Pharmaceutical combinations of quinoline derivatives and antibodies

Abstract

The invention provides a pharmaceutical composition of a quinoline derivative and an antibody, the pharmaceutical composition comprises an immune checkpoint inhibitor and a tyrosine kinase inhibitor, and the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof. The pharmaceutical composition provided by the invention has good anti-lung tumor and liver tumor activity.

Description

[0001] This application is a divisional application of the patent application with the application number 202310003034.5 and the application name "Pharmaceutical combination of quinoline derivatives and antibodies", the filing date of which is July 18, 2019. TECHNICAL FIELD

[0002] The present application belongs to the technical field of medicine, and relates to a combination therapy that can be used for anti-tumor. Specifically, the present application relates to a combination based on quinoline derivatives and antibodies and its use in anti-tumor. BACKGROUND

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

[0004] Anlotinib is a quinoline derivative tyrosine kinase inhibitor, which acts as a multi-target tyrosine kinase inhibitor (TKI) in affecting tumor angiogenesis and proliferation signaling, and the main targets include: receptor tyrosine kinase 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) alpha and beta, and stem cell factor receptor (SCFR) 7, 8 and 9. A phase 2 trial showed that anlotinib can improve the progression-free survival and has potential benefits for overall survival (Han B, et al. Br J Cancer. 2018; 118(5): 654-661). A multi-center, double-blind, phase 3 randomized clinical trial showed that in Chinese patients, anlotinib resulted in prolonged overall survival and progression-free survival, which indicated that anlotinib is 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] The document WO2008112407 discloses a tyrosine kinase inhibitor of quinoline derivatives 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl] cyclopropylamine and its preparation method in Example 24, and its structural formula is shown as formula I:

[0006]

[0007] The innate immune system, which includes T lymphocytes, has a powerful anticancer capacity with a broad capacity and exquisite specificity to respond to a wide variety of tumor antigens. Emerging cancer immunotherapies enhance the anti-tumor immune response through the adoptive transfer of activated effector cells, immunization against relevant antigens, or the provision of non-specific immune stimulants. In the past nearly 20 years, researchers have worked to develop specific immune checkpoint inhibitors and hope to provide new immunotherapy regimens for the treatment of cancer, including the development of antibodies that bind to and inhibit CTLA-4 (Antibody) Ipilimumab for the treatment of 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, ), which specifically bind to the 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 and B lymphocytes and mediates immune suppression, and its ligand includes at least PD-L1 and PD-L2. PD-L1 (Programmed death-ligand 1), also known as CD274 or B7-H1, is a 40 kDa type 1 transmembrane protein encoded by the CD274 gene, which is a ligand for PD-1. PD-L1 and PD-1 both belong to the immunoglobulin superfamily and both consist of two extracellular Ig domains, i.e. N-terminal V domain and C-terminal constant domain. The binding interface of PD-L1 with programmed death receptor-1 (PD-1) and B7-1 (CD80) is 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, i.e. immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). After T cell stimulation, PD-1 recruits tyrosine phosphatase SHP-2 to the ITSM motif in its cytoplasmic tail, resulting in dephosphorylation of effector molecules involved in CD3+ T cell signaling cascade, such as CD3ζ, PKCθ and ZAP70 (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 many tumor cells, and the malignant degree and poor prognosis of tumors are closely related to the expression level of PD-L1. There is clinical data showing that high tumor expression of PD-L1 is associated with increased tumor invasiveness and poor prognosis.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 failure or anergy among cognate antigen-specific T cells (Hofmeyer et al. (2011) Journal of Biomedicine and Biotechnology 2011: 1-9); promotes Th1 cell differentiation into Foxp3+regulatory T cells (Armanath et al. (2011) Science Trans Med 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 leads to upregulated T cell responses and generation of autoreactive T cells (Latchman et al. (2004) PNAS 101: 10691-10696). Antibody blockade of PD-1 or PD-L1 leads to increased antitumor immunity (Iwai et al. (2002) PNAS 99: 12293-12297).

[0008] The biggest challenge encountered by the prior art in the process of tumor immunotherapy is poor efficacy due to tumor immune tolerance and escape. Therefore, it has important theoretical significance and application value to break the immune tolerance of the body to tumor cells by using the combination of small molecule anti-tumor compounds and anti-PD-1 / PD-L1 antibodies. SUMMARY

[0009] It is at least an object of the present application to provide a pharmaceutical combination comprising a tyrosine kinase inhibitor, and an inhibitor of the interaction between PD-1 and its ligand PD-L1. In some aspects, the tyrosine kinase inhibitor is a compound of Formula I or a pharmaceutically acceptable salt thereof, and in some specific embodiments, the tyrosine kinase inhibitor is a hydrochloride salt of the compound of Formula I, i.e., 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 antigen-binding portion thereof that binds to Programmed Death Receptor 1 (PD-1) and / or inhibits the activity of PD-1, or is an antibody or antigen-binding portion thereof that binds to Programmed Death Ligand 1 (PD-L1) and / or inhibits the activity of PD-L1, e.g., is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the antibody or antigen-binding portion thereof is (a) a monoclonal antibody or 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 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 or murine antibody.

[0013] In some embodiments, the anti-PD-1 antibody can be selected from any one or more of Nivolumab, Pembrolizumab, Durvalumab, Toripalimab (JS-001), Cemiplimab (IBI308), Camrelizumab, Tislelizumab (BGB-A317), 14C12H1L1 (Kang Biotech), Genolimzumab (GB226), Lizumab (LZM009), HLX-10, BAT-1306, AK103 (HX008), AK104 (Kang Biotech), CS1003, SCT-I10A, F520, SG001, GLS-010.

[0014] In some embodiments, 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.

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

[0016] a heavy chain variable region comprising an amino acid sequence selected from the group consisting of 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 at least 80% identical, preferably at least 85% identical, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto; and

[0017] a light chain variable region comprising an amino acid sequence selected from the group consisting of 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 at least 80% identical, preferably at least 85% identical, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto.

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

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

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

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

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

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

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

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

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

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

[0028] i. a heavy chain as depicted in SEQ ID NO: 17 and a light chain as depicted in SEQ ID NO: 18;

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

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

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

[0032] m. a heavy chain as depicted in SEQ ID NO: 37 and a light chain as depicted in SEQ ID NO: 40;

[0033] n. a heavy chain as depicted in SEQ ID NO: 38 and a light chain as depicted in SEQ ID NO: 41; or

[0034] o. a heavy chain as depicted in SEQ ID NO: 39 and a light chain as depicted in SEQ ID NO: 42.

[0035] In some aspects, the anti-PD-1 antibody is an isolated antibody or antibody fragment that binds PD-1, comprising CDR sequences derived from the 6F5 antibody, which 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 aspects, the anti-PD-1 antibody is an isolated antibody or antibody fragment that binds PD-1 comprising 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 aspects, the compound of Formula I can be present in a pharmaceutically acceptable salt or a pharmaceutically acceptable formulation thereof, preferably in the form of a hydrochloride salt thereof.

[0050] In some 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, i.e., anlotinib hydrochloride.

[0051] In some aspects, the pharmaceutical combination comprises: a compound of Formula I or a hydrochloride salt (e.g., dihydrochloride salt) thereof; and sintyrosin or an antigen binding fragment thereof, InVivoMAb anti-mouse PD-1 monoclonal antibody or an antigen binding fragment thereof, or 14C12H1L1 or an antigen binding fragment thereof.

[0052] It is also an object of the present application to provide an anti-tumor use of a pharmaceutical 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 a hydrochloride salt thereof, 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 the group consisting of 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, and SEQ ID NO: 33; and a light chain variable region having an amino acid sequence selected from the group consisting of 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, and SEQ ID NO: 36.

[0053] The present application also provides a method for treating a subject afflicted with a cancer or a tumor, comprising 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 a hydrochloride salt thereof. 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 Programmed Death Receptor 1 (PD-1) and / or inhibits PD-1 activity, e.g., 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 afflicted with a cancer or a tumor, the method comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor alone and a therapeutically effective amount of an antibody or an antigen-binding portion thereof that inhibits PD-L1 and / or PD-L1 binding activity alone.

[0055] The present application also provides a method of treating a subject having a cancer or a tumor, the cancer or tumor being lung cancer, the method comprising: (i) measuring the level 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 antigen-binding portion thereof.

[0056] The present application provides a method for treating a subject having a cancer or a tumor. In certain embodiments, the subject is a patient diagnosed with lung cancer, e.g., a patient diagnosed with non-small cell lung cancer, or a patient diagnosed with small cell lung cancer, whose cancer can 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 herein is squamous non-small cell carcinoma, and in other specific embodiments, the non-small cell lung cancer described herein is non-squamous non-small cell carcinoma. In some specific embodiments, the non-small cell lung cancer described herein is lung adenocarcinoma, lung squamous cell carcinoma, or lung large cell carcinoma.

