Combination of a PD-1 antagonist, a VEGFR / FGFR / RET tyrosine kinase inhibitor, and a CBP / β-catenin inhibitor for treating cancer

Abstract

The present disclosure describes a combination therapy comprising an antagonist of the programmed death 1 receptor (PD-1), lenvatinib or a pharmaceutically acceptable salt thereof, and (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, and the use of said combination therapy for the treatment of cancer.

Description

Technical Field

[0001] A combination therapy useful for the treatment of cancer is disclosed. A combination therapy is disclosed that includes lenvatinib, a multi-receptor tyrosine kinase (multi-RTK) inhibitor or a pharmaceutically acceptable salt thereof, which is an antagonist of the programmed death 1 (PD-1) protein, and E7386, which is a CBP / β-catenin inhibitor that inhibits the interaction between CBP and β-catenin, or a pharmaceutically acceptable salt thereof. Also disclosed is an antitumor agent containing a combination of an anti-PD-1 antibody, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof.

Background Art

[0002] PD-1 is recognized as an important player in immune regulation and the maintenance of peripheral tolerance. PD-1 is moderately expressed in naive T-, B-, and natural killer T (NKT)-cells and is upregulated by T / B cell receptor signaling in lymphocytes, monocytes, and myeloid cells (1).

[0003] Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers that occur in various tissues. For example, in large sample sets of cancers of the ovary, kidney, colon, pancreas, liver, and melanoma, PD-L1 expression has been shown to correlate with poor prognosis and to reduce overall survival regardless of subsequent treatment (2 - 13). Similarly, PD-1 expression in tumor-infiltrating lymphocytes has been found to mark dysfunctional T cells in breast cancer and melanoma (14 - 15), and to correlate with poor prognosis in renal cancer (16). It has been proposed that PD-L1-expressing tumor cells contribute to the impairment of the immune response against tumors by interacting with PD-1-expressing T cells and attenuating T cell activation and the avoidance of immune surveillance. Therefore, antibodies against the PD-1 receptor or the PD-L1 ligand can inhibit the binding between them and increase the immune effect on tumor cells (23).

[0004] Several monoclonal antibodies that inhibit the interaction of PD-1 with one or both of its ligands, PD-L1 and PD-L2, have been approved for use by the FDA, and additional monoclonal antibodies are in clinical development for the treatment of cancer. Such antibodies may be more effective if administered in combination with other approved or experimental cancer therapies, such as radiation, surgery, chemotherapeutic agents, targeted therapies, agents that inhibit other signaling pathways that are dysregulated in tumors, and other immune enhancing agents.

[0005] Pembrolizumab is an anti-PD-1 antibody that is approved in the United States for monotherapy or in combination with certain other agents for the treatment of several tumor types. Such tumor types include melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial cancer, high-frequency microsatellite instability cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), and endometrial cancer.

[0006] Tyrosine kinases are important targets for cancer therapy because they are involved in the regulation of growth factor signaling. Lenvatinib is an oral receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activity of vascular endothelial growth factor (VEGF) receptors (VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4)), as well as fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4, in addition to other angiogenesis-promoting pathway-related RTKs and oncogenesis pathway-related RTKs (including platelet-derived growth factor (PDGF) receptor PDGFRα; KIT; and RET proto-oncogene (RET)) that are involved in tumor growth. In particular, lenvatinib has a new binding mode (type V) to VEGFR2, as confirmed by X-ray crystallographic analysis, and exhibits rapid and potent inhibition of kinase activity according to kinetic analysis.

[0007] The chemical name of lenvatinib is 4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide and it has the following structure: [Chemical formula]

[0008] Lenvatinib mesylate has been approved in the United States for the treatment of at least (a) patients with locally recurrent or metastatic progressive radioactive iodine-refractory differentiated thyroid cancer, (b) in combination with everolimus, for the treatment of patients with advanced renal cell carcinoma (RCC) after one prior anti-angiogenic therapy, (c) as first-line treatment for patients with unresectable hepatocellular carcinoma (HCC), and (d) in combination with pembrolizumab, for the treatment of patients with advanced endometrial cancer that is not microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR), whose disease has progressed after prior systemic therapy and who are not candidates for curative surgery or radiation. Lenvatinib mesylate is in clinical trials for use as monotherapy for additional tumor types or in combination therapy with anti-PD-1 antibodies. Additional tumor types include bladder cancer, melanoma, head and neck squamous cell carcinoma, urothelial cancer, breast cancer, gastric cancer, ovarian cancer, colorectal cancer (CRC), glioblastoma, and cholangiocarcinoma.

[0009] Some cancer cells have been observed to have β-catenin that is activated by the Wnt signal. Inhibitors of the Wnt signaling pathway have been studied as anti-cancer agents; however, none have reached practical use.

[0010] The CBP / β-catenin inhibitor under clinical development as an anticancer agent is less toxic in humans than conventional Wnt inhibitors with other mechanisms (J. Clin. Oncol. Vol. 31, 2013 (Separate volume; Abstract 2501)). As its mechanism, after inhibition of the binding between CBP (CREB-binding protein) and β-catenin, P300, which is highly similar to CBP, binds to β-catenin instead of CBP, and such a change is thought to suppress cancer growth and induce differentiation (The EMBO Journal 2013, Vol. 32: 1977-1989).

[0011] It has been shown that the main pathway of cholangiocarcinoma may be tumor growth via the Wnt-β-catenin pathway; ICG-001, a CBP / β-catenin inhibitor, can suppress tumor growth (J. Clin. Invest. March 2, 2015; Vol. 125 (3): 1269-1285. doi:10.1172 / JCI76452).

[0012] Conventional Wnt inhibitors block signals based on mechanisms such as inhibiting the production of Wnt ligands, blocking the function of receptors, and promoting the degradation of β-catenin. Due to such mechanisms, toxicity problems have emerged in preclinical studies and clinical trials, and development has, in most cases, been interrupted. The Wnt signaling pathway is a highly conserved pathway, associated with disorders, and involves β-catenin. The Wnt / β-catenin pathway is important for normal development. Although not bound by theory, CBP / β-catenin binding is thought to reduce Wnt signaling, while p300 / β-catenin is thought to activate Wnt signaling.

[0013] Furthermore, β-catenin is also known to suppress T cell activation by suppressing T cell differentiation (J. Immunol. 2011; Vol. 186: 784-790). Therefore, CBP / β-catenin inhibitors are thought to promote T cell differentiation and T cell activation.

[0014] E7386, whose chemical name is (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide, is a potential CBP / β-catenin inhibitor and has the following structure: [Chemical formula]

[0015] E7386 can exhibit antitumor effects alone (U.S. Patent Nos. 10,259,817 and 9,174,998), or in combination therapy with an anti-PD-1 antibody (U.S. Patent Application Publication No. 2018 / 0185395), or in combination therapy with lenvatinib (Canadian Patent Application Publication No. 3044658).

[0016] Generally, tumor therapeutics are often not effective in all patients when administered individually. Therefore, attempts have been made to increase the cure rate of such therapeutics by combining them (22). Summary of the Invention

[0017] As disclosed herein, administration of a combination of (i) a PD-1 antagonist other than atezolizumab, (ii) lenvatinib or a pharmaceutically acceptable salt thereof, and (iii) E7386 or a pharmaceutically acceptable salt thereof achieves an unexpectedly excellent antitumor effect.

[0018] A method of treating cancer in an individual, the method comprising administering to the individual a combination therapy comprising a PD-1 antagonist other than atezolizumab, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof. In some examples, the individual is a human. The cancer may be a solid tumor, such as renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, urothelial cancer, and head and neck squamous cell carcinoma. The cancer may be advanced cancer or metastatic cancer.

[0019] The PD-1 antagonist of the above method may be a monoclonal antibody or an antigen-binding fragment thereof. In some examples, the antagonist is an anti-PD-1 antibody. The antagonist may be pembrolizumab or nivolumab.

[0020] In some examples, the PD-1 antagonist of the above method is pembrolizumab, semiprimab, or nivolumab, preferably pembrolizumab. Administration of pembrolizumab may occur after administration of lenvatinib or a pharmaceutically acceptable salt thereof, and / or E7386 or a pharmaceutically acceptable salt thereof in some treatment regimens. In some examples, lenvatinib or a pharmaceutically acceptable salt thereof is administered after pembrolizumab and / or E7386 or a pharmaceutically acceptable salt thereof.

[0021] A method of treating a human individual diagnosed with cancer, the method comprising administering a combination therapy to the individual for at least 24 weeks. The combination therapy comprises pembrolizumab, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof. Lenvatinib or a pharmaceutically acceptable salt thereof may each be administered as lenvatinib at a daily dose of 24 mg, 20 mg, 18 mg, 12 mg, or 8 mg, and pembrolizumab may be administered at a dose of 200 mg Q3W or 400 mg Q6W. E7386 or a pharmaceutically acceptable salt thereof can be administered at a dose ranging from about 0.01 to 1000 mg / kg of body weight per day per individual.

[0022] There is provided an agent comprising a PD-1 antagonist for use in a combination of lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof for treating cancer.

[0023] There is also provided an agent comprising lenvatinib or a pharmaceutically acceptable salt thereof for use in a combination of a PD-1 antagonist and E7386 or a pharmaceutically acceptable salt thereof for treating cancer.

[0024] There is also provided an agent comprising E7386 or a pharmaceutically acceptable salt thereof for use in combination with a PD-1 antagonist and lenvatinib or a pharmaceutically acceptable salt thereof for treating cancer.

[0025] There is also provided the use of a treatment combination for treating cancer, the treatment combination comprising a PD-1 antagonist, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof.

[0026] Also provided is the use of a PD-1 antagonist in the manufacture of a medicament for treating cancer in an individual when administered in combination with lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof. Also provided is the use of lenvatinib or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in an individual when administered in combination with a PD-1 antagonist, and E7386 or a pharmaceutically acceptable salt thereof. Also provided is the use of E7386 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in an individual when administered in combination with a PD-1 antagonist, and lenvatinib or a pharmaceutically acceptable salt thereof.

[0027] Also provided is the use of a PD-1 antagonist, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in an individual. The medicament can comprise a kit, and also, the kit can comprise an accompanying document including instructions for using a PD-1 antagonist in combination with lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof for treating cancer in an individual.

[0028] In all of the therapies, medicaments, and uses, the PD-1 antagonist inhibits the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD-1. In some of the above-mentioned therapies, medicaments, and uses, the PD-1 antagonist is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to PD-1 or to PD-L1 to block the binding of PD-L1 to PD-1. For example, the PD-1 antagonist can be an anti-PD-1 antibody comprising a heavy chain and a light chain, and the heavy chain and the light chain comprise the amino acid sequences shown in Figure 6 (SEQ ID NO: 21 and SEQ ID NO: 22). Also provided is a method for treating a tumor, which can include the combined use of an anti-PD-1 antibody, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof.

