Precision therapy for cancer treatment

Abstract

The present invention provides a RAF inhibitor and a MEK inhibitor for use in combination in the treatment of solid tumors, and based on the identification of certain RAS mutations, a method for identifying the suitability of a patient with a solid tumor for treatment with a combination of a RAF inhibitor and a MEK inhibitor, a corresponding method of treatment, and related aspects or embodiments of the invention, the description is detailed as follows. The RAS mutation is in the mutation of a codon Q61 or G13R.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 315,763, filed on March 2, 2022, which is incorporated by reference in its entirety into this application. Technical Field

[0003] The present invention provides RAF inhibitors and MEK inhibitors for combined use in the treatment of solid tumors, and a method for identifying the response of patients with solid tumors described herein to combined treatment with a RAF inhibitor (e.g., naborafenib or bevafenib) and a MEK inhibitor (e.g., trametinib or cobimetinib) based on the identification of certain RAS mutations that can serve as prognostic markers for response to a given therapy, the corresponding treatment methods and related inventive aspects or embodiments are described in detail below.

[0004] In particular, the present invention provides a combination of a RAF inhibitor and a MEK inhibitor for use in treating solid tumors having a RAS codon Q61 mutation or having a G13R mutation.

[0005] In one aspect, the solid tumor to be treated is a solid tumor with a RAS codon Q61 mutation or with a G13R mutation, which is not a melanoma, or is not a NRAS mutant melanoma. In another aspect, the solid tumor to be treated is a solid tumor with a RAS codon Q61 mutation or with a G13R mutation, which is not a rhabdomyosarcoma. In another aspect, the solid tumor to be treated is a melanoma with a codon Q61 mutation in NRAS or a melanoma with a G13R mutation in NRAS. Background Art

[0006] RAS mutant cancers

[0007] RAS is the most frequently mutated gene family in cancer. In addition to NRAS, both KRAS and, to a lesser extent, HRAS proto-oncogenes are frequently mutated in many different types of human cancer. The incidence of RAS mutations varies widely among cancers; however, activating mutations in KRAS are common in lung, colorectal, and pancreatic cancers (Cox et al. 2014; Nat. Rev. Drug Discov. 13, 828–851).

[0008] In the vast majority of cases, activating mutations in the KRAS, NRAS, and HRAS genes change amino acids G12, G13, and Q61 in their respective proteins. However, the frequency of each amino acid change, the specific amino acid substitutions, and which RAS paralogs are changed vary significantly between different cancer types. For example, in lung adenocarcinoma, the G12C variant represents >40% of the total RAS variants, while G12C represents approximately 6% and 2% of the KRAS variants found in colorectal and pancreatic cancers, respectively (Cook et al. 2021). Some of these KRAS variant imbalances arise from differences in mutagenic pressure between tissue types (e.g., smoking in lung cancer). However, differences in mutagenic pressure alone do not appear to explain most of these allelic imbalances, suggesting that different RAS variants may have unique biological properties that intersect with the etiology of different cancer types (Cook et al. 2021).

[0009] Despite the fact that RAS is the most commonly mutated gene family in cancer and is associated with poor outcomes, there is currently only one approved therapy targeting a RAS mutation. Sotolacib, a KRAS G12C inhibitor, demonstrated 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 variants, there remains a significant unmet need for patients with advanced cancer who have progressed on available standard of care, or for whom there are no available standard treatment options.

[0010] Melanoma

[0011] Melanoma is the most aggressive of all skin cancers. The global incidence of melanoma is approximately 324,635 new cases per year, with 57,043 deaths (Sung et al. 2020; Global Cancer Statistics 2020: GLOBOCAN Estimates of Incide nce and Mortality Worldwide for 36Cancers in 185Countries. Available at: https: / / doi.org / 10.3322 / caac.21660 (accessed on August 2, 2021)). Among cancers in patients under 40 years old, the incidence of melanoma is second only to breast cancer in women and prostate cancer in men. Most melanoma patients are diagnosed at an early stage, when surgical resection can be curative, with an extremely high survival rate (approximately 90% at 5 years). In contrast, the prognosis for patients with distant metastasis is very poor, 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).

[0012] Primary 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 combined with anti-CTLA-4 therapy (i.e., ipilimumab). For melanoma patients with BRAF V600 mutations, which occur in approximately 40-60% of cases (Robert et al. 2019; The Lancet Oncology, Vol. 20, No. 9, KEYNOTE-006), treatment also includes targeted therapy with BRAF inhibitors combined with MEK inhibitors (e.g., dabrafenib and trametinib) (NCCN Melanoma 2nd edition 2021, https: / / www.nccn.org / professionals / physician_gls / pdf / colon.pdf; ESMO guidelines Michielin et al. 2019; Ann Oncol; 30(12):1884-1901).

[0013] In addition to BRAF mutations, the second most common mitogen-activated protein kinase (MAPK) pathway abnormality in melanoma is NRAS mutations. Melanomas with NRAS mutations are associated with a worse prognosis compared with other melanoma subtypes (Devitt et al. 2011).

[0014] Selective pharmacological inhibition of NRAS remains technically challenging because its GTPase activity has so far eluded the successful design of specific small-molecule antagonists. Several clinical trials are currently underway to evaluate alternative strategies that combine inhibitors of molecules downstream of NRAS, such as BRAF and MEK inhibitor combinations and PI3K pathway inhibitors in combination with drugs that inhibit the ERK pathway. The efficacy of these different approaches has yet to be determined.

[0015] In fact, there are currently no approved targeted therapies for NRAS-mutated 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. In addition, the LAG-3 blocking antibody relalizumab combined with anti-PD-1 recently showed a statistically significant PFS benefit over anti-PD-1 monotherapy in a phase 3 trial in patients with metastatic melanoma. If approved by health authorities, this regimen could become the new standard of care for this patient population. There is currently no established standard of care for patients with NRAS-mutated unresectable or metastatic melanoma who have progressed or are refractory to PD-1 inhibitors (with or without ipilimumab).

[0016] Current NCCN guidelines recommend a variety of systemic therapies for second-line and subsequent multi-line therapy for patients with metastatic melanoma who have failed prior anti-PD(L)1 therapy, either as monotherapy or in combination with ipilimumab, including ipilimumab monotherapy, high-dose IL-2, and cytotoxic agents such as dacarbazine, albumin-bound paclitaxel, temozolomide, or paclitaxel plus carboplatin (NCCN, supra). The ESMO guidelines acknowledge that second-line NRAS-mutated metastatic melanoma treatment options are very limited. Recommendations include treatment with MEK inhibitors (although limited activity is known), inclusion in clinical trials, or immunotherapy re-stimulation (e.g., if ipilimumab has not been used previously). If no clinical trials or new compounds are available, cytotoxic drugs such as dacarbazine or temozolomide can be administered (ESMO Guidelines Michielin et al. 2019, supra).

[0017] Ipilimumab as monotherapy was evaluated in a retrospective study of 162 patients with unresectable stage III / IV metastatic melanoma resistant to anti-PD(L)-1 therapy regardless of mutational status and demonstrated a limited 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) – two smaller retrospective studies also observed limited efficacy of ipilimumab monotherapy. A randomized, controlled, phase 3 study evaluated dacarbazine versus the MEK inhibitor bemetinib in 402 patients with NRAS-mutant melanoma, including both untreated patients (~79%) and those previously treated with immunotherapy (~21%). The ORR was 7% vs. 15%, mPFS was 1.7 months vs. 2.8 months, and mOS was 10.1 months vs. 11 months, respectively (Dummer et al. 2017; Lancet Oncol 2017; 18:435-45). Other treatments have shown only limited success.

[0018] The lack of standard of care and approved therapies, coupled with the limited efficacy of available therapies, highlights the unmet medical need for patients with NRAS-mutant melanoma, particularly those with unresectable or metastatic cutaneous melanoma with NRAS mutations. Summary of the invention

[0019] The present invention now provides a method of treating a solid tumor having a mutation in RAS other than KRAS G12 (eg other than KRAS G12C and / or other than KRAS G12D).

[0020] It has now been found that mutations in RAS codon Q61 and / or mutations in RAS G31R, whether the mutations occur in HRAS, KRAS or NRAS, can predict the response of solid tumors containing the mutations to combined treatment with RAF inhibitors and MEK inhibitors. This discovery therefore provides for the treatment of any tumor, in particular solid tumors, as long as the tumor contains a RAS codon Q61 mutation or in particular a RAS G13R mutation, thereby meeting a significant need and providing an accurate method for predicting response to treatment and treating identified tumors. Therefore, the tumor to be treated does not have to be organ or tissue specific (e.g. lung cancer or pancreatic cancer or colorectal cancer or breast cancer).

[0021] In other words, whether the mutation occurs in HRAS, KRAS or NRAS, in a tumor or in a tumor sample obtained from a patient with a tumor, and regardless of the tissue origin of the tumor in a sample obtained from a patient with a tumor (e.g., a solid tumor), the detection and presence of a mutation in RAS codon Q61 and / or a mutation in RAS G31R allows for a prognosis of whether a patient with such a tumor will benefit and therefore respond to a combination therapy with a RAF inhibitor and a MEK inhibitor (e.g., a combination of naborafenib and trametinib, or a combination of bevafenib and cobinmetinib). Thus, the present invention provides treatments for solid tumors containing such mutations.

[0022] A new and in-depth review of preclinical data from clinical studies of naborafenib and other agents acting on the MPAK pathway, as well as past and current emerging clinical research data, found that the combination of RAF inhibitors and MEK inhibitors could be particularly beneficial for treating patients with solid tumors harboring pan-RAS codon Q61 or G13R mutations.

[0023] For example, three-quarters of patients with non-small cell lung cancer (NSCLC) whose tumors harbored KRAS codon Q61 or G13R mutations responded to the combination of naborafenib and trametinib, compared with one in 43 patients with NSCLC whose tumors harbored KRAS mutations other than codon Q61 or G13R mutations. In addition, clinical data from the first-in-human study of LXH254 (CLXH254X2101) showed that patients with solid tumors (salivary gland tumors and NSCLC) harboring pan-RAS codon Q61 or G13R mutations were found to be excellent responders to LXH254 monotherapy. In addition, nonclinical models have shown consistent activity of LXH254 as a single agent or in combination with trametinib in pan-RAS codon Q61 / G13R mutant models across a variety of tumor types (NSCLC, melanoma, PDAC, rhabdomyosarcoma), independent of the specific mutant RAS gene (pan-RAS) and tissue of origin of the tumor (pan-tissue). Overall, the sum of these data suggests that pan-RAS codon Q61 / G13R mutations may represent a predictive biomarker for response to a combination of RAF and MEK inhibitors.

[0024] This data, together with preclinical evidence, and clinical data using another RAF inhibitor, bevafenib, rationalizes the use of codon Q61 / G13R (pan-)RAS mutations as a predictive biomarker for response to the combination of a RAF inhibitor (such as naborafenib or bevafenib) and a MEK inhibitor (such as trametinib or cobimetinib).

[0025] Furthermore, in a molecular dynamics simulation analysis performed by Novartis, G13R was found to overlap with Q61 and behave similarly in kinetics and may adopt a conformation similar to Q61. Therefore, mutations in RAS Q61 and G13R in RAS genes, whether selected from HRAS, KRAS, NRAS genes and their combination, are suitable biomarkers that allow the identification of tumor subtypes that strongly rely on MAPK and may benefit from combination therapy. Once the MAPK pathway is activated, homo- or heterodimers of RAF activate MEK1 and MEK2, which are the main downstream signaling nodes. Given that RAF and MEK are vertically integrated in the same pathway, combined inhibition of these two nodes may provide more effective inhibition of this pathway and delay the development of drug resistance.

[0026] The present invention is therefore based on the fact that by using the RAS codon Q61 or in particular the RAS G13R mutation as a predictive biomarker for solid tumors that can be treated by a combination of a RAF inhibitor and a MEK inhibitor, patients whose tumors contain this mutation can be selected and treated. It was found herein that the G13R mutation is an important predictive biomarker.

[0027] The present invention therefore provides the following embodiments:

[0028] Embodiment 1. A method of selectively treating a patient with a solid tumor, comprising selectively administering to the patient a therapeutically effective amount of a combination of a RAF inhibitor and a MEK inhibitor based on whether the tumor has a RAS codon Q61 mutation or a G13R mutation.

[0029] Embodiment 2. A method for selectively treating a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation using a combination of a RAF inhibitor and a MEK inhibitor, comprising:

[0030] a) selecting patients for treatment with a combination of a RAF inhibitor and a MEK inhibitor based on the fact that their tumor has a RAS codon Q61 mutation or a G13R mutation; and

[0031] b) Thereafter, administering to the patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor.

[0032] Embodiment 3. A method for selectively treating a patient having a solid tumor with a RAS codon Q61 mutation or a G13R mutation, comprising:

[0033] a) assaying a biological sample or a tumor sample from a patient for the presence of a RAS codon Q61 mutation or a G13R mutation in a solid tumor; and

[0034] b) Thereafter, optionally administering to the patient any of the following:

[0035] i. when there is a RAS codon Q61 mutation or a G13R mutation, administering a therapeutically effective amount of a combination of a RAF inhibitor and a MEK inhibitor; or

[0036] ii. Based on the absence or non-detection of RAS codon Q61 mutation or G13R mutation in the patient's biological or tumor sample, administering a therapeutically effective amount of a drug other than the combination of RAF inhibitor and MEK inhibitor.

