Pharmaceutical compositions containing anti-CTLA4 anti-PD-1 bispecific antibodies and uses thereof

Abstract

A pharmaceutical composition comprising an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine, and optionally paclitaxel (e.g., albumin-bound paclitaxel), which has a good anti-tumor effect.

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 202310213266.3 (filing date: March 7, 2023), the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field This disclosure relates to the fields of tumor therapy and molecular immunology, and to pharmaceutical combinations containing anti-CTLA4 anti-PD-1 bispecific antibodies and uses thereof. In particular, this disclosure relates to pharmaceutical combinations containing anti-CTLA4 anti-PD-1 bispecific antibodies, gemcitabine, and paclitaxel (e.g., albumin-bound paclitaxel) and uses thereof. [Background technology]

[0003] The transmembrane receptor PD-1 (programmed cell death protein 1) belongs to the CD28 gene family and is expressed on activated T cells, B cells, and myeloid cells. PD-1's ligands, PDL1 (programmed cell death 1 ligand 1 or PDL-1) and PDL2 (programmed cell death 1 ligand 2 or PDL-2), belong to the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, whereas PDL2 is expressed exclusively on antigen-presenting cells, such as dendritic cells and macrophages.

[0004] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune evasion. PD-1 is primarily expressed on immune cells such as T cells, and its ligand, PDL1, is highly expressed in several human tumor tissues. Blockade of the PD-1 / PDL1 signaling pathway may activate inhibited T cells to attack cancer cells. Blockade of PD-1 / PDL1 signaling promotes the proliferation of tumor antigen-specific T cells, which plays a role in tumor cell killing and further inhibits local tumor growth (Julie R et al., 2012, N Engl J Med., 366:2455-2465). Furthermore, tumors with high PDL1 expression are associated with cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA, 104:3360-5). An effective method is the in vivo injection of anti-PD-1 antibodies to regulate PD-1 expression. 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).

[0005] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and CD28 molecules are highly similar in gene structure, chromosomal location, sequence homology, and gene expression. Both molecules are receptors for the costimulatory molecule B7 and are expressed primarily on the surface of activated T cells. Binding of CTLA4 to B7 inhibits T cell activation in both mice and humans, negatively regulating T cell activation.

[0006] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can block the binding of CTLA4 to its natural ligand, blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing T cell reactivity to various antigens. In this regard, in vivo and in vitro studies are essentially consistent. Currently, there are CTLA4 monoclonal antibodies in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun., 13:5).

[0007] Interleukin-2 (IL2) is produced by T cells. It is a growth factor that regulates T cell subpopulations and is an important factor in regulating immune responses. It promotes the proliferation of activated B cells and is involved in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL2 has been approved by the US FDA for the treatment of malignancies, including melanoma and renal tumors, and clinical trials for the treatment of chronic viral infections are underway (Chavez, AR, et al., 2009, Ann. NY Acad. Sci., 1182:14-27). CTLA4 and CTLA4 antibodies are important factors affecting T cell function and interfering with the immune microenvironment in the body. CTLA4 antibodies can specifically alleviate CTLA4-mediated immunosuppression, activate T cells, and induce IL2 production, demonstrating their potential for broad application in gene therapy for diseases such as tumors and parasitic infections, in vitro and in vivo.

[0008] CTLA4 antibodies can provide specific therapeutic effects against diseases and show remarkable efficacy, and can be used to supplement conventional pharmaceuticals and explore new avenues of gene therapy.

[0009] Bispecific antibodies, also known as 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 offers certain advantages, such as flexibility in optimizing binding sites, exploring synthetic forms, and yields. Currently, over 45 antibodies have been demonstrated (Dafne Müller, Kontermann R E., 2010, BioDrugs, 24(2): 89-98). Many 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 been demonstrated to be one of the ideal forms of bifunctional antibodies due to its similarity to the naturally occurring IgG form and advantages in antibody production, 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).

[0010] Gemcitabine is a first-line chemotherapy drug commonly used to treat various solid tumors, including non-small cell lung cancer, pancreatic cancer, breast cancer, and bladder cancer. It can also be used to treat malignant lymphoma, ovarian cancer, and nasopharyngeal carcinoma. This drug is a difluoronucleoside antimetabolite antitumor drug that inhibits deoxyribonucleic acid synthesis, interfering with the replication and transcription of cellular DNA, thereby suppressing tumor cell proliferation and metastasis. However, gemcitabine monotherapy for cancer has limitations, the biggest of which is its short half-life, which means that effective concentrations can only be maintained with continuous intravenous administration at high doses. This inevitably leads to severe toxicity and side effects, significantly reducing patients' quality of life. The chemical structure of gemcitabine is shown below in Formula I. Gemcitabine hydrochloride, commonly used clinically, has the structural formula shown below in Formula II.

[0011] [ka]

[0012] Paclitaxel is a new antitumor drug derived from natural plants. It is a monomeric diterpenoid compound extracted from the bark of the yew tree (Taxus brevifolia) at the National Cancer Institute (NCI) in the late 1960s and was hailed as one of the three major advances in antitumor drugs in the 1990s. Paclitaxel has been widely used in the treatment of cancers such as ovarian cancer, breast cancer, rectal cancer, non-small cell lung cancer, and Kaposi's sarcoma (Anil K. Singla, et al. (2002) Int. J. Pharm. 235: 179-192). The structural formula of paclitaxel is shown in Formula III below.

[0013] [ka]

[0014] Albumin-bound paclitaxel is a novel chemotherapy drug in which paclitaxel is bound to albumin. Compared with conventional solvent-based paclitaxel, albumin-bound paclitaxel has better efficacy and solubility, making it more convenient for clinical application. Albumin-bound paclitaxel increases the drug concentration outside the tumor via the albumin receptor (Gp60) transcytosis pathway and the tumor extracellular matrix-associated secretory protein acidic-rich cysteine ​​(SPARC) pathway, thereby enhancing antitumor activity. Experiments have shown that the binding between paclitaxel and albumin is dominated by electrostatic and hydrophobic interactions (Chen Dongxiao, Zhang Juanmei, Li Jianye et al., Research Status and Progress of Paclitaxel Albumin Nanoparticles [J]. Journal of Henan University (Medical Science), 2017, 36(04): 296-300. DOI:10.15991 / j.cnki.41-1361 / r.2017.04.020.).

[0015] Due to the limited efficacy of single chemotherapeutic drugs, the toxicity and side effects caused by high doses, and drug resistance, the disease of many tumor patients remains uncontrolled for a long time after treatment with chemotherapeutic drugs.Therefore, the development of more effective therapeutic methods or combined treatment methods of antibodies and chemotherapeutic drugs is of great clinical significance, as it can reduce the occurrence of drug resistance, improve the in vivo kinetics of drugs, improve the therapeutic effect of drugs, and / or reduce the side effects of drugs. Summary of the Invention

[0016] Through intensive research and creative efforts, the inventors have creatively and appropriately combined anti-CTLA4 anti-PD-1 antibody with gemcitabine and albumin-bound paclitaxel, and found that the resulting combination drug has good anti-tumor effects.

[0017] The details of this disclosure are as follows: One aspect of this disclosure relates to a pharmaceutical combination comprising an anti-CTLA4 anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof, Preferably, the pharmaceutical combination further comprises paclitaxel.

[0018] In some aspects of this disclosure, pharmaceutical combinations are provided in which the amounts of anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and / or paclitaxel are effective to treat or prevent an indication of this disclosure.

[0019] In some aspects of this disclosure, the pharmaceutical combination consists of an anti-CTLA4 anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof.

