Drug combination of quinoline derivatives and antibodies

Abstract

The present application provides a pharmaceutical combination of a quinoline derivative and an antibody, which comprises an immune checkpoint inhibitor and a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof. The pharmaceutical combination in the present application shows good anti-lung tumor and anti-liver tumor activities.

Description

[0001] This application is a divisional application of the invention patent application with the application date of July 18, 2019, application number 201980047782.5, and invention title "Drug combination of quinoline derivative and antibody". Technical Field

[0002] This application belongs to the field of pharmaceutical technology and relates to combination therapies that can be used for anti-tumor treatment. Specifically, this application relates to combinations based on quinoline derivatives and antibodies and their use in anti-tumor treatment. Background Art

[0003] Tyrosine kinases are a group of enzymes that catalyze the phosphorylation of tyrosine residues in proteins and play an important role in intracellular signal transduction. They are involved in the regulation, signal transmission, and development of normal cells and are also closely related to the proliferation, differentiation, migration, and apoptosis of tumor cells. Many receptor tyrosine kinases are associated with tumor formation and can be classified into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), etc. according to the different structures of their extracellular regions.

[0004] Anlotinib is a quinoline derivative tyrosine kinase inhibitor that acts as a multi-target tyrosine kinase inhibitor (TKI) in affecting tumor angiogenesis and proliferation signal transduction. Its main targets include: receptor tyrosine kinases vascular endothelial growth factor receptor (VEGFR) 1 to 3, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR) 1 to 4, platelet-derived growth factor receptor (PDGFR) α and β, and stem cell factor receptor (SCFR) 7, 8, and 9. A phase 2 trial showed that anlotinib could improve progression-free survival and had potential benefits for overall survival (Han B, et al. Br J Cancer. 2018; 118(5): 654-661). A multicenter, double-blind, phase 3 randomized clinical trial showed that in Chinese patients, anlotinib led to extended overall survival and progression-free survival, and this finding indicated that anlotinib was well tolerated and was a potential third-line or further treatment for patients with advanced NSCLC (Han B, et al. JAMA Oncol. 2018 Nov; 4(11): 1569-1575).

[0005] Document WO2008112407 discloses a tyrosine kinase inhibitor 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropanamine of the quinoline derivative class and its preparation method in Example 24. Its structural formula is shown as Formula I:

[0006]

[0007] The innate immune system, which includes T lymphocytes, has a powerful anti-cancer ability with broad capabilities and precise specificity, thus responding to various tumor antigens. Emerging cancer immunotherapies enhance the anti-tumor immune response by adoptive transfer of activated effector cells, immunization against relevant antigens, or provision of non-specific immune stimulants. In the past nearly 20 years, researchers have strived to develop specific immune checkpoint inhibitors and expect to provide new immunotherapy regimens for treating cancer, including the development of the antibody ipilimumab that binds to and inhibits CTLA-4 for treating patients with advanced melanoma (Hodi et al. (2010) N Engl J Med 363: 711-23), and the development of antibodies such as nivolumab, ) and pembrolizumab, ) They specifically bind to programmed death receptor-1 (PD-1) and block the inhibitory PD-1 / PD-1 ligand pathway (Topalian et al. (2012a) N Engl J Med 366: 2443-54). Among them, PD-1 (programmed death-1, PD-1) is a key immune checkpoint receptor expressed by activated T lymphocytes and B lymphocytes and mediates immunosuppression, and its ligands include at least PD-L1 and PD-L2. PD-L1 (Programmed death-ligand1), also known as CD274 or B7-H1, is a 40 kDa type I transmembrane protein encoded by the CD274 gene and is a ligand of PD-1. Both PD-L1 and PD-1 belong to the immunoglobulin superfamily and are both composed of two extracellular Ig domains, namely the N-terminal V domain and the C-terminal constant domain. The binding interfaces of PD-L1 with programmed death receptor-1 (PD-1) and B7-1 (CD80) are on the IgV-like domain (Lin et al. (2008) PNAS 105: 3011-3016). PD-L1 contains a conserved short intracellular tail region (about 30 amino acids), and PD-1 contains two cytoplasmic tyrosine-based signaling motifs, namely immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). After T cell stimulation, PD-1 recruits the tyrosine phosphatase SHP-2 to the ITSM motif in its cytoplasmic tail, resulting in dephosphorylation of effector molecules (such as CD3ζ, PKCθ, and ZAP70) involved in the CD3+ T cell signaling cascade (Freeman et al. (2000) J Exp Med 192: 1027-34; Latchman et al. (2001) Nat Immunol 2: 261-8; Carter et al. (2002) Eur J Immunol 32: 634-43). PD-L1 is not only widely distributed on leukocytes and non-hematopoietic cells in lymphoid and non-lymphoid tissues, but also widely distributed in various cancer cells, highly expressed on the surface of a variety of tumor cells, and the malignancy and poor prognosis of tumors are closely related to the expression level of PD-L1. There is clinical data indicating that high tumor expression of PD-L1 is associated with increased tumor invasiveness and poor prognosis.The formation of the PD-1 / PD-L1 complex transmits inhibitory signals and negatively regulates T cell immune responses; it inhibits TCR-mediated T cell activation, cytokine production, and T cell proliferation (Fife et al. (2011) Nature Immunology 10:1185-1193); induces exhaustion or anergy among antigen-specific T cells (Hofmeyer et al. (2011) Journal of Biomedicine and Biotechnology 2011:1-9); promotes the differentiation of Th1 cells into Foxp3+ regulatory T cells (Armanath et al. (2011) Science TransMed 3:1-13; Francisco et al. (2009) J. Exp. Med. 206:3015-3029); and induces apoptosis of effector T cells. Disruption of the PD-L1 gene results in upregulated T cell responses and the generation of autoreactive T cells (Latchman et al. (2004) PNAS 101:10691-10696). Antibody blockade of PD-1 or PD-L1 leads to increased anti-tumor immunity (Iwai et al. (2002) PNAS 99:12293-12297).

[0008] The biggest challenge encountered by predecessors in the process of tumor immunotherapy was the poor efficacy due to tumor immune tolerance and escape. Therefore, it has important theoretical significance and application value to break the immune tolerance established by the body against tumor cells through the combined use of small molecule anti-tumor compounds and anti-PD-1 / PD-L1 antibodies. Summary of the Invention

[0009] The purpose of the present application is at least to provide a drug combination, which comprises a tyrosine kinase inhibitor and an inhibitor of the interaction between PD-1 and its ligand PD-L1. In some embodiments, the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof. In some specific embodiments, the tyrosine kinase inhibitor is the hydrochloride salt of the compound of formula I, namely anlotinib hydrochloride.

[0010] In some embodiments, the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an antibody or an antigen-binding portion thereof that binds to programmed death receptor 1 (PD-1) and / or inhibits the activity of PD-1, or an antibody or an antigen-binding portion thereof that binds to programmed death ligand 1 (PD-L1) and / or inhibits the activity of PD-L1, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. In some specific embodiments, the antibody or its antigen-binding portion is (a) a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human PD-1 and blocks the binding of human PD-L1 to human PD-1; or (b) a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 and blocks the binding of human PD-L1 to human PD-1.

[0011] In some embodiments, the anti-PD-1 or PD-L1 antibody is an anti-PD-1 or PD-L1 monoclonal antibody.

[0012] In some embodiments, the anti-PD-1 or PD-L1 antibody is a human-derived antibody or a murine-derived antibody.

[0013] In some embodiments, the anti-PD-1 antibody may be selected from any one or more of Nivolumab, Pembrolizumab, Durvalumab, Toripalimab (JS-001), Sintilimab (IBI308), Camrelizumab, Tirzepatide (BGB-A317), 14C12H1L1 (Kangfang Bio), Genolimab (GB226), Livzon Mab (LZM009), HLX-10, BAT-1306, AK103 (HX008), AK104 (Kangfang Bio), CS1003, SCT-I10A, F520, SG001, GLS-010.

[0014] In some embodiments, the anti-PD-L1 antibody may be selected from any one or more of Atezolizumab, Avelumab, Durvalumab, KL-A167, SHR-1316, BGB-333, JS003, STI-A1014 (ZKAB0011), KN035, MSB2311, HLX-20, CS-1001.

[0015] In some embodiments, the anti-PD-1 antibody comprises:

[0016] A heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33, or an amino acid sequence having at least 80% identity, preferably at least 85% identity, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto; and

[0017] A light chain variable region comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36, or an amino acid sequence having at least 80% identity, preferably at least 85% identity, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0018] In some embodiments, the anti-PD-1 antibody comprises: a heavy chain selected from SEQ ID NO:17, SEQ ID NO:37 or SEQ ID NO:39; and a light chain selected from SEQ ID NO:18, SEQ ID NO:40 or SEQ ID NO:42.

[0019] In some embodiments, the anti-PD-1 antibody comprises:

[0020] a. A heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:4;

[0021] b. A heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:5;

[0022] c. A heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:6;

[0023] d. A heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:9;

[0024] e. The heavy chain variable region as shown in SEQ ID NO:8 and the light chain variable region as shown in SEQ ID NO:10;

[0025] f. The heavy chain variable region as shown in SEQ ID NO:11 and the light chain variable region as shown in SEQ ID NO:14;

[0026] g. The heavy chain variable region as shown in SEQ ID NO:12 and the light chain variable region as shown in SEQ ID NO:15;

[0027] h. The heavy chain variable region as shown in SEQ ID NO:13 and the light chain variable region as shown in SEQ ID NO:16;

[0028] i. The heavy chain as shown in SEQ ID NO:17 and the light chain as shown in SEQ ID NO:18;

[0029] j. The heavy chain variable region as shown in SEQ ID NO:31 and the light chain variable region as shown in SEQ ID NO:34;

[0030] k. The heavy chain variable region as shown in SEQ ID NO:32 and the light chain variable region as shown in SEQ ID NO:35;

[0031] l. The heavy chain variable region as shown in SEQ ID NO:33 and the light chain variable region as shown in SEQ ID NO:36;

[0032] m. The heavy chain as shown in SEQ ID NO:37 and the light chain as shown in SEQ ID NO:40;

[0033] n. The heavy chain as shown in SEQ ID NO:38 and the light chain as shown in SEQ ID NO:41; or

[0034] o. The heavy chain as shown in SEQ ID NO:39 and the light chain as shown in SEQ ID NO:42.

[0035] In some embodiments, the anti-PD-1 antibody is a isolated antibody or antibody fragment that binds to PD-1 and comprises CDR sequences derived from the 6F5 antibody, and the CDR sequences of the 6F5 antibody are as follows:

[0036] HCDR1: GFTFSSYG (SEQ ID NO:19);

[0037] HCDR2: ISGGGSDT (SEQ ID NO:20);

[0038] HCDR3: ARQLNYAWFAY (SEQ ID NO:21);

[0039] LCDR1: ESVDNYGISF (SEQ ID NO:22);

[0040] LCDR2: TSS (SEQ ID NO:23); and

[0041] LCDR3: QQSKEVPWT (SEQ ID NO:24).

