Precision Therapies for the Treatment of Cancer

Abstract

The present invention provides RAF and MEK inhibitors for combined use in the treatment of solid tumors, as well as methods for identifying patients suffering from solid tumors for suitability for combined treatment with RAF and MEK inhibitors based on identifying certain RAS mutations, corresponding methods of treatment, and related aspects or embodiments of the invention as described in detail below. The RAS mutations are codon Q61 or G13R mutations.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,763, filed March 2, 2022, the entirety of which is incorporated herein by reference.

[0002] The present invention provides RAF inhibitors and MEK inhibitors for combined use in the treatment of solid tumors, as well as methods for identifying patients suffering from the solid tumors described herein for responsiveness to combined treatment with a RAF inhibitor (e.g., naporafenib or belbalafenib) and a MEK inhibitor (e.g., trametinib or cobimetinib) based on the identification of certain RAS mutations that can serve as prognostic markers of responsiveness to a given therapy, corresponding methods of treatment, and related aspects or embodiments of the invention as described in detail below.

[0003] In particular, the present invention provides a combination of a RAF inhibitor and a MEK inhibitor for use in the treatment of solid tumors that harbor RAS codon Q61 or G13R mutations.

[0004] In one embodiment, the solid tumor to be treated is the solid tumor that has RAS codon Q61 mutation or G13R mutation and is not melanoma or NRAS mutant melanoma.In another embodiment, the solid tumor to be treated is the solid tumor that has RAS codon Q61 mutation or GI3R mutation and is not rhabdomyosarcoma.In another embodiment, the solid tumor to be treated is the melanoma that has NRAS codon Q61 mutation or the melanoma that has NRAS G13R mutation. [Background technology]

[0005] RAS-mutated cancer RAS is the most frequently mutated gene family in cancer. In addition to NRAS, both the proto-oncogenes KRAS and, to a lesser extent, HRAS are frequently mutated in several different types of human cancer. The occurrence of RAS mutations varies widely across cancers, but activating mutations of KRAS are frequently found in lung, colon, and pancreatic cancers (Cox et al, 2014; Nat. Rev. Drug Discov. 13, 828-851).

[0006] In the vast majority of cases, activating mutations in the KRAS, NRAS, and HRAS genes result in changes to the amino acids G12, G13, and Q61 in the respective proteins. However, the frequency of each amino acid change, the specific amino acid substitutions, and which RAS paralogs the changes occur in vary substantially between cancer types. For example, in lung adenocarcinoma, G12C mutations represent more than 40% of all RAS mutations, whereas in colorectal and pancreatic cancers, G12C represents approximately 6% and 2% of KRAS mutations, respectively (Cook et al., 2021). This imbalance in KRAS mutations arises, in part, from differences in mutagenicity between tissue types (e.g., in lung cancer, smoking). However, differences in mutagenicity alone do not appear to explain the majority of such allelic imbalances, suggesting that different RAS mutations may have unique biological properties that intersect with the pathogenesis of different cancer types (Cook et al., 2021).

[0007] Despite the fact that RAS is the most frequently mutated gene family in cancer and is associated with poor outcomes, there is currently only one approved therapy targeting RAS mutations: sotorasib, a KRAS G12C inhibitor, has shown anticancer activity in patients with locally advanced or metastatic NSCLC harboring KRAS codon G12C mutations and was granted accelerated approval by the FDA in 2021. For other solid tumor types and RAS mutations, there remains a significant unmet need in patients with advanced cancer who have progressed on currently available standard therapies or for whom no standard treatment options are currently available.

[0008] melanoma Melanoma is the most aggressive form of all skin cancers. The global incidence of melanoma is approximately 324,635 new cases and 57,043 deaths per year (Sung et al. 2020; Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. Available at: https: / / doi.org / 10.3322 / caac.21660 (Accessed: 2 August 2021)). The incidence of melanoma is the second highest among cancers in patients under the age of 40 years, behind only breast cancer in women and prostate cancer in men. Most patients with melanoma are diagnosed at an early stage when surgical resection is considered curative, and have excellent survival rates (approximately 90% at 5 years). In contrast, patients with distant metastases have a very poor prognosis, with a 5-year survival rate of approximately 27% (American Cancer Society 2021; Cancer facts and figures 2021. ACS website https: / / www.nccn.org / professionals / physician_gls / pdf / cutaneous_melanoma.pdf, published in 2021).

[0009] First-line systemic therapy for patients with unresectable or metastatic melanoma includes anti-programmed cell death protein-1 (anti-PD-1) monotherapy (i.e., nivolumab or pembrolizumab) or anti-PD-1 in combination with anti-CTLA-4 therapy (i.e., ipilimumab). In patients with melanomas harboring BRAF V600 mutations, which occur in approximately 40-60% of cases (Robert et al. 2019; The Lancet Oncology, Volume 20, Issue 9., KEYNOTE-006), treatments include targeted therapy with a combination of BRAF and MEK inhibitors (e.g., dabrafenib and trametinib) (NCCN Guidelines® Melanoma Version 2 2021, https: / / www.nccn.org / professionals / physician_gls / pdf / colon.pdf; ESMO guidelines Michielin et al. 2019; Ann Oncol; 30(12):1884-1901).

[0010] Besides BRAF mutations, the second most common mitogen-activated protein kinase (MAPK) pathway abnormality in melanoma is NRAS mutation. Compared with other melanoma subtypes, melanomas with NRAS mutations are associated with a poorer prognosis (Devitt et al, 2011).

[0011] Selective pharmacological inhibition of NRAS remains technically challenging, as its GTPase activity has so far prevented the successful design of specific small molecule antagonists. Several clinical trials are underway to evaluate alternative strategies that combine inhibitors of downstream molecules of NRAS, such as BRAF and MEK inhibitors, or PI3K and ERK pathway inhibitors. The efficacy of these various approaches remains to be established.

[0012] Indeed, currently, no targeted therapy is approved for NRAS mutant melanoma, and patients with metastatic disease typically receive anti-PD-1 monotherapy or anti-PD-1 in combination with anti-CTLA4 (ipilimumab) as first-line treatment. Furthermore, leratolimab, a LAG-3-blocking antibody in combination with anti-PD-1, recently demonstrated a statistically significant PFS benefit over anti-PD-1 monotherapy in a phase 3 study in patients with metastatic melanoma. If this regimen is approved by health authorities, it may become the new standard of care for this patient population. There is no established standard of care for patients with NRAS mutant unresectable or metastatic melanoma who have progressed on or are resistant to PD-1 inhibitors (with or without ipilimumab).

[0013] Current NCCN guidelines recommend a variety of systemic therapies as second and subsequent lines in patients with metastatic melanoma who have failed prior anti-PD(L)1 as monotherapy or in combination with ipilimumab, including ipilimumab monotherapy, high-dose IL-2 and cytotoxic agents such as dacarbazine, nab-paclitaxel, temozolomide or paclitaxel + carboplatin (NCCN, cited above). The ESMO guidelines recognize that second-line treatment options for NRAS mutant metastatic melanoma are very limited. Recommendations include treatment with MEK inhibitors (despite known limited activity), inclusion in clinical trials, or re-administration of immunotherapy (e.g., ipilimumab if ipilimumab was not administered prior). In the absence of clinical trials or new compounds available, cytotoxic drugs such as dacarbazine or temozolomide may be administered (ESMO guidelines, Michielin et al. 2019, cited above).

[0014] Ipilimumab has been evaluated as monotherapy in a retrospective study in 162 patients with unresectable stage III / IV metastatic melanoma resistant to anti-PD(L)-1 therapy, regardless of mutational status, showing an objective response rate (ORR) of 13%, a median progression-free survival (PFS) of 2.6 months, and a median overall survival (mOS) of 8.8 months (Da Silva et al 2021; Lancet Oncol; 22(6):836-847), and two smaller retrospective studies also observed limited efficacy of ipilimumab monotherapy. A randomized controlled phase 3 study evaluated dacarbazine versus the MEK inhibitor binimetinib in 402 patients with NRAS mutant melanoma, including treatment-naïve patients (~79%) and patients previously treated with immunotherapy (~21%). Respectively, ORR was 7% vs 15%, mPFS was 1.7 vs 2.8 months, and mOS was 10.1 vs 11 months (Dummer et al 2017; Lancet Oncol 2017;18:435-45). Other treatments have shown only limited success.

[0015] The limited efficacy of available therapies, combined with the lack of standard or approved therapies, highlights an unmet medical need in patients with NRAS mutant melanoma, particularly NRAS mutant unresectable or metastatic cutaneous melanoma. Summary of the Invention

[0016] Here, the present invention provides a method of treatment for solid tumors with mutations in RAS other than KRAS G12, for example, mutations in KRAS other than KRAS G12C and / or mutations in KRAS other than KRAS G12D.

[0017] Here, it is found that RAS codon Q61 mutation and / or RAS G31R mutation, regardless of whether the mutation occurs in HRAS, KRAS or NRAS, predict the responsiveness of solid tumors with said mutations to the combined treatment of RAF inhibitors and MEK inhibitors.Therefore, this discovery fulfills a major need by providing the treatment of any tumor, especially solid tumors, with RAS codon Q61 mutation or especially RAS G13R mutation, and provides a precise method for predicting responsiveness to treatment and allows the identified tumors to be treated.Therefore, the tumor to be treated does not need to be organ-specific or tissue-specific (for example, lung cancer or pancreatic cancer or colon cancer or breast cancer).

[0018] In other words, the detection and presence of RAS codon Q61 mutation and / or RAS G31R mutation, regardless of whether the mutation occurs in HRAS, KRAS, or NRAS in the tumor or tumor sample taken from the patient affected by the tumor, and regardless of the tissue origin of the tumor in the sample taken from the patient affected by the tumor (e.g., solid tumor), is beneficial for the patient affected by such tumor, and allows prognosis of whether or not the patient will respond to the combination treatment of RAF inhibitor and MEK inhibitor (e.g., naporafenib and trametinib, or velbalafenib and cobinmetinib).Therefore, the present invention provides a treatment for solid tumors with such mutations.

[0019] Here, a fresh and thorough analysis of preclinical data, as well as new and current clinical trial data from napolafenib and other agents acting on the MPAK pathway, shows that a combination of a RAF inhibitor and a MEK inhibitor may be particularly beneficial for the treatment of patients with pan-RAS codon Q61 or G13R mutant solid tumors.

[0020] For example, 3 of 4 non-small cell lung cancer (NSCLC) patients with tumors harboring either KRAS codon Q61 or G13R mutations responded to treatment with a combination of naporafenib and trametinib, whereas only 1 of 43 NSCLC patients with tumors harboring KRAS mutations other than either codon Q61 or G13R mutations responded to treatment. Furthermore, as shown by clinical data from the LXH254 first-in-human trial (CLXH254X2101), patients with solid tumors (salivary gland tumors and NSCLC) harboring pan-RAS codon Q61 or G13R mutations were found to be exceptional responders to LXH254 monotherapy. Furthermore, preclinical models showed that the activity of LXH254 as a single agent or in combination with trametinib was consistent in pan-RAS codon Q61 / G13R mutant models across tumor types (NSCLC, melanoma, PDAC, rhabdomyosarcoma), regardless of the specific RAS gene harboring the mutation (pan-RAS) or the tumor's tissue origin (tissue-agnostic). Overall, this body of data suggests that pan-RAS codon Q61 / G13R mutations may represent a predictive biomarker for response to RAF and MEK inhibitor combinations.

[0021] This data, plus preclinical evidence and clinical data with another RAF inhibitor, belbalafenib, convincingly suggests that the codon Q61 / G13R (pan)RAS mutation represents a predictive biomarker for response to a combination of a RAF inhibitor (e.g., naporafenib or belbalafenib) and a MEK inhibitor (e.g., trametinib or cobimetinib).

[0022] Also, in the molecular dynamics simulation analysis performed at Novartis, it was found that G13R overlaps with Q61, behaves dynamically similarly, and is predicted to adopt a similar conformation to Q61. Therefore, the mutations RAS Q61 and G13R in RAS genes are suitable biomarkers that allow the identification of tumor subtypes that are highly dependent on MAPK and likely to benefit from combination therapy, whether it is RAS genes selected from HRAS, KRAS, NRAS genes, and their combinations. When the MAPK pathway is activated, homodimers or heterodimers of RAF activate the main downstream signaling nodes MEK1 and MEK2. As RAF and MEK are vertically integrated into the same pathway, the combined inhibition of these two nodes can more effectively inhibit the pathway and delay the development of resistance.

[0023] It is therefore the basis of the present invention that by using RAS codon Q61 or especially RAS G13R mutations as predictive biomarkers for solid tumors treatable by combination therapy with RAF and MEK inhibitors, it is possible to select and treat patients suffering from tumors carrying such mutations, where GBR mutations have been found to be important predictive biomarkers.

[0024] Therefore, the present invention provides the following embodiments.

[0025] Embodiment 1. A method of selectively treating a patient afflicted with a solid tumor, comprising selectively administering to said patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on the tumor having a RAS codon Q61 mutation or a G13R mutation.

