Combination therapy with RAF inhibitors and PD-1 axis inhibitors

Abstract

Combination therapies comprising a RAF inhibitor and a PD-1 axis inhibitor for the treatment of cancers characterized by an mutated MAPK signaling pathway are provided.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing in computer readable form that was submitted with this application via EFS-Web. The Sequence Listing, created on March 31, 2022, is 30,720 bytes in size. This Sequence Listing is incorporated herein by reference in its entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 173,207, filed April 9, 2021, the entire contents of which are incorporated by reference into this application.

[0003] Field of Disclosure The field of the invention relates generally to cancer treatment with a combination of a RAF inhibitor and a PD-1 axis inhibitor. [Background technology]

[0004] background RAS genes are the most frequently mutated oncogenes in human cancers. Among RAS isoforms, KRAS is the most frequently mutated (86%), followed by NRAS (11%), and is mutated mainly in cutaneous melanomas (28%). See Cox AD, Fesik SW, Kimmelman AC, et al, “Drugging the undruggable RAS: Mission possible?”, Nat Rev Drug Discov 13:828-51, 2014; Hilmi Kodaz, Osman Kostek, Muhammet Bekir Hacioglu, et al., “Frequency of RAS Mutations (KRAS, NRAS, HRAS) in Human Solid Cancer”, EJMO 1:1-7, 2017; and Cancer Genome Atlas N, “Genomic Classification of Cutaneous Melanoma”, Cell 161:1681-96, 2015. Preclinical models of RAS mutant-driven cancers have demonstrated a role for KRAS and NRAS in tumor initiation and maintenance, however, to date there has been limited clinical success in treating RAS mutant tumors by targeting their downstream effector pathways, such as inhibiting PI3K and MEK.

[0005] The RAF kinase family, consisting of three subtypes (A-RAF, B-RAF, C-RAF), is a key component of the MAPK signaling pathway downstream of RAS. Mutations in the RAF gene, particularly BRAF at codon V600, have been identified in a variety of cancers, including malignant melanoma, colorectal cancer, thyroid cancer, and lung cancer. See Davies H, Bignell GR, Cox C, et al., “Mutations of the BRAF gene in human cancer”, Nature 417:949-54, 200. BRAF V600 mutations allow BRAF to signal as a monomer, thereby constitutively activating the downstream MAPK signaling pathway.

[0006] The discovery of BRAF monomer inhibitors, such as vemurafenib, dabrafenib, and encorafenib, has V600 Although these have led to significant advances in the treatment of patients with BRAF mutant tumors, durability of treatment responses has nevertheless been limited due to a variety of resistance mechanisms, including BRAF amplification, BRAF splice variants, and RAS mutations, which largely converge on BRAF dimerization, as well as resistance to BRAF V600 monomeric therapy. See Sullivan RJ, Flaherty KT, “Resistance to BRAF-targeted therapy in melanoma,” Eur J Cancer 49:1297-304, 2013. Furthermore, these BRAF V600 Inhibitors have also been shown to paradoxically activate the MAPK signaling pathway in BRAF wild-type and KRAS mutant cell lines, resulting in dimerization of BRAF and CRAF and activation of MEK and ERK signaling in a RAS-dependent manner. See Heidorn SJ, Milagre C, Whittaker S, et al., "Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF", Cell 140:209-21, 2010; and Blasco RB, Francoz S, Santamaria D, et al., "c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma", Cancer Cell 19:652-63, 2011. Problematically, BRAF V600 Between 5% and 20% of treated patients develop squamous cell carcinoma (SCC), likely via paradoxical activation of the MAPK pathway.

[0007] Thus, there is a need for improved treatments for cancers that harbor KRAS, NRAS and RAF mutations. Summary of the Invention

[0008] Short Description The present disclosure provides a method of treating a subject having a cancer characterized by an mutated MAPK signaling pathway, the method comprising (i) administering to the subject a treatment consisting essentially of (ii) a therapeutically effective amount of a RAF inhibitor and (iii) a therapeutically effective amount of a PD-1 axis inhibitor.

[0009] In some embodiments, a subject is treated with (i) a RAF inhibitor at a dose of about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg twice daily, and (ii) a PD-1 axis inhibitor at a dose of about 400 mg to about 1200 mg, about 600 mg to about 1000 mg, about 700 mg to about 900 mg, or about 840 mg.

[0010] In some particular embodiments, the RAF inhibitor is belbalafenib or a pharma- ceutically acceptable salt thereof, and / or the PD-1 axis inhibitor is a PD-L1 inhibitor, hi some embodiments, the PD-L1 inhibitor is atezolizumab.

[0011] As used herein, HM95573 refers to belbalafenib. [Brief description of the drawings]

[0012] [Figure 1] 1 is a plot of mouse body weight change versus time for the K1735 syngeneic model for belbalafenib monotherapy (7.5 mg / kg and 15 mg / kg), atezolizumab monotherapy (10 mg / kg), combination treatment of belbalafenib at 7.5 mg / kg and atezolizumab at 10 mg / kg, and combination treatment of belbalafenib at 15 mg / kg and atezolizumab at 10 mg / kg of the present disclosure. Vehicle is represented by the overline. Points: mean relative body weight; Bars, SEM

[0013] [Diagram 2]FIG. 2 is a plot of mouse tumor volume versus time for the K1735 syngeneic model for belbalafenib monotherapy (7.5 mg / kg and 15 mg / kg), atezolizumab monotherapy (10 mg / kg), and belbalafenib / atezolizumab combination therapy ((i) 7.5 mg / kg and 10 mg / kg and (ii) 15 mg / kg and 10 mg / kg) of the present disclosure. In FIG. 2: * indicates P<0.05; **** indicates P<0.0001; § indicates P<0.05 compared to atezolizumab 10 mg / kg; # indicates P<0.05 compared to belbalafenib 15 mg / kg. P values ​​were calculated using two-way ANOVA. Vehicle is represented by an overline. Points: mean tumor volume; Bars: SEM

[0014] [Diagram 3] Plots of CD3+CD8+ T cells in the K1735 syngeneic mouse model for belbalafenib monotherapy (15 mg / kg), atezolizumab monotherapy (10 mg / kg), and belbalafenib / atezolizumab combination therapy (15 mg / kg and 10 mg / kg) in the K1735 syngeneic mouse model. In Figure 3: *** indicates P<0.001 compared to vehicle control; ### indicates P<0.001 compared to belbalafenib 15 mg / kg; § indicates P<0.05 compared to atezolizumab 10 mg / kg. P values ​​were calculated using one-way ANOVA. Points, mean CD3+CD8+ T cells; Bars, SEM

[0015] [Figure 4A] Plot of tumor volume (mm3) versus day in the CT26 syngeneic mouse model of KRASG12D CRC for oral treatment with vehicle, where the light lines are the results for individual animals within a group and the dark lines are the group mean. [Figure 4B] Plot of tumor volume versus day in a syngeneic mouse model for oral treatment with Mu IgG1(6E11)WT at 5 mg / kg twice weekly for 3 weeks; light lines are results for individual animals in the group, dark solid lines are averages for the group, and dark dashed lines are baseline fits. [Figure 4C]Plot of tumor volume versus day in a syngeneic mouse model for oral treatment with 10 mg / kg GDC-5573 (bervalafenib) daily for 3 weeks; light lines are results for individual animals in the group, dark solid lines are averages for the group, and dark dashed lines are baseline fits. [Figure 4D] Plot of tumor volume versus day in a syngeneic mouse model for the combination of oral treatment with Mu IgG1 (6E11)WT at 5 mg / kg twice weekly for 3 weeks and oral treatment with GDC-5573 (bervalafenib) at 10 mg / kg daily for 3 weeks; light lines are results for individual animals in the group, dark solid lines are the average for the group, and dark dashed lines are baseline fits.

[0016] [Figure 5A] Plot of % body weight change versus day in the CT26 syngeneic mouse model of KRASG12D CRC for oral treatment with vehicle, light lines are results for individual animals within a group, and dark lines are the group mean. [Figure 5B] Plot of body weight change (%) versus day in a syngeneic mouse model for oral treatment with Mu IgG1(6E11)WT at 5 mg / kg twice weekly for 3 weeks; light lines are results for individual animals within the group and dark lines are averages for the group. [Figure 5C] Plot of % body weight change versus day in a syngeneic mouse model orally treated with 10 mg / kg GDC-5573 (bervalafenib) daily for 3 weeks; light lines are results for individual animals within a group, and dark lines are the average for the group. [Figure 5D] Plot of body weight change (%) versus day in a syngeneic mouse model for combination of oral treatment with Mu IgG1(6E11)WT at 5 mg / kg twice weekly for 3 weeks and oral treatment with GDC-5573 (bervalafenib) at 10 mg / kg daily for 3 weeks; light lines are results for individual animals within the group and dark lines are averages for the group.

