Medicamentous combinations comprising KRAS G12C inhibitors and their use for the treatment of cancer

Abstract

The present invention relates to a pharmaceutical combination comprising a KRAS G12C inhibitor and one or more therapeutic agents selected from agents targeting the MAPK pathway or agents targeting parallel pathways; and a pharmaceutical composition comprising the same. The present invention also relates to a KRAS G12C inhibitor alone or in combination for use in a method for treating cancer or tumor, particularly where the cancer or tumor is a KRAS G12C mutant.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, said ASCII copy being created on Jun. 22, 2022, entitled PAT059141-WO-PCT SQL_ST25, 2,471 bytes in size, filed herewith and incorporated herein by reference.

[0002] The present invention relates to a KRAS G12C inhibitor and its use in combination with one or two additional therapeutically active agents in the treatment of cancer, particularly KRAS G12C mutant cancer (e.g., lung cancer, non-small cell lung cancer, colorectal cancer, pancreatic cancer or solid tumors). The present invention relates to a combination pharmaceutical comprising (i) a KRAS G12C inhibitor, e.g., Compound A, or a pharmaceutical acceptable salt thereof, and a second therapeutic agent selected from agents targeting the MAPK pathway or parallel pathways, e.g., the PI3K / AKT pathway. The second therapeutic agent may be selected from EGFR inhibitors, SOS inhibitors, SHP2 inhibitors (e.g., TNO155, or a pharmaceutical acceptable salt thereof), Raf inhibitors, ERK inhibitors, MEK inhibitors, AKT inhibitors, PI3K inhibitors, mTOR inhibitors, CDK4 / 6 inhibitors, FGFR inhibitors, and combinations thereof. The present invention also relates to a triple combination comprising a KRAS G12C inhibitor, e.g., Compound A, or a pharma- ceutically acceptable salt thereof, and a second therapeutic agent which is a SHP2 inhibitor (e.g., TNO155, or a pharma- ceutically acceptable salt thereof), and a third therapeutic agent, optionally wherein the third therapeutic agent may be selected from an EGFR inhibitor, an SOS inhibitor, a Raf inhibitor, an ERK inhibitor, a MEK inhibitor, an AKT inhibitor, a PI3K inhibitor, an mTOR inhibitor, a CDK4 / 6 inhibitor, and an FGFR inhibitor.

[0003] The invention also relates to pharmaceutical compositions containing same; and to methods of using such combinations and compositions in the treatment or prevention of cancer or solid tumours, in particular KRAS G12C mutant cancers or KRAS G12C solid tumours. [Background technology]

[0004] Cancer growth is driven by many diverse and complex mechanisms. It is inevitable that resistance to a given therapy occurs in some cancers. Inhibition of MAPK pathway induces feedback mechanisms and pathway rewiring that cause its subsequent reactivation. One common mechanism is, for example, the activation of receptor tyrosine kinases (RTKs).

[0005] In addition, despite recent successes of targeted and immunotherapeutic therapies, some cancers, especially metastatic cancers, remain largely incurable.

[0006] KRAS oncoprotein is a GTPase that plays a key role as a regulator of intracellular signaling pathways, including the MAPK, PI3K, and Ral pathways, and is involved in proliferation, cell survival, and tumorigenesis. Oncogenic activation of KRAS occurs predominantly through missense mutations in codon 12. KRAS gain-of-function mutations are found in approximately 30% of all human cancers. The KRAS G12C mutation is a specific submutation that is found in approximately 13% of lung adenocarcinomas, 4% (3-5%) of colon adenocarcinomas, and a smaller portion of other cancer types.

[0007] In normal cells, KRAS alternates between an inactive GDP-bound state and an active GTP-bound state. Mutations of KRAS at codon 12, such as G12C, impair GTP hydrolysis stimulated by GTPase-activating proteins (GAPs). Thus, in this case, KRAS G12C converts GTP to the GDP form very slowly. As a result, KRAS G12C shifts to the active GTP-bound state, thus promoting oncogenic signaling.

[0008] CDKN2A, also known as cyclin-dependent kinase inhibitor 2A, is the gene encoding the INK4 family members p16 (or p16INK4a) and p14arf, which act as tumor suppressors by regulating the cell cycle. p16 inhibits cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), thus activating the retinoblastoma (Rb) protein family, which blocks the G1 to S phase transition. p14ARF (also known as p19ARF in mice) activates the p53 tumor suppressor. CDKN2A is believed to be the second most commonly inactivated gene in cancer, after p53.

[0009] Mutations in CDKN2A have been described in cancers such as melanoma, gastric lymphoma, Burkitt's lymphoma, head and neck squamous cell carcinoma, oral cancer, pancreatic adenocarcinoma, non-small cell lung cancer, esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, epithelial ovarian cancer and prostate cancer.

[0010] The PIK3CA gene (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) is a gene that encodes p110, which is involved in cell proliferation, growth, differentiation, motility, and survival. Mutations in the PIK3CA gene produce abnormal p110 protein at increased rates. PIK3CA gene mutations have been found in breast cancer, ovarian cancer, lung cancer, stomach cancer, gastric cancer, and brain cancer.

[0011] Lung cancer remains the most common cancer type worldwide and is the leading cause of cancer deaths in many countries, including the United States. NSCLC accounts for approximately 85% of all lung cancer diagnoses. KRAS mutations are detected in approximately 25% of patients with lung adenocarcinoma (Sequist et al., 2011). These are most commonly found at codon 12, with KRAS G12C mutations being the most common in both adenocarcinoma and squamous NSCLC (40% overall) (Liu et al., 2020). The presence of KRAS mutations indicates poor prognosis for survival and is associated with reduced responsiveness to EGFR TKI treatment.

[0012] Standard of care treatment for patients with KRAS G12C mutated NSCLC consists of platinum-based chemotherapy and immune checkpoint inhibitors. Sotorasib (a KRAS G12C inhibitor) recently received accelerated approval from the FDA for this indication and for adult patients who have received at least one prior systemic therapy, and further confirmatory studies are currently ongoing. Sotorasib received accelerated approval by the US FDA (Food and Drug Administration) in May 2021 and conditional marking authorization by the European Commission (EC) in January 2022 in patients with KRAS G12C mutated locally advanced or metastatic non-small cell lung cancer (NSCLC). In this patient population in a phase 2 single-arm trial of 126 patients, sotorasib demonstrated an ORR of 37% (95%CI 28.6-46.2), a median DOR of 11.1 months, a median PFS of 6.8 months, and a median OS of 12.5 months (Skoulidis et al,N Engl J Med;384:2371-81). Adaglasib, another KRAS G12C inhibitor, is also in clinical development in KRAS G12C mutant malignancies, with a preliminary ORR of 45% in patients with NSCLC (Janne et al 2019,Presented at AACR-NCI-EORTC International Conference on Molecular Targets,28 October2019).

[0013] Immunotherapy of NSCLC with immune checkpoint inhibitors has demonstrated promise, with some NSCLC patients experiencing sustainable disease control for many years. However, such long-term non-progressors are uncommon, and therapeutic strategies that could increase the proportion of patients who respond to therapy and achieve durable remission are urgently needed.