[0020] In some aspects of this disclosure, the pharmaceutical combination consists of an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0021] In some aspects of this disclosure, the pharmaceutical combination consists of an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel.

[0022] In some aspects of this disclosure, the pharmaceutical combination consists of an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, paclitaxel, and one or more pharmaceutically acceptable excipients.

[0023] In some aspects of this disclosure, a combination pharmaceutical is provided in which the pharmaceutically acceptable salt of gemcitabine is gemcitabine hydrochloride.

[0024] In some aspects of this disclosure, pharmaceutical combinations are provided wherein the paclitaxel is albumin-bound paclitaxel.

[0025] In some aspects of this disclosure, the pharmaceutical combination consists of an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and albumin-bound paclitaxel.

[0026] In some aspects of this disclosure, the pharmaceutical combination consists of an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, albumin-bound paclitaxel, and one or more pharmaceutically acceptable excipients.

[0027] In some embodiments of the present disclosure, there is provided albumin-bound paclitaxel, wherein the mass ratio of paclitaxel to albumin is (1:15) to (5:1), (1:15) to (3:1), (1:15) to (2:1), (1:10) to (3:1), (1:10) to (2:1), 1:(5 to 15), 1:(8 to 12), 1:8, 1:9, 1:10, 1:11, or 1:12.

[0028] In some embodiments of this disclosure, a combination drug is provided in which the unit dose of the anti-CTLA4 anti-PD-1 bispecific antibody 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.

[0029] In some embodiments of this disclosure, a combination pharmaceutical is provided in which the anti-CTLA4 anti-PD-1 bispecific antibody is administered at a unit dose of 0.1 to 100 mg / kg body weight, preferably 1 to 15 mg, 1 to 12 mg, 1 to 10 mg, or 6 to 10 mg / kg body weight.

[0030] In some embodiments of this disclosure, there is provided a combination medicine wherein the unit dose of gemcitabine or a pharmaceutically acceptable salt thereof is 100 to 2000 mg, 500 to 1500 mg, 600 to 1300 mg, 800 to 1200 mg, 800 to 1000 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, or 1500 mg.

[0031] In some embodiments of this disclosure, gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100 mg / body surface area of ​​the subject. 2 ) based on 500 to 1500 mg / m 2 , 600-1300mg / m 2 , 800-1250mg / m 2 , 800-1000mg / m 2 , 800 mg / m 2 , 850 mg / m 2 , 900 mg / m 2 , 950 mg / m 2 , 1000 mg / m 2 , 1050 mg / m 2 , 1100 mg / m 2 , 1150 mg / m 2 , 1200 mg / m 2 or 1250 mg / m 2 The pharmaceutical combination is provided in a unit dose of:

[0032] In some embodiments of this disclosure, there is provided a combination drug wherein the unit dose of albumin-bound paclitaxel is 200 to 2000 mg, 500 to 1500 mg, 800 to 1200 mg, 900 to 1100 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg (based on the mass of albumin-bound paclitaxel).

[0033] In some embodiments of this disclosure, there is provided a combination pharmaceutical comprising a unit dose of paclitaxel of 20 to 300 mg, 50 to 250 mg, 80 to 200 mg, 100 to 180 mg, 120 to 180 mg, 125 to 175 mg, 135 to 175 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, or 175 mg (based on the mass of paclitaxel).

[0034] In some embodiments of this disclosure, albumin-bound paclitaxel is administered in a dose-dependent manner, based on the mass of albumin-bound paclitaxel (mg) / body surface area of ​​the subject (m 2 ) based on 80 to 300 mg / m 2 , 100-280 mg / m 2 , 120-260 mg / m 2 , 120-200 mg / m 2 , 120-180 mg / m 2 , 125-175 mg / m 2 , 125 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 140 mg / m 2 , 145 mg / m 2 , 150 mg / m 2 , 155 mg / m 2 , 160 mg / m 2 , 165 mg / m 2 , 170 mg / m 2 , 175 mg / m 2 or 260 mg / m 2 The pharmaceutical combination is provided in a unit dose of:

[0035] In some embodiments of this disclosure, paclitaxel is administered in a dose of 100 mg / body surface area of ​​the subject. 2 ) based on 20 to 300 mg / m 2 , 50-250 mg / m 2 , 80-200 mg / m 2 , 100-180 mg / m 2 , 120-180 mg / m 2 , 125-175 mg / m 2 , 135-175 mg / m 2 , 125 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 140 mg / m 2 , 145 mg / m 2 , 150 mg / m 2 , 155 mg / m 2 , 160 mg / m 2 , 165 mg / m 2 , 170 mg / m 2 or 175 mg / m 2 The pharmaceutical combination is provided in a unit dose of:

[0036] In some aspects of this disclosure, The pharmaceutical combination is a fixed combination (e.g., a pharmaceutical composition); the pharmaceutical combination is a loose combination, for example, the anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel in the loose combination are each present in separate pharmaceutical compositions; or The gemcitabine or a pharmaceutically acceptable salt thereof is in a fixed combination (e.g., a pharmaceutical composition); and the anti-CTLA4 anti-PD-1 bispecific antibody is in a separate pharmaceutical composition; Pharmaceutical combinations are provided.

[0037] In some aspects of this disclosure, a pharmaceutical combination is provided, wherein the pharmaceutical compositions are independently a solid pharmaceutical composition or a liquid pharmaceutical composition.

[0038] In some aspects of this disclosure, a pharmaceutical combination is provided in which the pharmaceutical composition further comprises one or more pharmaceutically acceptable additives (e.g., carriers and / or excipients).

[0039] In some aspects of this disclosure, a combination drug is provided, wherein the pharmaceutical composition does not contain any active pharmaceutical ingredients other than the anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel.

[0040] In some embodiments of this disclosure, 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 A pharmaceutical combination comprising: the first protein functional region is an immunoglobulin, the second protein functional region is a single-chain variable region fragment, the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the single-chain variable region fragment comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the single-chain variable region fragment comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 36 to 38, respectively; Or, the first protein functional region is a single-chain variable region fragment, the second protein functional region is an immunoglobulin, the heavy chain variable region of the single-chain variable region fragment comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the single-chain variable region fragment comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 36 to 38, respectively; Preferably, the immunoglobulin subtype is human IgG1, and the heavy chain constant region of the immunoglobulin has mutations at two or three positions, 234, 235, and 237, according to the EU numbering system, and the affinity constant of the bispecific antibody for FcγRIIIa and / or C1q after the mutations is reduced compared to before the mutations, preferably as measured using a Fortebio Octet molecular interaction system.

[0041] In one or more embodiments of the disclosure, there is provided a combination pharmaceutical comprising an anti-CTLA4, anti-PD-1 bispecific antibody in which the affinity constant for FcγRIIIa, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb of the bispecific antibody after the above-described mutations is reduced compared to before the mutations, preferably as measured using a Fortebio Octet molecular interaction instrument.

[0042] In some embodiments of this disclosure, the heavy chain constant region of the immunoglobulin comprises the following mutations, according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A; The present invention provides a pharmaceutical combination comprising an anti-CTLA4 anti-PD-1 bispecific antibody having the formula:

[0043] In this disclosure, 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.