[0017] [Figure 6A] An overlay that matches the tumor volumes of Figures 4A-4D. [Figure 6B] An overlay that matches the weight changes in Figures 5A-5D.

[0018] [Figure 7A] Plot of tumor volume (mm3) versus day in the EMT6 syngeneic mouse model of KRASG12D TNBC for oral treatment with vehicle, where the light lines are the results for individual animals in the group and the dark lines are the group average. [Figure 7B] Plot of tumor volume versus day in a syngeneic mouse model for oral treatment with Mu IgG1(6E11)WT at 5 mg / kg twice weekly for 3 weeks; light lines are results for individual animals in the group, dark solid lines are averages for the group, and dark dashed lines are baseline fits. [Figure 7C] Plot of tumor volume versus day in a syngeneic mouse model for oral treatment with 10 mg / kg GDC-5573 (bervalafenib) daily for 3 weeks; light lines are results for individual animals in the group, dark solid lines are averages for the group, and dark dashed lines are baseline fits. [Figure 7D] Plot of tumor volume versus day in a syngeneic mouse model for the combination of oral treatment with Mu IgG1 (6E11)WT at 5 mg / kg twice weekly for 3 weeks and oral treatment with GDC-5573 (bervalafenib) at 10 mg / kg daily for 3 weeks; light lines are results for individual animals in the group, dark solid lines are the average for the group, and dark dashed lines are baseline fits.

[0019] [Figure 8A] Plot of body weight change (%) versus day in the EMT6 syngeneic mouse model of KRASG12D TNBC for oral treatment with vehicle, where the light lines are the results for individual animals in the group and the dark lines are the group average. [Figure 8B]Plot of body weight change (%) versus day in a syngeneic mouse model for oral treatment with Mu IgG1(6E11)WT at 5 mg / kg twice weekly for 3 weeks; light lines are results for individual animals within the group and dark lines are averages for the group. [Figure 8C] Plot of % body weight change versus day in a syngeneic mouse model orally treated with 10 mg / kg GDC-5573 (bervalafenib) daily for 3 weeks; light lines are results for individual animals within a group, and dark lines are the average for the group. [Figure 8D] Plot of body weight change (%) versus day in a syngeneic mouse model for combination of oral treatment with Mu IgG1(6E11)WT at 5 mg / kg twice weekly for 3 weeks and oral treatment with GDC-5573 (bervalafenib) at 10 mg / kg daily for 3 weeks; light lines are results for individual animals within the group and dark lines are averages for the group.

[0020] [Figure 9A] An overlay of the tumor volumes of Figures 7A-7D is shown. [Figure 9B] An overlay is shown that matches the weight changes in Figures 8A-8D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description The present disclosure is directed to the treatment of cancers characterized by mutated MAPK signaling pathways with a combination of a RAF inhibitor and a PD-1 axis inhibitor, more particularly a combination of a RAF inhibitor and a PD-L1 inhibitor, and even more particularly a combination of belbalafenib and atezolizumab.

[0022] definition As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells.

[0023] 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 aspects, the patient or subject is a human.

[0024] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural history of the individual or cells being treated during the course of clinical pathology. Desirable 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 survival of the individual.

[0025] 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, ameliorate, 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 alleviate to some extent one or more symptoms associated with cancer. 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, as used herein, may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the drug to induce a desired response in an individual. A therapeutically effective amount is also an amount in which the toxic or detrimental effects of treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dose of another drug required to treat the disease, enhancing the effect of another drug (e.g., by targeting), delaying the progression of the disease, and / or extending survival time. In the case of a 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 to some extent or desirably stopping) invasion of cancer cells into peripheral organs, inhibiting (i.e., slowing to some extent or desirably stopping) tumor metastasis, inhibiting to some extent tumor growth, and / or alleviating to some extent one or more of the symptoms associated with the disorder.A therapeutically effective amount can be administered in one or more doses. For the 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 a 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" can be considered in the context of administration of one or more therapeutic agents, and a single agent can be considered to be given in a therapeutically effective amount if a desired result can or is achieved in combination with one or more other agents.

[0026] 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.

[0027] As used herein, the term "pharmaceutical formulation" refers to a preparation in such a form that the biological activity of the active ingredient is effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such a formulation is sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be appropriately administered to a target mammal to provide an effective dose of the active ingredient used.

[0028] As used herein, "C" in terms of maximum, minimum, or other metric refers to the drug concentration in plasma.

[0029] As used herein, "area under concentration curve" (AUC) refers to the area under the fitted plasma concentration versus time curve. AUC0-∞ refers to the area under the curve baseline-infinity. AUC0-T is the total exposure.

[0030] As used herein, "inhibit" refers to a decrease in the activity of a target enzyme compared to the activity of the target enzyme 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 100%. In some embodiments, inhibition refers to a decrease in activity of about 95% to 100%, for example, a decrease in activity of 95%, 96%, 97%, 98%, 99%, or 100%. Such a decrease can be measured using a variety of techniques that can be recognized by those skilled in the art.

[0031] As used herein, "progression free survival" (PFS) refers to the time from disease treatment to the first occurrence of disease progression or recurrence.

[0032] As used herein, "partial response" (PR) refers to at least a 30% reduction in the sum of the diameters of target lesions, referenced to the baseline sum of the diameters.

[0033] As used herein, "complete response" (CR) refers to the disappearance of all target lesions.

[0034] As used herein, "delaying progression" of a disease means postponing, preventing, slowing, delaying, stabilizing, and / or prolonging the onset of a disease (such as cancer). This delay can be of various durations depending on the disease history and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can essentially encompass prevention that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0035] As used herein, "progressive disease" (PD) refers to at least a 20% increase in the sum of the diameters of the target lesions, referenced to baseline and a study minimum sum (nadir) that includes an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression.

[0036] As used herein, "overall response rate" (ORR) refers to the rate of confirmed PR or CR occurring after randomization and 28 days or more, as determined by the investigator using RECIST v1.1.

[0037] 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 RECIST v1.1 or death from any cause on study, whichever occurs first.

[0038] As used herein, the term "RAF inhibitor" refers to a molecule that inhibits at least one of the three subtypes (A-RAF, B-RAF, C-RAF) in the MAPK signaling pathway downstream of RAS.

[0039] As used herein, the term "MAPK" refers to the mitogen-activated protein kinase pathway or signal transduction pathway. The Ras-Raf-MEK-ERK pathway, also called the MAPK 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) ERK 1 and ERK 2 (MAPK3 and MAPK1). MAPK phosphorylates ribosomal protein S6 kinase (RPS6KA1; RSK).

[0040] 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 any 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.

[0041] As used herein, the term "PD-1 inhibitor" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1 and / or 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 prevent signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1 inhibitor reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-1, rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.

[0042] As used herein, the term "PD-L1 inhibitor" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling 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 prevent signaling 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 inhibitor reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that are mediated by signaling through PD-L1, so as to render dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0043] As used herein, the term "PD-L2 inhibitor" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L2 with one or more of its binding partners, e.g., 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, a PD-L2 inhibitor includes anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L2 with one or more of its binding partners, e.g., PD-1. In one embodiment, a PD-L2 inhibitor reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediated 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.

[0044] 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 the other ingredients that make up the formulation and / or with the mammal 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 acids 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, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, 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.

[0045] 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.

[0046] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with the antibody's experimental, diagnostic, or therapeutic uses, including enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions, for example, using Coomassie blue or silver staining. An isolated antibody includes an antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0047] A "native antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0048] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CH1, CH2, and CH3 domains of the heavy chain (collectively, CH) and the CHL (or CL) domain of the light chain.

[0049] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0050] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) of both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration connected by three HVRs that form loops that connect, and in some cases form part of, a beta-sheet structure. The HVRs within each chain are held in close proximity to each other by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular toxicity.

[0051] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0052] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0053] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0054] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as described below. These terms specifically refer to an antibody having a heavy chain that includes an Fc region.