[0026] Embodiment 2. A method of selectively treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor, the method comprising: a) selecting patients for treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on tumors having a RAS codon Q61 mutation or a G13R mutation; b) then administering to said patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor. A method comprising:

[0027] Embodiment 3. A method of selectively treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation, the method comprising: a) evaluating a patient-derived biological sample or tumor sample for the presence of a RAS codon Q61 mutation or a G13R mutation in said solid tumor; b) Then, i. If a RAS codon Q61 mutation or G13R mutation is present, a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor in therapeutically effective amounts, or ii. administering to the patient a therapeutically effective amount of a drug other than a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on the absence or absence of detection of a RAS codon Q61 mutation or a G13R mutation in the biological sample or the tumor sample from the patient. selectively administering to said patient; A method comprising:

[0028] Embodiment 4. A method of selectively treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor, the method comprising: a) evaluating a patient-derived biological or tumor sample for the presence of a RAS codon Q61 or G13R mutation; b) then selecting a patient for treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on said biological sample or said tumor sample from said patient having a RAS codon Q61 mutation or a G13R mutation; c) then administering to said patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor. A method comprising:

[0029] Embodiment 5. The method of any one of embodiments 3 or 4, wherein the biological sample is selected from the group consisting of synovial fluid, blood, serum, feces, plasma, urine, tears, saliva, cerebrospinal fluid, a white blood cell sample, and a tumor sample (also called a tumor biopsy).

[0030] Embodiment 6. The method of any one of embodiments 3 to 5, wherein the evaluating step comprises a technique selected from the group consisting of Northern blot analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), TaqMan-based assays, direct sequencing, dynamic allele-specific hybridization, high-density oligonucleotide SNP arrays, restriction fragment length polymorphism (RFLP) assays, primer extension assays, oligonucleotide ligase assays, single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography, high-resolution melt analysis, DNA mismatch binding protein assays, SNPLex®, capillary electrophoresis, Southern blots, immunoassays, immunohistochemistry, ELISA, flow cytometry, Western blots, HPLC, and mass spectrometry.

[0031] Embodiment 7. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation, characterized in that the pharmaceutical combination of a RAF inhibitor and a MEK inhibitor is administered to the patient based on the patient having a solid tumor having a RAS codon Q61 mutation or a G13R mutation.

[0032] Embodiment 8. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient suffering from a solid tumor harboring a RAS codon Q61 mutation or a G13R mutation, said treatment comprising: a) evaluating a sample from a patient; b) determining whether the sample indicates the presence of a solid tumor having a RAS codon Q61 mutation or a G13R mutation; c) if a RAS codon Q61 mutation or a G13R mutation is present, administering to said patient a therapeutically effective amount of said pharmaceutical combination of a RAF inhibitor and a MEK inhibitor. 23. The pharmaceutical combination comprising:

[0033] Embodiment 9. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation, comprising: a) a patient is selected for treatment with said pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on the patient having a solid tumor with a RAS codon Q61 mutation or a G13R mutation; b) then administering to said patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor. 13. A pharmaceutical combination comprising:

[0034] Embodiment 10. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation, comprising: a) evaluating a biological sample from the patient for the presence of a RAS codon Q61 mutation or a G13R mutation; b) selectively administering to said patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on said biological sample from said patient having a RAS codon Q61 mutation or a G13R mutation. 13. A pharmaceutical combination comprising:

[0035] Embodiment 11. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient suffering from a solid tumor having a RAS codon Q61 mutation or a G13R mutation, comprising: a) evaluating a biological or tumor sample from the patient for the presence of a RAS codon Q61 mutation or a G13R mutation; b) selecting a patient for treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on said biological sample from said patient having a RAS codon Q61 mutation or a G13R mutation; c) selectively administering to said patient a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor in a therapeutically effective amount; 13. A pharmaceutical combination comprising:

[0036] Embodiment 12. A method for predicting the likelihood that a patient suffering from a solid tumor harboring a RAS codon Q61 mutation or a G13R mutation will respond to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor, comprising evaluating a biological sample or tumor sample from said patient for the presence or absence of a RAS codon Q61 mutation or a G13R mutation, wherein: a) the presence of a RAS codon Q61 mutation or a G13R mutation indicates that the patient is more likely to respond to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor; b) the absence of a RAS codon Q61 mutation or a G13R mutation indicates that the patient is less likely to respond to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor.

[0037] Embodiment 13 The method of embodiment 12, further comprising the step of obtaining the biological sample from the patient, wherein the obtaining step occurs before the detecting step.

[0038] Embodiment 14. The method of any one of embodiments 12-13, wherein the biological sample is selected from the group consisting of synovial fluid, blood, serum, feces, plasma, urine, tears, saliva, cerebrospinal fluid, a white blood cell sample, a tissue sample, and a tumor sample.

[0039] Embodiment 15. The method of any one of embodiments 12 to 14, wherein the evaluating step comprises a technique selected from the group consisting of Northern blot analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), TaqMan-based assays, direct sequencing, dynamic allele-specific hybridization, high-density oligonucleotide SNP arrays, restriction fragment length polymorphism (RFLP) assays, primer extension assays, oligonucleotide ligase assays, single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography, high-resolution melt analysis, DNA mismatch binding protein assays, SNPLex®, capillary electrophoresis, Southern blots, immunoassays, immunohistochemistry, ELISA, flow cytometry, Western blots, HPLC, and mass spectrometry.

[0040] Embodiment 16. A method for generating information in a transmittable form for predicting the responsiveness of a patient suffering from a solid tumor to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor, the method comprising: a) determining that the patient has an increased likelihood of responding to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor based on the presence of a RAS codon Q61 mutation or a G13R mutation in the tumor; b) recording the results of said determining step in a tangible or intangible media format for use in transmitting; A method comprising:

[0041] Embodiment 17. A kit for use in predicting the likelihood that a patient suffering from a solid tumor will respond to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor, the kit comprising: a) at least one probe capable of detecting the presence of a RAS codon Q61 mutation or a RAS codon G13R mutation; b) instructions for using said probe to evaluate a biological sample from said patient for the presence of a RAS codon Q61 mutation or a G13R mutation; Including, wherein the presence of a RAS codon Q61 mutation or a G13R mutation indicates that the patient is increased likely to respond to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor, and the absence of a RAS codon Q61 mutation or a G13R mutation indicates that the patient is decreased likely to respond to treatment with a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor.

[0042] Embodiment 18. A kit for use in treating a patient suffering from a solid tumor, comprising: a) a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor; b) at least one probe capable of detecting the presence of a RAS codon Q61 mutation or a G13R mutation in a solid tumor; c) instructions for using said probe to evaluate a biological or tumor sample from said patient for the presence of a RAS codon Q61 or G13R mutation; and d) instructions for administering to the patient the pharmaceutical combination of a RAF inhibitor and a MEK inhibitor if the biological sample or the tumor sample from the patient indicates the presence of a RAS codon Q61 mutation or a G13R mutation; e) optionally, a means for administering said pharmaceutical combination of a RAF inhibitor and a MEK inhibitor to said patient. Including the kit.

[0043] Embodiment 19. The kit of any one of embodiments 17 or 18, wherein the probe is an oligonucleotide that specifically hybridizes to a region of a nucleic acid encoding a RAS codon Q61 mutation or a G13R mutation, an antibody that detects a polypeptide product of a RAS codon Q61 mutation or a G13R mutation, or an oligonucleotide that specifically hybridizes to a region of a nucleic acid encoding an equivalent genetic marker of a RAS codon Q61 mutation or a G13R mutation.

[0044] In the above embodiment, the solid tumor with a RAS codon Q61 mutation or G13R mutation is a solid tumor that is not a melanoma or an NRAS mutant melanoma; Non-rhabdomyosarcoma solid tumors, a solid tumor that is a melanoma with a NRAS codon Q61 mutation or a melanoma with a NRAS G13R mutation; A solid tumor selected from non-small cell lung cancer, pancreatic cancer, thyroid cancer, and colorectal cancer. may be selected from: [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 is a graph showing that KRAS GBR protein dynamically overlaps with KRAS Q61R but not G13D. [Diagram 2] FIG. 2 is a diagram outlining the study design for the study on patients with mutant melanoma and solid tumors. [Diagram 3] FIG. 3 shows the antitumor activity of naporafenib and trametinib across 10 patients from a mouse NRASmut melanoma tumor xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] In one embodiment, the invention provides an agent or probe capable of identifying a RAS codon Q61 or in particular a RAS G13R mutation for suitability for combination treatment with a Raf inhibitor and a MEK inhibitor for diagnosing (or in particular for use in diagnosing) a solid tumor, in particular a non-melanoma, even more preferably a non-melanoma or a non-rhabdomyosarcoma, said diagnosis comprising applying said agent to assess a sample of said solid tumor for the presence or absence of a RAS codon Q61 or a RAS G13R mutation as a biomarker for said suitability.

[0047] In one embodiment, the invention provides a method for identifying a patient afflicted with a solid tumor, particularly a solid tumor other than melanoma, even more preferably a solid tumor other than melanoma or a solid tumor other than rhabdomyosarcoma, for an increased likelihood of response to combination treatment with a Raf inhibitor and a MEK inhibitor, the method comprising: (a) evaluating a tumor sample obtained from the patient for the presence of a RAS codon Q61 or RAS G13R mutation using the agent described in the preceding paragraph; (b) selecting patients whose tumor samples tested positive for the presence of the RAS codon Q61 or RAS G13R mutation in step (a) as candidates for the combination treatment; Includes.

[0048] In another embodiment, the invention provides a method for selecting a treatment for a patient suffering from a solid tumor, the method comprising screening a tumor sample from the patient for a RAS codon Q61 or (particularly) a RAS G13R mutation, the presence of which in the tumor sample identifies the patient as one who may benefit from a treatment comprising a Raf inhibitor and a MEK inhibitor.

[0049] In another embodiment, the present invention provides a method of treating a patient afflicted with a solid tumor, the method comprising: (a) testing a tumor sample from a patient suffering from a tumor for the presence or absence of a RAS codon Q61 / or RAS G13R mutation; (b) administering a therapeutically effective amount of a Raf inhibitor and a MEK inhibitor in combination therapy if the tumor sample is determined to have the mutation. Includes.

[0050] Another embodiment of the invention provides a method of treating a patient afflicted with a solid tumor, particularly a non-melanoma, in need of treatment, the method comprising administering to the patient, whose solid tumor has been identified as having a RAS codon Q61 or a RAS G13R mutation, an effective amount of a combination of a RAF inhibitor and a MEK inhibitor.

[0051] Another embodiment of the invention provides a kit comprising an agent for identifying a RAS codon Q61 or a RAS G13R mutation and reagents for the method according to any one of the preceding paragraphs.

[0052] Further embodiments of the invention provide a Raf inhibitor and a MEK inhibitor for combined use in the treatment of solid tumors, particularly non-melanomas and / or non-rhabdomyosarcomas harboring an NRAS codon Q61 mutation, in patients whose tumors have tested positive for and been identified as having a RAS codon Q61 or RAS G13R mutation.

[0053] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for use in combination as described in the preceding paragraph, wherein the Raf inhibitor is encorafenib, sorafenib, vemurafenib, dabrafenib, GDC-0879, PLX-4720, PLX8394, belbalafenib, CCT3833 / BAL3833, LY3009120, LSN3074753, lifirafenib, BG B659, RO5126766, AZ-628, MLN2480, BeiGene-283, RXDX-105, BAL3833, INU152, regorafenib, toborafenib, Day101, TAK580, MLN2480, TAK632, and in particular naporafenib (LXH254; Type II), or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, respectively.

[0054] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use as described in any one of the previous two paragraphs, wherein the Raf inhibitor is naporafenib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

[0055] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any one of the preceding paragraphs, wherein the MEK inhibitor is selected from the group consisting of binimetinib, trametinib, selumetinib, cobimetinib, CI-1040, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honoliol, AZD8330, PD318088, BIX 02188, pimasertib, mirdametinib, refametinib, BI-847325, GDC0623, G-573, trametiglue, RO5126766, and pimisertib, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0056] Preferred MEK inhibitors include trametinib, binimetinib, and cobmimetinib.

[0057] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any one of the previous four paragraphs, wherein the MEK inhibitor is trametinib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

[0058] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any one of the preceding paragraphs, wherein the solid tumor tests positive for harboring a RAS G13R mutation.

[0059] A further embodiment of the present invention provides the combined use of a Raf inhibitor and a MEK inhibitor, particularly as defined in more detail herein, in particular the combined use of naporafenib and trametinib, or a pharma- ceutically acceptable salt, hydrate or solvate thereof, in the treatment of solid tumors in patients whose tumors have tested positive for a RAS codon Q61 or a RAS G13R mutation.

[0060] A further embodiment of the present invention provides the use of a Raf inhibitor and a MEK inhibitor, particularly as defined in more detail herein, in the preparation of a medicament (particularly a combination), for the combined treatment of a solid tumor in a patient determined to have a RAS codon Q61 or a RAS G13R mutation, in particular the use of naporafenib and trametinib, or a pharma- ceutically acceptable salt, hydrate or solvate thereof, such as a kit for combined use or a fixed combination formulation comprising both active ingredients.

[0061] Another embodiment of the present invention provides a composition comprising a Raf inhibitor and a MEK inhibitor, particularly as defined in more detail herein, in particular naporafenib and trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof, respectively, and a pharmaceutically acceptable carrier, in the form of separate formulations or a kit or fixed formulation, for use in the treatment of rhabdomyosarcoma identified as having a RAS G13R mutation, or different solid tumors identified as having a RAS codon Q61 or a RAS G13R mutation, in particular a solid tumor selected from other than melanoma, or a corresponding method of treatment comprising administering a combined therapeutically effective amount of said Raf inhibitor and MEK inhibitor to a patient suffering from said rhabdomyosarcoma or other solid tumor in need of such treatment.