[0044] In some embodiments of this disclosure, 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 A pharmaceutical combination comprising: the first protein functional region is an immunoglobulin, the second protein functional region is a single-chain variable region fragment, the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the single-chain variable region fragment comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the single-chain variable region fragment comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 36 to 38, respectively; Or, the first protein functional region is a single-chain variable region fragment, the second protein functional region is an immunoglobulin, the heavy chain variable region of the single-chain variable region fragment comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 27 to 29, respectively, and the light chain variable region of the single-chain variable region fragment comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 30 to 32, respectively; the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the sequences shown in SEQ ID NOs: 33 to 35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the sequences shown in SEQ ID NOs: 36 to 38, respectively; Preferably, the immunoglobulin subtype is human IgG1, and the heavy chain constant region of the immunoglobulin has the following combination of mutations according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A; The present invention has one of the following features.

[0045] In some embodiments of this disclosure, the heavy chain constant region of the immunoglobulin is 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 The present invention provides a pharmaceutical combination comprising an anti-CTLA4 anti-PD-1 bispecific antibody further comprising one or more mutations selected from:

[0046] In one or more embodiments of this disclosure, 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., Morrison format.

[0047] In some aspects of this disclosure, the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the sequences set forth in 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 the sequences set forth in SEQ ID NO:16 and SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is selected from the sequences set forth in 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 variable region fragment is selected from the sequences set forth in 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 the sequences set forth in 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 the sequences set forth in 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 variable region fragment is selected from the sequences set forth in SEQ ID NO:14 and SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is selected from the sequences set forth in SEQ ID NO:16 and SEQ ID NO:20; Pharmaceutical combinations comprising anti-CTLA4 anti-PD-1 bispecific antibodies are provided.

[0048] In some embodiments of the present disclosure, the anti-CTLA4 anti-PD-1 bispecific antibody is selected from the group consisting of the following (1) to (20): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 4; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 8; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 12; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 4; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 8; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 12; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 42; (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 44; (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 42; (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 44; (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 16; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 20; and, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence set forth in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence set forth in SEQ ID NO: 20; The present invention provides a combination pharmaceutical composition selected from any one of the following:

[0049] In some embodiments of this disclosure, a pharmaceutical combination is provided that includes an anti-CTLA4, anti-PD-1 bispecific antibody, wherein the amino acid sequence of the immunoglobulin heavy chain is the sequence set forth in SEQ ID NO:40 and the amino acid sequence of the immunoglobulin light chain is the sequence set forth in SEQ ID NO:24.

[0050] In some aspects of this disclosure, pharmaceutical combinations are provided that include an anti-CTLA4, anti-PD-1 bispecific antibody in which a first protein functional domain is linked, either directly or via a linker fragment, to a second protein functional domain, and / or a heavy chain variable domain of the single chain variable domain fragment is linked, either directly or via a linker fragment, to a light chain variable domain of the single chain variable domain fragment.

[0051] In some aspects of the disclosure, there is provided a combination drug comprising an anti-CTLA4, anti-PD-1 bispecific antibody, wherein the linker fragment is the polypeptide set forth in SEQ ID NO: 45 (GGGGS), or a polypeptide in which multiple (e.g., 2, 3, 4, 5, or 6) polypeptides set forth in SEQ ID NO: 45 are linked in tandem.

[0052] In some aspects of this disclosure, pharmaceutical combinations are provided that include an anti-CTLA4 anti-PD-1 bispecific antibody, wherein the number of first protein functional domains and second protein functional domains are each independently one, two, or more.

[0053] In one or more embodiments of this disclosure, a pharmaceutical combination is provided that includes an anti-CTLA4 anti-PD-1 bispecific antibody, wherein the number of first protein functional regions is 1 and the number of second protein functional regions is 2.

[0054] In one or more embodiments of this disclosure, a pharmaceutical combination is provided that includes an anti-CTLA4-anti-PD-1 bispecific antibody in which a single-chain variable region fragment is linked to the C-terminus of an immunoglobulin heavy chain. Because an immunoglobulin has two heavy chains, two single-chain variable region fragment molecules are linked to one immunoglobulin molecule. Preferably, the two single-chain variable region fragment molecules are identical. Preferably, the single-chain variable region fragment is linked to the C-terminus of the immunoglobulin heavy chain by forming an amide bond via the linker fragment described above.

[0055] In one or more embodiments of this disclosure, the immunoglobulin constant regions are humanized, e.g., the heavy chain constant region is the Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is the Ig kappa chain C region, ACCESSION: P01834.

[0056] In one or more embodiments of this disclosure, the anti-CTLA4 anti-PD-1 bispecific antibody is a CTLA4 protein and / or a PD-1 protein, -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9M or 10 -10 K less than M or less D The present invention provides a pharmaceutical combination that binds

[0057] In one or more embodiments of this disclosure, a pharmaceutical combination is provided in which the anti-CTLA4 anti-PD-1 bispecific antibody is a monoclonal antibody.

[0058] In one or more embodiments of this disclosure, a pharmaceutical combination is provided in which the anti-CTLA4 anti-PD-1 bispecific antibody is a humanized antibody.

[0059] In some embodiments of this disclosure, a pharmaceutical combination is provided that includes an anti-CTLA4 anti-PD-1 bispecific antibody in which a single-chain variable region fragment is linked to the C-terminus of each of the two heavy chains of the immunoglobulin.

[0060] In some embodiments of this disclosure, 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 A pharmaceutical combination comprising: the number of said first protein functional domains is 1 and the number of said second protein functional domains is 2; the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain variable domain fragment; the heavy chain amino acid sequence of said immunoglobulin is the sequence set forth in SEQ ID NO: 40 and the light chain amino acid sequence of said immunoglobulin is the sequence set forth in SEQ ID NO: 24; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 44; the single-chain variable region fragment 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 variable region fragment is linked to the light chain variable region of the single-chain variable region fragment via a second linker fragment; the first linker fragment and the second linker fragment may be the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are each independently selected from the sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 26; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are the sequences shown in SEQ ID NO:26.

[0061] Another aspect of this disclosure relates to a kit containing a combination pharmaceutical according to any of the embodiments of this disclosure and product instructions.

[0062] In some embodiments of this disclosure, the kit includes a first product, a second product, and a third product in separate packages; the first article of manufacture comprises an anti-CTLA4 anti-PD-1 bispecific antibody according to any of the embodiments of this disclosure; the second product comprises gemcitabine or a pharmaceutically acceptable salt thereof; the third product comprises albumin-bound paclitaxel; Preferably, the first product, the second product, and the third product independently further comprise one or more pharmaceutically acceptable excipients; Preferably, the kit further comprises a product leaflet.

[0063] In one or more embodiments of this disclosure, a kit is provided in which 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.

[0064] Yet another aspect of this disclosure relates to the use of a pharmaceutical combination according to any of the aspects of this disclosure in the manufacture of a medicament for treating or preventing a tumor; Preferably, the tumor is selected from one or more of pancreatic cancer, 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 and nasopharyngeal cancer; Preferably, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma; Preferably, the pancreatic cancer is advanced pancreatic cancer or metastatic pancreatic 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 non-small cell lung cancer is intermediate-stage or advanced-stage non-small cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

[0065] There is provided a pharmaceutical combination according to any of the aspects of this disclosure for use in treating or preventing a tumor, comprising: Preferably, the tumor is selected from one or more of pancreatic cancer, 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 and nasopharyngeal cancer; Preferably, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma; Preferably, the pancreatic cancer is advanced pancreatic cancer or metastatic pancreatic 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 non-small cell lung cancer is intermediate-stage or advanced-stage non-small cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

[0066] Yet another aspect of this disclosure 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 aspects of this disclosure: Preferably, the tumor is selected from one or more of pancreatic cancer, 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 and nasopharyngeal cancer; Preferably, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma; Preferably, the pancreatic cancer is advanced pancreatic cancer or metastatic pancreatic 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 non-small cell lung cancer is intermediate-stage or advanced-stage non-small cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

[0067] In one or more embodiments of this disclosure, a method for treating or preventing a tumor is provided, wherein an effective amount of an anti-CTLA4 anti-PD-1 bispecific antibody is administered to a subject in need thereof before or after surgery and / or before or after radiation therapy.