[0055] An "antibody fragment" includes a portion of an intact antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0056] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0057] An "Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may associate in a "dimeric" structure similar to that in the two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0058] Fab fragments contain heavy and light chain variable domains, and also contain a light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0059] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0060] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) of the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains of another chain to generate two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully, for example, in EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0061] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogenous antibodies, e.g., the individual antibodies that make up the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence may be further modified, e.g., to improve affinity to the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, and create a multispecific antibody, and an antibody that includes a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0062] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced using, for example, hybridoma methods (e.g., those in Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al. Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Nature, 352:624-628 (1991); al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al., J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);and Lee et al.,J.Immunol.Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al.,Nature 362:255-258(1993);Bruggemann et al.,Year in Immunol.7:33(1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016;Marks et al.,Bio / Technology 10:779-783(1992);Lonberg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al.,Nature Biotechnol.14:845-851(1996);Neuberger,Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0063] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies, whose antigen-binding regions are derived from antibodies produced, for example, by immunizing macaque monkeys with the antigen of interest.

[0064] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0065] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. The methods described in Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985), Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). Also, for human antibodies produced by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0066] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homologue of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically" binds to a human antigen (e.g., has a binding affinity (Kd) value of about 1×10-7M or less, preferably about 1×10-8M or less, preferably about 1×10-9M or less), but has a binding affinity for a homologue of the antigen from a second non-human mammalian species that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than the binding affinity for the human antigen. A species-dependent antibody can be any of the various types of antibodies defined above, but is preferably a humanized or human antibody.

[0067] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is sequence hypervariable and / or forms structurally defined loops. Generally, an antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 show the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring excellent specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies, consisting only of heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0068] Several HVR delineations are used and encompassed herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of available complex crystal structures. Residues from each of these HVRs are shown below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0069] HVRs may include the following "extended HVRs": in the VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and in the VH, 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0070] "Framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0071] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al. (see above). Using this numbering scheme, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and may contain inserted residues after heavy chain FR residue 82 (e.g. residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0072] The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is commonly used when referring to residues in immunoglobulin heavy chain constant regions (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0073] The term "linear antibody" refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0074] As used herein, the terms "bind", "specifically bind to", or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, such as biological molecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for longer than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0075] The term "detection" includes any means of detection, including direct and indirect detection.

[0076] Therapeutic Agents The present disclosure uses a combination of a RAF inhibitor and a PD-1 axis inhibitor to treat a cancer characterized by an mutated MAPK signaling pathway in a subject. In some embodiments, (i) the RAF inhibitor is belbalafenib or a pharma- ceutically acceptable salt thereof, and (ii) the PD-1 axis inhibitor is a PD-L1 inhibitor, more specifically, the PD-L1 inhibitor is atezolizumab (trade name TECENTRIQ®).

[0077] 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, intratumorally, or intranasally.

[0078] It is understood that the appropriate dose of the active compound depends on many factors within the knowledge of a physician 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 combinations.

[0079] 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. RAF inhibitors

[0080] Examples of RAF inhibitors within the scope of the present disclosure include berbalafenib, vemurafenib, dabrafenib, and encorafenib.

[0081] Belvalafenib 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)), which has the following formula: Formula (I) It has the chemical structure TIFF2024513246000002.tif18170.

[0082] Bervalafenib has been found to be well tolerated and effective in treating certain brain cancers in subjects.Subjects within the scope of the present disclosure include, but are not limited to, humans or non-human mammals, such as cows, horses, dogs, sheep or cats.In some embodiments, the subject is a human.

[0083] Berbarafenib is a highly potent and selective type II RAF dimer inhibitor (pan-RAF inhibitor) that provides selective inhibition of BRAF and CRAF isoforms. In contrast to BRAFV600 selective monomeric inhibitors, Berbarafenib does not activate the MAPK pathway in non-BRAFV600 mutant cells, but instead maintains MAPK signaling suppression by inhibiting BRAF and CRAF dimers, resulting in reduced cell proliferation and increased antitumor activity in both BRAFV600 and RAS mutant tumors.

[0084] Berbalafenib inhibits the phosphorylation of MEK and ERK in the MAPK pathway in BRAF or RAS mutant melanoma, NSCLC, and CRC cell lines. Berbalafenib has been demonstrated to inhibit the growth of BRAF or RAS mutant melanoma, NSCLC, CRC, and thyroid cancer cell lines in vitro.

[0085] Berbarafenib is a potent and selective inhibitor of RAF kinases, including BRAFV600E mutant (IC50=7nM), BRAF wild type (IC50=41nM) and RAF-1 (CRAF) (IC50=2nM) in vitro. When tested in a panel of 189 kinase assays, Berbarafenib demonstrated inhibitory activity against seven other receptor tyrosine kinases (RTKs) with >90% inhibition at 1μM: colony-stimulating factor 1 receptor (CSF1R), former McDonough feline sarcoma (FMS) homolog, discoidin domain receptor tyrosine kinase 1 (DDR1), discoidin domain receptor tyrosine kinase 2 (DDR2), EPHA2, EPHA7, EPHA8 and EPHB2.

[0086] The in vitro antitumor efficacy of berbalafenib translated into efficacy in various mouse xenograft models. Berbalafenib shows dose-dependent inhibition of tumor growth in mouse xenograft models as monotherapy against BRAF and NRAS mutant melanoma, against KRAS mutant non-small cell lung cancer (NSCLC), and against BRAF mutant colorectal cancer (CRC) mouse xenograft models.

[0087] Bervalafenib has been shown in clinical trials to provide a safe and effective treatment for several cancers.

[0088] For example, a completed open-label Phase Ia dose escalation study examined several doses and schedules of belbalafenib in patients with solid tumors carrying mutations in the BRAF, KRAS, or NRAS genes. Efficacy was analyzed for 67 of 72 subjects who received 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), with partial response (PR) as the 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 with belbalafenib at or above the 100 mg once daily dose level. 59 (88.06%) subjects developed an event (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 of the median (7.12 weeks, 13.38 weeks). In the 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 subjects with BRAF mutant melanoma 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, with DOR in subjects at 100.29 weeks.

[0089] 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 post-baseline tumor assessment. 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 these, the two BRAF mutant melanoma responders had a median DOR of 22.49 weeks.

[0090] Another phase I, single-dose, randomized, crossover relative bioavailability and food effect study in healthy subjects evaluated the effect of changing formulation from phase I to phase II tablets on belbalafenib exposure. 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 between treatments. There was a positive effect of food on belbalafenib exposure in the fed state compared to the fasted state. Belbalafenib exposure, Cmax, and AUC0-inf were increased by approximately 2.2-fold and 2.8-fold, respectively, when belbalafenib was administered in the fed state to healthy subjects at a single dose of 200 mg, compared to the fasted state. No serious adverse events, adverse events of special interest, or deaths were reported in this study.

[0091] Bervalafenib or a pharma- ceutically acceptable salt thereof is suitably administered at about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, or about 1500 mg per day, and any range derived therefrom, the dosage being based on the active ingredient. Suitable daily dose ranges of berbalafenib may be about 100 mg to about 1500 mg, about 250 mg to about 1250 mg, about 500 mg to about 1000 mg, or about 700 mg to about 900 mg. In some embodiments, the subject is treated with berbalafenib or a pharma- ceutically acceptable salt thereof twice per day to achieve a total daily dose. In some such embodiments, the subject is treated with about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg of berbalafenib or a pharma- ceutically acceptable salt thereof twice per day. Other dosing regimens can be used to achieve a total daily dose, such as three times per day or four times per day. In any such dosing regimen, such as two, three, or four times per day, each dose may suitably be approximately equal. For example, if the daily dose is 900 mg, then two daily doses of 450 mg each or three daily doses of 300 mg each can be used.

[0092] 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.

[0093] PD-1 axis inhibitors According to the present disclosure, a PD-1 axis inhibitor may more specifically refer to a PD-1 inhibitor, a PD-L1 inhibitor, or a PD-L2 inhibitor. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0094] In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 inhibitor is a molecule that inhibits the binding of PD-L1 to its binding partner. In certain aspects, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, a PD-L2 inhibitor is a molecule that inhibits the binding of PD-L2 to its binding partner. In certain aspects, the PD-L2 binding partner is PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0095] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, lambrolizumab, and CT-011. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0096] In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). In still further embodiments, an isolated anti-PD-1 antibody is provided that comprises a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 1 and / or a light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO: 2. In still further embodiments, an isolated anti-PD-1 antibody is provided that comprises a heavy chain sequence and / or a light chain sequence, (a) The heavy chain sequence is: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN or has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence of AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1); (b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence of: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0097] In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). In still further embodiments, an isolated anti-PD-1 antibody is provided that comprises a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 3 and / or a light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO: 4. In still further embodiments, an isolated anti-PD-1 antibody is provided that comprises a heavy chain sequence and / or a light chain sequence, (a) the heavy chain sequence: QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRDYRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKTYTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVK GFYPSDIAVE has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence of WESNGQPENN YKTTPPVLDSDGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 3); (b) the light chain sequence: EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRL LIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 4).