[0062] The more specific definitions used herein, in particular the following, may be used to define further embodiments of the invention by replacing one, more or all of the broader terms, features or expressions of the embodiments of the invention herein.

[0063] Suitability for combined / combined treatment indicates that a favorable effect on tumor disease can be expected (see below for the definition of such disease), i.e., this means that the patient can benefit from such treatment and can have an improved prognosis.

[0064] RAS herein refers to HRAS, NRAS, and KRAS.

[0065] RAF inhibitors The Raf inhibitor referred to herein includes any RAF inhibitor, including specific (e.g., ARAF, BRAF, or CRAF specific) inhibitors, but mainly includes inhibitors against two or all three of ARAF, BRAF, and CRAF.Preferred classes of RAF inhibitors include type I inhibitors, type 1.5 inhibitors (including so-called paradox blockers), and type II inhibitors.

[0066] Paradox blockers are a subtype of type 1.5 inhibitors. In cells, type 1.5 inhibitors, such as encorafenib, vemurafenib, and dabrafenib, activate wild-type signaling but not BRAFV600E mutant BRAF signaling. This surprising activation by the inhibitors was termed paradoxical activation. "Paradox blockers" are type 1.5 inhibitors that did not induce this paradoxical activation.

[0067] Preferred RAF inhibitors are the so-called type II inhibitors.

[0068] Preferred Raf inhibitor compounds are sorafenib (BAY43-9006), belvarafenib (HM95573), CCT3833 / BAL3833, LY3009120, LSN3074753, lifirafemb (BGB-283), BGB659, RO5126766, AZ-628, MLN2480, BeiGene-283 (BGB283), RXDX-105, BAL3833, INU152, regorafenib, toborafenib, Day101, TAK580, MLN 2480, TAK632, and in particular naporafenib (LXH254), or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, respectively (some known types are described in parentheses).

[0069] More preferably, the RAF inhibitor compound may be selected from sorafenib (BAY43-9006; type II), velbalafenib (HM95573; type II; preferred), CCT3833 / BAL3833, LSN3074753, lifirafenib (BGB-283; type II), BGB659, RO5126766, AZ-628, MLN2480 (type II), BeiGene-283 (BGB283), RXDX-105, BAL3833, INU152, regorafenib (type II), toborafenib, Day101, TAK580, MLN 2480, TAK632, and in particular naporafenib (LXH254; type II), or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, respectively.

[0070] Preferred RAF inhibitors include: Examples include napolafenib (LXH254).

[0071] Napolafenib is a compound of formula I

[0072] [ka] has.

[0073] This compound is also known as N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide or N-{3-[2-(2-hydroxyethoxy)-6-(morpholin-4-yl)pyridin-4-yl]-4-methylphenyl}-2-(trifluoromethyl)pyridine-4-carboxamide. Whenever naporafenib is mentioned herein, this refers to the free compound itself, and in addition, is further defined herein to include its pharma- ceutically acceptable salts, hydrates, and / or solvates. Particularly preferred is naporafenib in the free base form, or the monohydrate form H Aand characterized in that the monohydrate form H has an X-ray powder diffraction pattern having at least one, two, three, four, or five peaks having refractive angles of 2-theta (θ) values ​​selected from 7.3, 10.7, 16.3, 16.7, 17.4, 23.0, 24.3, 25.3, 28.3, 32.0, said values ​​being ±0.2° 2θ, when measured using CuKα radiation. A Napolafenib is further described in WO 2020 / 230028 and may be prepared as described therein.

[0074] Napolafenib is primarily an adenosine triphosphate (ATP)-competitive inhibitor of BRAF and CRAF protein kinases. Napolafenib can inhibit both monomers and dimers of BRAF and CRAF, thus inducing minimal paradoxical activation and being effective in blocking mutant RAS-driven signaling and cell proliferation. Napolafenib has demonstrated efficacy in a wide range of MAPK pathway-driven human cancer cell lines and in vivo tumor xenografts, including models with activating lesions in KRAS, NRAS, and BRAF oncogenes.

[0075] The compound of formula (I) and its pharma- ceutically acceptable salts are described in WO 2014 / 151616, the entirety of which is incorporated herein by reference, and its preparation is described, for example, in Example 1156. Napolafenib may also be used in its polymorphic forms, for example, as described in WO 2020 / 230028.

[0076] In cell-based assays, the compound of formula (I) exhibited anti-proliferative activity in cell lines containing various mutations that activate MAPK signaling. In vivo, treatment with the compound of formula (I) caused tumor regression in multiple KRAS mutant models. Taken together, the in vitro and in vivo MAPK pathway inhibition and anti-proliferative activity observed with the compound of formula (I) at well-tolerated doses makes it likely that the compound of formula (I) will have anti-tumor activity in patients with tumors that harbor activating lesions in the MAPK pathway. Furthermore, the compound of formula (I) is a type 2 ATP-competitive inhibitor of both B-RAF and C-RAF that keeps the kinase pocket in an inactive conformation, thereby blocking mutant RAS-driven signaling and cell proliferation. Compounds of formula (I) have demonstrated efficacy in a number of MAPK-driven human cancer cell lines, as well as xenograft tumors representing model tumors harboring human lesions of the KRAS, NRAS, and BRAF oncogenes (see, e.g., International Publication No. WO 2018 / 203219, incorporated herein in its entirety).

[0077] Further evidence of the utility of the present invention may be derived from clinical data obtained from LXH254, e.g. ● CLXH254X2101: A first-in-human study of naporafenib as a single agent in patients with advanced solid tumors harboring MAPK pathway mutations and in combination with the checkpoint inhibitor spartalizumab (PDR001) in patients with advanced or metastatic KRAS mutant non-small cell lung cancer or NRAS mutant melanoma. ●CLXH254X2102: A Phase 1b, open-label, multicenter study of naporafenib in combination with LTT462 in adult patients with advanced or metastatic KRAS mutant or BRAF mutant non-small cell lung cancer, or naporafenib in combination with trametinib in adult patients with advanced or metastatic KRAS mutant or BRAF mutant non-small cell lung cancer, or NRAS mutant melanoma, or naporafenib in combination with ribociclib in adult patients with advanced or metastatic NRAS mutant melanoma. ● CLXH254C12201: A randomized, open-label, multi-arm, two-part, Phase II study to evaluate the efficacy and safety of multiple combinations of naporafenib in patients with previously treated unresectable or metastatic BRAF V600 mutant or NRAS mutant melanoma.

[0078] Belbarafenib Belvalafenib (HM95573, GDC5573, RG6185) has the formula

[0079] [ka] has.

[0080] Berbalafenib, named 4-amino-N-(1-((3-chloro-2-fluorophenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide, is a potent pan-RAF inhibitor with antitumor activity, particularly against BRAF, BRAF V600E, and CRAF. Its synthesis is described in WO 2013 / 100632 A1. The compound shows high selectivity for BRAF mutations and CRAF kinases. A phase 1b trial of berbalafenib in combination with cobimetinib in patients with advanced solid tumors is ongoing: https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2021.39.15_suppl.3007

[0081] MEK inhibitors The term "MEK inhibitor" is defined herein to refer to compounds that target, decrease or inhibit the activity of at least one of MAP / ERK kinases 1 and 2 (MEK1 / 2). MEK stands for "mitogen-activated protein kinase."

[0082] MEK inhibitors referred to herein include inhibitors of both MEK and the MEK / RAF heterodimer complex.

[0083] Preferred MEK inhibitor compounds are Arry-162 = MEK162 (binimetinib), AZD6244 (selumetinib), XL518 = GDC0973 (cobimetinib), CI-1040 = PD184352, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honoliol, AZD8330, PD318088, BIX 02188, AS703026=MSC1936369B (pimasertib), SL327, PD-325901=PD0325901 (mirdametinib), TAK-733, BAY86-9766 (refametinib), BI-847325, GDC0623, G-573, RAF / MEK heterodimer breaker, trametiglue (RAF / MEK complex inhibitors), CH4987655 = RO4987655, WX-554, HL-o85, CH5126766 = RO5126766 (RAF / MEK complex inhibitors) (preferred), and in particular trametinib (GSK1120212, an advanced RAF inhibitor, sold under the trade name Mekinist®).

[0084] Preferred MEK inhibitors include trametinib, binimetinib, and cobmimetinib.

[0085] Preferably, the MEK inhibitor is trametinib, or its pharma- ceutically acceptable salt or solvate. In some embodiments, trametinib is in the form of a sodium salt. Suitably, trametinib is in the form of a solvate selected from hydrate, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentancol, isopropyl alcohol, ethylene glycol, and 3-methyl-1-butanol. In some preferred embodiments, trametinib is in the form of a dimethylsulfoxide solvate. These solvates and salt forms can be prepared by those skilled in the art from the description in WO2005 / 121142.

[0086] Trametinib is a compound of formula II

[0087] [ka] has.

[0088] This compound is also known as (N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide) and is also called JPT-74057 or GSK1120212 or TMT212.

[0089] Trametinib is disclosed in Example 4-1 of PCT Publication No. 2005 / 121142, the entirety of which is incorporated herein by reference.

[0090] Whenever "trametinib" is referred to in the present disclosure, it refers to the free form per se, but is further defined herein to include pharma- ceutically acceptable salts, hydrates, and / or solvates thereof, in particular the dimethylsulfoxide solvate.

[0091] RO5126766 is of formula III

[0092] [ka] has.

[0093] The compound has the name N-(3-fluoro-4-((4-methyl-2-oxo-7-(7-(2-pyrimidinyloxy)-2H-1-benzopyran-3-yl)methyl-2-pyridinyl)-N'-methyl-sulfamide. RO5126766 is a dual RAF / MEK inhibitor with IC50 of 8.2 nM, 19 nM, 56 nM, and 160 nM for BRAF V600E, BRAF, CRAF, and MEK1, respectively.

[0094] "Furthermore" or "further" indicates that the feature following either of these words is less preferred than a feature not having that attribute.

[0095] As used herein, the terms "a" and "an" and "the" and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context, in the context of describing the invention. When the plural is used in reference to compounds, salts, and the like, it is construed to mean a single compound, salt, or the like.

[0096] The term "or" is used herein to mean and is used interchangeably with the term "and / or", unless the context clearly indicates otherwise, e.g., a reference to a compound or a pharma- ceutically acceptable salt thereof provides for the free compound, a pharma- ceutically acceptable salt, or a mixture of the free compound with one or more salts thereof.

[0097] "About" and "approximately" generally refer to an acceptable degree of error in the amount measured, taking into account the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. When a dosage is described herein with a specified amount with "about", the actual dosage may vary up to 10% from the described amount, and it is understood from such usage of "about" that the exact amount in a given dosage form may differ slightly from the intended amount for various reasons without substantially affecting the in vivo effect of the administered compound.

[0098] When numerical values ​​are stated without the term "about" immediately preceding them, it is understood that there is a degree of variation generally accepted in the art associated with the given value, e.g., the variation may be ±10%, e.g. ±5%, in particular ±1% of the respective given numerical value.

[0099] The terms "comprising" and "including" or "with" are used herein in their open-ended and non-limiting sense unless otherwise stated, and "consisting of" is limited to the specifically described compounds or components. A pharmaceutical combination consisting of (a) naporafenib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, and (b) trametinib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, refers to a combination therapy in which these two components are the only pharma- ceutically active ingredients, with or without one or more pharma- ceutically acceptable carrier materials.

[0100] "Combination" or "pharmaceutical combination" (these terms are synonymous unless otherwise indicated), or "in combination with" or "combination therapy" are not intended to imply that the therapies or therapeutics must be physically mixed, administered simultaneously, and / or formulated to be delivered together, and these delivery methods are within the scope of the present description. The therapeutics in these combinations (herein, this term primarily refers to RAF inhibitors and / or MEK inhibitors, particularly naporafenib and / or trametinib) may also be administered in parallel with, before, or after one or more other additional therapies or therapeutics, or may be administered without such additional therapies or therapeutics (active ingredients). The therapeutics may be administered in any order. In general, each agent is administered at a dose and / or time schedule that has been determined for that agent. It will be further understood that the additional therapeutic agents utilized in this combination may be administered together in a single composition and / or separately in different compositions, particularly when their time intervals allow the combination partners to show a cooperative effect, for example, a synergistic effect, particularly based on Bliss data or other data that can confirm synergistic effects.In general, it is expected that the additional therapeutic agents utilized in combination will be utilized at levels that do not exceed the levels at which these therapeutic agents are utilized individually.In some embodiments, the level of each related therapeutic agent=drug=active pharmaceutical ingredient (API) utilized in combination is lower than the level utilized as a monotherapy.

[0101] The term "synergy," as used herein, refers to the action of a RAF inhibitor, particularly naporafenib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, with a MEK inhibitor, particularly trametinib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, that results in an effect, e.g., slowing the progression of cancer or a symptom thereof, that is greater than the simple sum of the effects of each drug administered by itself or that requires lower dosages of either or both therapeutic agents.