[0068] In one or more embodiments of this disclosure, The anti-CTLA4 anti-PD-1 bispecific antibody is administered in a unit dose of 0.1 to 100 mg / kg body weight, preferably 1 to 15 mg, 1 to 12 mg, 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), or 6 to 10 mg / kg body weight; alternatively, the anti-CTLA4 anti-PD-1 bispecific antibody is administered in a unit dose of 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), preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg; Preferably, administration occurs 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; Methods for treating or preventing tumors are provided.

[0069] In some embodiments, the anti-CTLA4 anti-PD-1 bispecific antibody is administered 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, e.g., the anti-CTLA4 anti-PD-1 bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).

[0070] Gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100 mg / body surface area of ​​the subject (m 2 ) based on 500 to 1500 mg / m 2 , 600-1300mg / m 2 , 800-1250mg / m 2 , 800-1000mg / m 2 , 800 mg / m 2 , 850 mg / m 2 , 900 mg / m 2 , 950 mg / m 2 , 1000 mg / m2 , 1050 mg / m 2 , 1100 mg / m 2 , 1150 mg / m 2 , 1200 mg / m 2 or 1250 mg / m 2 administered in unit doses of; Preferably, administration occurs once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week or 2 weeks; Preferably, the route of administration is intravenous drip or intravenous injection.

[0071] In some embodiments, gemcitabine or a pharmaceutically acceptable salt thereof is administered once per week for three consecutive weeks, followed by a one-week break, as a course of treatment; alternatively, gemcitabine or a pharmaceutically acceptable salt thereof is administered once per week for two consecutive weeks, followed by a one-week break, as a course of treatment. Preferably, treatment is administered in two to four consecutive courses.

[0072] Albumin-bound paclitaxel was measured as the mass of albumin-bound paclitaxel (mg) / subject's body surface area (m 2 ) based on 80 to 300 mg / m 2 , 100-280 mg / m 2 , 120-260 mg / m 2 , 120-200 mg / m 2 , 120-180 mg / m 2 , 125-175 mg / m 2 , 125 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 140 mg / m 2 , 145 mg / m 2 , 150 mg / m 2 , 155 mg / m 2 , 160 mg / m 2 , 165 mg / m 2 , 170 mg / m 2 , 175 mg / m 2 or 260 mg / m 2 is administered in a unit dose of

[0073] Paclitaxel was administered in a dose-dependent manner using a 2-dose system, with the dose being calculated as the mass of paclitaxel (mg) / subject's body surface area (m 2 ) based on 20 to 300 mg / m 2 , 50-250 mg / m 2 , 80-200 mg / m 2 , 100-180 mg / m 2 , 120-180 mg / m 2 , 125-175 mg / m 2 , 135-175 mg / m 2 , 125 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 140 mg / m 2 , 145 mg / m 2 , 150 mg / m 2 , 155 mg / m 2 , 160 mg / m 2 , 165 mg / m 2 , 170 mg / m 2 or 175 mg / m 2 is administered in a unit dose of

[0074] Preferably, administration occurs once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks or 4 weeks; Preferably, the route of administration is intravenous drip or intravenous injection.

[0075] In one or more embodiments of this disclosure, methods for treating or preventing tumors are provided in which an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine, and paclitaxel (e.g., albumin-bound paclitaxel) are administered simultaneously or sequentially.

[0076] In one or more embodiments of this disclosure, a method for treating or preventing a tumor having a first and second stage is provided, wherein in the first stage, an anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel (e.g., albumin-bound paclitaxel) are administered.

[0077] In one or more embodiments of this disclosure, a method for treating or preventing tumors is provided in which, in a second step, an anti-CTLA4 anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof are administered.

[0078] In one or more embodiments of this disclosure, a method of treating or preventing tumors is provided in which an anti-CTLA4 anti-PD-1 bispecific antibody is administered in a second phase of treatment.

[0079] The first stage is a combination therapy stage that includes 1 to 10 courses, 2 to 8 courses, or 4 to 6 courses of treatment, each course lasting 2 to 4 weeks, preferably 4 weeks.

[0080] The second phase is the maintenance treatment phase.

[0081] In the first and second stages, the dosage and frequency of administration of each pharmaceutical ingredient may be determined by referring to the previous explanations and instructions for use, or by following the advice of a doctor.

[0082] Antibody drugs, especially monoclonal antibodies (mAbs), have achieved excellent efficacy in treating various diseases. Conventional methods for producing these therapeutic antibodies include immunizing animals with an antigen and obtaining antibodies targeting the antigen from the immunized animals, or modifying antibodies with low affinity for the antigen by affinity maturation.

[0083] The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain has three hypervariable regions, i.e., complementarity-determining regions (CDRs) (heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include LCDR1, LCDR2, and LCDR3; see the definitions in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda, Md.).

[0084] The amino acid sequences of the CDR regions of the following monoclonal antibodies (1) to (9) were analyzed using technical means well known to those skilled in the art, such as the VBASE2 database, and the results are as follows: (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 (SEQ ID NO: 29). The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDINTY (SEQ ID NO: 30) LCDR2:RAN (SEQ ID NO: 31) LCDR3: LQYDEFPLT (sequence number 32).

[0085] (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 the same as those of 14C12. The amino acid sequences of the three CDR regions of the light chain variable region are the same as those of 14C12.

[0086] (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 (SEQ ID NO: 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).

[0087] (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 the same as those of 4G10. The amino acid sequences of the three CDR regions of the light chain variable region are the same as those of 4G10.

[0088] (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 the same as those of 4G10. The amino acid sequences of the three CDR regions of the light chain variable region are the same as those of 4G10.

[0089] (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 (SEQ ID NO: 38). The amino acid sequences of the three CDR regions associated with the light chain variable region are as follows: LCDR1: QDINTY (SEQ ID NO: 30) LCDR2:RAN (SEQ ID NO: 31) LCDR3: LQYDEFPLT (sequence number 32).

[0090] (7) BiAb002(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are the same as those of BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as those of BiAb001(M).

[0091] (8) BiAb003(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are the same as those of BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as those of BiAb001(M).

[0092] (9) BiAb004(M) The amino acid sequences of the nine CDR regions associated with the heavy chain variable region are the same as those of BiAb001(M). The amino acid sequences of the three CDR regions associated with the light chain variable region are the same as those of BiAb001(M).

[0093] In the antibody BiAb004(hG1™) of this disclosure, amino acid mutations are introduced in a region other than the variable region of BiAb004(M). The amino acid mutations are introduced at positions 234, 235, and 237 according to the EU numbering system. 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 heavy chain hinge region.

[0094] In this disclosure, unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the experimental procedures used herein in cell culture, molecular genetics, nucleic acid chemistry, and immunology are routine procedures widely practiced in the corresponding fields. For a better understanding of this disclosure, the following definitions and explanations of relevant terms are provided.

[0095] In this specification, reference to the amino acid sequence of CTLA4 protein (cytotoxic T-lymphocyte-associated antigen 4) includes, but is not limited to, the full-length CTLA4 protein or the extracellular fragment of CTLA4, the CTLA4 ECD, or a fragment containing the CTLA4 ECD; fusion proteins of CTLA4 ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG, are also included. 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 the CTLA4 protein may be naturally generated or artificially introduced without affecting its biological function. Therefore, in this disclosure, the term "CTLA4 protein" includes all sequences, including natural or artificial variants thereof. Furthermore, reference to a fragment of the sequence of the CTLA4 protein also includes a fragment of the corresponding sequence in the natural or artificial variant.

