll-Paque™ PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare. Human IL-2 ELISA kit was purchased from Dakewe Biotech Co., Ltd. RPMI1640 medium, DMEM medium, trypsin-EDTA (0.25%), phenol red, and blasticidin were all purchased from Gibco. FBS was purchased from Excel bio.

[0131] The sequence of the isotype control antibody, human anti-hen egg lysozyme IgG (anti-HEL antibody, or human IgG, abbreviated as hIgG), was derived from the variable region sequence of Fab F10.6.6 in the study titled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" reported by Acierno et al. (Acierno et al., J Mol Biol., 2007; 374(1): 130-146).

[0132] In the following examples of the present invention, the chiauranib used was obtained from Shenzhen Chipscreen Biosciences Co., Ltd.

[0133] Preparation Example 1: Sequence design of anti-CTLA4 antibody The amino acid sequences and encoding nucleotide sequences of the heavy and light chains of anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to the sequences of 4G10, 4G10H1L1, and 4G10H3L3, respectively, in Chinese Patent Publication No. CN106967172A.

[0134] (1) 4G10 heavy and light chain variable region sequences Nucleotide sequence of the heavy chain variable region: (372bp) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAAG TTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (SEQ ID NO: 1)

[0135] Encoded amino acid sequence: (124aa) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO: 2)

[0136] Nucleotide sequence of the light chain variable region: (378bp) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTC TCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC (SEQ ID NO: 3)

[0137] Encoded amino acid sequence: (126aa) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO: 4)

[0138] (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 4G10H1L1 Nucleotide sequence of the heavy chain variable region (4G10H1V): (345bp) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCACCAA CTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT (SEQ ID NO: 5)

[0139] Encoded amino acid sequence: (115aa) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 6)

[0140] Nucleotide sequence of the light chain variable region (4G10L1V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACAATCG GGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG (SEQ ID NO: 7)

[0141] Encoded amino acid sequence: (109aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 8)

[0142] (3) Heavy and light chain variable region sequences of humanized monoclonal antibody 4G10H3L3 Nucleotide sequence of the heavy chain variable region (4G10H3V): (345bp) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACTAA CTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA (SEQ ID NO: 9)

[0143] Encoded amino acid sequence (4G10H3V): (115aa) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 10)

[0144] Nucleotide sequence of the light chain variable region (4G10L3V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAACAA GGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (SEQ ID NO: 11)

[0145] Encoded amino acid sequence (4G10L3V): (109aa) QAVVTQEPSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 12)

[0146] Preparation Example 2: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 The amino acid sequences and encoding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are identical to the sequences of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication No. CN106967172A.

[0147] (1) 14C12 heavy and light chain variable region sequences Nucleotide sequence of the heavy chain variable region: (354bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATC CTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 13)

[0148] Encoded amino acid sequence: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 14)

[0149] Nucleotide sequence of the light chain variable region: (321bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 15)

[0150] Encoded amino acid sequence: (107aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 16)

[0151] (2) Heavy and light chain variable region sequences, and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 Nucleotide sequence of the heavy chain variable region: (354bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 17)

[0152] Encoded amino acid sequence: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 18)

[0153] Nucleotide sequence of the light chain variable region: (321bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 19)

[0154] Encoded amino acid sequence: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK ((SEQ ID NO: 20)

[0155] DNA sequence of 14C12H1L1 heavy chain (14C12H1): (1344bp)

[0156] Encoded amino acid sequence: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22)

[0157] DNA sequence of 14C12H1L1 light chain (14C12L1): (642bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 23)

[0158] Encoded amino acid sequence: (214aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24)

[0159] Preparation Example 3: Sequence design of bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) The structural pattern of the bifunctional antibodies BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) is the Morrison format (IgG-scFv), i.e., the C-terminus of the two heavy chains of one IgG antibody is linked to the scFv fragment of another antibody via a linker fragment. The components of the heavy and light chain design are shown in Table A below. [Table A]

[0160] In Table A above, The amino acid sequence of linker 1 is (GGGGS)3 (SEQ ID NO:25). The amino acid sequence of linker 2 is (GGGGS)4 (SEQ ID NO:26).

[0161] In Table A above, the scFv fragments 4G10H1V(M), 4G10L1V(M), 4G10H3V(M), and 4G10L3V(M) of the BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) antibodies contain mutations in individual amino acids in the framework regions based on 4G10H1V, 4G10L1V, 4G10H3V, and 4G10L3V, respectively, which effectively optimizes the antibody structure and improves its efficacy.