[0102] The term "combination therapy" preferably refers to the administration of two or more therapeutic agents to treat the oncological disease or disorder described in the present invention. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single formulation with a fixed ratio of active ingredients, or a separate formulation for each active ingredient (e.g., capsules, tablets, and / or additional intravenous formulations). Additionally or alternatively, such administration also includes the use of each type of therapeutic agent in a sequential or separate manner, either at about the same time or at different times. Regardless of whether the active ingredients are administered as a single formulation or in separate formulations, the drugs are administered to the same patient as part of the same course of therapy. In either case, this treatment regimen provides beneficial effects in the treatment of oncological diseases or disorders described herein.

[0103] Simultaneous therapeutic use within the meaning of the present invention means in particular the administration of at least two active ingredients by the same route and at the same or substantially the same time.

[0104] Separate use, within the meaning of the present invention, in particular means the administration of at least two active ingredients by different routes or formulations at the same or substantially the same time.

[0105] Sequential therapeutic use especially refers to the administration of at least two active ingredients at different times, with the same or different routes of administration or formulations, more particularly to a method of administration in which a course of administration of one active ingredient is followed by the administration of another (or yet another).

[0106] As used herein, the interchangeable terms "fixed combination", "fixed dose" and "single formulation" refer to one dosage form that is preferably formulated to deliver to a patient an amount of both therapeutic agents that are jointly therapeutically effective for the treatment of cancer. The single vehicle is designed to deliver an amount of each agent together with any pharma- ceutically acceptable carrier (excipient). In some embodiments, the vehicle is a tablet, capsule, pill, sachet, or patch. In further embodiments, the vehicle is a solution or suspension.

[0107] Additionally or alternatively, the term combination includes a "non-fixed combination" or "kit of parts," which means that both therapeutic agents of the combination of the invention are administered to a patient as separate components simultaneously, in parallel, or sequentially without any specific time limit, in particular so that they are jointly therapeutically effective and such administration provides therapeutically effective levels of the two compounds in the body of a patient in need thereof. Furthermore, the latter term also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0108] The term "pharmacologically acceptable" as used herein refers to compounds, materials, excipients, compositions and / or dosage forms that are within the scope of sound medical judgment, suitable for contact with the tissues of a patient, e.g., an adult patient or a human patient, without excessive toxicity, irritation, allergic response, and other problems or complications, commensurate with a reasonable and acceptable benefit / risk ratio.

[0109] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, glidants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, that would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical composition is included.

[0110] The term "pharmaceutical composition" (= pharmaceutical preparation) is defined herein to refer to a mixture or solution containing at least one therapeutic agent intended for administration to a patient, e.g., a mammal or human, to treat a particular disease or disorder affecting the patient. The pharmaceutical combination of the present invention can be formulated into pharmaceutical compositions suitable for enteral or even parenteral administration, such as film-coated tablets (e.g., sugar-coated tablets), tablets, capsules or suppositories, sachets or ampoules. If not otherwise indicated, they are prepared in a manner known per se, for example by various conventional mixing, grinding, direct compression, granulation, coating (e.g., sugar-coating), dissolving, lyophilization processes or manufacturing techniques readily apparent to those skilled in the art. It will be understood that the unit content of the combination partner (therapeutic agent) contained in each individual dose of each dosage form does not necessarily constitute an effective amount per se, since the required effective amount may be reached by administering multiple dosage units. The pharmaceutical composition may contain about 0.1% to about 99.9%, preferably about 1% to about 60%, of any single therapeutic agent, and may contain about 0.1% to about 99.9%, preferably about 1% to about 60%, of each of the multiple therapeutic agents, if the fixed combination is in an amount of 0.2% to 70% in total. One of ordinary skill in the art may select one or more of the aforementioned carriers with respect to the particular desired properties of the dosage form by routine experimentation and without any undue burden. The amount of each carrier used may vary within conventional limits in the art. The following references disclose techniques and excipients used to formulate oral dosage forms: The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2003).These optional additional conventional carriers may be incorporated into the oral dosage form by incorporating one or more conventional carriers into the initial mixture prior to or during granulation, or by combining one or more conventional carriers with granules containing the therapeutic combination or individual therapeutics of the therapeutic combination in the oral dosage form. In the latter embodiment, the combined mixture may be further blended, for example, in a V-blender, and then compressed or molded into a tablet, for example a monolithic tablet, or enclosed in a capsule, or filled into a sachet.

[0111] When "%" is mentioned, this refers to weight percent, unless otherwise indicated.

[0112] A particular formulation relates to film-coated tablets of the active ingredients in separate or even fixed combination formulations used in the combination therapy of the invention.

[0113] The pharmaceutical combination or pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or "effective amount" of a RAF inhibitor, particularly naporafenib, and a MEK inhibitor, particularly trametinib. The term "pharmaceutical effective amount", "therapeutically effective amount", or "clinically effective amount" of a therapeutic combination is a sufficient amount, at the dosage and for the period of time required, to provide an observable or clinically significant improvement over baseline in the clinically observable signs and symptoms of the disorder being treated by the combination. A therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably modulates a measurable parameter, such as tumor growth rate or disease progression, in a desired manner. The ability of a therapeutic agent to modulate a measurable parameter may be evaluated in an animal model system predictive of efficacy in human sarcoma to facilitate establishing suitable dosing levels and schedules. Alternatively, this property of the combination may be evaluated by using in vitro assays known to those skilled in the art, or by examining the ability of the compounds to modulate undesirable parameters, particularly in clinical trials with patients, particularly in clinical trials with patients in need of treatment.

[0114] Preferably, for the treatment of human patients, the following dosages are used for the combination of a RAF inhibitor according to the invention, in particular naporafenib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, and a MEK inhibitor, in particular trametinib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, in which in each case the free form of the respective API is referred to:

[0115] The unit dosage of active ingredient may be administered once a day, or twice a day, or three times a day, or four times a day, with the actual dosage and timing of administration being determined by criteria such as the age, weight, and sex of the patient, the extent and severity of the cancer being treated, and the judgment of the treating physician, to result in a total appropriate daily dosage, such as those suggested below. Oral dosage forms are preferred, particularly tablets (preferably film-coated tablets).

[0116] When dose or dosage is mentioned herein, the amount mentioned refers to the amount of therapeutic agent (active ingredient, here in free form).For example, if 2mg dosage of trametinib is administered, and trametinib is administered in a tablet containing trametinib dimethyl sulfoxide, the tablet contains trametinib dimethyl sulfoxide equivalent to 2mg of trametinib in free form.The same reasoning is also valid for the monohydrate of naporafenib.

[0117] The RAF inhibitor is preferably administered orally. In a preferred embodiment, the RAF inhibitor, particularly napolafenib or a pharma- ceutically acceptable salt thereof, is administered at a total daily dose (TTD) of about 100 mg to about 1200 mg, respectively. The total daily dose of napolafenib is administered in one or more dosage units per day. Preferably, the total daily dose of napolafenib is administered twice per day. Thus, napolafenib may be administered at about 100 mg to about 600 mg twice per day.

[0118] In some embodiments, naporafenib is administered (preferably orally) once daily (QD) at a dose of about 100 mg, 125 mg, 150 mg, 175 mg, 200 mg (preferred), 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, or especially 400 mg, respectively.

[0119] In certain embodiments, naporafenib, or a pharma- ceutically acceptable salt, hydrate, and / or solvent thereof, is administered orally at a dosage of about 200 mg or 400 mg twice daily (BID or bid), based on the free form.

[0120] The MEK inhibitors as part of a combination or combination treatment according to the invention are administered in therapeutically effective amounts to a subject in need thereof.

[0121] In a preferred embodiment, the MEK inhibitor trametinib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, administered to a subject in need thereof as part of the combination according to the invention is in an amount selected from about 0.125 mg to about 10 mg per day, preferably selected from about 0.5 mg to about 2 mg per day, preferably about 1 mg or 2 mg per day. In a preferred embodiment, trametinib, or a pharma- ceutically acceptable salt or solvate thereof, is administered in a daily dose of 0.5 mg or 1 mg per day, in particular 1 mg per day. Preferably, the daily dosage (daily dosage) is administered once a day (qd).

[0122] Most preferably, the Raf inhibitor, particularly naporafenib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, is administered in a dosage of 400 mg BID and the MEK inhibitor, particularly trametinib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, is administered in a dosage of 0.5 mg QD, or even more preferably, the Raf inhibitor, particularly naporafenib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, is administered in a dosage of 200 mg BID and the MEK inhibitor, particularly trametinib, or a pharma- ceutically acceptable salt, hydrate, and / or solvate thereof, is administered in a dosage of 1.0 mg QD, each based on the free form.

[0123] In some embodiments, trametinib, or its pharma- ceutically acceptable salt or solvate, is administered orally. In one embodiment, trametinib may be prepared for administration by oral delivery and used in a solvated form in dimethylsulfoxide. In some embodiments, the compound is prepared in tablet form for oral administration, particularly as a film-coated tablet. Tablets may be manufactured in various dosages for flexible administration.

[0124] The unit dosage of trametinib, or a pharma- ceutically acceptable salt or solvate thereof, may be administered once a day, or twice a day, or three times a day, or four times a day. The total daily dose of trametinib, or a pharma- ceutically acceptable salt or solvate thereof, such as the dimethylsulfoxide solvate, may be administered once a day or even twice a day.

[0125] For example, as part of combination therapy, the compound of formula (I) or its pharma- ceutically acceptable salt, such as monohydrate or preferably in free form, may be administered at a total daily dose of about 200mg, about 300mg, about 400mg, or about 500mg, and trametinib, for example in dimethylsulfoxide solvate form, may be administered at a total daily dose of about 0.5 or 1.0mg. The compound of formula (1) may be administered at a daily dose once or twice. Thus, the compound of formula (I) may be administered at a dose of about 200mg twice a day (total daily dose of about 400mg), and trametinib may be administered at a dose of about 1.0mg or about 2.0mg once a day. Alternatively, a compound of formula (I) at a dose of about 200 mg may be administered twice daily (total daily dose of about 400 mg) and trametinib at a dose of about 0.5 or about 1.0 mg may be administered twice daily.

[0126] Further benefits may include the ability to use lower doses of the therapeutic agents of the combination of the invention, e.g., often resulting in lower dosages as well as less frequent application and / or reduced incidence of side effects observed with only one of the combination partners, consistent with the desires and requirements of the patient being treated.

[0127] The term "jointly therapeutically active" or "joint therapeutic effect" as used herein means that the therapeutic agents may be given jointly, separately or sequentially at a time interval such that the patient, particularly the human being, being treated still exhibits a (preferably synergistic) interaction (joint therapeutic effect). In particular, whether this is the case can be determined by tracking the blood levels of the compounds, which show that both compounds are present in the blood of the human being being treated for at least a certain time interval, or by the fact that the subsequent treatment produces better results than monotherapy with only one of the active ingredients.

[0128] The term "active ingredient" or "therapeutic agent" includes RAF inhibitors and MEK inhibitors as defined for the combination according to embodiments of the present invention.

[0129] In one embodiment, the disease or disorder suitable for or to be treated by the combination treatment according to the invention is cancer. The term "cancer" is used herein to mean a tumor selected from a wide range of tumors, including all solid tumors. Cancer may be at early, intermediate, or late stage. Cancer may be locally advanced or metastatic. "Cancer" or "tumor" are used interchangeably herein, and these identifiers are equivalent to "tumor disease" or "cancer disease", providing in the case of solid tumors, particularly advanced tumors / tumor diseases.

[0130] The cancer to be treated with the combination therapies described herein may be one for which progression has occurred following standard therapy or for which no effective standard therapy exists.

[0131] In one embodiment, the solid tumor is non-benign.In one embodiment, the solid tumor is selected from the group consisting of melanoma, or more specifically any solid tumor other than melanoma, such as lung cancer, particularly non-small cell lung cancer (NSCLC), colon cancer (CRC), such as colorectal adenocarcinoma, pancreatic cancer, such as pancreatic ductal adenocarcinoma (PADC), or even head and neck cancer, such as head and neck squamous cell carcinoma, bladder urothelial carcinoma, MUTYH-associated polyposis (MAP), cervical and ovarian cancer, esophageal cancer, kidney cancer, mesothelioma, thyroid cancer, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, Merkel cell carcinoma, or even sarcoma, such as rhabdomyosarcoma.

[0132] In one embodiment, the tumor is a melanoma.

[0133] In one embodiment, the tumor is a lung cancer, such as non-small cell lung cancer (NSCLC).

[0134] In one embodiment, the tumor is a colon cancer.

[0135] In one embodiment, the tumor is a pancreatic cancer.

[0136] In one embodiment, the tumor is a head and neck cancer.

[0137] In one embodiment, the tumor is bladder urothelial carcinoma.

[0138] In one embodiment, the proliferative disease is colorectal cancer (CRC), including MUTYH-associated polyposis (MAP).

[0139] In one embodiment, the tumor is a cervical cancer.

[0140] In one embodiment, the tumor is an ovarian cancer.

[0141] In one embodiment (particularly for the G13R mutation), the tumor is a rhabdomyosarcoma.

[0142] The solid tumor to be treated includes NRAS mutant melanoma.When referring to melanoma, this particularly refers to "NRAS mutant melanoma".When referring to "NRAS mutant melanoma", this particularly refers to ocular melanoma or cutaneous melanoma, unless the context clearly indicates otherwise.Preferably, this term refers to cutaneous melanoma, particularly unresectable and / or metastatic cutaneous melanoma.

[0143] The present invention provides a pharmaceutical combination for use in treating melanoma in a patient, where the melanoma has been previously treated, for example by surgical removal or other therapy, but has progressed following such therapy.