[0014] Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related deaths in the United States. The number of new cases of CRC was approximately 150,000 in the United States in 2019, while more than 300,000 patients are expected to be diagnosed with CRC in the EU in 2020 (European Cancer Information System 2020). Despite observed improvements in the overall incidence of CRC, the incidence in patients under 50 years of age has increased in recent years (Bailey et al. 2015), and the authors estimate that the incidence of colon and rectal cancer could increase by 90% and approximately 124%, respectively, by 2030 for patients aged 20–34 years. Systemic therapies for metastatic CRC include a variety of agents used alone or in combination, including, for example, 5-fluorouracil / leucovorin, capecitabine, oxaliplatin, and irinotecan; antiangiogenic agents, such as bevacizumab and ramucirumab; chemotherapy, including anti-EGFR agents, including cetuximab and panitumumab for KRAS / NRAS wild-type cancer; and immunotherapy, including nivolumab and pembrolizumab. However, despite multiple aggressive therapies, metastatic CRC remains incurable. Mismatch repair-deficient (MSI-high) CRC shows high response rates to immune checkpoint inhibitor therapy, whereas mismatch repair-competent CRC does not. This subtype of CRC is particularly in need of improved therapy, as KRAS-mutated CRC is typically mismatch repair-competent and not a candidate for anti-EGFR therapy.

[0015] Tumor profiling data indicate that there exists a subset of solid tumors other than NSCLC and CRC that harbor KRAS G12C mutations. KRAS G12C is present in approximately 1-2% of malignant solid tumors, including approximately 1% of all pancreatic cancers (Biernacka et al. 2016; Zehir et al. 2017). KRAS G12C mutations have also been found in appendix, small intestine, hepatobiliary, bladder, ovarian, and cancers of unknown primary site (Hassar et al., N Engl Med 2021 384;2 185-187).

[0016] Several targeted therapies are currently in clinical trials that aim to treat patients with KRAS mutations by inhibiting the RAS pathway. However, the benefit of these therapies in tumors with KRAS G12C mutations remains uncertain at present, as not all patients have responded and in some cases, the reported duration of responses is short, likely due to the emergence of resistance mediated at least in part by RAS gene mutations that abolish inhibitor binding and reactivation of downstream pathways.

[0017] Acquired resistance to monotherapy eventually occurs in most patients treated with KRAS G12C inhibitors. For example, among 38 patients included in a study with adagrasib: 27 with non-small cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer, putative resistance mechanisms to adagrasib were detected in 17 patients (45% of the cohort), of which 7 (18% of the cohort) had multiple concordant mechanisms. Acquired KRAS alterations included G12D / R / V / W, G13D, Q61H, R68S, H95D / Q / R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass resistance mechanisms included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN (Awad et al, Acquired Resistance to KRASG12C Inhibition in Cancer, N Engl J Med 2021;384:2382-93. Tanaka et al. (Cancer Discov 2021;11:1913-22) describe a novel KRAS Y96D mutation that affects the switch II pocket where adagrasib and other inactive KRAS G12C inhibitors bind, disrupting a key protein-drug interaction and conferring resistance to these inhibitors in engineered and patient-derived KRASG12C cancer models.

[0018] Therefore, additional therapeutic options are needed to overcome resistance mechanisms that arise during treatment with KRAS inhibitors, such as adagrasib or sotorasib.

[0019] Thus, there remains a high unmet medical need for new treatment options for patients suffering from cancer (including advanced and / or metastatic cancers, including lung cancer (including NSCLC), colorectal cancer, pancreatic cancer and solid tumors), particularly where the cancer or solid tumor has a KRAS G12C mutation. It is also important to provide potentially beneficial novel therapies for patients with KRAS G12C mutant tumors who have already received and failed standard of care therapy for incurable disease, particularly for the indication, or who are intolerant or ineligible for approved therapies, and therefore have limited treatment options. Summary of the Invention

[0020] The present invention seeks to provide new treatment options for patients suffering from cancer (including advanced and / or metastatic cancer) and to improve outcomes, particularly for patients with cancers driven by KRAS G12C.

[0021] Provided herein are compounds and combinations of compounds and their use in methods of treating cancer, including lung cancer (including NSCLC), colorectal cancer, pancreatic cancer and solid tumors, particularly where the cancer or solid tumor has a KRAS G12C mutation. The invention also provides potentially beneficial novel investigational therapies for patients with KRAS G12C mutant tumors who have already undergone and failed standard of care therapy for incurable disease, particularly for the indication, or who are intolerant or ineligible to approved therapies, and therefore have limited treatment options.

[0022] Additionally, the present invention also provides Compound A, alone or in combination with one or more additional therapeutic agents, for use in a method of treatment of cancer patients who have developed resistance to other therapies, e.g., prior treatment with other KRAS inhibitors, e.g., adagrasib and sotorasib; more preferably, prior treatment with sotorasib.

[0023] Compound A is a selective covalent irreversible inhibitor of KRAS G12C that utilizes a unique interaction with KRAS G12C to display a novel binding mode. In particular, Compound A traps KRAS G12C in a GDP-bound inactive state while avoiding direct interaction with H95, a recognized resistance pathway (Awad MM, et al. New Engl J Med 2021;384:2382-2392). Compound A potently inhibited the double mutant KRAS G12C H95Q, which mediates resistance to adagrasib in clinical trials.

[0024] Compound A demonstrates potent antitumor activity and favorable pharmacokinetic properties in preclinical models. Compound A is orally bioavailable, achieves exposure in the range predicted to confer antitumor activity, and is well tolerated.

[0025] Preliminary data (Phase Ib) from the KontRASt-01 trial (NCT04699188) showed that compound A, a selective, covalent, and orally bioavailable KRAS G12C inhibitor, demonstrated antitumor activity, high systemic exposure at its recommended dose, and a favorable safety profile based on early clinical data in patients with KRAS G12C-mutated solid tumors.

[0026] KRAS G12C inhibitors are specifically designed to inhibit KRAS G12C.However, many tumors have KRAS WT, HRAS and NRAS proteins that are not inhibited by KRAS G12C inhibitors.During KRAS G12C inhibitor treatment, for example, reactivated RTKs may enter the MAPK pathway through these proteins, thus preventing antitumor activity.Similarly, many RTKs and RAS proteins directly activate parallel pathways, for example, PI3K / AKT pathway.

[0027] The data and examples herein demonstrate that the addition of another therapeutically active agent targeting the MAPK pathway or a parallel pathway, such as the PI3K / AKT pathway, to a KRAS G12C inhibitor in a combination therapy has the potential to increase the depth and durability of anti-tumor responses.

[0028] For example, inhibitors of SHP2 have the potential to synergize with KRAS G12C inhibitors, such as Compound A. Inhibition of SHP2 inhibits the growth of KRAS mutant cancer cell lines in part by shifting the pool of KRAS to an inactive GDP-loaded state. When Compound A binds exclusively to GDP-bound KRAS G12C, the combination of SHP2 and KRAS G12C inhibition is predicted to be synergistic due to an increase in the target pool for irreversible Compound A binding.

[0029] As can be seen in the examples, the highest synergy scores were obtained in the presence of a combination of a PI3K inhibitor with a KRAS G12C inhibitor alone or in the presence of a SHP2 inhibitor in a cell viability assay. Thus, the present invention also provides triple or quadruple combinations as described herein.

[0030] As shown in the examples, compound A, a KRAS G12C inhibitor, shows deep tumor in xenograft model, especially in cancer xenograft model with one or more mutations selected from KRAS G12C, PIK3CA and CDKN2A. The antitumor response of KRAS G12C inhibitor as a single agent is improved with each of the combination partners tested, and some tumors even regress with combination treatment. Triple and quadruple combinations are considered to further improve response.

[0031] In summary, it can be seen that compound A, having unique properties and tolerability and safety profile, may be particularly useful for treating cancer, particularly the cancers described herein, either alone or in combination with one or more (e.g., one, two or three) therapeutic agents described herein.