[0057] In some embodiments of the present application, the subject has previously received surgery, chemotherapy, and / or radiation therapy. In some specific embodiments, the subject has experienced disease progression after achieving a complete remission following surgery, chemotherapy, and / or radiation therapy. In some specific embodiments, the subject has failed to achieve a complete remission or has failed to achieve a partial remission following surgery, chemotherapy, and / or radiation therapy.

[0058] In some embodiments of the present application, the subject has not previously received systemic chemotherapy. In some embodiments, the subject has previously received surgery, radiation therapy, induction chemotherapy, and / or adjuvant chemotherapy, or the subject is receiving concurrent chemotherapy. In some specific embodiments, the subject has not previously received systemic chemotherapy, but has received surgery, radiation therapy, induction chemotherapy, and / or adjuvant chemotherapy or will receive concurrent chemotherapy. In some specific embodiments, the subject has experienced disease progression after achieving a complete remission following surgery, radiation therapy, induction chemotherapy, concurrent chemotherapy, and / or adjuvant chemotherapy. In some specific embodiments, the subject has failed to achieve a complete remission or has failed to achieve a partial remission following surgery, radiation therapy, induction chemotherapy, concurrent chemotherapy, and / or adjuvant chemotherapy. In some specific embodiments, the subject has experienced metastasis of the cancer following surgery, radiation therapy, induction chemotherapy, concurrent chemotherapy, and / or adjuvant chemotherapy.

[0059] In some embodiments of the application, the cancer or tumor is lung cancer or a lung malignancy. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In other embodiments, the cancer is small cell lung cancer (SCLC). In some embodiments, the cancer is a relapsed or refractory lung cancer. In some embodiments, the lung cancer is relapsed. In some embodiments, the lung cancer is refractory. In some embodiments, the lung cancer is metastatic. In some embodiments, the cancer treatment is a first line treatment for relapsed or refractory non-small cell lung cancer. In some embodiments, the cancer treatment is a first line treatment for metastatic non-small cell lung cancer, such as a 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 application, the lung cancer is selected from non-small cell lung cancer. In some embodiments, the lung cancer comprises lung squamous carcinoma or lung adenocarcinoma. In some embodiments, the lung cancer is an advanced lung cancer. In some embodiments, the lung cancer is an EGFR, ALK and / or ROS1 wild-type non-small cell lung cancer. In some embodiments, the lung cancer is selected from advanced squamous carcinoma type non-small cell lung cancer, advanced adenocarcinoma type non-small cell lung cancer. In some embodiments, the lung cancer is selected from i) EGFR, ALK and / or ROS1 wild-type squamous carcinoma type non-small cell lung cancer, and ii) EGFR, ALK and / or ROS1 wild-type adenocarcinoma type non-small cell lung cancer. In some embodiments, the lung cancer is an advanced (IIIB / IV stage) lung cancer that has failed or is intolerant to first line standard chemotherapy.

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

[0062] In some embodiments of the application, the pharmaceutical combination is for treating or preventing a lung malignancy, which can be a primary lung tumor or a secondary lung tumor.

[0063] In some embodiments of the application, the lung malignancy is a metastatic lung cancer. In other embodiments, the metastatic lung cancer is a metastatic cancer metastasized from lung cancer, gastric cancer, rectal cancer, colon cancer, large intestine cancer, pancreatic cancer or breast cancer.

[0064] In some aspects of the application, the subject is a locally advanced (stage IIIB), metastatic or recurrent (stage IV) NSCLC patient who is driver gene negative (i.e., EGFR, ALK, and ROS1 mutation negative). In some examples, the patient is histologically or cytologically confirmed to be inoperable and cannot receive definitive concurrent chemoradiation. In some non-limiting examples, the EGFR mutation includes, but is not limited to, exon 19 or 21 mutations.

[0065] In some aspects of the application, the pharmaceutical combination is useful for treating or preventing a liver malignancy, which can be a primary liver tumor or a secondary liver tumor.

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

[0067] In some aspects of the application, the liver malignancy is metastatic liver cancer. In other aspects, 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.

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

[0069] In some aspects of the application, the pharmaceutical combination is a non-fixed combination. In some aspects, 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 aspects, a kit for a pharmaceutical combination for treating a lung tumor or a liver malignancy is also 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 application, the kit contains the compound of Formula I in an amount of 56 to 168 mg, such as 84 to 168 mg. In some aspects, 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 foregoing values. In some aspects, the kit contains the compound of Formula I in an amount of 112 mg to 168 mg. In some embodiments, the compound of Formula I in the above dosage range can be packaged together as a whole. In other aspects, the compound of Formula I in the above dosage range can be packaged separately in unit dosages, such as 8 mg, 10 mg, or 12 mg unit dosages.

[0072] It is also an object of the present application 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 concurrently) to a subject in need thereof. In some embodiments, the tyrosine kinase inhibitor is a compound of Formula I or a hydrochloride salt thereof. In some embodiments, the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is an antibody or antigen-binding portion thereof that binds to Programmed Death Receptor 1 (PD-1) and / or inhibits PD-1 activity, e.g., is an anti-PD-1 antibody or an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the cancer or tumor is selected from a liver tumor (e.g., a liver malignancy such as hepatocellular carcinoma) or a lung tumor (e.g., a lung cancer such as non-small cell lung cancer).

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

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

[0075] It is also an object of the present application to provide, at least, a pharmaceutical pack comprising in separate containers a single packaged pharmaceutical composition each, wherein a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof is contained in one container and a pharmaceutical composition comprising a PD-1 antagonist or a PD-L1 antagonist is contained in a second container.

[0076] In some embodiments of the present application, the pharmaceutical composition comprises a compound of Formula I in an amount of 56 to 168 mg, e.g., 84 to 168 mg. In some aspects, the pharmaceutical composition comprises a 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 foregoing values. In some aspects, the pharmaceutical composition comprises a compound of Formula I in an amount of 112 mg to 168 mg.

[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 flat doses effective to treat the cancer. In some embodiments, the flat dose is in the range of about 10 mg to about 1000 mg of anti-PD-1 antibody. In some embodiments, the flat dose is selected from about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg of anti-PD-1 antibody. In some embodiments, the flat dose is selected from about 200 mg of anti-PD-1 antibody.

[0079] In some embodiments, the administration of the anti-PD-1 antibody is treated in cycles of 2 weeks (14 days) or 3 weeks (21 days), preferably with the anti-PD-1 antibody being administered intravenously 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 an article of manufacture 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 an article of manufacture comprising a container containing a fixed dose of an anti-PD-1 antibody for treating cancer. In some 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 an anti-PD-1 antibody. In some embodiments, the article of manufacture further comprises a package insert or package insert instructing the user to administer the fixed dose of the anti-PD-1 antibody to a cancer patient. In some embodiments, the product comprises one or more vials containing approximately 50 mg, 100 mg, 200 mg, 300 mg, 350 mg, 400 mg, 500 mg, or 600 mg of an anti-PD-1 antibody. In some embodiments, the product comprises one vial containing approximately 50 mg of an anti-PD-1 antibody. In some embodiments, the product comprises one vial containing approximately 200 mg of an anti-PD-1 antibody. In some embodiments, the product comprises one vial containing approximately 350 mg of an anti-PD-1 antibody. In some embodiments, the product is packaged as a 50 mg / 5 mL / vial, 100 mg / 10 mL / vial, 200 mg / 10 mL / vial, or 350 mg / 35 mL / vial anti-PD-1 antibody solution.

[0081] The present application also aims to provide a drug combination for preventing or treating cancer or tumors, 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. 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 PD-1 activity, 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 a liver tumor (e.g., a liver malignancy, such as hepatocellular carcinoma) or a lung tumor (e.g., lung cancer, such as non-small cell lung cancer).

[0082] It is also an object of the present application to provide the use of a pharmaceutical combination in the manufacture of a medicament for the prevention or treatment of a 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 a hydrochloride salt thereof. In some embodiments, the inhibitor of the interaction between 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 PD-1 activity, e.g., 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 a liver tumor (e.g., a liver malignancy such as hepatocellular carcinoma) or a lung tumor (e.g., a lung cancer such as non-small cell lung cancer).

[0083] It is also an object of the present application to provide a unit formulation, wherein the unit formulation comprises: a compound component, 6-12 mg of a compound of Formula I or a hydrochloride salt thereof; 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.