[0029] In some aspects of the combination therapy, treatment method, agent, and use, the individual is a human, the cancer is a solid tumor, and in some examples, the solid tumor is renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, urothelial carcinoma, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, and head and neck squamous cell carcinoma, or any cancer disclosed herein.

[0030] Also, if the cancer shows a positive response for the expression of one or both of PD-L1 and PD-L2, any of the combination therapy, treatment method, agent, and use can be utilized. In still other examples, the cancer has an elevated PD-L1 expression.

[0031] In some aspects of the combination therapy, treatment method, agent, and use, the individual can be a human, the cancer shows a positive response for human PD-L1, and is selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, and head and neck squamous cell carcinoma. In one embodiment, the bladder cancer is urothelial carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0033] Abbreviations. Throughout the detailed description and examples, the following abbreviations are used: Best Overall Response of BOR Once-daily dose twice a day of BID CREB-binding protein of CBP Clinical benefit rate of CBR Complementary determining region of CDR Chinese hamster ovary of CHO Complete response of CR Colorectal cancer of CRC Disease control rate of DCR Disease-free survival of DFS Dose-limiting toxicity of DLT Duration of response of DOR Durable stable rate of DSDR Formalin-fixed, paraffin-embedded of FFPE Framework region of FR Hepatocellular carcinoma of HCC Immunoglobulin G of IgG Immunohistochemistry or immunohistochemical of IHC Immune-related response criteria of irRC Intravenous of IV Merkel cell carcinoma of MCC Maximum tolerated dose of MTD National Center for Biotechnology Information of NCBI National Cancer Institute of NCI Natural killer T cell of NKT Non-small cell lung cancer of NSCLC Overall response rate of ORR Overall survival of OS Progressive disease of PD Programmed cell death 1 of PD-1 Programmed cell death 1 ligand 1, also known as B7-H1 of PD-L1 Programmed cell death 1 ligand 2, also known as B7-DC of PD-L2 Progression-free survival of PFS Partial response of PR Once-daily dose every two weeks of Q2W Once-daily dose every three weeks of Q3W Once-daily dose per day of QD Renal cell carcinoma of RCC Response Evaluation Criteria in Solid Tumors of RECIST Receptor tyrosine kinase of RTK Small cell lung cancer (SCLC) Stability (SD) Vascular endothelial growth factor (VEGF) Variable region of immunoglobulin heavy chain (VH) Variable region of immunoglobulin kappa light chain (VK)

[0034] Definitions. To facilitate a better understanding of the methods, compositions, and uses, certain technical and scientific terms are specifically defined below. Unless otherwise defined elsewhere in this specification, all other technical and scientific terms used herein shall have the meanings commonly understood by those skilled in the art.

[0035] As used herein to modify a numerically defined parameter (e.g., the dosage of a PD-1 antagonist such as (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, or lenvatinib or a pharmaceutically acceptable salt thereof, or the length of the treatment time by the combination therapy described herein), "about" means that the parameter can vary by less than 10% or more than 10% of the explicitly stated numerical value for that parameter. For example, a dosage of about 20 mg may vary between 18 mg and 22 mg.

[0036] "Preferably" means a more desired choice. For example, when used to modify a numerically defined parameter, "preferably" indicates that the preferred parameter results in an improvement over another value for that parameter. This meaning of "preferably" applies only outside the United States. For the United States, any sentence using "preferably" should be read as if the term were not present.

[0037] As used herein, the singular forms of words such as "a," "an," and "the," including the appended claims, include the corresponding plural references unless the context clearly states otherwise.

[0038] "Administration" and "treatment" as used in connection with an animal, human, subject, cell, tissue, organ, or biological fluid refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell and contact of a reagent with a fluid (where the fluid is in contact with the cell). Also, "administration" and "treatment" mean, for example, in vitro and ex vivo treatment of a cell with a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, and rabbit), and most preferably a human.

[0039] The term "antibody" as used herein refers to any form of an antibody that exhibits the desired biological or binding activity. Thus, the term is used in its broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, chimeric antibodies, and camelized single domain antibodies. A "parent antibody" is an antibody obtained by exposure of the immune system to an antigen prior to modification of the antibody for its intended use, e.g., humanization of the antibody for use as a human therapeutic.

[0040] Generally, the basic antibody structural unit contains a tetramer. Each tetramer contains two identical pairs of polypeptide chains, and each pair has one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is mainly responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region that is mainly responsible for effector functions. Typically, human light chains are classified as kappa light chains and lambda light chains. Further, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of more than about 10 amino acids. Generally, see CHAPTER 7 OF FUNDAMENTAL IMMUNOLOGY (edited by Paul, W., 2nd edition, Raven Press, N.Y. (1989)).

[0041] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, generally, an intact antibody has two binding sites. Except for bifunctional antibodies or bispecific antibodies, the two binding sites are generally the same.

[0042] Typically, the variable domains of both the heavy and light chains, also called complementarity-determining regions (CDRs), contain three hypervariable regions, which are located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions to enable binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both the light-chain variable domain and the heavy-chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain usually follow the definitions of SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publication No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al. (1977) J. Biol. Chem. 252:6609-6616; Chothia et al. (1987) J Mol. Biol. 196:901-917, or Chothia et al. (1989) Nature 342:878-883.

[0043] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable regions include amino acid residues derived from "complementary determining regions" or "CDRs" (i.e., CDRL1, CDRL2, and CDRL3 in the light-chain variable domain and CDRH1, CDRH2, and CDRH3 in the heavy-chain variable domain). See Kabat et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of antibodies by sequence); also see Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies by structure). As used herein, the term "framework" or "FR" residues refers to the variable domain residues other than the CDR residues and the hypervariable region residues as defined herein.

[0044] As used herein, the "variable region" or "V region" refers to a segment of an IgG chain whose sequence is variable between different antibodies. The "variable region" or "V region" extends to Kabat residue 109 in the light chain and 113 in the heavy chain.

[0045] Unless otherwise specified, the "antibody fragment" or "antigen-binding fragment" used herein refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by the full-length antibody, for example, a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab’, F(ab’) 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments, and bispecific antibodies.

[0046] An antibody that "specifically binds" to a designated target protein is an antibody that exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered to be "specific" for its intended target if its binding is a determinant of the presence of the target protein in a sample without resulting in undesired outcomes such as false positives. An antibody or its binding fragment binds to a target protein with an affinity that is at least 2-fold greater, preferably at least 10-fold greater, more preferably at least 20-fold greater, and most preferably at least 100-fold greater than its affinity for non-target proteins. An antibody used herein is said to specifically bind to a polypeptide containing a given amino acid sequence, for example, the amino acid sequence of the mature human PD-1 or human PD-L1 molecule, if it binds to the polypeptide containing that sequence but does not bind to a protein lacking that sequence.

[0047] A "chimeric antibody" refers to an antibody in which part of the heavy chain and / or light chain is derived from a particular species (e.g., human), or is identical or homologous to the corresponding sequence in an antibody belonging to a particular antibody class or subclass, while the remainder of the chain(s) is derived from another species (e.g., mouse), or is identical or homologous to the corresponding sequence in an antibody belonging to another antibody class or subclass, or a fragment of such an antibody, as long as it exhibits the desired biological activity.

[0048] A "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse carbohydrate chains if produced in a mouse, in mouse cells, or in a hybridoma derived from mouse cells. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin sequences or rat immunoglobulin sequences, respectively.

[0049] A "humanized antibody" refers to a form of an antibody that contains sequences derived from a non-human (e.g., mouse) antibody and a human antibody. Such an antibody contains the minimum sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one, typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of the non-human immunoglobulin and all or substantially all of the FR regions being those of the human immunoglobulin sequence. Also, a humanized antibody optionally contains the immunoglobulin constant region (Fc), typically at least a portion of the Fc of a human immunoglobulin. When necessary to distinguish a humanized antibody from the parental rodent antibody, the prefix "hum", "hu", or "h" is added to the antibody clone designation. The humanized form of a rodent antibody usually contains the same CDR sequences as the parental rodent antibody, but may include specific amino acid substitutions for reasons such as increasing affinity, increasing the stability of the humanized antibody, or other reasons.

[0050] "Isolated antibody" and "isolated antibody fragment" refer to a purified state, and in such context, the named molecule is substantially free from other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Usually, the term "isolated" is not intended to refer to either the complete absence of such materials or the absence of water, buffer, or salts, so long as such materials are not present in an amount that substantially interferes with the experimental or therapeutic use of the binding compounds described herein.

[0051] As used herein, "Kabat" refers to the immunoglobulin alignment and numbering system originated by Elvin A. Kabat ((1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md.).

[0052] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a substantially homogeneous population of antibodies. That is, the antibody molecules comprising the population are identical in amino acid sequence except for naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a multiplicity of different antibodies having different amino acid sequences in the variable domains, particularly within the CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the property of antibodies obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibodies by any particular method. For example, the monoclonal antibodies to be used in accordance with the therapeutic methods, agents, and uses disclosed may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant deoxyribonucleic acid (DNA) methods (see, e.g., U.S. Patent No. 4,816,567). The term "monoclonal antibody" may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also, e.g., Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0053] As used herein, "CDR" or "CDRs" means the complementarity determining region(s) of the immunoglobulin variable region as defined using the Kabat numbering system, unless otherwise specified.

[0054] When referring to a cancer patient treated by a treatment plan such as the combination therapy described in this specification, "anti-tumor response" means at least one positive treatment effect, for example, a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, a decrease in the rate of tumor metastasis or tumor growth rate, or progression-free survival. Positive treatment effects in cancer can be measured in several ways (W.A. Weber, J. Null. Med. 50: 1S - 10S (2009); Eisenhauer et al., supra). In some examples, the anti-tumor response to the combination therapy described in this specification is evaluated using the RECIST 1.1 criteria (response evaluation criteria in solid tumors), two-dimensional irRC (immune-related response criteria), or one-dimensional irRC. In some examples, the anti-tumor response is any of SD, PR, CR, PFS, or DFS.

[0055] "Two-dimensional irRC" refers to a set of criteria described in Wolchok JD et al., "Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria", Clin Cancer Res. 15(23): 7412 - 7420 (2009). These criteria utilize two-dimensional tumor measurements of target lesions, which are obtained by multiplying the longest diameter and the longest perpendicular diameter of each lesion (cm 2 )).

[0056] "Biological therapeutic agent" means a biomolecule, such as an antibody or a fusion protein, that blocks ligand / receptor signaling in any biological pathway that supports tumor maintenance and / or growth or suppresses the anti-tumor immune response. Classes of biological therapeutic agents include, but are not limited to, antibodies against VEGF, epidermal growth factor receptor (EGFR), Her2 / neu, other growth factor receptors, CD20, CD40, CD - 40L, CTLA - 4, OX - 40, 4 - 1BB, and ICOS.