[0032] In particular, the combination of KRAS G12C inhibitors (e.g., Compound A) with other inhibitors of the MAPK pathway or inhibitors of the PI3K / AKT pathway has the potential to further improve anti-tumor response and overcome potential resistance. Such combination therapy may be useful in the treatment of cancer, particularly cancer driven by KRAS G12C mutation. The second therapeutic agent may be selected from EGFR inhibitors, SOS inhibitors, SHP2 inhibitors (e.g., TNO155, or a pharma- ceutically acceptable salt thereof), Raf inhibitors, ERK inhibitors, MEK inhibitors, AKT inhibitors, PI3K inhibitors, mTOR inhibitors, CDK4 / 6 inhibitors, and combinations thereof.

[0033] Therefore, the combination and method of the present invention may also provide clinical benefit in patients with acquired resistance to KRAS G12C inhibitors, for example, by reactivating the RTK-MAPK pathway, which bypasses KRAS G12C and signals through WT KRAS, NRAS and / or HRAS.In addition, inhibition of EGFR may target the KRAS signaling pathway upstream of KRAS, and improve the antitumor activity of KRAS G12C inhibitors, such as Compound A, in KRAS G12C mutant cancers.Cancers to be treated by the combination and method of the present invention include cancers or solid tumors with one, two or three mutations selected from KRAS G12C, PIK3CA and CDKN2A, and combinations thereof; for example, cancers with KRAS G12C and CDKN2A mutations; and cancers with KRAS G12C, PIK3CA and CDKN2A mutations.

[0034] Thus, the present invention also provides a pharmaceutical combination comprising a KRAS G12C inhibitor, such as Compound A, or a pharma- ceutically acceptable salt thereof, and at least one additional therapeutically active agent. The additional therapeutically active agent may be an agent targeting the MAPK pathway or an agent targeting a parallel pathway.

[0035] Thus, the present invention also provides a pharmaceutical combination comprising a KRAS G12C inhibitor, e.g., Compound A, or a pharma- ceutically acceptable salt thereof, and a therapeutically active agent selected from the group consisting of an EGFR inhibitor, an SOS inhibitor, an SHP2 inhibitor (e.g., TNO155, or a pharma- ceutically acceptable salt thereof), a Raf inhibitor, an ERK inhibitor, a MEK inhibitor, an AKT inhibitor, a PI3K inhibitor, an mTOR inhibitor, a CDK4 / 6 inhibitor, and combinations thereof.

[0036] Thus, the present invention also relates to KRAS G12C inhibitors, such as compound A, or a pharma- ceutically acceptable salt thereof, SHP2 inhibitors (e.g., TNO155, or a pharma- ceutically acceptable salt thereof), as well as EGFR inhibitors (e.g., cetuximab, panitumab, afatinib, lapatinib, erlotinib, gefitinib, osimertinib, or nazartinib), SOS inhibitors (e.g., BAY-293, BI-3406, or BI-1701963), Raf inhibitors (e.g., belbalafenib or LXH254 (napolafenib)), ERK inhibitors (e.g., LTT462 (linetelquib), GDC-0994, KO-947, Vtx-11, e.g., SCH-772984, MK2853, LY3214996 or ulixertinib), MEK inhibitors (e.g., pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib), AKT inhibitors (e.g., capivasertib (AZD5363) or ipatasertib), PI3K inhibitors (e.g., AMG511, buparlisib, alpelisib), mTOR inhibitors (e.g., everolimus or temsirolimus), and CDK4 / 6 inhibitors (e.g., ribociclib, palbociclib or alemaciclib).

[0037] The present invention also relates to a method for producing a [ka] 1-{6-[(4M)-4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one or a pharma- ceutically acceptable salt thereof, and EGFR inhibitors (e.g., cetuximab, panitumumab, erlotinib, gefitinib, osimertinib, or nazartinib), SOS inhibitors (e.g., BAY-293, BI-3406, or BI-1701963), Raf inhibitors (e.g., belbalafenib or LXH254 (napolafenib)), ERK inhibitors (e.g., LTT462 (linetelquib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996, or urixel a second therapeutically active agent selected from an anti-cancer ... The present invention provides a pharmaceutical combination comprising:

[0038] The present invention also relates to compound A, or a pharma- ceutically acceptable salt thereof, as well as a Raf inhibitor (e.g., belbalafenib or LXH254 (napolafenib)), an ERK inhibitor (e.g., LTT462 (linetelquib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib), a MEK inhibitor (e.g., pimasertib, The present invention provides a pharmaceutical combination comprising a second therapeutically active agent selected from a PI3K inhibitor (e.g., PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib), a PI3K inhibitor (e.g., AMG511, buparlisib, alpelisib), an mTOR inhibitor (e.g., everolimus or temsirolimus), and a CDK4 / 6 inhibitor (e.g., ribociclib, palbociclib or alemaciclib).

[0039] In an embodiment of the present invention, the second therapeutically active agent may be selected from FGFR inhibitors, such as infigratinib (BGJ398), pemigatinib, erdafitinib, derazantinib; and futibatinib. The present invention also relates to a method for treating or preventing rheumatoid arthritis, comprising administering to a patient a therapeutically effective amount of Compound A, or a pharma- ceutical agent comprising the steps of: (a) Compound A, or a pharma- ceutical acceptable salt thereof; (b) an SHP2 inhibitor (e.g., TNO155, or a pharma-ceutical acceptable salt thereof); and (c) Raf inhibitors (e.g., belvarafenib or LXH254 (napolafenib)), ERK inhibitors (e.g., LTT462 (lineterkib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib), MEK inhibitors (e.g., pimasertib, PD-032 5901, selumetinib, trametinib, binimetinib or cobimetinib), a PI3K inhibitor (e.g., AMG511, buparlisib, alpelisib), an mTOR inhibitor (e.g., everolimus or temsirolimus), and a CDK4 / 6 inhibitor (e.g., ribociclib, palbociclib or alemaciclib).

[0040] In an embodiment of the invention, the third therapeutically active agent may be selected from an FGFR inhibitor, such as infigratinib (BGJ398), pemigatinib, erdafitinib, derazantinib; and futibatinib.

[0041] The present invention also relates to (a) Compound A, or a pharma- ceutically acceptable salt thereof; (b) TNO155, or a pharma- ceutically acceptable salt thereof; and (c) a third therapeutically active agent selected from a Raf inhibitor (e.g., berbalafenib or LXH254 (napolafenib)), an ERK inhibitor (e.g., LTT462 (linetelquib), GDC-0994, KO-947, Vtx-11e, SCH-772984, MK2853, LY3214996 or ulixertinib), a MEK inhibitor (e.g., pimasertib, PD-0325901, selumetinib, trametinib, binimetinib or cobimetinib), a PI3K inhibitor (e.g., AMG511, buparlisib, alpelisib), an mTOR inhibitor (e.g., everolimus or temsirolimus), and a CDK4 / 6 inhibitor (e.g., ribociclib, palbociclib or alemaciclib).