[0144] The melanoma to be treated with the combination may be one that is resistant or refractory to previous treatment with another therapy. Thus, the combination of the present invention may be useful as a second, third, or fourth line treatment for melanoma.

[0145] Previous treatments for melanoma patients include: - treatment with talimogene laherparepvec (also known by the trade names T-Vec, Imlygic or Oncovex), a biopharmaceutical drug for treating unresectable melanoma, which is injected directly into a subset of metastatic lesions; - Standard of care chemotherapy (e.g., dacalzavin), Treatment with cytotoxic agents such as nitrosurea and / or mitomycin C, Immunotherapy (e.g., pembrolizumab, ipilimumab, or nivolumab, and combinations thereof), -Targeted therapy (e.g., dabrafenib and trametinib, vemurafenib and cobimetinib, and encorafenib and binimetinib) Examples include:

[0146] Thus, patients to be treated include those with NRAS mutant melanoma, particularly those who have undergone prior therapy including standard of care chemotherapy (e.g., dacalzavin), immunotherapy (e.g., pembrolizumab, ipilimumab, or nivolumab, and combinations thereof), targeted therapy (e.g., dabrafenib and trametinib, vemurafenib and cobimetinib, and encorafenib and binimetinib) and have progressed on prior therapy. Patients may also be those with NRAS mutant melanoma, particularly those that are unresectable and / or metastatic cutaneous melanoma.

[0147] In another embodiment, the NRAS mutant melanoma is resistant or refractory to standard treatment.

[0148] In another embodiment, the NRAS mutant melanoma is resistant or refractory to standard treatment with dacarbazine.

[0149] In another embodiment, the melanoma is resistant or refractory to treatment with cytotoxic agents, such as nitrosurea and / or mitomycin C.

[0150] In another embodiment, the melanoma is resistant or refractory to treatment with immunotherapy treatment, including therapy with one or more immune checkpoint inhibitors.

[0151] Thus, in one embodiment, the melanoma to be treated with the combination of the present invention is an NRAS mutant melanoma that is resistant to treatment with immunotherapy PD-1 (programmed cell death receptor 1) or PD-L1 (ligand for PD-1), alone or in combination with an anti-CTLA4 (cytotoxic T-lymphocyte associated protein) antibody (e.g., ipilimumab). See, e.g., Tsai et al, Human Vaccines & Immunotherapeutics 10:11, 3111--3116; November 2014.

[0152] Thus, in one embodiment, the melanoma (including NRAS mutant melanoma) to be treated is resistant or refractory to treatment with one or more therapeutic agents selected from ipilimumab, spartalizumab, nivolumab, pembrolizumab, pididilumab, BMS-9365559, MEDI4736, and MSB0010718C.

[0153] Thus, melanomas to be treated with the combination of the invention include NRAS mutant melanomas that are resistant to anti-PD-1 monotherapy (e.g., pembrolizumab or nivolumab) or the combination of an anti-PD-1 agent with ipilimumab.

[0154] Genetic assessment of BRAF, NRAS, and NF1 mutations in patients may be performed according to methods known in the art, for example using SNaPshot or DFCI Oncopanel as previously described (Sholl LM, et al. JCI Insight 2016;1:e87062; Zheng Z, et al., Nat Med 2014;20:1479-84). Current iterations of both assays utilize next generation sequencing, while earlier versions of SNaPshot relied on multiplex PCR. The current version of SNaPshot interrogates exons 11 and 15 of BRAF, exons 2-5 of KRAS and NRAS, and exons 1-58 of NF1. Oncopanel detects alterations involving all exons of BRAF, KRAS, NRAS, and NF1.

[0155] A subject suffering from cancer / tumor is defined as having progressed or no longer responding to one or more drug therapy, or being intolerant to one or more drugs, when the cancer that the subject suffers from progresses.Cancer progression may be monitored by methods well known to those skilled in the art.For example, progression may be monitored by visual inspection of the cancer, for example, by means such as X-ray, CT scan, or MRI, or by tumor biomarker detection.For example, an increase in cancer growth indicates cancer progression.Progression of cancer, for example NSCLC or tumor, may be suggested by detection of new tumor or detection of metastasis, or by cessation of tumor shrinkage. Tumor evaluation may be performed based on RECIST criteria (Therasse et al 2000), New Guidelines to Evaluate the Response to Treatment in Solid Tumors, Journal of National Cancer Institute, Vol. 92; 205-16 and revised RECIST guidelines (version 1.1) (Eisenhauer et al 2009) European Journal of Cancer; 45: 228-247. For example, the overall response rate (ORR) or any other parameter according to the Response Evaluation Criteria In Solid Tumors (RECIST v1.1) may be used to indicate the treatment effect.

[0156] A complete response is defined as the disappearance of all nonnodal target lesions. In addition, any pathological lymph nodes assigned as target lesions must have a short-axis reduction to <10 mm1.

[0157] A partial response is defined as at least a 30% reduction in the sum of the diameters of all target lesions, taken as the baseline sum of diameters.

[0158] Other response criteria can be inferred from the table below (based on RECIST v1.1):

[0159] [Table 1]

[0160] Tumor progression, increase or decrease in tumor burden, and response to treatment with the inhibitor combination according to the invention may be monitored by methods well known to those skilled in the art. Thus, progression and response to treatment may be monitored by visual inspection of the tumor, for example by means of X-ray, CT scan, or MRI, or by tumor biomarker detection.

[0161] Tumor progression may be determined by comparing the tumor status between time points after initiation of treatment, or by comparing the tumor status between a time point after initiation of treatment and a time point before initiation of the relevant treatment.

[0162] The combinations and combination therapies according to the invention are particularly useful for the treatment of cancers with one or more mitogen-activated protein kinase (MAPK) pathway mutations, including HRAS, or in particular KRAS mutant tumors and / or NRAS mutant tumors, in particular tumors expressing at least one gain-of-function mutation in RAS as described herein, in particular KRAS or NRAS, selected from, but not limited to, at least one gain-of-function mutation in RAF at codon Q61, such as a Q61H, Q61K mutation, or (in particular) a G13R mutation.

[0163] In another embodiment, the cancer is resistant or refractory to treatment with a MEK inhibitor, such as trametinib.

[0164] The present invention, as already mentioned, relies on the important discovery that certain RAS mutations, in particular codon Q61 or the mutation G13R, are suitable biomarkers for the suitability of tumor patients for combined treatment with RAF and MEK inhibitors.

[0165] When a RAS, e.g. KRAS, HRAS or NRAS (codon) mutation is mentioned in this specification, it relates, on the one hand, to a mutation at the corresponding point in the polynucleotide (e.g. gene, cDNA or mRNA) and, on the other hand, to the corresponding mutant protein having an altered amino acid sequence at the mentioned position (Q61 or G13R).

[0166] Suitable assays, methods and agents (in particular binding reagents that bind to the biomarkers of interest) that allow screening, testing or identifying the presence of RAS, e.g. KRAS, codon Q61 or G13R mutations, are in principle known and available to those skilled in the art.

[0167] "KRAS activating mutation" is any mutation in the KRAS gene (i.e., nucleic acid mutation) or any mutation in the Kras protein (i.e., amino acid mutation) that results in an abnormal Kras protein function associated with increased activity and / or constitutive activity by supporting the active GTP-bound state of the Kras protein. The mutation is conserved at the site that supports GTP binding and is a constitutively active Kras protein. The mutation in the use according to the invention is in a specific codon of the KRAS gene that codes for KRAS, including KRAS-G13R (particularly preferred), or KRAS-Q61E, KRAS-Q61P, KRAS-Q61H, KRAS-Q61K (preferred), KRAS-Q61L (preferred), or KRAS-Q61R (preferred).

[0168] "NRAS activating mutation" is any mutation in the NRAS gene (i.e., nucleic acid mutation) or any mutation in the Nras protein (i.e., amino acid mutation) that results in an abnormal Nras protein function associated with increased activity and / or constitutive activity by supporting the active GTP-bound state of the Nras protein. This mutation is conserved at the site that supports GTP binding and is a constitutively active Nras protein. The mutation in the use according to the present invention is in a specific codon of the NRAS gene that codes for KRAS, including the NRAS-G13R mutation (particularly preferred), or the NRAS-Q61E, NRAS-Q61P, NRAS-Q61H, NRAS-Q61K (preferred), NRAS-Q61L (preferred), or NRAS-Q61R (preferred) mutation.

[0169] The method of identifying RAS codon Q61 or RAS G13R mutation is known in the art, for example by using "next-generation sequencing".The agent or probe that can identify RAS codon Q61 or RAS G13R mutation is known in the art, and can be preferably selected from antibodies and polynucleotides, each of which can bind to the site of mutation either in expressed protein or preferably in the gene DNA, cDNA or mRNA that codes for it.

[0170] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0171] "Polynucleotide" or "nucleic acid" (these terms are used synonymously herein) refers to a polymer of nucleotides of any length, including either or both DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Thus, for example, the term includes, but is not limited to, single-stranded and double-stranded DNA, DNA containing single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA containing single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded or contain single-stranded and double-stranded regions. For nucleic acid or polynucleotide mutations (= those that can be prognostic for the suitability of a tumor (and therefore a patient) for successful combination treatment with RAF inhibitors and MEK inhibitors) of use according to the invention, DNA and RNA, especially cDNA or mRNA, are preferred.

[0172] In particular, in the case of polynucleotides (particularly oligonucleotides) or nucleic acids used according to the invention for identifying mutations in RAS DNA or RNA from tumor samples for use according to the invention (e.g. as probes or primers), modifications to the nucleotide structure, if any, may be added before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Such polynucleotides may be further modified after polymerization, for example by conjugation with a (e.g. fluorescent or isotopic) label. Other types of modifications include, for example, "caps" that replace one or more of the naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged bonds (such as methylphosphonates, phosphotriesters, phosphoamidates, or carbamates) and / or those with charged bonds (such as phosphorothioates or phosphorodithioates), those that contain pendant moieties, such as proteins (such as nucleases, toxins, antibodies, signal peptides, or poly-L-lysine), those that contain intercalating agents (such as acridines or psoralens), those that contain chelating agents (such as metals, radioactive metals, boron, or metal oxides), those that contain alkylating agents, those that contain modified bonds (such as alpha-anomeric nucleic acids), and unmodified forms of polynucleotides. Any one or more of the hydroxyl groups normally present in the sugar of the nucleotide backbone may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds to additional nucleotides, or may be conjugated to solid or semi-solid supports. The terminal OH may be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.These polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. Such polynucleotides may contain one or more different types of modifications and / or multiple modifications of the same type described herein. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0173] As used herein, "oligonucleotide" generally refers to a short, single-stranded polynucleotide, usually, but not necessarily, less than about 250 nucleotides in length, e.g., 2-80, e.g., 2-50 nucleotides in length. Oligonucleotides (particularly when used as probes, e.g., primers) may be synthetic.

[0174] The term "primer" refers to a single-stranded oligonucleotide or polynucleotide that can hybridize to a nucleic acid (especially DNA or RNA from a tumor sample) and generally provides a free 3'-OH group to allow polymerization of a complementary nucleic acid.

[0175] As used herein, the term "biomarker" refers to any part of a patient's molecule that is testimony to RAS G13R (particularly preferred), Q61E, Q61P, Q61H, Q61K, Q61L, or Q61R mutation. A biomarker may be a predictive biomarker and may serve as an indicator of the likelihood of suitability (sensitivity or benefit) of a patient with a solid tumor disease whose tumor has one of the described RAS mutations, thereby predicting whether the patient is suitable for treatment with a RAF, such as a Raf inhibitor, such as a Raf dimer, in combination with a MEK inhibitor. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA (e.g., mRNA)), alterations in polynucleotide copy number (e.g., DNA copy number), polypeptides (e.g., corresponding Ras polypeptides), modified polypeptides or polynucleotides (e.g., post-translational modifications). In some embodiments, as mentioned above or below, the biomarker is a gene carrying a mutation of interest (e.g., KRAS or NRAS genes).

[0176] In particular, in the case of DNA or RNA that is to be tested for the presence of a solid tumor or patient-derived biomarker (a mutation of interest according to the invention), isolation of the DNA or RNA is preferably followed by amplification of at least the region of its sequence (template sequence) that contains the mutation of interest.

[0177] Prospective or retrospective genetic assessment of RAS mutations in patients may be performed according to methods known in the art, for example using SNaPshot or DFCI Oncopanel as previously described (Sholl LM, et al. JCI Insight 2016;1:e87062; Zheng Z, et al., Nat Med 2014;20:1479-84). Current iterations of both assays utilize next generation sequencing (see, e.g., Boland GM, et al. Clinical next generation sequencing to identify actionable aberrations in a phase I program. Oncotarget. 2015;6(24):20099-20110), while earlier versions of SNaPshot relied on multiplex PCR. The current version of SNaPshot interrogates exons 2-5 of KRAS and NRAS. Oncopanel detects alterations involving all exons of RAS, specifically KRAS and NRAS.

[0178] As used herein, "amplifying" or "amplification" generally refers to a procedure for generating multiple copies of a desired nucleotide sequence. The "copies" usually (but do not necessarily) have perfect sequence complementarity or identity to the template sequence. For example, the copies may contain nucleotide analogs such as deoxyinosine, deliberate sequence modifications (e.g., sequence modifications introduced via primers that contain sequences that are hybridizable but not complementary to the template), and / or sequence errors that occur during amplification.

