Combination therapy with berbarafenib and cobimetinib, or combination therapy with berbarafenib, cobimetinib, and atezolizumab

Abstract

Combination therapies including belvarafenib and cobimetinib, and combination therapies including belvarafenib, cobimetinib and atezolizumab are provided for the treatment of NRAS-mutated melanoma.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 171,461, filed April 6, 2021, the entire contents of which are incorporated by reference into this application. [Technical field]

[0002] The field of the invention relates generally to cancer therapy using combinations of berbalafenib and cobimetinib, and combinations of berbalafenib, cobimetinib and atezolizumab to treat NRAS mutant melanoma. [Background technology]

[0003] Melanoma is a potentially life-threatening form of skin cancer that originates from melanocytes. While early-diagnosed superficial tumors have a good outcome, metastatic melanoma is associated with high mortality and disease-related morbidity.

[0004] The RAS / RAF / MEK / ERK mitogen-activated protein kinase (MAPK) signaling cascade is an important intracellular signaling network that transmits multiple signals from the extracellular environment to the cell nucleus to activate cell proliferation and differentiation (Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:1911-2; Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 2007;26:3291-310). This pathway is closely involved in the pathogenesis of melanoma. Approximately 40% to 50% of melanomas have activating mutations in BRAF (Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002, 417:949-54; Curtin JA, Fridyland J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med 2005, 353:2135-47; Jakob JA, Bassett Jr RL, Ng CS, et al. NRAS mutation status is an independent prognostic factor in metastatic melanoma. Cancer 2012, 118:4014-23), and 29% of melanomas have mutations in NRAS (Moore AR, Rosenberg SC, McCormick F, et al. RAS-targeted therapies: is the undruggable drugged? Nat Rev Drug Discov 2020;19:533-52).

[0005] Melanoma cells are highly immunogenic and therefore a suitable target for immunotherapy (Zhu F., Liang Yu, Chen D, et al. Melanoma antigen gene family in the cancer immunotherapy. Cancer Transl Med 2016;2:85-9.). The advent of immunotherapy has dramatically changed the outcomes of melanoma patients. Over the past decade, the overall survival (OS) of patients with advanced melanoma has improved from 9 months to more than 5 years due to the effects of immunotherapy agents. Several phase III trials have compared single-agent anti-PD-1 inhibitors with anti-CTLA4 inhibitors or chemotherapy and shown improvements in objective response rate (ORR), progression-free survival (PFS) and OS, with OS of approximately 3 years and PFS rates ranging from 4 to 7 months (Robert C, Schachter J, Long G, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med 2015,372:2521-32; Schachter J, Ribas A, Long GV. Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a multicentre, randomised, open-label phase 3 study. Lancet 2017,390,1853-62). Combination immunotherapy provides even more robust benefits. In a phase III trial of 1296 patients, patients randomized to nivolumab and ipilimumab had both increased PFS and OS compared with patients receiving nivolumab alone. PFS was 11.5 months in the nivolumab + ipilimumab group and 6.9 months in the nivolumab monotherapy group, with a hazard ratio (HR) of 0.42 (95% CI: 0.35 to 0.51).At the shortest follow-up of 5 years, median OS was not reached, more than 60.0 months (95% CI: 38.2 to "not reached") with nivolumab plus ipilimumab and 36.9 months (95% CI: 28.2 to 58.7) with nivolumab alone, with a HR of 0.052 (95% CI: 0.42 to 0.64) (Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Five-year survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl J Med 2019;381:1535-46). Of note, more than 50% of patients experienced grade 3 or higher adverse events with nivolumab plus ipilimumab, making completion of treatment difficult, with 36.4% of patients discontinuing treatment due to adverse events.

[0006] No phase III trials have been conducted exclusively in patients whose disease had progressed after treatment with anti-PD-1 agents. Patients who received low-dose ipilimumab with pembrolizumab immediately after progression on PD-1 antibodies in melanoma showed significant antitumor activity in a phase II trial. In patients who have previously received anti-PD-1 therapy, response rates with repeated use of immunotherapy range from approximately 15% for ipilimumab (Robert C, Ribas A, Schachter J, et al. Pembrolizumab versus ipilimumab in advanced melanoma (KEYNOTE-006): post-hoc 5-year results from an open-label, multicentre, randomised, controlled, phase 3 study. Lancet Oncol 2019;20:1239-51) to 27% (immune-related response criteria) for the combination of pembrolizumab and ipilimumab, with a PFS of 5 months (Olson D, Luke J, Poklepovic AS, et al. Significant antitumor activity for low-dose ipilimumab (IPI) with pembrolizumab (PEMBRO) immediately following progression on PD1 Ab in melanoma (MEL) in a phase II trial. J Clin Oncol 2020;38:10004).

[0007] Patients with melanoma have BRAF V600Mutations in BRAF are further identified for appropriate treatment. Patients without BRAF mutations are collectively referred to as BRAF wild-type (WT). These cancers may include melanomas with NRAS mutations, melanomas with NF1 mutations, and melanomas with no identified mutations or "triple WT". Approved treatments for patients with BRAF WT melanoma include immunotherapies, chemotherapy, and T-VEC. No targeted therapy has been identified for patients with BRAF WT melanoma. Approved treatments for patients with BRAF mutant melanoma include targeted therapy (BRAF inhibitors alone or in combination with MEK inhibitors), immunotherapy, and chemotherapy. The optimal treatment sequence (i.e., targeted therapy followed by immunotherapy or vice versa) for patients with BRAF mutant tumors is not known.

[0008] Within the BRAF WT subset, several common mutations have been described, including NRAS mutations, which occur in approximately 29% of all melanoma patients (Moore et al., 2020). NRAS mutations occur at either residue glycine 12 (G12), glycine 13 (G13), or glutamine 61 (Q61). Approximately 85% of NRAS mutant melanomas harbor mutations at NRAS Q61 (enriched at Q61R, Q61K, Q61L, and Q61H) (Moore et al., 2020), with a much smaller percentage of melanomas harboring mutations at NRAS G12 or G13. The most common NRAS non-Q61 mutations are NRAS G12D, G13R and G13D (Li S, Balmain A, Counter CM. A model for RAS mutation patterns in cancers: finding the sweet spot. Nat Rev Cancer 2018;18:767-7). In a tissue-specific conditional knock-in mouse model of melanoma, expression of NRAS Q61R has been shown to drive melanoma formation, and mechanistic studies have demonstrated that NRAS Q61R mutations exhibit distinct nucleotide binding ability, stability and GTPase resistance, which likely drive melanoma-forming properties (Burd CE, Liu W, Huynh MV, et al. Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma. Cancer Discov 2014;4:1418-29). The importance of MAPK signaling, specifically BRAF and CRAF kinases downstream of RAS, in NRAS mutant melanoma has also been demonstrated. Q61KThis has also been confirmed in an inducible melanoma mouse model, where conditional loss of the BRAF and CRAF genes led to a complete blockade of tumor growth (Dorard C, Estrada C, Barbotin C, et al. RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma. Nat Commun 2017;8:1-13).

[0009] Despite the prevalence of NRAS mutations and the severity of the resulting disease, few treatment options are available.

[0010] Treatment of NRAS mutant melanoma in patients with disease progression during or after treatment with anti-PD-1 agents represents a significant unmet medical need. These patients with identified activating MAPK pathway mutations require novel targeted treatment approaches. Summary of the Invention

[0011] The present disclosure provides methods of treating a subject with NRAS mutant melanoma.

[0012] In some aspects, the method comprises (i) administering to the subject a therapy consisting essentially of (ii) a therapeutically effective amount of berbalafenib, or a pharma- ceutical acceptable salt thereof, and (iii) a therapeutically effective amount of cobimetinib, or a pharma-ceutical acceptable salt thereof.

[0013] In some embodiments, the subject is administered (i) about 200 mg to about 1300 mg, about 400 mg to about 1200 mg, about 600 mg to about 1200 mg, or about 800 mg to about 1000 mg of berbalafenib or a pharma- ceutically acceptable salt thereof per day, and (ii) about 20 mg to about 100 mg of cobimetinib or a pharma- ceutically acceptable salt thereof per day.

[0014] In some aspects, the method includes (i) administering to the subject a therapy consisting essentially of (ii) a therapeutically effective amount of berbalafenib or a pharmaceutically acceptable salt thereof, (iii) a therapeutically effective amount of cobimetinib or a pharmaceutically acceptable salt thereof, and (iv) a therapeutically effective amount of atezolizumab.

[0015] In some embodiments, the subject is administered (i) about 200 mg to about 1300 mg, about 400 mg to about 1200 mg, about 600 mg to about 1200 mg, or about 800 mg to about 1000 mg of belvarafenib or a pharma- ceutically acceptable salt thereof per day; (ii) about 20 mg to about 100 mg of cobimetinib or a pharma- ceutically acceptable salt thereof per day; and (iii) about 500 mg to about 2000 mg, about 500 mg to about 1000 mg, about 750 mg to about 1000 mg, about 750 mg to about 2000 mg, about 1000 mg to about 2000 mg, or about 1500 mg to about 1750 mg of atezolizumab. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 is a schematic diagram of a design study for an embodiment of the present disclosure for the treatment of NRAS mutant melanoma with (i) a combination of belbalafenib and cobimetinib, and (ii) a combination of belbalafenib, cobimetinib, and atezolizumab. In Figure 1: A is atezolizumab; B is belbalafenib; BID is twice daily; C is cobimetinib 21 days out of 28 days; DLT is dose-limiting toxicity; QD is daily; Q4W is every 4 weeks; x refers to the dose (and schedule) to be determined. The dotted line indicates the de-escalation group. Additionally, a indicates the inclusion of 5 patients who consent to mandatory serial biopsies approximately 6 weeks after day 1 of the first cycle and upon disease progression (if deemed safe). Additionally, b indicates that up to 25 patients will be enrolled in group 2, which will receive belbalafenib and cobimetinib at the same doses.

[0017] [Figure 2A]FIG. 2A is a Western blot of in vitro MEK / ERK / RSK signaling pathway inhibition by belvarafenib (Belv), cobimetinib (Cobi) or their combination in SK-MEL-30 (NRASQ61K) mutant melanoma cell lines.

[0018] [Figure 2B] FIG. 2B is a Western blot of in vitro MEK / ERK / RSK signaling pathway inhibition by belvarafenib (Belv), cobimetinib (Cobi) or their combination in IPC-298 (NRASQ61L) mutant melanoma cell lines.

[0019] [Diagram 3] FIG. 3 depicts the results of a colony formation assay in IPC-298 (NRASQ61L) melanoma cell line treated with berbalafenib, cobimetinib, or their combination.