[0084] It is also an object of the present application to provide a method for the prevention or treatment of a cancer or a tumor, wherein one or more of the above unit formulations are administered to a subject in need thereof. Preferably, the compound component and the antibody component in the unit formulation are administered separately.

[0085] Anlotinib pharmaceutical composition

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

[0087] In some embodiments of the present application, a 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, the total dose of the anlotinib pharmaceutical composition comprises 84-168 mg per treatment cycle according to a 2-week-on, 1-week-off treatment cycle. In some embodiments, the total dose of the anlotinib pharmaceutical composition comprises a value selected from 84 mg, 112 mg, 140 mg, 168 mg, or a range formed by any of the above values. In some embodiments, the total dose of the anlotinib pharmaceutical composition preferably comprises 112-168 mg.

[0089] Anlotinib

[0090] As used herein, 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, which has the structural formula as follows:

[0091]

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

[0093] Cintirelimab

[0094] As used herein, Sintilimab (IBI308, IBI-308) is an anti-PD-1 monoclonal antibody, the sequence and structure of which can be found in the antibody D of document CN108473977A. On December 27, 2018, the PD-1 antibody drug "Sintilimab Injection" of Innovent Biologics was officially approved for marketing by the National Medical Products Administration (NMPA) of China, and was used for the treatment of at least two lines of systemic chemotherapy for recurrent or refractory classical Hodgkin's lymphoma.

[0095] The full-length sequence of the heavy chain of Sintilimab:

[0096] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0097] Full length sequence of the light chain of cintredegin:

[0098] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0099] 14C12H1L1

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

[0101] Definitions and explanations

[0102] The following terms as used in the present application have the following meanings unless otherwise indicated. A particular term should not be construed as indefinite or unclear in the absence of a specific definition, but should be understood in accordance with the 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 active ingredient thereof.

[0103] As used herein, the term "antibody" refers to an antigen-binding protein having at least one antigen-binding domain. Antibodies and fragments thereof of the present application can be whole antibodies or any fragment thereof. Accordingly, antibodies and fragments thereof of the present application include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, 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 fragments thereof can also include recombinant polypeptides, fusion proteins, and bispecific antibodies. Anti-PD-L1 antibodies and fragments thereof disclosed herein can be of IgGl, IgG2, IgG3, or IgG4 isotype.

[0104] The term "isotype" refers to the class of antibody that is coded for by heavy chain constant region genes. In one embodiment, anti-PD-1 / PD-L1 antibodies and fragments thereof disclosed herein are of IgGl or IgG4 isotype. Anti-PD-1 / PD-L1 antibodies and fragments thereof of the present application can be derived from any species, including but not limited to mouse, rat, rabbit, primate, llama, and human. PD-1 / PD-L1 antibodies and fragments thereof 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 a human immunoglobulin sequence. Humanized antibodies can include substitutions in the framework region such that the framework can not be an exact copy of the expressed human immunoglobulin or germline gene sequence.

[0106] An "isolated antibody" means an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-1 / PD-L1 is substantially free of antibodies that specifically bind antigens other than PD-1 / PD-L1). An isolated antibody that specifically binds to PD-1 / PD-L1 may, however, have cross-reactivity to other antigens, such as PD-1 / PD-L1 molecules from different species. Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals.

[0107] The term "monoclonal antibody" ("mAb") refers to antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or, in the case of bispecific monoclonal antibodies, displays dual binding specificity for two different epitopes. mAbs are 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 (Opdivo®, Bristol-Myers Squibb), Pembrolizumab (Keytruda®, Merck), Durvalumab (Imfinzi®, AstraZeneca), Avelumab (Bavencio®, Pfizer), Toripalimab (JS-001, Junshi Biosciences), Sintilimab (IBI308, Beijing Inspire Pembrolizumab (Keytruda®, Merck) Durvalumab (Imfinzi®, AstraZeneca), Avelumab (Bavencio®, Pfizer), Toripalimab (JS-001, Junshi Biosciences), Sintilimab (IBI308, Beijing Inspire), Camrelizumab (SHR-1210, Hengrui Medicine, see CN105026428B or WO2015085847A1), Tislelizumab (BGB-A317, BeiGene), 14C12H1L1 (Zhongshan Kalong), Genolimzumab (GB226, JHBB), Lizumab (LZM009, Lizhu Pharmaceutical), HLX-10 (Hengrui Biotech), BAT-1306 (Baoan Tai), HX008 (AK103, Kangfeng Biotech / HanZhong Biotech), AK104 (Zhongshan Kalong), CS1003 (Guangzhou Sparx Biopharm), SCT-I10A (Shengzhou Cell), F520 (Shandong Xindada Pharmaceutical / Lunan Pharmaceutical), SG001 (ShangJian Biotech), GLS-010 (Yuheng Pharmaceutical), Atezolizumab (Tecentriq®, Genentech / Roche), Avelumab (Bavencio®, Pfizer), Durvalumab (Imfinzi®, AstraZeneca) Roche), Avelumab (Bavencio®, Pfizer) Merck / Pfizer), Durvalumab (Imfinzi®, AstraZeneca Asana (AstraZeneca), KL-A167 (Colliery), SHR-1316 (Hengrui Medicine), BGB-333 (Beijing Junshi), JS003 (Junshi Biosciences), STI-A1014 (ZKAB0011, ZKbio), KN035 (Canning Jayvee / Siluodi), MSB2311 (Maibos Bio), HLX-20 (Fuhong Hanlin), CS-1001 (Generex), and the like.

[0108] An "antigen-binding portion" of an antibody (also called an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by the whole antibody.

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

[0110] As used herein, the term "EC50" refers to the effective concentration, 50% maximal response of an antibody. As used herein, the term "IC50" refers to the inhibitory concentration, 50% maximal response of an antibody. 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 any action taken to obtain a pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. As used herein, "treatment" covers any treatment of a patient for a disease, including: (a) preventing the disease or symptom from occurring in a patient that is predisposed or does not yet experience the disease or symptom; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing the disease or symptom to regress.

[0112] As used herein, the term "systemic treatment" refers to a treatment in which a drug substance is delivered through the bloodstream to 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 a component of a multimodal treatment, wherein 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, a feline, a canine, and a primate. Preferably, the subject according to the present application is a human.

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

[0116] As used herein, " adverse event " (AE) is any unfavorable and usually unintentional or undesirable sign (including abnormal laboratory findings), symptom or disease relevant to the application of medical treatment.For example, adverse event can be relevant to the activation of the immune system or the amplification of immune system cells (for example, T cells) in response to treatment.Medical treatment can have one or more related AEs, and each AE can have the same or different levels of severity.Reference to the method that can " modify adverse event " refers to the treatment scheme that reduces the incidence and / or severity of one or more AEs relevant to the application of different treatment schemes.

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

[0118] The term "dosing frequency" as used herein refers to the frequency of administration of a dosage of a formulation disclosed herein at a given time. The dosing frequency can be indicated as the number of administrations per given time, for example, once a week or once every two weeks.

[0119] The term "flat dose" is used to refer to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). Thus, a flat dose is specified as an absolute amount of an 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 would receive the same dose of antibody (e.g., 240 mg of anti-PD-1 antibody).

[0120] The use of the term "fixed dose" with respect to the compositions of the present application means that two or more different antibodies in a single composition are present in the composition in a specific (fixed) ratio to each other. In certain embodiments, the fixed dose is based on the weight of the antibodies (e.g., mg). In certain embodiments, the fixed dose is based on the concentration of the antibodies (e.g., mg / ml). In certain embodiments, the ratio of mg first antibody:mg 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 a first antibody and a 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 referred to herein means a dose calculated based on the weight of a patient that is administered to the patient. For example, when a patient having 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 draw the appropriate amount of the anti-PD-1 antibody (i.e., 180 mg) and the anti-CTLA-4 antibody (i.e., 60 mg) from a 3: 1 ratio fixed dose formulation of the anti-PD-1 antibody and the anti-CTLA-4 antibody at one time.

[0122] The term "immunotherapy" means treatment of a subject having a disease or at risk of infection or suffering from a relapse of a disease by a method that includes inducing, enhancing, suppressing or otherwise altering an immune response. "Treatment" or "therapy" of a subject means any type of intervention or process performed on the subject, or the administration of an active agent to the subject, with the object of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or a biochemical indicia associated with a disease.

[0123] As used herein, "PD1 / PD-L1 positive" is used interchangeably with "at least about 1% PD-1 / PD-L1 expression." In one embodiment, PD-1 / PD-L1 expression is measured 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)" means an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly 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, isoforms, and species homologs of hPD-1, 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) that downregulate 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 hamsters. In certain embodiments, the subject is a human. The terms "subject," "subject," and "patient" are 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 conjunction with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by determining the activity of the agent in in vitro assays.