[0179] A preferred variation for identifying mutations of interest according to the invention involves a polymerase chain reaction step.

[0180] The term "polymerase chain reaction" or "PCR" technique (most important and preferred herein), as used herein, generally refers to a procedure in which minute specific pieces of nucleic acid, RNA, and / or DNA, are amplified, as described, for example, in U.S. Pat. No. 4,683,195; Schuller M., et al., (eds.), "PCR for Clinical Microbiology - an Australian and International Perspective, Springer 2010; BY Chan and HW Janes (eds.), PCR Cloning Protocols, 2nd ed., Humana press 2002; or Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987); and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Generally, sequence information from the region of interest must be available so that oligonucleotide primers can be designed, the sequences of which are identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of these two primers may match the ends of the material to be amplified. PCR may be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, and the like. Any of the nucleic acid polymerase reaction methods of amplifying a nucleic acid test sample may be used, including using known nucleic acids (DNA or RNA) as primers and utilizing a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or a specific piece of nucleic acid complementary to a specific nucleic acid.

[0181] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acid taken from a single source (e.g., a solid tumor sample from one individual) using two or more primer sets for the purpose of amplifying two or more DNA sequences in a single reaction.

[0182] "Quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a specific form of PCR in which the amount of PCR product is measured at each step of the PCR reaction, as appropriate. This technique is described in various publications, including, for example, Cronin et al., Am. J. Pathol. 164(1):35-42 (2004) and Ma et al., Cancer Cell 5:607-616 (2004).

[0183] The disclosed methods and assays provide a simple, efficient, and potentially cost-effective means for obtaining data and information useful in evaluating appropriate or effective therapies for treating a patient 40. For example, a patient can provide a tumor sample as a tissue sample (e.g., a tumor biopsy) that can be tested by various in vitro assays to determine whether the patient's cells are sensitive to the combination treatment before a combination treatment with a RAF inhibitor, such as a RAF inhibitor in combination with a MEK inhibitor, is considered a treatment option.

[0184] In any of the above methods, the identification of the specific mutation status of HRAS, or particularly KRAS and / or NRAS gene, mRNA, or protein product in the sample taken from an individual may be carried out by any one or more methods well known to those skilled in the art.For example, the identification of mutation may be achieved by cloning HRAS, particularly KRAS and / or particularly NRAS gene, or a part thereof, and sequencing using techniques known in the art.Alternatively, gene sequence may be amplified from genomic DNA or cDNA, for example, using PCR, and from the sequenced product.Some non-limiting methods for analyzing patient DNA for mutations at a given locus are described below.

[0185] DNA microarray technology can be used, for example, DNA chip device, and high-density microarray for high-throughput screening applications, and low-density microarray for example, DNA microarray hybridization.Additional methods include interference RNA microarray, and the combination of microarray with other methods, for example, laser capture microdissection (LCM), comparative genomic hybridization (CGH), and chromatin immunoprecipitation (ChiP).See, for example, He et al. (2007) Adv. Exp. Med. Biol. 593: 117-133, and Heller (2002) Annu. Rev. Biomed. Eng. 4: 129-153.

[0186] Other methods include PCR, xMAP, Invader assay, mass spectrometry, and pyrosequencing (Wang et al. (2007) 593:105-106).

[0187] Another detection method is allele-specific hybridization using probes that overlap the polymorphic site and have about 5, or alternatively 10, or alternatively 20, or alternatively 25, or alternatively 30 nucleotides around the region where the mutation site is found. For example, a plurality of probes that can specifically hybridize to a particular mutation (e.g., KRAS-G13R, which corresponds to the KRAS c.38G>A nucleotide substitution point mutation) are attached to a solid support, such as a "chip."

[0188] Oligonucleotides may be attached to solid supports by a variety of methods, including lithography. Mutation detection analysis using these chips containing oligonucleotides, also called "DNA probe arrays", is described, for example, in Cronin et al. (1996) Human Mutation 7:244.

[0189] Other detection methods require first amplifying at least a portion of the gene before identifying the mutant. Amplification may be performed, for example, by PCR and / or LCR, or other methods well known in the art.

[0190] In some cases, the presence of a particular mutation in DNA from a subject may be demonstrated by restriction enzyme analysis. For example, a particular mutation may result in a nucleotide sequence that contains a restriction site that is not present in another mutant or wild-type version of the gene.

[0191] In further embodiments, protection from cleavage agents (e.g., those containing nucleases, hydroxylamine, or osmium tetroxide, and piperidine) may be used to detect mismatched bases in RNA / RNA, DNA / DNA, or RNA / DNA heteroduplexes (see, e.g., Myers et al. (1985) Science 230:1242). In general, the technique of "mismatch cleavage" begins by providing a heteroduplex formed by hybridizing a control nucleic acid, e.g., RNA or DNA, containing the nucleotide sequence of an allelic variant of a gene, optionally labeled with a sample nucleic acid, e.g., RNA or DNA, taken from a tumor sample. The double-stranded duplex is treated with an agent that cleaves single-stranded regions of the duplex, such as the duplex formed based on base pair mismatches between the control strand and the sample strand. For example, RNADNA duplexes may be treated with RNase, and DNADNA hybrids may be treated with S1 nuclease, to enzymatically digest the mismatched regions. Alternatively, either DNA / DNA or RNADNA duplexes can be treated with hydroxylamine or osmium tetroxide, or with piperidine, to digest mismatched regions. After digestion of mismatched regions, the resulting material is then size-separated on a denaturing polyacrylamide gel to determine whether the control nucleic acid and the sample nucleic acid have the same nucleotide sequence or which nucleotides are different. See, for example, U.S. Patent No. 6,455,249; Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Meth. Enzymol. 217:286-295.

[0192] In addition, changes in electrophoretic mobility may be used to identify specific allelic mutations. For example, single-strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and renatured. The secondary structure of single-stranded nucleic acids varies with sequence, and even single base changes can be detected by the resulting changes in electrophoretic mobility. DNA fragments may be labeled with labeled probes and detected. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), whose secondary structure is more sensitive to sequence changes. In another embodiment, the subject method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on alterations in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

[0193] The identity of the mutant may also be determined by analyzing the migration of nucleic acids containing the polymorphic region in polyacrylamide gels containing a gradient of denaturing agent, assessed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). If DGGE is used as the analysis method, the DNA is modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high melting temperature GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used instead of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:1275).

[0194] Examples of techniques for detecting at least one nucleotide difference between two nucleic acid molecules (e.g., DNA or RNA molecules) include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, an oligonucleotide probe with a known polymorphic nucleotide centrally located may be prepared (allele-specific probe) and hybridized to a target DNA under conditions that permit hybridization only if there is a perfect match (Saiki et al. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele-specific oligonucleotide hybridization techniques may be used to detect nucleotide changes in polymorphic regions of genes. For example, an oligonucleotide having the nucleotide sequence of a specific allele mutation is bound to a hybridization membrane, which is then hybridized with a labeled sample nucleic acid. Analysis of the hybridization signal then reveals the identity of the nucleotides of the 5 sample nucleic acid.

[0195] Alternatively, allele-specific amplification techniques that rely on selective PCR amplification may be useful in conjunction with the present invention.

[0196] Oligonucleotides used as primers for specific amplification may carry the allelic mutation of interest in the middle of the molecule (so that amplification relies on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437-2448) or at the 3' end of one primer, where, under appropriate conditions, mismatches can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238, and Newton et al. (1989) Nucl. Acids Res. 17:2503). This technique is also called "PROBE", for Probe Oligo Base Extension. In addition to this, it may be desirable to introduce a new restriction site in the region of the mutation for truncated base detection (Gasparini et al. (1992) Mot. Cell. Probes 6:1).

[0197] In another embodiment, the identification of mutants is performed using an oligonucleotide ligation assay (OLA), for example as described in U.S. Pat. No. 4,998,617 and Laridegren, U. et al. Science 241:1077-1080 (1988). The OLA protocol uses two oligonucleotides that are designed to hybridize to adjacent sequences of a single strand of a target. One of the oligonucleotides is bound to a separation marker, e.g., biotinylated, and the other is detectably labeled. If the exact complementary sequence is found in the target molecule, the oligonucleotides will hybridize with their ends adjacent, creating a ligation substrate. Ligation then allows the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson et al. describe a nucleic acid detection assay that combines the properties of PCR with the attributes of OLA (Nickerson, DA et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927). In this method, PCR is used to achieve exponential amplification of target DNA, which is then detected using OLA.

[0198] A method is described for detecting single nucleotide mutations in a given gene, for example, RAS gene, for example, KRAS gene or NRAS gene or HRAS gene.Because single nucleotide changes are adjacent to regions of invariant sequence, their analysis does not require more than determining the identity of single mutant nucleotides, and does not require determining the complete gene sequence of each patient.Several methods have been developed to facilitate the analysis of such mutations.

[0199] Single base mutations may be detected by using specialized exonuclease-resistant nucleotides, for example as disclosed in U.S. Pat. No. 4,656,127. According to this method, a primer complementary to the mutated sequence immediately 3' to the mutation site is allowed to hybridize to a target molecule taken from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, that derivative is incorporated at the end of the hybridized primer. Such incorporation renders the primer resistant to exonucleases, thereby allowing its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the discovery that the primer has become resistant to exonucleases reveals that the nucleotide present at the mutation site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of unrelated sequence data.

[0200] A solution-based method may be used to determine the identity of the nucleotide at the mutation site (WO 91 / 02087). As above, a primer is used that is complementary to the mutated sequence immediately 3' to the mutation site. This method determines the identity of the nucleotide at that site using a labeled dideoxynucleotide derivative, which is incorporated onto the end of the primer if it is complementary to the nucleotide at the mutation site.

[0201] An alternative method is described in WO 92 / 15712. This method uses a mixture of a labeled terminator at the mutation or polymorphism site and a primer that is complementary to the sequence 3'. The labeled terminator that is incorporated is therefore determined by and complementary to the nucleotide present at the mutation site of the target molecule being evaluated. This method is usually a heterogeneous phase assay in which the primer or the target molecule is immobilized on a solid phase.

[0202] Many other primer-directed nucleotide incorporation procedures for analyzing mutation sites in DNA have been described (Komher, J Set al. (1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B. P. (1990) Nucl. Acids Res. 18:3671; Syvanen, A.-C., et al. (1990) Genomics 8:684-692; Kuppuswamy, M Net al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147; Prezant, T. R. et al. (1992) Hum. Mutat. 1:159-164; Ugozzoli, L. et al. (1992) GA TA 9:107-112; Nyren, P. et al. (1993) Anal. Biochem. 208:171-175). All of these methods rely on the incorporation of labeled deoxynucleotides to distinguish bases at the mutation site.

[0203] Generally, the presence and / or amount of the biomarker genes described herein can be analyzed by any one of numerous methods, including those described above, as well as many others known in the art and understood by those of skill in the art, such as Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR), next generation sequencing, and other amplification-based detection methods such as branched DNA, SISBA, TMA, etc.), RNA-Seq, microarray analysis, NanoString, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as a wide variety of assays that can be performed by protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis).

[0204] Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD") may be used.

[0205] Additionally, the presence and / or amount of the biomarker proteins (i.e., gene products) described herein can be analyzed by a number of methods, including immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, spectroscopy, molecular binding assays, HPLC, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration (ELIFA), fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescent in situ hybridization (FISH), and protein sequence analysis. In certain instances, the method includes contacting a biological sample from an individual with an antibody that specifically binds to a protein biomarker described herein under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo methods. In some cases, the protein expression level of a biomarker (e.g., KRAS-G13D protein or an NRAS protein with an activating mutation, as described herein above) is determined in tumor cells (e.g., from a biopsy).

[0206] Additionally, any of the above methods for detecting mutations in a gene or gene product may be used to monitor the course of a treatment or therapy (e.g., a combination treatment that includes a RAF inhibitor, e.g., a Raf inhibitor and a MEK inhibitor).

[0207] The methods described herein may be carried out, for example, by utilizing a pre-packaged diagnostic kit, such as those described below, that includes at least one probe or primer nucleic acid that can be conveniently used to determine whether a subject is likely to benefit from a combination treatment that includes a RAF inhibitor, e.g., a Raf inhibitor and a MEK or PI3K inhibitor.

[0208] In all of the above or herein-referred screening methods, mutations in one or more RAS genes (e.g., HRAS, KRAS, or NRAS) or their protein products can generally be identified by determining the nucleic acid sequence (e.g., DNA or RNA sequence) or protein sequence (i.e., amino acid sequence) in a tumor sample taken from an individual (=patient) and comparing the sequence with a reference sequence (e.g., wild-type sequence). In certain cases, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combination of multiple samples from the same subject or individual taken at one or more time points different from the time point at which the test sample is taken. For example, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is taken from the same subject or individual at an earlier time point than the time point at which the test sample is taken. Such reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue can be useful when the reference sample is taken during the initial diagnosis of cancer and then the test sample is taken when the cancer becomes metastatic.

[0209] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0210] For further details regarding the data and information shown in the figures, reference should also be made to the examples.

[0211] The following examples illustrate the above invention. However, these examples are not intended to limit the scope of the invention in any way. The beneficial effect of the pharmaceutical combination of the present invention may also be determined by other test models known to those skilled in the art. EXAMPLES

[0212] The following examples are presented to facilitate understanding of the invention, but are not intended, and should not be construed as, in any way limiting its scope.