[0037] Embodiment 4. A method for selectively treating a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation using a combination of a RAF inhibitor and a MEK inhibitor, comprising:

[0038] a) assaying a biological sample or a tumor sample from a patient for the presence of a RAS codon Q61 mutation or a G13R mutation;

[0039] b) subsequently selecting patients for treatment with a combination of a RAF inhibitor and a MEK inhibitor based on biological or tumor samples of patients having a RAS codon Q61 mutation or a G13R mutation; and

[0040] c) Subsequently, administering to the patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor.

[0041] Embodiment 5. The method according to 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 leukocyte sample, and a tumor sample (also called a tumor biopsy).

[0042] Embodiment 6. The method according to any one of embodiments 3-5, wherein the determining step comprises an assay selected from Northern blot analysis, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), TaqMan-based assay, direct sequencing, dynamic allele-specific hybridization, high-density oligonucleotide SNP array, restriction fragment length polymorphism (RFLP) assay, primer extension assay, oligonucleotide ligase assay, single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography, high resolution melting analysis, DNA mismatch binding protein assay, Techniques of capillary electrophoresis, Southern blotting, immunoassay, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry.

[0043] Embodiment 7. A combination of a RAF inhibitor and a MEK inhibitor for treating a patient having a solid tumor with a RAS codon Q61 mutation or a G13R mutation, characterized in that the combination of a RAF inhibitor and a MEK inhibitor is administered to the patient on the basis that the patient has a solid tumor with a RAS codon Q61 mutation or a G13R mutation.

[0044] Embodiment 8. A combination of a RAF inhibitor and a MEK inhibitor for treating a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation, comprising a) performing an assay on a sample of the patient,

[0045] b) determine whether the sample shows the presence of a solid tumor with a RAS codon Q61 mutation or a G13R mutation, and

[0046] c) if a RAS codon Q61 mutation or a G13R mutation is present, administering to the patient a therapeutically effective amount of a combination of a RAF inhibitor and a MEK inhibitor.

[0047] Embodiment 9. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation, characterized in that:

[0048] a) selecting a patient for treatment with a 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; and

[0049] b) Thereafter, administering to the patient a therapeutically effective amount of a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor.

[0050] Embodiment 10. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation, characterized in that:

[0051] a) testing a biological sample of the patient for a RAS codon Q61 mutation or a G13R mutation; and

[0052] b) selectively administering a therapeutically effective amount of a combination of a RAF inhibitor and a MEK inhibitor to a patient based on a biological sample of the patient having a RAS codon Q61 mutation or a G13R mutation.

[0053] Embodiment 11. A pharmaceutical combination of a RAF inhibitor and a MEK inhibitor for use in treating a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation, characterized in that:

[0054] a) testing a biological sample or tumor sample of a patient for the presence of a RAS codon Q61 mutation or a G13R mutation;

[0055] b) selecting patients to be treated with a combination of a RAF inhibitor and a MEK inhibitor based on biological samples of patients having a RAS codon Q61 mutation or a G13R mutation; and

[0056] c) selectively administering to the patient a therapeutically effective amount of a combination of a RAF inhibitor and a MEK inhibitor.

[0057] Embodiment 12. A method for predicting the likelihood that a patient with a solid tumor having a RAS codon Q61 mutation or a G13R mutation will respond to treatment with a combination of a RAF inhibitor and a MEK inhibitor, comprising assaying a biological sample or a tumor sample of the patient for the presence or absence of a RAS codon Q61 mutation or a G13R mutation, wherein:

[0058] a) the presence of a RAS codon Q61 mutation or a G13R mutation indicates an increased likelihood that the patient will respond to treatment with a combination of a RAF inhibitor and a MEK inhibitor; and

[0059] b) The absence of a RAS codon Q61 mutation or a G13R mutation indicates a reduced likelihood that the patient will respond to combination therapy with a RAF inhibitor and a MEK inhibitor.

[0060] Embodiment 13. The method according to embodiment 12, further comprising the step of obtaining a biological sample from the patient, wherein the obtaining step is performed before the detecting step.

[0061] Embodiment 14. The method according to 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 leukocyte sample, a tissue sample, and a tumor sample.

[0062] Embodiment 15. The method according to any one of embodiments 12-14, wherein the determining step comprises an assay selected from Northern blot analysis, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), TaqMan-based assay, direct sequencing, dynamic allele-specific hybridization, high-density oligonucleotide SNP array, restriction fragment length polymorphism (RFLP) assay, primer extension assay, oligonucleotide ligase assay, single-stranded conformation polymorphism analysis, temperature gradient gel electrophoresis (TGGE), denaturing high performance liquid chromatography, high resolution melting analysis, DNA mismatch binding protein assay, Techniques in capillary electrophoresis, Southern blotting, immunoassays, immunohistochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry.

[0063] Embodiment 16. A method for generating information in a transmittable form predicting a response of a patient with a solid tumor to treatment with a combination of a RAF inhibitor and a MEK inhibitor, comprising:

[0064] a) determining that the patient has an increased likelihood of responding to treatment with a 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; and

[0065] b) recording the results of the determining step in a tangible or intangible medium for transmission purposes.

[0066] Embodiment 17. A kit for use in predicting the likelihood that a patient with a solid tumor will respond to treatment with a combination of a RAF inhibitor and a MEK inhibitor, comprising:

[0067] a) at least one probe capable of detecting the presence of a RAS codon Q61 mutation or a G13R mutation; and

[0068] b) an indication of using the probe to determine the presence of a RAS codon Q61 mutation or a G13R mutation in a biological sample of a patient, wherein the presence of the RAS codon Q61 mutation or the G13R mutation indicates an increased likelihood that the patient will respond to a combination treatment with a RAF inhibitor and a MEK inhibitor, and the absence of the RAS codon Q61 mutation or the G13R mutation indicates a decreased likelihood that the patient will respond to a combination treatment with a RAF inhibitor and a MEK inhibitor.

[0069] Embodiment 18. A kit for use in treating a patient with a solid tumor, comprising:

[0070] a) a therapeutically effective amount of a combination of a RAF inhibitor and a MEK inhibitor;

[0071] b) at least one probe capable of detecting the presence of a RAS codon Q61 mutation or a G13R mutation in a solid tumor;

[0072] c) instructions for using the probe to determine the presence of a RAS codon Q61 mutation or a G13R mutation in a biological sample or a tumor sample from a patient,

[0073] d) instructions for administering to the patient a combination of a RAF inhibitor and a MEK inhibitor if the patient's biological sample or tumor sample indicates the presence of a RAS codon Q61 mutation or a G13R mutation; and

[0074] e) Optionally, means for administering to the patient a pharmaceutical combination of a RAF inhibitor and a MEK inhibitor.

[0075] Embodiment 19. The kit according to any one of embodiments 17 or 18, wherein the probe is an oligonucleotide that specifically hybridizes to a nucleic acid region 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 nucleic acid region encoding an equivalent genetic marker of a RAS codon Q61 mutation or a G13R mutation.

[0076] In the above embodiments, the solid tumor having RAS codon Q61 mutation or having G13R mutation can be selected from:

[0077] Solid tumors other than melanoma or non-NRAS mutant melanoma;

[0078] Solid tumors other than rhabdomyosarcoma;

[0079] A solid tumor that is a melanoma with a codon Q61 mutation in NRAS or a melanoma with a G13R mutation in NRAS;

[0080] A solid tumor selected from non-small cell lung cancer, pancreatic cancer, thyroid cancer and colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 is a graphical representation of KRAS G13R protein dynamics overlapping with KRAS Q61R but not G13D.

[0082] Figure 2 is a diagram showing an overview of the study design for studies in patients with mutant melanoma and solid tumors.

[0083] Figure 3 The antitumor activity of naborafenib and trametinib was demonstrated in ten patient-derived NRAS-mutant melanoma tumor xenograft mouse models. DETAILED DESCRIPTION

[0084] In one embodiment, the present invention provides an agent or probe capable of recognizing RAS codon Q61 or, in particular, RAS G13R mutation, for use (or particularly for this purpose) in diagnosing the suitability of solid tumors, in particular solid tumors other than melanoma, but more preferably solid tumors other than melanoma or rhabdomyosarcoma, for combined treatment with a Raf inhibitor and a MEK inhibitor, wherein the diagnosis comprises the use of the agent to determine whether a sample of the solid tumor contains RAS codon Q61 or RAS G13R mutation as a biomarker for the suitability.

[0085] In one embodiment, the invention provides a method of identifying a patient with a solid tumor, particularly a solid tumor other than melanoma, yet more preferably a solid tumor other than melanoma or a solid tumor other than rhabdomyosarcoma, who has an increased likelihood of responding to a combination treatment with a Raf inhibitor and a MEK inhibitor, the method comprising:

[0086] (a) assaying a tumor sample obtained from a patient for the presence of a RAS codon Q61 or RAS G13R mutation using an agent according to the preceding paragraph; and

[0087] (b) selecting patients whose tumor samples tested positive under (a) for the presence of said RAS codon Q61 or RAS G13R mutation as candidates for said combination treatment.

[0088] In another embodiment, the invention provides a method for selecting a treatment for a patient with a solid tumor, the method comprising screening a tumor sample from the patient for a RAS codon Q61 or (particularly) a RAS G13R mutation, wherein the presence of the mutation in the tumor sample identifies the patient as a patient who may be expected to benefit from a treatment comprising a Raf inhibitor and a MEK inhibitor.

[0089] In another embodiment, the present invention provides a method of treating a patient suffering from a solid tumor, the method comprising

[0090] (a) examining whether a tumor sample from a patient having a tumor contains a RAS codon Q61 and / or RAS G13R mutation, and,

[0091] (b) where the tumor sample is determined to contain the mutation, administering a therapeutically effective amount of a Raf inhibitor and a MEK inhibitor as a combination therapy.

[0092] Another embodiment of the invention provides a method of treating a patient having a solid tumor, particularly a solid tumor other than melanoma, in need of such treatment, comprising administering to the patient an effective amount of a combination of a RAF inhibitor and a MEK inhibitor, where the solid tumor is identified as containing a RAS codon Q61 or RAS G13R mutation.

[0093] Another embodiment of the present invention provides a kit comprising an agent for identifying RAS codon Q61 or RASG13R mutation and a reagent for the method according to any of the preceding paragraphs.

[0094] Another embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use in the treatment of a solid tumor in a patient, particularly a solid tumor other than melanoma and / or rhabdomyosarcoma containing a NRAS codon Q61 mutation, wherein the tumor is tested positive and identified as containing a RAS codon Q61 or RAS G13R mutation.

[0095] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to the preceding paragraphs, wherein the Raf inhibitor is selected from conafenib; sorafenib, vemurafenib, dabrafenib, GDC-0879, PLX-4720, PLX8394, bevafenib, CCT3833 / BAL3833, LY3009120, LSN3074753, rifolafenib, BGB659, RO5126766, AZ-628, MLN2480, BeiGene-283, RXDX-105, BAL3833, INU152, regorafenib, tovofenib, Day101, TAK580, MLN2480, TAK632, and in particular nabolafenib (LXH254; Type II); or their respective pharmaceutically acceptable salts, hydrates or solvates.

[0096] A further embodiment of the present invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any one of the two preceding paragraphs, wherein the Raf inhibitor is naborafenib, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0097] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any of the preceding paragraphs, wherein the MEK inhibitor is selected from bemetinib, trametinib, selumetinib, cobimetinib, CI-1040, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, Honoliol, AZD8330, PD318088, BIX 02188, pimacitinib, midametinib, refactinib, BI-847325, GDC0623, G-573, trametiglue, RO5126766 and pimacitinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0098] Preferred MEK inhibitors include trametinib, bemetinib and cobimetinib.

[0099] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any one of the four preceding paragraphs, wherein the MEK inhibitor is trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0100] A further embodiment of the invention provides a Raf inhibitor and a MEK inhibitor for combined use according to any of the preceding paragraphs, wherein the solid tumor is tested positive for containing a RAS G13R mutation.

[0101] A further embodiment of the invention provides the combined use of a Raf inhibitor and a MEK inhibitor, in particular as defined in more detail herein, in particular naborafenib and trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in the treatment of a solid tumor in a patient, wherein the tumor is tested positive for containing a RAS codon Q61 or RAS G13R mutation.

[0102] A further embodiment of the invention provides the use of a Raf inhibitor and a MEK inhibitor, in particular as defined in more detail herein, in particular naborafenib and trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in the preparation of a (in particular combined) medicament, such as a kit for combined use or a fixed combination preparation comprising both active ingredients, for the combined treatment of a solid tumor in a patient, wherein the solid tumor has been determined to contain a RAS codon Q61 or RAS G13R mutation.