[0162] (1) 4G10H1V(M): (115 aa, the mutation sites are underlined in the amino acid sequence based on 4G10H1V) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ C LEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 41)

[0163] (2) 4G10L1V(M): (110 aa, the mutation sites are underlined in the amino acid sequence based on 4G10L1V) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFG C GTKLTVL R (SEQ ID NO:42)

[0164] (3) 4G10H3V(M): (115 aa, the mutation sites are underlined in the amino acid sequence based on 4G10H3V) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ C LEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 43)

[0165] (4) 4G10L3V(M): (110 aa, the mutation sites are underlined in the amino acid sequence based on 4G10L3V) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFG C GTKLTVL R (SEQ ID NO:44)

[0166] Hereinafter, to distinguish it from the mutant antibody, BiAb004(M) is also referred to as BiAb004(hG1WT) in the examples of the present invention. The above BiAb004(M) is a "wild type" and contains an Ig gamma-1 chain C region (accession number P01857) as a heavy chain constant region and an Ig kappa chain C region (accession number P01834) as a light chain constant region.

[0167] Preparation Example 4: Design of mutations in non-variable region amino acids based on humanized bifunctional antibody BiAb004 Based on BiAb004(hG1WT) obtained in Preparation Example 3, the present inventors introduced a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the heavy chain to obtain BiAb004(hG1TM).

[0168] DNA sequence of the heavy chain of the immunoglobulin portion of BiAb004(hG1TM): (1344bp, mutation sites are underlined) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCT ACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCT AGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGC AAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGA GCC(SEQ ID NO:39)

[0169] Amino acid sequence of the heavy chain of the immunoglobulin portion of BiAb004(hG1TM): (448aa, mutation sites are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0170] BiAb004(hG1TM) and BiAb004(hG1WT) share the same DNA sequence and the same encoded amino acid sequence of the light chain, the specific sequence of which is shown in Preparation Example 3.

[0171] Experimental Example 1: Affinity assay of BiAb004(hG1WT) and BiAb004(hG1TM) for the receptor FcγRI The Fc receptor FcγRI, also known as CD64, can bind to the Fc fragment of IgG antibodies and is involved in antibody-dependent cellular cytotoxicity (ADCC). The ability of a therapeutic monoclonal antibody to bind to Fc receptors affects the safety and efficacy of the antibody.

[0172] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRI were measured using a Fortebio Octet molecular interaction instrument to assess the potential ADCC and ADCP activities of the antibodies.

[0173] The method for measuring the affinity constant of the corresponding antibody to FcγRI by the Fortebio Octet molecular interaction device is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween 20, 0.1% BSA, pH 7.4. A concentration of 1 μg / mL of FcγRI solution (purchased from Sinobio) was added to the HIS1K sensor to immobilize FcγRI on the sensor surface for 50 seconds. Both the binding and dissociation constants of the antibody to FcγRI were measured in buffer with antibody concentrations ranging from 3.12 to 50 nM (serial two-fold dilution). The antigen-immobilized sensor was equilibrated with buffer for 60 seconds, and then the binding of FcγRI to the antibody immobilized on the sensor was measured for 120 seconds. The dissociation of FcγRI from the antibody was measured for 120 seconds. The temperature was 30 °C and the frequency was 0.3 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constant of the antibody to FcγRI.

[0174] The results of the affinity constant assay for FcγRI of BiAb004(hG1TM) and BiAb004(hG1WT) are shown in Table 1 below and in FIGS. [Table 1]

[0175] The results show that BiAb004(hG1WT) bound to FcγRI with an affinity constant of 5.92E-09M. On the other hand, no corresponding data was obtained for BiAb004(hG1TM) since no or only a very weak binding signal to FcγRI was detected, indicating that there was no binding to FcγRI.

[0176] The results show that the binding activity of BiAb004(hG1TM) to FcγRI is effectively eliminated compared to BiAb004(hG1WT).

[0177] Experimental Example 2: Affinity constant assay of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_V158 The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) binds the Fc fragment of IgG antibodies and can mediate ADCC.

[0178] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_V158 were measured using a Fortebio Octet molecular interaction device to evaluate the ADCC activity of the antibodies.