[0213] Example 1: Analysis of Data from Study CLXH254X2102 (see https: / / clinicaltrials.gov / ct2 / show / NCT02974725) As of May 2021, 94 patients with KRAS / BRAF mutant non-small cell lung cancer and NRAS mutant melanoma were treated with naporafenib on a bid schedule and in combination with trametinib on a qd schedule at the following dose levels: naporafenib 200 mg and trametinib 1 mg (39 patients), naporafenib 400 mg and trametinib 1 mg (5 patients), naporafenib 200 mg and trametinib 0.5 mg (7 patients), naporafenib 400 mg and trametinib 0.5 mg (37 patients), and naporafenib 400 mg and trametinib 1 mg (6 patients), 2 weeks on, 2 weeks off. The RDE for the combinations of naporafenib and trametinib was determined to be naporafenib 400 mg bid + trametinib 0.5 mg qd and naporafenib 200 mg bid + trametinib 1 mg qd.

[0214] As of September 1, 2021, efficacy data for NRAS mutant melanoma was available for 30 patients in the expansion part, 15 of whom were treated with naporafenib 200 mg bid and trametinib 1 mg qd, and the other 15 patients were treated with naporafenib 400 mg bid and trametinib 0.5 mg qd. Overall, patients had received up to seven prior lines of therapy, with six patients (40%) receiving one prior line of therapy in the 200 mg / 1 mg group and four patients (26.7%) receiving up to one prior line of therapy in the 400 mg / 0.5 mg group. Confirmed ORR was 46.7% vs. 13.3%, and mPFS was 5.52 vs. 4.44 months in the 200 mg / 1 mg and 400 mg / 0.5 mg groups, respectively. Only three of the 30 patients with NRAS mutant melanoma did not have a confirmed Q61 or G13R codon mutation, and no complete or partial responses were confirmed in these three patients. See Table 1.

[0215] [Table 2]

[0216] N: Total number of subjects in the treatment group (denominator for calculating percentages (%)) n: number of patients in the corresponding category. Bid=2 times a day Qd = once a day ORR = objective response rate DCR = disease control rate DOR = duration of response mDOR = median duration of response mPFS = median progression-free survival CR=complete response PR=partial response SD=stable disease mOS = median overall survival OSR=overall survival rate Clopper-Pearson was used to calculate 95% CI for frequency distributions of each variable.

[0217] As of November 24, 2021, efficacy data were also available for 49 patients with KRAS mutant NSCLC, 27 of whom were treated with naporafenib 200 mg bid and trametinib 1 mg qd, and the other 22 patients were treated with naporafenib 400 mg bid and trametinib 0.5 mg qd. Napolafenib in combination with trametinib also showed greater efficacy in patients with KRAS mutant NSCLC harboring codon Q61 or G13R mutations. Three of four NSCLC patients with tumors harboring codon Q61 or G13R KRAS mutations responded (CR or PR) to naporafenib in combination with trametinib, whereas one of 45 NSCLC patients with tumors harboring KRAS mutations other than codon Q61 or GI3R mutations responded (CR or PR).

[0218] This unpublished data, as well as the preclinical evidence described in the Background section above, and clinical data with another Raf inhibitor, berbalafenib (see Example X below), convince us that codon Q61 / G13R pan-RAS mutation represents a predictive biomarker for response to naporafenib and trametinib combination.One hypothesis is that the activity of naporafenib in combination with trametinib seen in NRAS mutant melanoma may be due to the natural abundance of codon Q61 mutations, rather than due to the presence of NRAS mutations themselves.The presence of mutations is identified using next generation sequencing (NGS).

[0219] Example 2: Similar effects of G13R mutations versus codon Q61 mutations on RAS Molecular dynamics simulation analysis performed at Novartis showed that G13R overlaps with Q61, suggesting that G13R behaves dynamically similarly to Q61 and probably adopts a similar conformation / pose as Q61 (RAF-1 proficient).

[0220] Molecular dynamics simulations were performed using the Amber20 package (Case et al., AMBER 2020, University of California, San Francisco, 2020). For the G13R simulations, the x-ray crystal structure of GMPPNP-bound KRAS-G13D (PDB code 6XGV) was used. For the Q61R simulations, the x-ray crystal structure of GMPPNP-bound KRAS-Q61R (PDB code 6XGU) was used. Starting coordinates were prepared using the CCG MOE molecular editor, and in the case of G13R, D13 was mutated to R13 (CCG “Molecular Operating Environment (MOE), Inc., CCG “Molecular Operating Environment (MOE), 101 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015). All simulations were performed with the GTP nucleotide and a coordinated Mg2+ ion bound. The system was embedded in a water box with a 12 Å buffer containing charge-neutral Na+ ions. The protein was modeled using the Amber force field ff19SB (Tian, ​​C., et al, J. Chem. Theory Comput. 2020, 16, 528-552), water was modeled using TIP3P (Jorgensen et al., J. Chem. Phys 1983, 79, 926-935), and ions were modeled using JC parameters (Joung et al., J. Phys. Chem. B 1983, 79, 926-935). 2008, 112, 9020-9041). Each system was first equilibrated in the NPT ensemble for 100 ns before starting a 1 μs NPT generation run. Simulations were run in quadruplicate, corresponding to a generation time of 4 μs per KRAS mutation.

[0221] Molecular dynamics trajectory analysis was performed using CPPTRAJ (Roe et al., J. Chem. Theory Comput 2013, 9, 3084-3095). The root mean square fluctuation (RMSF) per residue per generation run was calculated, and the final result was taken as the average over four runs for each mutation.

[0222] Analyzing the RMSF data, we found that G13R and Q61R exhibited similar dynamics during the simulations, both with a mobile "switch-I" loop and a more constrained "switch-II" region. Therefore, we can conclude that the G13R and Q61R mutations dynamically overlap and likely adopt similar conformations.

[0223] In Figure 1 , the corresponding results are shown, confirming that the KRAS G13R protein dynamically overlaps with KRAS Q61R but not G13D.

[0224] Example 3: Study of the combination of naporafenib and trametinib for the treatment of solid tumors harboring RAS codon Q61 or G13R mutations The following clinical trials may be conducted: an open-label, non-randomized, Phase II study to evaluate the efficacy and safety of naporafenib in combination with trametinib in subjects with unresectable or metastatic melanoma with codon Q61 or G13R NRAS mutations who have received prior anti-PD(L)-1 with or without anti-CTLA-4 or anti-LAG-3, and in subjects with advanced solid tumors with RAS codon Q61 or G13R mutations who have progressed on available standard treatments or for whom no standard treatment options are available. The primary endpoint, ORR, may be based on a blinded independent review committee (BIRC) using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. PFS, OS, and DoR are secondary endpoints. A single central diagnostic test may select patients with NRAS mutant melanoma for enrollment in the clinical trial. This investigational clinical trial assay is intended to be a test enrolled as a companion diagnostic.

[0225] You can assign targets as follows: Cohort 1: Adults (>18 years of age) with unresectable or metastatic melanoma harboring codon Q61 or G13R NRAS mutations who have progressed on treatment with a PD-(L)-1 inhibitor alone or in combination with a CTLA4 or LAG3 inhibitor. Cohort 2: Adults (>18 years of age) with progressive metastatic solid tumors harboring codon Q61 or G13R RAS mutations who have progressed on available standard therapies or for whom no standard treatment options are available, as determined by local / regional standard of care and treating physician discretion.

[0226] Subjects may receive naporafenib 200 mg orally twice daily and trametinib 1 mg orally once daily until progressive disease per RECIST 1.1, unacceptable toxicity or death, loss to follow-up, or withdrawal of consent.

[0227] This study design aims to further evaluate the novel combination of naporafenib and trametinib, which showed promising activity and tolerable toxicity in the Phase 1b study CLXH254X2102 (see Example 1), in a population with high unmet medical need, characterized by poor prognosis and the above-mentioned mutations.

[0228] Clinical trials and unpublished internal data indicate that both trametinib and naporafenib have limited activity when administered alone in patients with NRAS mutant melanoma (Falchook et al. 2012; Lancet Oncol. 13(8):782-9). Single-agent MEK inhibitors, such as binimetinib from the NEMO trial (Dummer et al. 2017; Lancet Oncol. 19,1315-27), also show limited antitumor activity in NRAS mutant melanoma.

[0229] However, in light of the data and information herein, it is shown that the combination of naporafenib and trametinib has potential in the treatment of solid tumors (and (particularly) unresectable) melanoma).

[0230] Mutations in NRAS activate intracellular signaling through various pathways, including the RAS-RAF-MAPK pathway. These mutations are also known to activate the MAP kinase pathway to the same extent as BRAF V600 mutations. Because RAF and MEK are vertically integrated into the same pathway, combined inhibition of these two nodes can provide more effective inhibition, as supported by preclinical and clinical data. This study evaluates the efficacy of naporafenib (a RAF inhibitor) in combination with trametinib (a MEK inhibitor) in subjects with unresectable or metastatic melanoma harboring codon Q61 or G13R NRAS mutations who have progressed on treatment with PD-(L)-1 inhibitors as single agents and / or in combination with CTLA-4 or LAG3 inhibitors, particularly in subjects with advanced metastatic solid tumors harboring codon Q61 or G13R RAS mutations who have progressed on available standard therapies or for which no standard treatment options are available.

[0231] The main characteristics of the target population are as follows: Main selection criteria Cohort 1: Histologically confirmed unresectable stage IIIB / C / D or metastatic / stage IV melanoma using AJCC 8th edition Previously Treated for Unresectable or Metastatic Melanoma: Subjects must have received prior systemic therapy for unresectable or metastatic melanoma with an anti-PD-(L)-1 agent, with or without anti-CTLA-4 or anti-LAG (≤2 lines of anti-PD(L)-1-based regimens). Codon Q61 or G13R NRAS mutations in tumor tissue were confirmed in the laboratory even if local test results were available. All subjects must provide fresh or archived tumor specimens in the metastatic setting for testing for NRAS mutations. Cohort 2: Histologically confirmed solid tumor with advanced disease (AJCC 8th ed.) for which no standard treatment options are available as determined by the local / regional standard of care and the treating physician's discretion. Codon Q61 or G13R RAS (including HRAS, KRAS, and NRAS) mutations in tumor tissue are confirmed by a Novartis-designated laboratory even if local test results are available. All subjects must provide fresh tumor or archival specimens in the metastatic setting for testing of RAS mutations. All cohorts: ● Be 18 years of age or older (male or female) at the time of informed consent ECOG performance status of 0, 1, or 2 The last dose of prior therapy must have been administered ≥4 weeks prior to randomization. Patients must have at least one measurable lesion according to RECIST v1.1.

[0232] Main Exclusion Criteria Cohort 1: The subjects have uveal melanoma or mucosal melanoma. - Received prior systemic therapy for unresectable or metastatic melanoma with any investigational agent or any agent other than anti-PD-(L)-1 monotherapy, or anti-PD-(L)-1 in combination with anti-CTLA-4 or anti-LAG-3 Cohort 2: The subjects have NRAS melanoma mutations. Prior treatment with chemotherapy, immunotherapy, biologic therapy, or chemoirradiation to delay toxicity within 4 weeks prior to enrollment. Prior treatment with an investigational product within 4 weeks or 5 half-lives (whichever is longer) prior to enrollment. For all: Upfront systemic therapy with BRAF inhibitor and / or MEK inhibitor Prior solid organ or stem cell transplant Radiotherapy within 4 weeks prior to the start of study treatment (note: palliative radiotherapy for bone lesions is permitted) or active CTCAE grade >2 for radiotherapy-related toxicity within 2 weeks prior to the start of study treatment Major surgery prior to enrollment <14 days prior. Surgery-related AEs must have resolved prior to enrollment ● Primary CNS tumor or clinically active or unstable brain metastases present at screening, or symptomatic or untreated leptomeningeal or spinal cord compression. Note: Subjects with previously unstable brain lesions who have been definitively treated with stereotactic body radiation therapy, surgery, or gamma knife radiosurgery are eligible. Subjects with brain lesions who have not been treated (including newly discovered brain lesions during screening) or have received intracranial radiation must have documented stable disease on two consecutive assessments, at least 2 weeks apart, and must not have required high-dose steroids for at least 4 weeks prior to randomization / enrollment. History of other malignancies, including hematological malignancies Note: Subjects with another malignancy are eligible if: Have you been disease-free for three years? Have a history of completely excised non-melanoma skin cancer; and / or Any type of in situ carcinoma has been completely removed Clinically significant GI abnormalities that may alter absorption, such as malabsorption syndromes or extensive gastric or intestinal resections History or current evidence / risk of retinal vein occlusion (RVO) or central serous retinopathy - Systemic chronic steroid therapy (≥10 mg prednisone or equivalent per day) or any immunosuppressive therapy within 7 days prior to the scheduled first dose of study treatment. Topical, inhaled, nasal, and ophthalmic steroids are permitted. Current use of prohibited substances or prohibited substance requirements during the trial Cardiac disease or cardiac repolarization abnormalities, including any of the following: Congestive heart failure requiring treatment (New York Heart Association grade ≥2), LVEF <50% as measured by multiple gated acquisition (MUGA) scan or echocardiogram (ECHO), or uncontrolled hypertension defined as resting blood pressure ≥140 (systolic) / 90 (diastolic) mmHg despite medical therapy. - History of myocardial infarction, angina, or coronary artery bypass graft surgery within 6 months prior to starting study treatment History or presence of clinically significant arrhythmias (e.g., ventricular tachycardia), complete left bundle branch block, or high-degree AV block (e.g., bifascicular block, Mobitz type II block, and third-degree AV block) Long QT syndrome, family history of idiopathic sudden death or congenital long QT syndrome, or any of the following: Risk factors for polymorphic ventricular tachycardia, including uncorrected hypokalemia or hypomagnesemia, history of heart failure, or clinically significant / symptomatic bradycardia Concomitant medications with a known risk for polymorphic ventricular tachycardia that cannot be discontinued or substituted with a safe alternative within 7 days prior to initiating treatment QTcF interval cannot be measured Abnormal ECG as measured by the average of triple ECG and assessed in the central laboratory at the time of screening: Resting heart rate is less than 60 bpm or more than 90 bpm QTcF ≥ 450 msec (men) or ≥ 460 msec (women) at screening (using Fridericia correction).