[0098] In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 inhibitor is selected from the group consisting of YW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874. Antibody YW243.55.S70 (heavy and light chain variable regions set forth in SEQ ID NOs: 5 and 6, respectively) is an anti-PD-L1 antibody described in WO 2010 / 077634. ​​MEDI4736 is an anti-PD-L1 antibody described in WO 2011 / 066389 and US Patent Application Publication No. 2013 / 034559.

[0099] Examples of anti-PD-L1 antibodies useful in the methods of the invention, and methods for making them, are described in PCT Patent Application WO 2010 / 077634 A1 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.

[0100] In some embodiments, the PD-1 axis inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

[0101] Anti-PD-L1 antibodies useful in the present invention include compositions containing such antibodies, such as those described in WO 2010 / 077634. ​​In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 or 8 (Infra) and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9 (Infra).

[0102] In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region polypeptide comprising the sequences HVR-H1, HVR-H2, and HVR-H3; (a) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 10); (b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 11); (c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 12); Furthermore, X1 is D or G, X2 is S or L, and X3 is T or S.

[0103] In one particular embodiment, X1 is D, X2 is S, and X3 is T. In another embodiment, the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the following formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another embodiment, the framework sequences are derived from a human consensus framework sequence. In a further embodiment, the framework sequence is a VH subgroup III consensus framework. In yet a further embodiment, at least one of the framework sequences is selected from the following: HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13) HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 14) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 15) HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 16) It is.

[0104] In yet a further aspect, the heavy chain polypeptide is further combined with a light chain variable region comprising HVR-L1, HVR-L2 and HVR-L3, (a) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 17); (b) HVR-L2 sequence is SASX9LX 10 S, (SEQ ID NO: 18); (c) HVR-L3 sequence, QQX 11 X 12 X 13 X 14 PX 15 T (SEQ ID NO: 19); Further, X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; 10 is Y or A; X 11 is Y, G, F or S; X 12 is L, Y, F or W; X 13is Y, N, A, T, G, F or I; X 14 is H, V, P, T or I, and X 15 is A, W, R, P or T.

[0105] In still further embodiments, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; and X 10 is Y;X 11 is Y;X 12 is L;X 13 is Y;X 14 is H;X 15 is A. In a still further embodiment, the light chain further comprises a variable region light chain framework sequence juxtaposed between the HVRs according to the following formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a still further embodiment, the framework sequence is derived from a human consensus framework sequence. In a still further embodiment, the framework sequence is a VL kappa I consensus framework. In a still further embodiment, at least one of the framework sequences is selected from the following: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 20) LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 21) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 22) LC-FR4 is FGQGTKVEIKR (SEQ ID NO:23) It is.

[0106] In another embodiment, there is provided an isolated anti-PD-L1 antibody or antigen-binding fragment comprising a heavy chain variable region sequence and a light chain variable region sequence, The heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, and further comprises (i) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 10) (ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 11) (iii) the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO: 12) The light chain comprises HVR-L1, HVR-L2, and HVR-L3, and further comprises (i) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 17) (ii) HVR-L2 sequence is SASX9LX 10 S; and (SEQ ID NO: 18) (iii) HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 T; (SEQ ID NO: 19),

[0107] Further, X1 is D or G; X2 is S or L; X3 is T or S; X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X10 is Y or A; X11 is Y, G, F or S; X12 is L, Y, F or W; X13 is Y, N, A, T, G, F or I; X14 is H, V, P, T or I; and X15 is A, W, R, P or T.

[0108] In certain embodiments, X1 is D, X2 is S, and X3 is T. In other embodiments, X4 is D;X5 is V;X6 is S;X7 is A;X8 is V;X9 is F;X10 is Y;X11 is Y;X12 is L;X13 is Y;X14 is H; and X15 is A. In yet other embodiments, X1 is D;X2 is S, X3 is T, X4 is D;X5 is V;X6 is S;X7 is A;X8 is V;X9 is F;X10 is Y;X11 is Y;X12 is L;X13 is Y;X14 is H, and X15 is A.

[0109] In a further aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a still further aspect, the framework sequences are derived from a human consensus framework sequence. In a still further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequences are VH subgroup III consensus framework. In a still further aspect, the one or more heavy chain framework sequences are the following: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 14) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 15) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 16) As follows.

[0110] In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In a still further aspect, the light chain framework sequence is a VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is selected from the following: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 20) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 21) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 22) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 23) As follows.

[0111] In a still further particular aspect, the antibody further comprises a human or mouse constant region. In a still further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further particular aspect, the human constant region is IgG1. In a still further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further aspect, the mouse constant region is IgG2A. In a still further particular aspect, the antibody has reduced or minimal effector function. In a still further particular aspect, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In a still further embodiment, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0112] In yet another embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence: (a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 24), AWISPYGGSTYYADSVKG (SEQ ID NO: 25), and RHWPGGFDY (SEQ ID NO: 12), respectively; or (b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (sequence number 26), SASFLYS (sequence number 27), and QQYLYHPAT (sequence number 28), respectively.

[0113] In particular aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet further embodiments, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet further embodiments, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet further embodiments, the one or more heavy chain framework sequences are selected from the following: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 14) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 15) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 16) As follows.

[0114] In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In a still further aspect, the light chain framework sequence is a VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is selected from the following: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 20) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 21) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 22) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 23) As follows.

[0115] In yet further particular aspects, the antibody further comprises a human or mouse constant region. In still further aspects, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In still further particular aspects, the human constant region is IgG1. In still further aspects, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In still further aspects, the mouse constant region is IgG2A. In still further particular aspects, the antibody has reduced or minimal effector function. In still further particular aspects, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In still further embodiments, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0116] In yet a further embodiment, there is provided an isolated anti-PD-L1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence, (a) the heavy chain sequence has at least 85% sequence identity to the following heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 29); or (b) the light chain sequence has at least 85% sequence identity to the light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9).

[0117] In particular aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a further aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In a still further aspect, the heavy chain framework sequences are VH subgroup III consensus frameworks. In a still further aspect, one or more of the heavy chain framework sequences are selected from the following: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 14) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 15) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 16) As follows.

[0118] In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In a still further aspect, the light chain framework sequence is a VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is selected from the following: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 20) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 21) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 22) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 23) As follows.

[0119] In yet further particular aspects, the antibody further comprises a human or mouse constant region. In still further aspects, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In still further particular aspects, the human constant region is IgG1. In still further aspects, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In still further aspects, the mouse constant region is IgG2A. In still further particular aspects, the antibody has reduced or minimal effector function. In still further particular aspects, the minimal effector function results from production in a prokaryotic cell. In still further particular aspects, the minimal effector function results from an "effector-less Fc mutation" or aglycosylation. In still further embodiments, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0120] In another further embodiment, there is provided an isolated anti-PD-L1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence, (a) the heavy chain sequence is the following heavy chain sequence: Has at least 85% sequence identity with EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7); or (b) the light chain sequence has at least 85% sequence identity to the light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9).

[0121] In yet a further embodiment, there is provided an isolated anti-PD-L1 antibody comprising a heavy chain variable region sequence and a light chain variable region sequence, (a) the heavy chain sequence has at least 85% sequence identity to the following heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWI SPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8); or (b) the light chain sequence has at least 85% sequence identity to the following light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9).

[0122] In particular aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a further embodiment, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In a still further embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In a still further embodiment, one or more of the heavy chain framework sequences are selected from the following: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 14) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 15) HC-FR4 WGQGTLVTVSS (SEQ ID NO: 30) As follows.

[0123] In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In a still further aspect, the light chain framework sequence is a VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is selected from the following: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 20) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 21) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 22) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 23) As follows.