[0179] The method for measuring the affinity constants of the corresponding antibodies to FcγRIIIa_V158 by the Fortebio Octet molecular interaction instrument is briefly described as follows. The sample dilution buffer was a solution of PBS, 0.02% Tween 20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 s. The sensor was equilibrated in the buffer for 60 s, and the binding of antibodies with concentrations from 31.25 to 500 nM (serial 2-fold dilutions) to the FcγRIIIa_V158 immobilized on the sensor was measured for 60 s. The antibodies were dissociated in the buffer for 60 s. The sensor was refreshed with 10 mM glycine (pH 1.5) four times, 5 s each. The temperature was 30 °C, and the frequency was 0.3 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0180] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIIa_V158 are shown in Table 2 below and in Figures 3 and 4. [Table 2]

[0181] The results show that BiAb004(hG1WT) bound to FcγRIIIa_V158 with an affinity constant of 1.77E-07 M. On the other hand, for BiAb004(hG1TM), no or only a very weak binding signal to FcγRIIIa_V158 was detected, and therefore no corresponding data was obtained, indicating that there was no binding to FcγRIIIa_V158.

[0182] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIIa_V158 is effectively eliminated compared to BiAb004(hG1WT).

[0183] Experimental Example 3: Affinity constant assay of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_F158 The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) binds the Fc fragment of IgG antibodies and can mediate ADCC.

[0184] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_F158 were measured using a Fortebio Octet molecular interaction device to evaluate the ADCC activity of the antibodies.

[0185] The method for measuring the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_F158 by the Fortebio Octet molecular interaction device is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween 20, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 seconds. The sensor was equilibrated in the buffer for 60 seconds, and the binding of antibodies with concentrations of 31.25 to 500 nM (serial two-fold dilutions) to the FcγRIIIa_F158 immobilized on the sensor was measured for 60 seconds. The antibodies were dissociated in the buffer for 60 seconds. The sensor was refreshed with 10 mM glycine (pH 1.5) four times, 5 seconds each. The temperature was 30 °C, and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0186] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIIa_F158 are shown in Table 3 below and in Figures 5 and 6. [Table 3]

[0187] The results show that BiAb004(hG1WT) bound to FcγRIIIa_F158 with an affinity constant of 2.21E-07 M. On the other hand, for BiAb004(hG1TM), no or only a very weak binding signal to FcγRIIIa_F158 was detected, and therefore no corresponding data was obtained, indicating that there was no binding to FcγRIIIa_F158.

[0188] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIIa_F158 is effectively eliminated compared to BiAb004(hG1WT).

[0189] Experimental Example 4: Affinity constant assay of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_H131 The Fc receptor FcγRIIIa_H131 (also known as CD32a_H131) binds the Fc fragment of IgG antibodies and can mediate ADCC.

[0190] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_H131 were measured using a Fortebio Octet molecular interaction instrument to evaluate the ADCC activity of the antibodies.

[0191] The method for measuring the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_H131 by Fortebio Octet molecular interaction device is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween 20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween 20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at a fixation height of approximately 1.0 nm. The sensor was blocked and equilibrated in a buffer of PBS, 0.02% Tween 20, 0.02% casein, and 0.1% BSA, pH 7.4, for 300 seconds, and the binding of FcγRIIa_H131 immobilized on the sensor with antibodies at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was measured for 60 seconds. The antibodies were dissociated in the buffer for 60 seconds. The sensor was refreshed with 10 mM glycine (pH 1.7) and 10 nM nickel sulfate. The temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0192] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIa_H131 are shown in Table 4 below and in Figures 7 and 8. [Table 4]

[0193] The results show that BiAb004(hG1WT) bound to FcγRIIa_H131 with an affinity constant of 2.22E-08 M. On the other hand, for BiAb004(hG1TM), no or only a very weak binding signal to FcγRIIa_H131 was detected, and therefore no corresponding data was obtained, indicating that there was no binding to FcγRIIIa_H131.

[0194] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIa_H131 is effectively eliminated compared to BiAb004(hG1WT).

[0195] Experimental Example 5: Affinity constant assay of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_R131 The Fc receptor FcγRIIIa_R131 (also known as CD32a_R131) binds the Fc fragment of IgG antibodies and can mediate ADCC.

[0196] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIIa_R131 were measured using a Fortebio Octet molecular interaction device to evaluate the ADCC activity of the antibodies.