[0128] As used herein, "subtherapeutic 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 to treat a hyperproliferative disease (e.g., cancer).

[0129] As an example, an "anti-cancer agent" promotes regression of cancer or prevents further tumor growth in a subject. In certain embodiments, a therapeutically effective amount of the agent promotes regression of the cancer to the point of eliminating the cancer. To "promote regression of cancer" means that administration of an effective amount of the agent, alone or in combination with an anti-neoplastic agent, results in a decrease in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of symptom-free periods, or a prevention of impairment or disability due to disease affliction. In addition, the terms "effective" and "effectiveness" with respect to a treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness indicates the ability of the agent to promote regression of cancer in the patient. Physiological safety indicates the level or other adverse physiological effects (adverse effects) at the cellular, organ and / or organism level resulting from administration of the agent.

[0130] As an example for treating a tumor, a therapeutically effective amount of an anti-cancer agent 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 untreated subjects, or, in certain embodiments, relative to patients treated with standard-of-care therapies. In other embodiments of the application, tumor regression can be observed for a period of at least about 20 days, at least about 40 days, or at least about 60 days. Despite these ultimate measures of therapeutic effectiveness, evaluation of immunotherapeutic agents must also take into account the "immune-related" pattern of response.

[0131] The "immune-related" pattern of response indicates a pattern of clinical response frequently observed in cancer patients treated with immunotherapeutic agents that produce anti-tumor effects by inducing cancer-specific immune responses or by altering natural immune processes. This pattern of response is characterized by beneficial therapeutic effects following an initial increase in tumor burden or appearance of new lesions, which would be classified as progressive disease in the evaluation of traditional chemotherapeutic agents and would be synonymous with drug failure. Thus, proper evaluation of immunotherapeutic agents can require long-term monitoring of the effects of these agents on the target disease.

[0132] A therapeutically effective amount of an agent includes a "prophylactically effective amount", which is any amount of the agent that, when administered alone or in combination with an anti-neoplastic agent to a subject at risk of developing a cancer (e.g., a subject with a precancerous condition) or a subject at risk of recurrence of a cancer, inhibits the development or recurrence of the cancer. In certain embodiments, a prophylactically effective amount completely prevents the development or recurrence of the cancer. To "inhibit" the development or recurrence of the cancer means to reduce the likelihood of the development or recurrence of the cancer, or to completely prevent the development or recurrence of the cancer.

[0133] A "recurrent" cancer is a cancer that regrows, either at the original site or at a distant site, after an initial treatment, e.g., surgery. A "locally recurrent" cancer is a cancer that appears, after treatment, in the same location as the previously treated cancer.

[0134] A "non-resectable" cancer is one that cannot be removed by surgery.

[0135] A "metastatic" cancer refers to a cancer that spreads from one part of the body (e.g., the lung) to another part of the body.

[0136] The use of the alternative (e.g., "or") should be understood to mean either one, but not both, of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to mean "one or more" of any listed or enumerated component.

[0137] The terms "about," "approximately" or "substantially" mean a value or composition that is acceptable within the margins of error of the determining the particular value or composition by a person of ordinary skill in the art, which will vary based on the context and the manner in which the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about," "approximately" or "substantially" can mean within one or more than one standard deviation in the practice in the art. Alternatively, "about" or "substantially" can mean a range of up to 10% or 20% (i.e., ±10% or ±20%) of a parameter or value modified by the term. For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%) or 2.4 mg and 3.6 mg (for 20%). Further, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5-fold of a value. When a particular value or composition is provided in the application and claims, the meaning of "about" or "substantially" should be assumed to be within the acceptable error range of that particular value or composition, unless otherwise noted.

[0138] As used herein, the term "about once a week," "about once every two weeks," or any other like dosing interval term refers to an approximation. "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, e.g., 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 week 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 particular day of the first week (e.g., Monday), and then the second dose is administered on the same day of the sixth week or twelfth week (i.e., Monday), respectively. Similar principles apply to phrases including, but not limited to, "about once every 2 weeks," "about once a month," and the like.

[0139] As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include values that are both within and outside of the recited range, and to include fractions of the values within the recited range (e.g. integers within the range, and tenths and hundredths of the values within the range), where appropriate.

[0140] Unless otherwise indicated, "about" or "approximately" as used herein will be understood by those of ordinary skill in the art to refer to a range of values that one of ordinary skill in the art would consider as the equivalent to the recited value (e.g. within 5% of the recited value, preferably within 2% of the recited value, and more preferably within 1% of the recited value). 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" is used in reference 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 problem or complication, commensurate with a reasonable benefit / risk ratio.

[0142] The term "pharmaceutically acceptable salt" includes a salt formed between an alkali ion and a free acid or a salt formed between an acid ion and a free base, for example, including a hydrochloride, a hydrobromide, a nitrate, a sulfate, a phosphate, a formate, an acetate, a trifluoroacetate, a fumarate, an oxalate, a maleate, a citrate, a succinate, a methanesulfonate, a benzenesulfonate, or a p-toluenesulfonate, preferably a hydrochloride, a hydrobromide, a sulfate, a formate, an acetate, a trifluoroacetate, a fumarate, a maleate, a methanesulfonate, a p-toluenesulfonate, a sodium salt, a potassium salt, an ammonium salt, an amino acid salt, and the like. In the present application, when a pharmaceutically acceptable salt is formed, the molar ratio of the free acid to the alkali 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 a pharmaceutically acceptable salt is formed, the molar ratio of the free base to the acid 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 ingredients (e.g., an anti-PD-1 antibody or a compound of Formula I) are co-administered to a subject in a fixed dose ratio, or in a single entity, pharmaceutical composition, or formulation.

[0144] The term "fixed combination" means that the active ingredients (e.g., an anti-PD-1 antibody or a compound of Formula I) are co-administered to a subject in a fixed dose ratio, or in a single entity, pharmaceutical composition, or formulation.

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

[0146] The term "pharmaceutical composition" refers to a mixture of one or more active ingredients (e.g., an anti-PD-1 antibody or a compound of Formula I) or a pharmaceutical combination thereof of the present application with a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of a compound or a pharmaceutical combination of the present application to a subject.

[0147] The term "synergistic effect" refers to an effect (e.g., inhibition of colon cancer growth, or relief of colon cancer symptoms) produced by two or more components (e.g., an anti-PD-1 antibody or a compound of Formula I) that is greater than the simple addition of the effects produced by the components administered alone.

[0148] Modes of administration

[0149] The following is not intended to limit the manner in which the pharmaceutical combination of the present application is administered.

[0150] The components of 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 administration.

[0151] The components of the pharmaceutical combination of the present application can be administered separately, or some or all of them can be administered together. The components of the pharmaceutical combination of the present application can be administered substantially at different times, or some or all of them can be administered substantially at the same time.

[0152] The components of the pharmaceutical combination of the present application can be administered independently, or some or all of them can be administered together, by any suitable route, including but not limited to, orally or parenterally (by intravenous, intramuscular, topical or subcutaneous route). 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 of the pharmaceutical combination of the present application can be administered independently, or some or all of them can be administered together, in any suitable dosage form, including but not limited to, tablets, troches, pills, capsules (e.g., hard capsules, soft capsules, enteric capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions, and dosage forms for sustained release for oral or non-oral administration.

[0154] The components of the pharmaceutical combination of the present application can be administered independently, or some or all of them can be administered together, with a pharmaceutically acceptable carrier and / or excipient.

[0155] The pharmaceutical combination of the present application can further comprise an additional therapeutic agent. 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 investigates the therapeutic effect of anlotinib hydrochloride, anti-PD-1 antibody alone or in combination on lung tumors. The experimental results surprisingly found that anlotinib hydrochloride and anti-PD-1 antibody can have a significant synergistic effect, breaking the established immune tolerance of the body to tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0157] Figure 1 The effects of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with anti-mPD-1 antibody on the growth of subcutaneous transplanted H22 hepatoma in mice are shown.

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

[0159] Figure 3 The individual tumor volume (upper panel) and individual tumor weight (lower panel) of each treatment group of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with anti-mPD-1 antibody at the end of the experiment (D17) are shown.

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

[0161] Figure 5 The photos of tumors showing the efficacy of anlotinib hydrochloride, lenvatinib mesylate alone or in combination with anti-mPD-1 antibody on subcutaneous transplanted H22 hepatoma are shown.