[0103] Another embodiment of the present invention provides a composition comprising a Raf inhibitor and a MEK inhibitor, in particular as defined in more detail herein, in particular naborafenib and trametinib, or their respective pharmaceutically acceptable salts, hydrates or solvates, and a pharmaceutically acceptable carrier, in the form of a separate formulation or a kit or a fixed formulation for the treatment of solid tumors selected from rhabdomyosarcoma identified as containing a RAS G13R mutation, or different solid tumors identified as containing RAS codon Q61 or RAS G13R mutations, in particular solid tumors other than melanoma; or a corresponding method of treatment, comprising administering the mentioned Raf inhibitor and MEK inhibitor in a combined therapeutically active amount to a patient suffering from said rhabdomyosarcoma or other solid tumor in need of such treatment.

[0104] The more specific definitions used herein, particularly below, may be used to replace one, more or all of the broader terms, features or expressions of the inventive embodiments herein, thereby defining further inventive embodiments.

[0105] Suitability for combination / combined therapy indicates that a positive effect on the neoplastic disease - as defined below - can be expected. In other words, it means that the patient may benefit from this treatment, allowing for an improved prognosis.

[0106] RAS in this article refers to HRAS, NRAS, and KRAS.

[0107] RAF inhibitors

[0108] Raf inhibitors mentioned herein include any RAF inhibitor, for example specific (e.g., ARAF, BRAF or CRAF specific) inhibitor, but mainly inhibitors for two or all three of ARAF, BRAF and CRAF. Preferred RAF inhibitor classes include type I inhibitors, type 1.5 inhibitors (including so-called paradox breakers) and type II inhibitors.

[0109] Paradox breakers are a subtype of type 1.5 inhibitors. In cells, type 1.5 inhibitors such as contrafenib, vemurafenib, and dabrafenib activate signaling through wild-type BRAF, but not BRAFV600E mutant BRAF. This unexpected activation by inhibitors is called paradoxical activation. A "paradox breaker" is a type 1.5 inhibitor that does not induce this paradoxical activation.

[0110] Preferred RAF inhibitors are the so-called type II inhibitors.

[0111] Preferred Raf inhibitory compounds are selected from (with some known types mentioned in brackets):

[0112] Sorafenib (BAY43-9006), bevafenib (HM95573), CCT3833 / BAL3833, LY3009120, LSN3074753, rifampinib (BGB-283), BGB659, RO5126766, AZ-628, MLN2480, BeiGene-283 (BGB283), RXDX-105, BAL3833, INU152, regorafenib, tovofenib, Day101, TAK580, MLN2480, TAK632, and in particular naborafenib (LXH254), or their respective pharmaceutically acceptable salts, hydrates or solvates.

[0113] More preferably, the RAF inhibitor compound can be selected from sorafenib (BAY43-9006; Type II), bevafenib (HM95573; Type II; preferred), CCT3833 / BAL3833, LSN3074753, rifampinib (BGB-283; Type II), BGB659, RO5126766, AZ-628, MLN2480 (Type II), BeiGene-283 (BGB283), RXDX-105, BAL3833, INU152, regorafenib (Type II), tovofenib, Day101, TAK580, MLN2480, TAK632, and especially naborafenib (LXH254; Type II); or their respective pharmaceutically acceptable salts, hydrates or solvates.

[0114] Preferred RAF inhibitors include:

[0115] Nabolafenib (LXH254)

[0116] Nabolafenib has the chemical formula I

[0117]

[0118] The 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. Where naborafenib is mentioned herein, it refers to the free compound itself, but it is further defined herein to include its pharmaceutically acceptable salts, hydrates and / or solvates. Particularly preferred is the free form or as a monohydrate such as the monohydrate form H ANabolafenib, characterized in that when measured using CuKα radiation, the X-ray powder diffraction pattern has at least one, two, three, four or five peaks, the refraction angle 2theta (θ) values ​​of these peaks are selected from 7.3, 10.7, 16.3, 16.7, 17.4, 23.0, 24.3, 25.3, 28.3, 32.0, wherein the value is plus or minus 0.2° 2θ. Monohydrate form H of Nabolafenib A Further described in and can be prepared as described in WO / 2020 / 230028.

[0119] Nabolafenib is an adenosine triphosphate (ATP) competitive inhibitor that primarily targets BRAF and CRAF protein kinases. Because nabolafenib inhibits both monomeric and dimerized BRAF and CRAF, it induces minimal paradoxical activation and effectively blocks mutant RAS-driven signaling and cell proliferation. Nabolafenib has demonstrated efficacy in a wide range of MAPK pathway-driven human cancer cell lines and in vivo tumor xenografts, including models containing activating lesions within the KRAS, NRAS, and BRAF proto-oncogenes.

[0120] The compound of formula (I) and its pharmaceutically acceptable salts are described in WO2014 / 151616, which is hereby incorporated by reference in its entirety, and methods for their preparation have been described, for example, in Example 1156 thereof. Naborafenib can also be used in its polymorphic form, for example, as described in WO / 2020 / 230028.

[0121] In cell-based assays, the compound of formula (I) exhibits antiproliferative activity in cell lines containing multiple mutations that activate MAPK signal transduction. In vivo, treatment with the compound of formula (I) produces tumor regression in several KRAS mutant models. In general, the in vitro and in vivo MAPK pathway inhibition and antiproliferative activity of the compound of formula (I) observed at well-tolerated doses rationalize that the compound of formula (I) can have antitumor activity in tumor patients with MAPK pathway activation lesions. In addition, 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 signal transduction and cell proliferation. The compound of formula (I) has shown efficacy in a variety of MAPK-driven human cancer cell lines and xenograft tumors representing model tumors containing human lesions in KRAS, NRAS, and BRAF proto-oncogenes (see, for example, WO / 2018 / 203219, which is incorporated herein in its entirety).

[0122] Further evidence of the utility of the present invention can be derived from the clinical data obtained on LXH254, e.g.

[0123] CLXH254X2101: A first-in-human study of naborafenib as a single agent in patients with advanced solid tumors harboring MAPK pathway alterations 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.

[0124] CLXH254X2102: A Phase Ib, open-label, multicenter study of naborafenib in combination with LTT462 in adult patients with advanced or metastatic KRAS or BRAF mutant NSCLC, or in combination with trametinib in adult patients with advanced or metastatic KRAS or BRAF mutant NSCLC or NRAS mutant melanoma, or in combination with ribociclib in adult patients with advanced or metastatic NRAS mutant melanoma.

[0125] CLXH254C12201: A randomized, open-label, multi-arm, two-part, phase II study to evaluate the efficacy and safety of multiple nabolafenib combinations in patients with previously treated unresectable or metastatic BRAF V600 or NRAS mutant melanoma.

[0126] Bevafenib

[0127] Bevafenib (HM95573, GDC5573, RG6185) has the following formula

[0128]

[0129] Bevafenib is named 4-amino-N-(1-((3-chloro-2-fluorophenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide and is a potent pan-RAF inhibitor with antitumor activity, particularly against BRAF, BRAF V600E and CRAF. Its synthesis has been described in WO2013 / 100632A1. The compound is highly selective for BRAF mutants and CRAF kinases. A Phase Ib trial of bevafenib 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

[0130] MEK inhibitors

[0131] The term "MEK inhibitor" is defined herein to mean a compound that targets, decreases or inhibits at least one activity of MAP / ERK kinases 1 and 2 (MEK1 / 2). MEK stands for "mitogen-activated protein kinase".

[0132] Reference herein to MEK inhibitors includes inhibitors of both MEK and the MEK / RAF heterodimer complex.

[0133] Preferred MEK inhibiting compounds are selected from:

[0134] Arry-162 = MEK162 (bemetinib), AZD6244 (selumetinib), XL518 = GDC0973 (cobimetinib), CI-1040 = PD184352, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, honokiol, AZD8330, PD318088, BIX 02188, AS703026 = MSC1936369B (pimacitinib), SL327, PD-325901 = PD0325901 (midametinib), TAK-733, BAY86-9766 (refatinib), BI-847325, GDC0623, G-573, RAF / MEK heterodimer disruptor), trimethoprim (RAF / MEK complex inhibitor), CH4987655 = RO 4987655, WX-554, HL-o85, CH5126766 = RO5126766 (RAF / MEK complex inhibitor) (preferred), and especially trametinib (GSK1120212; advanced RAF inhibitor; marketed under the trade name meginin marketing.

[0135] Preferred MEK inhibitors include trametinib, bemetinib and cobimetinib.

[0136] Preferably, the MEK inhibitor is trametinib, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, trametinib is in the form of a sodium salt. Suitably, trametinib is in the form of a solvate selected from the following: hydrate, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentanol, isopropanol, ethylene glycol and 3-methyl-1-butanol. In some preferred embodiments, trametinib is in the form of a dimethyl sulfoxide solvate. Those skilled in the art can prepare these solvates and salt forms according to the description in WO 2005 / 121142.

[0137] Trametinib has the formula II

[0138]

[0139] This compound is known by another name 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 known as JPT-74057 or GSK1120212 or TMT212.

[0140] Trametinib is disclosed in Example 4-1 of PCT Publication No. WO 2005 / 121142, which disclosure is hereby incorporated by reference in its entirety.

[0141] Where "trametinib" is mentioned in the present disclosure, it refers to its free form itself, but it is also defined herein to include its pharmaceutically acceptable salts, hydrates and / or solvates, in particular dimethyl sulfoxide solvates.

[0142] RO5126766 has the formula III,

[0143]

[0144] The compound is named N-(3-fluoro-4-((4-methyl-2-oxo-7-(7-(2-pyrimidinyloxy)-2H-1-benzopyran-3-yl)methyl-2-pyridinyl)-N'-methyl-sulfonamide. RO5126766 is a dual RAF / MEK inhibitor with IC50 of 8.2nM, 19nM, 56nM and 160nM for BRAF V600E, BRAF, CRAF and MEK1, respectively.

[0145] "Also" or "further" means that the feature following either of these words is less preferred than the feature without this attribute.

[0146] As used herein, the terms "a", "an", "the", and similar references in the context of describing the present invention should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. When the plural form is used for compounds, salts, etc., this should also be understood as a single compound, salt, etc.

[0147] 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., reference to a compound or a pharmaceutically acceptable salt thereof provides a free compound, a pharmaceutically acceptable salt, or a mixture of the free compound and one or further salts thereof.

[0148] "About" and "approximately" shall generally indicate an acceptable degree of error in the quantity measured, taking into account the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%) of a given value or range of values, typically within 10%, and more typically within 5%. When a dose is described herein as "about" a particular amount, the actual dose may vary from the specified amount by up to 10%: This use of "about" indicates that the precise amount in a given dosage form may vary slightly from the expected amount for a variety of reasons without having a substantial effect on the in vivo effects of the administered compound.

[0149] It will be understood that when a numerical value is given, without the word "about" preceding it, there will be a certain degree of variation relative to the given value, as is generally accepted in the art; for example, the variation may be ±10%, such as ±5%, and in particular ±1%, respectively, of the given value.

[0150] As used herein, the terms "comprising" and "including" or "and" are used in their open and non-limiting sense, unless otherwise indicated, while "consisting of" limits the compounds or components to those specifically mentioned. A pharmaceutical combination consisting of naborafenib or a pharmaceutically acceptable salt, hydrate or solvate thereof and (b) trametinib or a pharmaceutically acceptable salt, hydrate or solvate thereof refers to a combination therapy in which these two components are the only pharmaceutically active ingredients; with or without one or more pharmaceutically acceptable carrier materials.

[0151] "Combination" or "combination of agents" (these terms are synonymous if not otherwise specified) or "combined with..." or "combination therapy" is not only intended to imply that the therapy or therapeutic agents must be physically mixed or administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope described herein. The therapeutic agents in these combinations (the term mainly refers to RAF inhibitors and / or MEK inhibitors herein, especially naborafenib and / or trametinib) can be further administered at the same time, before or after one or more other additional therapies or additional therapeutic agents, or in particular when there is no such additional therapy or additional therapeutic agent (active ingredient). The therapeutic agents can be administered in any order. Typically, each agent will be administered at a dose and / or schedule determined for the agent. It should be further understood that the additional therapeutic agents used in the combination can be administered together in a single composition and / or administered separately in different compositions, particularly when these time intervals allow the combination partners to exhibit cooperative (e.g., synergistic) effects, particularly based on Bliss data or other data confirming synergy. Typically, it is expected that the level of use of the additional therapeutic agent used in the combination does not exceed the level at which it is used alone. In some embodiments, the level of each relevant therapeutic agent = drug = active pharmaceutical ingredient (API) used in combination will be lower than the level used as a single agent treatment.

[0152] The term "synergistic effect" as used herein refers to the effect of a RAF inhibitor (particularly naborafenib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof) and a MEK inhibitor (particularly trametinib, a pharmaceutically acceptable salt, hydrate and / or solvate thereof) to produce an effect, such as a slowing of symptom progression of cancer or its symptoms, that is greater than the simple addition of the effects of each drug when administered alone or that requires a lower dose of either or both therapeutic agents.