[0096] Throughout this specification, references to the amino acid sequence of the PD-1 protein include, but are not limited to, the full-length PD-1 protein (NCBI GenBank: NM_005018) or the extracellular fragment of PD-1, the PD-1 ECD, or fragments containing the PD-1 ECD; fusion proteins of the PD-1 ECD, such as fragments fused with the Fc protein fragments (mFc or hFc) of mouse or human IgG, are also included. 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 the PD-1 protein may be naturally generated or artificially introduced without affecting its biological function. Therefore, in this disclosure, the term "PD-1 protein" includes all sequences, including natural or artificial variants thereof. Furthermore, references to fragments of the PD-1 protein sequence also include fragments of the corresponding sequences in natural or artificial variants thereof.

[0097] In this specification, unless otherwise specified, B7 refers to B7-1 and / or B7-2, the specific protein sequences of which are publicly known and can be found in existing literature or in GenBank (e.g., B7-1 (CD80) has NCBI Gene ID: 941; B7-2 (CD86) has NCBI Gene ID: 942).

[0098] In this specification, the term EC 50 refers to the concentration of 50% of the maximum effect, i.e., the concentration capable of producing 50% of the maximum effect.

[0099] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. In general, heavy chains can be understood as the larger polypeptide chains in an antibody, while light chains refer to the smaller polypeptide chains 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 connected by a "J" region of about 12 or more amino acids, and 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 consists of a light chain variable region (V L ) and light chain constant region (C L The light chain constant region consists of one domain, C L The 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) to the first component (C1q) of the classical complement system. H and V L The region can be further subdivided into hypervariable regions (called complementarity determining regions or CDRs), with conserved regions called framework regions (FRs) distributed between each CDR. 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, and FR4. H and V L) form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions in the 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 (e.g., 6, 9, or 12). For example, in the bifunctional antibody disclosed herein, the heavy chain can be formed by linking the C-terminus of the heavy chain of an IgG antibody to the scFv of another antibody, in which case the heavy chain comprises nine CDRs. The term "antibody" is not limited by the specific method of production of the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

[0100] Antigen-binding fragments of antibodies (e.g., fragments of the antibodies described above) can be obtained from a given antibody by conventional techniques known to those of skill in the art (e.g., DNA recombination, enzymatic or chemical cleavage), and the antigen-binding fragments of antibodies are screened for specificity in the same manner as intact antibodies.

[0101] In this specification, unless expressly defined otherwise, the term "antibody" includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0102] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody fragment derived from a population of highly homologous antibody molecules, i.e., derived from a population of antibody molecules that are identical except for natural mutations that may occur spontaneously. Monoclonal antibodies are highly specific to a single epitope of an antigen. Compared to monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes of an antigen. Monoclonal antibodies are usually obtained by the hybridoma method first described by Kohler et al. (Kohler et al., Nature, 256:495, 1975), but can also be obtained using DNA recombination (see, e.g., U.S. Pat. No. 4,816,567).

[0103] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by substituting all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody). The donor antibody may be a non-human (e.g., mouse, rat, or rabbit) antibody with the expected specificity, affinity, or reactivity. Furthermore, to further improve or optimize antibody performance, amino acid residues in the framework region (FR) of the recipient antibody can be substituted with corresponding amino acid residues in the non-human antibody or with amino acid residues in another antibody. For details on humanized antibodies, see, for example, 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).

[0104] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to in the art as "antigenic determinant." Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules, such as amino acids, carbohydrate, or sugar side chains, and usually have specific three-dimensional structural and charge characteristics. For example, epitopes typically contain 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 three-dimensional space and are either "linear" or "conformational." See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are located linearly along the amino acid sequence of the protein. In a conformational epitope, the sites of interaction are located at amino acid residues of the protein that are separated from one another.

[0105] As used herein, the term "isolated" refers to something obtained by artificial means from a natural state. When an "isolated" substance or component occurs in nature, its natural environment may have been altered, it may have been isolated from its natural environment, or both. For example, a non-isolated polynucleotide or polypeptide may naturally occur in a particular living animal; the same polynucleotide or polypeptide isolated in high purity from such a natural state would be referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic materials or other impurities that do not affect the activity of the substance.

[0106] As used herein, the term "E. coli expression system" refers to an expression system consisting of an E. coli strain and a vector, where the E. coli strain is derived from a commercially available strain, such as, but not limited to, GI698, ER2566, BL21(DE3), B834(DE3), and BLR(DE3).

[0107] As used herein, the term "vector" 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. A vector can be introduced into a host cell by transformation, transduction, or transfection, resulting in the expression of the genetic material carried by the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain a replication origin.

[0108] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0109] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one in which the antibody binds to an antigen within about 10 -5 Less than M, e.g., about 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K D ) means binding to an antigen. In some aspects of this disclosure, the term "targeting" refers to specific binding.

[0110] In this specification, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10, as measured, for example, with a BIACORE surface plasmon resonance (SPR) instrument or a Fortebio Octet molecular interaction instrument. -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (K D ) binds to the antigen.

[0111] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably; and the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, as used herein, amino acids are generally represented by their one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0112] As used herein, the term "pharmaceutically acceptable auxiliary material" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. Such auxiliary substances are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th Ed., Pennsylvania, Mack Publishing Company, 1995) and 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 buffer; surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants (e.g., Tween® 80); and ionic strength enhancers include, but are not limited to, sodium chloride.

[0113] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to an organism simultaneously with or prior to an antigen, can enhance or change the type of immune response of the organism to the antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant, incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is frequently used in clinical trials.

[0114] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactically effective amount for a disease (e.g., a disease (e.g., tumor) associated with CTLA4-B7 binding or CTLA4 overactivity) is an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a disease (e.g., tumor) associated with CTLA4-B7 binding or CTLA4 overactivity); a therapeutically effective amount is an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. Determining such effective amounts is undoubtedly within the capabilities of one skilled in the art. For example, a therapeutically effective amount will depend on the severity of the disease being treated, the overall state of the patient's immune system, the patient's general condition, such as age, weight, and sex, the route of administration, and other concurrent therapies.

[0115] A "recurrent" cancer is one that grows back at the original site or a distant site after responding to previous treatment (e.g., surgery). A "locally recurrent" cancer is one that grows back at the same site as the previously treated cancer after treatment.

[0116] "Metastatic" cancer refers to cancer that has spread from one part of the body (e.g., the lungs) to another.

[0117] In this disclosure, the terms "first" (e.g., first protein functional domain, first linker fragment, first product) and "second" (e.g., second protein functional domain, second linker fragment, second product) are used to distinguish or clarify the language and do not imply any particular order unless otherwise specified.

[0118] In this disclosure, unless otherwise specified, "about" or "approximately" means that the specified value or physical quantity varies within a range of 10%, 20%, or 30%. For example, about 100 minutes or approximately 100 minutes may be 90 to 110 minutes, 80 to 120 minutes, or 70 to 130 minutes.