[0020] [Figure 4] FIG. 4 shows the results of a colony formation assay in Mel-Juso (NRASQ61L) melanoma cell line treated with berbalafenib, cobimetinib, or a combination thereof.

[0021] [Diagram 5] FIG. 5 shows the results of a colony formation assay in SK-MEL-30 (NRASQ61K) melanoma cell line treated with berbalafenib, cobimetinib, or a combination thereof.

[0022] [Figure 6] FIG. 6 shows the in vivo tumor volume antitumor activity of belbalafenib (designated HM95573) or cobimetinib alone and in combination in mice xenografted with the SK-MEL-30 melanoma cancer cell line, measured on days 1, 4, 8, 11, and 15 of a 14-day oral drug administration regimen.

[0023] [Figure 7] FIG. 7 shows changes in mouse body weight in an evaluation of in vivo tumor volume antitumor activity of belbalafenib (designated HM95573) or cobimetinib alone and in combination in mice xenografted with SK-MEL-30 melanoma cancer cell line, measured on days 1, 4, 8, 11, and 15 for a 14-day oral drug administration regimen.

[0024] [Figure 8] FIG. 8 shows the antitumor activity of belbalafenib (designated HM95573) or binimetinib (designated MEK162) alone and in combination in mice xenografted with the SK-MEL-30 melanoma cancer cell line, measured on days 1, 4, 7, 11, 14, 18, and 21 of a 21-day oral drug administration regimen.

[0025] [Figure 9] FIG. 9 depicts the change in body weight associated with belbalafenib (designated HM95573) or binimetinib (designated MEK162) alone and in combination, measured on days 1, 4, 7, 11, 14, 18, and 21 of a 21-day oral drug administration regimen in mice xenografted with the SK-MEL-30 melanoma cancer cell line.

[0026] [Figure 10] FIG. 1 shows the in vivo antitumor activity induced by either belbalafenib (designated HM95573) or selumetinib (designated AZD6244), alone or in combination, orally administered for 17 days in mice xenografted with calu-6 non-small cell lung cancer cell line.

[0027] [Figure 11]FIG. 11 is a graph of the body weight change induced by orally administered belbalafenib (designated HM95573) or selumetinib (designated AZD6244), either alone or in combination, for 17 days in mice xenografted with the calu-6 non-small cell lung cancer cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In one aspect, the disclosure relates to the treatment of NRAS mutant melanoma cancer by the combined administration of berbalafenib and cobimetinib.

[0029] In one aspect, the present disclosure relates to the treatment of NRAS mutant melanoma cancer by the combined administration of berbalafenib, cobimetinib, and atezolizumab. definition

[0030] As used herein, "NRAS mutation" refers to the NRAS (NRAS proto-oncogene GTPase) oncogene with a DNA sequence alteration (mutation). The NRAS gene codes for the synthesis of the N-Ras protein, which is involved in the regulation of cell division. Without being bound to any particular theory, it is believed that NRAS mutations result in impaired GTPase activity and lock NRAS into its activated (GTP-bound) state, independent of upstream RTK activation (see Normanno, N., OncologyPRO, 2015). NRAS mutations in melanoma are associated with aggressive disease and poor prognosis. NRAS, mutated primarily at codon 61, is believed to be involved in up to 30% of all melanomas. NRAS oncogene mutations are also believed to be present in codons 12 and 13.

[0031] As used herein, the terms "patient" and "subject" refer to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In certain embodiments, the patient or subject is a human.

[0032] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of the individual or cells being treated during the course of clinical pathology. The desired effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease state, and improving remission or prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, reducing (or destroying) the proliferation of cancerous cells, reducing symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required to treat the disease, and / or prolonging the individual's survival.

[0033] As used herein, the phrase "therapeutically effective amount" refers to an amount of one or more drug compounds that (i) treat or prevent a particular disease, condition, or disorder, (ii) attenuate, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder, or (iii) prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. In the case of cancer, a therapeutically effective amount of drug may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow down to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or relieve one or more symptoms associated with cancer to some extent. To the extent that the drug may prevent the growth of existing cancer cells and / or kill them, the drug may be cytostatic and / or cytotoxic. In the case of cancer treatment, efficacy may be measured, for example, by assessing the overall response rate (ORR). The therapeutically effective amount herein may vary according to factors such as disease state, age, sex and weight of the patient, and the ability of the drug to induce a desired response in an individual. The therapeutically effective amount is also an amount in which the toxic or harmful effects of treatment outweigh the therapeutically beneficial effects. In the case of prophylactic use, the beneficial or desired result includes results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including the biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes that appear during the development of disease. In the case of therapeutic use, the beneficial or desired result includes clinical results such as reducing one or more symptoms caused by disease, improving the quality of life of the person suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug (e.g., by target), delaying the progression of disease, and / or extending survival time.In the case of cancer or tumor, a therapeutically effective amount of a drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing or preferably stopping) the invasion of cancer cells into peripheral organs, inhibiting (i.e., slowing or preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more of the symptoms associated with the disorder. A therapeutically effective amount may be administered in one or more administrations. For purposes of the present invention, a therapeutically effective amount of a drug, compound, pharmaceutical composition, or pharmaceutical preparation is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in the clinical context, a therapeutically effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, a therapeutically effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in a therapeutically effective amount if, in combination with one or more other agents, a desired result can be achieved or is achieved.

[0034] As used herein, "in combination with" refers to the administration of one treatment modality in addition to another treatment modality. Thus, "in combination with" refers to the administration of one treatment modality before, during, or after the administration of the other treatment modality to an individual.

[0035] As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a subject to provide an effective amount of the active ingredient used.

[0036] As used herein, "C" in terms of maximum, minimum, or other criteria refers to the drug concentration in plasma.

[0037] As used herein, "area under the curve" (AUC) refers to the area under the fitted plasma concentration versus time curve. AUC 0-∞ refers to the area under the curve baseline minus infinity. AUC 0-T is the total exposure.

[0038] As used herein, "inhibit" refers to a decrease in the activity of a target enzyme or other protein compared to the activity of that enzyme (or protein) in the absence of an inhibitor. In some embodiments, the term "inhibit" refers to a decrease in activity of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In other embodiments, inhibit refers to a decrease in activity of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 100%. In some embodiments, inhibit refers to a decrease in activity of about 95% to about 100%, e.g., a decrease in activity of 95%, 96%, 97%, 98%, 99%, or 100%. Such a decrease can be measured using a variety of techniques that would be recognizable by one of skill in the art.

[0039] As used herein, "progression-free survival" (PFS) refers to the time from disease treatment to the first occurrence of disease progression or recurrence.

[0040] As used herein, "partial response" (PR) refers to at least a 30% reduction in the sum of the diameters of target lesions relative to the sum of the diameters of the baseline lesions.

[0041] As used herein, "complete response" (CR) refers to the disappearance of all target lesions.

[0042] As used herein, "progressive disease" (PD) refers to an increase of at least 20% in the sum of the diameters of target lesions, with an absolute increase of at least 5 mm, relative to the smallest lesion diameter (nadir) during the study including baseline. The appearance of one or more new lesions is also considered progression.

[0043] As used herein, "overall response rate" (ORR) refers to the rate of PR or CR occurring after randomization and confirmed 28 days or more later, as determined by the investigator using RECIST v1.1.

[0044] As used herein, "duration of response" (DOR) refers to the time from the first occurrence of a documented objective response to recurrence as determined by the investigator using RECISTv1.1 or death from any cause on study, whichever occurs first.

[0045] As used herein, the term "RAF inhibitor" refers to a molecule that inhibits at least one of the three receptor subtypes (A-RAF, B-RAF, C-RAF) in the MAPK signaling pathway downstream of RAS.

[0046] As used herein, the term "MAPK" refers to the mitogen-activated protein kinase pathway or signal transduction pathway. The MAPK pathway, also called the Ras-Raf-MEK-ERK pathway, is a chain or pathway of proteins within a cell that transmits signals from receptors on the surface of the cell to DNA in the nucleus of the cell. In the MAPK pathway, activated RAS activates the protein kinase activity of RAF kinase, which phosphorylates and activates MEK (MEK1 and MEK2), which in turn phosphorylates and activates mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (MAPK3 and MAPK1). MAPK phosphorylates ribosomal protein S6 kinase (RPS6KA1; RSK).

[0047] As used herein, the term "PD-1 axis inhibitor" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis inhibitors include PD-1 inhibitors, PD-L1 inhibitors, and PD-L2 inhibitors.

[0048] As used herein, the term "PD-1 inhibitor" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1 and PD-L2). In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In certain aspects, a PD-1 inhibitor inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 inhibitors include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 inhibitor reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that are mediated by signaling through PD-1, thereby relieving dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.

[0049] As used herein, the term "PD-L1 inhibitor" refers to a molecule that reduces, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. In some embodiments, a PD-L1 inhibitor is a molecule that inhibits the binding of PD-L1 to its binding partners. In certain aspects, a PD-L1 inhibitor inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 inhibitors include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-L1, alleviating the dysfunctional state of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some aspects, the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0050] As used herein, the term "PD-L2 inhibitor" refers to a molecule that reduces, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with any one or more of its binding partners (such as PD-1). In some embodiments, a PD-L2 inhibitor is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In certain aspects, a PD-L2 inhibitor inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 inhibitors include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with any one or more of its binding partners (such as PD-1). In one embodiment, the PD-L2 inhibitor reduces the negative costimulatory signal mediated by or through a cell surface protein expressed on T lymphocytes that is mediated by signaling through PD-L2, rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 inhibitor is an immunoadhesin.

[0051] The term "pharmaceutically acceptable salt" refers to a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts. The phrase "pharmaceutically acceptable" indicates that a substance or composition is chemically and / or toxicologically compatible with other ingredients comprising a formulation and / or the subject being treated therewith. Acid addition salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid "mesylate", ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Base addition salts are formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Salts derived from pharma- ceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins.

[0052] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0053] The term "detection" includes any means of detection, including direct and indirect detection. Treatment

[0054] In one aspect, the present disclosure relates to a combination of berbalafenib, or a pharma- ceutically acceptable salt thereof, and cobimetinib, or a pharma- ceutically acceptable salt thereof, for the treatment of NRAS mutant melanoma.

[0055] In one aspect, the present disclosure relates to a combination of berbalafenib, or a pharmaceutically acceptable salt thereof, cobimetinib, or a pharmaceutically acceptable salt thereof, and atezolizumab for treating NRAS mutant melanoma.

[0056] The compounds disclosed herein can be administered in any suitable manner known in the art.In some aspects, the compounds can be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, intratumorally or intranasally.