[0057] The terms "cancer", "cancerous", "tumor", or "malignant" refer to or describe a physiological state in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, such as urothelial carcinoma, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, and head and neck squamous cell carcinoma. Another specific example of cancer is renal cell carcinoma (RCC). A further specific example of cancer is clear cell renal carcinoma. Cancer can be primary cancer, but is likely to be advanced in the cancer staging classification that includes metastatic disease (e.g., lymphatic disorder or other organ disorder). Cancers that can be treated according to the disclosed therapies, agents, and disclosed uses include cancers characterized by high expression of one or both of PD-L1 and PD-L2 in a tissue sample being tested.

[0058] "CBR" or "clinical benefit rate" means CR + PR + durable SD.

[0059] "Chemotherapeutic agent" is a compound useful in the treatment of cancer. Classes of chemotherapeutic agents that can be used in combination with the therapeutic combinations, methods, and uses described herein include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antineoplastic antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterones, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR (epidermal growth factor receptor) inhibitors, VEGF (vascular endothelial growth factor) inhibitors, VEGFR (vascular endothelial growth factor receptor) inhibitors, and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents useful in the therapies disclosed herein include cell growth inhibitors and / or cytotoxic agents.

[0060] As used herein, "Chothia" refers to the antibody numbering system described in Al-Lazikani et al., JMB 273:927-948 (1997).

[0061] "Comprising" or variations thereof, such as "comprise", "comprises", or "comprised of", is used throughout this specification and the claims in an inclusive sense, that is, specifying the presence of the stated features, but not excluding the presence or addition of further features that may materially enhance any operation or utility of the disclosed therapeutic methods, agents, and disclosed uses, unless the context requires a different interpretation due to express language or necessary implication.

[0062] "Conservative modification variant" or "conservative substitution" refers to substitutions in a protein's amino acids by other amino acids having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone higher-order structure, and rigidity, among others), which can frequently cause changes without altering the protein's biological activity or other desired properties, such as antigen affinity and / or specificity. One of ordinary skill in the art generally recognizes that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224 (4th edition)). Also, substitutions of structurally or functionally similar amino acids are less likely to impair biological activity. Exemplary conservative substitutions are shown in Table 1 below.

[0063] [Table 1]

[0064] As used throughout this specification and the claims, "consists essentially of", and variations thereof, such as "consist essentially of" or "consisting essentially of", indicate the inclusion of any recited element or group of elements, and the optional inclusion of other elements of similar or different nature that do not materially change the basic and novel characteristics of the recited dosage regimen, method, or composition. By way of non-limiting example, a PD-1 antagonist consisting essentially of the recited amino acid sequence may also include one or more amino acids containing substitutions of one or more amino acid residues that do not materially affect the properties of the binding compound.

[0065] "DCR" or "disease control rate" means CR + PR + SD.

[0066] "Diagnostic anti-PD-L monoclonal antibody" means an mAb that specifically binds to the mature form of the designated PD-L (PD-L1 or PDL2) expressed on the surface of certain mammalian cells. Mature PD-L lacks the presecretory leader sequence, also referred to as the leader peptide. The terms "PD-L" and "mature PD-L" are used interchangeably herein and will be understood to mean the same molecule unless otherwise specified or readily apparent from the context.

[0067] As used herein, diagnostic anti-human PD-L1 mAb or anti-hPD-L1 mAb refers to a monoclonal antibody that specifically binds to mature human PD-L1. The mature human PD-L1 molecule consists of amino acids 19 - 290 of the following sequence: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 25).

[0068] Specific examples of diagnostic anti-human PD-L1 mAbs useful as diagnostic mAbs for IHC detection of PD-L1 expression in FFPE tumor tissue sections are antibody 20C3 and antibody 22C3, which are described in International Application PCT / US13 / 075932 filed on December 18, 2013 (published as International Publication No. 2014 / 100079 on June 26, 2014). Another anti-human PD-L1 mAb (Chen, B.J. et al., Clin. Cancer Res. 19:3462-3473 (2013)), which has been reported to be useful for IHC detection of PD-L1 expression in FFPE tissue sections, is a rabbit anti-human PD-L1 mAb publicly available from Sino Biological, Inc. (Beijing, P.R. China; Catalog No. 10084-R015).

[0069] "DSDR" or "Durable Stability Rate" means SD for ≧ 23 weeks.

[0070] As used herein, "framework region" or "FR" means the immunoglobulin variable region excluding the CDR regions.

[0071] "Identity" refers to the sequence similarity between two polypeptide sequences when the two polypeptide sequences are optimally aligned. If the positions in both of the two compared sequences are occupied by the same amino acid monomer subunit, for example, if the positions within the light chain CDRs of two different Abs are occupied by alanine, then the two Abs are identical at that position. The percentage of identity is the number of identical positions shared by the two sequences divided by the total number of positions being compared, multiplied by 100. For example, if 8 out of 10 positions in two sequences match or are identical when the sequences are optimally aligned, then the two sequences are 80% identical. Usually, the comparison is made when the two sequences are aligned to give the maximum percent identity. For example, the comparison can be performed by the Basic Local Alignment Search Tool (BLAST®) algorithm, which is a registered trademark of the National Library of Medicine, and the parameters of the algorithm are selected to give the largest match between each pair of sequences over the full length of each reference sequence.

[0072] The following representative references are related to the BLAST (registered trademark) algorithms that are widely used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al. (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al. (1993) Nature Genet. 3: 266-272; Madden, T.L. et al. (1996) Meth. Enzymol. 266: 131-141; Altschul, S.F. et al. (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al. (1997) Genome Res. 7: 649-656; Wootton, J.C. et al. (1993) Comput. Chem. 17: 149-163; Hancock, J.M. et al. (1994) Comput. Appl. Biosci. 10: 67-70; ATLAS OF PROTEIN SEQUENCE AND STRUCTURE (1978) Vol. 5, Suppl. 3, M.O. Dayhoff (ed.), 345-352, Natl. Biomed. Res. Found., Washington, DC. ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O. et al., "A model of evolutionary change in proteins."; ATLAS OF PROTEIN SEQUENCE AND STRUCTURE (1978) Vol. 5, Suppl. 3, M.O. Dayhoff (ed.), 353-358, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M. et al., "Matrices for detecting distant relationships."; Altschul, S.F. (1991) J. Mol. Biol. 219: 555-565; States, D.J. et al. (1991) Methods 3: 66-70; Henikoff, S. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, S.F. et al. (1993) J. Mol. Evol. 36: 290-300; ALIGNMENT STATISTICS: Karlin, S. et al. (1990) Proc.Natl. Acad. Sci. USA, Vol. 87, pp. 2264 - 2268; Karlin, S. et al. (1993) Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 5873 - 5877; Dembo, A. et al., (1994) Ann. Prob. 22:2022 - 2039; and Altschul, S.F. "Evaluating the statistical significance of multiple distinct local alignments." in THEORETICAL AND COMPUTATIONAL METHODS IN GENOME RESEARCH (edited by S. Suhai), (1997), pp. 1 - 14, Plenum, New York.

[0073] When referring to a "non - responder patient" in the context of treatment by the combination therapies described herein for a specific anti - tumor response, it means that the patient did not show an anti - tumor response.

[0074] "ORR" or "overall response rate" sometimes refers to CR + PR, and ORR (week 24) refers to CR and PR measured by irRECIST for each patient in the cohort 24 weeks after treatment with lenvatinib mesylate combined with pembrolizumab.

[0075] "Patient" or "subject" or "individual" refers to any single subject for whom treatment is desired, or who is participating in a clinical trial, an epidemiological study, or is used as a control, including humans, as well as veterinary patients of mammals, such as cows, horses, dogs, and cats.

[0076] PD-1 antagonist. The anti-PD1 antagonist is as follows. A "PD-1 antagonist" means any compound or biomolecule that blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells, or NKT cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to immune cell-expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include the following: PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In any of the therapies, agents, and disclosed uses for which a human individual is to be treated, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD-1. The human PD-1 amino acid sequence can be found at NCBI locus number: NP_005009. The human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbers: NP_054862 and NP_079515, respectively. The PD-1 antagonist is not the anti-PD-L1 monoclonal antibody atezolizumab.

[0077] Examples of PD-1 antagonists useful in any of the therapies, agents, and disclosed uses include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to PD-1 or PD-L1, preferably specifically bind to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. The human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and preferably, the human constant region is an IgG1 or IgG4 constant region. In some examples, the antigen-binding fragment is selected from the group consisting of Fab, Fab’-SH, F(ab’) 2 , scFv, and Fv fragments.

[0078] Any monoclonal antibody that binds to a PD-1 polypeptide, a PD-1 polypeptide fragment 10, a PD-1 peptide, or a PD-1 epitope and blocks the interaction between PD-1 and its ligand PD-L1 or PD-L2 can be used. In some embodiments, the anti-human PD-1 monoclonal antibody binds to a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope and blocks the interaction between PD-1 and PD-L1. In other embodiments, the anti-human PD-1 monoclonal antibody binds to a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope and blocks the interaction between PD-1 and PD-L2. In still other embodiments, the anti-human PD-1 monoclonal antibody binds to a PD-1 polypeptide, a PD-1 polypeptide fragment, a PD-1 peptide, or a PD-1 epitope and blocks the interaction between PD-1 and PD-L1 and the interaction between PD-1 and PD-L2.

[0079] Also, any monoclonal antibody that binds to a PD-L1 polypeptide, a PD-L1 polypeptide fragment 20, a PD-L1 peptide, or a PD-L1 epitope and blocks the interaction between PD-L1 and PD-1 can be used.

[0080] In certain embodiments, the anti-human PD-1 monoclonal antibody is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, pidilizumab (U.S. Patent No. 7,332,582), AMP-514 (MedImmune LLC, Gaithersburg, MD), PDR001 (U.S. Patent No. 9,683,048), BGB-A317 (U.S. Patent No. 8,735,553), and MGA012 (MacroGenics, Rockville, MD).

[0081] In one embodiment, the anti-human PD-1 monoclonal antibody is pembrolizumab. In another embodiment, the anti-human PD-1 monoclonal antibody is nivolumab. In another embodiment, the anti-human PD-1 monoclonal antibody is semiprimab. In yet another embodiment, the anti-human PD-1 monoclonal antibody is pidilizumab. In one embodiment, the anti-human PD-1 monoclonal antibody is AMP-514. In another embodiment, the anti-human PD-1 monoclonal antibody is PDR001. In yet another embodiment, the anti-human PD-1 monoclonal antibody is BGB-A317. In yet another embodiment, the anti-human PD-1 monoclonal antibody is MGA012.