[0042] The present invention also relates to compound A, or a pharma- ceutically acceptable salt thereof, and (i) LXH254 (napolafenib), or a pharma- ceutically acceptable salt thereof; (ii) trametinib, a pharma- ceutically acceptable salt or solvate thereof, such as a DMSO solvate thereof; (iii) LTT462 (linetelquib), or a pharma- ceutically acceptable salt thereof, e.g., the HCl salt thereof; (iv) BYL719 (alpelisib), or a pharma- ceutically acceptable salt thereof; (v) LEE011 or a pharma- ceutically acceptable salt thereof, e.g., the succinate salt thereof; and (vi) Everolimus (RAD001) or a pharma- ceutically acceptable salt thereof The present invention provides a combination comprising a second agent selected from:

[0043] The present invention also relates to a compound comprising: (a) Compound A, or a pharma- ceutically acceptable salt thereof; (b) TNO155, or a pharma- ceutically acceptable salt thereof; and (i) naporafenib (LXH254), or a pharma- ceutically acceptable salt thereof; (ii) trametinib, a pharma- ceutically acceptable salt or solvate thereof, such as a DMSO solvate thereof; (iii) linetelqib (LTT462), or a pharma- ceutically acceptable salt thereof, e.g., its HCl salt; (iv) alpelisib (BYL719), or a pharma- ceutically acceptable salt thereof; (v) ribociclib (LEE011), or a pharma- ceutically acceptable salt thereof, e.g., the succinate salt thereof; and (vi) Everolimus (RAD001) or a pharma- ceutically acceptable salt thereof The present invention provides a combination comprising a third agent selected from:

[0044] It will be understood that reference herein to "a combination of the invention" or "the combination(s) of the invention" is intended to include each of those combination pharmaceuticals individually, and all of those combinations as a group.

[0045] In particular, reference to "combinations of the invention" is intended to include combinations of a KRAS G12C inhibitor and a SHP2 inhibitor (e.g., Compound A and TNO155); combinations of a KRAS G12C inhibitor and a PI3K inhibitor (e.g., Compound A and alpelisib (BYL719)); combinations of a KRASG 12C inhibitor and a CDK4 / 6 inhibitor (e.g., Compound A and ribociclib).

[0046] Triple combinations are also included within the definition of "combinations of the invention." Preferred embodiments include: (i) the combination of Compound A, TNO155 and alpelisib, and (ii) the combination of Compound A, TNO155 and ribociclib.

[0047] The present invention provides pharmaceutical combinations for use in the treatment of cancer as described herein.

[0048] The efficacy of the treatment method of the present invention can be determined by methods well known in the art, such as by determining best overall response (BOR) by RECIST v.1.1, overall response rate (ORR), duration of response (DOR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). Thus, the present invention provides a combination drug of the present invention that improves KRAS G12C inhibitor therapy, for example, by measuring one or more increases in best overall response (BOR) by RECIST v.1.1, overall response rate (ORR), duration of response (DOR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS).

[0049] In another embodiment of the combination of the present invention, compound A, or a pharma- ceutically acceptable salt thereof, the second therapeutically active agent, and the third therapeutically active agent (if present) are present in separate formulations.

[0050] In another embodiment, a combination of the invention is for simultaneous or sequential administration (in any order).

[0051] In another embodiment, there is a method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination of the present invention.

[0052] In an embodiment of the invention, the cancer or tumor to be treated is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer, and squamous lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendix cancer, small intestine cancer, esophageal cancer, hepatobiliary cancer (including liver cancer and bile duct cancer), bladder cancer, ovarian cancer, and solid tumors, particularly where the cancer or tumor has a KRAS G12C mutation.

[0053] In an embodiment of the invention, the cancer or tumor to be treated is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendix cancer, small intestine cancer, esophageal cancer, hepatobiliary cancer (including liver cancer, bile duct cancer and bile duct carcinoma), bladder cancer, ovarian cancer, ampullary duodenal carcinoma and solid tumors, particularly where the cancer or tumor has a KRAS G12C mutation.

[0054] In an embodiment of the invention, the cancer or tumor to be treated is selected from non-small cell lung cancer, colorectal cancer, bile duct cancer, ovarian cancer, ampullary duodenal cancer and pancreatic cancer.

[0055] Cancers of unknown primary site but which exhibit KRAS G12C mutations may also benefit from treatment with the methods of the present invention.

[0056] In an embodiment of the method of the present invention, the cancer is selected from non-small cell lung cancer, colorectal cancer, pancreatic cancer and solid tumors.

[0057] In a further embodiment of the method, the cancer is a solid tumor.

[0058] In a further embodiment of the method, the cancer is colorectal cancer.

[0059] In a further embodiment of the method, the cancer is non-small cell lung cancer.

[0060] In a further embodiment of the method, the cancer is pancreatic cancer.

[0061] In a further embodiment of the method, the cancer is a solid tumor.

[0062] In a further embodiment of the method, the cancer is appendiceal cancer.

[0063] In a further embodiment of the method, the cancer is small intestine cancer.

[0064] In a further embodiment of the method, the cancer is esophageal cancer.

[0065] In a further embodiment of the method, the cancer is hepatobiliary cancer.

[0066] In a further embodiment of the method, the cancer is bladder cancer.

[0067] In a further embodiment of the method, the cancer is ovarian cancer.

[0068] In a further embodiment of the method, the cancer is cholangiocarcinoma.

[0069] In a further embodiment of the method, the cancer is papillary duodenal carcinoma.

[0070] In a further embodiment, the invention provides a combination of the invention for use in the manufacture of a medicament for treating a cancer selected from non-small cell lung cancer, colorectal cancer, pancreatic cancer and a solid tumor, optionally wherein the cancer or solid tumor is a KRAS G12C mutation. In another embodiment there is a pharmaceutical composition comprising a combination of the invention.

[0071] In further embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable excipients described herein.

[0072] Figures 1-5 are waterfall plots depicting the efficacy of KRAS G12C inhibitors alone and in combination with other agents in CRC and lung cancer patient-derived xenograft models. Each figure shows the response to a particular treatment for each individual mouse model shown as % best average response (Best Avg.Resp.) on the (vertical) y-axis. The best average effect is the smallest average effect (average volume change across all time points from day 0 to day X - this is similar to a cumulative sum or area under the curve. It captures the speed, intensity, and durability of the response into a single value). [Brief description of the drawings]