[0057] It is understood that the appropriate dose of the active compound depends on several factors within the knowledge of a person skilled in the art. It is understood that the dose of the active compound will vary depending on, for example, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion and any drug combination.

[0058] It will also be understood that the effective dosage of the disclosed compounds or pharma- ceutically acceptable salts, prodrugs, metabolites or derivatives thereof used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become evident from the results of diagnostic assays.

[0059] Belbarafenib

[0060] Berbalafenib is disclosed in PCT Application WO 2013 / 100632 and has the chemical name 4-amino-N-(1-((3-chloro-2-fluorophenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide (referred to herein as formula (I)) and has the following chemical structure: Formula (I) TIFF2024514112000002.tif18170

[0061] Berbalafenib is a highly potent and selective type II RAF dimer inhibitor (pan-RAF inhibitor) that provides selective inhibition of BRAF and CRAF isoforms. V600 In contrast to selective monomeric inhibitors, belbalafenib does not activate the MAPK pathway in non-BRAFV600 mutant cells, instead maintaining suppression of MAPK signaling by inhibiting BRAF and CRAF dimers, leading to the suppression of BRAF V600 and RAS mutant tumors showed decreased cell proliferation and increased antitumor activity.

[0062] Bervalafenib inhibits the phosphorylation of MEK and ERK in the MAPK pathway in BRAF or RAS mutant melanoma, NSCLC, and CRC cell lines. Bervalafenib has been demonstrated to inhibit the proliferation of BRAF or RAS mutant melanoma, NSCLC, CRC, and thyroid cancer cell lines in vitro.

[0063] Belbarafenib inhibited the in vitro activity of BRAF V600E mutant (IC 50 =7nM), BRAF wild type (IC50 = 41 nM), and WT RAF-1 (CRAF) (IC 50 Berbarafenib is a potent and selective inhibitor of RAF kinases, including BRAF / CRAF kinase (Knockout inhibitor) and BRAF kinase (Knockout inhibitor) ...

[0064] Berbarafenib demonstrated significant inhibition of tumor cell proliferation across a panel of BRAF- or KRAS-mutant non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and thyroid cancer cell lines, as well as NRAS- or BRAF-mutant melanoma cell lines. Berbarafenib demonstrated inhibitory effects on cell viability in BRAF-mutant and NRAS-mutant melanoma, with IC 50 ) were 340 nM and 82 nM, respectively. Belbarafenib inhibited the NRAS mutant melanoma xenograft model SK-MEL-30 (NRAS Q61K In patients with refractory leukemia (TMS) who had refractory leukemia or refractory leukemia (TMS), it demonstrated tumor growth inhibition (TGI) of up to 80% as a single agent and up to 89.8% in combination with cobimetinib.

[0065] Based on the experimental evidence of this disclosure, it was found that belbalafenib exhibited a maximum TGI of 130% in a panel of NRAS mutant melanoma patient-derived xenograft models as follows: NRAS in Patient 1 Q61K In mouse xenografts of mutant melanoma cells, the maximum TGI was 85%. Q61R In mouse xenografts of mutant melanoma cells, the maximum TGI was 107%. G12CIn mouse xenografts of mutant melanoma cells, the maximum TGI was 90%. Q61R In mouse xenografts of mutant melanoma cells, the maximum TGI was 127%. Q61R In mouse xenografts of mutant melanoma cells, the maximum TGI was 130%. Q61K In mouse xenografts of mutant melanoma cells, the maximum TGI was 110%. Q61R In mouse xenografts of mutant melanoma cells, the maximum TGI was 124%. Q61R In mouse xenografts of mutant melanoma cells, the maximum TGI was 115%. Q61K In mouse-derived xenografts of mutant melanoma cells, the maximum TGI was 125%.

[0066] The in vitro antitumor efficacy of berbarafenib translated into efficacy in various mouse xenograft models. Berbarafenib shows dose-dependent inhibition of tumor growth in mouse xenograft models as monotherapy against BRAF- and NRAS-mutated melanoma, against KRAS-mutated non-small cell lung cancer (NSCLC), and in BRAF-mutated colorectal cancer (CRC) mouse xenograft models.

[0067] Bervalafenib has been shown in clinical trials to provide a safe and effective treatment for several cancers. For example, a completed open-label Phase Ia dose-escalation study investigated several doses and schedules of bervalafenib in patients with solid tumors harboring mutations in the BRAF, KRAS, or NRAS genes (Clinical Trial NCT02405065). Efficacy was analyzed in 67 of 72 subjects who underwent at least one post-baseline tumor assessment. The best overall response rate (BORR) was 8.96% (6 / 67 subjects), the objective response rate (ORR) was 4.48% (3 / 67 subjects), and partial response (PR) as confirmed best overall response (2 subjects with melanoma and 1 subject with gastrointestinal stromal tumor). Disease control was observed in 50.57% (34 / 67) of subjects treated at or above the bervalafenib 100 mg once daily dose level. Fifty-nine (88.06%) subjects developed events (disease progression or death), all of which were reported as progressive disease (PD). Furthermore, the median progression-free survival was 11.53 weeks, with a 95% CI for the median of [7.12 weeks, 13.38 weeks]. In updated results, the BORR was 10.45% (7 / 67 subjects), with a 95% CI of [4.30%, 20.35%]. The ORR remained at 4.48% (3 / 67 subjects). Furthermore, a subgroup reanalysis for BRAF-mutated melanoma subjects showed a BORR of 7.69% (1 / 13 subjects), with DCR, median PFS, and time to progression unchanged in all subjects. The median DOR increased to 30.18 weeks in the 800 mg twice daily group and 23.99 weeks in the combined group, which included subjects with a DOR of 100.29 weeks.

[0068] Another open-label Phase Ib dose-expansion study evaluated belbalafenib at a dose of 450 mg twice daily in patients with solid tumors harboring mutations in the BRAF, KRAS, or NRAS genes. Efficacy was analyzed in 59 of 63 subjects who received at least one dose of belbalafenib after enrollment and had at least one tumor assessment after baseline. BORR was 11.86% (7 / 59 subjects) and ORR was 6.78% (4 / 59 subjects) with PR as confirmed best overall response (3 subjects with melanoma and 1 subject with CRC). Disease control was observed in 35.59% (21 / 59) of subjects. Fifty of the 59 subjects (84.75%) developed an event (disease progression or death), all of which were reported as PD except for one death. Furthermore, the median progression-free survival (PFS) was 7.83 weeks, with a 95% confidence interval of the median (7.26 weeks, 8.26 weeks). The median duration of response (DOR) for all responses in this study was 15.66 weeks from the seven responders. Of those, the two BRAF-mutant melanoma responders showed a median DOR of 22.49 weeks.

[0069] Another phase I, single-dose, randomized, crossover relative bioavailability and food effect study in healthy subjects evaluated the effect of a formulation change from phase I to phase II tablets on belbalafenib exposure (clinical trial GP41348). A total of 18 healthy subjects were enrolled in the study and received the following randomized treatments: one 150 mg and one 50 mg phase I tablet in the fed state, two 100 mg phase II tablets in the fed state, or two 100 mg phase II tablets in the fasted state, with an 18-day washout period between treatments. There was a positive effect of food on belbalafenib exposure in the fed state compared to the fasted state. Belbalafenib exposure, C max and AUC 0-inf were increased by approximately 2.2-fold and 2.8-fold, respectively, when belvarafenib was administered to healthy subjects in a single 200 mg dose in the fed state compared with the fasted state. No serious adverse events, adverse events of special interest, or deaths were reported in this study.

[0070] Bervalafenib or a pharma- ceutically acceptable salt thereof is suitably administered at about 200 mg to about 1300 mg, about 400 mg to about 1200 mg, about 600 mg to about 1200 mg, or about 800 mg to about 1000 mg per day on an active ingredient basis. In some such embodiments, the subject is administered about 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, about 600mg, about 650mg, about 700mg, about 750mg, about 800mg, about 850mg, about 900mg, about 950mg, about 1000mg, about 1050mg, about 1100mg, about 1150mg, about 1200mg, about 1250mg or about 1300mg of berbalafenib or its pharmaceutically acceptable salt per day.In some other such embodiments, the subject is administered about 250mg, about 300mg, about 350mg, about 400mg, about 450mg or about 500mg of berbalafenib or its pharmaceutically acceptable salt twice a day ("BID"). In some other such embodiments, subject is administered about 300mg or about 400mg of belbalafenib or its pharmaceutically acceptable salt twice a day.Other dosing regimens can be used to achieve total daily dose, such as 3 times a day or 4 times a day.In any such dosing regimen, such as 2 times a day, 3 times a day or 4 times a day, each dose is suitably about equal.For example, if daily dose is 900mg, each dose of 450mg can be used twice a day or each dose of 300mg can be used 3 times a day.

[0071] In some embodiments, berbalafenib may be administered on days 1-21 of a 28 day cycle. In some embodiments, berbalafenib may be administered on days 1-28 of a 28 day cycle. Cobimetinib

[0072] Cobimetinib has the chemical name (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl]methanone and has the following structure (II): TIFF2024514112000003.tif63170

[0073] Cotellic® is the fumarate salt of cobimetinib. Cobimetinib is described in U.S. Patent Nos. 7,803,839 and 8,362,002, each of which is incorporated by reference in its entirety. Cobimetinib is a reversible, potent and highly selective inhibitor of MEK1 and MEK2 (the central components of RAS / RAF / MEK / ERK (MAPK)) pathways, and has single-agent antitumor activity in multiple human cancer models.

[0074] Cobimetinib is a potent and highly selective inhibitor of MEK1 and MEK2, central components of the MAPK pathway that signal downstream through ERK to promote normal cell proliferation. Upregulation of the pathway leads to constitutive signaling and malignant transformation, and occurs in the majority of tumors, often due to oncogenic activating mutations in RAS and BRAF (Bamford S, Dawson E, Forbes S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 2004;91:355-8). BRAF V600Cancer cells transformed by BRAF are exceptionally sensitive to MEK inhibition in vitro. Allosteric MEK inhibitors can induce G1 growth arrest in melanoma cells (Solit DB, Garraway L, Pratilas C, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 2006,439:358-62; Haass NK, Sproesser K, Nguyen TK, et al. The mitogen-activated protein / extracellular signal-regulated kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel. Clin Cancer Res 2008,14:230-9).