[0197] The method for measuring the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIa_R131 by Fortebio Octet molecular interaction device is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween 20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween 20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIa_R131 was immobilized on the NTA sensor at a fixation height of approximately 1.0 nm. The sensor was blocked and equilibrated in a buffer of PBS, 0.02% Tween 20, 0.02% casein, and 0.1% BSA, pH 7.4, for 300 seconds, and the binding of FcγRIIa_R131 immobilized on the sensor with antibodies at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was measured for 60 seconds. The antibodies were dissociated in the buffer for 60 seconds. The sensor was refreshed with 10 mM glycine (pH 1.7) and 10 nM nickel sulfate. The temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0198] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIa_R131 are shown in Table 5 below and in Figures 9 and 10. [Table 5]

[0199] The results show that both BiAb004(hG1WT) and BiAb004(hG1TM) bound to FcγRIIa_R131 with affinity constants of 1.43E-08M and 4.20E-08M, respectively.

[0200] The results suggest that both BiAb004(hG1WT) and BiAb004(hG1TM) have binding activity to FcγRIIa_R131. However, the results in Figures 9 and 10 show that BiAb004(hG1WT) has a stronger binding signal and thus a higher affinity than BiAb004(hG1TM).

[0201] Experimental Example 6: Affinity constant assay of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIb The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc fragment of IgG antibodies and negatively regulates the function of immune cells, inhibiting immune cell activation and proliferation, and inhibiting cytokine secretion.

[0202] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIb were measured using a Fortebio Octet molecular interaction instrument to evaluate the binding ability of BiAb004(hG1WT) and BiAb004(hG1TM) to Fc receptors.

[0203] The method for measuring the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for FcγRIIb using a Fortebio Octet molecular interaction device is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween 20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of PBS, 0.02% Tween 20, 0.02% casein, and 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIb was immobilized on the NTA sensor at a fixed height of approximately 1.0 nm. The sensor was blocked and equilibrated with a buffer of PBS, 0.02% Tween 20, 0.02% casein, and 0.1% BSA, pH 7.4 for 300 seconds, and the binding of FcγRIIb immobilized on the sensor with antibodies at concentrations of 12.5 to 200 nM (serial two-fold dilutions) was measured for 60 seconds. The antibodies were allowed to dissociate in the buffer for 60 seconds. The sensor was refreshed with 10 mM glycine (pH 1.7) and 10 nM nickel sulfate. The temperature was 30 °C and the frequency was 0.6 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0204] The results of the affinity constant assay of BiAb004(hG1TM) and BiAb004(hG1WT) for FcγRIIb are shown in Table 6 below and in Figures 11 and 12. [Table 6]

[0205] The results show that BiAb004(hG1WT) bound to FcγRIIb with an affinity constant of 5.61E-08 M. On the other hand, for BiAb004(hG1TM), no or only a very weak binding signal to FcγRIIb was detected, and therefore no corresponding data was obtained, indicating that there was no binding to FcγRIIb.

[0206] The results suggest that the binding activity of BiAb004(hG1TM) to FcγRIIb is effectively eliminated compared to BiAb004(hG1WT).

[0207] Experimental Example 7: Affinity assay of BiAb004(hG1WT) and BiAb004(hG1TM) for C1q Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate CDC. The binding ability of therapeutic monoclonal antibodies to C1q affects the safety and efficacy of the antibodies.

[0208] In this experiment, the affinity constants of BiAb004(hG1WT) and BiAb004(hG1TM) for C1q were measured using a Fortebio Octet molecular interaction device to evaluate the CDC activity of the antibodies. In this example experiment, the anti-PDL1 antibody 5C10H2L2-IgG1mt was used as a control antibody, the preparation of which is described in PCT Publication No. WO2017148424A1.

[0209] The method for measuring the affinity constant of the corresponding antibody for C1q by the Fortebio Octet molecular interaction instrument is briefly described as follows: The sample dilution buffer was a solution of PBS, 0.02% Tween 20, and 0.1% BSA, pH 7.4. 50 μg / mL of antibody was immobilized on the FAB2G sensor at a fixation height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s, and the binding of the antibody immobilized on the sensor with antigen C1q at concentrations ranging from 1.25 to 20 nM (serial two-fold dilutions) was measured for 60 s at 1000 s. The antigen-antibody was dissociated in the buffer for 60 s. The sensor was regenerated with 10 mM glycine (pH 1.7) four times, 5 s each. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 0.6 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 7.0 and the data analysis software was Fortebio Data Analysis 7.0.