[0073] [Figure 1-1] FIG. 1: Waterfall plot showing the efficacy of combinations of KRAS G12C inhibitors and agents targeting the MAPK pathway in CRC patient-derived xenograft models, presented as best mean efficacy results. [Figure 1-2] (As stated above.) [Diagram 2] Waterfall plot showing the efficacy of combinations of KRAS G12C inhibitors and agents targeting parallel pathways in CRC patient-derived xenograft models, presented as best mean efficacy results. [Figure 3-1] Figure 3: Waterfall plot showing the efficacy of triple combinations including KRAS G12C inhibitors in NSCLC patient-derived xenograft models, presented as best mean efficacy results. [Figure 3-2] (As stated above.) [Figure 4-1] FIG. 4: Waterfall plot showing the efficacy of combinations of KRAS G12C inhibitors and agents targeting the MAPK pathway in NSCLC patient-derived xenograft models, presented as best mean efficacy results. [Figure 4-2] (As stated above.) [Diagram 5] Waterfall plot showing the efficacy of combinations of KRAS G12C inhibitors and agents targeting parallel pathways in NSCLC patient-derived xenograft models, presented as best mean efficacy results. [Figure 6] Spider plot showing % tumor volume change over time. Fragments of CRC or lung cancer are implanted into mice, and when the tumors reach the required volume (T=0, on the x-axis of the spider plot), control mouse models are assigned to groups and tumor volumes are monitored. Spider plots show % tumor volume change over time for each tumor model for untreated controls after enrollment. Fragments of CRC or lung cancer are implanted into mice, and when the tumors reach the required volume (T=0, on the x-axis of the spider plot), control mice are assigned as controls and tumor volumes are monitored. [Figure 7] Kaplan-Meier plot of time to tumor volume doubling in patient-derived NSCLC and CRC xenografts. A benefit of combination treatment was observed for time to tumor volume doubling. [Figure 8] Compound A potently inhibited KRAS G12C cell signaling and proliferation in a mutant-selective manner and demonstrated dose-dependent antitumor activity with efficacy driven by daily AUC. A. Aggregate best tumor growth inhibition in six KRAS G12C tumor models. JDQ443 efficacy was evaluated after oral dosing at 10, 30 and 100 mg / kg / day in six human KRAS G12C mutant CDX models in mice. In dark grey, NSCLC cell line models are shown, while in light grey, PDAC (MIA Paca-2) and esophageal (KYSE-410) cancer cell line models are shown. Data are averages from 2-11 independent in vivo studies. B-G. CDX-bearing mice bearing KRAS G12C mutant (C-G) and non-KRAS G12C mutant (NCI-441, KRAS G12V; B) tumors were treated orally with JDQ443 at the doses and schedules indicated. G. LU99 tumor-bearing mice were treated with JDQ443 by continuous intravenous infusion using a minipump. H-I. Simulated pop-PKPD metrics (H) Daily AUC of JDQ443 in mouse blood and (I) mean free KRAS G12C levels in tumors at steady state correlate with observed efficacy in LU99 (T / C or % regression). Points correspond to the mean and error bars correspond to simulated PK / PD metrics ±1 S.D. based on 100 simulated and observed efficacy metrics. *p<0.05 vs. vehicle, #p<0.05 vs. reciprocal by one-way ANOVA. [Figure 9]Effect of Compound A (JDQ443), Sotorasib (AMG510) and Adaglasib (MRTX-849) on the proliferation of KRAS G12C / H95 double mutants. Ba / F3 cells expressing the indicated FLAG-KRASG12C single or double mutants were treated with the indicated compound concentrations for 3 days and inhibition of proliferation was assessed by Cell titer glo viability assay. The y-axis shows the % growth of treated cells on day 3 of treatment and the x-axis shows the log concentration in μM of KRAS G12C inhibitors. [Figure 10] Western blot analysis of ERK phosphorylation to assess the effect of Compound A (JDQ443), sotorasib (AMG510) and adagrasib (MRTX-849) on KRAS G12C / H95 double mutant signaling. Ba / F3 cells expressing the indicated FLAG-KRASG12C single or double mutants were treated with the indicated compound concentrations for 30 min and inhibition of the MAPK pathway was assessed by probing cell lysates for reduction of pERK by Western blot. [Figure 11-1]FIG. 11: Synergy scores (SS) obtained in a 3-day cell viability assay in NCI H23 cells. Matrix combination proliferation assays (treatment time 3 days, cell titer glow assay) were performed in KRAS G12C mutant H23 cell lines using a KRASG12C inhibitor (labeled "KRASG12Ci" in FIG. 11) as single agent or in combination with 10 μM SHP099, an SHP2 inhibitor (labeled "SHP2i" in FIG. 11), in the presence of an upstream receptor kinase inhibitor BGJ398, an FGFR inhibitor (labeled "FGFRi" in FIG. 11), and either erlotinib, an EGFR inhibitor (labeled "EGFRi" in FIG. 11) or trametinib, a MEK inhibitor (labeled "MEKi" in FIG. 11) or a PI3K effector arm inhibitor alpelisib (labeled "PI3Kαi" in FIG. 11) and GDC0941, a pan-PI3K inhibitor (labeled "panPI3Ki" in FIG. 11). The synergy score (SS) is shown above each grid as an "SS" value. The values ​​in the grid are % growth inhibition values: values ​​higher than 100% indicate cell death. The values ​​on the x-axis of each grid indicate the concentration (μM) of the KRAS G12c inhibitor used. The values ​​on the y-axis of each grid indicate the concentration (μM) of the second agent (i.e., FGFR inhibitor, EGFR inhibitor, MEK inhibitor, PI3αK inhibitor and pan-PI3K inhibitor, respectively). [Figure 11-2] (As stated above.) [Figure 12] PI3K+ / -CDK4 inhibition improves KRAS G12C+SHP2 combination therapy. Double and higher order combinations of Compound A (JDQ443) improve single agent activity in LU99 lung xenografts (KRAS G12C, PIK3CAmut, CDKN2Adel). Combination of Compound A with SHP2 inhibitors, PI3K inhibitors or CDK4 / 6 inhibitors delays time to progression (TTP) compared to single agent treatment with Compound A. Time to progression increased from single agent to quadruple combination (TTP: single agent < double combination < triple combination < quadruple combination). [Figure 13] Dose response of compound A (JDQ443) in combination with EGFR inhibitors in NSCLC and CRC cell lines. [Figure 14] In vitro survival of colorectal cancer cell lines and lung cancer was assessed using CellTiterGlo after 7 days of treatment with KRAS G12C inhibitor Compound A ("NVP-JDQ443" in Figure 14) in combination with SOS1 inhibitor BI-3406. % growth inhibition: 0-99=proliferation retardation, 100=growth arrest / quiescence, 101-200=reduction in cell number / cell death. [Figure 15] PK and target occupancy profiles of JDQ443 RD 200 mg BID. Top panels show PK profiles at steady state. Error bars indicate standard deviation for the PK profile at each time point. Bottom panels show predicted target occupancy profiles, with lines indicating simulated medians and shaded areas indicating 5th to 95th percentile prediction intervals. [Figure 16] The top panel shows the best overall response across dose levels and indications for JDQ443 monotherapy. Waterfall plot: 37 (94.9%) patients with usable change from baseline tumor assessment; data is plotted from N=39 JDQ443 monotherapy patients. Best overall response is assessed by the investigator according to RECIST v1.1. Three (7.7%) patients had uPR contributing to ORR (confirmed and unconfirmed). uPR=unconfirmed PR with treatment continued without PD, pending confirmation. Intrapatient dose escalation per protocol was performed from 200mg QD to 200mg BID in four patients. The bottom panel shows the best overall response across doses in all patients with NSCLC. Waterfall plot: 19 (95.0%) NSCLC patients with usable change from baseline tumor assessment; data is plotted from N=20 NSCLC patients in the JDQ443 monotherapy cohort. [Figure 17] PET scan showing significant reduction in 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (18-F-FDG) avidity of the tumor mass after four cycles of treatment with Compound A administered at 200 mg BID in a patient with NSCLC. CT: computed tomography; PET, positron emission tomography. Arrow indicates location of tumor. [Figure 18] Serial axial CT / PET images and steady-state (Cycle 1, Day 14) JDQ443 PK exposure for combination therapy with Compound A. The combination of Compound A and a SHP2 inhibitor is efficacious. Efficacy of Compound A and TNO155 in patients with papillary duodenal carcinoma. Arrows indicate the location of the tumor.

[0074] KRAS G12C inhibitors Examples of KRAS G12C inhibitors useful in the combinations and methods of the invention include Compound A, sotorasib (Amgen), adagrasib (Mirati), D-1553 (InventisBio), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Lilly), BI1823911 (Boehringer), AS KRAS G12C (Ascentage Pharma), SF KRAS G12C (Sanofi), RMC032 (Revolution Medicine), JAB-21822 (Jacobio Pharmaceuticals), AST-KRAS G12C (Allist Pharmaceuticals), AZ KRAS G12C (Astra Zeneca), NYU-12VC1 (New York and RMC6291 (Revolution Medicines), or a pharma- ceutically acceptable salt thereof.