[0075] In vitro, MEK inhibitors inhibit BRAF V600 Reduced cell proliferation, soft agar colony formation and Matrigel invasion of BRAF mutant-positive melanoma cells V600 It is also effective against NRAS mutant-positive melanoma xenografts (Solit et al., 2006). NRAS mutant melanoma is highly dependent on MAPK pathway signaling, and MAPK signaling is required for tumor progression in NRAS mutant melanoma mouse models (Dorard C, Estrada C, Barbotin C, et al. RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma. Nat Commun 2017;8:1-13). In non-clinical trials, CI-1040 (MEK inhibitor) inhibited SK-MEL-130 (NRAS Q61RARRY-162, a novel MEK1 / 2 inhibitor, inhibits tumor growth regardless of KRas / Raf pathway mutations. EORTC-NCI-AACR, Berlin [poster]. 2010).

[0076] The pharmacokinetics (PK) of cobimetinib administered as a single agent has been characterized in cancer patients after single and multiple doses of oral administration in the Phase Ia dose-escalation study MEK4592g, which included evaluation of a cobimetinib dose of 60 mg per day in patients with BRAF, NRAS, or KRAS mutations. Overall, six patients (all of whom had melanoma; 6.2%) had a confirmed partial response (PR), 28 patients (28.9%) had stable disease (SD), and 40 patients (41.2%) had progressive disease. Of the 14 patients with colorectal cancer (CRC), all patients experienced progressive disease (PD). Study MEK4592g stage III enrolled 18 patients, and best overall response was assessed for 14 of the 18 patients. Four patients (22.2%) had SD as their best overall response, and two patients (11.1%) had unconfirmed tumor responses.

[0077] Cobimetinib has a moderate absorption rate (time to maximum concentration [t max[median t1 / 2] 1-3 hours) and mean terminal half-life (t1 / 2) of 48.8 hours (range 23.1-80 hours). Cobimetinib binds to plasma proteins in a concentration-independent manner (95%). Cobimetinib exhibits linear pharmacokinetics over a dose range of 0.05 mg / kg (approximately 3.5 mg / kg for a 70 kg adult) to 80 mg, and in a study MEK4952g in healthy subjects, absolute bioavailability was determined to be 45.9% (90% CI: 39.74%, 53.06%). The pharmacokinetics of cobimetinib are unchanged when administered in the fed state compared to administration in the fasted state in healthy subjects. Food does not alter the pharmacokinetics of cobimetinib, so cobimetinib may be administered with or without food. The proton pump inhibitor rabeprazole appears to have minimal effects on the pharmacokinetics of cobimetinib, whether administered in the presence or absence of a high-fat meal, compared with cobimetinib alone in the fasted state. Thus, increases in gastric pH do not affect the pharmacokinetics of cobimetinib, indicating that it is insensitive to changes in gastric pH.

[0078] Cobimetinib salts, crystalline forms and prodrugs are within the scope of the present disclosure.Cobimetinib, its preparation method and therapeutic use are disclosed in International Publication Nos. WO2007 / 044515, WO2014 / 027056 and WO2014 / 059422, each of which is incorporated herein by reference in its entirety.For example, in some embodiments of the present disclosure, MEK inhibitor is crystalline cobimetinib hemifumarate polymorph form A.

[0079] Dosages of cobimetinib or a pharma- ceutically acceptable salt thereof within the scope of the present disclosure are from about 20 mg to about 100 mg, from about 40 mg to about 80 mg, or about 60 mg per day on an active ingredient basis. In some embodiments, the dose of cobimetinib is about 60 mg, about 40 mg, or about 20 mg.

[0080] Cobimetinib is preferably administered once daily. In some embodiments, cobimetinib is administered once daily for 21 consecutive days of a 28-day treatment cycle. In some embodiments, cobimetinib is administered once daily on days 1-21 of a 28-day treatment cycle. In some embodiments, cobimetinib is administered once daily on days 3-23 of a 28-day treatment cycle. Atezolizumab

[0081] Atezolizumab is also known as MPDL3280A (CAS Registry Number: 1380723-44-3). Atezolizumab is a humanized IgG1 monoclonal antibody that targets PD-L1 and inhibits the interaction between PD-L1 and its receptors PD-1 and B7-1 (also known as CD80, both of which function as inhibitory receptors expressed on T cells). Therapeutic blockade of PD-L1 binding with atezolizumab has been shown to enhance the magnitude and quality of tumor-specific T cell responses, resulting in improved antitumor activity (Fehrenbacher L, Spira A, Ballinger M, et al. Atezolizumab versus docetaxel for patients with previously treated non-small-cell lung cancer (POPLAR): a multicentre, open label, phase 2 randomised controlled trial. Lancet 2016, 387: 1837-46; Rosenberg JE, Hoffman-Censits J, Powles T, et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet 2016, 387: 1909-20). Atezolizumab minimally binds to Fc receptors, thus eliminating detectable Fc effector function and the associated antibody-mediated clearance of activated effector T cells.

[0082] The pharmacokinetics of atezolizumab administered as a single agent have been characterized based on clinical data from study PCD4989g and are consistent with the ongoing Phase III study WO29522 in first-line treatment of TNBC. Atezolizumab antitumor activity has been observed across doses of 1-20 mg / kg. Overall, atezolizumab exhibits pharmacokinetics that are linear and consistent with a typical IgG1 antibody for doses of 1 mg / kg or greater every 3 weeks (q3w). Pharmacokinetic data (Bai S, Jorga K, Xin Y, et al., A guide to rational dosing of monoclonal antibodies, Clin Pharmacokinet 2012;51:119-35, incorporated herein by reference in its entirety) suggest no clinically meaningful differences in exposure at fixed doses or doses adjusted for body weight. Atezolizumab was tested with q3w and q2w dosing schedules. A fixed dose of 800 mg atezolizumab every 2 weeks (q2w) (corresponding to a weight-based dose of 10 mg / kg q2w) provides equivalent exposure to a phase III dose of 1200 mg administered every 3 weeks (q3w). The q3w schedule has been used in multiple phase III trials of atezolizumab monotherapy across multiple tumor types, with q2w primarily used in combination with chemotherapy regimens. In study PCD4989g, the overall 24-week progression-free survival (PFS) by Kaplan-Meier estimate was 33% (95% CI: 12%, 53%).

[0083] Atezolizumab doses of the present disclosure are suitably about 500 mg to about 2000 mg, about 500 mg to about 1000 mg, about 750 mg to about 1000 mg, about 750 mg to about 2000 mg, about 1000 mg to about 2000 mg, about 1500 mg to about 1750 mg. In some such embodiments, the subject is administered about 840 mg of atezolizumab. In some such embodiments, the subject is administered about 1680 mg of atezolizumab. In some such embodiments, the subject is administered atezolizumab every 14 days of a 28-day treatment cycle. In some other embodiments, the subject is administered atezolizumab on days 1 and 15 of a 28-day treatment cycle. In other embodiments, the subject is administered atezolizumab every 4 weeks of a 28-day treatment cycle. In other embodiments, the subject is administered atezolizumab on day 1 of a 28-day treatment cycle. In one embodiment, the subject is administered about 1680 mg of atezolizumab on day 1 of a 28-day treatment cycle. Melanoma

[0084] The melanoma within the scope of the present disclosure has NRAS mutation. In some embodiments, the melanoma progresses as defined by American Joint Committee on Cancer, 8th revised edition (Amin MB, Edge S, Greene F, et al. (eds.). AJCC Cancer Staging Manual (8th edition). Springer International Publishing: American Joint Commission on Cancer; 2017) with NRAS activating mutation. The determination of NRAS mutation positive status can be carried out by using clinical mutation test approved by national health authorities, such as, but not limited to, the US Food and Drug Administration, the College of American Pathologists, or CE marked (European Conformity) in vitro diagnostics in EU countries. In some embodiments, NRAS mutation positive status is defined as a mutation occurring in NRAS gene codons 12, 13 of exon 2 and codon 61 of exon 3.

[0085] In some embodiments, the melanoma is NRASQ61K Mutation, NRAS Q61R Mutation, NRAS G12C Mutation, NRAS Q61L Mutation, NRAS G13D In some other embodiments, the melanoma has an NRAS mutation, a NRAS mutation, or any combination thereof. G12D Mutation, NRAS G12C mutations, or combinations thereof.

[0086] In some embodiments, the melanoma is metastatic or unresectable.

[0087] In some embodiments, the melanoma continued to progress in the subject following treatment with immunotherapy, BRAF V600E therapy, or a combination of immunotherapy and BRAF V600E therapy.

[0088] In some embodiments, the melanoma continues to progress in a subject who has previously been administered a course of treatment with an anti-PD-1 or anti-PD-L1 drug. Combination therapy

[0089] The combination therapy of (i) berbalafenib and cobimetinib, and (ii) berbalafenib, cobimetinib and atezolizumab will be evaluated according to the protocol shown in FIG. 1 and described herein.

[0090] This protocol describes a Phase Ib, open-label, multicenter study to evaluate the safety, pharmacokinetics, and preliminary antitumor activity of belbalafenib as a single agent in combination with either cobimetinib or cobimetinib + atezolizumab in patients with NRAS-mutant metastatic or unresectable locally advanced cutaneous melanoma who have received up to two prior systemic anticancer therapies, including anti-PD-1 / PD-L1 therapy. Patients may have been treated with anti-PD-1 or anti-PD-L1 in the adjuvant setting.

[0091] The study will evaluate three treatment regimens in each arm: a belbalafenib monotherapy arm (Belva arm) of up to 15 patients; a belbalafenib + cobimetinib arm of up to approximately 43 patients, enrolling an initial dose-finding phase (9-24 patients) followed by expansion to the selected dose of up to 25 patients; a belbalafenib + cobimetinib + atezolizumab arm of approximately 25 total patients; and a belbalafenib + cobimetinib + atezolizumab arm of approximately 25 total patients, with a safety run-in phase (10 patients) followed by an expansion phase.

[0092] In some embodiments, berbalafenib is administered with food.

[0093] In some aspects, the combination therapy of the present disclosure is characterized by the absence of incidence of squamous cell carcinoma in human subjects.

[0094] Belbarafenib monotherapy group

[0095] Up to 15 patients will be enrolled in the Belva arm and will receive 400 mg of belvarafenib BID in tablet form on days 1-28 of each 28-day cycle.

[0096] Plasma samples for PK characterization of belvalafenib are collected as outlined in Table 1. Sampling schedules for all three groups are designed to allow for characterization of belvalafenib pharmacokinetics using non-compartmental analysis and / or population PK (popPK) methods. Belvalafenib PK data from the combination groups (where belvalafenib is co-administered with either cobimetinib or cobimetinib + atezolizumab) will be compared to the belvalafenib monotherapy group or single-agent belvalafenib in a previous Phase I study to evaluate whether belvalafenib exposure has changed. In Table 1: "C" refers to cycle, "D" refers to day; "PBMC" refers to peripheral blood mononuclear cells, and "PK" refers to pharmacokinetics.