[0233] Study treatment This study evaluates the combination of naporafenib (a Raf inhibitor) and trametinib (a MEK inhibitor) in patients with unresectable or metastatic codon Q61 or G13R NRAS mutant melanoma and in subjects with codon Q61 or G13R RAS mutant advanced metastatic solid tumors. Different RAS mutations display distinct patterns of interaction with downstream effector molecules. Considerable combinatorial activity with naporafenib and trametinib was confirmed in NRAS melanoma cell lines. The combination of naporafenib and trametinib was synergistic in all systems tested, with the strongest synergy observed in MM127 (NRASG13R), SK-MEL-2 (NRASQ61R), and MEL-JUSO (NRASQ61L). The combination of naporafenib and trametinib was superior to either drug alone in reducing phosphorylation of MEK1 / 2, ERK1 / 2, and RSK3. Furthermore, the combination of low-dose trametinib and naporafenib was equivalent to or superior to a 10-fold greater dose of single-agent naporafenib. In a mouse xenograft (PTX) model, the combination of naporafenib with a MEK inhibitor (trametinib) achieved better tumor growth inhibition than either drug alone (3. Figure 2 illustrates the results of the antitumor activity of naporafenib and trametinib across 10 patients from a mouse NRASmut melanoma tumor xenograft model.

[0234] Moreover, clinical data support the preclinical findings: trametinib has shown limited single-agent activity in RAS-driven tumors (i.e., a phase II randomized trial failed to demonstrate superiority over docetaxel, the second-line standard of care in KRAS G12, G13C, and G13D mutant NSCLC - Blumenschein et al 2015, Ann. Oncol. 26(5), 894-901). A phase 1 first-in-human (FIH) dose-escalation study evaluated single-agent trametinib in 97 patients with advanced melanoma, 7 of whom had NRAS mutations. In these seven patients, trametinib monotherapy (sequential doses of 2 mg, 2.5 mg, 3 mg; 2-week lead-in at dose escalations of 1 / 2 mg, 0.5 / 2.5 mg, and 0.5 / 2.5 mg; 3 weeks on, 1 week off, 2 mg) showed limited activity, with no response and, in two patients, stable disease (SD) (Falchook et al. 2012; Lancet Oncol. 13(8),789-9). Similarly, the phase 1 FIH trial CLXH254X2101 of naporafenib monotherapy in patients with solid tumors harboring MAPK mutations enrolled 10 patients with NRAS mutant melanoma and showed no response and four SD across a range of doses (400–1200 qd and 200–800 bid) (data cut May 31, 2021). These studies suggest that, consistent with preclinical findings, both trametinib and naporafenib have limited activity when administered alone.

[0235] In contrast, in patients with NRAS mutant advanced or metastatic melanoma, study CLXH254X2102 investigated the combination of naporafenib 200mg and trametinib 1mg. Sixteen patients were treated with the combination of naporafenib 200mg and trametinib 1mg. This combination resulted in a confirmed overall response rate (ORR) of 43.8%, a median duration of response (DOR) of 3.76 months, mPFS of 5.5 months, mOS of 8.8 months, and a 12-month OS rate of 48.9% (see Table 1 in Example 1).

[0236] The above data support the combination of naporafenib and trametinib versus each agent alone in patients with NRAS mutant melanoma.

[0237] Based on this data and the data of Example 1, a particularly preferred dosing regimen useful in the methods of the invention is isonaporafenib 200 mg bid + trametinib 1 mg qd.

[0238] Endpoints and Estimates To avoid potential investigator bias, the primary endpoint will be ORR assessed by a blinded independent review committee (BIRC) according to RECIST v1.1 (Response Evaluation Criteria in Solid Tumors, version 1.1).

[0239] ORR is selected as the primary endpoint because it is a direct measurement of drug antitumor activity that can be evaluated in single-arm trials (FDA Guidance for Industry, 2018; Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics (fda.gov). Source URL: https: / / www.fda.gov / media / 71195 / download).

[0240] PFS, OS, and duration of response (DOR) are secondary endpoints. PFS is defined as the time from randomization to disease progression or death from any cause as documented by BIRC per RECIST 1.1, whichever occurs first. OS is defined as the time from randomization to death from any cause.

[0241] DOR is also an important factor in evaluating study outcome and, for the subset of subjects with a confirmed overall response by BIRC, is defined as the time from first radiological evidence of a confirmed overall response by BIRC to disease progression documented by BIRC per RECIST 1.1 or death from any cause, whichever occurs first.

[0242] The present invention provides a method for treating solid tumors, in which a pharmaceutical combination of naporafenib and trametinib is administered to a subject suffering from unresectable or metastatic Q61 / G13R NRAS mutant melanoma that has progressed on anti-PD-(L)-1 (alone or in combination with anti-CTLA-4 or anti-LAG-3), or a RAS Q61 / G13R mutant progressive metastatic solid tumor that has progressed on available standard therapy or for which no standard treatment option is available. Thus, the patients treated by the method and pharmaceutical combination of the present invention include patients suffering from unresectable or metastatic Q61 or G13R NRAS mutant melanoma that has progressed on anti-PD-(L)-1 therapy (alone or in combination with anti-CTLA-4 or anti-LAG-3), or a RAS Q61 / G13R mutant progressive metastatic solid tumor that has progressed on available standard therapy or for which no standard treatment option is available.

[0243] Prior therapy for such patients includes:

[0244] Overall, these patients progressed on anti-PD-(L)-1 and subsequently received treatment with ipilimumab (ORR 13%, Da Silva 2021; Lancet Oncol. 22(6), 836-847), IL-2 (ORR 16%, Atkins et al 1999; J. Clin. Oncol. 17(7), 2105-16), paclitaxel / carboplatin (ORR 26%, Rao et al 2006; Cancer 106(2), 375-82), dacarbazine (ORR 7-20%, Dummer et al 2017, cited above; Serrone et al 2000; J. Exp. Clin. Cancer Res. 19(1), 21-34), and temazolamide (ORR 13.5% of patients with solid tumors are treated with gliomas-resistant Staphylococcus aureus (Rao et al 2006, cited above). Alternative treatments approved for other types of solid tumors are not RAS isoform specific, with the exception of the KRAS G12C inhibitor (sotorasib) for NSCLC, which has an ORR of 36% and a median DoR of 10 months.

[0245] INCORPORATION BY REFERENCE All clinical trials, publications, patents, and deposit numbers referred to in this specification are incorporated by reference in their entirety into this specification, to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein.

[0246] Equivalent While specific embodiments of the present invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the present invention will become apparent to those skilled in the art upon consideration of this specification and the following claims. The full scope of the present invention should be determined by reference to the claims as well as the full scope of their equivalents, and to the specification as well as such variations. Even if the deletion of this paragraph is made due to a rejection by the Patent Office or the court against this wording, it does not mean that the scope of protection, including the protection by the doctrine of equivalents, is narrowed.

Claims

1. 1. An agent or probe capable of identifying RAS codon Q61 and / or (particularly) G13R mutations for suitability for combination therapy with a Raf inhibitor and a MEK inhibitor for the diagnosis (or particularly for use in the diagnosis) of a solid tumor, particularly a non-melanoma, even more preferably a non-melanoma or a non-rhabdomyosarcoma, said diagnosis comprising applying said agent to assess a sample of said solid tumor for the presence or absence of a RAS codon Q61 or a RAS G13R mutation as a biomarker for said suitability.

2. 1. A method for diagnosing a patient suffering from a solid tumor, particularly one other than melanoma, even more preferably one other than melanoma or one other than rhabdomyosarcoma, for suitability for combined treatment with a Raf inhibitor and a MEK inhibitor, the method comprising: (a) evaluating a tumor sample taken from said patient for the presence of a RAS codon Q61 or RAS G13R mutation with an agent as described in the preceding paragraph; (b) selecting patients whose tumor samples tested positive in step (a) for the presence of the RAS codon Q61 or RAS G13R mutation as candidates for combination therapy; A method comprising:

3. 1. A method for selecting a treatment for a patient suffering from a solid tumor, the method comprising screening a tumor sample from the patient for a RAS codon Q61 or a RAS G13R mutation, the presence of which in the tumor sample identifies the patient as one who may benefit from a treatment comprising a Raf inhibitor and a MEK inhibitor.

4. 1. A method of treating a patient suffering from a solid tumor, the method comprising: (a) testing a tumor sample from said patient suffering from said solid tumor for the presence or absence of a RAS codon Q61 or RAS G13R mutation; (b) administering a therapeutically effective amount of a Raf inhibitor and a MEK inhibitor in combination therapy if the tumor sample is determined to have the mutation. A method comprising:

5. 1. A method of treating a patient afflicted with a solid tumor, particularly a non-melanoma, in need of treatment, comprising administering to said patient an effective amount of a combination of a RAF inhibitor and a MEK inhibitor, if said solid tumor is identified as having a RAS codon Q61 or a RAS G13R mutation.

6. A kit comprising an agent for identifying a RAS codon Q61 or a RAS G13R mutation and reagents for the method according to any one of the preceding paragraphs.

7. 1. A Raf inhibitor and a MEK inhibitor for use in combination in the treatment of a solid tumor, particularly a non-melanoma and / or a non-rhabdomyosarcoma having an NRAS Q61 mutation, in a patient identified as having a RAS codon Q61 or a RAS G13R mutation, wherein said solid tumor tests positive for having a RAS codon Q61 or a RAS G13R mutation.

8. The Raf inhibitors include encorafenib, sorafenib, vemurafenib, dabrafenib, GDC-0879, PLX-4720, PLX8394, belbarafenib, CCT3833 / BAL3833, LY3009120, LSN3074753, lifirafenib, BGB659, RO5126766, AZ-628, MLN2480, and BeiGene-2. 83, RXDX-105, BAL3833, INU152, regorafenib, toborafenib, Day101, TAK580, MLN2480, TAK632, and in particular naporafenib (LXH254; Type II), or a pharma- ceutically acceptable salt, hydrate, or solvate thereof, respectively.

9. 9. The Raf inhibitor and MEK inhibitor of claim 7 or 8, wherein the Raf inhibitor is napolafenib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

10. 10. The Raf inhibitor and MEK inhibitor of any one of claims 7 to 9, wherein the MEK inhibitor is selected from the group consisting of binimetinib, trametinib, selumetinib, cobimetinib, CI-1040, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honoliol, AZD8330, PD318088, BIX 02188, pimasertib, mirdametinib, refametinib, BI-847325, GDC0623, G-573, trametiglue, RO5126766, and pimisertib, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

11. The Raf inhibitor and MEK inhibitor of any one of claims 7 to 10, wherein the MEK inhibitor is trametinib, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

12. The Raf inhibitor and MEK inhibitor of any one of claims 7 to 11, wherein the solid tumor has tested positive for a G13R mutation.

13. The use of a Raf inhibitor in combination with a MEK inhibitor, particularly as defined in more detail herein, in particular the use of naporafenib in combination with trametinib, or a pharma- ceutical acceptable salt, hydrate or solvate thereof, in the treatment of a solid tumour in a patient where said solid tumour has tested positive for a RAS codon Q61 or a RAS G13R mutation.

14. 13. The use of a Raf inhibitor and a MEK inhibitor, particularly as defined in claims 8 and 10, respectively, in particular naporafenib and trametinib, or a pharma- ceutically acceptable salt, hydrate or solvate thereof, for the combined treatment of a solid tumor in a patient, said solid tumor being determined to harbor a RAS codon Q61 or a RAS G13R mutation, in the preparation of a medicament (particularly a combination), for the combined treatment of said solid tumor in a patient, said solid tumor being determined to harbor a RAS codon Q61 or a RAS G13R mutation, e.g. a kit for combined use or a fixed combination preparation comprising both active ingredients.

15. A composition comprising a Raf inhibitor and a MEK inhibitor, particularly as more particularly defined in claims 10 and 12, respectively, in particular naporafenib and trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof, respectively, and a pharmaceutically acceptable carrier, in the form of separate formulations or a kit or fixed formulation, for use in the treatment of rhabdomyosarcoma identified as having a RAS codon G13R mutation, or different solid tumors identified as having a RAS codon Q61 or a RAS G13R mutation, in particular a solid tumor selected from those other than melanoma, or a corresponding method of treatment comprising administering a combined therapeutically effective amount of said Raf inhibitor and said MEK inhibitor to a patient suffering from said rhabdomyosarcoma or other solid tumor in need of such treatment.

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