[0075] KRAS G12C inhibitors also include the compounds detailed in "KRAS G12C inhibitors", including those compounds detailed in WO 2013 / 155223, WO 2014 / 143659, WO 2014 / 152588, WO 2014 / 160200, WO 2015 / 054572, WO 2016 / 044772, WO 2016 / 049524, WO 2016164675, WO 2016168540 ... International Publication No. 2017 / 058805, International Publication No. 2017015562, International Publication No. 2017058728, International Publication No. 2017058768, International Publication No. 2017058792, International Publication No. 2017058805, International Publication No. 2017058807, International Publication No. 2017058902, International Publication No. 2017058915, International Publication No. 2017087528, International Publication No. 2017100546 Brochure, International Publication No. 2017 / 201161 Brochure, International Publication No. 2018 / 064510 Brochure, International Publication No. 2018 / 068017 Brochure, International Publication No. 2018 / 119183 Brochure, International Publication No. 2018 / 217651 Brochure, International Publication No. 2018 / 140512 Brochure, International Publication No. 2018 / 140513 Brochure, International Publication No. 2018 / 140514 Brochure, International Publication No. 2018 / 140598 Brochure, International Publication No. 2018 / 140599 Brochure Lett., International Publication No. 2018 / 140600, International Publication No. 2018 / 143315, International Publication No. 2018 / 206539, International Publication No. 2018 / 218070, International Publication No. 2018 / 218071, International Publication No. 2019 / 051291, International Publication No. 2019 / 099524, International Publication No. 2019 / 110751, International Publication No. 2019 / 141250, International Publication No. 2019 / 150305,The compound is selected from the compounds detailed in WO 2019 / 155399, WO 2019 / 213516, WO 2019 / 213526, WO 2019 / 217307 and WO 2019 / 217691. Examples are 1-(4-(6-chloro-8-fluoro-7-(3-hydroxy-5-vinylphenyl)quinazolin-4-yl)piperazin-1-yl)prop-2-en-1-one-methane(1 / 2) (compound 1); (S)-1-(4-(6-chloro-8-fluoro-7-(2-fluoro-6-hydroxyphenyl)quinazolin-4-yl)piperazin-1-yl)prop-2-en-1-one (compound 2); and 2-((S)-1-acrylolyl-4-(2-(((S)-1-methylpyrrolidin-2-yl)methoxy)-7-(naphthalen-1-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl)piperazin-2-yl)acetonitrile (compound 3).

[0076] KRAS G12C inhibitor compound A A preferred KRAS G12C inhibitor of the present invention is Compound A, which is 1-{6-[(4M)-4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one, or a pharma- ceutically acceptable salt thereof. Compound A is also known by the name "(R)-1-(6-(4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptan-2-yl)prop-2-en-1-one".

[0077] The synthesis of Compound A is described in the Examples below or in Example 1 of PCT Application WO 2021 / 124222, published June 24, 2021. The use of Compound A alone or in combination with additional therapeutic agents is described in PCT / CN2021 / 139694, filed December 20, 2021. Compound A is also known as "JDQ443" or "NVP-JDQ443".

[0078] The structure of compound A is as follows: [ka]

[0079] Alternatively, the structure of compound A can be depicted as follows: [ka]

[0080] Compound A is a potent and selective small molecule inhibitor of KRAS G12C that covalently binds to mutant Cys12 and traps KRAS G12C in an inactive GDP-bound state. Compound A is structurally unique compared to sotorasib or adagrasib; its binding mode is a novel approach to reach residue C12 and does not have a direct interaction with residue H95.

[0081] Preclinical data show that Compound A binds to KRAS G12C with low reversible binding affinity to the RAS SWII pocket and specifically inhibits downstream cell signaling and proliferation driven by KRAS G12C in cell lines, but not in KRAS wild-type (WT) or MEK Q56P mutant cell lines. Compound A demonstrated deep and persistent target occupancy leading to antitumor activity in different KRAS G12C mutant xenograft models.

[0082] SHP2 Inhibitors Examples of SHP2 inhibitors useful in the combinations and methods of the invention include TNO155, JAB3068 (Jacobio), JAB3312 (Jacobio), RLY1971 (Roche), SAR442720 (Sanofi), RMC4450 (Revolution Medicines), BBP398 (Navire), BR790 (Shanghai Blueray), SH3809 (Nanjing Sanhome), PF0724982 (Pfizer), ERAS601 (Erasca), RX-SHP2 (Redx Pharma), ICP189 (InnoCare), HBI2376 (HUYA Bioscience), ETS001 (Shanghai ETERN Biopharma), TAS-ASTX (Taiho Oncology) and X-37-SHP2 (X-37), or a pharma- ceutically acceptable salt thereof.

[0083] Examples of SHP2 inhibitors useful in the combinations and methods of the invention, particularly in dual combinations and methods of treating cancer using dual combinations as described herein, include JAB3068 (Jacobio), JAB3312 (Jacobio), RLY1971 (Roche), SAR442720 (Sanofi), RMC4450 (Revolution Medicines), BBP398 (Navire), BR790 (Shanghai Blueray), SH3809 (Nanjing Sanhome), PF0724982 (Pfizer), ERAS601 (Erasca), RX-SHP2 (Redx Pharma), ICP189 (InnoCare), HBI2376 (HUYA Bioscience), ETS001 (Shanghai ETERN Biopharma), TAS-ASTX (Taiho Oncology), and X-37-SHP2 (X-37).

[0084] Particularly preferred SHP2 inhibitors for use according to the invention, in particular in the triple combinations and methods of using the triple combinations of the invention, may be selected from: [ka]

[0085] A particularly preferred SHP2 inhibitor for use according to the invention, particularly in the triple combinations and methods of using the triple combinations of the invention, is (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (TNO155), or a pharma- ceutically acceptable salt thereof. TNO155 is synthesized according to Example 69 of WO 2015 / 107495, which is incorporated by reference in its entirety. A preferred salt of TNO155 is the succinate salt.

[0086] Further, as SHP2 inhibitors, there are disclosed, for example, those described in WO 2015 / 107493, WO 2015 / 107494, WO 2015 / 107495, WO 2016 / 203406, WO 2016 / 203404, WO 2016 / 203405, WO 2017 / 216706, WO 2017 / 156397, WO 2020 / 063760, WO 2018 / 172984, and the like. Brochure, International Publication No. 2017 / 211303 Brochure, International Publication No. 21 / 061706 Brochure, International Publication No. 2019 / 183367 Brochure, International Publication No. 2019 / 183364 Brochure, International Publication No. 2019 / 165073 Brochure, International Publication No. 2019 / 067843 Brochure, International Publication No. 2018 / 218133 Brochure, International Publication No. 2018 / 081091 Brochure, International Publication No. 2018 / 057884 Brochure, International Publication No. 2020 / 247643 Brochure, International Publication International Publication No. 2020 / 076723, International Publication No. 2019 / 199792, International Publication No. 2019 / 118909, International Publication No. 2019 / 075265, International Publication No. 2019 / 051084, International Publication No. 2018 / 136265, International Publication No. 2018 / 136264, International Publication No. 2018 / 013597, International Publication No. 2020 / 033828, International Publication No. 2019 / 213318, International Publication No. 2019 / 158 International Publication No. 019, International Publication No. 2021 / 088945, International Publication No. 2020 / 081848, International Publication No. 21 / 018287, International Publication No. 2020 / 094018, International Publication No. 2021 / 033153, International Publication No. 2020 / 022323, International Publication No. 2020 / 177653, International Publication No. 2021 / 073439, International Publication No. 2020 / 156243, International Publication No. 2020 / 156242,Examples of the compounds include those described in International Publication No. WO 2020 / 249079, International Publication No. WO 2020 / 033286, International Publication No. WO 2021 / 061515, International Publication No. WO 2019 / 182960, International Publication No. WO 2020 / 094104, International Publication No. WO 2020 / 210384, International Publication No. WO 2020 / 181283, International Publication No. WO 2021 / 043077, International Publication No. WO 2021 / 028362, International Publication No. WO 2020 / 259679, International Publication No. WO 2020 / 108590, and International Publication No. WO 2019 / 051469.