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

[0163] Figure 7 The SLD changes of 13 patients in an open-label, multicenter Ib / II phase clinical study of 14C12H1L1 in combination with anlotinib hydrochloride as first-line treatment for unresectable hepatocellular carcinoma over time are shown. DETAILED DESCRIPTION

[0164] The present application will be further described with reference to the following specific examples. However, these examples are set forth merely to illustrate but not to limit the scope of the present application. Also, the present application is not limited to any specific preferred embodiments described herein. Those skilled in the art should understand that equivalent substitutions, or corresponding modifications of the technical features of the present application still fall within the scope of the present application. Unless otherwise specified, the reagents used in the following examples are commercially available products, and the preparation of solutions can be performed using conventional techniques in the art.

[0165] Table 1 Abbreviation Table

[0166]

[0167]

[0168]

[0169] Example 1 Anlotinib hydrochloride combined drug treatment of advanced non-small cell lung cancer

[0170] This example discloses the results of anlotinib hydrochloride combined drug treatment of advanced non-small cell lung cancer. The main target population is histologically or cytologically confirmed patients with locally advanced (IIIB), metastatic or recurrent (stage IV) NSCLC who cannot be treated by surgery and cannot receive radical concurrent chemoradiotherapy, and are negative for driver genes (i.e. EGFR, ALK and ROS1 mutation negative).

[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 due to any cause. In days, subjects who were lost to follow-up, the last follow-up time was calculated as the time of death.

[0175] Key inclusion criteria: Chinese patients with stage IIIB / IV NSCLC; never received systemic treatment; clear molecular typing; 18-75 years old; PS score: 0-1; expected survival time more than 3 months; sufficient organ reserve function; no symptomatic brain metastasis.

[0176] Key exclusion criteria: previously received systemic anticancer treatment; multiple factors affecting oral medication; known symptomatic brain metastasis, spinal cord compression, carcinomatous meningitis, or disease in the brain or soft meninges found by CT or MRI examination at the time of screening; central hollow squamous cell carcinoma or presence of bleeding tendency; presence of any severe and uncontrolled disease in patients.

[0177] Anlotinib hydrochloride: light yellow crystalline powder, content 98.9%, batch number 17316007, stored at 2-8℃ away from light. Anlotinib hydrochloride was provided by Zhejiang Hisun Pharmaceutical Co., Ltd. Anlotinib hydrochloride was prepared and diluted to the required concentration with distilled water.

[0178] Dose:

[0179] Anti-PD-1 antibody: administer sintilimab injection, 200 mg, intravenous infusion, once every 21 days on the first day (D1) of each cycle (21 days in total).

[0180] Anlotinib hydrochloride capsules (active ingredient: Anlotinib dihydrochloride): Anlotinib hydrochloride capsules 12 mg are taken on an empty stomach within ± 5 min of the start of infusion of anti-PD-1 antibody injection, and are continuously taken orally for 2 weeks and stopped for 1 week, i.e. 21 days for a treatment cycle.

[0181] Before the start of the test, the test scheme of the test, the copy of the electronic case report form, the sample of the informed consent form, etc. were submitted to the clinical research responsible unit and the ethics committee of each participating unit for examination, and only after the approval of the ethics committee for clinical trials could the clinical trials be carried out. Before the start of the clinical trial, the researchers explained in detail to all the subjects the purpose of the test, the risks and benefits, ensured that all the subjects had informed consent before the test, signed the informed consent form, and fully protected the rights and interests of the subjects during the test. The researchers strictly followed the ethical guidelines of human medical research in the Helsinki Declaration (2013) and the provisions of the "Good Clinical Practice (2003)" and other relevant laws and regulations to perform the duties of the researchers.

[0182] As of January 25, 2019, 11 patients had met the efficacy evaluation conditions. The basic conditions and efficacy evaluation results of the 11 patients participating in the study are shown in Table 2, and 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 conditions and efficacy evaluation results of patients participating in the study

[0184]

[0185] The results of this exploratory data study of anlotinib combined with first-line treatment of advanced non-small cell lung cancer suggest surprising efficacy, ORR: 63.6% for anlotinib combined with sintilimab, DCR: 100% (see Table 3), and PFS and OS are promising. At the same time, the anlotinib first-line combination mode can show high patient tolerance (detailed results not shown).

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

[0187]

[0188] Example Two Anlotinib Hydrochloride Combined Drug Treatment of Hepatocellular Carcinoma

[0189] 1. Drug information

[0190] Anlotinib hydrochloride: light yellow crystalline powder, content 98.9%, batch number 17316007, valid until December 2018, stored at 2-8℃ and protected from light.

[0191] Lenvatinib mesylate: white crystalline powder, content 98.7%, batch number 19916003, valid until June 2019, 2-8℃, avoid light, save.

[0192] Anti-mouse PD-1 (abbreviation: mPD-1): colorless transparent liquid, purity >95%, concentration 7.83mg / ml, batch number 665418F1, 2-8℃, avoid light, save.

[0193] 2. Providing unit

[0194] Anlotinib hydrochloride and Lenvatinib mesylate were provided by Sinotau Pharmaceutical Group Co., Ltd.

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

[0196] 3. Preparation method

[0197] Anlotinib hydrochloride and Lenvatinib mesylate were prepared with distilled water and diluted to the required concentration; mPD-1 was diluted with normal saline to the required concentration, and prepared for use.

[0198] 4. Cells

[0199] H22 cells were cultured in 10-cm culture dishes, and the culture conditions were RPMI 1640 medium with 10% fetal bovine serum and penicillin, streptomycin, and cultured in a 37℃, 5% CO2 air incubator. Subculture twice a week, when the cells were in exponential growth, the cells were collected, counted, and inoculated.

[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 qualification certificate No. 311620400013790. Raising environment: SPF level.

[0202] The use and welfare of the experimental animals in this experiment were carried out in accordance with the regulations of the International Assessment and Recognition Committee of Experimental Animals (AAALAC). The health status and death of the animals were monitored every day, and routine inspection included observation of the effects of test substances or drugs on the daily behavior of animals such as behavior activity, body weight change, appearance, and other signs.

[0203] 6. Experimental index

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

[0205] Tumor volume (V) was calculated by the formula:

[0206] Tumor volume (V) was calculated by the formula:

[0207] V = 1 / 2 x a x b 2 wherein a and b represent length and width, respectively.

[0208] T / C (%) = (T - T0) / (C - C0) x 100 wherein T and C represent tumor volume at the end of the experiment in the test group and the control group animals, respectively; T0and C0represent tumor volume at the beginning of the experiment in the test group and the control group animals, respectively.

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

[0210] When the tumor regressed, tumor inhibition rate (TGI) (%) = 100 - (T - T0) / T0x 100

[0211] If the tumor volume was reduced compared to the initial volume, i.e. T < T0or C < C0, it was defined as partial tumor regression (PR); if the tumor completely disappeared, it was defined as complete tumor regression (CR).

[0212] 7. Experimental implementation

[0213] Each mouse was subcutaneously inoculated with H22 cells, and when the tumor grew to 100-200 mm 3 in size, the mice were randomly divided into groups and administered drugs; the mice were administered drugs by gavage (i.g.) or intraperitoneal injection (IP); the solvent group was administered the same volume of distilled water by gavage; the administration volume was 0.1 mL / 10 g of body weight; the specific administration dose and administration schedule are shown in Table 4.

[0214] Table 4 Administration dose and administration schedule

[0215]

[0216] Note: Randomly divided into groups, the first administration time was D0; IP: intraperitoneal injection; i.g., gavage administration; QD, once a day.

[0217] After the end of the experiment, or when the tumor volume of the animals reached the euthanasia endpoint of 1500 mm 3 , the animals were sacrificed by carbon dioxide inhalation, and then the tumor tissue was dissected and weighed and photographed.

[0218] 8. Experimental results

[0219] Anlotinib hydrochloride (1 mg / kg, gavage, QD x 17) inhibited the growth of H22 subcutaneous hepatocellular carcinoma xenografts in mice, with an inhibition rate of 61.7%. Lenvatinib mesylate (5 mg / kg, gavage, QD x 17) had an inhibition rate of 79%. mPD-1 (1 mg / kg, IP, twice weekly for 5 times) had an inhibition rate of 29.3% for H22, with complete regression of 2 / 10 tumors. Combination therapy of anlotinib hydrochloride and mPD-1 increased the inhibition rate to 79.5%, with partial regression and complete regression of 1 / 10 tumors. Combination therapy of lenvatinib mesylate and mPD-1 had an inhibition rate of 63.4% for H22, with partial regression of 1 / 10 tumors. Tumor-bearing mice tolerated the combination of anlotinib hydrochloride and mPD-1 well, without significant weight loss or other symptoms. In comparison, the combined use of anlotinib hydrochloride and mPD-1 was more effective in treating H22 subcutaneous liver cancer xenografts than either anlotinib hydrochloride or mPD-1 alone. The results are shown in Table 5.