[0210] The results of the affinity constant assay for C1q of BiAb004(hG1TM), BiAb004(hG1WT), and 5C10H2L2-IgG1mt are shown in Table 7 below and in Figures 13, 14, and 15. [Table 7]

[0211] The results show that BiAb004(hG1WT) bound to C1q with an affinity constant of 2.53E-09 M. On the other hand, no corresponding data was obtained for BiAb004(hG1TM) since no or only a very weak signal was detected for binding to C1q, indicating no binding to C1q.

[0212] The results suggest that the binding activity of BiAb004(hG1TM) to C1q is effectively eliminated compared to BiAb004(hG1WT).

[0213] Experimental Example 8: ADCC activity assay of BiAb004(hG1WT) and BiAb004(hG1TM) against 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens ADCC refers to the effect of effector immune cells with killing activity recognizing the Fc fragment of an antibody bound to an antigen on a target cell via the Fc receptor (FcR) expressed on the cell's surface, and directly killing the target cell. To test the ADCC effect of the anti-CTLA4-anti-PD-1 bifunctional antibodies BiAb004(hG1WT) and BiAb004(hG1TM), the present inventors established a co-culture system of 293T-CTLA4-PD1 cells expressing PD-1 and CTLA4 antigens and primary PBMCs to test the ADCC activity of the antibodies.

[0214] The method for assaying the ADCC activity of BiAb004(hG1WT) and BiAb004(hG1TM) against 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens is as follows:

[0215] Normal human PBMCs were isolated according to the Ficoll peripheral blood mononuclear cell isolation instructions. The isolated PBMCs were resuspended in RPMI-1640 complete medium, stained with AO / PI, counted and frozen. One day before the experiment, the PBMCs were thawed, counted and viability was measured. The cells were incubated overnight in a carbon dioxide incubator at 37°C with 5% CO2. On the day of the experiment, 293T-CTLA4-PD1 cells and PBMCs were collected, centrifuged at 800 rpm or 1200 rpm for 5 minutes, resuspended in RPMI-1640 (containing 1% FBS; hereafter referred to as medium) and washed twice. The cells were counted and viability was measured. The cell density was adjusted to an appropriate range using medium. According to the experimental design, 100 μL of 293T-CTLA4-PD1 cells were added to a 96-well plate at a density of 3.0E+04 cells / well. 50 μL of antibody was added and the cells were pre-incubated at room temperature for 1 hour. After 1 hour, 50 μL of PBMCs were added at 9.0E+05 cells / well, the mixture was mixed well and incubated at 37°C in a carbon dioxide incubator with 5% CO2 for 4 hours. The cells were centrifuged at 250×g for 5 minutes. 100 μL of cell supernatant was transferred to a new 96-well flat-bottom microplate (cell pellets should not be pipetted). 100 μL of freshly prepared reaction solution was added to each well according to the instructions of the cytotoxicity detection kit. The cells were incubated at room temperature in the dark for 30 minutes. The OD values ​​were measured at 490 nm and 650 nm. The ADCC percentage was calculated for each group according to the formula ADCC (%) = (treated group - negative control group) / (maximum LDH release in target cells - spontaneous LDH release in target cells) × 100%.

[0216] The ADCC activities of BiAb004(hG1WT) and BiAb004(hG1TM) against 293T-CTLA4-PD1 cells expressing CTLA4 and PD-1 antigens were expressed as ADCC percentage, and the results are shown in FIG.

[0217] The results show that in a mixed culture system of PBMC and 293T-CTLA4-PD1, the ADCC percentage induced by BiAb004(hG1TM) was significantly lower than that induced by BiAb004(hG1WT) at the same dose level. The results suggest that BiAb004(hG1TM) has no ADCC activity.