[0042] In some embodiments, the anti-PD-1 antibody is an isolated antibody or antibody fragment that binds to PD-1 and comprises CDR sequences derived from the 14C12 antibody. The CDR sequences of the 6F5 antibody are as follows:

[0043] HCDR1: GFAFSSYD (SEQ ID NO:25);

[0044] HCDR2: ISGGGRYT (SEQ ID NO:26);

[0045] HCDR3: ANRYGEAWFAY (SEQ ID NO:27);

[0046] LCDR1: QDINTY (SEQ ID NO:28);

[0047] LCDR2: RAN (SEQ ID NO:29); and

[0048] LCDR3: LQYDEFPLT (SEQ ID NO:30).

[0049] In some embodiments, the compound of formula I may exist in the form of a pharmaceutically acceptable salt or a pharmaceutically acceptable formulation thereof, preferably in the form of its hydrochloride salt.

[0050] In some specific embodiments, the compound is the hydrochloride salt of 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropanamine, namely anlotinib hydrochloride.

[0051] In some embodiments, the pharmaceutical combination comprises: a compound of formula I or its hydrochloride salt (such as the dihydrochloride); and sintilimab or its antigen-binding fragment, InVivoMAb anti-mouse PD-1 monoclonal antibody or its antigen-binding fragment, or 14C12H1L1 or its antigen-binding fragment.

[0052] The object of the present application also lies at least in providing an anti-tumor use of a drug combination, the drug combination comprising a tyrosine kinase inhibitor and an inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1. In some embodiments, the tyrosine kinase inhibitor is a compound of formula I or its hydrochloride salt, and the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an anti-PD-1 antibody or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some specific embodiments, the anti-PD-1 monoclonal antibody comprises: a heavy chain variable region having an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33; and a light chain variable region having an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.

[0053] The present application also provides a method for treating a subject suffering from cancer or a tumor, which comprises administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1. In some embodiments, the tyrosine kinase inhibitor is a compound of formula I or its hydrochloride salt. In some embodiments, the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an antibody or an antigen-binding portion thereof that binds to programmed death receptor 1 (PD-1) and / or inhibits the activity of PD-1, such as an anti-PD-1 antibody or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0054] The present application also provides a combination therapy for treating a subject suffering from cancer or a tumor, the method comprising separately administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor and separately administering a therapeutically effective amount of an antibody or an antigen-binding portion thereof that inhibits PD-L1 and / or PD-L1 binding activity.

[0055] The present application also provides a method for treating a subject suffering from cancer or a tumor, wherein the cancer or tumor is lung cancer, and the method comprises: (i) measuring the levels of PD-1 and / or PD-L1 in a sample of the subject, wherein the subject is PD-1 and / or PD-L1 positive, and (ii) administering to the subject a therapeutically effective amount of an anti-PD-1 and / or PD-L1 antibody or an antigen-binding portion thereof.

[0056] The present application provides a method for treating a subject suffering from cancer or a tumor. In certain embodiments, the subject is a patient diagnosed with lung cancer, such as a patient diagnosed with non-small cell lung cancer or a patient diagnosed with small cell lung cancer, and the cancer may be refractory, recurrent or metastatic lung cancer. For example, in certain patients, the lung cancer is recurrent; in certain patients, the lung cancer is metastatic; in certain patients, the lung cancer is refractory. In some specific embodiments, the non-small cell lung cancer described in the present application is squamous non-small cell carcinoma, and in other specific embodiments, the non-small cell lung cancer described in the present application is non-squamous non-small cell carcinoma. In some specific embodiments, the non-small cell lung cancer described in the present application is lung adenocarcinoma, lung squamous cell carcinoma or large cell lung cancer.

[0057] In some embodiments of the present application, the subject has previously received surgery, chemotherapy and / or radiotherapy. In some specific embodiments, the subject experiences disease progression again after achieving a complete remission after surgery, chemotherapy and / or radiotherapy. In some specific embodiments, the subject fails to achieve a complete remission or a partial remission after surgery, chemotherapy and / or radiotherapy.

[0058] In some embodiments of the present application, the subject has not previously received systemic chemotherapy. In some embodiments, the subject has previously received surgical treatment, radiotherapy, induction chemotherapy and / or adjuvant chemotherapy, or the subject receives concurrent chemotherapy. In some specific embodiments, the subject has not previously received systemic chemotherapy, but has received surgical treatment, radiotherapy, induction chemotherapy and / or adjuvant chemotherapy or will receive concurrent chemotherapy. In some specific embodiments, the subject experiences disease progression again after achieving a complete remission after surgical treatment, radiotherapy, induction chemotherapy, concurrent chemotherapy and / or adjuvant chemotherapy. In some specific embodiments, the subject fails to achieve a complete remission or a partial remission after surgical treatment, radiotherapy, induction chemotherapy, concurrent chemotherapy and / or adjuvant chemotherapy. In some specific embodiments, the cancer metastasizes after surgical treatment, radiotherapy, induction chemotherapy, concurrent chemotherapy and / or adjuvant chemotherapy.

[0059] In some embodiments of the present application, the cancer or tumor is lung cancer or malignant lung tumor. In some specific embodiments, the cancer is non-small cell lung cancer (NSCLC). In some other specific embodiments, the cancer is small cell lung cancer (SCLC). In some specific embodiments, the cancer is recurrent or refractory lung cancer. In some specific embodiments, the lung cancer is recurrent. In some specific embodiments, the lung cancer is refractory. In some specific embodiments, the lung cancer is metastatic. In some specific embodiments, the cancer treatment is the first-line treatment for recurrent or refractory non-small cell lung cancer. In some specific embodiments, the cancer treatment is the first-line treatment for metastatic non-small cell lung cancer, such as the first-line treatment for non-small cell lung cancer with lymph node metastasis, brain metastasis, and / or bone metastasis.

[0060] In some embodiments of the present application, the lung cancer is selected from non-small cell lung cancer. In some embodiments, the lung cancer includes squamous cell lung cancer or adenocarcinoma of the lung. In some specific embodiments, the lung cancer is advanced lung cancer. In some specific embodiments, the lung cancer is non-small cell lung cancer with wild-type EGFR, ALK, and / or ROS1. In some specific embodiments, the lung cancer is selected from advanced squamous cell non-small cell lung cancer and advanced adenocarcinoma non-small cell lung cancer. In some specific embodiments, the lung cancer is selected from i) squamous cell non-small cell lung cancer with wild-type EGFR, ALK, and / or ROS1, and ii) adenocarcinoma non-small cell lung cancer with wild-type EGFR, ALK, and / or ROS1. In some embodiments, the lung cancer is advanced (stage IIIB / IV) lung cancer that has failed first-line standard chemotherapy or is intolerant to chemotherapy.

[0061] In some embodiments of the present application, the cancer is recurrent and / or metastatic non-small cell lung cancer. In some embodiments, the cancer is recurrent and / or metastatic small cell lung cancer.

[0062] In some embodiments of the present application, the drug combination is used for treating or preventing malignant lung tumors, which can be primary or secondary lung tumors.

[0063] In some embodiments of the present application, the malignant lung tumor is metastatic lung cancer. In some other embodiments, the metastatic lung cancer is metastatic cancer metastasized from lung cancer, gastric cancer, rectal cancer, colon cancer, colorectal cancer, pancreatic cancer, or breast cancer.

[0064] In some embodiments of the present application, the subject is a patient with locally advanced (stage IIIB), metastatic or recurrent (stage IV) NSCLC who is negative for driver genes (i.e., negative for EGFR, ALK, and ROS1 mutations). In some instances, the patient is histologically or cytologically confirmed to be inoperable and unable to receive radical concurrent chemoradiotherapy. In some non-limiting examples, the EGFR mutations include, but are not limited to, exon 19 or exon 21 mutations.

[0065] In some embodiments of the present application, the drug combination can be used to treat or prevent liver malignancies, which can be primary liver tumors or secondary liver tumors.

[0066] In some embodiments of the present application, the liver malignancy is hepatocellular carcinoma.

[0067] In some embodiments of the present application, the liver malignancy is metastatic liver cancer. In some other embodiments, the metastatic liver cancer is a metastatic carcinoma metastasized from lung cancer, gastric cancer, rectal cancer, colon cancer, large intestine cancer, pancreatic cancer, or breast cancer.

[0068] In some embodiments of the present application, the drug combination is a fixed combination. In some embodiments, the fixed combination is in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition.

[0069] In some embodiments of the present application, the drug combination is a non-fixed combination. In some embodiments, the anti-PD-1 / PD-L1 antibody and the compound of formula I in the non-fixed combination are each in the form of a pharmaceutical composition.

[0070] In some embodiments, a kit for a drug combination for treating lung tumors or liver malignancies is further provided, which contains (a) a first pharmaceutical composition containing an anti-PD-1 / PD-L1 antibody as an active ingredient; and (b) a second pharmaceutical composition containing a compound of formula I as an active ingredient.

[0071] In some embodiments of the present application, the kit contains the compound of formula I in an amount of 56-168 mg, such as 84-168 mg. In some embodiments, the kit contains the compound of formula I in an amount selected from 56 mg, 70 mg, 84 mg, 112 mg, 140 mg, 168 mg, or a range formed by any of the above values. In some embodiments, the kit contains the compound of formula I in an amount of 112 mg-168 mg. In some embodiments, the compound of formula I within the above dose range can be packaged together as a whole. In some other embodiments, the compound of formula I within the above dose range can be separately packaged in unit doses, for example, separately packaged in unit doses of 8 mg, 10 mg, or 12 mg.

[0072] The object of the present application is also to provide a method for preventing or treating cancer or tumor, wherein a therapeutically effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 are administered (e.g., sequentially or simultaneously) to a subject in need. In some embodiments, the tyrosine kinase inhibitor is a compound of formula I or its hydrochloride. In some embodiments, the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an antibody or an antigen-binding portion thereof that binds to programmed death receptor 1 (PD-1) and / or inhibits the activity of PD-1, such as an anti-PD-1 antibody or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer or tumor is selected from liver tumors (e.g., liver malignancies such as hepatocellular carcinoma) or lung tumors (e.g., lung cancer such as non-small cell lung cancer).

[0073] The object of the present application is also at least to provide the use of an antagonist of PD-1 or PD-L1 for treating cancer, which is used in combination with a compound of formula I or a pharmaceutically acceptable salt thereof.

[0074] The object of the present application is also at least to provide the use of a compound of formula I or a pharmaceutically acceptable salt thereof for treating cancer, which is used in combination with an antagonist of PD-1 or PD-L1. In some embodiments, the PD-1 antagonist is a PD-1 monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to human PD-1 and blocks the binding of human PD-L1 to human PD-1, and / or the PD-L1 antagonist is a PD-L1 monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to human PD-1 and blocks the binding of human PD-L1 to human PD-1.