[0097] [Table 1]

[0098] Belbarafenib and cobimetinib group

[0099] Patients will be enrolled in the Belva+Cobi arm in two phases: an initial dose-finding phase, followed by an expansion phase.

[0100] The combination of belbalafenib and cobimetinib appears to more profoundly suppress ERK production and produce greater TGI in tumors that exhibit MAPK pathway dependency (e.g., KRAS, NRAS, or BRAF mutations) compared with MEK inhibitors alone or RAF inhibitors. It is further believed that the synergistic effect between RAF inhibitors and MEK inhibitors may be due to the increased dependency of RAS mutant tumors on RAF kinase signaling in the presence of MEK inhibitors.

[0101] The aim of the dose-finding phase is to identify the recommended doses of belbalafenib and cobimetinib when used in combination. Approximately 9–24 patients will be enrolled in this phase. Each dose group of 3–6 patients will be treated according to the treatment regimen, with 300 or 400 mg belbalafenib BID on days 1–28 of each 28-day cycle, and in combination with 20, 40 or 60 mg cobimetinib QD on days 1–21 of each treatment cycle. The dose-finding phase will consist of a conventional 3 × 3 schema with a 28-day dose-limiting toxicity (DLT) assessment window (1 cycle). As belbalafenib is a potent RAF inhibitor and cobimetinib is a potent MEK inhibitor, dose-finding using DLT criteria in the belbalafenib + cobimetinib group will be performed to characterize the potential combined effect of MAPK pathway inhibition.

[0102] DLT is defined as any one of the following adverse events determined by the investigator to be possibly related to belvarafenib and / or cobimetinib, regardless of outcome, unless such event is considered by the investigator to be attributable to another clearly identifiable cause (e.g., documented disease progression, concomitant or preexisting medications, or intercurrent illness). Events are as follows: ≥ 1 ... Severe hepatotoxicity defined as: Grade 3 or higher elevation of total bilirubin or ALT / AST, or ALP lasting >7 days, with the following exceptions: For patients with Grade 2 hepatic transaminases or ALP at baseline as a result of metastases, liver transaminases or ALP of 10 × ULN are considered DLTs; liver transaminases (ALT or AST) >3x baseline and direct bilirubin >2x upper limit of normal or with clinical jaundice, in the absence of cholestasis or other contributing factors (e.g., worsening metastatic disease, concurrent exposure to known hepatotoxic agents, or documented infectious etiology) (per Hy's law); QTc interval (QTcF) corrected using Fridericia's method increased by >60 ms compared to baseline (pre-dose) and / or absolute QTcF value >500 ms (confirmed by repeat measurements);Other non-hematological / non-hepatic major organ adverse events of Grade ≥ 3, except for the following: Grade 3 rash that resolves to Grade ≤ 2 within 7 days with appropriate supportive care; Grade ≥ 3 fatigue that resolves to Grade ≤ 2 within 7 days; Grade 3 fever (defined as > 40°C) for ≤ 24 hours; Grade 3 laboratory abnormalities that are asymptomatic and deemed clinically insignificant by the investigator; Alopecia of any grade. Death clearly not attributable to underlying disease or external causes.

[0103] In the dose-finding phase of the combination of belbalafenib and cobimetinib, the starting dose of belbalafenib will be 300 mg PO BID in the first cohort. In subsequent cohorts, the dose of belbalafenib will be increased to 400 mg BID. The starting dose of cobimetinib in combination with belbalafenib will be 20 mg daily for the first 21 days of each 28-day cycle in the first two cohorts of the dose-finding phase. The dose of cobimetinib will be increased by 20 mg increments to a maximum dose of 60 mg daily for the first 21 days of each 28-day cycle or until a safety threshold is observed. After the doses of belbalafenib and cobimetinib are selected in the dose-finding phase, these doses will be further evaluated in up to 25 patients enrolled in the belbalafenib + cobimetinib arm during the expansion phase.

[0104] Dose determinations are made according to the following rules, regardless of the duration of the DLT window: In each titration cohort, a minimum of three patients are initially enrolled. If none of the first three DLT-evaluable patients experience a DLT, enrollment of the next cohort at the next highest dose level proceeds. If one of the first three DLT-evaluable patients experiences a DLT, the cohort is expanded to a minimum of six patients. If there are no further DLTs in the first six DLT-evaluable patients, enrollment of the next cohort at the next highest dose level proceeds. If two or more of the first six DLT-evaluable patients in a cohort experience a DLT, the MTD has been exceeded and dose escalation is stopped. The previous cohort is also expanded to a minimum of six patients unless six patients have already been evaluated at that dose level. If the MTD is exceeded at any dose level, the highest dose at which fewer than two of the first six DLT-evaluable patients (i.e., fewer than 33%) experience a DLT is the MTD. If the MTD is not exceeded at any dose level, the highest combination of belbalafenib and cobimetinib doses administered in this study will be the Maximum Administered Dose (MAD). Any dose level may be escalated in the absence of DLTs if justified based on the sponsor's and investigator's assessment of non-DLT adverse events.

[0105] If there are two DLTs in the first 6 patients in the first titration cohort (300 mg belvarafenib BID + 20 mg cobimetinib QD every first 21 days of each 28-day cycle), de-escalation of cobimetinib from QD to TIW will occur. A de-escalation cohort will be opened for enrollment with escalation to 400 mg belvarafenib BID and dose reduction of cobimetinib to 20 mg TIW for the first 21 days of each 28-day cycle (see Figure 4).

[0106] If there are two DLTs in the first 6 patients in the de-escalation cohort (400 mg belvarafenib BID + 20 mg cobimetinib TIW), a dose reduction of belvarafenib will be permitted in a new cohort with 300 mg belvarafenib BID + 20 mg cobimetinib TIW for the first 21 days of each 28-day cycle. No further dose reduction cohorts will be initiated for consideration beyond that point.

[0107] Patients in the titration phase who do not complete the 28-day DLT assessment window for reasons other than a DLT are considered non-evaluable for DLT assessment and may be replaced by additional patients at the same dose level. Additionally, patients who do not experience a DLT but miss more than 14 doses of berbalafenib on a 20 mg QD schedule, more than 5 doses of cobimetinib, and / or more than 10 doses of cobimetinib on a 20 mg TIW schedule, regardless of the contiguity of the missed doses, may be considered non-evaluable for DLT assessment and replaced by additional patients at the same dose level. The dose and / or frequency of berbalafenib and / or cobimetinib may be reduced during the DLT assessment window to manage DLTs in consultation with the medical monitor. Patients who do not experience a DLT and are receiving supportive care during the DLT assessment window that confounds the assessment of a DLT are considered non-evaluable for DLT assessment and may be replaced at the discretion of the medical monitor.

[0108] Plasma samples for cobimetinib PK characterization will be collected as outlined in Tables 2 and 3. The sampling schedule for dose finding in the belbalafenib + cobimetinib group will be designed to allow characterization of cobimetinib pharmacokinetics using non-compartmental analysis and / or popPK methodologies. The sampling schedule will transition to a sparse sampling schedule in the belbalafenib + cobimetinib expansion group and the belbalafenib + cobimetinib + atezolizumab group, and will be designed to allow estimation of key cobimetinib PK parameters using popPK methods. Cobimetinib PK data from the belbalafenib + cobimetinib and belbalafenib + cobimetinib + atezolizumab groups will be compared to single-agent cobimetinib to assess whether cobimetinib exposure is altered. In Tables 2 and 3: "C" refers to cycle, "D" refers to day; "PBMC" refers to peripheral blood mononuclear cells, and "PK" refers to pharmacokinetics.

[0109] [Table 2]

[0110] [Table 3]

[0111] Belbarafenib, cobimetinib, and atezolizumab group

[0112] Patients will be enrolled in two phases in the belbalafenib + cobimetinib + atezolizumab arm: an initial safety run-in phase followed by an expansion phase.

[0113] The combination of berbalafenib, cobimetinib, and atezolizumab appears to have increased antitumor activity compared to monotherapy. More specifically, the combination of berbalafenib and atezolizumab provides simultaneous inhibition of RAF isoforms and the PD-1 pathway, which appears to improve efficacy in tumors harboring mutations in RAS and RAF.

[0114] After the recommended doses of belbalafenib and cobimetinib have been determined, approximately 10 patients will be enrolled in the safety run-in phase. The arm will be activated only after dose determination has been performed, the assessment of the corresponding safety data from the belbalafenib + cobimetinib dose-finding arm has been completed, and all relevant data have been reviewed.

[0115] After all patients in the safety lead-in phase have been followed for 28 days, the sponsor's safety committee will review the safety data to determine whether the expansion phase should begin. Enrollment in the expansion phase will begin if fewer than 80% of patients in the safety lead-in phase experience grade 3 or higher adverse events during the 28-day safety lead-in period.

[0116] During the expansion phase, after the first 10 patients have received study treatment for at least 90 days, the sponsor's safety committee will reassess the safety data, which will allow for a thorough evaluation of toxicity, including time course, delayed events, and reversibility. The expansion phase will enroll up to 25 patients.

[0117] In each 28-day cycle, patients will receive belbalafenib at the recommended dose BID on days 1-28, cobimetinib at the recommended dose on days 1-21, and atezolizumab at 1680 mg by IV infusion every 4 weeks (Q4W).

[0118] Serum samples for PK characterization of atezolizumab will be collected as outlined in Table 4. The sampling schedule in the bevalafenib + cobimetinib + atezolizumab group is designed to allow estimation of key atezolizumab PK parameters using popPK methodology. Atezolizumab PK data from the bevalafenib + cobimetinib + atezolizumab group will be compared to atezolizumab single agent to evaluate whether atezolizumab exposure is altered. In Table 4: "C" refers to cycle, "D" refers to day; "PBMC" refers to peripheral blood mononuclear cells, and "PK" refers to pharmacokinetics. The sampling schedule for assessment of atezolizumab anti-drug antibodies (ADA) is believed to allow evaluation of the immunogenicity of atezolizumab given on a Q4W schedule when administered in combination with bevalafenib and cobimetinib.

[0119] [Table 4]

[0120] PK and biomarker evaluation

[0121] The concentrations of belvarafenib will be analyzed from samples collected at various time points as described above. For the belvarafenib and belvarafenib + cobimetinib groups, the following PK parameters will be calculated from the plasma concentrations of belvarafenib versus time from dosing using non-compartmental methods, as appropriate, for cycle 1, day 1, and steady state: C max , t max , the area under the concentration-time curve (AUC) from nominal time 0 to time t (AUC 0-t). In addition, belvalafenib plasma concentrations are reported as individual values ​​for all groups and summarized by treatment group and cycle when appropriate and data permit. Individual and mean belvalafenib concentrations are plotted by treatment group and day. Belvalafenib concentration data may be pooled with data from other studies using established population PK models to derive PK parameters such as clearance, volume of distribution and AUC that are warranted by the data. Potential correlations of relevant PK parameters with dose, safety, efficacy or biomarker outcomes may be examined.