[0124] In yet further particular aspects, the antibody further comprises a human or mouse constant region. In still further aspects, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In still further particular aspects, the human constant region is IgG1. In still further aspects, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In still further aspects, the mouse constant region is IgG2A. In still further particular aspects, the antibody has reduced or minimal effector function. In still further particular aspects, the minimal effector function results from production in a prokaryotic cell. In still further particular aspects, the minimal effector function results from an "effector-less Fc mutation" or aglycosylation. In still further embodiments, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0125] In yet another embodiment, the anti-PD-L1 antibody is atezolizumab or MPDL3280A (CAS Registry Number: 1380723-44-3). In yet a further embodiment, the anti-PD-L1 antibody is atezolizumab or MPDL3280A (CAS Registry Number: 1380723-44-3). In yet a further embodiment, the anti-PD-L1 antibody is atezolizumab or MPDL3280A (CAS Registry Number: 1380723-44-3). In yet a further embodiment, the anti-PD-L1 antibody is atezolizumab or MPDL3280A (CAS Registry Number: 1380723-44-3). and a light chain variable region comprising the amino acid sequence of: LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9). In yet a further embodiment, an isolated anti-PD-L1 antibody is provided, comprising a heavy chain sequence and / or a light chain sequence: (a) does the heavy chain sequence have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the following heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31), and / or (b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the following light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32).

[0126] In yet a further embodiment, there is provided an isolated nucleic acid encoding the light chain variable region sequence or the heavy chain variable region sequence of an anti-PD-L1 antibody, (a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 24), AWISPYGGSTYYADSVKG (SEQ ID NO: 25), and RHWPGGFDY (SEQ ID NO: 12), respectively; (b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 26), SASFLYS (SEQ ID NO: 27), and QQYLYHPAT (SEQ ID NO: 28), respectively.

[0127] In particular aspects, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In one aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4) and the light chain variable region comprises one or more framework sequences juxtaposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a further embodiment, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In a still further embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In a still further embodiment, one or more of the heavy chain framework sequences are selected from the following: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 14) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 15) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 16) As follows.

[0128] In yet a further aspect, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In a still further aspect, the light chain framework sequence is a VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is selected from the following: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 20) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 21) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 22) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 23) As follows.

[0129] In still further particular aspects, the antibodies described herein (such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies) further comprise a human or mouse constant region. In still further aspects, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In still further particular aspects, the human constant region is IgG1. In still further aspects, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In still further aspects, the mouse constant region is IgG2A. In still further particular aspects, the antibody has reduced or minimal effector function. In still further particular aspects, the minimal effector function results from production in a prokaryotic cell. In still further particular aspects, the minimal effector function results from an "effector-less Fc mutation" or aglycosylation. In still further aspects, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region.

[0130] In still yet further aspects, provided herein is a nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the aforementioned anti-PD-L1, anti-PD-1, or anti-PD-L2 antibodies. In still further particular aspects, the vector further comprises a host cell suitable for expressing the nucleic acid. In still further particular aspects, the host cell is a eukaryotic cell or a prokaryotic cell. In still further particular aspects, the eukaryotic cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO).

[0131] The antibodies or antigen-binding fragments thereof may be produced using methods known in the art, for example by a process which comprises culturing a host cell containing nucleic acid encoding any of the above-mentioned anti-PD-L1, anti-PD-1, or anti-PD-L2 antibodies, or antigen-binding fragments, in a form suitable for expression, under conditions suitable for the production of such antibody or fragment, and recovering the antibody or fragment.

[0132] In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of the antibody is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from the antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) is removed. This alteration can be made by substitution of an asparagine, serine, or threonine residue within a glycosylation site with another amino acid residue (eg, glycine, alanine, or a conservative substitution).

[0133] In this regard, it should be noted that the pharmacokinetics of atezolizumab administered as a single agent has been characterized based on clinical data from study PCD4989g and is consistent with WO 29522, an ongoing Phase III study in the first-line treatment of triple-negative breast cancer (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). The 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 by reference herein in its entirety) do not suggest clinically meaningful differences in exposure following fixed doses or doses adjusted for body weight. Atezolizumab dosing schedules of q3w and q2w were tested. 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 studies of atezolizumab monotherapy across multiple tumor types, with q2w being used primarily in combination with chemotherapy regimens. In study PCD4989g, the Kaplan-Meier estimated overall 24-week progression-free survival (PFS) was 33% (95% CI: 12%, 53%).

[0134] The PD-1 axis inhibitor dose of the present disclosure is suitably about 400 mg to about 1200 mg, about 600 mg to about 1000 mg, about 700 mg to about 900 mg, or about 840 mg. In some embodiments, the PD-1 axis inhibitor is a PD-L1 inhibitor, more specifically, atezolizumab administered at a dose of about 840 mg.

[0135] In certain embodiments, the PD-1 axis inhibitor, or more specifically, the PD-L1 inhibitor, is administered intravenously every 14 days of a 28-day treatment cycle. In some aspects, the subject is treated with the PD-1 axis inhibitor, or more specifically, the PD-L1 inhibitor, on days 1 and 15 of a 28-day treatment cycle.

[0136] Cancers characterized by mutated MAPK signaling pathways In some embodiments, the cancer with a mutated MAPK signaling pathway for treatment by the methods of the present disclosure is selected from melanoma, lung, breast, colorectal (CRC), bladder, gallbladder, nephroblastoma, gastrointestinal stromal tumor (GIST), prostate, glioblastoma, myeloid leukemia, multiple myeloma, thyroid, bile duct, adenocarcinoma, choriocarcinoma, sarcoma, and combinations thereof. In some embodiments, the cancer is selected from melanoma, nephroblastoma, GIST, CRC, sarcoma, gallbladder cancer, bladder cancer, and combinations thereof.

[0137] In some embodiments, the cancer has an NRAS mutation, a KRAS mutation, or a RAF mutation. In some such embodiments, the cancer has at least one mutation selected from a BRAF V600E mutation, a KRAS G12V mutation, a KRAS G12D mutation, a KRAS G12C mutation, a KRAS Q61H mutation, a NRAS G13D mutation, a NRAS G12D mutation, a NRAS Q61K mutation, a NRAS Q61R mutation, and a NRAS G12C mutation.

[0138] In some embodiments, the cancer has a RAF mutation. In some such embodiments, the cancer has a BRAF V600E mutation. In some such embodiments, the cancer is selected from BRAF V600E nephroblastoma, BRAF V600E mutated melanoma, BRAF V600E mutated GIST, BRAF V600E mutated CRC, and combinations thereof. In other such embodiments, the cancer is BRAF V600E mutated melanoma, BRAF V600E mutated nephroblastoma, BRAF V600E mutated GIST, and combinations thereof. In other such embodiments, the cancer is selected from BRAF V600E mutated melanoma, BRAF V600E mutated GIST, and combinations thereof. In other such embodiments, the cancer is selected from BRAF V600E mutated melanoma, BRAF V600E mutated GIST, and combinations thereof. In some such embodiments, the melanoma is metastatic or unresectable.

[0139] In some NRAS / KRAS embodiments, the cancer is metastatic or unresectable melanoma.

[0140] In some such embodiments, the cancer is NRAS mutated melanoma.

[0141] In some NRAS / KRAS embodiments, the cancer is selected from KRAS G12V mutated sarcoma, NRAS G13D mutated melanoma, NRAS G12D, NRAS Q61K mutated melanoma, NRAS Q61R mutated melanoma, NRAS G12C mutated melanoma, KRAS G12D mutated gallbladder cancer, KRAS G12C mutated CRC, KRAS Q61H mutated CRC, KRAS G12D mutated CRC, KRAS G12D mutated bladder cancer, KRAS G12V mutated bladder cancer, and combinations thereof.

[0142] In other NRAS / KRAS aspects, the cancer is a sarcoma with a KRAS G12V mutation, a melanoma with a NRAS G13D mutation, a melanoma with a NRAS G12D mutation, a melanoma with a NRAS G12C mutation, a gallbladder cancer with a KRAS G12D mutation, a CRC with a KRAS G12C mutation, a CRC with a KRAS Q61H mutation, a CRC with a KRAS G12D mutation, a bladder cancer with a KRAS G12D mutation, a bladder cancer with a KRAS G12V mutation, and combinations thereof. In some such aspects, the cancer is selected from a sarcoma with a KRAS G12V mutation, a melanoma with a NRAS G13D mutation, a melanoma with a NRAS Q61K mutation, a melanoma with a NRAS Q61R mutation, and combinations thereof.