[0082] Examples of mAbs that bind to human PD-1 and are useful for the therapeutic methods, agents, and uses disclosed are described in U.S. Patent No. 7,488,802, U.S. Patent No. 7,521,051, U.S. Patent No. 8,008,449, U.S. Patent No. 8,354,509, and U.S. Patent No. 8,168,757; International Publication No. 2004 / 004771, International Publication No. 2004 / 072286, and International Publication No. 2004 / 056875, and U.S. Patent Application Publication No. 2011 / 0271358. Specific anti-human PD-1 mAbs useful as PD-1 antagonists in the therapeutic methods, agents, and uses disclosed include the following: pembrolizumab (also known as MK-3475), a humanized IgG4 mAb (the structure is described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013) and includes the heavy-chain amino acid sequence and light-chain amino acid sequence shown in Figure 6), nivolumab (BMS-936558), a human IgG4 mAb (the structure is described in WHO Drug Information, Vol. 27, No. 1, pp. 68-69 (2013) and includes the heavy-chain amino acid sequence and light-chain amino acid sequence shown in Figure 7), pidilizumab, a humanized monoclonal antibody, AMP-224, and AMP-514; the humanized antibodies h409A11, h409A16, and h409A17 (described in International Publication No. 2008 / 156712), and AMP-514 (developed by MedImmune).

[0083] Examples of mAbs that bind to human PD-L1 and are useful for the therapies, agents, and uses disclosed are described in WO 2013 / 019906, WO 2010 / 077634 A1, and US Patent No. 8,383,796. Specific anti-human PD-L1 mAbs useful as PD-1 antagonists for the therapies, agents, and uses disclosed include BMS-936559, pembrolizumab, MEDI4736, MSB0010718C, and antibodies comprising the heavy chain variable region and light chain variable region of SEQ ID NO: 24 and SEQ ID NO: 21 of WO 2013 / 019906, respectively.

[0084] Other PD-1 antagonists useful for any of the therapies, agents, and uses disclosed include immunoadhesins that specifically bind to PD-1 or PD-L1, preferably human PD-1 or human PD-L1, such as fusion proteins containing the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region such as the Fc region of an immunoglobulin molecule. Examples of immunoadhesin molecules that specifically bind to PD-1 are described in WO 2010 / 027827 and WO 2011 / 066342. A specific fusion protein useful as a PD-1 antagonist for the therapies, agents, and uses described herein is the PD-L2-Fc fusion protein, and AMP-224 (also known as B7-DCIg) that binds to human PD-1.

[0085] The therapies, agents, and uses disclosed contemplate PD-1 antagonists that are monoclonal antibodies or antigen-binding fragments thereof comprising (a) light chain CDR SEQ ID NOs: 1, 2, and 3, and heavy chain CDR SEQ ID NOs: 4, 5, and 6; or (b) light chain CDR SEQ ID NOs: 7, 8, and 9, and heavy chain CDR SEQ ID NOs: 10, 11, and 12.

[0086] The treatment methods, agents, and disclosed uses contemplate a PD-1 antagonist that specifically binds to human PD-1 and is a monoclonal antibody or an antigen-binding fragment thereof that comprises (a) a heavy-chain variable region comprising SEQ ID NO: 13 or a variant thereof, and (b) a light-chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15 or a variant thereof; SEQ ID NO: 16 or a variant thereof; and SEQ ID NO: 17 or a variant thereof. A variant of the heavy-chain variable region sequence is identical to the reference sequence except having up to 17 conservative amino acid substitutions in the framework region (i.e., outside the CDRs), preferably having 10, 9, 8, 7, 6, or less than 5 conservative amino acid substitutions in the framework region. A variant of the light-chain variable region sequence is identical to the reference sequence except having up to 5 conservative amino acid substitutions in the framework region (i.e., outside the CDRs), preferably having 4, 3, or less than 2 conservative amino acid substitutions in the framework region.

[0087] The PD-1 antagonist for any of the treatment methods, agents, and disclosed uses can be a monoclonal antibody that specifically binds to human PD-1 and comprises (a) a heavy chain comprising SEQ ID NO: 14, and (b) a light chain comprising SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0088] The treatment methods, agents, and disclosed uses contemplate a PD-1 antagonist that specifically binds to human PD-1 and is a monoclonal antibody that comprises (a) a heavy chain comprising SEQ ID NO: 14, and (b) a light chain comprising SEQ ID NO: 18.

[0089] Table 2 below shows a list of the amino acid sequences of exemplary anti-PD-1 mAbs used in the treatment methods, agents, and disclosed uses, and the sequences are shown in FIGS. 1-5B.

[0090]

Table 2

[0091] As used herein, the expression of "PD-L1" or "PD-L2" means any detectable level of expression of the designated PD-L protein on the cell surface or the designated PD-L mRNA in cells or tissues. PD-L protein expression can be detected by a diagnostic PD-L antibody in an IHC assay of tumor tissue sections or by flow cytometry. In addition, PD-L protein expression by tumor cells can be detected by positron emission tomography (PET) imaging using a binder (e.g., antibody fragment, affibody, etc.) that specifically binds to the desired PD-L target, such as PD-L1 or PD-L2. Techniques for detecting and measuring PD-L mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0092] Several approaches for quantifying PD-L1 protein expression by IHC assay of tumor tissue sections have been described. See, for example, Thompson, R. H. et al., PNAS 101(49):17174-17179 (2004); Thompson, R. H. et al., Cancer Res. 66:3381-3385 (2006); Gadiot, J. et al., Cancer 117:2192-2201 (2011); Taube, J. M. et al., Sci Transl Med 4:127-37 (2012); and Toplian, S. L. et al., New Eng. J Med. 366(26):2443-2454 (2012).

[0093] As one approach, a simple binary endpoint of positive or negative for PD-L1 expression was used. A positive result was defined as the percentage of tumor cells showing histological evidence of cell-surface membrane staining. Tumor tissue sections were counted as positive for PD-L1 expression if at least 1%, preferably 5%, of the total tumor cells were positive.

[0094] In another approach, PD-L1 expression in tumor tissue sections is quantified in tumor cells and in infiltrating immune cells mainly comprising lymphocytes. The percentages of tumor cells and infiltrating immune cells showing membrane staining are quantified separately as <5%, 5 - 9%, and in 10% increments up to 100%. For tumor cells, PD-L1 expression is counted as negative if the score is <5% and positive if the score is ≧5%. PD-L1 expression in the immune infiltrate is reported as a semi-quantitative measurement called the adjusted inflammation score (AIS), which is determined by multiplying the percentage of membrane-stained cells by the intensity of the infiltrate. The semi-quantitative measurement is graded as none (0), mild (score of 1, rare lymphocytes), moderate (score of 2, focal infiltration of the tumor by lymphohistiocytic aggregates), or severe (score of 3, diffuse infiltration). If the AIS is ≧5, the tumor tissue section is counted as positive for PD-L1 expression by the immune infiltrate.

[0095] The level of PD-L mRNA expression may be compared to the mRNA expression levels of one or more reference genes frequently used in quantitative RT-PCR, such as ubiquitin C.

[0096] In some examples, the level of PD-L1 expression (protein and / or mRNA) by malignant cells and / or infiltrating immune cells within the tumor is determined to be "overexpressed" or "elevated" based on comparison to the level of PD-L1 expression (protein and / or mRNA) by an appropriate control. For example, the control PD-L1 protein or mRNA expression level may be the level quantified in non-malignant cells of the same type or in sections from matched normal tissue (i.e., non-malignant tissue). PD-L1 expression in the tumor sample is preferably determined to be elevated if the PD-L1 protein (and / or PD-L1 mRNA) in the sample is at least 10%, 20%, or 30% greater than in the control.

[0097] "Pembrolizumab biosimilar" means a biological product manufactured by an entity other than Merck & Co., Inc. d.b.a. Merck Sharp and Dohme (MSD), and has been approved by the regulatory authorities of any country for sale and purchase as a pembrolizumab biosimilar. The pembrolizumab biosimilar may contain, as the active ingredient, a pembrolizumab variant or antibody having the same amino acid sequence as pembrolizumab.

[0098] As used herein, "pembrolizumab variant" means a monoclonal antibody that contains the same heavy-chain and light-chain sequences as those within pembrolizumab, except that it has 3, 2, or 1 conservative amino acid substitutions at positions outside the light-chain CDRs and 6, 5, 4, 3, 2, or 1 conservative amino acid substitutions at positions outside the heavy-chain CDRs. For example, the variant positions are located within the FR regions and / or the constant regions. In other words, pembrolizumab and the pembrolizumab variant contain the same CDR sequences but differ from each other due to having conservative amino acid substitutions at no more than 3 or 6 other positions within the full-length light-chain and heavy-chain sequences, respectively. The pembrolizumab variant is substantially the same as pembrolizumab with respect to the following characteristics: binding affinity for PD-1 and the ability to block the binding of each of PD-L1 and PD-L2 to PD-1.

[0099] Definition of patient / cancer / response. As used herein, "RECIST 1.1 response criteria" means the definitions set forth by Eisenhauer E.A. et al., Eur. J. Cancer 45:228-247 (2009) for target or non-target lesions, as appropriate, based on the context in which the response is being measured.

[0100] When referring to a "responder patient" in the context of a specific anti-tumor response to treatment by the combination therapies described herein, it means that the patient has shown an anti-tumor response.

[0101] "Sustained response" means a continuing therapeutic effect after cessation of treatment with a therapeutic agent or combination therapy as described herein. In some examples, the sustained response has a duration that is at least as long as, or at least 1.5, 2.0, 2.5, or 3 times longer than, the treatment period.

[0102] "Tissue section" refers to a single part or piece of a tissue sample, e.g., a thin slice of tissue cut from a sample of normal tissue or a tumor.