[0220] Table 5 The therapeutic effect on H22 subcutaneous transplanted tumor in mice

[0221]

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

[0223] like Figure 1 As shown, the average tumor volume of mice in each treatment group increased with the number of days of treatment from D0 to D17, but the growth rate varied among the groups. The average tumor volume of mice in the solvent control group increased the fastest, reaching the largest volume on D17. The average tumor volume of the other groups on D17 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] like Figure 2 As shown, the tumor growth of mice in each group was similar from D0 to D17 after administration. Figure 1 The results were relatively consistent. The solvent control group showed a rapid growth trend, while the growth of anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg) was the slowest.

[0225] like Figure 3As shown in the table, the individual tumor volume and tumor weight of mice in each dose group at the end of the experiment (D17): 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 shown in the table, the body weight of mice in the solvent control group and the mPD-1 group showed a steady increase from D0 to D17, and no significant body weight loss or other symptoms occurred in the 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) groups. Figure 4 As shown in the table, the body weight of mice in the solvent control group and the mPD-1 group showed a steady increase from D0 to D17, and no significant body weight loss or other symptoms occurred in the 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) groups.

[0227] Figure 5 As shown in the table, the body weight of mice in the solvent control group and the mPD-1 group showed a steady increase from D0 to D17, and no significant body weight loss or other symptoms occurred in the 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) groups. Figure 1

[0228] Lenvatinib mesylate (5 mg / kg, i.g., QD 17) significantly inhibited the growth of H22 subcutaneous transplanted tumors in mice, and anlotinib hydrochloride (1 mg / kg) + mPD-1 (1 mg / kg) could achieve basically the same tumor inhibition effect, and was superior to lenvatinib mesylate in inhibiting tumor growth, but the dose could be greatly reduced, which was conducive to reducing the risk of drug side effects; mPD-1 had a certain effect on H22, but the combination of anlotinib hydrochloride and mPD-1 could significantly improve the H22 tumor inhibition rate, and the tumor-bearing mice could show good tolerance to the combined drugs.

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

[0230] Establishment of PD-1 Hybridoma Cell Strain

[0231] 1. The fusion protein PD-1 with the corresponding amino acid sequence was prepared according to the method of molecular biology. The PD-1 fusion protein prepared by the above method was used as an antigen, and BALB / C mice were immunized with Freund's adjuvant. After the mice produced an immune response, their spleen cells were fused with mouse myeloma cells to form hybridoma cells, and the hybridoma cells were cultured using a 96-well plate.

[0232] 2. The antibodies secreted by each hybridoma cell strain were coated with PD-1-hFc as an antigen to coat the enzyme-labeled plate, and the enzyme-labeled plate was blocked with PBS buffer containing 1% BSA. The coated enzyme-labeled plate was used to screen hybridoma cells secreting new antibodies specifically binding to PD-1 by indirect ELISA method.​​

[0233] 3. Screening the hybridoma cells secreting monoclonal antibodies capable of competing with PD-L1 for binding to PD-1 from the hybridoma cells screened by indirect ELISA by competitive ELISA.

[0234] 4. Subcloning the screened cell strains to screen monoclonal stable cell strains secreting monoclonal antibodies capable of competing with PD-L1 for specific binding to PD-1. The specific method is as follows: counting the viable cells of the cells to be subcloned, diluting and inoculating the cells into 96-well cell culture plates with IMDM medium containing 15% fetal bovine serum according to the number of viable cells, and the theoretical cell density of inoculation is 1 cell / well. After the cells grow into monoclonal cell clusters, screening is performed by ELISA method, and after multiple subcloning and screening, stable monoclonal cell strains are obtained.

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

[0236] Design of antibody humanization

[0237] To construct a humanized antibody, the variable region amino acid sequence of the murine antibody is aligned with the variable region gene sequence of a human. By selectively mutating part of the murine amino acid sequence to a humanized amino acid sequence, a plurality of humanized antibodies are designed.

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

[0239] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKSLEWVATISGGGSDTYYPDSVKG RFTISRDNAKNNLYLQMSSLRSEDTALYYCARQLNYAWFAYWGQGTLVTVSAAKTTPPSVYRSSKGNSST LAAVTS (SEQ ID NO: 1)

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

[0241] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYTSSNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIKR (SEQ ID NO: 4)

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

[0243] The heavy chain variable region sequence of humanized antibody 1 is:

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

[0245] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYTSSNQGTGVPARFSGSGSGTDFTLNINPMEADDTAMYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 5)

[0246] The heavy chain variable region sequence of humanized antibody 3 is:

[0247] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISGGGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQLNYAWFAYWGQGTLVTVSS (SEQ ID NO: 3)

[0248] The light chain variable region sequence of humanized antibody 3 is:

[0249] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWYQQKPGQPPKLLIYTSSNKDTGVPARFSGSGSGTDFTLTINPMEAEDTAVYYCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 6)

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

[0251] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 7)

[0252] The light chain variable region sequence of humanized antibody 4 is:

[0253] DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLEL (SEQ ID NO: 9)

[0254] The heavy chain variable region sequence of humanized antibody 5 is:

[0255] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKG RFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 8)

[0256] The light chain variable region sequence of humanized antibody 5 is:

[0257] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 10)

[0258] The heavy chain variable region sequence of humanized antibody 6 is:

[0259] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPEKGLDWVATISGGGRDTYYPDSVKG RFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYW GQGTLVTVSS (SEQ ID NO: 11) The light chain variable region sequence of humanized antibody 6 is:

[0260] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNIHPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 14)

[0261] The heavy chain variable region sequence of humanized antibody 7 is:

[0262] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQKGEAWFAYWGQGTLVTVSS (SEQ ID NO: 13)

[0263] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 15)

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

[0265] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQKGEAWFAYWGQGTLVTVSS (SEQ ID NO: 13)

[0266] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWYQQKPGQPPKLLIYAASNKATGVPARFSGSGSGTDFTLNINPMEANDTAVYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 16)

[0267] The heavy chain sequence of humanized antibody 8A is:

[0268] EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHHYTQKSLSLSLGK (SEQ ID NO: 17)

[0269] The light chain sequence of humanized antibody 8A is:

[0270] DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLNINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18)

[0271] The heavy chain variable region sequence of humanized antibody 9 is:

[0272] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKF KNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 31) The light chain variable region sequence of humanized antibody 9 is:

[0273] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK (SEQ ID NO: 34)

[0274] The heavy chain sequence of humanized antibody 9 is:

[0275] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 37)

[0276] The light chain sequence of humanized antibody 9 is:

[0277] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40)

[0278] The heavy chain variable region sequence of humanized antibody 10 is:

[0279] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVK GRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 32)

[0280] The light chain variable region sequence of humanized antibody 10 is:

[0281] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 35)

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

[0283] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 38)

[0284] The light chain sequence of humanized antibody 10 is:

[0285] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41)

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

[0287] EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSS (SEQ ID NO: 33)

[0288] The light chain variable region sequence of humanized antibody 11 is:

[0289] DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFR (SEQ ID NO: 36)

[0290] The heavy chain sequence of humanized antibody 11 is:

[0291] EVQLLESGGVLVQPGGSLRLSCAASGFTFSNFGMTWVRQAPGKGLEWVSGISGGGRDTYFADSVKGRFTISRDNSKNTLYLQMNSLKGEDTAVYYCVKWGNIYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 39)

[0292] The light chain sequence of humanized antibody 11 is:

[0293] DIQMTQSPSSLSASVGDSITITCRASLSINTFLNWYQQKPGKAPNLLIYAASSLHGGVPSRFSGSGSGTDFTLTIRTLQPEDFATYYCQQSSNTPFTFGPGTVVDFRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42)

[0294] The nucleic acid sequence encoding the above humanized antibody is synthesized entirely, and constructed into an expression vector. The expression vector DNA is extracted, and transfected into mammalian cells 293 cells. After transfection of the cells, the antibody is expressed in the mammalian cells, and secreted outside the cells. The expressed antibody is purified by antibody A affinity chromatography column, and the humanized antibody protein is obtained. These experimental operations are all conventional operations known in the art.

[0295] Example Four Clinical Study of 14C12H1L1 Combined with Anlotinib Hydrochloride for First-line Treatment of Unresectable Hepatocellular Carcinoma

[0296] This example discloses the results of an open-label, multicenter Ib / II phase clinical study of early 14C12H1L1 combined with anlotinib hydrochloride as first-line treatment for unresectable hepatocellular carcinoma.

[0297] The present study is an open-label, multicenter Ib / II phase clinical study, the purpose of which is to evaluate the effectiveness of 14C12H1L1 combined with anlotinib as first-line treatment for unresectable hepatocellular carcinoma, i.e. safety.