[0075] The object of the present application is also at least to provide a drug package, which separately contains drug compositions in single packages in separate containers, wherein one container contains a drug composition containing a compound of formula I or a pharmaceutically acceptable salt thereof, and the second container contains a drug composition containing a PD-1 antagonist or a PD-L1 antagonist.

[0076] In some embodiments of the present application, the drug composition contains 56-168 mg, such as 84-168 mg, of the compound of formula I. In some embodiments, the drug composition contains the compound of formula I in an amount selected from 56 mg, 70 mg, 84 mg, 112 mg, 140 mg, 168 mg or a range formed by any of the above values. In some embodiments, the drug composition contains 112 mg-168 mg of the compound of formula I.

[0077] In some embodiments, the anti-PD-1 antibody is administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 9 mg / kg, 10 mg / kg body weight.

[0078] In some embodiments, the anti-PD-1 antibody is administered at one or more uniform doses effective to treat the cancer. In some specific embodiments, the uniform dose ranges from about 10 mg to about 1000 mg of anti-PD-1 antibody. In some specific embodiments, the uniform dose is selected from about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg of anti-PD-1 antibody. In some specific embodiments, the uniform dose is selected from about 200 mg of anti-PD-1 antibody.

[0079] In some embodiments, the administration treatment of the anti-PD-1 antibody is carried out in a cycle of 2 weeks (14 days) or 3 weeks (21 days), and preferably the anti-PD-1 antibody is intravenously administered on the first day (D1) of each cycle. That is, the anti-PD-1 antibody is administered at a frequency of once every two weeks (q2w) or once every three weeks (q3w).

[0080] The present invention provides a product comprising a container containing a fixed dose of an anti-PD-1 antibody. The present invention also provides the use of an anti-PD-L1 antibody in the preparation of a product for treating cancer, the product comprising a container containing a fixed dose of an anti-PD-1 antibody. In some specific embodiments, the container is a vial. The fixed dose is selected from about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 500 mg, or about 1000 mg of the anti-PD-1 antibody. In some specific embodiments, the product further comprises a package insert or a drug label instructing the user to administer the fixed dose of the anti-PD-1 antibody to a cancer patient. In some specific embodiments, the product comprises one or more vials containing about 50 mg, 100 mg, 200 mg, 300 mg, 350 mg, 400 mg, 500 mg, or 600 mg of the anti-PD-1 antibody. In some specific embodiments, the product comprises one vial containing about 50 mg of the anti-PD-1 antibody. In some specific embodiments, the product comprises one vial containing about 200 mg of the anti-PD-1 antibody. In some specific embodiments, the product comprises one vial containing about 350 mg of the anti-PD-1 antibody. In some specific embodiments, the product is packaged in the form of an anti-PD-1 antibody solution at 50 mg / 5 mL / vial, 100 mg / 10 mL / vial, 200 mg / 10 mL / vial, or 350 mg / 35 mL / vial.

[0081] The object of the present application is also to provide a pharmaceutical combination for preventing or treating cancer or a tumor, wherein the pharmaceutical combination comprises a tyrosine kinase inhibitor and an inhibitor of the interaction between PD-1 and its ligand PD-L1. In some embodiments, the tyrosine kinase inhibitor is a compound of formula I or its hydrochloride. In some embodiments, the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an antibody or an antigen-binding portion thereof that binds to programmed death receptor 1 (PD-1) and / or inhibits the activity of PD-1, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer or tumor is selected from liver tumors (such as liver malignancies, such as hepatocellular carcinoma) or lung tumors (such as lung cancer, such as non-small cell lung cancer).

[0082] The present application also aims to provide the use of a drug combination in the preparation of a preparation for preventing or treating cancer or tumor, wherein the drug combination comprises a tyrosine kinase inhibitor and an inhibitor of the interaction between PD-1 and its ligand PD-L1. In some embodiments, the tyrosine kinase inhibitor is a compound of formula I or its hydrochloride salt. In some embodiments, the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an antibody or an antigen-binding portion thereof that binds to programmed death receptor 1 (PD-1) and / or inhibits the activity of PD-1, such as an anti-PD-1 antibody or an anti-PD-L1 antibody or an antigen-binding fragment thereof. In some embodiments, the cancer or tumor is selected from liver tumors (such as malignant liver tumors, such as hepatocellular carcinoma) or lung tumors (such as lung cancer, such as non-small cell lung cancer).

[0083] The present application also aims to provide a unit dosage form, wherein the unit dosage form comprises: a compound component, 6 to 12 mg of a compound of formula I or its hydrochloride salt; and an antibody component, 50 to 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof; wherein the compound component and the antibody component are separately packaged.

[0084] The present application also aims to provide a method for preventing or treating cancer or tumor, wherein one or more of the above-mentioned unit dosage forms are administered to a subject in need thereof. Preferably, the compound component and the antibody component in the unit dosage form are administered separately.

[0085] Anlotinib pharmaceutical composition

[0086] In the present application, the anlotinib pharmaceutical composition is any pharmaceutical composition comprising anlotinib or anlotinib hydrochloride (i.e., a compound of formula I or its hydrochloride salt) as an active ingredient.

[0087] In some embodiments of the present application, the unit dose of the anlotinib pharmaceutical composition comprises 2 mg, 6 mg, 8 mg, 10 mg, or 12 mg of anlotinib.

[0088] In some embodiments of the present application, according to a treatment cycle of administering for 2 weeks and stopping for 1 week, the total dose of the anlotinib pharmaceutical composition administered in each treatment cycle comprises 84 to 168 mg. In some regimens, the total dose of the anlotinib pharmaceutical composition comprises a range selected from 84 mg, 112 mg, 140 mg, 168 mg or any value formed by the above. In some regimens, the total dose of the anlotinib pharmaceutical composition preferably comprises 112 mg to 168 mg.

[0089] Anlotinib

[0090] As used in the present application, the chemical name of the anlotinib (i.e., the compound of Formula I) is 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropanamine, and it has the following structural formula:

[0091]

[0092] As used in the present application, the anlotinib includes its non-salt form (e.g., free acid or free base), and also includes its pharmaceutically acceptable salts, and both the non-salt form and the salts are within the scope of protection of the present application. For example, the pharmaceutically acceptable salt of the anlotinib may be hydrochloride or dihydrochloride. The doses of anlotinib or its salts involved in the present application are calculated based on anlotinib free base unless otherwise specified.

[0093] Sintilimab

[0094] As used in the present application, sintilimab (IBI308, IBI-308) is an anti-PD-1 monoclonal antibody, and its sequence and structure can be referred to Antibody D in Document CN108473977A. On December 27, 2018, the PD-1 antibody drug "Sintilimab Injection" of Innovent Biologics was officially approved by the National Medical Products Administration (NMPA) of China for the treatment of relapsed or refractory classical Hodgkin lymphoma that has undergone at least second-line systemic chemotherapy.

[0095] Full-length sequence of the heavy chain of sintilimab:

[0096] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0097] Full-length sequence of the light chain of Sintilimab:

[0098] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0099] 14C12H1L1

[0100] As used in the present application, 14C12H1L1 is an anti-PD-1 monoclonal antibody, and its sequence and structure can be found in the literature (CN106977602A). In the 14C12H1L1 monoclonal antibody, HCDR1 contains the sequence GFAFSSYD (SEQ ID NO: 25), HCDR2 contains the sequence ISGGGRYT (SEQ ID NO: 26), HCDR3 contains the sequence ANRYGEAWFAY (SEQ ID NO: 27), LCDR1 contains the sequence QDINTY (SEQ ID NO: 28), LCDR2 contains the sequence RAN (SEQ ID NO: 29), and LCDR3 contains the sequence LQYDEFPLT (SEQ ID NO: 30).

[0101] Definitions and explanations

[0102] Unless otherwise specified, the following terms used in the present application have the following meanings. A particular term should not be considered indefinite or unclear without a specific definition, but should be understood according to its ordinary meaning in the art. When a trade name appears in the present application, it is intended to refer to the corresponding product, composition, or its active ingredient.

[0103] As used herein, the term "antibody" refers to an antigen-binding protein having at least one antigen-binding domain. The antibodies and fragments thereof in the present application can be whole antibodies or any fragments thereof. Therefore, the antibodies and fragments thereof in the present application include monoclonal antibodies or their fragments and antibody variants or their fragments, as well as immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab)' fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv), and other antibody fragments known in the art. Antibodies and their fragments can also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The anti-PD-L1 antibodies and their fragments disclosed herein can be of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0104] The term "isotype" refers to the class of antibodies encoded by the heavy chain constant region genes. In one embodiment, the anti-PD-1 / PD-L1 antibodies and their fragments disclosed herein are of the IgG1 or IgG4 isotype. The anti-PD-1 / PD-L1 antibodies and their fragments in the present application can be derived from any species, including but not limited to mice, rats, rabbits, primates, camels, and humans. The PD-1 / PD-L1 antibodies and their fragments can be chimeric antibodies, humanized antibodies, or fully human antibodies.

[0105] The term "humanized antibody" refers to an antibody in which the antigen-binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. A humanized antibody may contain substitutions in the framework region such that the framework may not be an exact copy of the expressed human immunoglobulin or germline gene sequence.

[0106] "Isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 / PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-1 / PD-L1). However, an isolated antibody that specifically binds to PD-1 / PD-L1 may have cross-reactivity with other antigens, such as PD-1 / PD-L1 molecules from different species. In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0107] The term "monoclonal antibody" ("mAb") refers to an antibody molecule of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope, or in the case of a bispecific monoclonal antibody, exhibits dual binding specificities for two different epitopes. An mAb is an example of an isolated antibody. mAbs can be produced by hybridoma technology, recombinant technology, transgenic technology, or other techniques known to those skilled in the art. Examples of isolated anti-PD-1 / PD-L1 monoclonal antibodies include, but are not limited to, Nivolumab Pembrolizumab Durvalumab, Avelumab, Toripalimab (JS-001, Junshi Biosciences), Sintilimab (IBI308, Innovent Biologics), Camrelizumab (SHR-1210, Hengrui Medicine, see CN105026428B or WO2015085847A1),Tislelizumab (BGB-A317, BeiGene), 14C12H1L1 (Sunshine Konfronta), Genolimab (GB226, Jiahe Biologics), Livzon Mab (LZM009, Livzon Pharmaceutical), HLX-10 (Fosun Pharma Henlius), BAT-1306 (Bio-Thera Solutions), HX008 (AK103, Sunshine Konfronta / Hanzhong Biotech), AK104 (Sunshine Konfronta), CS1003 (CStone Pharmaceuticals), SCT-I10A (Shenzhou Cell), F520 (Shandong New Times Pharmaceutical / Lunan Pharmaceutical), SG001 (Sunny Health Biotech), GLS-010 (YHG Pharma), Atezolizumab ( Roche), Avelumab ( Merck / Pfizer), Durvalumab ( AstraZeneca) KL-A167 (Sichuan Kelun Pharmaceutical Co., Ltd.), SHR-1316 (Jiangsu Hengrui Medicine Co., Ltd.), BGB-333 (BeiGene, Ltd.), JS003 (Shanghai Junshi Biosciences Co., Ltd.), STI-A1014 (ZKAB0011, Zhaoke Pharmaceutical Co., Ltd.), KN035 (Conintech Therapeutics Inc. / Shanghai Sirtex Medical Co., Ltd.), MSB2311 (Shanghai MabSpace Biosciences Co., Ltd.), HLX-20 (Shanghai Fosun Pharmaceutical (Group) Co., Ltd. - Fosun Pharma Hanlon Biologics), CS-1001 (CStone Pharmaceuticals Co., Ltd.), etc.