[0143] Combination treatment The combination therapy of RAF inhibitors and PD-1 axis inhibitors targets the MAPK signaling pathway, and based on the present experimental evidence, it is believed that the combination therapy will produce synergistic anti-tumor activity in cancers characterized by mutated MAPK signaling pathways.It is further believed that the combination therapy of the present disclosure can extend the median progression-free survival of subjects with such cancers.It is believed that the combination therapy is particularly useful for treating metastatic and / or unresectable melanomas with RAF mutations, such as BRAF V600E mutations.

[0144] Furthermore, the combination of a RAF inhibitor, such as belbalafenib, and a PD-1 axis inhibitor, such as atezolizumab, is believed to provide patients with cancers characterized by mutated MAPK signaling pathways with aggressive treatment with reduced toxicity compared to chemotherapy-based regimens. Moreover, since the mechanism of action of the combination treatment of the present invention is different from conventional chemotherapy regimens, it is further believed that the activity of additional standard treatments will not be significantly affected, allowing patients with progressive disease to continue treatment.

[0145] In this regard, it should be noted that any combination of the recited dosage ranges of the recited components of the combination may be used without departing from the intended scope of the present disclosure. In some embodiments of the present disclosure, a combination of cancer therapeutics is provided that includes: (i) a RAF inhibitor at a twice-daily dose of about 250 mg to about 500 mg, or about 350 mg to about 450 mg of berbalafenib or a pharma- ceutically acceptable salt thereof; (ii) a PD-1 axis inhibitor at a dose of about 400 mg to about 1200 mg, about 600 mg to about 1000 mg, about 700 mg to about 900 mg, or about 840 mg. In a particular embodiment, the RAF inhibitor is berbalafenib and the PD-L1 inhibitor is atezolizumab.

[0146] When a subject is administered a drug combination (i.e., a RAF inhibitor and a PD-1 axis inhibitor) on the same day, the drugs can be administered separately in any order. In some embodiments, the RAF inhibitor and the PD-1 axis inhibitor are each administered on the same day, and the RAF inhibitor is administered before, after, or simultaneously with the PD-1 axis inhibitor. The administration of each drug of the drug combination can be separated by a period of time, such as 0.5 hours, 1 hour, 2 hours, 3 hours, or 4 hours. In some specific embodiments, berbalafenib can be administered orally and atezolizumab can be administered intravenously. In such embodiments, berbalafenib can be administered before or after atezolizumab, or they can be administered simultaneously or closely separated in time. In some embodiments, the RAF inhibitor and the PD-1 axis inhibitor are administered on days 1 and 15, respectively, of a 28-day treatment cycle, and the RAF inhibitor is administered on days 1 to 21 of a 28-day treatment cycle or days 1 to 28 of a 28-day treatment cycle.

[0147] In some embodiments, the RAF inhibitor is administered with food.

[0148] In some embodiments, the subject has previously received a course of treatment with an anti-PD-1 agent or an anti-PD-L1 agent.

[0149] In some embodiments, prior to treatment with a method according to the present disclosure, the subject experienced disease progression following treatment with immunotherapy, BRAF V600E therapy, or a combination of immunotherapy and BRAF V600E therapy.

[0150] In some embodiments, the methods for treating cancer are characterized by the absence of occurrence of squamous cell carcinoma in a human subject. EXAMPLES

[0151] The Animal Care and Use Committee of Hanmi Research Center approved the animal experiment protocols of the Examples.

[0152] Example 1

[0153] In Example 1, the efficacy of belbalafenib monotherapy, atezolizumab monotherapy, and combination therapy of belbalafenib and atezolizumab was evaluated in a mutant K1735 subcutaneous mouse model.

[0154] The mouse strain was C3H / HeNCrlOri, which is widely used for the K1735 syngeneic model. Mice were supplied by Orient Bio Inc. (Korea). Mice were female, 9-11 weeks old at the start of dosing, and had a weight range of 19-26 grams.

[0155] The cell line was K1735 and was provided by the American Type Culture Collection (ATCC). K1735 cells are melanoma cells with NRASG13D mutation. The cell culture in vitro medium was Roswell Park Memorial Institute (RPMI) 10% fetal bovine serum (FBS), and incubation was at 5% CO2 and 37°C. For the K1735 syngeneic model, cancer cells (1.5 x 108 cells / 10 mL) were mixed with Hank's balanced salt solution (HBSS) and injected subcutaneously at 0.1 mL / head. Seven animals per group were treated with vehicle (control), 7.5 mg / kg belbalafenib monotherapy, 15 mg / kg belbalafenib monotherapy, 10 mg / kg atezolizumab monotherapy, belbalafenib (7.5 mg / kg) and atezolizumab (10 mg / kg) combination therapy, and belbalafenib (15 mg / kg) and atezolizumab (10 mg / kg) combination therapy. Experiments were performed using first generation tumor tissue.

[0156] Mice were kept in conventional animal laboratory cages for 14 days for acclimatization before the start of the experiment. During the acclimatization period, mice were observed daily for health and disease signs. Mice were housed in a clean barrier room in polysulfone cages 1291H (W425 × D266 × H185 mm, Techniplast, Italy). Ten mice were housed in each cage at a temperature of 22 ± 2 °C, a relative humidity of 50 ± 20%, an air change frequency of 10-15 times / h, a 12-h light / dark cycle, a light intensity of 150-300 lux, and at least weekly cage changes. Mice were fed Picolab Rodent chow (5053, Lab Diet, USA) with abundant tap water.

[0157] Berbalafenib dihydrochloride, 99.6% purity, stored at room temperature was used. Dosing was based on the active ingredient free base and corrected for assay and water content. Dosing vehicles were DMSO (5%), Cremophor EL (5%) and deionized water (90%). Berbalafenib for dosing was dissolved in the vehicle. Berbalafenib oral doses of 7.5 mg / kg and 15 mg / kg were evaluated as monotherapy and in combination with atezolizumab. Berbalafenib was administered daily for 21 days.

[0158] Atezolizumab (TECENTRIQ) stored at 2-8°C (登録商標) ) was used. The dosing vehicle was saline and the dosing concentration was 5 mL / kg. An intravenous dose of 10 mg / kg atezolizumab was evaluated as monotherapy and in combination with 7.5 mg / kg and 15 mg / kg belbalafenib. Atezolizumab was administered three times weekly for three weeks.

[0159] The following observations and measurements were made:

[0160] Clinical Signs. General clinical signs and mortality were observed at least once daily during the dosing period.

[0161] Body weight. Body weight measurements were performed twice weekly during the treatment period.

[0162] Relative weight. Relative weight (%) = weight (g) / initial weight (g) x 100.

[0163] Tumor size. Tumor size was assessed twice a week during the treatment period by digital calipers (MITUTOYO CD-15CPX, MonotaRO Singapore, Japan) and calculated using the formula for ellipsoidal spheroid (V=L×S2 / 2=mm3, where L=longer diameter, S=shorter diameter). Tumor size was recorded on the day of weight measurement, and the data was recorded on the tumor volume record sheet.

[0164] Relative tumor volume (RTV). RTV(%)=tumor volume / initial tumor volume×100.

[0165] Tumor growth (TG). TG(%)=RTVdayx / RTVday0×100.

[0166] Relative tumor growth (RTG). The ratio of the mean tumor volume on the last measurement day to the mean tumor volume on day 0.

[0167] Percent inhibition (IR). Inhibition of tumor growth was calculated relative to vehicle-treated controls. IR (%) = (1 - mean relative tumor weight of treatment group / mean relative tumor weight of control group) x 100.

[0168] Maximum Inhibition Rate (MIR). The MIR was the highest recorded IR (%) among the inhibition rates (%) measured.

[0169] Mean Tumor Weight (MTW). MTW was at the start of the study and at the end of the study.

[0170] Maximum Weight Loss (MWL). The MWL was the highest recorded weight loss (%) among the weight losses (%) measured.