[0103] As used herein, "treating" or "treatment" of cancer means administering to a subject having or diagnosed with cancer a combination therapy of a PD-1 antagonist, lenvatinib, or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof, to achieve at least one positive therapeutic effect, such as a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell infiltration into peripheral organs, or a reduction in the rate of tumor metastasis or tumor growth. Positive therapeutic effects in cancer can be measured in several ways (see W.A. Weber, J. Nucl. Med. 50:1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI criteria, T / C ≤ 42% is the minimum level of antitumor activity. T / C < 10% is considered a high level of antitumor activity (T / C (%) = median tumor volume of treatment / median tumor volume of control × 100). In some examples, the response to the combination therapies described herein is evaluated using a treatment achieved by a combination of RECIST 1.1 criteria or irRC (two-dimensional or one-dimensional), and lenvatinib or a pharmaceutically acceptable salt thereof, (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, and a PD-1 antagonist, and is any of PR, CR, OR, PFS, DFS, and OS. PFS, also referred to as "time to tumor progression," indicates the length of time during and after treatment during which the cancer does not grow, and includes the amount of time the patient experiences CR or PR, and the amount of time the patient experiences SD. DFS refers to the length of time during and after treatment during which the patient remains disease-free. OS refers to an increase in mean survival compared to naive or untreated individuals or patients.In some cases, the response to a combination of lenvatinib or a pharmaceutically acceptable salt thereof, (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, and a PD-1 antagonist is any one of PR, CR, PFS, DFS, OR, and OS evaluated using the RECIST 1.1 response criteria. The treatment plan for the disclosed combination, which is effective for treating cancer patients, may vary according to factors such as the patient's disease state, age, and weight, and the ability of the treatment to induce an anti-cancer response in the subject. The treatment method, drug, and disclosed use do not have to be effective for achieving a positive treatment effect for each subject, and should be effective in a statistically significant number of subjects determined by any statistical test known in the art, such as Student's t-test, chi. 2 test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0104] The terms "treatment plan", "administration protocol", and "administration plan" are used interchangeably to refer to the dosage and timing of administration of each therapeutic agent in a combination of lenvatinib or a pharmaceutically acceptable salt thereof, (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, and a PD-1 antagonist.

[0105] The "tumor" used in a subject diagnosed with cancer or suspected of having cancer refers to a malignant or potentially malignant neoplasm or mass of tissue of any size, including primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid regions. The various types of solid tumors are named for the type of cells that form the solid tumor. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.

[0106] The "tumor burden", also referred to as "tumor load", refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor(s) in the body, including lymph nodes and bone marrow. Tumor burden can be determined by various methods known in the art, for example, by measuring the dimensions of the tumor(s) using calipers, for example, at the time of removal from the subject, or in vivo, using imaging techniques such as ultrasound, bone scan, computed tomography (CT), or magnetic resonance imaging (MRI) scan.

[0107] The term "tumor size" refers to the total size of the tumor, which can be measured as the overall length and width of the tumor. Tumor size can be determined by various methods known in the art, for example, by measuring the dimensions of the tumor(s) using calipers, for example, at the time of removal from the subject, or in vivo, using imaging techniques such as bone scan, ultrasound, CT, or MRI scan.

[0108] "One-dimensional irRC" refers to a set of criteria described in Nishino M, Giobbie-Hurder A, Gargano M, Suda M, Ramaiya NH, Hodi FS. "Developing a Common Language for Tumor Response to Immunotherapy: Immune-related Response Criteria using Unidimensional measurements", Clin. Cancer Res. 2013, Vol. 19 (No. 14): pp. 3936-3943. These criteria utilize the longest diameter (cm) of each lesion.

[0109] "Multi-RTK inhibitor" means a small molecule compound that inhibits the receptor tyrosine kinase (RTK) activity of at least each of the following RTKs: (i) VEGFR2, and (ii) at least one FGFR selected from the group consisting of FGFR1, 2, 3, and 4. An exemplary multi-RTK inhibitor is lenvatinib or a pharmaceutically acceptable salt thereof.

[0110] β-catenin functions as a mediator of Wnt signaling, binds to the transcription factor Tcf / Lef (T cell factor / lymphocyte enhancer factor), promotes the expression of various genes involved in Wnt signaling (such as cyclin D1, C-Myc, etc.), and controls cell proliferation and differentiation (He et al., Science 281: 1509-1512, 1998; Kolligs et al., Mol. Cell. Biol. 19: 5696-5706, 1999; Crawford et al., Oncogene 18: 2883-2891, 1999; Shtutman et al., Proc. Natl. Acad. Sci. USA, 11: 5522-5527, 1999; Tetsu and McCormick, Nature 398: 422-426, 1999).

[0111] CBP (cyclic AMP response element-binding protein (CREB)-binding protein) directly interacts with β-catenin within the CREB-binding domain to promote the transcriptional activation of Tcf / Lef (Ken-Ichi Takemaru and Randall T. Moon, 2000, J. Cell. Biol. 149(2):249-254). A CBP / β-catenin inhibitor is not particularly limited as long as it inhibits the interaction between CBP and catenin, particularly β-catenin. As a result of inhibiting the binding of β-catenin and CBP, an embodiment in which gene expression by the β-catenin complex is suppressed is preferred.

[0112] Inhibition of CBP / β-catenin can be measured by binding assays (such as radio-binding assays and others), reporter assay methods, and other in vitro assays and in vivo assays that are known per se. Inhibition can be confirmed by measuring gene expression of Wnt signaling by the reporter assay method described in International Publication No. 2009 / 148192.

[0113] The CBP / β-catenin inhibitor of the present invention is not particularly limited as long as it is as defined above. The CBP / β-catenin inhibitor of the present invention is preferably an α-helix mimetic compound having CBP / β-catenin inhibitory activity. Examples thereof include α-helix mimetic compounds and pharmaceutically acceptable salts thereof as described in International Publication No. 2003 / 031448, International Publication No. 2004 / 093828, International Publication No. 2005 / 116032, International Publication No. 2009 / 148192, International Publication No. 2010 / 044485, International Publication No. 2010 / 128685, and International Publication No. 2012 / 115286. An exemplary CBP / β-catenin inhibitor is (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386).

[0114] In the combination therapies disclosed herein, each of the PD-1 antagonist, lenvatinib, and E7386 may be administered alone or in a medicament / formulation (also referred to herein as a pharmaceutical composition) comprising a therapeutic agent and one or more pharmaceutically acceptable carriers, excipients, and diluents, in accordance with standard pharmaceutical practice. Each therapeutic agent may be prepared by formulating lenvatinib or a pharmaceutically acceptable salt thereof, an anti-PD-1 antibody, and / or E7386 or a pharmaceutically acceptable salt thereof, and may be administered simultaneously or separately. Further, the formulations may be placed in a single package to provide a so-called kit formulation.

[0115] Lenvatinib or a pharmaceutically acceptable salt can be produced by the method described in Reference 17. Examples of pharmaceutically acceptable salts include salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with acidic or basic amino acids. Preferred examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Preferred examples of salts with organic acids include salts with acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and the like. Preferred examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; and ammonium salts. Preferred examples of salts with organic bases include salts with diethylamine, diethanolamine, meglumine, and N,N-dibenzylethylenediamine, and the like. Preferred examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid, and the like. Preferred examples of salts with basic amino acids include salts with arginine, lysine, ornithine, and the like. More preferred pharmaceutically acceptable salts are salts with organic acids, and particularly preferred pharmaceutically acceptable salts are salts with methanesulfonic acid.

[0116] In the combination therapies disclosed herein, the PD-1 antagonist, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof may be administered simultaneously (i.e., by the same agent), concomitantly (i.e., one agent is administered immediately after the other in any order separately), or sequentially in any order. Sequential administration is particularly useful when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and the other is a sterile liquid), and / or are administered on different dosing schedules, for example, a chemotherapeutic agent administered at least daily, and a biological therapeutic agent administered at a lower frequency, for example, once a week, once every two weeks, or once every three weeks.

[0117] In some examples, lenvatinib or a pharmaceutically acceptable salt thereof is administered prior to the administration of the PD-1 antagonist and / or the CBP / β-catenin inhibitor, while in other examples, the multi-RTK inhibitor is administered after the administration of the PD-1 antagonist and / or E7386 or a pharmaceutically acceptable salt thereof.

[0118] In some examples, at least one of the therapeutic agents in the combination therapy is administered using the same dosing regimen (dose, frequency, and duration of treatment) typically used when the agent is used as a monotherapy for treating the same cancer. In other examples, the patient receives at least one of the therapeutic agents in the combination therapy at a lower total dose, for example, a lower dose, a less frequent dose, and / or a shorter treatment duration than when the agent is used as a monotherapy.

[0119] Each small molecule therapeutic agent in the combination therapies disclosed herein can be administered orally in the form of a solid preparation, for example, tablets, granules, fine granules, powders, or capsules, or in the form of a liquid, jelly, or syrup, etc. Each small molecule therapeutic agent in the combination therapies disclosed herein may also be administered parenterally, and examples of parenteral administration routes include intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal administration.

[0120] The combination therapies disclosed herein may be used before or after a surgical procedure to remove a tumor and may also be used before, during, or after radiation therapy.

[0121] In some examples, the combination therapies disclosed herein are administered to patients who have not been previously treated with a biological or chemotherapeutic agent, i.e., treatment-naïve patients. In other examples, the combination therapy is administered to patients who have not been able to achieve a durable response after previous treatment with a biological or chemotherapeutic agent (i.e., treatment-experienced patients).

[0122] The combination therapies disclosed herein are typically used to treat tumors that are large enough to be found by palpation or by imaging techniques well known in the art, such as magnetic resonance imaging (MRI), ultrasound, or computed axial tomography (CAT) scans.

[0123] The combination therapies disclosed herein are preferably administered to human patients having cancer that shows a positive response for PD-L1 expression. PD-L1 expression is preferably detected by IHC assay of FFPE or frozen tissue sections of tumor samples removed from the patient using a diagnostic anti-human PD-L1 antibody or an antigen-binding fragment thereof. Typically, the patient's physician will order a diagnostic test to determine PD-L1 expression in a tumor tissue sample removed from the patient prior to initiation of treatment with a PD-1 antagonist, E7386 or a pharmaceutically acceptable salt thereof, and lenvatinib or a pharmaceutically acceptable salt thereof, but it is contemplated that the physician may order an initial or subsequent diagnostic test at any time after initiation of treatment, e.g., after completion of a treatment cycle.

[0124] The selection of a dosing schedule (also referred to herein as an administration schedule) for the combination therapies disclosed herein is determined by several factors including the turnover rate of the entity's serum or tissue, the level of the disease manifestation, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the individual to be treated. Preferably, the dosing schedule maximizes the amount of each therapeutic agent delivered to the patient without conflicting with an acceptable level of side effects. Thus, the dosage and frequency of administration of each biological and chemotherapeutic agent in the combination is determined in part by the particular therapeutic agent, the severity of the cancer to be treated, and the patient characteristics. Guidance is available for selecting appropriate dosages of antibodies, cytokines, and small molecules. See, for example, Wawrzynczak (1996) ANTIBODY THERAPY, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) MONOCLONAL ANTIBODIES, CYTOKINES AND ARTHRITIS, Marcel Dekker, New York, NY; Bach (ed.) (1993) MONOCLONAL ANTIBODIES AND PEPTIDE THERAPY IN AUTOIMMUNE DISEASES, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348: 601-608; Milgrom et al. (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344: 783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342: 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602; PHYSICIANS’ DESK REFERENCE 2003 (Physicians’ Desk Reference, 57th ed.); Medical Economics Company; ISBN: 1563634457; 57th ed. (November 2002).The determination of an appropriate dosing schedule may be made by a clinician, for example, using parameters or factors that are known or suspected in the art to affect or be predictive of affecting treatment, and may depend, for example, on the patient's medical history (e.g., previous treatments), the type and stage of the cancer to be treated, and biomarkers of response to one or more of the therapeutic agents in combination therapy.