[0108] The "antigen-binding portion" (also referred to as "antigen-binding fragment") of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind the antigen to which the intact antibody binds.

[0109] As used herein, the term "derived", when used in reference to a molecule or polypeptide relative to a reference antibody or other binding protein, means a molecule or polypeptide capable of specifically binding the same epitope as the reference antibody or other binding protein.

[0110] As used herein, the term "EC50" refers to the effective concentration at which 50% of the maximum response of the antibody is achieved. As used herein, the term "IC50" refers to the inhibitory concentration at which 50% of the maximum response of the antibody is achieved. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.

[0111] The term "treatment" generally refers to an operation to obtain the desired pharmacological and / or physiological effect. This effect can be prophylactic, according to the complete or partial prevention of a disease or its symptoms; and / or therapeutic, according to the partial or complete stabilization or cure of the disease and / or the side effects resulting from the disease. "Treatment" as used herein encompasses any treatment of a patient's disease, including: (a) preventing the occurrence of a disease or symptoms in a patient who is susceptible to the disease or symptoms but has not been diagnosed as having the disease; (b) inhibiting the symptoms of the disease, i.e., preventing its progression; or (c) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

[0112] As used herein, the term "systemic treatment" refers to a treatment in which a pharmaceutical substance is delivered through the bloodstream to reach and affect cells throughout the body.

[0113] As used herein, the term "systemic chemotherapy" refers to systemic chemotherapy that does not include chemotherapy for locally advanced disease as part of a multimodal treatment, where chemotherapy for locally advanced disease includes induction chemotherapy, chemotherapy concurrent with radiotherapy, and adjuvant chemotherapy.

[0114] As used herein, the term "subject" refers to a mammal, such as a rodent, feline, canine, and primate. Preferably, the subject according to the present application is a human.

[0115] "Administering" means physically introducing a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Routes of administration of immune checkpoint inhibitors (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies) include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In certain embodiments, the immune checkpoint inhibitor (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) is administered by a non-parenteral route, and in certain embodiments, orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, such as, intranasally, vaginally, rectally, sublingually, or topically. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended time periods.

[0116] As used herein, "adverse event" (AE) is any adverse and generally unintended or undesired sign (including abnormal laboratory findings), symptom, or disease associated with the application of a medical treatment. For example, an adverse event can be associated with activation of the immune system or expansion of immune system cells (e.g., T cells) in response to treatment. A medical treatment can have one or more associated AEs, and each AE can have the same or different levels of severity. Reference to a method that "modifies an adverse event" refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the application of different treatment regimens.

[0117] As used herein, "dosing interval" refers to the amount of time elapsed between multiple doses of a formulation disclosed herein administered to a subject. Thus, the dosing interval can be indicated as a range.

[0118] As used herein, the term "dosing frequency" refers to the frequency of administration of doses of a formulation disclosed herein over a given period of time. The dosing frequency can be indicated as the number of administrations per given time period, e.g., once per week or once every two weeks.

[0119] The application of the term "flat dose" refers to a dose administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, a flat dose is specified as an absolute amount of a pharmaceutical agent (e.g., an anti-PD-1 antibody), rather than as a mg / kg dose. For example, a 60 kg person and a 100 kg person will receive the same dose of an antibody (e.g., 240 mg of an anti-PD-1 antibody).

[0120] The application of the term "fixed dose" to the compositions of the present application means that two or more different antibodies in a single composition are present in the composition at a specific (fixed) ratio to each other. In certain embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In certain embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg / ml). In certain embodiments, the ratio of mg of the first antibody:mg of the second antibody is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1 or about 2:1. For example, a 3:1 ratio of the first antibody to the second antibody can mean that a vial can contain about 240 mg of the first antibody and 80 mg of the second antibody, or about 3 mg / ml of the first antibody and 1 mg / ml of the second antibody.

[0121] The term "weight-based dose" as used herein refers to a dose administered to a patient that is calculated based on the weight of the patient. For example, when a patient with a body weight of 60 kg requires 3 mg / kg of an anti-PD-1 antibody and 1 mg / kg of an anti-CTLA-4 antibody, one can withdraw an appropriate amount of the anti-PD-1 antibody (i.e., 180 mg) and the anti-CTLA-4 antibody (i.e., 60 mg) in one draw from a fixed-dose formulation with a 3:1 ratio of the anti-PD-1 antibody to the anti-CTLA-4 antibody.

[0122] The term "immunotherapy" refers to a method of treating a subject suffering from a disease or at risk of infection or disease recurrence, the method including inducing, enhancing, suppressing, or otherwise altering the immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on the subject, or the administration of an active agent to the subject, with the aim of reversing, alleviating, improving, suppressing, slowing down, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or diseases, or biochemical markers associated with the disease.

[0123] As used herein, "PD1 / PD-L1 positive" can be used interchangeably with "at least about 1% PD-1 / PD-L1 expression". In one embodiment, PD-1 / PD-L1 expression can be used by any method known in the art. In another embodiment, PD-1 / PD-L1 expression is measured by automated IHC. In certain embodiments, "PD-1 / PD-L1 positive" means the presence of at least 100 cells expressing PD-1 / PD-L1 on the cell surface.

[0124] "Programmed death receptor-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants of hPD-1, allotypes and species homologs, and analogs having at least one common epitope with hPD-1.

[0125] "Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1.

[0126] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In certain embodiments, the subject is a human. The terms "subject", "test subject", and "patient" may be used interchangeably in certain contexts herein.

[0127] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of the disease-free symptom stage, or the prevention of damage or disability caused by the affliction of the disease. The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to a skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0128] As used herein, a "sub-therapeutic dose" refers to a dose of a therapeutic compound (e.g., an antibody) that is lower than the usual or typical dose of the therapeutic compound when administered alone for the treatment of a proliferative disease (e.g., cancer).

[0129] As an example, an "anticancer drug" promotes cancer regression in a subject or prevents further tumor growth. In certain embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means administering an effective amount of the drug, either alone or in combination with an anti-tumor agent, that results in a decrease in tumor growth or size, necrosis of the tumor, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of a disease symptom-free phase, or prevention of damage or disability caused by the affliction of the disease. Additionally, the terms "effective" and "efficacy" with respect to treatment include pharmacological efficacy and physiological safety. Pharmacological efficacy represents the ability of the drug to promote cancer regression in a patient. Physiological safety represents the level of toxicity or other adverse physiological effects (adverse reactions) at the cellular, organ, and / or organism level caused by the administration of the drug.

[0130] As an example for treating a tumor, a therapeutically effective amount of an anticancer drug can inhibit cell growth or tumor growth by at least about 10%, at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to an untreated subject or, in certain embodiments, relative to a patient treated with standard of care therapy. In other embodiments of the present application, tumor regression can be observed and persist for a period of at least about 20 days, at least about 40 days, or at least about 60 days. Despite these end measurements of treatment efficacy, the evaluation of immunotherapeutic drugs must also consider "immune-related" response patterns.

[0131] An "immune-related" response pattern represents a clinical response pattern frequently observed in cancer patients treated with an immunotherapeutic agent that exerts an anti-tumor effect by inducing a cancer-specific immune response or by altering the innate immune process. This response pattern is characterized by a beneficial treatment effect following an initial increase in tumor burden or the appearance of new lesions, which would be classified as disease progression and synonymous with drug failure in the evaluation of traditional chemotherapeutic agents. Thus, the appropriate evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

[0132] A therapeutically effective amount of a drug includes a "preventive effective amount", which is any amount of the drug that inhibits the occurrence or recurrence of cancer when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject at risk of cancer recurrence. In certain embodiments, the preventive effective amount completely prevents the occurrence or recurrence of cancer. "Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of the occurrence or recurrence of cancer or completely preventing the occurrence or recurrence of cancer.

[0133] "Recurrent" cancer is cancer that regenerates at the original site or at a distant site after responding to initial treatment (e.g., surgery). "Local recurrent" cancer is cancer that appears at the same location as the previously treated cancer after treatment.

[0134] "Unresectable" cancer cannot be removed by surgery.

[0135] "Metastatic" cancer refers to cancer that spreads from one part of the body (e.g., the lungs) to another part of the body.

[0136] The use of alternatives (e.g., "or") shall be understood to mean any one, two, or any combination of the alternatives. The indefinite article "a" or "an" as used herein shall be understood to mean "one or more / one or more kinds" of any listed or enumerated components.

[0137] The terms "about", "approximately", or "substantially comprise" mean a value or a composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about", "approximately", or "substantially comprise" may mean within 1 or more standard deviations in accordance with the practice in the art. Alternatively, "about" or "substantially comprise" may mean a range that differs from the parameter or numerical value being modified by at most 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg may include any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%). In addition, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the numerical value. When a specific value or composition is provided in this application and the claims, unless otherwise stated, the meaning of "about" or "substantially comprise" shall be assumed to be within the acceptable error range of that specific value or composition.

[0138] As used herein, the terms "about once a week", "about once every two weeks", or any other similar dosing interval terms refer to approximations. "About once a week" can include every 7 days ± 1 day, i.e., every 6 days to every 8 days. "About once every two weeks" can include every 14 days ± 3 days, i.e., every 11 days to every 17 days. Similar approximations apply to, for example, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, and about once every 12 weeks. In certain embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose can be administered on any day of the first week, and then the second dose can be administered on any day of the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose is administered on a specific day of the first week (e.g., Monday), and then the second dose is administered on the same day (i.e., Monday) of the sixth or twelfth week, respectively. Similar principles apply to phrases including, but not limited to, "about once every 2 weeks", "about once a month", etc.

[0139] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values within the recited range, and when appropriate, include its fractions (such as tenths and hundredths of an integer), unless otherwise indicated.

[0140] Unless otherwise specified, "about" or "approximately" in this application means fluctuating within ±5% of the given specific numerical range, preferably within ±2%, and more preferably within ±1%. For example, a pH of about 5.5 means a pH of 5.5 ± 5%, preferably a pH of 5.5 ± 2%, and more preferably a pH of 5.5 ± 1%.