[0119] Beneficial Effects of This Disclosure This disclosure achieves one or more of the following technical effects (1) to (6). (1) The combination drug of this disclosure has a favorable effect on the treatment or prevention of tumors. (2) In the pharmaceutical combinations disclosed herein, the anti-CTLA4 anti-PD-1 bispecific antibody has a synergistic effect with gemcitabine and / or paclitaxel (e.g., albumin-bound paclitaxel), thereby achieving a synergistic effect in the treatment or prevention of tumors, which exhibit superior efficacy compared to the anti-CTLA4 anti-PD-1 bispecific antibody alone or the combination of other anti-PD-1 monoclonal antibodies (e.g., Opdivo®) + anti-CTLA4 monoclonal antibodies (e.g., Yervoy®) + gemcitabine and / or paclitaxel (e.g., albumin-bound paclitaxel), wherein the tumors include, but are not limited to, pancreatic cancer, 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, and nasopharyngeal cancer. (3) This disclosure may be beneficial in reducing the amount and / or effective concentration of gemcitabine. (4) This disclosure may be beneficial in reducing the toxicity and side effects of gemcitabine. (5) This disclosure may be beneficial in reducing the amount and / or effective concentration of paclitaxel. (6) This disclosure may be beneficial in reducing the toxicity and side effects of paclitaxel. [Brief explanation of the drawings]

[0120] [Figure 1] FIG. 1 shows the promotion of INFγ secretion by the combination of an anti-CTLA4 anti-PD-1 bispecific antibody with gemcitabine and albumin-bound paclitaxel in a mixed lymphocyte reaction system. DETAILED DESCRIPTION OF THE INVENTION

[0121] Detailed Description Hereinafter, the embodiments of this disclosure will be described in detail with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative of this disclosure and should not be construed as limiting the scope of this disclosure. Examples that do not specify specific techniques or conditions are carried out according to the techniques or conditions described in publications in the art (see, for example, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the product instructions. Reagents or equipment used are commercially available products unless the manufacturer is specified. [Example]

[0122] In the following examples of this disclosure: Human peripheral blood mononuclear cells were isolated and prepared by Akeso Biopharma Inc. Raji-PDL1 are human PD-L1-expressing cells constructed by Akeso Biopharma Inc. from the human B cell line Raji by transfection. Ficoll-Paque™ PLUS (or Ficoll-Paque PLUS) was purchased from Cytiva. RPMI1640 medium, DMEM medium, trypsin-EDTA (0.25%), phenol red, and blasticidin were all purchased from Gibco. Staphylococcus aureus enterotoxin antigen (SEB) was purchased from Toxin Technology (catalog number BT202). FBS was purchased from Excell Bio. Gemcitabine (abbreviated as Gem) was purchased from MCE (catalog number HY-17026). Albumin-bound paclitaxel (also known as Abraxane, abbreviated as Abra) was purchased from MCE (catalog number HY-P99974).

[0123] Production Example 1: Sequence design of anti-CTLA4 antibody The amino acid sequences of the heavy and light chains and the nucleotide sequences encoding them of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to those of 4G10, 4G10H1L1 and 4G10H3L3 in Chinese Patent Application Publication No. 106967172, respectively.

[0124] (1) Sequences of the heavy and light chain variable regions of 4G10 Nucleotide sequence of the heavy chain variable region: (372 bp) CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAAG TTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT (SEQ ID NO: 1) Encoded amino acid sequence: (124 aa) QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO: 2) Nucleotide sequence of the light chain variable region: (378bp) CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTC TCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC (SEQ ID NO: 3) Encoded amino acid sequence: (126 aa) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC (SEQ ID NO: 4)

[0125] (2) Sequences of the heavy and light chain variable regions of humanized monoclonal antibody 4G10H1L1 Nucleotide sequence of the heavy chain variable region (4G10H1V): (345bp) CAGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCACCAA CTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT (SEQ ID NO: 5) Encoded amino acid sequence: (115 aa) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 6) Nucleotide sequence of the light chain variable region (4G10L1V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACAATCG GGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG (SEQ ID NO: 7) Encoded amino acid sequence: (109 aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 8)

[0126] (3) Sequences of the heavy and light chain variable regions of humanized monoclonal antibody 4G10H3L3 Nucleotide sequence of the heavy chain variable region (4G10H3V): (345bp) CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACTAA CTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA (SEQ ID NO: 9) Encoded amino acid sequence (4G10H3V): (115 aa) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 10) Nucleotide sequence of the light chain variable region (4G10L3V): (327bp) CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAACAA GGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG (SEQ ID NO: 11) Encoded amino acid sequence (4G10L3V): (109aa) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO: 12)

[0127] Production Example 2: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 The amino acid sequences of the heavy and light chains and the nucleotide sequences encoding them of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are identical to those of 14C12 and 14C12H1L1 in Chinese Patent Application Publication No. 106967172, respectively.

[0128] (1) Sequences of the heavy and light chain variable regions of 14C12 Nucleotide sequence of the heavy chain variable region: (354 bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATC CTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 13) Encoded amino acid sequence: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 14) Nucleotide sequence of the light chain variable region: (321 bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 15) Encoded amino acid sequence: (107aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 16)

[0129] (2) Sequences of the heavy and light chain variable regions of humanized monoclonal antibody 14C12H1L1, and sequences of the heavy and light chains Nucleotide sequence of the heavy chain variable region: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 17) Encoded amino acid sequence: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 18) Nucleotide sequence of the light chain variable region: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 19) Encoded amino acid sequence: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 20) DNA sequence of 14C12H1L1 heavy chain (14C12H1): (1344 bp) Encoded amino acid sequence: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22) DNA sequence of 14C12H1L1 light chain (14C12L1): (642bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 23) Encoded amino acid sequence: (214aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24)

[0130] Production 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) belongs to the Morrison format (IgG-scFv), i.e., the C-termini of the two heavy chains of an IgG antibody are linked to the scFv of another antibody via linker fragments. The components of the heavy and light chain designs are shown in Table A.

[0131] [Table A]

[0132] In Table A, 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).

[0133] In Table A, the scFv fragments 4G10H1V(M), 4G10L1V(M), 4G10H3V(M) and 4G10L3V(M) of antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) contain mutations in individual amino acids of the framework regions based on 4G10H1V, 4G10L1V, 4G10H3V and 4G10L3V, respectively, which effectively optimize the structure of the antibody and improve its efficacy. (1) 4G10H1V(M): (115 aa, underlined are the mutation sites from the amino acid sequence of 4G10H1V) QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQ C LEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 41) (2) 4G10L1V(M): (110 aa, underlined are the mutation sites from the amino acid sequence of 4G10L1V) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 42) (3) 4G10H3V(M): (115 aa, underlined are the mutation sites from the amino acid sequence of 4G10H3V) QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ C LEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA (SEQ ID NO: 43) (4) 4G10L3V(M): (110 aa, underlined are the mutation sites from the amino acid sequence of 4G10L3V) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFG C GTKLTVL R (SEQ ID NO: 44) In the Examples of this disclosure, BiAb004(M) is also referred to as BiAb004(hG1WT) to distinguish it from mutant antibodies. BiAb004(M) is a "wild-type" antibody that contains the Igγ1 chain C region (ACCESSION: P01857) as a heavy chain constant region and the Igκ chain C region (ACCESSION: P01834) as a light chain constant region.

[0134] Production Example 4: Design of amino acid mutations outside the variable region based on humanized bifunctional antibody BiAb004 Based on BiAb004(hG1WT) obtained in Preparation Example 3, the inventors produced BiAb004(hG1TM) 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 heavy chain.