[0122] Cobimetinib concentrations will be analyzed from samples taken at various time points as described above. For the belbalafenib + cobimetinib arm, the following PK parameters will be calculated from cobimetinib plasma concentrations using non-compartmental methods on Day 1 of Cycle 1 and at steady state, if appropriate: max , t max , and AUC 0-t For the belbalafenib + cobimetinib and belbalafenib + cobimetinib + atezolizumab groups, cobimetinib plasma concentrations are reported as individual values ​​and summarized by treatment group and cycle, where appropriate and data permit. Individual cobimetinib concentrations and mean cobimetinib mean concentrations are plotted by treatment group and day. Cobimetinib concentration data may be pooled with data from other studies using established population PK models to derive PK parameters such as clearance, volume of distribution and AUC, as warranted by the data. Potential correlations of relevant PK parameters with dose, safety, efficacy or biomarker outcomes may be examined.

[0123] Atezolizumab concentrations are measured as outlined above. Atezolizumab serum concentrations are reported as individual values ​​and summarized by treatment group and cycle where appropriate and data permit (mean, standard deviation, coefficient of variation, median, range, geometric mean, and geometric mean coefficient of variation). Individual and median serum atezolizumab concentrations are plotted by treatment group and day. Atezolizumab concentration data may be pooled with data from other studies using established population PK models to derive PK parameters such as clearance, volume of distribution, and AUC, as justified by the data. Potential correlations of relevant PK parameters with dose, safety, efficacy, or biomarker outcomes may be examined.

[0124] ADA analysis will be performed on the immunogenicity analysis population of patients enrolled in the belbalafenib + cobimetinib + atezolizumab group. The immunogenicity analysis will include all patients who had at least one ADA assessment for atezolizumab. The number and percentage of ADA-positive and ADA-negative patients at baseline (baseline prevalence) and after drug administration (post-baseline incidence) will be summarized by treatment group. The baseline prevalence summary will be based on patients with at least one evaluable baseline ADA assessment, and the post-baseline incidence summary will be based on treated patients with at least one evaluable post-baseline ADA assessment. When determining post-baseline incidence, patients will be considered ADA-positive if they are ADA-negative or have missing data at baseline but develop an ADA response after study drug exposure (treatment-induced ADA response), or if they are ADA-positive at baseline and the titer of one or more post-baseline samples is at least 0.60 titer units greater than the titer of the baseline sample (treatment-augmented ADA response). A patient is considered ADA negative if they are ADA negative or have missing data at baseline and all post-baseline samples are negative, or if they are ADA positive at baseline but have no post-baseline samples with a titer at least 0.60 titer units greater than the titer of the baseline sample (treatment unaffected). Relationships between ADA status and safety, activity, PK, and biomarker endpoints may be analyzed and reported by descriptive statistics.

[0125] Biomarker evaluation will be performed to understand the mechanism of action of belvarafenib as a single agent and in combination with cobimetinib and / or atezolizumab, and to identify prognostic and predictive biomarkers to assess disease progression and response to treatment.

[0126] Blood samples will be collected at baseline and throughout the study to assess changes in biomarkers, including those related to T-cell activation and lymphocyte subpopulations.

[0127] To identify optimal dose combinations of belbalafenib, cobimetinib, and atezolizumab for future trials, we will evaluate the modulation of circulating tumor DNA (ctDNA) to monitor the efficacy of treatment. There is growing evidence that cell-free DNA obtained from blood specimens of patients with cancer contains ctDNA, which represents the DNA and mutational status of cells within the tumor (Diehl et al. 2008; Maheswaran et al. 2008). Blood samples will be collected at baseline and during the study to evaluate changes in tumor mutation status in ctDNA as an indicator of treatment efficacy or emergence of resistance to the belbalafenib combination. Analysis of ctDNA collected from patients before treatment, at various time points during treatment, and after the study has progressed may help identify mechanisms of response and acquired resistance to belbalafenib.

[0128] Correlations between biomarkers and efficacy endpoints are investigated to identify blood-based biomarkers that may predict which patients are more likely to benefit from the drug combinations of the present disclosure.

[0129] Tissue and blood samples for RNA sequencing analysis and DNA extraction will be collected to allow for whole genome sequencing (WGS) or whole exome sequencing (WES) to identify variants that may predict response to the study drug, be associated with acquired resistance to the study drug, or increase knowledge and understanding of disease biology and understand the contribution of the immune system to the response. WGS and WES provide comprehensive characterization of the genome and exome, respectively. Together with the clinical data collected in this study, WGS and WES may increase opportunities to develop new therapeutic approaches or new methods to monitor efficacy and safety or to predict which patients are more likely to respond to a drug or develop adverse events.

[0130] To characterize tumor heterogeneity among patients and its relationship to clinical response, tumor tissue is collected at baseline (archived tissue or pretreatment biopsy), during treatment, and at disease progression (fresh biopsy performed at either first evidence of progression or confirmation of progression, whichever is closest to the last day of study treatment administration). Tumor tissue is evaluated for tumor immune landscape, such as PD-L1 expression, and other components of tumor immunity. Additionally, DNA and / or RNA are extracted from these tumor samples to allow for next generation sequencing (NGS) of DNA and / or RNA. These molecular characterizations are analyzed in relation to clinical response to identify patients more likely to benefit from treatment. As these biomarkers may also have prognostic value, their potential association with disease progression is also investigated.

[0131] Archival tumor tissue or fresh tumor biopsies are collected at baseline to allow for analysis of tumor tissue biomarkers associated with resistance, disease progression, and clinical benefit of subsequent drug treatments during the study. Tumor tissues are collected at baseline (archived tissue or pre-treatment biopsy), during treatment, and at disease progression (fresh biopsy taken at either first evidence of progression or confirmation of progression, whichever is closest to the last day of study treatment administration) to characterize tumor heterogeneity among patients and its relationship to clinical response. Tumor tissues are evaluated for tumor immune status, such as PD-L1 expression, and other components of tumor immunity. Additionally, DNA and / or RNA are extracted from these tumor samples to allow for next generation sequencing (NGS) of DNA and / or RNA. These molecular characterizations are analyzed in relation to clinical response to identify patients more likely to benefit from treatment. As these biomarkers may also have prognostic value, such as predicting response and resistance to the drug combinations of the present disclosure, their potential association with disease progression is also explored. Comparison of biomarkers between tissues obtained before treatment and tissues obtained at progression will further elucidate potential mechanisms of acquired resistance to this combination. Detailed mutational and immune profiles from biopsies taken at the time of disease progression may also provide data to consider subsequent treatment options.

[0132] DNA and RNA sequencing technologies such as targeted NGS and whole exome, genome or single nucleus RNA sequencing may provide unique opportunities to identify biomarkers of response and / or resistance to belbalafenib when administered alone and in combination with cobimetinib and / or atezolizumab. Sequencing of cancer-related genes may result in the identification of de novo and acquired mechanisms of resistance to belbalafenib. NGS technologies can generate large amounts of sequencing data. Tumor DNA may contain both reported and unreported chromosomal alterations due to the tumorigenesis process. To help control for sequencing calls in previously unreported genomic alterations, pre-dose blood samples will be taken to determine whether the alterations are somatic. NGS will also be performed on tumor tissue samples to identify immune and stromal signatures and T-cell receptor clonality, in addition to tumor-specific somatic mutations to aid in understanding disease biology. Working Example

[0133] The Animal Care and Use Committee of Hanmi Research Center approved the animal experiment protocols of the Examples.

[0134] Example 1

[0135] NCT03284502 is a Phase 1a / b study investigating belbalafenib in combination with cobimetinib at various dose levels in patients with locally advanced or metastatic solid tumors harboring RAS or RAF mutations. A total of 67 subjects have been enrolled in the study, including: 7 subjects receiving 200 mg belbalafenib BID on days 1-28 of a 28-day cycle and 40 mg cobimetinib QD for 21 days of a 28-day cycle; 4 subjects receiving 100 mg belbalafenib BID on days 1-28 of a 28-day cycle and 20 mg cobimetinib QD for 21 days of a 28-day cycle; 1 subject receiving 200 mg belbalafenib BID on days 1-28 of a 28-day cycle and 1 subject receiving 20 mg cobimetinib QD for 21 days of a 28-day cycle; 27 subjects receiving 20 mg cobimetinib QD for 21 days of a 28-day cycle; 5 subjects receiving 300 mg belvarafenib BID on days 1-28 of a 28-day cycle and 20 mg cobimetinib QD for 21 days of a 28-day cycle; and 24 subjects receiving 300 mg belvarafenib BID on days 1-28 of a 28-day cycle and 20 mg cobimetinib QOD (3 times weekly) for 3 weeks of a 28-day cycle.

[0136] Dose-limiting toxicities (DLTs) (including grade 3 colitis, grade 3 diarrhea, and grade 3 nausea) were reported in 2 of 3 DLT-evaluable subjects treated with 200 mg belbalafenib BID + 40 mg cobimetinib QD. No DLTs were reported at other dose levels.

[0137] Sixty-one of 67 subjects (91%) experienced adverse events regardless of investigator causality assessment (TEAE). TEAEs occurring in 10% or more of subjects were dermatitis acneiform (26 subjects; 38.8%), diarrhea (24 subjects; 35.8%), rash (22 subjects; 32.8%), elevated blood CPK (18 subjects; 26.9%), anorexia (15 subjects; 22.4%), pyrexia (14 subjects; 20.9%), constipation (13 subjects; 19.4%), anemia (11 subjects; 16.4%), fatigue (10 subjects; 14.9%), asthenia (8 subjects; 11.9%), and chorioretinopathy (7 subjects; 10.4%). The majority of TEAEs were grade 1 (5 subjects; 7.5%) or grade 2 (31 subjects; 46.3%) in severity. Grade 3 or higher TEAEs were reported by 25 subjects (37.3%), and the events reported in 13 subjects were considered by the investigators to be related to both belbalafenib and cobimetinib. The most common grade 3 or higher treatment-related adverse events (TRAEs) associated with both belbalafenib and cobimetinib were elevated blood CPK, fatigue (3 subjects each, 4.5%), and diarrhea (2 subjects, 3.0%). During the study, two subjects (3.0%) died due to grade 5 events of cardiac arrest and prolonged anorexia. Grade 5 cardiac arrest occurred in a subject treated with 200 mg belbalafenib BID + 40 mg cobimetinib QD. This event was considered by the treating physician to be related to both belbalafenib and cobimetinib. An event of grade 5 prolonged anorexia occurred in a subject treated with 300 mg bevalafenib BID + 20 mg cobimetinib QD; the event was considered unrelated to study treatment by the treating physician.