[0141] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0142] The term "pharmaceutically acceptable salt" includes salts formed by basic ions and free acids or acidic ions and free bases, for example, including hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, trifluoroacetate, fumarate, oxalate, maleate, citrate, succinate, mesylate, benzenesulfonate or p-toluenesulfonate, preferably hydrochloride, hydrobromide, sulfate, formate, acetate, trifluoroacetate, fumarate, maleate, mesylate, p-toluenesulfonate, sodium salt, potassium salt, ammonium salt, amino acid salt, etc. In the present application, when forming a pharmaceutically acceptable salt, the molar ratio of the free acid to the basic ion is about 1:0.5 to 1:5, preferably 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8. In the present application, when forming a pharmaceutically acceptable salt, the molar ratio of the free base to the acidic ion is about 1:0.5 to 1:5, preferably 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8.

[0143] The term "fixed combination" means that the active components (such as anti-PD-1 antibody or compound of formula I) are administered to a subject simultaneously in a fixed total dose or dose ratio, or in the form of a single entity, pharmaceutical composition or preparation.

[0144] The term "non-fixed combination" means that two or more active components are administered to a subject simultaneously, in parallel or sequentially without a specific time limit as independent entities (such as pharmaceutical compositions, preparations), wherein the active ingredients administered to the subject reach a therapeutically effective amount level. Examples of non-fixed combinations that can be cited are cocktail therapies, for example, administering 3 or more active components. In a non-fixed combination, each of the active components can be packaged, sold or administered as a completely independent pharmaceutical composition. The "non-fixed combination" also includes the combined use between "fixed combinations", or between a "fixed combination" and an independent entity of any one or more active components.

[0145] As used herein, "administered in combination" or "used in combination" means that two or more active substances can be administered to a subject together in a mixture, simultaneously as a single preparation or sequentially in any order as a single preparation.

[0146] The term "pharmaceutical composition" refers to a mixture composed of one or more active ingredients (such as anti-PD-1 antibody or compound of formula I) of the present application or their pharmaceutical combinations and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds or their pharmaceutical combinations of the present application to a subject.

[0147] The term "synergistic effect" refers to the effect (such as inhibiting the growth of colon cancer or alleviating the symptoms of colon cancer) produced by two or more components (such as anti-PD-1 antibody or a compound of formula I) being greater than the simple addition of the effects of the components when administered alone.

[0148] Route of administration

[0149] The following does not limit the administration mode of the pharmaceutical combination of the present application.

[0150] The components in the pharmaceutical combination of the present application can be formulated separately, or some or all of them can be formulated together. In one embodiment, the pharmaceutical combination of the present application can be formulated into a pharmaceutical composition suitable for single or multiple administrations.

[0151] The components in the pharmaceutical combination of the present application can be administered separately, or some or all of them can be administered together. The components in the pharmaceutical combination of the present application can be administered substantially non-simultaneously, or some or all of them can be administered substantially simultaneously.

[0152] The components in the pharmaceutical combination of the present application can be administered independently or some or all of them can be administered together by various suitable routes, including but not limited to oral or parenteral (by intravenous, intramuscular, topical or subcutaneous routes). In some embodiments, the components of the pharmaceutical combination of the present application can be administered independently or some or all of them can be administered together orally or by injection, such as intravenous injection or intraperitoneal injection.

[0153] The components in the pharmaceutical combination of the present application can be independently or some or all of them can be together suitable dosage forms, including but not limited to tablets, lozenges, pills, capsules (such as hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersants and dosage forms of sustained-release preparations for oral or non-oral administration.

[0154] The components in the pharmaceutical combination of the present application can independently or some or all of them can be together contain pharmaceutically acceptable carriers and / or excipients.

[0155] The pharmaceutical combination of the present application can also contain additional therapeutic agents. In one embodiment, the additional therapeutic agent can be a cancer therapeutic agent known in the art, preferably a lung cancer therapeutic agent.

[0156] In some specific embodiments, the present application investigated the efficacy of anlotinib hydrochloride, anti-PD-1 antibody alone or in combination on lung tumors. Surprisingly, the experimental results found that anlotinib hydrochloride and anti-PD-1 antibody can have an obvious synergistic effect, breaking the immune tolerance of the body established against tumor cells. Brief Description of the Drawings

[0157] Figure 1 Shows the effects of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with an anti-mPD-1 antibody on the growth of subcutaneous transplanted tumors of mouse liver cancer H22.

[0158] Figure 2 Shows the individual tumor growth curves of mice in each treatment group of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with an anti-mPD-1 antibody.

[0159] Figure 3 Shows the individual tumor volumes (upper figure) and individual tumor weights (lower figure) of each treatment group of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with an anti-mPD-1 antibody at the end of the experiment (D17).

[0160] Figure 4 Shows the effects of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with an anti-mPD-1 antibody on the body weights of tumor-bearing mice.

[0161] Figure 5 Shows the photos of tumors of the efficacy of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with an anti-mPD-1 antibody on subcutaneous transplanted tumors of liver cancer H22.

[0162] Figure 6 Shows the changes in SLD (sum of the longest diameters of the smallest target lesions recorded after the start of treatment) of 13 patients in an open-label, multicenter phase Ib / II clinical study of 14C12H1L1 combined with anlotinib hydrochloride for first-line treatment of unresectable hepatocellular carcinoma.

[0163] Figure 7 Shows the changes in SLD over time of 13 patients in an open-label, multicenter phase Ib / II clinical study of 14C12H1L1 combined with anlotinib hydrochloride for first-line treatment of unresectable hepatocellular carcinoma. Detailed Description of the Preferred Embodiments

[0164] The present application will be further described below in conjunction with specific embodiments. However, these embodiments in the present application are only used for clarification and do not limit the scope of the present application. Similarly, the present application is not limited to any specific preferred implementation described herein. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the technical features of the present application still fall within the protection scope of the present application. Unless otherwise specified, the reagents used in the following examples are all commercially available products, and the preparation of solutions can adopt conventional techniques in the art.

[0165] Table 1 Abbreviation Table

[0166]

[0167]

[0168]

[0169] Example 1: Treatment of Advanced Non-Small Cell Lung Cancer with Anlotinib Hydrochloride in Combination with Other Drugs

[0170] This example discloses the exploratory research results of treating advanced non-small cell lung cancer with anlotinib hydrochloride in combination with other drugs. The main target population is patients with locally advanced (IIIB), metastatic or recurrent (IV stage) NSCLC who are histologically or cytologically confirmed to be inoperable and ineligible for radical concurrent chemoradiotherapy, with negative driver genes (i.e., negative for EGFR, ALK, and ROS1 mutations).

[0171] Primary study endpoints: Safety, ORR.

[0172] Secondary study endpoints: DCR, PFS, OS.

[0173] Progression-free survival (PFS): Defined as the time from the first dose until objective tumor progression or death.

[0174] Overall survival (OS): Defined as the time from the start of the first dose until death from any cause. In days, for subjects lost to follow-up, the last follow-up time is usually counted as the time of death.

[0175] Key inclusion criteria: Chinese patients with IIIB / IV stage NSCLC; not previously treated with systemic therapy; clear molecular typing; aged 18 - 75 years; PS score: 0 - 1; expected survival of more than 3 months; sufficient organ reserve function; asymptomatic brain metastases.

[0176] Key exclusion criteria: Previously received systemic anti-cancer therapy; having multiple factors affecting oral drugs; known symptomatic brain metastases, spinal cord compression, carcinomatous meningitis, or patients with brain or leptomeningeal diseases detected by CT or MRI during screening; central cavitary squamous cell carcinoma or having a bleeding tendency; patients with any severe and uncontrolled diseases.

[0177] Anlotinib Hydrochloride: Pale yellow crystalline powder, with a content of 98.9%, batch number 17316007, stored in the dark at 2 - 8°C. Anlotinib hydrochloride was provided by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Anlotinib hydrochloride was prepared with distilled water and diluted to the required concentration.

[0178] Dosage administered:

[0179] Anti-PD-1 antibody: Sintilimab injection was administered intravenously at a dose of 200 mg on the first day (D1) of each cycle (a total of 21 days), and administered once every 21 days.

[0180] Anlotinib Hydrochloride Capsules (the active ingredient is anlotinib dihydrochloride): Take 12 mg of anlotinib hydrochloride capsules on an empty stomach within ±5 minutes before or after the start of the infusion of the anti-PD-1 antibody injection. Take continuously for 2 weeks and then stop for 1 week, that is, 21 days is a treatment cycle.

[0181] Before the start of the trial, the trial protocol, copies of the electronic case report form, draft informed consent form, etc. of this trial were submitted to the clinical research responsible unit and the ethics committees of each participating research unit for review. Clinical trials can only be carried out after obtaining the approval of the ethics committee for conducting clinical trials. Before the start of the clinical trial, the researchers introduced the purpose, risks and benefits of this trial to all subjects in detail, ensured that all subjects were informed and consented before the trial, signed the informed consent form, and fully protected the rights and interests of the subjects during the trial. The researchers strictly followed the ethical guidelines for human medical research in the Declaration of Helsinki (2013) and the provisions of relevant laws and regulations such as the Good Clinical Practice for Pharmaceutical Clinical Trials (2003) to fulfill the responsibilities of the researchers.

[0182] As of January 25, 2019, 11 patients had met the conditions for efficacy evaluation. The basic information and efficacy evaluation results of these 11 patients participating in the study are shown in Table 2, where the efficacy evaluation times were the second cycle (about 42 days) and the fourth cycle (about 84 days) respectively; the statistical results of the efficacy evaluation are shown in Table 3.

[0183] Table 2 Basic information and efficacy evaluation results of patients participating in the study

[0184]

[0185] The exploratory data research results of this anlotinib combined with first-line treatment of advanced non-small cell lung cancer showed surprising efficacy. The ORR of anlotinib combined with sintilimab was 63.6%, and the DCR was 100% (see Table 3), and the PFS and OS were worth looking forward to. At the same time, the first-line combined mode of anlotinib could show a high degree of patient tolerance (detailed results not shown).

[0186] Table 3 Efficacy evaluation of anlotinib combined with sintilimab

[0187]

[0188] Example 2 Treatment of liver cancer with anlotinib hydrochloride combined medication

[0189] 1. Drug information

[0190] Anlotinib Hydrochloride: Light yellow crystalline powder, with a content of 98.9%, batch number 17316007, expiration date until December 2018, stored in the dark at 2-8°C.

[0191] Lenvatinib mesylate: off-white crystalline powder, content 98.7%, batch number 19916003, expiration date June 2019, store in the dark at 2 - 8°C.

[0192] Anti-mouse PD-1 (abbreviation: mPD-1): colorless transparent liquid, purity > 95%, concentration 7.83 mg / ml, batch number 665418F1, store in the dark at 2 - 8°C.

[0193] 2. Provider

[0194] Anlotinib hydrochloride and lenvatinib mesylate are provided by Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

[0195] mPD-1 is purchased from Bio X Cell, specification: InVivoMAb anti-mouse PD-1 (CD279).

[0196] 3. Preparation method

[0197] Anlotinib hydrochloride and lenvatinib mesylate are prepared and diluted to the required concentration with distilled water; mPD-1 is diluted to the required concentration with normal saline and prepared immediately before use.