[0218] Experimental Example 9: Evaluation of the activity of anti-CTLA4 / anti-PD-1 bispecific antibodies in combination with chiauranib in a lymphocyte-tumor cell mixed reaction system Freshly isolated PBMCs (from healthy donors) were allowed to adhere to the wall overnight. Mononuclear cells were collected. GM-CSF (2000U / mL, peprotech, catalog number 300-03) and IL-4 (2000U / mL, peprotech, catalog number 200-04) were added. After 3 days of culture, induction was performed. For the iDC group, half of the medium was replaced and GM-CSF (2000U / mL) and IL4 (2000U / mL) were added. For the mDC group, half of the medium was replaced and GM-CSF (2000U / mL), IFN-γ (50ng / mL, Sino Biological, catalog number 11725-HNAS-100), and LPS (100ng / mL, SIGMA, catalog number L6529) were added. The cells were cultured in an incubator for 2 days and frozen for later use.

[0219] A375 cells (purchased from ATCC, distributor: Zhongyuan Ltd., catalog number CRL-1619) were digested by conventional methods, collected by centrifugation, and plated at 2 × 10 6A375 cells were seeded at 1:10 cells / dish. Chiauranib (Chipscreen Biosciences, Lot No. 210604) was added to one of the dishes at a final concentration of 15 μM to set up a blank control group. The mixture was cultured in an incubator at 37°C and 5% CO2 for 48 hours (medium: DMEM+10%FBS). PBMCs from the same donor (autologous) or different donor (allogeneic) (from healthy donors) containing iDCs / mDCs were thawed the night before. iDCs (immature dendritic cells) and mDCs (mature dendritic cells) were thawed 2 hours before. Cells were counted and seeded in a 96-well flat-bottom plate at DC:PBMC=1:10. Untreated A375 cells and A375 cells treated with 15 μM Chiauranib for 48 hours were collected and added to the corresponding wells at 3×10 4 BiAb004(hG1TM) was added to the corresponding group of wells at 300nM / 50μL / well. The mixture was incubated in an incubator for 3-5 days. The cell supernatants were collected on the 3rd and 5th days separately. The secretion levels of IFN-γ (purchased from Dakewe, Cat. No. 1110202) and IL-2 (purchased from Dakewe, Cat. No. 1110202) were measured by ELISA kits.

[0220] The results of the IFN-γ secretion assay after mixed culture of DCs, PBMCs, and A375 cells are shown in Figure 17. The results of the IL-2 secretion assay after mixed culture of DCs, PBMCs, and A375 cells are shown in Figure 18.

[0221] As can be seen from the figure, the bifunctional antibody BiAb004 (hG1TM) single agent group, the chiauranib single agent group, and the combination group of the bifunctional antibody BiAb004 (hG1TM) and chiauranib can effectively promote the secretion of IFN-γ and IL-2 in the reaction system. Among these groups, the IFN-γ and IL-2 secretion promoting ability of the combination group of the bifunctional antibody BiAb004 (hG1TM) and chiauranib is separately and significantly stronger than the IFN-γ and IL-2 secretion promoting ability of the bifunctional antibody BiAb004 (hG1TM) single agent group and the chiauranib single agent group.

[0222] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that, according to all the teachings disclosed, various modifications and substitutions can be made to these details, and all these modifications are within the protection scope of the present invention. The full scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical combination comprising chiauranib or a pharma- ceutical acceptable salt thereof (e.g., the hydrochloride salt) or a crystalline form thereof (e.g., nonsolvated crystalline forms A, B, or C) and at least one (e.g., one, two, or three) anti-CTLA4-anti-PD-1 bispecific antibody.

2. 2. The pharmaceutical combination of claim 1, wherein the mass ratio of the chiauranib or a pharma- ceutically acceptable salt thereof or a crystalline form thereof to the anti-CTLA4-anti-PD-1 bispecific antibody is 1:(1-1000), preferably 1:(5-500), and more preferably 1:(10-100).

3. 3. The pharmaceutical combination of any one of claims 1 to 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody is in a unit dose of 100 mg to 1000 mg, 200 mg to 800 mg, 200 mg to 500 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg.

4. 4. The pharmaceutical combination of any one of claims 1 to 3, wherein the chiauranib or a pharma- ceutically acceptable salt thereof or a crystalline form thereof is in a unit dose of 0.1 mg to 100 mg, 0.5 mg to 50 mg, 1 mg to 20 mg, 2 mg to 15 mg, 3 mg to 12 mg, 4 mg to 8 mg, or 5 mg.