[0138] Pharmacokinetic analysis demonstrated that belbalafenib, in combination with cobimetinib, demonstrated overlapping steady-state exposure (AUC 0-24) was shown. Berbalafenib exposure was within the range of single-agent exposures, indicating no apparent drug-drug interactions with cobimetinib affecting berbalafenib exposure. Cobimetinib steady-state exposure was consistent with dose-normalized steady-state single-agent cobimetinib exposure, indicating no apparent drug-drug interactions with berbalafenib affecting cobimetinib exposure.

[0139] Example 2

[0140] In Example 2, we evaluated the in vitro signaling of belbalafenib, cobimetinib and their combination in NRAS mutant melanoma cells. SK-MEL-30 (NRAS Q61K ) and IPC-298 (NRAS Q61L ) melanoma cells were treated with 1 uM belvalafenib (Belva), 250 nM cobimetinib (Cobi), or a combination of both compounds (Belva+Cobi) and evaluated at 0, 3, 6, 24, and 48 hours. Cells were analyzed for MAPK signaling markers (phosphorylated MEK, ERK, and RSK) by Western blot. SK-MEL-30 (NRAS Q61K The results for IPC-298 (NRAS Q61L ) are shown in Figure 2B. The results show that the combination of belbalafenib and cobimetinib inhibits MAPK pathway signaling in NRAS mutant cell lines.

[0141] Example 3

[0142] In Example 3, in vitro colony formation growth assays of belvarafenib, cobimetinib, or combinations thereof in melanoma cells were evaluated. The following combinations were evaluated: 10 nM cobi + vehicle; 50 nM cobi + vehicle; 100 nM cobi + vehicle; 250 nM cobi + vehicle; 10 nM belv + vehicle; 100 nM belv + vehicle; 1000 nM belv + vehicle; 10,000 nM belv + vehicle; 10 nM cobi + 10 nM belv; 10 nM cobi + 100 nM belv; 10 nM cobi + 1000 nM belv; 10 nM cobi + 10,000 nM belv; 50 nM cobi + 10 nM belv. ;50nM cobi+100nM belv;50nM cobi+1000nM belv;50nM cobi+10,000nM belv;100nM cobi+10nM belv;100nM cobi+100nM belv;100nM cobi+1000nM belv;100nM cobi+10,000nM belv;250nM cobi+10nM belv;250nM cobi+100nM belv;250nM cobi+1000nM belv;250nM cobi+10,000nM belv. The following cell lines were evaluated: IPC-298(NRAS Q61L ) melanoma cell line; Mel-Juso (NRAS) treated with belvarafenib, cobimetinib, or their combination Q61L ) melanoma cell lines; and SK-MEL-30 (NRAS Q61K ) melanoma cell lines. Cells were treated with the indicated concentrations of berbalafenib and / or cobimetinib for 8 days and subsequently stained with crystal violet. The results are shown in Figures 3-5. The results show that the combination of berbalafenib and cobimetinib inhibits cell colony formation in NRAS mutant cell lines.

[0143] Example 4

[0144] KRAS mutant CRC cell line HCT116 (KRAS G13D ) and Lovo(KRASG13D ) and NSCLC cell line Calu-6 (KRAS Q61K ) the in vitro signaling of the pan RAF inhibitor berbarafenib was evaluated against the BRAF inhibitors vemurafenib and dabrafenib. IC in nM 50 To determine this, cell lines were exposed to berbalafenib, vemurafenib and dabrafenib over a range of concentrations and evaluated 2 hours after exposure.

[0145] The results are shown in Table 5 below. As shown in Table 5, belbarafenib, but not vemurafenib and dabrafenib, showed inhibitory effects on MEK and ERK phosphorylation in HCT116, Lovo and Calu-6 cell lines. In vitro cellular IC of belbarafenib on MEK and ERK phosphorylation 50 The values ​​were 2,698 nM and 253 nM, respectively, in HCT116; >10 μM (37% inhibition at 10 μM) and 267 nM in Lovo; and 367 nM and 590 nM, respectively, in Calu-6 cell line. The corresponding IC 50 Values ​​were greater than 10 μM in HCT116, Lovo, and Calu-6 cell lines.

[0146] [Table 5]

[0147] Example 5

[0148] In vitro cell growth inhibition of BRAF or KRAS mutant CRC and KRAS mutant NSCLC cell lines by vemurafenib was evaluated against the BRAF inhibitors vemurafenib and dabrafenib in the following: BRAF mutant CRC cell lines HT-29 and Colo-205 (both V600E );KRAS mutant CRC cell line LS174T (KRAS G12D ), LS513(KRAS G12D ), HCT116(KRAS G13D) and Lovo(KRAS G13D ); and the KRAS mutant NSCLC cell line Calu-6 (KRAS Q61K ) and Calu-1(KRAS G12C ).

[0149] The results are shown in Tables 6 and 7 below. Vemurafenib and vemurafenib inhibited the BRAF-mutated CRC cell lines HT-29 and Colo-205 (GI 50 range = 47–118 nM), whereas dabrafenib showed comparable activity in inhibiting cell proliferation with a GI of less than 0.1 nM. 50 Berbarafenib showed the most potent cell growth inhibitory effect in these cells. Berbarafenib inhibited GI in all KRAS mutant CRC cell lines tested in vitro, including LS174T, LS513, HCT116 and Lovo. 50 Belbarafenib inhibited cell proliferation with GI values ​​of 258, 62, 177, and 51 nM, respectively (Table 6). Activity of belbalafenib in inhibiting cell proliferation in KRAS mutant NSCLC cell lines was also observed in Calu-6 and Calu-1 (GI values ​​of 179 and 749 nM, respectively). 50 ) (Table 7). Dabrafenib also inhibited the progression-free survival (PFS) of patients with Lovo (KRAS mutant, CRC) cell lines (GI 50 = 214 nM) and Calu-6 and Calu-1 (KRAS mutant, NSCLC) cell lines (GI of 618 and 904 nM, respectively). 50 ) showed in vitro cell proliferation inhibition. However, the activity of dabrafenib was approximately 3-4 times weaker than that of vemurafenib, except in Calu-1 cells. Vemurafenib showed no activity in inhibiting proliferation in KRAS mutant cells.

[0150] [Table 6]

[0151] [Table 7]

[0152] Example 6

[0153] The in vitro cell growth inhibitory activity of vemurafenib versus other BRAF inhibitors, vemurafenib and dabrafenib, against BRAF or KRAS mutant cell lines was compared in BRAF mutant thyroid cell lines: SNU790, FRO, B-CPAP, NPA, 8505C, ARO (all BRAF V600E ), and SNU80(BRAF G468R ); and the KRAS mutant thyroid cancer cell line, CAL-62 (KRAS G12R The results are summarized in Table 8.

[0154] Berbarafenib and dabrafenib demonstrated activity in inhibiting cell proliferation in all seven BRAF-mutant thyroid cancer cell lines (GI <1 μM). 50 Vemurafenib has a GI of less than 1 μM in SNU790, B-CPAP and NPA, and BRAF-mutant thyroid cancer cell lines. 50 Furthermore, only belbarafenib, but not vemurafenib or dabrafenib, inhibited CAL-62 (KRAS G12R ) showed activity against cell proliferation inhibition in thyroid cancer cells and GI 50 The value was 479 nM.

[0155] [Table 8]

[0156] Example 7

[0157] NRAS Q61KThe in vivo antitumor activity of the combination of belbalafenib and cobimetinib as a combination therapy in a mouse model xenografted with the SK-MEL-30 human melanoma cell line harboring the mutation was investigated. Five animals per group were treated by oral gavage with vehicle (control), belbalafenib alone at a dose of 10 or 30 mg / kg once daily, cobimetinib alone at a dose of 10 mg / kg once daily, and the combination therapy up to day 14. Tumor volume (mm 3 ) and body weight changes were measured on days 1, 4, 9 and 15.

[0158] As shown in Figures 6 and 7 and Table 9, oral administration of belbalafenib resulted in dose-dependent antitumor activity, with maximum inhibition rates of 70.3% (day 15) and 80.0% (day 15) at 10 and 30 mg / kg qd, respectively. Maximum inhibition rates (max inhib (%)) were determined by (1-(mean relative tumor weight of treatment group / mean relative tumor weight of control group))*100. Cobimetinib alone at 10 mg / kg qd showed a maximum inhibition rate of 54.3% (day 15). Both belbalafenib and cobimetinib treatments were well tolerated with no weight loss; clinical signs of hair growth on the back were observed in the belbalafenib treatment group. Combination therapy with belbalafenib 10 or 30 mg / kg qd and cobimetinib 10 mg / kg demonstrated additive antitumor activity with a maximum inhibition rate of 89.8% (day 15) or 89.1% (day 15), respectively. No mortality or significant weight loss (>10%) was observed. Combination therapy with belbalafenib 10 or 30 mg / kg qd and cobimetinib 10 mg / kg demonstrated additive antitumor activity with a maximum inhibition rate of 89.8% (day 15) or 89.1% (day 15), respectively. No mortality or significant weight loss (>10%) was observed.

[0159] [Table 9]

[0160] Example 8

[0161] The in vivo antitumor activity of belbalafenib in mice bearing NRAS-mutated patient-derived xenografts (PDXs) was investigated. Ten animals per group were treated with vehicle (control) or belbalafenib at a dose of 20 mg / kg QD. Tumor volume and body weight changes were measured every 3-4 days. As shown in Tables 10-12C, oral administration of belbalafenib resulted in potent antitumor activity, and belbalafenib treatment was well tolerated without significant (>10%) body weight loss. The data in Tables 11A-12C were converted from graph form with an estimated error of + / - 10% (e.g., + / - 20 mm3 for Tables 11A-11C).

[0162] The overall results of Example 8 are shown in Table 10 below.

[0163] [Table 10]

[0164] Tables 11A-11C: Tumor volume results of efficacy studies of NRAS PDX melanoma models. For each PDX model, the dose of belbalafenib was 20 mg / kg QD.

[0165] [Table 11A]

[0166] [Table 11B]

[0167] [Table 11C]

[0168] Tables 12A-12C: % weight change results of efficacy studies of NRAS PDX melanoma models. For each PDX model, the dose of belbalafenib was 20 mg / kg QD.

[0169] [Table 12A]

[0170] [Table 12B]

[0171] [Table 12C]

[0172] Example 9

[0173] In vivo combination study of belvarafenib and the MEK inhibitor MEK162 (binimetinib) in the SK-MEL-30 xenograft model.