[0135] DNA sequence of the heavy chain of the immunoglobulin portion of BiAb004(hG1TM): (1344 bp, the mutated sites are underlined) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCCCCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAGTCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAA GCTGCT GGA GCC(SEQ ID NO: 39)

[0136] Amino acid sequence of the heavy chain of the immunoglobulin portion of BiAb004 (hG1™): (448 aa, mutation sites are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA G APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0137] BiAb004(hG1TM) and BiAb004(hG1WT) have the same light chain DNA sequence and encoded amino acid sequence. Specific sequences are shown in Production Example 3.

[0138] Example 1: Assay of the IFNγ secretion-enhancing biological activity of an anti-CTLA4 anti-PD-1 bispecific antibody in combination with gemcitabine and paclitaxel by mixed lymphocyte reaction (MLR) 1. PBMC treatment: Normal human PBMCs were isolated according to the instructions for the Ficoll-Paque™ Plus isolation solution. Two days before the experiment, the PBMCs were thawed and cultured in complete medium in a 5% carbon dioxide incubator at 37°C. After two hours, the PBMCs recovered and were stimulated with SEB (Staphylococcus aureus enterotoxin antigen, final concentration 0.5 μg / mL) for two days.

[0139] 2. BxPC-3 treatment: Two days before the experiment, human pancreatic cancer cells BxPC-3 (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were collected by conventional methods and centrifuged at 170 × g for 5 minutes. 6 BxPC-3 cells were seeded onto culture dishes and divided into the following four groups according to the experimental design: (1) untreated (negative control); (2) Treatment with gemcitabine at a final concentration of 100 nM for 48 hours; (3) Albumin-bound paclitaxel was added to a final concentration of 20 ng / mL for 24 hours; and (4) Gemcitabine was added to a final concentration of 100 nM and treated for 24 hours, and albumin-bound paclitaxel was added to a final concentration of 20 ng / mL and treated for another 24 hours.

[0140] 3. Raji-PDL1 treatment: On the day of the experiment, Raji-PDL1 cells were harvested, centrifuged at 110 × g for 5 minutes, resuspended (in Assay Medium 1640 + 10% FBS), and counted. The cell density was adjusted to 2,000,000 cells / mL, and MMC (mitomycin C, final concentration 2 μg / mL, purchased from Stressmarq (catalog number SIH-246-10MG)) was added. The cells were treated for 1 hour in a 37°C, 5% CO2 incubator.

[0141] 4. PBMCs after 2 days of SEB stimulation and Raji-PDL1 cells after MMC treatment were collected, washed twice with assay medium, resuspended, and counted; BxPC-3 cells treated with / without different chemotherapy drugs in step 2 were collected conventionally, centrifuged at 170 × g for 5 minutes, resuspended, and counted; cells were seeded into a 96-well U-shaped plate (3799) at 100,000 cells / well for PBMCs, 100,000 cells / well for Raji-PDL1, and 20,000 cells / well for BxPC-3 to obtain a mixed lymphocyte system.

[0142] 5. Antibodies were added as follows: BiAb004(hG1TM) was added separately to the BiAb004(hG1TM) group, gemcitabine + BiAb004(hG1TM) group, and gemcitabine + albumin-bound paclitaxel + BiAb004(hG1TM) group at final concentrations of 0.3, 3, and 30 nM, respectively.

[0143] Opdivo® at final concentrations of 0.3 / 3 / 30 nM, respectively, and Yervoy® at final concentrations of 0.3 / 3 / 30 nM, respectively, were added separately to the gemcitabine + Opdivo® + Yervoy® group and the gemcitabine + albumin-bound paclitaxel + Opdivo® + Yervoy® group.

[0144] No antibody was added to the negative control group (PBMC + Raji-PDL1 + BxPC-3 without chemotherapy treatment), the gemcitabine-only treatment group, the albumin-bound paclitaxel (Abraxane)-only treatment group, and the gemcitabine + albumin-bound paclitaxel (gemcitabine + Abraxane) treatment group.

[0145] The final volume of each system was 200 μL, and the cells were cultured for 3 days.

[0146] 6. After 3 days, the cells were centrifuged at 250 xg for 5 minutes (Beckman centrifuge), the cell supernatant was collected and assayed for IFNγ content using a Dakewe kit.

[0147] The results are shown in Figure 1.

[0148] In the mixed lymphocyte system analysis, the results showed that the INFγ production ability of the anti-CTLA4 anti-PD-1 bispecific antibody BiAb004(hG1™) + gemcitabine combination group was more potent than the BiAb004(hG1™) single agent group and the anti-PD-1 monoclonal antibody Opdivo® (purchased from BMS, batch number ABS3203) + anti-CTLA4 monoclonal antibody Yervoy® (purchased from BMS, batch number AAT3892) + gemcitabine combination group, and that the INFγ production ability of the BiAb004(hG1™) + gemcitabine + albumin-bound paclitaxel combination group was more potent than the anti-PD-1 monoclonal antibody Opdivo® + anti-CTLA4 monoclonal antibody Yervoy® + gemcitabine + albumin-bound paclitaxel combination group.

[0149] Although the specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made thereto according to all the teachings disclosed, and all of these modifications are included in the scope of protection of this invention. The full scope of this disclosure is defined by the claims and their equivalents.

Claims

1. A pharmaceutical combination comprising an anti-CTLA4 anti-PD-1 bispecific antibody and gemcitabine or a pharmaceutically acceptable salt thereof, preferably said pharmaceutical combination further comprising paclitaxel.

2. 2. The pharmaceutical combination of claim 1, wherein the pharmaceutically acceptable salt of gemcitabine is gemcitabine hydrochloride.

3. the paclitaxel is albumin-bound paclitaxel; The pharmaceutical combination according to claim 1 or 2, wherein the mass ratio of paclitaxel to albumin is preferably 1:(5-15), 1:(8-12), 1:8, 1:9, 1:10, 1:11 or 1:

12.

4. 4. The pharmaceutical combination of any one of claims 1 to 3, wherein the unit dose of the anti-CTLA4 anti-PD-1 bispecific antibody 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.

5. 5. The pharmaceutical combination of any one of claims 1 to 4, wherein the anti-CTLA4 anti-PD-1 bispecific antibody is administered at a unit dose of 0.1 to 100 mg / kg body weight, preferably at a unit dose of 1 to 15 mg, 1 to 12 mg, 1 to 10 mg or 6 to 10 mg / kg body weight.

6. Gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100 mg / 200 m2, where m2 is the mass of gemcitabine or a pharmaceutically acceptable salt thereof / body surface area of ​​the subject (m2). ２ ) based on 500 to 1500 mg / m ２ , 600-1300mg / m ２ , 800-1250mg / m ２ , 800-1000mg / m ２ , 800 mg / m ２ , 850 mg / m ２ , 900 mg / m ２ , 950 mg / m ２ , 1000 mg / m ２ , 1050 mg / m ２ , 1100 mg / m ２ , 1150 mg / m ２ , 1200 mg / m ２ or 1250 mg / m ２ or the unit dose of gemcitabine or a pharmaceutically acceptable salt thereof is 100-2000 mg, 500-1500 mg, 600-1300 mg, 800-1200 mg, 800-1000 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, or 1500 mg; The combination pharmaceutical according to any one of claims 1 to 5.

7. Albumin-bound paclitaxel was measured as the mass of albumin-bound paclitaxel (mg) / subject's body surface area (m ２ ) based on 80 to 300 mg / m ２ , 100-280mg / m ２ , 120-260mg / m ２ , 120-200mg / m ２ , 120-180mg / m ２ , 125-175mg / m ２ , 125 mg / m ２ , 130 mg / m ２ , 135 mg / m ２ , 140 mg / m ２ , 145 mg / m ２ , 150 mg / m ２ , 155 mg / m ２ , 160 mg / m ２ , 165 mg / m ２ , 170 mg / m ２ , 175 mg / m ２ or 260 mg / m ２ or the unit dose of albumin-bound paclitaxel is 200-2000 mg, 500-1500 mg, 800-1200 mg, 900-1100 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg; The pharmaceutical combination according to any one of claims 1 to 6.