[0087] TNO155 is an orally bioavailable allosteric inhibitor of Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2, encoded by the PTPN11 gene), which transmits signals from activated receptor tyrosine kinases (RTKs) to downstream pathways, including the mitogen-activated protein kinase (MAPK) pathway. SHP2 is also involved in immune checkpoint and cytokine receptor signaling. TNO155 has demonstrated efficacy in a broad range of RTK-dependent human cancer cell lines and in vivo tumor xenografts.

[0088] PI3K inhibitors Examples of PI3K inhibitors useful in the combinations and methods of the present invention include dactolisib, apitolisib, gedatolisib buparlisib, duvelisib, copanlisib, idelalisib, alpelisib taselisib and pictilisib.Preferred PI3K inhibitors of the present invention include AMG511, buparlisib and alpelisib.In a preferred embodiment of the present invention, alpelisib is the PI3K inhibitor.

[0089] In the combination of the present invention, each of the therapeutically active agents may be administered separately, simultaneously or sequentially in any order.

[0090] In the combination of the present invention, Compound A and / or TNO155 may be administered in an oral dosage form.

[0091] In another embodiment, there is provided a pharmaceutical composition comprising a pharmaceutical combination of the present invention and at least one pharma- ceutically acceptable carrier.

[0092] Cancers to be treated by the combinations and methods of the present invention Therefore, the combination of the present invention can be useful in the treatment of cancer and in the cancer or tumor that is KRAS G12C mutation.The combination of the present invention can be useful in the treatment of cancer or tumor selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer, and squamous lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendix cancer, small intestine cancer, esophageal cancer, hepatobiliary cancer (including liver cancer and bile duct cancer), bladder cancer, ovarian cancer, and solid tumors, especially when the cancer or tumor has KRAS G12C mutation.Cancer that is of unknown primary site but shows KRAS G12C mutation may also benefit from treatment by the method of the present invention.

[0093] The cancer or tumor to be treated may be selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendix cancer, small intestine cancer, esophageal cancer, hepatobiliary cancer (including liver cancer, cholangiocarcinoma and bile duct carcinoma), bladder cancer, ovarian cancer, ampullary duodenal carcinoma and solid tumors, particularly where the cancer or tumor has a KRAS G12C mutation.

[0094] The cancer or tumor to be treated may be selected from non-small cell lung cancer, colorectal cancer, bile duct cancer, ovarian cancer, ampullary duodenal cancer and pancreatic cancer, particularly where the cancer or tumor has a KRAS G12C mutation.

[0095] Other cancers to be treated by the compounds, combinations and methods of the invention include gastric cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, and Hodgkin's lymphoma, particularly where the cancer harbors a KRAS G12C mutation.

[0096] In particular, the present invention provides methods of treating and combinations for use in treating a cancer selected from the group consisting of lung cancer (e.g., lung adenocarcinoma and non-small cell lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma) and solid tumours, particularly where the cancer or tumour harbours a KRAS G12C mutation.

[0097] As shown in the examples, compound A and combination of the present invention show antitumor activity in xenograft models with one, two or three mutations selected from KRAS G12C, PIK3CA and CDKN2A.Therefore, the cancer to be treated by the combination and method of the present invention includes cancer or solid tumor with one, two or three mutations selected from KRAS G12C, PIK3CA and CDKN2A and combinations thereof; for example, cancer with KRAS G12C and CDKN2A mutation; and cancer with KRAS G12C, PIK3CA and CDKN2A mutation.For example, the cancer to be treated can be lung cancer with KRAS G12C and CDKN2A mutation (e.g., non-small cell lung cancer); or lung cancer with KRAS G12C, PIK3CA and CDKN2A mutation (e.g., non-small cell lung cancer).

[0098] Cancers with one, two or three mutations selected from KRAS G12C, PIK3CA and CDKN2A may also be selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma), appendix cancer, small intestine cancer, esophageal cancer, hepatobiliary cancer (including liver cancer, cholangiocarcinoma and bile duct carcinoma), bladder cancer, ovarian cancer, ampullary duodenal carcinoma and solid tumors, particularly where the cancer or tumor has a KRAS G12C mutation.

[0099] In an embodiment of the invention, the cancer to be treated by compound A or by combination or in the method of the invention is selected from the group consisting of melanoma, gastric lymphoma, Burkitt's lymphoma, head and neck squamous cell carcinoma, oral carcinoma, pancreatic adenocarcinoma, non-small cell lung cancer, esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, epithelial ovarian cancer and prostate cancer; optionally the cancer has a KRAS G12C mutation and / or a CDKN2A mutation; or the cancer has a KRAS G12C, PIK3CA and CDKN2A mutation.

[0100] In an embodiment of the invention, the cancer to be treated by compound A or by combination or in the method of the invention is selected from the group consisting of breast cancer, ovarian cancer, lung cancer, gastric cancer, stomach cancer and brain cancer; optionally the cancer has a KRAS G12C mutation and / or a PIK3CA mutation; or the cancer has a KRAS G12C, PIK3CA and CDKN2A mutation.

[0101] The cancer may be early stage, mid-stage, late stage, or may be metastatic cancer. In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer is a refractory cancer. In some embodiments, the cancer is a relapsing cancer. In some embodiments, the cancer is an unresectable cancer.

[0102] The cancer can be early stage, intermediate stage, late stage, or metastatic cancer.

[0103] Compound A and combinations of the invention may also be useful in the treatment of solid malignancies characterised by mutations in RAS.

[0104] Compound A and the combinations of the present invention may also be useful in the treatment of solid malignant tumours characterised by one or more mutations in KRAS, in particular the G12C mutation in KRAS.

[0105] The present invention provides compounds A and combinations of the invention for use in the treatment of cancers or solid tumors characterized by acquired KRAS alterations selected from G12D / R / V / W, G13D, Q61H, R68S, H95D / Q / R, Y96C, Y96D and high level amplification of the KRASG12C allele, or characterized by acquired bypass resistance mechanisms, including MET amplification, activating mutations in NRAS, BRAF, MAP2K1 and RET, oncogenic fusions involving ALK, RET, BRAF, RAF1 and FGFR3, and loss-of-function mutations in NF1 and PTEN.

[0106] Thus, as a further embodiment, the present invention provides the combination of the present invention for use in therapy. The present invention also provides a triple combination consisting of compound A, or a pharma- ceutically acceptable salt thereof, an SHP2 inhibitor, such as TNO155, or a pharma- ceutically acceptable salt thereof, and a third therapeutically active agent. As a further embodiment, the present invention provides the combination of the present invention for use in therapy. In a preferred embodiment, the therapy or the therapy for which the medicament is useful is selected from diseases that can be treated by inhibition of RAS mutant proteins, in particular KRAS, HRAS or NRAS G12C mutant proteins. In another embodiment, the present invention provides a method for treating a disease that is treated by inhibition of RAS mutant proteins, in particular G12C mutants of any of KRAS, HRAS or NRAS proteins, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the combination of the present invention.

[0107] In a more preferred embodiment, the disease is selected from the above list, preferably non-small cell lung cancer, colorectal cancer and pancreatic cancer.

[0108] In a preferred embodiment, the therapy is for a disease that can be treated by inhibition of a RAS mutant protein, in particular a G12C mutant of either the KRAS, HRAS or NRAS protein. In a more preferred embodiment, the disease is selected from the above list, preferably non-small cell lung cancer, colorectal cancer and pancreatic cancer, characterized by a G12C mutation of either the KRAS, HRAS or NRAS protein.