[0125] The biological therapeutic agents (i.e., PD-1 antagonist, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof) in the combination therapies disclosed herein may be administered by continuous infusion or, for example, daily, every other day, three times per week, or weekly, bi-weekly, tri-weekly, monthly, every other month, or at other intervals. The total weekly dose is usually at least 0.05 μg / kg body weight, 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, or more. See, for example, Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (2003) Cancer Immunol. Immunother. 52:133-144. Cemiplimab-rwlc (LIBTAYO®) is another PD-1 antagonist that can be administered intravenously, with 350 mg administered once every three weeks (Q3W) over 30 minutes.

[0126] The dosage of lenvatinib or a pharmaceutically acceptable salt thereof can be appropriately selected according to the degree of symptoms, the age, sex, and weight of the patient, the sensitivity, route, time difference, administration interval of administration, and / or the type of pharmaceutical formulation, etc. Typically, when oral administration is carried out for an adult (60 kg body weight), the dosage is 1 to 600 mg per day, preferably 5 to 400 mg, more preferably 5 to 200 mg. The dosage may be administered once or divided into smaller dosages and given 2 to 3 times per day.

[0127] In some examples of using an anti-human PD-1 mAb as a PD-1 antagonist in combination therapy, the dosing schedule includes administering the anti-human PD-1 mAb at a dose of 1, 2, 3, 5, or 10 mg / kg at intervals of about 14 days (±2 days), or about 21 days (±2 days), or about 30 days (±2 days) throughout the course of treatment. The dosage of the anti-PD-1 antibody can be appropriately selected in the same manner as described above. Typically, when intravenous administration is carried out for an adult (60 kg body weight), the dosage can be 2 mg / kg with respect to a schedule of once every 3 weeks (a total of 2 dosages) in a 6-week cycle. The antibody can be administered for 1 to 10 cycles at appropriate intervals.

[0128] In other examples of using an anti-human PD-1 mAb as a PD-1 antagonist in combination therapy, the dosing schedule includes administering the anti-human PD-1 mAb at a dose ranging from about 0.005 mg / kg to about 10 mg / kg by dose escalation within the patient. The interval between dosages can be gradually shortened, for example, about 30 days (±2 days) between the first dose and the second dose, and about 14 days (±2 days) between the second dose and the third dose. In certain embodiments, for doses after the second dose, the administration interval is about 14 days (±2 days).

[0129] In certain examples, a subject can be administered an intravenous (IV) infusion of an agent comprising any of the PD-1 antagonists described herein.

[0130] In combination therapy, the PD-1 antagonist is preferably nivolumab in some cases, and is administered intravenously at a dose selected from the group consisting of 1 mg / kg Q2W, 2 mg / kg Q2W, 3 mg / kg Q2W, 5 mg / kg Q2W, 10 mg Q2W, 1 mg / kg Q3W, 2 mg / kg Q3W, 3 mg / kg Q3W, 5 mg / kg Q3W, and 10 mg Q3W. The PD-1 antagonist may also be semiprimab-rwlc administered intravenously at a dose of 350 mg Q3W.

[0131] In combination therapy, the PD-1 antagonist is preferably pembrolizumab, a pembrolizumab variant, or a pembrolizumab biosimilar in some cases, and is administered by a liquid formulation at a dose selected from the group consisting of 1 mg / kg Q2W, 2 mg / kg Q2W, 3 mg / kg Q2W, 5 mg / kg Q2W, 10 mg Q2W, 1 mg / kg Q3W, 2 mg / kg Q3W, 3 mg / kg Q3W, 5 mg / kg Q3W, 10 mg Q3W, and the flat dose equivalents of any of these doses, i.e., 200 mg Q3W and 400 mg Q6W, etc. In some examples, pembrolizumab is given as a liquid formulation containing 25 mg / ml pembrolizumab, 7% (w / v) sucrose, and 0.02% (w / v) polysorbate 80 in 10 mM histidine buffer pH 5.5.

[0132] In some examples, the selected dose of pembrolizumab is administered by IV infusion over a period of 25 - 40 minutes, or about 30 minutes.

[0133] For pembrolizumab in combination with lenvatinib or a pharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) and a CBP / β-catenin inhibitor (e.g., E7386), the optimal dosage can be determined by dose escalation or dose de-escalation of one or both of these agents. In some examples, the combination therapy includes a 21-day treatment cycle in which pembrolizumab is administered IV at 200 mg Q3W (or 400 mg IV Q6W), and the CBP / catenin inhibitor lenvatinib mesylate is administered orally at (a) 24 mg per day as lenvatinib, (b) 20 mg per day as lenvatinib, or (c) 14 mg per day as lenvatinib, respectively.

[0134] Patients can be treated initially with IV pembrolizumab at 200 mg Q3W (or 400 mg IV Q6W) and lenvatinib mesylate orally at 24 mg per day (as lenvatinib), the daily dose of the CBP / β-catenin inhibitor, until at least one DLT is observed, and then the dosage of lenvatinib mesylate can be reduced to 20 or 14 mg per day (as lenvatinib, respectively), while the pembrolizumab dosage can be continued at 200 mg of pembrolizumab Q3W (or 400 mg IV Q6W), and the CBP / β-catenin inhibitor can be continued at the same daily dosage or reduced.

[0135] As an example of the dosing schedule, lenvatinib or a pharmaceutically acceptable salt thereof can be orally administered once daily with water, approximately at the same time each day, in a 21-day cycle, with or without food. Lenvatinib or a pharmaceutically acceptable salt thereof can be provided as 4 mg and 10 mg (as lenvatinib each) capsules. On day 1 (D1) of each cycle, lenvatinib or a pharmaceutically acceptable salt thereof can be administered within approximately 1 hour after completion of pembrolizumab administration and / or administration of E7386 or a pharmaceutically acceptable salt thereof. Pembrolizumab may be provided in a single-use vial as a preservative-free white to off-white sterile lyophilized powder. Each vial can be reconstituted and diluted for intravenous use. Each 2 mL of the reconstituted solution may contain approximately 50 mg of pembrolizumab. In some examples, pembrolizumab may be provided as a preservative-free, clear to slightly opalescent, colorless to slightly yellow sterile solution that requires dilution for intravenous use. Each vial may contain 100 mg of pembrolizumab in a 4 mL solution. Each 1 mL of the solution may contain 25 mg of pembrolizumab. Pembrolizumab may be administered as a 30-minute intravenous infusion, as a dose of 200 mg Q3W (e.g., 25 minutes to 40 minutes).

[0136] When an oral solid preparation is prepared, a pharmaceutically acceptable vehicle, and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, and / or a flavoring agent or the like are added to the main component, that is, the CBP / β-catenin inhibitor and / or the anti-PD-1 antibody which is a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof according to a conventional method, and then tablets, granules, fine granules, powders, capsules or the like may be prepared. Examples of the vehicle include lactose, corn starch, sucrose, glucose, sorbitol, crystalline cellulose, and silicon dioxide. Examples of the binder include polyvinyl alcohol, ethyl cellulose, methyl cellulose, gum arabic, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose. Examples of the lubricant include magnesium stearate, talc, and silica. Examples of the coloring agent include titanium oxide, ferric sesquioxide, yellow ferric sesquioxide, cochineal, carmine, and riboflavin. Examples of the flavoring agent include cocoa powder, ascorbic acid, tartaric acid, peppermint oil, borneol, and cinnamon powder. These tablets and granules may be coated if necessary.

[0137] In some examples, the patient is treated with combination therapy for at least 24 weeks, for example, during eight 3-week cycles. In some examples, treatment with combination therapy continues until the patient shows evidence of PD or CR.

[0138] In some examples, the patient is selected for treatment with the combination therapy disclosed herein if diagnosed with renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, urothelial carcinoma, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, or head and neck squamous cell carcinoma.

[0139] The "therapeutic agent" or "combination therapy" for cancer comprises an immune checkpoint inhibitor (e.g., anti-PD-1 antibody), lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof. The drugs may be formulated so that each of the three drugs is separate and distinct, and separately, or in combinations of two of the three drugs, or as a whole, by a method suitable for the desired administration, e.g., oral, nasal, mucosal, rectal, vaginal, topical, intravenous, intraperitoneal, intradermal, subcutaneous, and intramuscular administration.

[0140] Determination of a therapeutically effective amount of a dosage and the timing of its administration of a combination therapy for cancer containing a combination of an immune checkpoint inhibitor, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof are well within the knowledge of those skilled in the art. For example, an initial effective amount can be envisioned from cell culture or other in vitro assays. The dosage can be determined by cell culture assays and / or animal models to produce a circulating concentration or tissue concentration, e.g., IC 50 concentration.

[0141] The method of administration is selected depending on the treatment and the condition under the therapeutic agent. An immune checkpoint inhibitor, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof can be administered by various methods. For example, one or more of the components can be administered to a subject via any of the following routes: subcutaneous, intravenous, intraperitoneal, intramuscular, and systemic administration, and, optionally, direct injection into a specific organ or tumor, etc. An immune checkpoint inhibitor, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof can be administered simultaneously or sequentially via a single route or several simultaneous routes as described herein.

[0142] E7386 or a pharmaceutically acceptable salt thereof may be administered once a day, twice a day, several times a day, or even multiple times a day, depending especially on the therapeutic index and the judgment of the prescribing physician.

[0143] The amount of an immune checkpoint inhibitor, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof, which is essential for imparting a therapeutic effect, can be determined experimentally according to conventional procedures for a specific subject. Usually, a pharmacologically effective dose of the cells is administered for the purpose of treatment. "Pharmacologically effective amount" or "pharmacologically effective dose" refers to an amount sufficient to produce the desired physiological effect or capable of achieving the desired result, for example, reducing or eliminating one or more symptoms or signs such as a disorder or disease, and treating a specific disorder or disease state.

[0144] The combination therapy may be a combination of an immune checkpoint inhibitor, lenvatinib or a pharmaceutically acceptable salt thereof, and E7386 or a pharmaceutically acceptable salt thereof, and these can be further combined with other cancer treatments, such as surgical resection, radiotherapy, chemotherapy, immunotherapy, and supportive therapy (for example, analgesics, diuretics, antidiuretics, antiviral drugs, antibiotics, nutritional supplements, anemia treatment, blood coagulation treatment, bone treatment, as well as psychopathological treatment and psychological treatment).

[0145] These and other aspects disclosed herein, including the exemplary specific therapies, agents, and uses described below, will become apparent from the teachings contained herein.

[0146] Specific Therapies, Agents, and Uses [1] A method of treating cancer in a human subject, comprising administering to an individual: (i) an antagonist of programmed cell death 1 protein (PD-1); (ii) lenvatinib having the structure:

Chem.