8. The pharmaceutical combination is a fixed combination, e.g., a pharmaceutical composition; the pharmaceutical combination is a loose combination, for example, the anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel in the loose combination are each present in separate pharmaceutical compositions; or gemcitabine or a pharmaceutically acceptable salt thereof and paclitaxel are a fixed combination, e.g., a pharmaceutical composition; and the anti-CTLA4 anti-PD-1 bispecific antibody is present in a separate pharmaceutical composition; The pharmaceutical combination according to any one of claims 1 to 7.

9. the pharmaceutical composition is, independently, a solid pharmaceutical composition or a liquid pharmaceutical composition; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition does not contain any active pharmaceutical ingredients other than the anti-CTLA4 anti-PD-1 bispecific antibody, gemcitabine or a pharmaceutically acceptable salt thereof, and paclitaxel. The pharmaceutical combination according to claim 8.

10. The anti-CTLA4 anti-PD-1 bispecific antibody a first protein functional region that targets PD-1; and Second Protein Functional Domain Targeting CTLA4 Including, the first protein functional region is an immunoglobulin, the second protein functional region is a single chain variable region fragment, the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the sequences set forth in SEQ ID NOS: 27 to 29, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the sequences set forth in SEQ ID NOS: 30 to 32, respectively; the heavy chain variable region of the single chain variable region fragment comprises HCDR1 to HCDR3 of the sequences set forth in SEQ ID NOS: 33 to 35, respectively, and the light chain variable region of the single chain variable region fragment comprises LCDR1 to LCDR3 of the sequences set forth in SEQ ID NOS: 36 to 38, respectively; Or, the first protein functional region is a single chain variable region fragment, the second protein functional region is an immunoglobulin, the heavy chain variable region of the single chain variable region fragment comprises HCDR1 to HCDR3 of the sequences set forth in SEQ ID NOS: 27 to 29, respectively, and the light chain variable region of the single chain variable region fragment comprises LCDR1 to LCDR3 of the sequences set forth in SEQ ID NOS: 30 to 32, respectively; the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the sequences set forth in SEQ ID NOS: 33 to 35, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the sequences set forth in SEQ ID NOS: 36 to 38, respectively; Preferably, the immunoglobulin subtype is human IgG1, and the heavy chain constant region of the immunoglobulin has the following combination of mutations according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A; having one of the following: The pharmaceutical combination according to any one of claims 1 to 9.

11. The anti-CTLA4 anti-PD-1 bispecific antibody is characterized in that the heavy chain constant region of the immunoglobulin is 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 The pharmaceutical combination according to any one of claims 1 to 10, further comprising one or more mutations selected from:

12. The anti-CTLA4 anti-PD-1 bispecific antibody the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the sequences set forth in 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 the sequences set forth in SEQ ID NO:16 and SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is selected from the sequences set forth in 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 variable region fragment is selected from the sequences set forth in 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 the sequences shown in 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 the sequences shown in 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 variable region fragment is selected from the sequences shown in SEQ ID NO:14 and SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is selected from the sequences shown in SEQ ID NO:16 and SEQ ID NO:

20. The pharmaceutical combination according to any one of claims 1 to 11.

13. The anti-CTLA4 anti-PD-1 bispecific antibody is selected from the following (1) to (20): (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 4; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 8; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 12; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 4; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 8; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 12; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO:16; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO:20; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 42; (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 16; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 44; (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 42; (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 44; (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 16; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 42; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; and, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 44; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 20; The pharmaceutical combination according to any one of claims 1 to 12, wherein the pharmaceutical combination is selected from any one of the following:

14. The anti-CTLA4 anti-PD-1 bispecific antibody The combination pharmaceutical according to any one of claims 1 to 13, wherein the amino acid sequence of the heavy chain of the immunoglobulin is the sequence shown in SEQ ID NO: 40 and the amino acid sequence of the light chain is the sequence shown in SEQ ID NO:

24.

15. The anti-CTLA4 anti-PD-1 bispecific antibody 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 variable region fragment is linked to the light chain variable region of the single-chain variable region fragment directly or via a linker fragment; Preferably, the linker fragment is a polypeptide set forth in SEQ ID NO: 45 or a polypeptide in which a plurality of (e.g., 2, 3, 4, 5, or 6) polypeptides set forth in SEQ ID NO: 45 are linked in tandem; The pharmaceutical combination according to any one of claims 1 to 14.

16. The anti-CTLA4 anti-PD-1 bispecific antibody The pharmaceutical combination according to any one of claims 1 to 15, wherein the number of said first protein functional domains and said second protein functional domains is each independently 1, 2 or more.

17. The anti-CTLA4 anti-PD-1 bispecific antibody The pharmaceutical combination according to any one of claims 1 to 16, wherein the single-chain variable region fragment is linked to each of the C-terminus of two heavy chains of the immunoglobulin.

18. The anti-CTLA4 anti-PD-1 bispecific antibody a first protein functional region that targets PD-1; and Second Protein Functional Domain Targeting CTLA4 Including, the number of said first protein functional domains is 1 and the number of said second protein functional domains is 2; where: the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain variable domain fragment; the heavy chain amino acid sequence of the immunoglobulin is the sequence set forth in SEQ ID NO: 40 and the light chain amino acid sequence of the immunoglobulin is the sequence set forth in SEQ ID NO: 24; the amino acid sequence of the heavy chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 43, and the amino acid sequence of the light chain variable region of the single-chain variable region fragment is the sequence shown in SEQ ID NO: 44; the single-chain variable region fragment 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 variable region fragment is linked to the light chain variable region of the single-chain variable region fragment via a second linker fragment; the first linker fragment and the second linker fragment may be the same or different; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are each independently selected from the sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 26; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are the sequences shown in SEQ ID NO: 26; The pharmaceutical combination according to any one of claims 1 to 17.

19. A kit comprising the pharmaceutical combination of any one of claims 1 to 18 and a product description.

20. Use of the pharmaceutical combination according to any one of claims 1 to 18 in the manufacture of a medicament for treating or preventing a tumor, Preferably, the tumor is selected from one or more of pancreatic cancer, 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 and nasopharyngeal cancer; Preferably, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma; Preferably, the pancreatic cancer is advanced pancreatic cancer or metastatic pancreatic 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 non-small cell lung cancer is intermediate-stage or advanced-stage non-small cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; use.

21. The pharmaceutical combination according to any one of claims 1 to 18, which is used for the treatment or prevention of a tumor, Preferably, the tumor is selected from one or more of pancreatic cancer, 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 and nasopharyngeal cancer; Preferably, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma; Preferably, the pancreatic cancer is advanced pancreatic cancer or metastatic pancreatic 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 non-small cell lung cancer is intermediate-stage or advanced-stage non-small cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Combination medicines.

22. A method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination according to any one of claims 1 to 18, Preferably, the tumor is selected from one or more of pancreatic cancer, 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 and nasopharyngeal cancer; Preferably, the pancreatic cancer is selected from pancreatic ductal adenocarcinoma and pancreatic adenosquamous carcinoma; Preferably, the pancreatic cancer is advanced pancreatic cancer or metastatic pancreatic 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 non-small cell lung cancer is intermediate-stage or advanced-stage non-small cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; method.

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