5. The pharmaceutical combination of any one of claims 1 to 4, wherein the pharmaceutical combination is a fixed combination, e.g. a pharmaceutical composition, or the pharmaceutical combination is a non-fixed combination, e.g., the chiauranib or a pharma- ceutically acceptable salt thereof or a crystalline form thereof and the anti-CTLA4-anti-PD-1 bispecific antibody in the non-fixed combination are each present in a separate pharmaceutical composition.

6. The pharmaceutical combination of claim 5, wherein said pharmaceutical compositions are independently a solid pharmaceutical composition or a liquid pharmaceutical composition.

7. The pharmaceutical combination according to any one of claims 5 to 6, wherein said pharmaceutical composition further comprises one or more pharma- ceutically acceptable adjuvants.

8. The pharmaceutical combination of any one of claims 5 to 7, wherein the pharmaceutical composition does not contain any active pharmaceutical ingredient other than the chiauranib or a pharma- ceutical acceptable salt thereof or a crystalline form thereof and the anti-CTLA4-anti-PD-1 bispecific antibody.

9. The pharmaceutical combination according to any one of claims 1 to 8, wherein said anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional region that targets PD-1; and a second protein functional region that targets CTLA4; Where: the first protein functional region is an immunoglobulin and the second protein functional region is a single chain fragment variable region, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:27-29, respectively, the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:30-32, respectively, the heavy chain variable region of the single chain fragment variable region comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:33-35, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:36-38, respectively; or the first protein functional region is a single chain fragment variable region and the second protein functional region is an immunoglobulin, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33-35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36-38, respectively, the heavy chain variable region of the single chain fragment variable region comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27-29, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30-32, respectively; the immunoglobulin is a human IgG1 subtype, and wherein the heavy chain constant region of said immunoglobulin has mutations at any two or three of positions 234, 235, and 237 according to the EU numbering system, and the affinity constant of said bispecific antibody for FcγRIIIa and / or C1q is decreased after the mutations compared to before the mutations; preferably, said affinity constant is measured by a Fortebio Octet molecular interaction instrument.

10. 10. The pharmaceutical combination according to any one of claims 1 to 9, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein, according to the EU numbering system, the heavy chain constant region of said immunoglobulin has the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, and G237A.

11. The pharmaceutical combination according to any one of claims 1 to 10, wherein said anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional region that targets PD-1; and Second Protein Functional Domain Targeting CTLA4 Where: the first protein functional region is an immunoglobulin and the second protein functional region is a single chain fragment variable region, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:27-29, respectively, the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:30-32, respectively, the heavy chain variable region of the single chain fragment variable region comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs:33-35, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs:36-38, respectively; or the first protein functional region is a single chain fragment variable region, and the second protein functional region is an immunoglobulin, wherein the heavy chain variable region of the immunoglobulin comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 33-35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 36-38, respectively, the heavy chain variable region of the single chain fragment variable region comprises HCDR1-HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 27-29, respectively, and the light chain variable region of the single chain fragment variable region comprises LCDR1-LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 30-32, respectively; the immunoglobulin is a human IgG1 subtype, According to the EU numbering system, said immunoglobulin comprises a heavy chain constant region having one of the following mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, and G237A.

12. 12. The pharmaceutical combination according to any one of claims 1 to 11, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein, according to the EU numbering system, the heavy chain constant region of said immunoglobulin further comprises one or more mutations selected from the following: 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.

13. A pharmaceutical combination according to any one of claims 1 to 12, in which the anti-CTLA4-anti-PD-1 bispecific antibody is provided: Where: the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:14 and SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:16 and SEQ ID NO:20, and the amino acid sequence of the heavy chain variable region of the single chain fragment variable part is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41, and SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single chain fragment variable part is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42, and SEQ ID NO:44; or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:41, and SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:42, and SEQ ID NO:44, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is selected from SEQ ID NO:14 and SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is selected from SEQ ID NO:16 and SEQ ID NO:

20.

14. The pharmaceutical combination according to any one of claims 1 to 13, wherein the anti-CTLA4-anti-PD-1 bispecific antibody is selected from any one of the following (1) to (20): （１） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

4. （２） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

8. （３） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

12. （４） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

4. （５） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

8. （６） the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 20, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is set forth in SEQ ID NO: 12; （７） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 16; （８） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 20; （９） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:6, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:8, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO:16; （１０） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:6, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO:8, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO:20; （１１） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 16; （１２） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 10, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 12, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

20. （１３） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

42. （１４） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 14, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 16, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

44. （１５） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 42; （１６） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 20, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 44; （１７） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 41, the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 42, the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

16. （１８） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 16; （１９） the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 42, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable part is shown in SEQ ID NO: 20; （２０） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO:

20.