[0153] The term "combination therapy" preferably refers to the administration of two or more therapeutic agents to treat the tumor disorders or conditions described in the present invention. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as with a single formulation having a fixed active ingredient ratio, or with a separate formulation (e.g., capsules, tablets, and / or other intravenous formulations) of each active ingredient. Additionally or alternatively, such administration also includes the use of each type of therapeutic agent in a sequential or separate manner (approximately at the same time or at different times). Whether the active ingredient is administered in a single formulation or in a separate formulation, the drug is administered to the same patient as part of the same course of treatment. In any case, the treatment regimen will provide a beneficial effect in treating the tumor disorders or conditions described herein.

[0154] Simultaneous therapeutic use within the meaning of the present invention refers in particular to the administration of at least two active ingredients by the same route and simultaneously or essentially simultaneously.

[0155] Within the meaning of the present invention, separate use refers in particular to the simultaneous or essentially simultaneous administration of at least two active ingredients by different routes or formulations.

[0156] Sequential therapeutic use refers in particular to administration of at least two active ingredients at different times, with the same or different administration routes or formulations. More specifically, an administration method refers to a method according to which all administration of one of the active ingredients is performed before starting administration of the other (or further other) active ingredient.

[0157] As used herein, the synonymous terms "fixed combination", "fixed dose" and "single formulation" preferably refer to a dosage form that is formulated to deliver to a patient an amount of two therapeutic agents that have a combined therapeutic effect for treating cancer. A single vehicle is designed to deliver an amount of each agent and any pharmaceutically acceptable carriers (excipients). In some embodiments, the vehicle is a tablet, capsule, pill, sachet or patch. In further embodiments, the vehicle is a solution or suspension.

[0158] The term combination also includes or alternatively includes "non-fixed combinations" or "kits of parts", which means that the therapeutic agents of the combination of the invention are all administered to a patient as separate entities simultaneously, concurrently or sequentially without specific time limits, particularly so that they have a combined therapeutic activity, wherein such administration provides therapeutically effective levels of both compounds in a patient in need thereof. The latter also applies to cocktail therapy, e.g., administration of three or more active ingredients.

[0159] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, excipients, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of patients (e.g., adult patients or human patients) without excessive toxicity, irritation, allergic response, and other problems or complications, commensurate with a reasonable and tolerable benefit / risk ratio.

[0160] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, diluents, disintegrants, glidants, lubricants, sweeteners, flavoring agents, dyes, etc. and combinations thereof as known to those skilled in the art. Except to the extent that any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0161] The term "pharmaceutical composition" (=pharmaceutical preparation) is defined herein to mean a mixture or solution containing at least one therapeutic agent for administration to a patient, such as a mammal or a human, to treat a specific disease or condition affecting the patient. The present pharmaceutical combination may be formulated into a suitable pharmaceutical composition for enteral or further parenteral administration, such as a film (e.g. sugar) coated tablet, tablet, capsule or suppository, sachet or ampoule. If not otherwise indicated, these are prepared in a manner known per se, such as by various conventional mixing, comminution, direct compression, granulation, (e.g. sugar) coating, dissolution, lyophilization processes or manufacturing techniques obvious to those skilled in the art. It should be understood that the unit content of the combination partner (therapeutic agent) contained in a single dose of each dosage form does not necessarily constitute an effective amount in itself, since the necessary effective amount can be achieved by administering a plurality of dosage units. The pharmaceutical composition may contain from about 0.1% to about 99.9%, preferably from about 0.1% to about 60% of any single or each therapeutic agent, with the sum totaling to 0.2% to 70% in the case of a fixed combination. One of ordinary skill in the art can select one or more of the above-mentioned carriers according to the specific desired characteristics of the dosage form by routine experiments, and without any unnecessary burden. The amount of each carrier used can vary within the conventional range of the art. The following references disclose techniques and excipients for preparing oral dosage forms: The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., ed., 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 can be incorporated into the initial mixture by incorporating one or more conventional carriers into the initial mixture before or during granulation, or by incorporating one or more conventional carriers into the oral dosage form with a single therapeutic agent combination of particles containing a therapeutic agent combination or a therapeutic agent combination in an oral dosage form. In the latter embodiment, the combined mixture can be further stirred, for example, by a V-blender, and then compressed or molded into a tablet, such as a single tablet, encapsulated in a capsule or filled in a sachet.

[0162] When "%" is mentioned, this refers to percent by weight unless otherwise stated.

[0163] A specific formulation concerns film-coated tablets of the active ingredients used in the present combination therapy alone or in a further fixed combination preparation.

[0164] The pharmaceutical combinations and compositions of the present invention may include a "therapeutically effective amount" or "effective amount" of a RAF inhibitor (particularly naborafenib) and a MEK inhibitor (particularly trametinib). The term "pharmaceutically effective amount", "therapeutically effective amount" or "clinically effective amount" of a therapeutic combination of agents refers to an amount sufficient to provide an observable or clinically significant improvement in the clinically observable signs and symptoms of the condition treated by the combination, at a dose and for a necessary period of time. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual. The therapeutically effective amount is also the amount in which any toxic or deleterious effects of the therapeutic agent are offset by the beneficial effects of the treatment. The "therapeutically effective dose" 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 can be assessed in an animal model system that predicts the efficacy of human sarcomas to help determine appropriate dosage levels and schedules. Alternatively, this property of the composition can be assessed by using in vitro assays known to skilled practitioners or, in particular, by examining the ability of the compound to modulate an undesirable parameter in a clinical study of a patient (particularly a patient in need of treatment).

[0165] Preferably, according to the present invention, in the context of the treatment of human patients, the following dosage combinations are used for a RAF inhibitor (especially naborafenib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof) and a MEK inhibitor (especially trametinib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof), in each case referring to the free form of the respective API:

[0166] The unit dose of active ingredient may be administered once daily, twice daily, three times daily or four times daily, totaling to produce an appropriate daily dose, for example as shown below, the actual dose used and the time of administration being determined by criteria such as: the age, weight and sex of the patient; the extent and severity of the cancer to be treated; and the judgment of the treating physician. Oral dosage forms, especially (preferably film-coated) tablets are preferred.

[0167] When a dose or dose is mentioned herein, the amount referred to is the amount of the therapeutic agent (active ingredient, here in free form). For example, when a 2 mg dose of trametinib is administered, and trametinib is administered as a tablet containing trametinib dimethyl sulfoxide, the tablet will contain trametinib dimethyl sulfoxide equivalent to 2 mg of trametinib in free form. Corresponding reasoning is valid for naborafenib monohydrate.

[0168] RAF inhibitors are preferably administered orally. In a preferred embodiment, RAF inhibitors, in particular naborafenib, or a pharmaceutically acceptable salt thereof, are administered at a total daily dose (TTD) of about 100 mg to about 1200 mg, respectively. The total daily dose of naborafenib is administered daily in one or more dosage units. The total daily dose of naborafenib is preferably administered twice daily. Therefore, naborafenib can be administered twice daily in an amount of about 100 mg to about 600 mg.

[0169] In some embodiments, naborafenib is administered once daily (QD) (preferably orally) at a dose of about 100 mg, 125 mg, 150 mg, 175 mg, 200 mg (preferably), 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg or in particular 400 mg, respectively.

[0170] In certain embodiments, naborafenib or a pharmaceutically acceptable salt, hydrate and / or solvent thereof is orally administered twice daily (BID or bid) at a dose of about 200 mg or 400 mg, based on the free form.

[0171] According to the present invention, a MEK inhibitor as part of a combination or combination therapy will be administered to a subject in need thereof in a therapeutically effective amount.

[0172] In a preferred embodiment, the MEK inhibitor trametinib or a pharmaceutically acceptable salt, hydrate or solvate thereof administered in a subject in need as part of a combination according to the present invention will be an amount selected from about 0.125 mg to about 10 mg / day; suitably, the amount will be selected from about 0.5 mg to about 2 mg / day; suitably, the amount will be about 1 mg or 2 mg / day. In a preferred embodiment, trametinib, trametinib or a pharmaceutically acceptable salt or solvate thereof is administered at a daily dose of 0.5 mg or 1 mg / day (especially 1 mg / day). Preferably, the dose / day (daily dose) is administered once a day (qd).

[0173] Most preferably, the Raf inhibitor (especially naborafenib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof) is administered at a dose of 400 mg BID and the MEK inhibitor (especially trametinib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof) is administered at a dose of 0.5 mg QD, based on the free form, respectively; or yet more preferably, the Raf inhibitor (especially naborafenib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof) is administered at a dose of 200 mg BID and the MEK inhibitor (especially trametinib, or a pharmaceutically acceptable salt, hydrate and / or solvate thereof) is administered at a dose of 1.0 mg QD.

[0174] In some embodiments, trametinib or a pharmaceutically acceptable salt or solvate thereof is administered orally. In one embodiment, trametinib is prepared for oral administration and can be used in a solvated form in dimethyl sulfoxide. In some embodiments, the compound is prepared in tablet form for oral administration, especially film-coated tablets. Tablets can be prepared in a variety of doses for flexible administration.

[0175] The unit dose of trametinib or a pharmaceutically acceptable salt or solvate thereof can be administered once a day, twice a day, three times a day, or four times a day. The total daily dose of trametinib or a pharmaceutically acceptable salt or solvate thereof (e.g., dimethyl sulfoxide solvate) can be administered once a day or further twice a day.

[0176] For example, as part of a combination therapy, a compound of formula (I) or a pharmaceutically acceptable salt thereof, such as a monohydrate of the compound or a preferred free form, can be administered at a total daily dose of about 200 mg, about 300 mg, about 400 mg or about 500 mg, and trametinib, such as a dimethyl sulfoxide solvate form, can be administered at a total daily dose of about 0.5 or 1.0 mg. The daily dose of the compound of formula (I) can be administered once or twice a day. Therefore, a compound of formula (I) at a dose of about 200 mg can be administered twice a day (total daily dose of about 400 mg), and trametinib at a dose of about 1.0 mg or about 2.0 mg can be administered once a day. Alternatively, a compound of formula (I) at a dose of about 200 mg can be administered twice a day (total daily dose of about 400 mg), and trametinib at a dose of about 0.5 or about 1.0 mg can be administered twice a day.

[0177] A further benefit may be that lower doses of the therapeutic agents of the combination of the invention may be used, for example, so that the dose may not only be often smaller, but may also be applied less frequently, or may be used to reduce the incidence of side effects observed when one of the combination partners is used alone. This is in line with the wishes and requirements of the patient to be treated.

[0178] The term "combined therapeutic activity" or "combined therapeutic effect" as used herein means that the therapeutic agents can be administered jointly, separately or sequentially within such a time interval that the patient to be treated, especially a human, still exhibits a (preferably synergistic) interaction (combined therapeutic effect). Whether this is the case can be determined, in particular, by following the blood levels of the compounds, indicating that both compounds are present in the blood of the human to be treated at least within a certain time interval, or by the fact that the subsequent treatment leads to a better result than a monotherapy with only one of the active ingredients.

[0179] The term "active ingredient" or "therapeutic agent" includes a RAF inhibitor and a MEK inhibitor as defined in combination according to embodiments of the present invention.

[0180] In one embodiment, the disease or condition suitable for or to be treated with the combination therapy according to the present invention is cancer. The term "cancer" is used herein to represent a tumor selected from a wide spectrum of tumors, including all solid tumors. Cancer can be in the early, middle or late stages. Cancer can be locally advanced or metastatic. "Cancer" or "tumor" are used synonymously herein, and these identifiers are equivalent to "tumor disease" or "cancer disease", and in the case of solid tumors, late stage tumors / tumor diseases are particularly provided.

[0181] The cancer to be treated by the combination therapies described herein may have progressed following standard of care or for which no effective standard therapy exists.

[0182] In one embodiment, the solid tumor is non-benign. In one embodiment, the solid tumor is selected from melanoma, or more particularly any solid tumor other than melanoma, selected from lung cancer (especially non-small cell lung cancer (NSCLC)), colorectal cancer (CRC) (e.g., colorectal adenocarcinoma), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PADC)), or further selected from head and neck cancer (e.g., head and neck squamous cell carcinoma), bladder urothelial carcinoma, MUTYH-associated polyposis (MAP), cervical cancer and ovarian cancer, esophageal cancer, renal cancer, mesothelioma, thyroid cancer, prostate cancer, glioblastoma, cervical cancer, thymic cancer, Merkel cell carcinoma, or further sarcoma, such as rhabdomyosarcoma.

[0183] In one embodiment, the tumor is melanoma.

[0184] In one embodiment, the tumor is lung cancer, such as non-small cell lung cancer (NSCLC).

[0185] In one embodiment, the tumor is colorectal cancer.

[0186] In one embodiment, the tumor is pancreatic cancer.

[0187] In one embodiment, the tumor is head and neck cancer.

[0188] In one embodiment, the tumor is bladder urothelial carcinoma.

[0189] In one embodiment, the proliferative disease is colorectal cancer (CRC), including MUTYH-associated polyposis (MAP).

[0190] In one embodiment, the tumor is cervical cancer.

[0191] In one embodiment, the tumor is ovarian cancer.

[0192] In one embodiment (particularly for the G13R mutation), the tumor is rhabdomyosarcoma.