[0174] MEK inhibitors as monotherapy appear to offer minimal benefit in patients previously treated with BRAF inhibitors. Combining BRAF and MEK inhibitors in NRAS-mutated tumors may prevent or overcome resistance to monotherapy and improve the safety profile of monotherapy. In this regard, NRAS Q61K The in vivo antitumor activity of berbarafenib in combination with binimetinib, a selective ATP-noncompetitive inhibitor of MEK1 / 2, was investigated using a mouse model xenografted with the mutant SK-MEL-30 melanoma cell line. Five animals per group were treated by oral gavage for 21 days with vehicle (control), berbarafenib alone at doses of 10 or 30 mg / kg once daily, binimetinib alone at doses of 10 or 30 mg / kg once daily, or their combinations (berbarafenib 10 mg / kg + binimetinib 10 mg / kg or berbarafenib 30 mg / kg + binimetinib 30 mg / kg) once daily.

[0175] As shown in Figures 8 and 9 and Table 13, oral administration of belbalafenib resulted in dose-dependent antitumor activity, with maximum inhibition rates of 36.7% (day 21) and 74.6% (day 21) at 10 and 30 mg / kg q.d., respectively. Binimetinib was also administered at 10 and 30 mg / kg q.d. and showed antitumor activity against this tumor model, with maximum inhibition rates of 13.6% (day 21) and 27.1% (day 21), respectively. Both belbalafenib and binimetinib treatments were well tolerated with little significant weight loss; clinical signs of hair growth on the back, abdomen and face were observed in belbalafenib-treated groups at doses of 10 mg / kg and above.

[0176] Combination therapy with belbalafenib 10 mg / kg qd and binimetinib 10 mg / kg showed enhanced antitumor activity with a maximum inhibition rate of 74.2% on day 21 and toxicities of facial edema or erythema that were not observed with either treatment alone. Coadministration of belbalafenib 30 mg / kg qd and binimetinib 30 mg / kg qd induced tumor shrinkage (77.7% on day 21) but was poorly tolerated; 2 / 5 animals were found dead on day 10 and other surviving animals showed clinical signs such as facial edema or erythema that were not observed with either treatment alone during the study period.

[0177] [Table 13]

[0178] Example 10

[0179] In vivo combination study of belbarafenib and AZD6244 using the Calu-6 xenograft model

[0180] KRAS Q61KThe in vivo antitumor activity of belbalafenib in combination with selumetinib (AZD6244), a selective ATP-noncompetitive inhibitor of MEK1 / 2, was investigated in a mouse model xenografted with the mutation-bearing Calu-6 NSCLC cell line.Five animals per group were treated by oral gavage for 17 days with vehicle (control), belbalafenib alone at doses of 3, 10, or 30 mg / kg once daily, selumetinib alone at doses of 5 mg / kg twice daily, or the combination (belbalafenib 3 or 10 mg / kg once daily + selumetinib 5 mg / kg twice daily).

[0181] As shown in Figures 10 and 11 and Table 14, oral administration of belbalafenib resulted in dose-dependent antitumor activity, with maximum inhibition rates of 53.5% (day 18), 79.3% (day 15) and 86.3% (day 12) at 3, 10 and 30 mg / kg q.d., respectively. Selumetinib was also administered at 5 mg / kg bid and showed significant antitumor activity against this tumor model, with a maximum inhibition rate of 59.0% on day 18. Treatment with belbalafenib or selumetinib was well tolerated with minimal weight loss, and clinical signs of hair growth on the back, abdomen and face regions were observed in the belbalafenib treatment group at doses of 10 mg / kg and above. Combination therapy with belbalafenib and selumetinib resulted in enhanced antitumor activity, with no additional toxicity observed with either treatment alone. The maximum inhibition rates with belvarafenib 3 or 10 mg / kg qd and selumetinib 5 mg / kg bid were 71.8% (day 18) and 85.5% (day 12), respectively.

[0182] [Table 14] Example 11

[0183] (i) The combination of belvarafenib and cobimetinib, and (ii) the combination of belvarafenib, cobimetinib and atezolizumab will be evaluated in clinical trials according to the protocol shown in Figure 1 and described above.

[0184] This specification uses examples to disclose the present invention. The patentable scope of the present invention is defined by the claims and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that have insubstantial differences from the literal language of the claims.

Claims

1. A pharmaceutical composition for treating melanoma in a subject having an NRAS mutated melanoma, comprising: (i) a therapeutically effective amount of belbalafenib or a pharma- ceutical acceptable salt thereof; and (ii) a therapeutically effective amount of cobimetinib or a pharma- ceutical acceptable salt thereof.

2. The melanoma is Ｑ６１Ｋ Mutation, NRAS Ｑ６１Ｒ Mutation, NRAS Ｇ１２Ｃ Mutation, NRAS Ｑ６１Ｌ Mutation, NRAS Ｇ１３Ｄ Mutation, NRAS Ｇ１２Ｄ 2. The pharmaceutical composition of claim 1, having a nucleotide sequence, ...

3. The pharmaceutical composition of claim 1 , wherein the melanoma is metastatic or unresectable.

4. The object is (i) receiving 200 mg to 1300 mg, 400 mg to 1200 mg, 600 mg to 1200 mg, or 800 mg to 1000 mg of berbalafenib or a pharma- ceutically acceptable salt thereof per day; or (ii) The pharmaceutical composition according to any one of claims 1 to 3, wherein 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg of berbalafenib or a pharma- ceutically acceptable salt thereof is administered twice daily.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein berbalafenib or a pharma- ceutically acceptable salt thereof is administered daily for 28 consecutive days of a 28-day treatment cycle.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein berbalafenib or a pharma- ceutically acceptable salt thereof is administered with food.

7. The object is (i) 20 mg to 100 mg of cobimetinib or a pharma- ceutically acceptable salt thereof per day; or (ii) 20 mg, 40 mg, or 60 mg of cobimetinib or a pharma- ceutically acceptable salt thereof per day. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is administered

8. Cobimetinib or a pharma- ceutically acceptable salt thereof (i) administered daily for 21 consecutive days of a 28-day treatment cycle; (ii) administered on days 1 to 21 of a 28-day treatment cycle; or (iii) The pharmaceutical composition according to any one of claims 1 to 3, administered on days 3 to 23 of a 28-day treatment cycle.

9. The pharmaceutical composition of any one of claims 1 to 3, wherein the subject has previously received a course of treatment with an anti-PD-1 drug or an anti-PD-L1 drug.

10. The pharmaceutical composition of any one of claims 1 to 3, wherein the subject has experienced disease progression following treatment with immunotherapy, BRAF V600E therapy, or a combination of immunotherapy and BRAF V600E therapy.

11. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that said pharmaceutical composition is characterized by the absence of occurrence of squamous cell carcinoma in human subjects.

12. A pharmaceutical composition for treating melanoma in a subject having an NRAS mutated melanoma, comprising: (i) a therapeutically effective amount of belbalafenib or a pharma- ceutical acceptable salt thereof; (ii) a therapeutically effective amount of cobimetinib or a pharma- ceutical acceptable salt thereof; and (iii) a therapeutically effective amount of atezolizumab.

13. The melanoma is Ｑ６１Ｋ Mutation, NRAS Ｑ６１Ｒ Mutation, NRAS Ｇ１２Ｃ Mutation, NRAS Ｑ６１Ｌ Mutation, NRAS Ｇ１３Ｄ Mutation, NRAS Ｇ１２Ｄ 13. The pharmaceutical composition of claim 12, having a mutation, a nucleotide sequence ...

14. The pharmaceutical composition of claim 12, wherein the melanoma is metastatic or unresectable.

15. The object is (i) receiving 200 mg to 1300 mg, 400 mg to 1200 mg, 600 mg to 1200 mg, or 800 mg to 1000 mg of berbalafenib or a pharma- ceutically acceptable salt thereof per day; or (ii) The pharmaceutical composition according to any one of claims 12 to 14, wherein 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg of berbalafenib or a pharma- ceutically acceptable salt thereof is administered twice daily.

16. The pharmaceutical composition according to any one of claims 12 to 14, wherein berbalafenib or a pharma- ceutically acceptable salt thereof is administered daily for 28 consecutive days of a 28-day treatment cycle.

17. The pharmaceutical composition according to any one of claims 12 to 14, wherein berbalafenib or a pharma- ceutically acceptable salt thereof is administered with food.

18. The object is (i) 20 mg to 100 mg of cobimetinib or a pharma- ceutically acceptable salt thereof per day; or (ii) 20 mg, 40 mg, or 60 mg of cobimetinib or a pharma- ceutically acceptable salt thereof per day. The pharmaceutical composition according to any one of claims 12 to 14, wherein the composition is administered

19. Cobimetinib or a pharma- ceutically acceptable salt thereof (i) administered daily for 21 consecutive days of a 28-day treatment cycle; (ii) administered on days 1 to 21 of a 28-day treatment cycle; or (iii) The pharmaceutical composition according to any one of claims 12 to 14, which is administered on days 3 to 23 of a 28 day treatment cycle.

20. The object is (i) 500 mg to 2000 mg, 500 mg to 1000 mg, 750 mg to 1000 mg, 750 mg to 2000 mg, 1000 mg to 2000 mg, or 1500 mg to 1750 mg of atezolizumab; or (ii) 840 mg or 1680 mg of atezolizumab The pharmaceutical composition according to any one of claims 12 to 14, wherein the composition is administered

21. The object is (i) atezolizumab administered every 14 days of a 28-day treatment cycle; (ii) atezolizumab administered on days 1 and 15 of a 28-day treatment cycle; (iii) receiving atezolizumab every 4 weeks of a 28-day treatment cycle; or (iv) The pharmaceutical composition according to any one of claims 12 to 14, wherein atezolizumab is administered on day 1 of a 28-day treatment cycle.

22. 15. The pharmaceutical composition of any one of claims 12-14, wherein the subject is administered 1680 mg of atezolizumab on day 1 of a 28 day treatment cycle.

23. The pharmaceutical composition of any one of claims 12 to 14, wherein the subject has previously been administered a course of treatment with an anti-PD-1 drug or an anti-PD-L1 drug.

24. The pharmaceutical composition of any one of claims 12 to 14, wherein the subject has experienced disease progression following treatment with immunotherapy, BRAF V600E therapy, or a combination of immunotherapy and BRAF V600E therapy.

25. The pharmaceutical composition according to any one of claims 12 to 14, characterized in that said pharmaceutical composition is characterized by the absence of occurrence of squamous cell carcinoma in a human subject.

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