Chem.

[10] Lenvatinib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 24 mg, 20 mg, 18 mg, 12 mg, or 8 mg; and pembrolizumab is administered once every three weeks at a dose of 200 mg for adults or 2 mg / kg (maximum 200 mg) for pediatric patients, the method of [9].

[11] The lenvatinib or a pharmaceutically acceptable salt thereof is lenvatinib mesylate; and the E7386 or a pharmaceutically acceptable salt thereof is E7386, by any one of the methods [1] to

[10] .

[12] A pharmaceutical composition for treating cancer, comprising (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, wherein the E7386 or a pharmaceutically acceptable salt thereof is administered in combination with (a) lenvatinib or a pharmaceutically acceptable salt thereof; and (b) an anti-PD-1 antibody.

[13] A pharmaceutical composition for treating cancer, comprising an anti-PD-1 antibody, wherein the anti-PD-1 antibody is administered in combination with (a) lenvatinib or a pharmaceutically acceptable salt thereof; and (b) (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof.

[14] A pharmaceutical composition for treating cancer, comprising lenvatinib or a pharmaceutically acceptable salt thereof, wherein the lenvatinib or a pharmaceutically acceptable salt thereof is administered in combination with (a) (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof; and (b) an anti-PD-1 antibody.

[15] The lenvatinib or a pharmaceutically acceptable salt thereof is lenvatinib mesylate; and the E7386 or a pharmaceutically acceptable salt thereof is E7386, the pharmaceutical composition according to any one of

[12] to

[14] .

[16] The cancer is a solid tumor, the pharmaceutical composition according to any one of

[12] to

[15] .

[17] The cancer is selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, urothelial cancer, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, and head and neck squamous cell carcinoma, the pharmaceutical composition according to any one of

[12] to

[15] .

[18] The cancer is RCC, the pharmaceutical composition of

[17] .

[19] The PD-1 antagonist is a monoclonal antibody or an antigen-binding fragment thereof, the pharmaceutical composition according to any one of

[12] to

[18] .

[20] The PD-1 antagonist is an anti-PD-1 antibody, the pharmaceutical composition according to any one of

[12] to

[18] .

[21] The PD-1 antagonist is pembrolizumab or nivolumab, the pharmaceutical composition according to any one of

[12] to

[20] .

[22] The PD-1 antagonist is pembrolizumab, the pharmaceutical composition according to any one of

[12] to

[21] .

[23] The lenvatinib or a pharmaceutically acceptable salt thereof is administered daily; and the PD-1 antagonist is pembrolizumab, which is administered once every three weeks, the pharmaceutical composition according to any one of

[12] to

[22] .

[24] The lenvatinib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 24 mg, 20 mg, 18 mg, 12 mg, or 8 mg; and pembrolizumab is administered once every three weeks, at a dose of 200 mg for adults or 2 mg / kg (maximum 200 mg) for children, the pharmaceutical composition of

[23] .

[25] The lenvatinib or a pharmaceutically acceptable salt thereof is lenvatinib mesylate; and the E7386 or a pharmaceutically acceptable salt thereof is E7386, the pharmaceutical composition according to any one of

[12] to

[24] . Use of any of the pharmaceutical compositions of

[12] to

[25] for the manufacture of a medicament for the treatment of cancer.

[27] Any of the pharmaceutical compositions of

[12] to

[25] for use in the treatment of cancer.

[0147] General methods. Standard methods in molecular biology are described in Sambrook, Fritsch, and Maniatis (2nd edition 1982 and 1989, 3rd edition 2001) MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) MOLECULAR CLONING, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) RECOMBINANT DNA, volume 217, Academic Press, San Diego, CA). Standard methods are also shown in Ausbel et al. (2001) Current Protocols in Molecular Biology, volumes 1 to 4, John Wiley and Sons, Inc. New York, NY, which explains cloning and DNA mutagenesis in bacterial cells (volume 1), cloning in mammalian cells and yeast (volume 2), glycoconjugate and protein expression (volume 3), and bioinformatics (volume 4).

[0148] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan et al. (2000) CURRENT PROTOCOLS IN PROTEIN SCIENCE, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation are described (e.g., Coligan et al. (2000) Current PROTOCOLS IN PROTEIN SCIENCE, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5 - 16.22.17; Sigma-Aldrich, Co. (2001) PRODUCTS FOR LIFE SCIENCE RESEARCH, St. Louis, MO; pp. 45 - 89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384 - 391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) CURRENT PROTCOLS IN IMMUNOLOGY, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) USING ANTIBODIES, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (e.g., see Coligan et al. (2001) CURRENT PROTOCOLS IN IMMUNOLOGY, Vol. 4, John Wiley, Inc., New York).

[0149] Monoclonal antibodies, polyclonal antibodies, and humanized antibodies can be prepared (for example, see Shepherd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 139-243; Carpenter et al. (2000) J. Immunol. 165:6205; He et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; see U.S. Patent No. 6,329,511).

[0150] As an alternative to antibody humanization, there are those that use a human antibody library displayed on phage or a human antibody library in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14: 309-314; Barbas (1995) Nature Medicine 1: 837-839; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol. Today 21: 371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17: 397-399).

[0151] Purification of the antigen is not essential for antibody production. Animals can be immunized with cells having the antigen of interest. Subsequently, splenocytes can be isolated from the immunized animals, and the splenocytes can be fused with a myeloma cell line to generate hybridomas (see, for example, Meyaard et al. (1997) Immunity 7: 283-290; Wright et al. (2000) Immunity 13: 233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163: 5157-5164).

[0152] Antibodies can be conjugated, for example, to small molecule drugs, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit, or other purposes, and examples include antibodies conjugated to dyes, radioisotopes, enzymes, or metals such as gold colloids (see, e.g., Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

[0153] Flow cytometry methods, including fluorescence-activated cell sorting (FACS), are available (see, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Nucleic acids, polypeptides, and fluorescent reagents suitable for modifying antibodies, such as nucleic acid primers and probes, which are used, for example, as diagnostic reagents, are available (Molecular Probesy (2003) catalog, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) catalog, St. Louis, MO).

[0154] Standard methods of immunological histology are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0155] For example, software packages and databases are available for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (see, e.g., GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16:741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0156] Table 3 shows a brief description of the sequences in the sequence listing.

[0157] [Table 3] [Examples]

[0158] Example 1: Antitumor effect of the triple combination of E7386, lenvatinib, and anti-PD-1 antibody The mouse renal cell carcinoma cell line RAG (ATCC number: CCL-142) was cultured using Eagle's Minimum Essential Medium (E-MEM) containing 10% fetal bovine serum (FBS) and penicillin / streptomycin (100 units / mL each). Logarithmically growing cells were collected from the flask using trypsin-EDTA. The cell suspension was centrifuged to remove the supernatant. Next, a cell suspension with a concentration of 2.5×10 7 cells / mL was prepared using Hank's Balanced Salt Solution (HBSS). The cell suspension was subcutaneously transplanted at a dose of 0.1 mL to the right outer part of the body of each 7-week-old mouse (BALB / cAnNCrlCrlj, female, Charles River Laboratories Japan, Inc.). Eight days after transplantation, the short and long diameters of the tumor of interest were measured using a digital electronic caliper (Digimatic (trademark) Caliper; Mitutoyo Corporation). The tumor volume TV and RTV were calculated using the following formulas. EQ.1: Tumor volume TV (mm 3 ) = long diameter (mm) × short diameter (mm) × short diameter (mm) / 2. EQ.2: Relative tumor volume RTV = TV on day n / TV on day 1.

[0159] Based on the tumor volume on the first day of administration, grouping was performed such that the average values of the tumor volumes were almost the same. A 1 mg / ml solution of lenvatinib was prepared using 3 mM HCl and orally administered once a day for 28 days at a dose of 0.2 mL per 20 g of mouse body weight. A 0.2 mL dosing sample containing 1.0 mg / mL anti-mouse PD-1 antibody (Clone: RMP1-14, BioXCell, catalog #: BE0146) diluted with PBS was intraperitoneally administered twice a week for a total of 8 times (on the 1st, 4th, 8th, 11th, 15th, 18th, 22nd, and 25th days (the day of grouping setting relative to the 1st day)) at a dosage of 200 μg / mouse. A 2.5 mg / ml solution of E7386 was prepared using 0.1 M HCl and orally administered once a day for 28 days at a dose of 0.2 mL per 20 g of mouse body weight. The control group was not administered anything. Experiments were conducted using each group containing 8 mice. Twice a week (on the 1st, 4th, 8th, 11th, 15th, 18th, 22nd, 25th, and 29th days), the tumor volume (TV) was determined for the control group, lenvatinib-administered group, anti-mouse PD-1 antibody-administered group, lenvatinib + anti-mouse PD-1 antibody-administered group, E7386 + lenvatinib-administered group, and triple combination group. Statistical analysis by repeated measures Dunnett's multiple comparison was performed using the values obtained by logarithmic transformation of the tumor volume. E7386 is (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide.

[0160] In the subcutaneous (s.c.) RAG transplantation model, the triple combination of E7386, lenvatinib, and anti-mouse PD-1 antibody showed a significantly higher anti-tumor effect than any of the agents when administered alone as a dual combination (i.e., the combination of lenvatinib + anti-PD-1 antibody, or the combination of lenvatinib + E7386), or monotherapy. For example, on day 29, the tumor volume in the triple combination group was less than 1 / 200 compared to the control group and the E7386 group. The triple combination had a tumor volume of less than 1 / 30 and less than 1 / 120 compared to the lenvatinib group and the anti-PD-1 antibody group, respectively. Also, the triple combination group had a tumor volume of less than 1 / 9 and less than 1 / 17 compared to the lenvatinib + anti-PD-1 antibody combination group and the E7386 + lenvatinib combination group, respectively.

[0161] In terms of the CR rate, the rate observed in the triple combination group (lenvatinib, pembrolizumab, and E7386) was superior to the rates observed in the other treatment groups (CR rate: among 8 mice in the control group, lenvatinib group, anti-PD-1 antibody group, E7386 group, lenvatinib + anti-PD-1 antibody group, E7386 + lenvatinib group, and triple combination group, there were 0, 0, 2, 1, 4, 1, and 7 mice, respectively).

[0162] The daily changes in tumor volume are shown in Table 4. Also, the time course of tumor volume changes during the administration of each group and the relative tumor volume on day 29 are shown in Figures 8 and 9, respectively.