[0193] Solid tumors to be treated include NRAS mutant melanomas. Where a melanoma is mentioned, this particularly relates to "NRAS mutant melanomas". Where a "NRAS mutant melanoma" is mentioned, this particularly relates to ocular or cutaneous melanomas - unless the context clearly dictates otherwise. Preferably, the term refers to cutaneous melanomas, in particular unresectable and / or metastatic cutaneous melanomas.

[0194] The present invention provides a combination of agents for use in treating melanoma in a patient, wherein the melanoma has been previously treated, such as by surgical resection, or other therapy, and has progressed following such therapy.

[0195] The melanoma to be treated by the combination may be a melanoma that is refractory or resistant to previous treatment with another therapy. The combination of the invention may therefore be used in the second, third or fourth line treatment of melanoma.

[0196] Previous treatments for patients with melanoma included:

[0197] - Treatment with talimogene laherparepvec (also known by the trade names T-Vec, Imlygic or Oncovex), a biopharmaceutical drug for the treatment of unresectable melanoma that can be injected directly into a subset of metastatic lesions;

[0198] - Standard of care chemotherapy (e.g., dacazabine);

[0199] - Treatment with cytotoxic agents such as nitrosoureas and / or mitomycin C;

[0200] - Immunotherapy (eg, pembrolizumab, ipilimumab, or nivolumab, and combinations thereof);

[0201] - Targeted therapies (e.g., dabrafenib and trametinib, vemurafenib and cobimetinib, and enisefenib and bemetinib).

[0202] Therefore, patients to be treated include patients with NRAS mutant melanoma, especially those who have received previous therapies, including standard of care chemotherapy (e.g., dacazabine), immunotherapy (e.g., pembrolizumab, ipilimumab or nivolumab and combinations thereof), targeted therapy (e.g., dabrafenib and trametinib, vemurafenib and cobimetinib and conafenib and bemetinib) and patients whose disease progresses after previous therapies. Patients can be patients with NRAS mutant melanoma, especially in the case where the melanoma is an unresectable and / or metastatic skin melanoma.

[0203] In another embodiment, the NRAS mutant melanoma is resistant or refractory to standard of care.

[0204] In another embodiment, the NRAS mutant melanoma is resistant or refractory to standard of care with dacarbazine.

[0205] In another embodiment, the melanoma is resistant or refractory to treatment with cytotoxic agents such as nitrosoureas and / or mitomycin C.

[0206] In another embodiment, the melanoma is resistant or refractory to immunotherapy treatment, including therapy with one or more immune checkpoint inhibitors.

[0207] Thus in one embodiment, the melanoma to be treated by the combination of the invention is a NRAS mutant melanoma that is resistant to immunotherapy PD-1 (programmed cell death 1 receptor) or PD-L1 (ligand for PD-1) treatment, either 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.

[0208] Thus in one embodiment, the melanoma to be treated (including NRAS mutant melanoma) is resistant or refractory to treatment with one or more therapeutic agents selected from ipilimumab, spartalizumab, nivolumab, pembrolizumab, pidizilumab, BMS-9365559, MEDI4736, and MSB0010718C.

[0209] Therefore, melanomas to be treated by 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 and ipilimumab.

[0210] Genetic assessment of patients for BRAF, NRAS, and NF1 mutations can be performed according to methods known in the art, for example, using the previously described SNaPshot or DFCI Oncopanel (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, whereas earlier versions of SNaPshot relied on multiplex PCR. The current version of SNaPshot detects exons 11 and 15 of BRAF, exons 2–5 of KRAS and NRAS, and exons 1–58 of NF1. Oncopanel detects alterations in all exons of BRAF, KRAS, NRAS, and NF1.

[0211] The object with cancer / tumor is defined as when the cancer he or she suffers from progresses, progresses on the therapy of one or more agents or no longer responds to it, or is intolerant to one or more agents. The progress of cancer can be monitored by methods well known to those skilled in the art. For example, progress can be monitored by visual inspection of cancer, such as by the method of X-ray, CT scan or MRI or by tumor biomarker detection. For example, the growth of cancer increases and indicates the progress of cancer. The progress of cancer (such as NSCLC) or tumor can be indicated by detecting a new tumor or detecting metastasis or tumor shrinkage. Tumor assessment can be based on RECIST standards (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 is carried out. For example, to show the therapeutic effect, the overall response rate (ORR) according to Response Evaluation Criteria in Solid Tumors (RECIST v1.1) or any other parameter may be used.

[0212] A complete response was defined as the disappearance of all non-nodal target lesions. In addition, any pathological lymph node designated as a target lesion had to be reduced to <10 mm in short axis.

[0213] A partial response was defined as at least a 30% reduction in the sum of the diameters of all target lesions, with the baseline sum of diameters as reference.

[0214] Other response criteria can be derived from the following table (based on RECIST v1.1):

[0215]

[0216] The progression of the tumor, the increase or decrease of the tumor burden, and the response to the treatment with the inhibitor combination according to the present invention can be monitored by methods well known to those skilled in the art. Thus, the progression and the response to treatment can be monitored by visual inspection of the tumor (e.g., by means of X-ray, CT scan or MRI) or by tumor biomarker detection.

[0217] Tumor progression can be determined by comparing tumor status between various time points after the start of treatment or by comparing tumor status between a time point after the start of treatment and a time point before the start of the relevant treatment.

[0218] The combinations and combination therapies according to the invention are particularly suitable for the treatment of cancers containing alterations in one or more mitogen-activated protein kinase (MAPK) pathways, including HRAS or especially KRAS mutant tumors and / or NRAS mutant tumors, and in particular tumors expressing at least one RAS (especially KRAS or NRAS) gain-of-function mutation, as described herein, selected from codon Q61, such as Q61H, Q61K mutations or (especially) G13R mutations, not limited to but also applicable to at least one RAF gain-of-function mutation.

[0219] In another embodiment, the cancer is resistant or refractory to treatment with a MEK inhibitor (eg, trametinib).

[0220] As mentioned above, the present invention is based on the important discovery that certain RAS mutations, in particular codon Q61 or G13R mutations, are biomarkers for suitability of tumor patients for combined treatment with RAF and MEK inhibitors.

[0221] Where a RAS (e.g. KRAS, HRAS or NRAS) (codon) mutation is mentioned herein, this relates on the one hand to the corresponding point mutation in a polynucleotide (e.g. a gene, cDNA or mRNA) and on the other hand to the corresponding mutated protein having an altered amino acid sequence at said position (Q61 or G13R).

[0222] Suitable assays, methods and reagents (especially binding agents that bind to the biomarker to be identified) allowing screening, detection or identification of the presence of RAS (eg KRAS) codon Q61 or G13R mutations are in principle known and available to the person skilled in the art.

[0223] "KRAS activating mutation" refers to any mutation of the KRAS gene (ie, nucleic acid mutation) or Kras protein (ie, amino acid mutation) that results in abnormal Kras protein function associated with increased and / or constitutive activity by favoring the active GTP-bound state of the Kras protein. The mutation is located at a conserved site that favors GTP binding and constitutively active Kras protein. The mutation used in accordance with the present invention is located at certain codons of the KRAS gene encoding KRAS, including KRAS-G13R (particularly preferred), or KRAS-Q61E, KRAS-Q61P, KRAS-Q61H, KRAS-Q61K (preferred), KRAS-Q61L (preferred) or KRAS-Q61R (preferred).

[0224] "NRAS activating mutation" is any mutation of the NRAS gene (i.e., nucleic acid mutation) or Nras protein (i.e., amino acid mutation) that results in abnormal Nras protein function associated with increased and / or constitutive activity by favoring the active GTP binding state of the Nras protein. The mutation is located at a conserved site that favors GTP binding and constitutively active Nras protein. The mutation used according to the present invention is located at certain codons of the NRAS gene encoding KRAS, including NRAS-G13R mutation (particularly preferred), or NRAS-Q61E, NRAS-Q61P, NRAS-Q61H, NRAS-Q61K (preferred), NRAS-Q61L (preferred) or NRAS-Q61R (preferred) mutations.

[0225] Methods for identifying RAS codon Q61 or RAS G13R mutations are known in the art, for example by using "next generation sequencing". Agents or probes capable of identifying RAS codon Q61 or RAS G13R mutations are known in the art and can preferably be selected from antibodies and polynucleotides, each capable of binding to the mutation site, whether in the expressed protein or, preferably, in the gene DNA, cDNA or mRNA encoding it.

[0226] The term "antibody" herein is used in the broadest sense and covers various 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.

[0227] "Polynucleotide" or "nucleic acid" (these terms are used as synonyms herein) refers to a polymer of nucleotides of any length, and includes one or both of DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by a 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 comprising single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA comprising single-stranded and double-stranded regions, including hybrid molecules of DNA and RNA, which may be single-stranded, or more typically double-stranded, or comprise single-stranded and double-stranded regions. For nucleic acid or polynucleotide mutations used according to the invention (= mutations that allow prediction of the suitability of a tumor (and therefore a patient) for successful combined treatment with a RAF inhibitor and a MEK inhibitor, DNA and RNA, in particular cDNA or mRNA, are preferred.

[0228] If present, in particular for polynucleotides (especially oligonucleotides) or nucleic acids used according to the invention for identifying mutations in RAS DNA or RNA in a tumor sample (e.g. as probes or primers), the nucleotide structure may be modified before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. Such polynucleotides may be further modified after synthesis, for example by incorporation of (e.g. fluorescent or isotopic) labels. Other types of modifications include, for example, "capping", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications, for example, modifications with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidites, or carbamates) and / or modifications with charged linkages (e.g., phosphorothioates or phosphorodithioates), modifications containing (covalently or otherwise bound) linking moieties (e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, or poly-L-lysine)), modifications with intercalators (e.g., acridine or psoralen), modifications containing chelators (e.g., metals, radioactive metals, boron, or oxidized metals), modifications containing alkylating agents, modifications with modified linkages (e.g., alpha anomeric nucleic acids), and unmodified forms of polynucleotides. Any one or more hydroxyl groups typically present in the sugar of the nucleotide backbone may be replaced, for example, by a phosphonate group, a phosphate group, protected by conventional protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to a solid or semisolid support. The terminal OH can be phosphorylated or partially replaced by an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyls can also be derived as standard protecting groups. These polynucleotides can also contain similar forms of ribose or deoxyribose generally 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, furanose, sedoheptulose, acyclic analogs and alkaline nucleoside analogs (such as methyl nucleosides). One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), "(O)NR2 ("amide"), P(O)R, P(O)OR', CO or CH2 ("acetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally containing ether (-O-) linkages, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. All linkages in a polynucleotide need not be identical. Such a polynucleotide may contain one or more different types of modifications described herein and / or multiple modifications of the same type. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0229] As used herein, "oligonucleotide" generally refers to a short single-stranded polynucleotide, which is generally (but not necessarily) less than about 250 nucleotides in length, such as 2 to 80, such as 2 to 50 nucleotides in length. Oligonucleotides, especially when used as probes (e.g., primers), can be synthetic.

[0230] The term "primer" refers to a single-stranded oligonucleotide or polynucleotide that is capable of hybridizing to a nucleic acid, particularly DNA or RNA from a tumor sample, and allows polymerization of a complementary nucleic acid, typically by providing a free 3'-OH group.

[0231] The term "biomarker" as used herein refers to any part of a molecule in a patient that provides evidence of a RASG13R (particularly preferred), Q61E, Q61P, Q61H, Q61K, Q61L or Q61R mutation. A biomarker can be a predictive biomarker and, as an indicator of the likelihood of suitability (=sensitivity or benefit) of a patient with a solid tumor disease whose tumor contains one of the mentioned RAS mutations, allows prediction of whether the patient is suitable for treatment with a combination of a RAF (e.g., a Raf inhibitor, such as a Raf dimer) and a MEK inhibitor. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA (e.g., mRNA)), polynucleotide copy number changes (e.g., DNA copy number), polypeptides (e.g., corresponding Ras polypeptides), modified polypeptides or polynucleotides (e.g., with post-translational modifications). In some embodiments, as cited above or below, a biomarker is a gene (e.g., a KRAS or NRAS gene) containing a mutation of interest.

[0232] In particular, when DNA or RNA is to be detected for the presence of a biomarker (mutation of interest according to the invention) in a solid tumor or patient, after isolating the DNA or RNA, preferably at least the region of its sequence (template sequence) containing the mutation of interest is amplified.

[0233] Patients can be prospectively or retrospectively genetically evaluated for RAS mutations according to methods known in the art, such as using the previously described SNaPshot or DFCI Oncopanel (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 detects exons 2-5 of KRAS and NRAS, and Oncopanel detects changes involving all exons of RAS (especially KRAS, NRAS).

[0234] As used herein, "amplifying" or "amplification" generally refers to the process of producing multiple copies of a desired nucleotide sequence. The "copies" generally have (but further do not necessarily need to have) perfect sequence complementarity or identity with the template sequence. For example, the copies may include nucleotide analogs (e.g., deoxyinosine), intentional sequence changes (e.g., sequence changes introduced by primers containing sequences that can hybridize to the template but are not complementary), and / or sequence errors that occur during the amplification process.

[0235] According to the present invention, identifying preferred variants of a mutation of interest comprises a polymerase chain reaction step.