[0198] 4. Cells

[0199] Culture H22 cells in a 10-cm culture dish, and the culture conditions are RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin, and culture in an incubator at 37°C with 5% CO2 air. Passage twice a week. When the cells are in exponential growth, collect the cells, count them, and inoculate.

[0200] 5. Experimental animals

[0201] KM mice, 6 - 7 weeks old, female, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. Production license number: SCXK (Shanghai) 2013 - 0006; Animal certificate number No. 311620400013790. Breeding environment: SPF level.

[0202] The use and welfare of the experimental animals in this experiment are carried out in accordance with the regulations of the "Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)". Monitor the health status and death of the animals every day, and routine inspections include observing the effects of the test substance or drug on the daily behavior of the animals, such as behavioral activities, weight changes, and appearance signs.

[0203] 6. Experimental indicators

[0204] The experimental indicators are to investigate the effect of the drug on tumor growth, and the specific indicators are T / C (%) or tumor growth inhibition rate TGI (%).

[0205] Measure the tumor diameter with a vernier caliper twice a week. The formula for calculating the tumor volume (V) is as follows:

[0206] The formula for calculating the tumor volume (V) is as follows:

[0207] V = 1 / 2 × a × b 2 where a and b represent the length and width respectively.

[0208] T / C(%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment in the test group and the control group animals respectively; T0 and C0 are the tumor volumes at the start of the experiment in the test group and the control group animals respectively.

[0209] Tumor growth inhibition rate (TGI)(%) = 100 - T / C(%).

[0210] When the tumor shows regression, Tumor growth inhibition rate (TGI)(%) = 100 - (T - T0) / T0 × 100

[0211] If the tumor shrinks compared to the initial volume, i.e., T < T0 or C < C0, it is defined as partial regression (PR) of the tumor; if the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0212] 7. Experimental implementation

[0213] Subcutaneously inoculate each mouse with H22 cells. When the tumor grows to 100 - 200 mm 3 , randomly divide the mice into groups and administer drugs; administer drugs to the mice by gavage (i.g.) or intraperitoneal injection (IP); the solvent group is given the same volume of distilled water by gavage; the drug administration volume is 0.1 mL / 10 g body weight; the specific drug administration doses and regimens are shown in Table 4.

[0214] Table 4 Drug administration doses and regimens

[0215]

[0216] Note: Random grouping, the first drug administration time is D0; IP: Intraperitoneal injection; i.g., Gavage administration; QD, once a day.

[0217] After the experiment ends, or when the tumor volume of the animal reaches the euthanasia endpoint of 1500 mm 3 , euthanize the animals by carbon dioxide inhalation, and then dissect to take the tumor tissue and weigh and photograph it.

[0218] 8. Experimental results

[0219] Anlotinib hydrochloride (1 mg / kg, i.g., QD×17) was able to inhibit the growth of subcutaneous transplanted tumors of mouse liver cancer H22, and the tumor inhibition rate was 61.7%; the tumor inhibition rate of lenvatinib mesylate (5 mg / kg, i.g., QD×17) against subcutaneous transplanted tumors of H22 was 79%; the tumor inhibition rate of mPD-1 (1 mg / kg, IP, twice a week for a total of 5 times) against H22 was 29.3%, and 2 / 10 tumors completely regressed; the combined use of anlotinib hydrochloride and mPD-1 increased the tumor inhibition rate to 79.5%, with 1 / 10 tumors partially regressing and 1 / 10 tumors completely regressing; the combined use of lenvatinib mesylate and mPD-1 had a tumor inhibition rate of 63.4% against H22, with 1 / 10 tumors partially regressing. Tumor-bearing mice could well tolerate the combined use of anlotinib hydrochloride and mPD-1, and no obvious symptoms such as weight loss occurred. In comparison, the combined use of anlotinib hydrochloride and mPD-1 had a stronger therapeutic effect on subcutaneous transplanted tumors of liver cancer H22 than the single-agent therapies of anlotinib hydrochloride or mPD-1. The results are shown in Table 5.

[0220] Table 5 Therapeutic effects on subcutaneous transplanted tumors of mouse liver cancer H22

[0221]

[0222] The tumor conditions of mice in each dosing group were analyzed, and the results were as follows.

[0223] As Figure 1 shown, from D0 to D17 after dosing, the average tumor volume of mice in each dosing group increased with the increase in the number of dosing days, but the growth rates of each group were different. The average tumor volume of mice in the solvent control group increased the fastest, and the volume was the largest on D17. The average tumor volume on D17 in other groups was as follows: mPD-1 (1 mg / kg) > anlotinib hydrochloride (1 mg / kg) > lenvatinib mesylate (5 mg / kg) + mPD-1 (1 mg / kg) > anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg) > lenvatinib mesylate (5 mg / kg).

[0224] As Figure 2 shown, from D0 to D17 after dosing, the individual tumor growth conditions of mice in each group were Figure 1 relatively consistent. The solvent control group showed a rapid growth trend, and the growth of anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg) was the gentlest.

[0225] As Figure 3As shown, at the end of the experiment (D17), the individual tumor volumes and tumor weights of mice in each dose group were as follows: solvent control group > mPD-1 (1 mg / kg) > lenvatinib mesylate (5 mg / kg) + mPD-1 (1 mg / kg) > anlotinib hydrochloride (1 mg / kg) > lenvatinib mesylate (5 mg / kg) > anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg).

[0226] As Figure 4 shown, during the administration period from D0 to D17, the body weights of mice in the solvent control group and the mPD-1 group increased steadily. There were no obvious symptoms such as significant weight loss in the groups of lenvatinib mesylate (5 mg / kg) + mPD-1 (1 mg / kg), anlotinib hydrochloride (1 mg / kg), lenvatinib mesylate (5 mg / kg), and anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg).

[0227] As Figure 5 shown, after the administration ended, the mice were dissected to remove the tumors, and it was found that the tumor growth situation was consistent with Figure 1 the results.

[0228] Lenvatinib mesylate (5 mg / kg, i.g., QD 17) significantly inhibited the growth of subcutaneous transplanted tumors of mouse liver cancer H22. Anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg) could achieve basically the same tumor inhibitory effect, and in terms of the tumor growth inhibitory effect, it was superior to lenvatinib mesylate, but the dosage used could be greatly reduced, which was beneficial to reducing the risk of drug side effects; mPD-1 had a certain therapeutic effect on H22, but when anlotinib hydrochloride was combined with mPD-1, the tumor inhibition rate of H22 could be significantly increased, and the tumor-bearing mice could show good tolerance to the combined drugs.

[0229] Example 3 Preparation of Anti-PD-1 Monoclonal Antibody

[0230] Establishment of PD-1 Hybridoma Cell Lines

[0231] 1. Prepare the fusion protein PD-1 with the corresponding amino acid sequence according to the molecular biology method. Use the PD-1 fusion protein prepared by the above method as an antigen and emulsify it with Freund's adjuvant to immunize BALB / C mice. After the mice generate an immune response, take their spleen cells and fuse them with mouse myeloma cells to form hybridoma cells, and culture the formed hybridoma cells using a 96-well plate.

[0232] 2. For the antibodies secreted by each hybridoma cell line, coat an enzyme-linked immunosorbent assay (ELISA) plate with PD-1-hFc as an antigen, block the ELISA plate with PBS buffer containing 1% BSA, and use the coated ELISA plate for screening hybridoma cells that secrete new antibodies that specifically bind to PD-1 by the indirect ELISA method.

[0233] 3. By competitive ELISA, hybridoma cells that can secrete monoclonal antibodies that competitively bind to PD-1 with PD-L1 were screened from the hybridoma cells obtained by indirect ELISA.

[0234] 4. The screened cell line was subcloned to obtain a monoclonal stable cell line that secretes antibodies that specifically bind to PD-1 competitively with PD-L1. The specific method is as follows: The cells to be subcloned were counted for viable cells, and according to the number of viable cells, they were diluted and inoculated into a 96-well cell culture plate with IMDM medium containing 15% fetal bovine serum for culture. The theoretical inoculation cell density was 1 cell / well. After the cells grew into monoclonal cell clusters, they were screened by ELISA method. After multiple subclonings and screenings, a stable monoclonal cell line was obtained.

[0235] 5. After obtaining the stable cell line, the stable cell line was cultured with low IgG fetal bovine serum containing 10%. After 7-10 days of culture, the cell culture supernatant was collected for antibody purification to obtain the corresponding anti-PD-1 antibody.

[0236] Humanization design of antibody

[0237] To construct a humanized antibody, the amino acid sequences of the variable regions of the murine antibody were aligned with the gene sequences of the human variable regions. By selectively mutating some murine amino acid sequences to humanized amino acid sequences, multiple humanized antibodies were designed respectively.

[0238] The heavy chain variable region sequence of humanized antibody 1 is:

[0239] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKSLEWVATISGGGSDT YYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARQLNYAWFAYWGQGTLVTVSA AKTTPPSVYRSSKGNSSTLAAVTS(SEQ ID NO:1)

[0240] The light chain variable region sequence of humanized antibody 1 is:

[0241] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYTSSNQGS GVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIKR(SEQ IDNO:4)

[0242] The heavy chain variable region sequence of humanized antibody 2 is:

[0243] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISGGGSDT YYPDSVKGRFTISRDNSKNNLYLQMSSLRAEDTAVYYCARQLNYAWFAYWGQGTLVTVSS(SEQ ID NO:2)

[0244] The light chain variable region sequence of humanized antibody 2 is:

[0245] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYTSSNQGT GVPARFSGSGSGTDFTLNINPMEADDTAMYFCQQSKEVPWTFGGGTKLEIK(SEQ ID NO:5) The heavy chain variable region sequence of humanized antibody 3 is:

[0246] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISGGGSDT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQLNYAWFAYWGQGTLVTVSS(SEQ ID NO:3)

[0247] The light chain variable region sequence of humanized antibody 3 is:

[0248] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWYQQKPGQPPKLLIYTSSNKDT GVPARFSGSGSGTDFTLTINPMEAEDTAVYYCQQSKEVPWTFGGGTKLEIK(SEQ ID NO:6) The heavy chain variable region sequence of humanized antibody 4 is:

[0249] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYT YYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA(SEQ ID NO:7)

[0250] The light chain variable region sequence of humanized antibody 4 is:

[0251] The heavy chain variable region sequence of humanized antibody 5 is: DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLEL (SEQ ID NO:9)

[0252] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYY PDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO:8)

[0253] The light chain variable region sequence of humanized antibody 5 is:

[0254] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO:10) The heavy chain variable region sequence of humanized antibody 6 is:

[0255] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPEKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYW GQGTLVTVSS (SEQ ID NO:11)

[0256] The light chain variable region sequence of humanized antibody 6 is:

[0257] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNIHPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO:14)

[0258] The heavy chain variable region sequence of humanized antibody 7 is:

[0259] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRD TYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVS S(SEQ ID NO:12)

[0260] The light chain variable region sequence of humanized antibody 7 is:

[0261] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGT GVPARFSGSGSGTDFTLNINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK(SEQ ID NO:15)

[0262] The heavy chain variable region sequence of humanized antibody 8 is:

[0263] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRD TYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQKGEAWFAYWGQGTLVTVS S(SEQ ID NO:13)

[0264] The light chain variable region sequence of humanized antibody 8 is:

[0265] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWYQQKPGQPPKLLIYAASNKA TGVPARFSGSGSGTDFTLNINPMEANDTAVYFCQQSKEVPWTFGGGTKLEIK(SEQ ID NO:16)

[0266] The heavy chain sequence of humanized antibody 8A is:

[0267] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHHYTQKSLSLSLGK(SEQ ID NO: 17)

[0268] The light chain sequence of the humanized antibody 8A is:

[0269] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 18)

[0270] The heavy chain variable region sequence of the humanized antibody 9 is:

[0271] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSN GGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTT VTVSS(SEQ ID NO: 31)

[0272] The light chain variable region sequence of the humanized antibody 9 is:

[0273] The heavy chain sequence of humanized antibody 9 is: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK(SEQ ID NO:34)

[0274] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:37)

[0275] The light chain sequence of humanized antibody 9 is:

[0276] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:40)

[0277] The heavy chain variable region sequence of humanized antibody 10 is:

[0278] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGS KRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS(SEQ ID NO:32)

[0279] The light chain variable region sequence of humanized antibody 10 is:

[0280] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK(SEQ ID NO:35) The heavy chain sequence of humanized antibody 10 is:

[0281] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:38)

[0282] The light chain sequence of humanized antibody 10 is:

[0283] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:41)

[0284] The heavy chain variable region sequence of humanized antibody 11 is:

[0285] EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRD TYFADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSS(SEQ ID NO:33)

[0286] The light chain variable region sequence of humanized antibody 11 is:

[0287] DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPS RFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFR(SEQ ID NO:36) The heavy chain sequence of humanized antibody 11 is:

[0288] EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:39)

[0289] The light chain sequence of the humanized antibody 11 is as follows:

[0290] DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:42)

[0291] The nucleic acid sequence encoding the above humanized antibody was synthesized by total gene synthesis and constructed into an expression vector. The expression vector DNA was extracted and transfected into mammalian cells, 293 cells. After cell transfection, the antibody was expressed in mammalian cells and secreted extracellularly. The expressed antibody was purified through an antibody A affinity chromatography column to obtain the humanized antibody protein. These experimental operations are all conventional operations known in the art.

[0292] Clinical study of 14C12H1L1 combined with anlotinib hydrochloride for first-line treatment of unresectable hepatocellular carcinoma

[0293] This example discloses the results of an open-label, multi-center Ib / II phase clinical study on the first-line treatment of unresectable hepatocellular carcinoma with 14C12H1L1 combined with anlotinib hydrochloride.

[0294] This study is an open-label, multi-center Ib / II phase clinical study, aiming to evaluate the efficacy and safety of 14C12H1L1 combined with anlotinib in the first-line treatment of unresectable hepatocellular carcinoma.

[0295] Primary endpoint: Objective response rate (ORR) evaluated according to RECISIT v1.1.

[0296] Secondary endpoints: (1) Disease control rate (DCR), duration of response (DOR), time to response (TTR), progression-free survival (PFS), and time to progression (TPP) evaluated by the investigator according to RECISIT v1.1; (2) Overall survival (OS); (3) Safety assessment: Incidence and severity of adverse events (AEs), and clinically significant abnormal laboratory test results.

[0297] Progression-free survival (PFS): Defined as the time from the first dose until objective tumor progression or death.

[0298] Overall survival (OS): Defined as the time from the first dose until death due to any cause. For subjects lost to follow-up, the last follow-up time is usually counted as the time of death, in days.

[0299] Duration of response (DOR): Defined as the time from the first assessment of CR or PR until the first assessment of PD or death.

[0300] Time to response (TTR): The time from the first administration of the drug to the first assessment of CR or PR in the subject.

[0301] Investigational drug

[0302] 14C12H1L1 injection, 100mg / 10ml solution, manufacturer: Kangfang Tiancheng (Guangdong) Pharmaceutical Co., Ltd., stored in the dark at 2 - 8°C.

[0303] Anlotinib hydrochloride capsule (Fukewei), 8mg / capsule, manufacturer: Chia Tai Tianqing Pharmaceutical Group Co., Ltd., sealed and stored in the dark.

[0304] Drug administration

[0305] Drug administration plan and dose selection

[0306] 14C12H1L1 combined with anlotinib, wherein 14C12H1L1: 200 mg, intravenous injection Q3W; anlotinib: once daily, 8 mg each time, take the drug for 14 days and stop for 7 days, and 21 days is a cycle.

[0307] Early research results

[0308] Therapeutic effect

[0309] As of July 15, 2019, a total of 13 subjects had received at least one tumor evaluation, and the median treatment cycle was 3 times. Among these patients, 3 achieved partial remission (PR), the objective response rate (ORR) was 23.1% (3 / 13), and the disease control rate (DCR) was 69.2% (9 / 13)( Figure 6 , Figure 7 ).

[0310] Adverse events

[0311] As of June 27, 2019, the incidence of treatment-related adverse events (TRAE) was 75%, and the TRAE of grade 3 and above was 12.5%. The results showed that the incidence of adverse events of 14C12H1L1 combined with anlotinib hydrochloride in the first-line treatment of unresectable hepatocellular carcinoma (see Table 6) was significantly lower than that of anlotinib monotherapy (data not shown) or 14C12H1L1 monotherapy (data not shown).

[0312] Table 6 Adverse events of 14C12H1L1 combined with anlotinib

[0313]

[0314] The exemplary embodiments described in this application can be described by the following numbered paragraphs:

[0315] 1. A drug combination comprising:

[0316] a) An inhibitor of the interaction between PD-1 and its ligand PD-L1, and

[0317] b) A tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof,

[0318]

[0319] 2. The drug combination according to paragraph 1, wherein the pharmaceutically acceptable salt of the compound of formula I is the hydrochloride salt, preferably the dihydrochloride salt, of 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropanamine.

[0320] 3. The pharmaceutical combination according to any one of paragraphs 1-2, wherein the inhibitor of the interaction between PD-1 and its ligand PD-L1 is an anti-PD-1 or anti-PD-L1 antibody, or an antigen-binding fragment thereof.

[0321] 4. The pharmaceutical combination according to paragraph 3, wherein the anti-PD-1 or anti-PD-L1 antibody is a monoclonal antibody against PD-1 or PD-L1.

[0322] 5. The pharmaceutical combination according to any one of paragraphs 3-4, wherein the anti-PD-1 antibody can be selected from any one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, 14C12H1L1, genolimab, livzon mab, HLX-10, BAT-1306, AK103, AK104, CS1003, SCT-I10A, F520, SG001, GLS-010.

[0323] 6. The pharmaceutical combination according to any one of paragraphs 3-4, wherein the anti-PD-L1 antibody can be selected from any one or more of atezolizumab, avelumab, durvalumab, KL-A167, SHR-1316, BGB-333, JS003, STI-A1014 (ZKAB0011), KN035, MSB2311, HLX-20, CS-1001.

[0324] 7. The pharmaceutical combination according to any one of paragraphs 3-4, wherein the anti-PD-1 antibody comprises:

[0325] a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33, or an amino acid sequence having at least 80% identity thereto; and

[0326] a light chain variable region containing an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36, or an amino acid sequence having at least 80% identity thereto.

[0327] 8. The drug combination according to paragraph 7, wherein the anti-PD-1 antibody comprises:

[0328] a. A heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:4;

[0329] b. A heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:5;

[0330] c. A heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:6;

[0331] d. A heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:9;

[0332] e. A heavy chain variable region as shown in SEQ ID NO:8 and a light chain variable region as shown in SEQ ID NO:10;

[0333] f. A heavy chain variable region as shown in SEQ ID NO:11 and a light chain variable region as shown in SEQ ID NO:14;

[0334] g. A heavy chain variable region as shown in SEQ ID NO:12 and a light chain variable region as shown in SEQ ID NO:15;

[0335] h. A heavy chain variable region as shown in SEQ ID NO:13 and a light chain variable region as shown in SEQ ID NO:16;

[0336] i. A heavy chain as shown in SEQ ID NO:17 and a light chain as shown in SEQ ID NO:18;

[0337] j. A heavy chain variable region as shown in SEQ ID NO:31 and a light chain variable region as shown in SEQ ID NO:34;

[0338] k. A heavy chain variable region as shown in SEQ ID NO:32 and a light chain variable region as shown in SEQ ID NO:35;

[0339] l. A heavy chain variable region as shown in SEQ ID NO:33 and a light chain variable region as shown in SEQ ID NO:36;

[0340] m. A heavy chain as shown in SEQ ID NO:37 and a light chain as shown in SEQ ID NO:40;

[0341] n. A heavy chain as shown in SEQ ID NO:38 and a light chain as shown in SEQ ID NO:41; or

[0342] o. A heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:42.

[0343] 9. The pharmaceutical combination according to any one of paragraphs 1 - 8, wherein the pharmaceutical combination is a fixed combination.

[0344] 10. The pharmaceutical combination according to paragraph 9, wherein the fixed combination is in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition.

[0345] 11. The pharmaceutical combination according to any one of paragraphs 1 - 8, wherein the pharmaceutical combination is a non - fixed combination.

[0346] 12. The pharmaceutical combination according to paragraph 11, wherein the anti - PD - 1 antibody and the compound of formula I or its pharmaceutically acceptable salt in the non - fixed combination are each in the form of a pharmaceutical composition.

[0347] 13. The pharmaceutical combination according to any one of paragraphs 1 - 12, comprising: a compound of formula I or its hydrochloride; and sintilimab or its antigen - binding fragment, InVivoMAb anti - mouse PD - 1 monoclonal antibody or its antigen - binding fragment, or 14C12H1L1 or its antigen - binding fragment.

[0348] 14. Use of the pharmaceutical combination according to any one of paragraphs 1 - 13 for the treatment or prevention of liver malignancies.

[0349] 15. The use according to paragraph 14, wherein the pharmaceutical combination is used for the treatment of primary liver tumors or secondary liver tumors.

[0350] 16. The use according to paragraph 14, wherein the liver malignancy is hepatocellular carcinoma.

[0351] 17. The use according to paragraph 14, wherein the liver malignancy is metastatic liver cancer.

[0352] 18. The use according to paragraph 17, wherein the metastatic liver cancer is a metastatic cancer metastasized from lung cancer, gastric cancer, rectal cancer, colon cancer, large intestine cancer, pancreatic cancer or breast cancer.

[0353] 19. Use of a combination of an inhibitor of the interaction between PD - 1 and its ligand PD - L1 and a compound of formula I or its pharmaceutically acceptable salt for the prevention or treatment of cancer.