[0171] Flow cytometry was used for tumor-infiltrating CD3+CD8+ T cell analysis using BD FACSCanto™ IL (BD Biosciences) and FlowJo™ v 10.6.2 software (BD Biosciences). At the end of the experiment, tumor tissues were collected for pharmacodynamic evaluation, except for the monotherapy group and combination therapy group with 7.5 mg / kg velvalafenib. Tumors were surgically dissected, washed twice with phosphate-buffered saline (PBS), and then chopped into 1 mm3 pieces. The chopped tumor pieces were digested in 7 mL of dissociation medium (RPMI medium containing 10% FBS, 2 mg / mL collagenase type II, 2 mg / mL collagenase type IV, and 1 mg / mL DNase I) at 37°C for 30 minutes and filtered through a 70 μm cell strainer (BD Pharm, USA). The filtered cells were then washed twice with PBS. The remaining red blood cells were lysed using ammonium-potassium chloride (ACK) solution. Dissociated single cells were stained with Fe block (anti-CD16 / 32, Ref. 14-0161-82, Invitrogen, USA) for 15 min at 4°C to prevent non-specific antibody binding. Cells were then stained with appropriate antibodies for 30 min at 4°C. To assess the presence of CD3+CD8+ T cell populations, cells were stained with PE Cy7-conjugated anti-CD3e (1:100 dilution) (clone: ​​145-2C11, Invitrogen, USA) and FITC-conjugated anti-CD8a (1:100 dilution) (clone: ​​53-6.7, Invitrogen, USA).

[0172] Statistical analysis of tumor size was performed using GraphPad Prism version 6 (GraphPad software, Inc., USA). Values ​​were expressed as ± SEM. The significance of differences between multiple groups was assessed using two-way ANOVA. Post-hoc pairwise comparisons between groups were tested for significance using Dennett's method.

[0173] No clinical signs were observed except for tumor necrosis in one mouse in a group of seven mice on day 21 of atezolizumab monotherapy.

[0174] The results are shown in Figures 1 to 3 and the following Tables 1 to 3.

[0175] Weight loss is shown in Figure 1, which is a plot of mouse weight change versus time for the K1735 syngeneic model for the present disclosure of belbalafenib monotherapy (7.5mg / kg and 15mg / kg), atezolizumab monotherapy (10mg / kg), combination treatment of belbalafenib at 7.5mg / kg and atezolizumab at 10mg / kg, and combination treatment of belbalafenib at 15mg / kg and atezolizumab at 10mg / kg. HM95573 refers to belbalafenib. Points, mean relative weight; Bar, SEM No specific weight loss or clinical symptoms were observed during administration.

[0176] Tumor volumes are shown in Figure 2, which is a plot of mouse tumor volume versus time for the K1735 syngeneic model for belbalafenib monotherapy (7.5 mg / kg and 15 mg / kg), atezolizumab monotherapy (10 mg / kg), belbalafenib / atezolizumab combination therapy ((i) 7.5 mg / kg and 10 mg / kg and (ii) 15 mg / kg and 10 mg / kg) of the present disclosure. In Figure 2: HM95573 refers to belbalafenib. * P < 0.05; **** indicates P<0.0001; § indicates P<0.05 compared to atezolizumab 10 mg / kg; # indicates P<0.05 compared to belvalafenib 15 mg / kg. P values ​​were calculated using two-way ANOVA. Points, mean tumor volume; bars, SEM

[0177] Figure 3 is a plot of CD3+CD8+ T cells for the K1735 syngeneic mouse model with belbalafenib monotherapy (15 mg / kg), atezolizumab monotherapy (10 mg / kg), and belbalafenib / atezolizumab combination therapy (15 mg / kg and 10 mg / kg). In Figure 3: HM95573 refers to belbalafenib. ***indicates P<0.001 compared to vehicle control; ### indicates P<0.001 compared to 15 mg / kg belvalafenib; § indicates P<0.05 compared to 10 mg / kg atezolizumab. P values ​​were calculated using one-way ANOVA. Points, mean of CD3+CD8+ T cells; bars, SEM

[0178] The results are summarized in Table 1. Maximum inhibition (%) is the highest value among IR (%). Maximum weight loss (%) is the highest recorded value among the measured weight loss (%). In Table 1, "Belv.Mono." refers to belvalafenib monotherapy; "Atezo.Mono." refers to atezolizumab monotherapy; "Belv. / Atezo.Comb." refers to belvalafenib / atezolizumab combination therapy; "QDx21" refers to daily oral doses for 21 days; "TIWx3" refers to intraperitoneal doses three times a week for 21 days; "Max Inhib." refers to maximum inhibition in %; "Max Wt." refers to maximum weight loss in %.

[0179] [Table 1]

[0180] Table 2 shows the antitumor activity of belvalafenib monotherapy, atezolizumab monotherapy, and belvalafenib + atezolizumab combination treatment in the K1735 syngeneic mouse model. In Table 2, "Belv." refers to belvalafenib monotherapy; "Atezo." refers to atezolizumab monotherapy; "Belv.+Atezo." refers to belvalafenib and atezolizumab combination treatment; "QDx21" refers to daily oral doses for 21 days; "TIWx3" refers to intraperitoneal doses three times a week for 21 days; "MTV0" refers to the mean tumor volume on day 1 of treatment; "MTV0" refers to the mean tumor volume on day 2 of treatment; "MTV0" refers to the mean tumor volume on day 3 of treatment ... 21 " refers to the average tumor volume on the 21st day of treatment; "RTG" refers to relative tumor growth; "" refers to maximum weight loss; "IR(Day 21)" refers to the inhibition rate on day 21; "MIR" refers to the maximum inhibition rate.

[0181] [Table 2]

[0182] Table 3 shows the CD of belbalafenib monotherapy, atezolizumab monotherapy, and belbalafenib + atezolizumab combination therapy in the K1735 syngeneic mouse model. 3 +CD8 + The percentage of T cells is shown.

[0183] [Table 3]

[0184] Tumor growth inhibition occurred. On day 21, belbalafenib (7.5 and 15 mg / kg) and atezolizumab (10 mg / kg) showed 48.2%, 54.7% and 53.4% ​​tumor growth inhibition, respectively. The combination of belbalafenib (7.5 or 15 mg / kg) and atezolizumab (10 mg / kg) showed 78.3% and 84.8% tumor growth inhibition, respectively, and the effect of the combination treatment was significant compared with the single-agent treatment (P<0.05).

[0185] The CD3+CD8+ T cells were generated. Berbalafenib monotherapy (15 mg / kg) and atezolizumab monotherapy (10 mg / kg) showed 1.27% and 6.52% of CD3+CD8+ T cells, respectively, which did not increase significantly compared to the control group. The combination of Berbalafenib (15 mg / kg) and atezolizumab (10 mg / kg) significantly increased CD3+CD8+ T cells to 16.0% compared to each monotherapy (P<0.001 vs. Berbalafenib (15 mg / kg) and P<0.05 vs. atezolizumab (10 mg / kg)).

[0186] The experimental results show that simultaneous administration of berbalafenib and atezolizumab in combination therapy synergistically inhibited tumor growth and induced infiltration of cytotoxic T cells compared with berbalafenib and atezolizumab monotherapy in the NRASG13D mutant K1735 syngeneic mouse model. Therefore, the results demonstrate that combination treatment of pan-RAF inhibitors and PD-L1 inhibitors may be an effective anti-cancer treatment in NRASG13D mutant melanoma patients with mutated MAPK signaling.

[0187] Example 2

[0188] In Example 2, the efficacy of berbalafenib monotherapy, Mu igG1 anti-PDL1 monotherapy, and combination therapy of berbalafenib and Mu igG1 anti-PDL1 was evaluated in a CT26 syngeneic mouse model (KRASG12D, CRC).

[0189] Mu igG1 anti-PDL1 (6E11) WT was administered orally (PO) at 5 mg / kg twice weekly (BIW) for 3 weeks, either alone or in combination with berbalafenib. Berbalafenib was administered PO at 10 mg / kg once daily (QD) for 21 days, either alone or in combination with Mu igG1 anti-PDL1 (6E11) WT. The vehicle was 5% dimethylsulfide / 5% Cremophor EL (100 μL), 0.5% (w / v) methylcellulose / 0.2% Tween 80™.

[0190] Tumor volumes were measured in two dimensions (length and width) using Ultra Cal-IV calipers (model 54-10-111; Fred V. Fowler Co.; Newton, Massachusetts) and analyzed using Excel, version 14.2.5 (Microsoft Corporation; Redmond, Wash.). Tumor volumes were calculated using the following formula: tumor size (mm3) = (longer measurement x shorter measurement2) x 0.5.

[0191] %TGI=percentage of tumor growth inhibition based on AUC.