[0236] As used herein (most importantly and most preferred herein) "polymerase chain reaction" or "PCR" technology generally refers to a procedure for amplifying trace amounts of specific nucleic acid segments, RNA and / or DNA, such as described in: U.S. Patent 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 Edition, 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). Typically, sequence information from the region of interest is required so that oligonucleotide primers can be designed whose sequences are identical or similar to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers can coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, etc. Any nucleic acid polymerase reaction method for amplifying nucleic acid detection samples can be used, including using known nucleic acids (DNA or RNA) as primers and using nucleic acid polymerases to amplify or generate specific nucleic acid fragments, or amplify or generate specific nucleic acid fragments complementary to specific nucleic acids.

[0237] The term "multiplex PCR" refers to the use of more than one primer set to perform a single PCR reaction on nucleic acid obtained from a single source (eg, a solid tumor sample from one individual) with the goal of amplifying two or more DNA sequences in a single reaction.

[0238] "Quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a special form of PCR that can measure the amount of PCR product at each step in the PCR reaction as needed. The technology has been described in a variety of publications, including, for example, Cronin et al., Am. J. Pathol. 164 (1): 35-42 (2004) and Ma et al., Cancer Ce / / 5: 607-616 (2004).

[0239] The disclosed methods and assays provide a convenient, efficient, and potentially cost-effective means for obtaining data and information that can be used to evaluate appropriate or effective therapies for treating 40 patients. For example, a patient can provide a tumor sample as a tissue sample (e.g., a tumor biopsy) before a combination therapy of a RAF inhibitor (e.g., a RAF inhibitor) in combination with a MEK inhibitor is considered a treatment option, and the sample can be examined by various in vitro assays to determine whether the patient's cells are sensitive to the combination therapy.

[0240] In any of the foregoing methods, the specific mutation status of HRAS or particularly KRAS and / or NRAS genes, mRNA or protein products in a sample obtained from an individual can be identified by any one or more methods well known to those skilled in the art. For example, the identification of mutations can be achieved by cloning HRAS, particularly KRAS and / or particularly NRAS genes or portions thereof and sequencing them using techniques well known in the art. Alternatively, gene sequences can be amplified from genomic DNA or cDNA, for example using PCR, and the products are sequenced. Several non-limiting methods for analyzing mutations in patient DNA at a given genetic locus are described below.

[0241] DNA microarray technology can be used, such as DNA chip devices and high-density microarrays and low-density microarrays for high-throughput screening applications, such as for DNA microarray hybridization. Other methods include interfering RNA microarrays and combinations of microarrays with other methods, such as 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.

[0242] Other methods include PCR, xMAP, invader assay, mass spectrometry, and pyrosequencing (Wang et al. (2007) 593: 105-106).

[0243] Another detection method is allele-specific hybridization, using a probe that overlaps the polymorphic site and has about 5, or alternatively 10, or alternatively 20, or alternatively 25, or alternatively 30 nucleotides around the region where the mutation site is sought. For example, several probes that can specifically hybridize to a specific mutation variant (e.g., KRAS-G13R, which corresponds to a KRAS C.38G>A nucleotide substitution point mutation) are attached to a solid support (e.g., a "chip").

[0244] Oligonucleotides can be bound to solid supports by a variety of methods, including photolithography. Mutation detection assays using these chips (also referred to as "DNA probe arrays") containing oligonucleotides are described in, for example, Cronin et al. (1996) Human Mutation 7:244.

[0245] In other detection methods, at least a portion of the gene must first be amplified before identifying mutant variants. Amplification can be performed, for example, by PCR and / or LCR or other methods well known in the art.

[0246] In some cases, the presence of a specific mutation in the subject's DNA can be shown by restriction enzyme analysis. For example, a specific mutation can result in a nucleotide sequence that includes a restriction site that is not present in the nucleotide sequence of another mutant variant or the wild-type version of the gene.

[0247] In another embodiment, protection against cleavage agents (e.g., nucleases, hydroxylamine or osmium tetroxide and piperidine) can 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 "mismatch cleavage" technique first provides a heteroduplex formed by hybridization of a control nucleic acid with a sample nucleic acid (e.g., RNA or DNA) obtained from a tumor sample, the control nucleic acid being optionally labeled, such as RNA or DNA, comprising the nucleotide sequence of an allelic variant of a gene. The double-stranded duplex is treated with an agent that cleaves the single-stranded region of the duplex (e.g., a duplex formed based on a base pair mismatch between the control strand and the sample strand). For example, the RNNDNA duplex can be treated with RNase, and the DNNDNA hybrid can be treated with S1 nuclease to enzymatically digest the mismatched region. Alternatively, DNA / DNA or RNNDNA duplexes can be treated with hydroxylamine or osmium tetroxide and piperidine to digest the mismatched regions. After digesting the mismatched regions, the resulting material is separated by size on a denaturing polyacrylamide gel to determine whether the control and sample nucleic acids have the same nucleotide sequence or at which nucleotides they differ. 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.

[0248] In addition, changes in electrophoretic mobility can also be used to identify specific allelic variants. For example, single-stranded conformation polymorphism (SSCP) can be used to detect electrophoretic mobility differences 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 acid are denatured and allowed to renature. The secondary structure of single-stranded nucleic acid varies according to the sequence, and the resulting change in electrophoretic mobility makes it possible to detect changes in even single bases. DNA fragments can be labeled or detected with labeled probes. The sensitivity of the assay can be enhanced by using RNA (rather than DNA) because the secondary structure of RNA is more sensitive to sequence changes. In another embodiment, the subject methods utilize heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

[0249] The identity of the mutant variant can also be determined by analyzing the movement of nucleic acids containing the polymorphic region in a polyacrylamide gel containing a gradient of denaturing agents, which is determined using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as an analytical method, the DNA will be modified to ensure that it is not completely denatured, for example by adding a GC clamp of about 40 bp of high melting point GC-rich DNA by PCR. In another embodiment, a temperature gradient is used instead of a denaturing agent gradient to identify differences in mobility between control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:1275).

[0250] 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 can be prepared in which a known polymorphic nucleotide is located at the center (allele-specific probe), and then hybridized with a target DNA under conditions that allow hybridization only when a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele-specific oligonucleotide hybridization techniques can be used to detect nucleotide changes in polymorphic regions of a gene. For example, an oligonucleotide having a nucleotide sequence of a specific allele variant is attached to a hybridization membrane, and then the membrane is hybridized with a labeled sample nucleic acid. Then, analysis of the hybridization signal will reveal the nucleotide identity of the sample nucleic acid.

[0251] Alternatively, allele-specific amplification techniques that rely on selective PCR amplification may be used in conjunction with the present invention.

[0252] Oligonucleotides used as specific amplification primers can contain the allelic variant of interest in the center of the molecule (so amplification depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17: 2437-2448) or at the extreme 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 known as "PROBE", i.e. probe oligonucleotide base extension. In addition, it may be necessary to introduce a new restriction site in the mutant region to create a cleavage-based assay (Gasparini et al. (1992) Mot. Cell. Probes 6: 1).

[0253] In another embodiment, oligonucleotide ligation assay (OLA) is used to identify mutant variants, such as U.S. Patent No. 4,998,617 and Laridegren, U. et al. Science 241: 1077-1080 (1988). The OLA scheme uses two oligonucleotides, which are designed to hybridize with the adjacent sequence of the single strand of the target. One of the oligonucleotides is connected to a separation marker (e.g., biotinylated), and the other oligonucleotide is detectably labeled. If an accurate complementary sequence is found in the target molecule, the oligonucleotides will hybridize so that their ends are adjacent and a connection substrate is generated. Then, the connection allows the use of avidin or another biotin ligand to recover the labeled oligonucleotides. Nickerson et al. describe a nucleic acid detection assay (Nickerson, DA et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8923-8927) that combines PCR and OLA attributes. In this method, PCR is used to achieve exponential amplification of target DNA, which is then detected using OLA.

[0254] Methods for detecting single nucleotide mutations in a given gene (e.g., a RAS gene, such as a KRAS gene or a NRAS gene or a HRAS gene) are described. Since single nucleotide changes are flanked by invariant sequence regions, analyzing them only requires determining the identity of a single variant nucleotide, without determining the complete gene sequence of each patient. Several methods have been developed to facilitate the analysis of such mutations.

[0255] Single base mutations can be detected using specialized exonuclease-resistant nucleotides, such as those disclosed in U.S. Pat. No. 4,656,127. According to this method, a primer complementary to the mutant sequence at the 3' position of the mutation site is allowed to hybridize with a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific exonuclease-resistant nucleotide derivative present, the derivative will be incorporated into the end of the hybridization primer. This incorporation makes the primer resistant to exonucleases and thereby allows its detection. Since the identity of the exonuclease-resistant derivative of the known sample, the discovery that the primer is resistant to exonucleases indicates that the nucleotides present in the mutation site of the target molecule are complementary to the nucleotides of the nucleotide derivative used in the reaction. The advantage of this method is that it does not require the determination of a large amount of irrelevant sequence data.

[0256] A solution-based method can also be used to determine the identity of the nucleotide at the mutation site (WO 91 / 02087). As described above, a primer complementary to the mutant sequence at the 3' position of the mutation site is used. The method uses a labeled dideoxynucleotide derivative to determine the identity of the nucleotide at the site, and if the nucleotide is complementary to the nucleotide at the mutation site, it will be incorporated into the end of the primer.

[0257] An alternative method is described in WO 92 / 15712. This method uses a mixture of labeled terminators and primers complementary to the sequence at the 3' position of the mutation or polymorphism site. Therefore, the incorporated labeled terminator is determined by the nucleotides present in the mutation site of the target molecule being evaluated and is complementary to it. This method is usually a heterogeneous assay, in which the primers or the target molecule are fixed on a solid phase.

[0258] Many other primer-directed nucleotide incorporation procedures for determining mutation sites in DNA have been described (Komher, JS et al. (1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, BP (1990) Nucl. Acids Res. 18:3671; Syvanen, A.-C., et al. (1990) Genomics 8:684-692; Kuppuswamy, MN et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147; Prezant, TR et al. (1992) Hum. Mutat. 1:159-164; Ugozzoli, L. et al. (1992) GATA 9:107-112; Nyren, P. et al. (1993) Anal. Biochem. 208:171-175). These methods all rely on the incorporation of labeled deoxynucleotides to distinguish the base at the mutation site.

[0259] In general, the presence and / or amount of the biomarker genes described herein can be analyzed by a variety of methods, including the above methods and many other methods known in the art and understood by those skilled 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 type 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"), and any of the various assays that can be performed by protein, gene and / or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are found in, for example, Ausubel et al., ed., 1995, Current Protocols In Molecular Biology, Unit 2 (Northern Blotting), Unit 4 (Southern Blotting), Unit 15 (Immunoblotting), and Unit 18 (PCR Analysis).

[0260] Multiplex immunoassays, such as those provided by Rules Based Medicine or Meso Scale Discovery ("MSD"), may also be used.

[0261] In addition, the presence and / or amount of the biomarker proteins (i.e., gene products) described herein can be analyzed by a variety of methods, including immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, spectroscopy, molecular binding assays, HPLC, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofiltration assays (ELIFA), fluorescence-activated cell sorting ("FAGS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescence in situ hybridization (FISH) and protein sequencing. In some cases, 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 biomarker binding, and detecting whether a complex is formed between the antibody and the biomarker. This method can be an in vitro or in vivo method. In some cases, the protein expression level of a biomarker (e.g., KRAS-G13R protein or NRAS protein with an activating mutation (e.g., mutation described above)) is determined in tumor cells (e.g., from a biopsy).

[0262] Furthermore, it will be appreciated that any of the methods described above for detecting mutations in a gene or gene product can also be used to monitor the course of a treatment or therapy (eg, a combination therapy comprising a RAF inhibitor (eg, a Raf inhibitor) and a MEK inhibitor).

[0263] The methods described herein can be performed, for example, by using a prepackaged diagnostic kit (e.g., the kit described below) comprising at least one probe or primer nucleic acid that can be conveniently used, for example, to determine whether a subject is likely to benefit from a combination therapy comprising a RAF inhibitor (e.g., a Raf inhibitor) and a MEK or PI3K inhibitor.

[0264] In all screening methods cited above or herein, mutations in one or more RAS genes (e.g., HRAS, KRAS or NRAS) or their protein products can generally be identified by determining a nucleic acid sequence (e.g., DNA or RNA sequence) or protein sequence (i.e., amino acid sequence) in a tumor sample obtained from an individual (= patient) and comparing the sequence to a reference sequence (e.g., wild-type sequence). In some cases, a reference level, a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue is a single sample or a combination of multiple samples from the same subject or individual, which are obtained at one or more time points different from when the test sample was obtained. For example, a reference level, a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue is obtained from the same subject or individual at an earlier time point than when the test sample was obtained. If a reference sample is obtained during the initial diagnosis of cancer and a test sample is obtained later when the cancer becomes metastatic, such a reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful.

[0265] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0266] See also the Examples for more details on the data and information presented in the figures.