[0298] Primary endpoint: Objective response rate (ORR) assessed according to RECISIT v1.1.

[0299] Secondary endpoints: (1) Disease control rate (DCR) assessed by the investigator according to RECISIT v1.1, duration of response (DOR): time to response (TTR), progression-free survival (PFS) and time to progression (TPP); (2) Overall survival (OS); (3) Safety evaluation: incidence and severity of AEs, clinically significant abnormal laboratory findings.

[0300] Progression-free survival (PFS): defined as the time from the first dose until objective tumor progression or death.

[0301] Overall survival (OS): defined as the time from the start of the first dose until death due to any cause. In days, subjects who were lost to follow-up, the last follow-up time was calculated as the time of death.

[0302] Duration of response (DOR): defined as the time from the first assessment of CR or PR until the first assessment of PD or death.

[0303] Time to response (TTR): the time from the start of the first dose until the first assessment of CR or PR.

[0304] Test drug

[0305] 14C12H1L1 injection, 100 mg / 10 ml solution, manufacturer: Kangfangtiancheng (Guangdong) Pharmaceutical Co., Ltd., stored at 2-8°C, away from light.

[0306] Anlotinib hydrochloride capsules (Fokewei), 8 mg / grain, manufacturer: Zhongda Tianqing Pharmaceutical Group Co., Ltd., sealed, away from light.

[0307] Dosing

[0308] Dosing regimen and dose selection

[0309] 14C12H1L1 in combination with Anlotinib, wherein 14C12H1L1: 200 mg, Q3W intravenous injection; Anlotinib: 8 mg once daily, 14 days on and 7 days off, 21 days as a cycle.

[0310] Early study results

[0311] Treatment effect

[0312] As of July 15, 2019, a total of 13 subjects received at least one tumor evaluation, with a median number of treatment cycles of 3. Among these patients, 3 achieved partial remission (PR), and 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 ).

[0313] Adverse events

[0314] As of June 27, 2019, the incidence of treatment-related adverse events (TRAEs) was 75%, and the incidence of Grade 3 or higher TRAEs was 12.5%. The results showed that the incidence of adverse events of 14C12H1L1 in combination with Anlotinib hydrochloride for 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).

[0315] Table 6 Adverse events of 14C12H1L1 in combination with Anlotinib treatment

[0316]

[0317] The exemplary embodiments described herein can be described by the following numbered paragraphs:

[0318] 1. A pharmaceutical combination comprising:

[0319] a) an inhibitor of the interaction between PD-1 and its ligand PD-L1, and

[0320] b) a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of Formula I or a pharmaceutically acceptable salt thereof,

[0321]

[0322] 2. The pharmaceutical 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.

[0323] 3. The pharmaceutical combination according to any 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.

[0324] 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.

[0325] 5. The pharmaceutical combination according to any of paragraphs 3-4, wherein the anti-PD-1 antibody can be selected from any one or several of Nivolumab, Pembrolizumab, Toripalimab, Sindilimab, Camrelizumab, Tislelizumab, 14C12H1L1, Genolimzumab, Libivimab, HLX-10, BAT-1306, AK103, AK104, CS1003, SCT-I10A, F520, SG001, GLS-010.

[0326] 6. The pharmaceutical combination according to any of paragraphs 3-4, wherein the anti-PD-L1 antibody can be selected from any one or several of Atezolizumab, Avelumab, Durvalumab, KL-A167, SHR-1316, BGB-333, JS003, STI-A1014 (ZKAB0011), KN035, MSB2311, HLX-20, CS-1001.

[0327] 7. The pharmaceutical combination according to any of paragraphs 3-4, wherein the anti-PD-1 antibody comprises:

[0328] a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11,

[0329] 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

[0330] a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14,

[0331] a light chain variable region of the amino acid sequence set forth in 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.

[0332] 8. The pharmaceutical combination according to paragraph 7, wherein the anti-PD-1 antibody comprises:

[0333] a. a heavy chain variable region as set forth in SEQ ID NO: 1 and a light chain variable region as set forth in SEQ ID NO: 4;

[0334] b. a heavy chain variable region as set forth in SEQ ID NO: 2 and a light chain variable region as set forth in SEQ ID NO: 5;

[0335] c. a heavy chain variable region as set forth in SEQ ID NO: 3 and a light chain variable region as set forth in SEQ ID NO: 6;

[0336] d. a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 9;

[0337] e. a heavy chain variable region as set forth in SEQ ID NO: 8 and a light chain variable region as set forth in SEQ ID NO: 10;

[0338] f. a heavy chain variable region as set forth in SEQ ID NO: 11 and a light chain variable region as set forth in SEQ ID NO: 14;

[0339] g. a heavy chain variable region as set forth in SEQ ID NO: 12 and a light chain variable region as set forth in SEQ ID NO: 15;

[0340] h. a heavy chain variable region as set forth in SEQ ID NO: 13 and a light chain variable region as set forth in SEQ ID NO: 16;

[0341] i. a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 18;

[0342] j. a heavy chain variable region as set forth in SEQ ID NO: 31 and a light chain variable region as set forth in SEQ ID NO: 34;

[0343] k. a heavy chain variable region as set forth in SEQ ID NO: 32 and a light chain variable region as set forth in SEQ ID NO: 35;

[0344] l. a heavy chain variable region as depicted in SEQ ID NO: 33 and a light chain variable region as depicted in SEQ ID NO: 36;

[0345] m. a heavy chain as depicted in SEQ ID NO: 37 and a light chain as depicted in SEQ ID NO: 40;

[0346] n. a heavy chain as depicted in SEQ ID NO: 38 and a light chain as depicted in SEQ ID NO: 41 ; or

[0347] o. a heavy chain as depicted in SEQ ID NO: 39 and a light chain as depicted in SEQ ID NO: 42.

[0348] 9. The pharmaceutical combination according to any one of paragraphs 1-8, which is a fixed combination.

[0349] 10. The pharmaceutical combination according to paragraph 9, which is in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition.

[0350] 11. The pharmaceutical combination according to any one of paragraphs 1-8, which is a non-fixed combination.

[0351] 12. The pharmaceutical combination according to paragraph 11, wherein the anti-PD-1 antibody and the compound of Formula I or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition.

[0352] 13. The pharmaceutical combination according to any one of paragraphs 1-12, comprising: a compound of Formula I or a hydrochloride salt thereof; and sintilimab or an antigen-binding fragment thereof, InVivoMAb anti-mouse PD-1 monoclonal antibody or an antigen-binding fragment thereof, or 14C12H1L1 or an antigen-binding fragment thereof.

[0353] 14. Use of the pharmaceutical combination according to any one of paragraphs 1-13 for the treatment or prevention of a liver malignancy.

[0354] 15. The use according to paragraph 14, wherein the pharmaceutical combination is used for the treatment of a primary liver tumor or a secondary liver tumor.

[0355] 16. The use according to paragraph 14, wherein the liver malignancy is hepatocellular carcinoma.

[0356] 17. The use according to paragraph 14, wherein the liver malignancy is metastatic liver cancer.

[0357] 18. The use according to paragraph 17, 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.

[0358] 19. Use of an inhibitor of the interaction between PD-1 and its ligand PD-L1 in combination with a compound of Formula I or a pharmaceutically acceptable salt thereof for the prevention or treatment of cancer.

[0359] 20. A pharmaceutical combination for the prevention or treatment of 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.

[0360] 21. The use of 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 antigen binding fragment thereof that specifically binds to human PD-1 or PD-L1 and blocks the binding of human PD-L1 to human PD-1.

[0361] 22. The use of any one of paragraphs 19-21, wherein the cancer or tumor is selected from a liver tumor (e.g., a liver malignancy such as hepatocellular carcinoma) or a lung tumor (e.g., a lung cancer such as non-small cell lung cancer).

[0362] 23. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination comprising:

[0363] an inhibitor of the interaction between PD-1 and its ligand PD-L1, and

[0364] a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0365] 24. The method of 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 antigen binding fragment thereof.

[0366] 25. The method of 24, wherein the anti-PD-1 antibody can be selected from any one or several of nivolumab, pembrolizumab, tepilumab, sintilimab, camrelizumab, toripalimab, 14C12H1L1, genolimzumab, lixivabtag, HLX-10, BAT-1306, AK103, AK104, CS1003, SCT-I10A, F520, SG001, GLS-010.

[0367] 26. The method of 24, wherein the anti-PD-Ll antibody can be selected from any one or several of Atezolizumab, Avelumab, Durvalumab, KL-A167, SHR-1316, BGB-333, JS003, STI-A1014, KN035, MSB2311, HLX-20, CS-1001.