[0354] 20. A pharmaceutical combination for preventing or treating cancer or tumor, wherein the pharmaceutical combination comprises a compound of formula I or a pharmaceutically acceptable salt thereof, and an inhibitor of the interaction between PD-1 and its ligand PD-L1.

[0355] 21. The use according to any one of paragraphs 19-20, wherein the inhibitor of the interaction between PD-1 and its ligand PD-L1 is an anti-PD-1 or anti-PD-L1 monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to human PD-1 or PD-L1 and blocks the binding of human PD-L1 to human PD-1.

[0356] 22. The use according to any one of paragraphs 19-21, wherein the cancer or tumor is selected from liver tumors (such as liver malignancies, such as hepatocellular carcinoma) or lung tumors (such as lung cancer, such as non-small cell lung cancer).

[0357] 23. A method for treating cancer in a subject, which comprises administering to the subject a therapeutically effective amount of a pharmaceutical combination comprising:

[0358] An inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1, and

[0359] A tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

[0360] 24. The method according to paragraph 23, wherein the inhibitor of the interaction between PD-1 and its ligand PD-L1 is an anti-PD-1 or anti-PD-L1 antibody, or an antigen-binding fragment thereof.

[0361] 25. The method according to 24, wherein the anti-PD-1 antibody can be selected from any one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, 14C12H1L1, genolimab, livzumab, HLX-10, BAT-1306, AK103, AK104, CS1003, SCT-I10A, F520, SG001, GLS-010.

[0362] 26. The method according to 24, wherein the anti-PD-L1 antibody can be selected from any one or more of atezolizumab, avelumab, durvalumab, KL-A167, SHR-1316, BGB-333, JS003, STI-A1014, KN035, MSB2311, HLX-20, CS-1001.

[0363] 27. The method according to 24, wherein the anti-PD-1 antibody comprises:

[0364] A heavy chain variable region comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33, or an amino acid sequence having at least 80% identity thereto; and

[0365] A light chain variable region comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36, or an amino acid sequence having at least 80% identity thereto.

[0366] 28. The method according to any one of 23-27, wherein an inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is administered approximately once a week (q1w), approximately once every 2 weeks (q2w), approximately once every 3 weeks (q3w), or approximately once every 4 weeks (q4w).

[0367] 29. The method according to any one of 23-28, wherein the method results in an objective response, preferably a complete response or a partial response.

[0368] 30. The method according to any one of 23-29, wherein the subject has not previously received systemic chemotherapy.

[0369] 31. The method according to any one of 23-30, wherein the subject has not previously received systemic chemotherapy, but has received one or more of surgical treatment, radiotherapy, induction chemotherapy and / or adjuvant chemotherapy, or the subject has received concurrent chemotherapy.

[0370] 32. The method according to any one of 23-30, wherein the subject has previously received systemic chemotherapy.

[0371] 33. The method according to any one of 23-32, wherein the cancer is driver gene negative.

[0372] 34. The method according to 33, wherein the driver gene negative means that one, two or three of the EGFR, ALK and ROS1 genes of the subject are mutation negative.

[0373] 35. The method according to any one of claims 26 - 32, wherein the EGFR, ALK, and ROS1 genes in the subject are all wild-type genes or all mutation-negative.

[0374] 36. The method according to any one of claims 26 - 35, wherein the cancer is lung cancer.

[0375] 37. The method according to any one of claims 23 - 36, wherein the cancer is recurrent and / or metastatic lung cancer.

[0376] 38. The method according to any one of claims 23 - 37, wherein the cancer is advanced lung cancer.

[0377] 39. The method according to any one of claims 23 - 38, wherein the cancer is small cell or non-small cell lung cancer.

[0378] 40. The method according to any one of claims 23 - 39, wherein the cancer treatment is first-line treatment for recurrent or metastatic non-small cell lung cancer.

[0379] 41. The method according to any one of claims 39 - 40, wherein the non-small cell lung cancer is lung adenocarcinoma, lung squamous cell carcinoma, or large cell lung cancer.

[0380] 42. The method according to any one of paragraphs 26 - 35, wherein the cancer is primary liver tumor or secondary liver tumor.

[0381] 43. The method according to paragraph 41, wherein the liver malignancy is hepatocellular carcinoma.

[0382] 44. The method according to paragraph 41, wherein the liver malignancy is metastatic liver cancer.

[0383] 45. The method according to paragraph 44, wherein the metastatic liver cancer is metastatic cancer metastasized from lung cancer, gastric cancer, rectal cancer, colon cancer, large intestine cancer, pancreatic cancer, or breast cancer.

[0384] 46. A drug combination for preventing or treating cancer or tumor, wherein the drug combination comprises a tyrosine kinase inhibitor and an inhibitor of the interaction between PD-1 and its ligand PD-L1, and wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

[0385] 47. The method or drug combination according to any one of claims 23 - 46, wherein the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 and the tyrosine kinase inhibitor are each in a pharmaceutical composition and can be administered simultaneously, sequentially, or at intervals.

[0386] 48. The method or pharmaceutical combination according to any one of 23 - 47, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is administered according to a treatment cycle of administering for 2 weeks (14 days) and then discontinuing for 1 week (7 days).

[0387] 49. The method or pharmaceutical combination according to 48, wherein the pharmaceutical combination administered in each treatment cycle contains about 84 - 168 mg of the compound of formula I or a pharmaceutically acceptable salt thereof.

[0388] 50. The method or pharmaceutical combination according to 48 or 49, wherein the pharmaceutical combination contains a compound of formula I or a pharmaceutically acceptable salt thereof selected from about 84 mg, about 112 mg, about 140 mg, about 168 mg, or within the range formed by any of the above values.

[0389] 51. The method or pharmaceutical combination according to 50, wherein the pharmaceutical combination contains about 112 mg of the compound of formula I or a pharmaceutically acceptable salt thereof.

[0390] 52. The method or pharmaceutical combination according to 50, wherein the pharmaceutical combination contains about 140 mg of the compound of formula I or a pharmaceutically acceptable salt thereof.

[0391] 53. The method or pharmaceutical combination according to 50, wherein the pharmaceutical combination contains about 168 mg of the compound of formula I or a pharmaceutically acceptable salt thereof.

[0392] 54. The method or pharmaceutical combination according to 50, wherein about 8 mg, 10 mg, or about 12 mg of the compound of formula I or a pharmaceutically acceptable salt thereof is administered daily, administering for 2 weeks and then discontinuing for 1 week.

[0393] 55. The method or pharmaceutical combination according to any one of 23 - 54, wherein about 100 - 600 mg of an anti - PD - 1 antibody is administered approximately once every 2 weeks (q2w) or approximately once every 3 weeks (q3w).

[0394] 56. The method or pharmaceutical combination according to any one of 23 - 55, wherein about 200 mg of an anti - PD - 1 antibody is administered approximately once every 2 weeks (q2w).

[0395] 57. The method or pharmaceutical combination according to any one of 23 - 56, wherein about 200 mg of an anti - PD - 1 antibody is administered approximately once every 3 weeks (q3w).

[0396] 58. A drug pack, which contains separately packaged pharmaceutical compositions in separate containers, wherein one container contains a pharmaceutical composition containing a compound of formula I or a pharmaceutically acceptable salt thereof, and the second container contains a pharmaceutical composition containing an inhibitor of the interaction between the PD - 1 receptor and its ligand PD - L1.

[0397] 59. The pharmaceutical package according to claim 58, wherein the drug comprises 84 - 168 mg of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0398] 60. The pharmaceutical package according to claim 59, wherein the drug comprises a compound of formula I or a pharmaceutically acceptable salt thereof selected from 84 mg, 112 mg, 140 mg, 168 mg or any value within the range formed by the above values.

[0399] 61. The pharmaceutical package according to claim 60, wherein the drug comprises 112 mg - 168 mg of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0400] 62. A kit for treating liver tumors, which contains (a) a first pharmaceutical composition containing an inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 as an active ingredient; and (b) a second pharmaceutical composition containing a compound of formula I or a pharmaceutically acceptable salt thereof as an active ingredient.

[0401] 63. The kit according to claim 62, wherein the kit comprises 84 - 168 mg of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0402] 64. The kit according to claim 63, wherein the kit comprises a compound of formula I or a pharmaceutically acceptable salt thereof selected from 84 mg, 112 mg, 140 mg, 168 mg or any value within the range formed by the above values.

[0403] 65. The kit according to claim 64, wherein the kit comprises 112 mg - 168 mg of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0404] 66. A unit dosage form, wherein the unit dosage form comprises: a compound component, 6 - 12 mg of a compound of formula I or its hydrochloride; and an antibody component, 50 - 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof; wherein the compound component and the antibody component are separately packaged.

[0405] 67. A method for preventing or treating cancer or tumor, wherein one or more unit dosage forms described in paragraph 66 are administered to a subject in need thereof.

[0406] 68. The method according to claim 67, wherein the cancer or tumor is selected from liver tumors (such as liver malignancies, such as hepatocellular carcinoma) or lung tumors (such as lung cancer, such as non-small cell lung cancer).

[0407] In accordance with the disclosure of the present application, although the compositions and methods of the present application are described according to preferred embodiments, those skilled in the art can make changes to the compositions and / or methods described herein and the steps or the order of the steps of the methods without departing from the concepts, spirit and scope of the present application.

[0408] The disclosures of all documents cited herein are incorporated herein by reference to the extent that they provide exemplary, procedural and other details to supplement the content described herein.

Claims

1. Use of an anti-PD-1 antibody in the preparation of a medicament for combined treatment of liver cancer with anlotinib hydrochloride, wherein the anti-PD-1 antibody comprises a heavy chain variable region shown in SEQ ID NO:8 and a light chain variable region shown in SEQ ID NO:10, and the anti-PD-1 antibody is an injection.

2. The use according to claim 1, wherein the anti-PD-1 antibody is 14C12H1L1.

3. The use according to claim 1, wherein the liver cancer is primary liver tumor or secondary liver tumor.

4. The use according to claim 1, wherein the liver cancer is hepatocellular carcinoma.

5. The use according to any one of claims 1-4, wherein the anti-PD-1 antibody and anlotinib hydrochloride are each in a pharmaceutical composition and can be administered simultaneously, sequentially or at intervals.

6. The use according to claim 5, wherein the anlotinib hydrochloride is administered according to a treatment cycle of 2 weeks of administration followed by 1 week of rest.

7. The use according to claim 6, wherein 84-168 mg of anlotinib hydrochloride is administered in each treatment cycle.

8. The use according to claim 5, wherein 8 mg, 10 mg or 12 mg of anlotinib hydrochloride is administered daily, with 2 weeks of administration followed by 1 week of rest.

9. The use according to claim 5, wherein 100-600 mg of the anti-PD-1 antibody is administered once every 2 weeks or once every 3 weeks.

10. The use according to claim 9, wherein 200 mg of the anti-PD-1 antibody is administered once every 2 weeks or once every 3 weeks.

11. The use according to claim 10, wherein 200 mg of the anti-PD-1 antibody is administered once every 3 weeks.

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