15. A pharmaceutical combination according to any one of claims 1 to 14, in which the anti-CTLA4-anti-PD-1 bispecific antibody is provided, The amino acid sequence of the heavy chain of said immunoglobulin is shown in SEQ ID NO: 40, and the amino acid sequence of the light chain is shown in SEQ ID NO:

24. Pharmaceutical combinations.

16. 16. The pharmaceutical combination according to any one of claims 1 to 15, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein The immunoglobulin or antigen-binding fragment thereof binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb by about 10 －７ Affinity constants greater than M, e.g., about 10 －６ M, 10 －５ M, 10 －４ M or 10 －３ binds with an affinity constant greater than or equal to M; preferably, said affinity constant is measured by a Fortebio Octet molecular interaction instrument; Preferably, said immunoglobulin or antigen-binding fragment thereof has no binding signal or has a binding signal of less than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb; preferably, said binding signal refers to a response value measured by a Fortebio Octet molecular interaction device.

17. 17. The pharmaceutical combination according to any one of claims 1 to 16, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein The immunoglobulin or antigen-binding fragment thereof binds to C1q at about 10 －９ Affinity constants greater than M, e.g., about 10 －８ M, 10 －７ M, 10 －６ M or 10 －５ binds with an affinity constant greater than or equal to M; preferably, said affinity constant is measured by a Fortebio Octet molecular interaction instrument; Preferably, said immunoglobulin or antigen-binding fragment thereof has no binding signal to C1q or has a binding signal of less than 0.1 nm; preferably, said binding signal refers to a response value measured by a Fortebio Octet molecular interaction device.

18. 18. The pharmaceutical combination according to any one of claims 1 to 17, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein the first protein functional region is linked to the second protein functional region directly or via a linker fragment, and / or the heavy chain variable region of the single chain fragment variable region is linked to the light chain variable region of the single chain fragment variable region directly or via a linker fragment; Pharmaceutical combinations.

19. 19. The pharmaceutical combination according to any one of claims 1 to 18, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein The linker fragment is (GGGGS)n, where n is a positive integer, preferably n is 1, 2, 3, 4, 5, or 6; Pharmaceutical combinations.

20. 20. The pharmaceutical combination according to any one of claims 1 to 19, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein The number of the first protein functional region and the second protein functional region is independently 1, 2, or 2 or more; Pharmaceutical combinations.

21. 21. The pharmaceutical combination of any one of claims 1 to 20, wherein said anti-CTLA4-anti-PD-1 bispecific antibody is provided, wherein said single chain fragment variable region is linked to the C-terminus of an immunoglobulin heavy chain.

22. The pharmaceutical combination according to any one of claims 1 to 21, wherein said anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional region that targets PD-1; and Second Protein Functional Domain Targeting CTLA4 the number of said first protein functional domains is 1 and the number of said second protein functional domains is 2; wherein the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain fragment variable domain; The amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO: 40, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 24; The amino acid sequence of the heavy chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain fragment variable region is shown in SEQ ID NO: 44; the single chain fragment variable region is linked to the C-terminus of the heavy chain of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment, the heavy chain variable region of the single chain fragment variable region is linked to the light chain variable region of the single chain fragment variable region 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 said first linker fragment and said second linker fragment are shown in SEQ ID NO:

26.

23. A kit product comprising the pharmaceutical combination of any one of claims 1 to 22 and a package insert.

24. Use of a pharmaceutical combination according to any one of claims 1 to 22 for the preparation of a medicament for the treatment or prevention of tumors, comprising Preferably, the tumor is selected from one or more of melanoma, renal cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, and nasopharyngeal cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors of MSI-H / dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

25. A pharmaceutical combination according to any one of claims 1 to 22 for use in the treatment or prevention of a tumor, comprising Preferably, the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, and nasopharyngeal cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, said tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, said tumor is selected from one or more of the following tumors of MSI-H / dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

26. A method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of a pharmaceutical combination according to any one of claims 1 to 22, Preferably, the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, pancreatic cancer, and nasopharyngeal cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, said tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors of MSI-H / dMMR phenotype: colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

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