[0267] The following examples illustrate the above invention; however, they are not intended to limit the scope of the invention in any way.The beneficial effects of the pharmaceutical combination of the present invention can also be determined by other test models known to those skilled in the art.

[0268] Example

[0269] The presentation of the following examples is intended to aid in the understanding of the present invention, but is not intended to, and should not be construed to, limit the scope thereof in any way.

[0270] Example 1: Analysis of data obtained in study CLXH254X2102 (see https: / / clinicaltrials.gov / ct2 / show / NCT02974725)

[0271] As of May 2021, 94 patients with KRAS / BRAF-mutant NSCLC and NRAS-mutant melanoma have been treated in study CLXH254X2102 with naborafenib (on a bid schedule) combined with trametinib (on a qd schedule) at the following dose levels: 200 mg naborafenib and 1 mg trametinib (39 patients), 400 mg naborafenib and 1 mg trametinib (5 patients), 200 mg naborafenib and 0.5 mg trametinib (7 patients), 400 mg naborafenib and 0.5 mg trametinib (37 patients), and 400 mg naborafenib and 1 mg trametinib, 2 weeks on and 2 weeks off (6 patients). The RDE for the combination of naborafenib and trametinib was determined to be naborafenib 400 mg bid + trametinib 0.5 mg qd and naborafenib 200 mg bid + trametinib 1 mg qd.

[0272] As of September 1, 2021, efficacy data for NRAS-mutant melanoma were available for 30 patients in the expansion portion; 15 patients were treated with nabolafenib 200 mg bid and trametinib 1 mg qd, and 15 patients were treated with nabolafenib 400 mg bid and trametinib 0.5 mg qd. Overall, patients had received up to 7 lines of therapy, with 6 (40%) patients in the 200mg / 1mg group having received first-line therapy and 4 (26.7%) patients in the 400mg / 0.5mg group having received up to 1 line of therapy. Confirmed ORRs were 46.7% and 13.3% in the 200mg / 1mg and 400mg / 0.5mg groups, respectively, and mPFS was 5.52 and 4.44 months, respectively. Of the 30 patients with NRAS mutant melanoma, only 3 did not have a confirmed Q61 or G13R codon mutation; these 3 patients did not have a confirmed complete or partial response. See Table 1:

[0273] Table 1: Full analysis set of patients with NRAS melanoma expansion stage treated with LXH254 (LXH) + trametinib (TMT) based on best overall response according to RECIST 1.1, based on investigator assessment and cohort confirmation

[0274]

[0275] N: Total number of subjects in the treatment group (denominator for percentage (%) calculation)

[0276] n: number of patients belonging to the corresponding category.

[0277] Bid = twice a day

[0278] Qd = once daily

[0279] ORR = objective response rate

[0280] DCR = disease control rate

[0281] DOR = Duration of Response

[0282] mDOR = mean duration of response

[0283] mPFS = mean progression-free survival

[0284] CR = complete remission

[0285] PR = partial response

[0286] SD = stable disease

[0287] mOS = mean overall survival

[0288] OSR = Overall Survival Rate

[0289] The 95% CI of the frequency distribution of each variable was calculated using Clopper-Pearson.

[0290] As of November 24, 2021, efficacy data for an additional 49 patients with KRAS mutant NSCLC were obtained; 27 patients were treated with naborafenib 200 mg bid and trametinib 1 mg qd, and 22 patients were treated with naborafenib 400 mg bid and trametinib 0.5 mg qd. The combination of naborafenib and trametinib also showed higher efficacy in patients with KRAS mutant NSCLC with codon Q61 or G13R mutations; 3 of 4 NSCLC patients whose tumors contained codon Q61 or G13R KRAS mutations responded (CR or PR) to the combination of naborafenib and trametinib, while 1 of 45 NSCLC patients whose tumors contained KRAS mutations other than codon Q61 or G13R mutations responded to the combination of naborafenib and trametinib.

[0291] These unpublished data, together with the preclinical evidence described above in the Background section and clinical data for another Raf inhibitor, bevafenib (see Example X below), rationalize the codon Q61 / G13R pan-RAS mutation as a predictive biomarker for response to the combination of naborafenib and trametinib. One hypothesis is that the activity shown by the combination of naborafenib and trametinib in NRAS mutant melanoma may be due to the natural enrichment of codon Q61 mutations, rather than the presence of NRAS mutations themselves. The presence of the mutation was identified using next generation sequencing (NGS).

[0292] Example 2: Similarity in the effects of the G13R mutation and the codon Q61 mutation on RAS

[0293] In molecular dynamics simulation analysis performed by Novartis, G13R was found to overlap with Q61 and behave similarly kinetically and may adopt a similar conformation / pause as Q61 (RAF-1 proficient).

[0294] Molecular dynamics simulations were performed using the Amber20 software package (Case et al., AMBER 2020, University of California, San Francisco, 2020). The x-ray crystal structure of GMPPNP bound to KRAS-G13D (PDB code 6XGV) was used for G13R simulations. The x-ray crystal structure of GMPPNP bound to KRAS-Q61R (PDB code 6XGU) was used for Q61R simulations. The CCG MOE molecular editor was used to prepare the starting coordinates, and in the case of G13R, D13 was mutated to R13 (Center for Molecular Dynamics Engineering, Inc., CCG "Molecular Operating Environment (MOE), 101 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015). All simulations had GTP nucleotides and bound coordinated Mg2+ ions. The system was embedded in a The buffer contains Na+ ions that neutralize the charge in the buffer tank. Proteins are modeled with the Amber force field ff19SB (Tian, ​​C., et al., J. Chem. Theory Comput. 2020, 16, 528-552), water is modeled with TIP3P (Jorgensen et al., J. Chem. Phys 1983, 79, 926-935), and ions are modeled with JC parameters (Joung et al., J. Phys. Chem. B 2008, 112, 9020-9041). Each system is first balanced in the NPT ensemble for 100 nanoseconds, and then a 1 μs NPT production run is started. The simulation is repeated four times, and the production time accumulated to each KRAS mutant is 4 μs.

[0295] Analysis of molecular dynamics trajectories was performed using CPPTRAJ (Roe et al., J. Chem. Theory Comput 2013, 9, 3084-3095). The root mean square fluctuation (RMSF) was calculated for each residue for each production run, and the final result was the average of four runs for each mutant.

[0296] When analyzing the RMSF data, it was found that G13R and Q61R exhibited similar dynamics in the simulations, with both having a mobile "switch-I" loop and a more restricted "switch-II" region. It can therefore be concluded that the G13R and Q61R mutant forms have overlapping dynamics and are likely to adopt similar conformations.

[0297] Figure 1 The corresponding results are shown and confirm that KRAS G13R protein dynamics overlap with KRAS Q61R but not G13D.

[0298] Example 3: Study on the combination of naborafenib and trametinib in the treatment of solid tumors with RAS codon Q61 or G13R mutations

[0299] The following clinical trials may be conducted: An open-label, non-randomized Phase II study to evaluate the efficacy and safety of naborafenib 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 of care or have no available standard treatment options. ORR as the primary endpoint 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. Patients with NRAS mutant melanoma may be selected for enrollment in the clinical study by a single central diagnostic test. This investigational clinical trial assay is intended to be a test registered as a companion diagnostic.

[0300] Objects can be allocated as follows:

[0301] Cohort 1: Adults (≥18 years) with unresectable or metastatic melanoma with codon Q61 or G13R NRAS mutations who have progressed following treatment with a PD-(L)-1 inhibitor alone or in combination with a CTLA4 or LAG 3 inhibitor.

[0302] Cohort 2: Adult subjects (≥18 years) with advanced metastatic solid tumors with codon Q61 or G13R RAS mutations who have progressed on available standard of care or have no standard treatment options based on local / regional standard practice and the treating physician's judgment.

[0303] Subjects could receive nabolafenib 200 mg orally twice daily and trametinib 1 mg orally once daily until disease progression according to RECIST 1.1, unacceptable toxicity or death, loss to follow-up, or withdrawal of consent.

[0304] This study was designed to further evaluate the novel combination of naborafenib plus trametinib, which showed encouraging activity and acceptable toxicity in the Phase Ib study CLXH254X2102 (see Example 1) in a population with a poor prognosis and high unmet medical need characterized by the above mutations.

[0305] Clinical studies and internal unpublished data suggest that both trametinib and naborafenib 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 bemetinib in the NEMO study (Dummer et al. 2017; Lancet Oncol. 19, 1315-27) also showed limited antitumor activity in NRAS mutant melanoma.

[0306] However, in view of the data and information herein, the combination of naborafenib and trametinib shows potential in the treatment of solid tumors, and also (especially unresectable) melanoma.

[0307] Mutations in NRAS activate intracellular signaling through multiple pathways, including the RAS-RAF-MAPK pathway. And these mutations are known to activate the MAP kinase pathway to the same extent as BRAF V600 mutations. Given that RAF and MEK are vertically integrated in the same pathway, combined inhibition of these two nodes may provide more effective inhibition, which is supported by preclinical and clinical data. This study evaluated the efficacy of naborafenib (RAF inhibitor) in combination with trametinib (MEK inhibitor) in subjects with unresectable or metastatic melanoma with codon Q61 or G13R NRAS mutations who have progressed after treatment with PD-(L)-1 inhibitors alone and / or in combination with CTLA-4 or LAG 3 inhibitors, and specifically in subjects with advanced metastatic solid tumors with codon Q61 or G13R RAS mutations who have progressed on available standard of care or have no standard treatment options.

[0308] The main characteristics of the target groups are:

[0309] Key inclusion criteria

[0310] Queue 1:

[0311] Histologically confirmed unresectable stage IIIB / C / D or metastatic / stage IV melanoma using AJCC 8th edition

[0312] ·Previously treated for unresectable or metastatic melanoma. Subjects must have received prior systemic therapy for unresectable or metastatic melanoma using an anti-PD-(L)-1 agent with or without anti-CTLA-4 or anti-LAG (no more than 2 lines of anti-PD(L)-1 based regimens).

[0313] Laboratory confirmation of the presence of codon Q61 or G13RNRAS mutations in tumor tissue is required, even with local test results. All subjects must provide fresh or archived tumor specimens from the metastatic setting for NRAS mutation testing.

[0314] Queue 2:

[0315] Solid tumors with histologically confirmed advanced disease (AJCC 8th edition) and no standard treatment options available, as determined by local / regional standards of care and the treating physician's judgment.

[0316] Codon Q61 or G13R RAS (including HRAS, KRAS and NRAS) mutations in tumor tissue must be confirmed in a Novartis-designated laboratory, even when local test results are available. All subjects must provide fresh or archived tumor samples from the metastatic setting for RAS mutation testing.

[0317] All queues:

[0318] Age at the time of informed consent (male or female) was 18 years or older

[0319] ECOG performance status 0, 1, or 2

[0320] The last dose of prior therapy must have been received more than four weeks before randomization

[0321] There must be at least one measurable lesion according to RECIST v1.1.

[0322] Key exclusion criteria

[0323] Queue 1:

[0324] Subjects with uveal or mucosal melanoma

[0325] 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

[0326] Queue 2:

[0327] Subjects with NRAS melanoma mutations

[0328] · Previous treatment with chemotherapy, immunotherapy, biological therapy, or chemoradiotherapy with delayed toxicity within 4 weeks before enrollment. Previous treatment with investigational products within 4 weeks or 5 half-lives (whichever is longer) before enrollment.

[0329] All objects:

[0330] Prior systemic therapy with BRAF inhibitors and / or MEK inhibitors

[0331] Previous solid organ transplant or stem cell transplant

[0332] ≤ 4 weeks of radiotherapy prior to the start of study treatment (note: palliative radiotherapy for bone lesions is allowed) or active CTCAE grade ≥ 2 radiotherapy-related toxicity ≤ 2 weeks prior to the start of study treatment

[0333] · Previous major surgery within 14 days before enrollment. Any surgery-related AE must be resolved before enrollment.

[0334] Primary CNS tumor or clinically active or unstable brain metastases at screening; or symptomatic or untreated leptomeningeal or spinal cord compression. Note: Subjects with prior unstable brain lesions who have definitively received stereotactic radiotherapy, surgery, or Gamma Knife therapy are eligible.

[0335] Subjects with brain lesions who were untreated (including newly discovered brain lesions during screening) or who received cranial radiation must have demonstrated stable disease by two consecutive assessments ≥2 weeks apart and not require high-dose steroids for at least ≥4 weeks prior to randomization / enrollment.

[0336] A history of other malignancies, including hematologic malignancies

[0337] Note: Subjects with other malignancies are eligible if they meet the following criteria:

[0338] 3 years of no illness, or

[0339] A history of completely excised non-melanoma skin cancer, and / or

[0340] Complete removal of any type of carcinoma in situ

[0341] Clinically significant gastrointestinal abnormalities that could alter absorption, such as malabsorption syndromes or major resection of the stomach or intestine

[0342] History or current evidence / risk of retinal vein occlusion (RVO) or central serous retinopathy

[0343] Receipt of chronic systemic steroid therapy (≥10 mg / day prednisone or equivalent) or any immunosuppressive therapy within 7 days prior to the planned date of the first dose of study treatment. Topical, inhaled, nasal, and ophthalmic steroids are permitted.