[0368] 27. The method of 24, wherein the anti-PD-1 antibody comprises:

[0369] a heavy chain variable region comprising an amino acid sequence selected from the group consisting of 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 at least 80% identical thereto; and

[0370] a light chain variable region comprising an amino acid sequence selected from the group consisting of 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 at least 80% identical thereto.

[0371] 28. The method of any one of 23-27, wherein the inhibitor of the interaction between the PD-1 receptor and its ligand PD-L1 is administered about once a week (q1w), about once every 2 weeks (q2w), about once every 3 weeks (q3w), or about once every 4 weeks (q4w).

[0372] 29. The method of any one of 23-28, wherein the method results in an objective response, preferably a complete response or a partial response.

[0373] 30. The method of any one of 23-29, wherein the subject has not previously received systemic chemotherapy.

[0374] 31. The method of any one of 23-30, wherein the subject has not previously received systemic chemotherapy, but has received one or several of surgical treatment, radiation treatment, induction chemotherapy, and / or adjuvant chemotherapy, or the subject receives concurrent chemotherapy.

[0375] 32. The method of any one of 23-30, wherein the subject has previously received systemic chemotherapy.

[0376] 33. The method of any one of 23-32, wherein the cancer is driver gene negative.

[0377] 34. The method of 33, wherein the driver gene negative comprises the subject being negative for 1, 2, or 3 of the EGFR, ALK, and ROS1 genes being mutant.

[0378] 35. The method of any one of 26-32, wherein the EGFR, ALK, and ROS1 genes in the subject are all wild-type genes or are all negative for mutation.

[0379] 36. The method of any one of 26-35, wherein the cancer is lung cancer.

[0380] 37. The method of any one of 23-36, wherein the cancer is recurrent and / or metastatic lung cancer.

[0381] 38. The method of any one of 23-37, wherein the cancer is advanced lung cancer.

[0382] 39. The method of any one of 23-38, wherein the cancer is small cell or non-small cell lung cancer.

[0383] 40. The method of any one of 23-39, wherein the cancer treatment is first line treatment for recurrent or metastatic non-small cell lung cancer.

[0384] 41. The method of any one of 39-40, wherein the non-small cell lung cancer is lung adenocarcinoma, lung squamous cell carcinoma, or lung large cell carcinoma.

[0385] 42. The method of any one of paragraphs 26-35, the cancer is a primary liver tumor or a secondary liver tumor.

[0386] 43. The method of paragraph 41, the liver malignancy is hepatocellular carcinoma.

[0387] 44. The method of paragraph 41, the liver malignancy is metastatic liver cancer.

[0388] 45. The method of paragraph 44, 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.

[0389] 46. A pharmaceutical combination for use in the prevention or treatment of 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, wherein the tyrosine kinase inhibitor is a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0390] 47. The method or pharmaceutical combination according to any one of 23-46, wherein the inhibitor of the interaction between PD-1 receptor and its ligand PD-L1 and the tyrosine kinase inhibitor are each in a pharmaceutical composition, and are administered simultaneously, sequentially or separately.

[0391] 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 2 weeks (14 days) on and 1 week (7 days) off.

[0392] 49. The method or pharmaceutical combination according to 48, wherein the pharmaceutical combination administered per treatment cycle contains about 84-168 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0393] 50. The method or pharmaceutical combination according to 48 or 49, wherein the pharmaceutical combination contains the compound of Formula I or a pharmaceutically acceptable salt thereof in an amount selected from about 84 mg, about 112 mg, about 140 mg, about 168 mg, or a range formed by any of the foregoing.

[0394] 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.

[0395] 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.

[0396] 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.

[0397] 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 per day for 2 weeks on and 1 week off.

[0398] 55. The method or pharmaceutical combination according to any one of 23-54, wherein 100-600 mg of the anti-PD-1 antibody is administered about once every 2 weeks (q2w) or about once every 3 weeks (q3w).

[0399] 56. The method or pharmaceutical combination according to any one of 23-55, wherein about 200 mg of the anti-PD-1 antibody is administered about once every 2 weeks (q2w).

[0400] 57. The method or pharmaceutical combination of any one of 23-56, wherein about 200 mg of the anti-PD-1 antibody is administered once about every 3 weeks (q3w).

[0401] 58. A pharmaceutical pack comprising separate containers containing separately packaged pharmaceutical compositions, wherein one container comprises a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof and a second container comprises a pharmaceutical composition comprising an inhibitor of the interaction between the PD-1 receptor and its ligand, PD-L1.

[0402] 59. The pharmaceutical pack of 58, comprising 84-168 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0403] 60. The pharmaceutical pack of 59, comprising the compound of Formula I or a pharmaceutically acceptable salt thereof in an amount selected from 84 mg, 112 mg, 140 mg, 168 mg, or a range formed by any of the foregoing values.

[0404] 61. The pharmaceutical pack of 60, comprising 112-168 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0405] 62. A kit for treating a liver tumor, comprising (a) a first pharmaceutical composition comprising, as an active ingredient, an inhibitor of the interaction between the PD-1 receptor and its ligand, PD-L1; and (b) a second pharmaceutical composition comprising, as an active ingredient, a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0406] 63. The kit of 62, comprising 84-168 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0407] 64. The kit of 63, comprising the compound of Formula I or a pharmaceutically acceptable salt thereof in an amount selected from 84 mg, 112 mg, 140 mg, 168 mg, or a range formed by any of the foregoing values.

[0408] 65. The kit of 64, comprising 112-168 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0409] 66. A unit formulation, wherein the unit formulation comprises: a compound component comprising 6-12 mg of a compound of Formula I or a hydrochloride salt thereof; and an antibody component comprising 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.

[0410] 67. A method for preventing or treating a cancer or a tumor, wherein one or more unitary formulations of paragraph 66 are administered to a subject in need thereof.

[0411] 68. The method of 67, wherein the cancer or tumor is selected from a liver tumor (e.g., a liver malignancy such as hepatocellular carcinoma) or a lung tumor (e.g., a lung cancer such as non-small cell lung cancer).

[0412] In light of the disclosure provided herein, while compositions and methods according to preferred embodiments are described, modifications can be made in the compositions and / or methods described and in the steps or the order of steps in the methods described herein, without departing from the concept, spirit and scope of the disclosure.

[0413] The disclosures of all documents cited herein are hereby incorporated by reference herein, to the extent that they provide exemplary, procedural and other details that supplement those described herein.

Claims

1. Use of an anti-PD-1 antibody in the preparation of a medicament for treating liver cancer and lung cancer in combination with anlotinib hydrochloride, the anti-PD-1 antibody comprising: a. The heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 4; b. the heavy chain variable region shown in SEQ ID NO: 2 and the light chain variable region shown in SEQ ID NO: 5; c. the heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO: 6; d. the heavy chain variable region as shown in SEQ ID NO: 7 and the light chain variable region as shown in SEQ ID NO: 9; e. the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO: 10; f. The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14; g. The heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 15; h. the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 16; i. a heavy chain as shown in SEQ ID NO: 17 and a light chain as shown in SEQ ID NO: 18; j. the heavy chain variable region shown in SEQ ID NO: 31 and the light chain variable region shown in SEQ ID NO: 34; k. the heavy chain variable region shown in SEQ ID NO: 32 and the light chain variable region shown in SEQ ID NO: 35; l. the heavy chain variable region shown in SEQ ID NO: 33 and the light chain variable region shown in SEQ ID NO: 36; m. a heavy chain as shown in SEQ ID NO: 37 and a light chain as shown in SEQ ID NO: 40; n. The heavy chain as shown in SEQ ID NO: 38 and the light chain as shown in SEQ ID NO: 41; or o. The heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO:

42.

2. The use according to claim 1, wherein the anti-PD-1 antibody comprises 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.

3. The use according to claim 1, wherein the lung cancer is recurrent and / or metastatic lung cancer. The use according to claim 1 , wherein the lung cancer is advanced lung cancer. The use according to claim 1 , wherein the lung cancer is small cell or non-small cell lung cancer. The use according to claim 5 , wherein the non-small cell lung cancer is lung adenocarcinoma, lung squamous cell carcinoma or lung large cell carcinoma. The use according to claim 1 , wherein the liver cancer is a primary liver tumor or a secondary liver tumor. The use according to claim 1 , wherein the liver cancer is hepatocellular carcinoma. The use according to claim 1 , wherein the liver cancer is metastatic liver cancer.

10. The use according to any one of claims 1 to 9, wherein the anti-PD-1 antibody and anlotinib hydrochloride are each in the form of a pharmaceutical composition and can be administered simultaneously, sequentially or intermittently.

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