[0163]

Table 4

[0164] Example 2: Anti-tumor effect by the triple combination of E7386, lenvatinib, and anti-PD-1 antibody Mouse cell lines (see Figures 10A - 10C, column A) were cultured using various media. Logarithmically growing cells were collected from flasks using trypsin - EDTA. The cell suspension was centrifuged to remove the supernatant. Next, a cell suspension with a specific cell concentration (see Figures 10A - 10C, column B) was prepared using Hank's Balanced Salt Solution (HBSS). The cell suspension was subcutaneously transplanted at a dose of 0.1 mL to the right outer part of the body of each 7 - week - old immunocompetent mouse (see Figures 10A - 10C, column C).

[0165] Several days after transplantation (see Figures 10A - 10C, column D), the short and long diameters of the tumors in the animals were measured using an electronic digital caliper (Digimatic (trademark) Caliper; Mitutoyo Corporation). The tumor volume TV and RTV were calculated using the following formulas. EQ.1: Tumor volume TV (mm 3 ) = long diameter (mm)×short diameter (mm)×short diameter (mm) / 2. EQ.2: Relative tumor volume RTV = TV on day n / TV on day 1.

[0166] Based on the tumor volume on the first day of administration, grouping was performed so that the average value of the tumor volume was almost the same. The 1 mg / ml solution of lenvatinib was prepared using 3 mM HCl and orally administered once a day for 28 days at a dose of 0.2 mL / mouse body weight of 20 g. A 0.2 mL administration sample containing 1.0 mg / mL anti-mouse PD-1 antibody (Clone: RMP1-14, BioXCell, catalog #: BE0146) diluted with PBS was intraperitoneally administered twice a week for 3 or 4 weeks (see Figures 10A to 10C, column E) (at a dosage of 200 μg / mouse). A 2.5 mg / ml solution of E7386 was prepared using 0.1 M HCl and orally administered once a day for 3 or 4 weeks (see Figures 10A to 10C, column E) at a dose of 0.2 mL / mouse body weight of 20 g. The control group was not administered anything. Experiments were conducted using each group containing 8 mice. The tumor volume (TV) of each group, namely the control group, the lenvatinib-administered group, the anti-mouse PD-1 antibody-administered group, the lenvatinib + anti-mouse PD-1 antibody-administered group, the E7386 + lenvatinib-administered group, and the triple combination therapy group, was determined twice a week for 3 or 4 weeks (see Figures 10A to 10C, column E). Statistical analysis by repeated measurement Dunnet's multiple comparison was performed using the values obtained by logarithmic transformation of the tumor volume. E7386 is (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide.

[0167] As shown in FIGS. 11 and 12A to 12G, the triple combination therapy of E7386, lenvatinib, and anti-mouse PD-1 antibody showed a higher anti-tumor effect than any of the individual agents when administered alone as a dual combination (i.e., the combination of lenvatinib + anti-PD-1 antibody, or the combination of lenvatinib + E7386), or as monotherapy.

[0168] The relative tumor volume (RTV) at time t was calculated according to the following formula: Equation 3: RTV = TV t / TV initial × 100%.

[0169] The inventors defined the Best Average Response as the minimum value of the average of (RTV - 100%) (Equation 4) for t ≥ 8d. This metric captures the combination of the speed, intensity, and durability of the response into a single value. The criteria for response (mRECIST) were applied from the RECIST criteria and defined as follows (used in this order): mCR, Best Average Response ≤ 95%; mPR, Best Average Response ≤ 50%; mSD, Best Average Response < 30%; mPD, not categorized otherwise.

[0170] References 1. Sharpe, A.H, Wherry, E.J., Ahmed R.,and Freeman G.J. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nature Immunology (2007); 8:239 - 245. 2. Dong H et al. Tumor - associated B7 - H1 promotes T - cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002 Aug;8(8):793 - 800. 3. Yang et al. PD - 1 interaction contributes to the functional suppression of T - cell responses to human uveal melanoma cells in vitro. Invest Ophthalmol Vis Sci. 2008 Jun;49(6 (2008): 49:2518 - 2525. 4. Ghebeh et al. The B7-H1 (PD-L1) Tlymphocyte-inhibitory molecule is expressed in breast cancer patients withinfiltrating ductal carcinoma: correlation with important high-risk prognosticfactors. Neoplasia (2006) 8: 190-198. 5. Hamanishi J et al. Programmed celldeath 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognosticfactors of human ovarian cancer. Proceeding of the National Academy of Sciences(2007): 104: 3360-3365. 6. Thompson RH et al. Significance ofB7-H1 overexpression in kidney cancer. Clinical genitourin Cancer (2006): 5:206-211. 7. Nomi, T. Sho, M., Akahori, T., et al.Clinical significance and therapeutic potential of the programmed death- 1 ligand / programmeddeath-1 pathway in human pancreatic cancer. Clinical Cancer Research(2007);13:2151-2157. 8. Ohigashi Y et al. Clinicalsignificance of programmed death-1 ligand-1 and programmed death-1 ligand 2expression in human esophageal cancer. Clin. Cancer Research (2005): 11:2947-2953. 9. Inman et al. PD-L1 (B7-H1) expressionby urothelial carcinoma of the bladder and BCG-induced granulomata:associations with localized stage progression. Cancer (2007): 109: 1499-1505. 10. Shimauchi T et al. Augmentedexpression of programmed death-1 in both neoplasmatic and nonneoplastic CD4+T-cells in adult T-cell Leukemia / Lymphoma. Int. J. Cancer (2007):121:2585-2590. 11. Gao et al. Overexpression of PD-L1significantly associates with tumor aggressiveness and postoperative recurrencein human hepatocellular carcinoma. Clinical Cancer Research (2009) 15: 971-979. 12. Nakanishi J. Overexpression of B7-H1(PD-L1) significantly associates with tumor grade and postoperative prognosisin human urothelial cancers. Cancer Immunol Immunother. (2007) 56: 1173-1182. 13. Hino et al. Tumor cell expression ofprogrammed cell death-1 is a prognostic factor for malignant melanoma. Cancer(2010): 00: 1-9. 14. Ghebeh H. Foxp3+ tregs andB7-H1+ / PD-1+ T lymphocytes co-infiltrate the tumor tissues of high-risk breastcancer patients: implication for immunotherapy. BMC Cancer. 2008 Feb 23; 8: 57. 15. Ahmadzadeh M et al. Tumorantigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1and are functionally impaired. Blood (2009) 114: 1537-1544. 16. Thompson RH et al. PD-1 is expressedby tumor infiltrating cells and is associated with poor outcome for patientswith renal carcinoma. Clinical Cancer Research (2007) 15: 1757-1761. 17. US Patent Application PublicationNo. 2018-0185395. 18. Canadian Application No. 3 044 658. 19. U.S. Patent 9,174,998. 20. U.S. Patent 10,259,817. 21. Keiichi Tamai, et al., “Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma,” Cancer Sci. 105(6): 667-674, 2014. Anthony B. El-Khoueiry, et al., “A phase I first-in-human study of PRI-724 in patients (pts) with advanced solid tumors,” J. Clin. Oncol. 31(15_supple(May 20,2013)): abstr 2501. 22. Renee van Amerongen, et al., “Break the loop, escape the cycle?” The EMBO Journal 2013, 32: 1977-1989.

[0171] All references cited in this specification are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent were specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by the applicant to relate to each and every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, and each of these is hereby clearly identified, even if such citation is not in close proximity to a dedicated statement of incorporation by reference, in accordance with 37 C.F.R. §1.57(b)(2). If there are any, the inclusion of dedicated statements of incorporation by reference in this specification does not in any way weaken this general statement of incorporation by reference. The citation of references in this specification is not intended as an admission that the references are prior art relevant thereto, nor does it constitute any admission as to the content or date of these publications or documents.

[0172] [Cross - reference to Sequence Listing] This application contains a Sequence Listing submitted electronically in ASCII format. The entire Sequence Listing is hereby incorporated by reference into this specification. A copy of the ASCII was created on October 27, 2020, named 213597_0005_00_ST25, and is 31,517 bytes in size.

[0173] [Cross - reference to Related Applications] This application claims the benefit and priority of U.S. Provisional Patent Application Nos. 62 / 927,334 and 62 / 927,576. Both of these provisional applications were filed on October 29, 2019, and their entire contents are hereby incorporated by reference into this specification.

Claims

1. A pharmaceutical composition for treating cancer, comprising (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof, wherein E7386 or a pharmaceutically acceptable salt thereof is administered in combination with (a) lenvatinib or a pharmaceutically acceptable salt thereof; and (b) an anti-PD-1 antibody.

2. A pharmaceutical composition for treating cancer, comprising an anti-PD-1 antibody, wherein the anti-PD-1 antibody is administered in combination with (a) lenvatinib or a pharmaceutically acceptable salt thereof; and (b) (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition for treating cancer, comprising lenvatinib or a pharmaceutically acceptable salt thereof, wherein lenvatinib or a pharmaceutically acceptable salt thereof is administered in combination with (a) (6S,9aS)-N-benzyl-8-({6-[3-(4-ethylpiperazin-1-yl)azetidin-1-yl]pyridin-2-yl}methyl)-6-(2-fluoro-4-hydroxybenzyl)-4,7-dioxo-2-(prop-2-en-1-yl)hexahydro-2H-pyrazino[2,1-c][1,2,4]triazine-1(6H)-carboxamide (E7386) or a pharmaceutically acceptable salt thereof; and (b) an anti-PD-1 antibody.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein lenvatinib or a pharmaceutically acceptable salt thereof is lenvatinib mesylate; and E7386 or a pharmaceutically acceptable salt thereof is E7386.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is a solid tumor.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), melanoma, bladder cancer, urothelial carcinoma, breast cancer, non-small cell lung cancer (NSCLC), endometrial cancer, and head and neck squamous cell carcinoma.

7. The pharmaceutical composition according to claim 6, wherein the cancer is RCC.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the anti-PD-1 antibody is pembrolizumab.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein lenvatinib or a pharmaceutically acceptable salt thereof is administered daily; and the anti-PD-1 antibody is pembrolizumab and is administered once every three weeks.

12. The pharmaceutical composition according to claim 11, wherein lenvatinib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 24 mg, 20 mg, 18 mg, 12 mg, or 8 mg; and pembrolizumab is administered once every three weeks at a dose of 200 mg for adult subjects or 2 mg / kg (maximum 200 mg) for pediatric subjects.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein lenvatinib or a pharmaceutically acceptable salt thereof is lenvatinib mesylate; and E7386 or a pharmaceutically acceptable salt thereof is E7386.

14. Use of the pharmaceutical composition according to any one of claims 1 to 13 for the manufacture of a medicament for the treatment of cancer.

15. The pharmaceutical composition according to any one of claims 1 to 13 for use in the treatment of cancer.

16. The pharmaceutical composition according to any one of claims 1 to 10, wherein lenvatinib or a pharmaceutically acceptable salt thereof is administered daily; and the anti-PD-1 antibody is pembrolizumab and is administered once every six weeks.

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