[0344] Currently using banned drugs or need to ban drugs during research

[0345] Heart disease or cardiac repolarization abnormalities, including any of the following:

[0346] Congestive heart failure requiring treatment (New York Heart Association class ≥2), LVEF < 200 mg / kg as determined by multi-gated acquisition (MUGA) scanning or echocardiography (ECHO)

[0347] 50%, or uncontrolled hypertension despite medical treatment, defined as resting blood pressure ≥140 (systolic) / 90 (diastolic) mmHg (average of 3 consecutive readings).

[0348] Myocardial infarction, angina pectoris, coronary artery bypass grafting within 6 months before starting study treatment

[0349] History of bridging surgery

[0350] The presence or history of clinically significant arrhythmias (e.g., ventricular tachycardia), complete left bundle branch block, or higher-degree AV block (e.g., bifascicular block, Mobitz type II, and third-degree AV block)

[0351] A family history of long QT syndrome, idiopathic sudden death, or congenital long QT syndrome, or

[0352] Any of the following:

[0353] Risk factors for torsades de pointes, including uncorrected hypokalemia or hypomagnesemia, a history of heart failure, or a history of clinically significant / symptomatic bradycardia

[0354] Concomitant medications known to carry a risk of torsades de pointes that cannot be discontinued or replaced with a safe alternative within 7 days prior to starting treatment

[0355] Unable to determine QTcF interval

[0356] Abnormal ECG determined by the average of three ECGs evaluated by the central laboratory at Screening:

[0357] Resting heart rate <60 or >90 bpm

[0358] QTcF ≥450 msec (males) or ≥460 msec (females) at screening (using Fridericia correction).

[0359] Study Treatment

[0360] The study evaluated the combination of naborafenib, a Raf inhibitor, and trametinib, a MEK inhibitor, in patients with unresectable or metastatic codon Q61 or G13R NRAS-mutated melanoma and patients with advanced metastatic solid tumors with codon Q61 or G13R RAS-mutated RAS. Different RAS variants exhibit different modes of interaction with downstream effector molecules.

[0361] Significant combined activity of naborafenib and trametinib was observed in NRAS melanoma cell lines. The combination of naborafenib and trametinib was synergistic in all lines tested, with significant activity in MM127 (NRASG13R), SK-MEL-2

[0362] The strongest synergy was observed in MEL-JUSO (NRASQ61R) and MEL-JUSO (NRASQ61L). The combination of naborafenib and trametinib was superior to either agent alone in reducing MEK1 / 2, ERK1 / 2, and RSK3 phosphorylation. Furthermore, the combination of low-dose trametinib and naborafenib was equivalent to or superior to a 10-fold higher single-agent dose of naborafenib. In a mouse xenograft (PTX) model, the combination of naborafenib with the MEK inhibitor trametinib controlled tumor growth better than either agent alone (3.

[0363] Figure 2 Results showing the antitumor activity of naborafenib and trametinib in ten patient-derived NRAS mutant melanoma tumor xenograft mouse models.

[0364] Clinical data further support preclinical findings. Trametinib showed limited single-agent activity in RAS-driven tumors (i.e., a phase II randomized trial failed to demonstrate superiority over standard care docetaxel in second-line KRAS G12C, 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 trametinib as a single agent in 97 patients with advanced melanoma, 7 of whom had NRAS mutations. In these 7 patients, trametinib monotherapy (continuous doses of 2 mg, 2.5 mg, 3 mg; 2-week lead-in with dose increases of 1 / 2 mg, 0.5 / 2.5 mg, and 0.5 / 2.5 mg; 3-week dosing and 1-week rest of 2 mg) showed limited activity, with no response and 2 patients with stable disease (SD) (Falchook et al. 2012; Lancet Oncol. 13(8), 789-9). Similarly, the Phase 1 FIH study of nabolafenib single agent in patients with solid tumors containing MAPK alterations, CLXH254X2101, enrolled 10 patients with NRAS mutant melanoma and showed no response and 4 SD at different doses (doses 400–1200qd and 200–800bid) (data cutoff May 31, 2021). These studies showed that trametinib and nabolafenib had limited activity when administered alone, which is consistent with the results of preclinical studies.

[0365] In contrast, study CLXH254X2102 explored the combination of 200 mg naborafenib and 1 mg trametinib in patients with advanced or metastatic melanoma with NRAS mutations. Sixteen patients were treated with a combination of 200 mg naborafenib and 1 mg trametinib. This combination resulted in a confirmed overall response rate (ORR) of 43.8%, a median duration of response (DOR) of 3.76 months, an mPFS of 5.5 months, an mOS of 8.8 months, and a 12-month OS rate of 48.9% (see Table 1 in Example 1).

[0366] These data support the use of the combination of naborafenib and trametinib in patients with NRAS-mutant melanoma rather than either agent alone.

[0367] Based on this data and those in Example 1, a particularly preferred dosing regimen useful in the methods of the present invention is naborafenib 200 mg bid + trametinib 1 mg qd

[0368] End Points and Estimates

[0369] ORR, assessed by a blinded independent review committee (BIRC) according to RECIST 1.1 Response Evaluation Criteria in Solid Tumors, version 1.1), was the primary endpoint to avoid potential investigator bias.

[0370] ORR was chosen as the primary endpoint because it is a direct measure of the anti-tumor activity of the drug, which can be evaluated in a single-arm trial (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).

[0371] PFS, OS, and duration of response (DOR) were secondary endpoints. PFS was defined as the time from randomization to the earliest disease progression or death due to any cause recorded by BIRC according to RECIST 1.1. OS was defined as the time from randomization to death due to any cause.

[0372] DOR was also an important factor in evaluating trial outcomes. For the subset of subjects with an overall response confirmed by BIRC, DOR was defined as the time from the first radiographic evidence of an overall response confirmed by BIRC to the earliest disease progression or death due to any cause recorded by BIRC according to RECIST 1.1.

[0373] The present invention provides a method of treating solid tumors, wherein a combination of naborafenib and trametinib is administered to subjects with unresectable or metastatic Q61 / G13R NRAS mutant melanoma who have progressed on anti-PD-(L)-1 (alone or in combination with anti-CTLA-4 or anti-LAG-3), or subjects with RAS Q61 / G13 mutant advanced metastatic solid tumors who have progressed under available standard care or have no available standard treatment options. Therefore, patients treated with the methods of the present invention and the combination of the agents of the present invention include patients with unresectable or metastatic Q61 or G13RNRAS mutant melanoma who have progressed on anti-PD-(L)-1 therapy (alone or in combination with anti-CTLA-4 or anti-LAG-3), or patients with RAS Q61 / G13 mutant advanced metastatic solid tumors who have progressed under available standard care or have no available standard treatment options.

[0374] Previous treatments for these patients included the following.

[0375] In general, these patients were treated 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, supra; Serrone et al. 2000; J. Exp. Clin. Cancer Res. 19(1), 21-34) and temazolamide (ORR 13.5%, Rao et al. 2006; supra) as metastatic melanoma in 2L / 3L, regardless of NRAS mutation, after progression on anti-PD-(L)-1. Alternative approved treatments for other solid tumor types are not specific for RAS isoforms, with the exception of the KRAS G12C inhibitor (sotoracib) for NSCLC, which has an ORR of 36% and a median DoR of 10 months.

[0376] Incorporation by reference

[0377] All clinical trials, publications, patents, and accession numbers mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0378] Equivalent

[0379] Although specific embodiments of the subject invention have been discussed, the foregoing description is illustrative only and not limiting. Many variations of the invention will become apparent to those skilled in the art upon reading this description and the following claims. The full scope of the invention should be determined by reference to the claims and their full scope of equivalents and the description and variations thereof. Deletion of this paragraph due to official or court objections to its wording does not imply a reduction in the scope of protection, including equivalent protection.

Claims

1. An agent or probe capable of recognizing RAS codon Q61 and / or (especially) G13R mutations, for use (or particularly for this purpose) in diagnosing the suitability of a solid tumor, in particular a solid tumor other than melanoma, but more preferably a solid tumor other than melanoma or other than rhabdomyosarcoma, for a combination treatment with a Raf inhibitor and a MEK inhibitor, wherein the diagnosis comprises using the agent to determine whether a sample of the solid tumor contains a RAS codon Q61 or RAS G13R mutation as a biomarker of the suitability.

2. A method of identifying the suitability of a patient having a solid tumor, particularly a solid tumor other than melanoma, yet more preferably a solid tumor other than melanoma or other than rhabdomyosarcoma, for combination treatment with a Raf inhibitor and a MEK inhibitor, the method comprising: (a) assaying a tumor sample obtained from the patient for the presence of a RAS codon Q61 or RAS G13R mutation using an agent according to the preceding paragraph; as well as (b) selecting patients whose tumor samples tested positive under (a) for the presence of said RAS codon Q61 or RAS G13R mutation as candidates for said combination treatment.

3. A method for selecting a treatment for a patient with a solid tumor, the method comprising screening a tumor sample from the patient for a RAS codon Q61 or RAS G13R mutation, wherein the presence of the mutation in the tumor sample identifies the patient as a patient who may be expected to benefit from a treatment comprising a Raf inhibitor and a MEK inhibitor.

4. A method of treating a patient suffering from a solid tumor, the method comprising (a) examining whether a tumor sample of said patient having said tumor contains a RAS codon Q61 or RAS G13R mutation, and, (b) in case the tumor sample is determined to contain the mutation, administering a therapeutically effective amount of a Raf inhibitor and a MEK inhibitor as a combination therapy.

5. A method of treating a patient in need of such treatment who has a solid tumor, particularly a solid tumor other than melanoma, comprising administering to said patient an effective amount of a combination of a RAF inhibitor and a MEK inhibitor, respectively, where said solid tumor is identified as containing a RAS codon Q61 or RAS G13R mutation.

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

7. A Raf inhibitor and a MEK inhibitor for combined use in the treatment of a solid tumor in a patient, in particular a solid tumor other than melanoma and / or rhabdomyosarcoma containing an NRAS Q61 mutation, wherein the tumor is tested positive and identified as containing a RAS codon Q61 or RAS G13R mutation.

8. A Raf inhibitor and a MEK inhibitor for combined use according to claim 7, wherein the Raf inhibitor is selected from conafenib; sorafenib, vemurafenib, dabrafenib, GDC-0879, PLX-4720, PLX8394, bevafenib, CCT3833 / BAL3833, LY3009120, LSN3074753, rifirafenib, BGB659, RO5126766, AZ-628, MLN2480, BeiGene-283, RXDX-105, BAL3833, INU152, regorafenib, tovofenib, Day101, TAK580, MLN2480, TAK632, and in particular naborafenib (LXH254; Type II); or their respective pharmaceutically acceptable salts, hydrates or solvates.

9. The Raf inhibitor and MEK inhibitor for combined use according to any one of claims 7 or 8, wherein the Raf inhibitor is naborafenib, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

10. A Raf inhibitor and a MEK inhibitor for combined use according to any one of claims 7 to 9, wherein the MEK inhibitor is selected from bemetinib, trametinib, selumetinib, cobimetinib, CI-1040, U0126-EtOH, PD198306, PD98059, BIX 02189, TAK-733, honokiol, AZD8330, PD318088, BIX 02188, pimacitinib, midametinib, refactinib, BI-847325, GDC0623, G-573, trimethoprim, RO5126766 and pimacitinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

11. A Raf inhibitor and a MEK inhibitor for combined use according to any one of claims 7 to 10, wherein the MEK inhibitor is trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

12. A Raf inhibitor and a MEK inhibitor for combined use according to any one of claims 7 to 11, wherein the solid tumor is tested positive for containing a G13R mutation.

13. Use of a combination of a Raf inhibitor and a MEK inhibitor, in particular as defined in more detail herein, in particular naborafenib and trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in the treatment of a solid tumor in a patient, wherein the tumor is tested positive for containing a RAS codon Q61 or RAS G13R mutation.

14. Use of a Raf inhibitor and a MEK inhibitor, in particular as defined in claims 8 and 10, respectively, in particular naborafenib and trametinib, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in the preparation of a (in particular combined) medicinal product, such as a kit for combined use or a fixed combination preparation comprising both active ingredients, for the combined treatment of a solid tumor in a patient, wherein the solid tumor has been determined to contain a RAS codon Q61 or RASG13R mutation.

15. A composition comprising a Raf inhibitor and a MEK inhibitor, in particular as defined in more detail in claim 10 and claim 12, respectively, in particular naborafenib and trametinib, or their respective pharmaceutically acceptable salts, hydrates or solvates, and a pharmaceutically acceptable carrier, in the form of a separate formulation or a kit or a fixed formulation for the treatment of solid tumors selected from rhabdomyosarcoma identified as containing the RAS codon G13R mutation, or different solid tumors identified as containing the RAS codon Q61 or RAS G13R mutation, in particular solid tumors other than melanoma; or a corresponding method of treatment, comprising administering the mentioned Raf inhibitor and MEK inhibitor in a combined therapeutically active amount to a patient suffering from said rhabdomyosarcoma or other solid tumor in need of such treatment.

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