[0192] Transformed tumor volumes over time were analyzed using generalized additive mixed models (GAMMs) because this approach addresses both repeated measurements from the same study subjects and moderate dropout before the end of the study (Lin et al. 1999 and Liang 2005). Because tumors generally exhibit exponential growth, tumor volumes were subjected to natural log transformation before analysis. Changes in tumor volume over time in each group are described by fits (i.e., regression splines with automatically generated spline basis) generated using customized functions in R version 3.4.2 (2017-09-28) (R Development Core Team 2008; R Foundation for Statistical Computing; Vienna, Austria), which integrates software from several open source packages, including ime4, mgcv, gamm4, multcomp, setting, plyr, and tidyverse, e.g., magrittr, dplyr, tidyr, and ggplot2.

[0193] Anti-tumor responses were observed during the study with a partial response (PR) being defined as a greater than 50% reduction from the initial tumor volume, and a complete response (CR) being defined as a 100% reduction in tumor volume, for the purposes of this example.

[0194] Animal weights were measured using an Adventura Pro AV812 scale (Ohaus Corporation; Pine Brook, NJ). Percent weight change was calculated using the following formula: Weight Change (%) = [(Current Weight / Initial Weight)-1) x 100]. Percent animal weight was tracked for each individual animal during the study, and the percent change in weight was calculated and plotted for each group.

[0195] Generalized additive mixed models (GAMMs) were also used to analyze live weight (i.e., grams) over time. After data fitting, live weight data at each time point from all individual animals and all group fits were normalized and replotted separately in two different ways: 1) normalized to starting weight and reported as a percentage resulting in % body weight change, and 2) normalized to maximum weight to date and reported as a percentage resulting in % body weight loss.

[0196] Efficacy estimates were obtained by calculating the percentage difference between the mean daily baseline-corrected AUC of the relevant groups fitted to the original (i.e., untransformed) scale over a common time period.

[0197] Tumor volume results are shown in Figure 4A for vehicle, Figure 4B for Mu igG1 anti-PDL1 (6E11) WT, Figure 4C for berbalafenib, Figure 4D for the combination of Mu igG1 anti-PDL1 (6E11) WT and berbalafenib, and Figure 6A for associated overlays.

[0198] Body weight change results are shown in Figure 5A for vehicle, Figure 5B for Mu igG1 anti-PDL1 (6E11) WT, Figure 5C for berbalafenib, and Figure 5D for the combination of Mu igG1 anti-PDL1 (6E11) WT and berbalafenib, with body weight changes fitted in Figure 6B for associated overlay.

[0199] Example 3

[0200] The protocol of Example 2 was repeated in the EMT6 syngeneic model (KRASwT, TNBC).

[0201] Tumor volume results are shown in Figure 7A for vehicle, Figure 7B for Mu igG1 anti-PDL1 (6E11) WT, Figure 7C for berbalafenib, Figure 7D for the combination of Mu igG1 anti-PDL1 (6E11) WT and berbalafenib, and Figure 9A for the associated overlay.

[0202] Body weight change results are shown in Figure 8A for vehicle, Figure 8B for Mu igG1 anti-PDL1 (6E11) WT, Figure 8C for berbalafenib, Figure 8D for the combination of Mu igG1 anti-PDL1 (6E11) WT and berbalafenib, with body weight changes fitted in Figure 9B for associated overlay.

[0203] 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. 1. A pharmaceutical composition for preventing or treating cancer in a subject having a cancer characterized by a mutated MAPK signaling pathway, the pharmaceutical composition comprising: (i) a therapeutically effective amount of a RAF inhibitor; and (ii) a therapeutically effective amount of a PD-1 axis inhibitor.

2. 2. The pharmaceutical composition of claim 1, wherein the cancer is selected from melanoma, lung cancer, breast cancer, colorectal cancer (CRC), bladder cancer, gallbladder cancer, nephroblastoma, gastrointestinal stromal tumor (GIST), prostate cancer, glioblastoma, myeloid leukemia, multiple myeloma, thyroid cancer, cholangiocarcinoma, adenocarcinoma, choriocarcinoma, sarcoma, and combinations thereof.

3. The pharmaceutical composition of claim 1 , wherein the cancer has a RAF mutation, an NRAS mutation, or a KRAS mutation.

4. 4. The pharmaceutical composition of claim 3, wherein the cancer has at least one mutation selected from a BRAF V600E mutation, a KRAS G12V mutation, a KRAS G12D mutation, a KRAS G12C mutation, a KRAS Q61H mutation, a NRAS G13D mutation, a NRAS G12D mutation, a NRAS Q61K mutation, a NRAS Q61R mutation, and a NRAS G12C mutation.

5. The cancer is selected from the group consisting of nephroblastoma having a BRAF V600E mutation, melanoma having a BRAF V600E mutation, GIST having a BRAF V600E mutation, CRC having a BRAF V600E mutation, sarcoma having a KRAS G12V mutation, melanoma having an NRAS G13D mutation, melanoma having an NRAS G12D mutation, melanoma having an NRAS Q61K mutation, melanoma having an NRAS Q61R mutation, melanoma having an NRAS G12C mutation, gallbladder cancer having a KRAS G12D mutation, CRC having a KRAS G12C mutation, CRC having a KRAS Q61H mutation, CRC having a KRAS G12D mutation, bladder cancer having a KRAS G12D mutation, and sarcoma having a KRAS G12V mutation.

4. The pharmaceutical composition of claim 3, wherein the cancer is selected from the group consisting of bladder cancer with G12V mutation, bladder cancer with G12V mutation, and combinations thereof.

6. The pharmaceutical composition of claim 2 , wherein the melanoma is metastatic or unresectable.

7. The pharmaceutical composition of claim 1 , wherein the RAF inhibitor is a pan-RAF inhibitor.

8. The pharmaceutical composition of claim 7, wherein the RAF inhibitor is berbalafenib or a pharma- ceutically acceptable salt thereof.

9. The pharmaceutical composition of claim 1, wherein the PD-1 axis inhibitor is a PD-L1 inhibitor.

10. 10. The pharmaceutical composition of claim 9, wherein the PD-L1 inhibitor is an antibody comprising a heavy chain comprising the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 24), the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 25), and the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 12), and a light chain comprising the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 26), the HVR-L2 sequence of SASFLYS (SEQ ID NO: 27), and the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 28).

11. The PD-L1 inhibitor, a heavy chain variable region comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 7); a light chain variable region comprising the amino acid sequence of: The pharmaceutical composition of claim 9, which is an antibody comprising:

12. The pharmaceutical composition of claim 9, wherein the PD-L1 inhibitor is atezolizumab.

13. 10. The pharmaceutical composition of claim 1, wherein the subject is treated with 100 mg to 1500 mg of the RAF inhibitor per day.

14. 2. The pharmaceutical composition of claim 1, wherein the RAF inhibitor is berbalafenib or a pharma- ceutical acceptable salt thereof, and further wherein the subject is (i) treated with 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, or 1300 mg of berbalafenib or a pharma- ceutical acceptable salt thereof per day, or (ii) treated with 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg of berbalafenib or a pharma- ceutical acceptable salt thereof twice daily.

15. 15. The pharmaceutical composition of claim 14, wherein berbalafenib is administered daily for 28 consecutive days of a 28-day treatment cycle.

16. 2. The pharmaceutical composition of claim 1, wherein the subject is (i) treated with 400 mg to 1200 mg of the PD-1 axis inhibitor on 2 days of a 28 day treatment cycle, (ii) treated with the PD-1 axis inhibitor every 14 days of a 28 day treatment cycle, or (iii) treated with the PD-1 axis inhibitor on days 1 and 15 of the 28 day treatment cycle.

17. The pharmaceutical composition of claim 1, wherein (i) the RAF inhibitor and the PD-1 axis inhibitor are administered on days 1 and 15, respectively, of a 28 day treatment cycle, or (ii) if the RAF inhibitor and the PD-1 axis inhibitor are administered on the same days, the RAF inhibitor is administered before, after, or simultaneously with administration of the PD-1 axis inhibitor.

18. The pharmaceutical composition of claim 1 , wherein the RAF inhibitor is administered with food.

19. The pharmaceutical composition of claim 1, wherein the subject has previously been administered a course of treatment with an anti-PD-1 drug or an anti-PD-L1 drug.

20. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is characterized by the absence of occurrence of squamous cell carcinoma in the human subject.

21. the cancer is melanoma; Prior to the treatment, the human subject experienced disease progression following treatment with immunotherapy, BRAF V600E therapy, or a combination of immunotherapy and BRAF V600E therapy. A pharmaceutical composition according to any one of claims 1 to 20.

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