[0109] In another embodiment, there is a method of treating (e.g., reducing, inhibiting, or delaying the progression of) a cancer or tumor in a subject, comprising administering to a subject in need thereof a pharmaceutical composition comprising Compound A, or a pharma- ceutically acceptable salt thereof, in combination with a second therapeutic agent described herein, and optionally in combination with a third combination.

[0110] Accordingly, the present invention provides a method of treating (e.g. reducing, inhibiting or delaying the progression of) a cancer or tumor in a patient in need thereof, comprising administering to a patient in need thereof a therapeutically active amount of a combination of the present invention, wherein the cancer is lung cancer (including lung adenocarcinoma and non-small cell lung cancer), colorectal cancer (including colorectal adenocarcinoma), pancreatic cancer (including pancreatic adenocarcinoma), uterine cancer (including endometrial cancer), rectal cancer (including rectal adenocarcinoma) and a solid tumor, optionally wherein the cancer is KRAS-, NRAS- or HRAS-G12C mutant.

[0111] Cancer or tumor refractory to KRAS G12C inhibitors The methods and combinations of the present invention may be particularly useful in treating cancers or tumors that are refractory or resistant to prior treatment with a KRAS G12C inhibitor. Examples of such KRAS G12C inhibitors include Compound A, sotorasib (Amgen), adagrasib (Mirati), D-1553 (InventisBio), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Lilly), BI1823911 (Boehringer), AS KRAS G12C (Ascentage Pharma), SF KRAS G12C (Sanofi), RMC032 (Revolution Medicine), JAB-21822 (Jacobio Pharmaceuticals), AST-KRAS G12C (Allist Pharmaceuticals), AZ KRAS G12C (Astra Zeneca), NYU-12VC1 (New York In one embodiment, the cancer, e.g., NSCLC, has already been treated with a KRAS G12C inhibitor (e.g., sotrasib, adagrasib, D-1553, and GDC6036).

[0112] Combination therapies comprising a KRAS G12C inhibitor (e.g., Compound A, or a pharma- ceutically active salt thereof, and a second therapeutically active agent, and optionally a third therapeutic agent, are expected to be particularly useful in overcoming this resistance.

[0113] The methods and combinations of the invention may be useful as a first line therapy (or as a second or more advanced line therapy). For example, the patient may be a treatment-naive patient or a patient who has progressed and / or relapsed on prior therapy.

[0114] For example, patients or subjects to be treated by the methods and combinations of the present invention include patients suffering from cancer, e.g., KRAS G12C mutant NSCLC, including advanced (metastatic or unresectable) KRAS G12C mutant NSCLC, optionally having progressed on prior therapy.

[0115] In an embodiment of the invention, a subject or patient who is treated with and is likely to benefit from treatment with Compound A monotherapy or combination therapy using the combination therapies described herein is selected from: - Patients with KRAS G12C mutant solid tumors (e.g., advanced (metastatic or unresectable) KRAS G12C mutant solid tumors) who have, optionally, received and failed standard of care therapy or who are intolerant or ineligible or refractory to prior investigational and / or approved therapy; - Patients with KRAS G12C mutant NSCLC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant NSCLC) who have optionally received and failed a platinum-based chemotherapy regimen and an immune checkpoint inhibitor therapy, either in combination or sequentially; - Patients with KRAS G12C mutant CRC (e.g., progressive (metastatic or unresectable) KRAS G12C mutant CRC) who have received and failed standard of care therapy, optionally including fluropyrimidine, oxaliplatin, and / or irinotecan-based chemotherapy; and - Patients with KRAS G12C mutant NSCLC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), optionally having been previously treated with a KRAS G12C inhibitor (e.g., sotrasib, adagrasib, GDC6036 or D-1553).

[0116] Compound A alone or in combination with another therapeutic agent described herein may be useful in the treatment of patients selected from: Patients with NSCLC (G12Ci-naive) whose tumors harbor a KRAS G12C tumor mutation and have received a prior platinum-based chemotherapy regimen and immune checkpoint inhibitor therapy, either in combination or sequentially; Patients with NSCLC whose tumors harbor a KRAS G12C tumor mutation and who have received a prior platinum-based chemotherapy regimen and immune checkpoint inhibitor therapy, either in combination or sequentially, followed directly by one line of treatment with a KRAS G12C inhibitor other than Compound A, e.g., sotorasib or adgrasib given as a single agent, which was discontinued within 6 months of the first day of study treatment in this study (G12Ci treatment); Patients with CRC whose tumors harbored a KRAS G12C tumor mutation and received fluoropyrimidine-, oxaliplatin-, or irinotecan-based chemotherapy.

[0117] In a further embodiment, Compound A, or a pharma- ceutically acceptable salt thereof, is administered to a patient in need thereof in an amount effective to treat cancer.

[0118] In an embodiment of the invention, the amount of Compound A, or a pharma- ceutically acceptable salt thereof, and the second and third therapeutic agents, if present, administered to a subject in need thereof is effective in an amount effective to treat cancer.

[0119] Dosage and Administration Regimen When Compound A is used as monotherapy, the total recommended daily dose of Compound A is 400 mg given once or twice daily, given continuously (i.e., no rest periods). The recommended dose for Compound A monotherapy is 100 mg BID given continuously based on observed safety, PK, and efficacy data.

[0120] When Compound A is used as monotherapy or in combination therapy, it is preferably taken with food, for example immediately (within 30 minutes) after a meal.

[0121] The doses of the KRAS G12C inhibitor and the second therapeutically active agent, as well as the third therapeutically active agent in the combination therapy according to the present invention are designed to be pharmacologically active and to produce an anti-tumor response.

[0122] When the KRAS G12C inhibitor is compound A in the combination of the present invention, compound A, or a pharma- ceutically acceptable salt thereof, is administered at a therapeutically effective dose ranging from 50 to 1600 mg per day, for example, 200 to 1600 mg per day, or 400 to 1600 mg per day or 50 to 400 mg per day. The total daily dose of compound A may be selected from 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, 1000, 1200 and 1600 mg. For example, the total daily dose of compound A may be selected from 100, 200, 300, 400, 600, 800, 1000, 1200 and 1600 mg.

[0123] The total daily dose of Compound A can be administered continuously in a QD (once a day) or BID (twice a day) regimen. For example, Compound A can be administered at a dose of 200mg BID (total daily dose of 400mg), 400mg QD (total daily dose of 400mg). Compound A can also be administered at a dose of 100mg BID (total daily dose of 200mg) or at a dose of 200mg QD (total daily dose of 200mg). PK / PD modeling predicts high levels of target occupancy that are sustained at the recommended dose of 200mg BID. Compound A at 100mg BID is also predicted to enable an adequate therapeutic window when combined with selected therapies.

[0124] When an SHP2 inhibitor is present in the combination of the present invention and TNO155 is an SHP2 inhibitor, the dose of TNO155 in the combination of the present invention is designed to be pharmacologically active and have the potential for synergistic antitumor effects while simultaneously minimizing the possibility of unacceptable toxicity due to the inhibitory activity of both agents on MAPK pathway signaling. Thus, TNO155 can be administered at a total daily dose ranging from 10 to 80 mg, or 10 to 60 mg. For example, the total daily dose of TNO155 can be selected from 10, 15, 20, 30, 40, 60 and 80 mg. The total daily dose of TNO155 can be administered continuously QD (once a day) or BID (twice a day), and can be administered QD or BID on a 2 week on / 1 week off schedule. The total daily dose of TNO155 can be administered continuously QD (once a day) or BID (twice a day), and can be administered continuously (ie, with no breaks in administration) QD or BID.

[0125] In the combinations of the present invention, Compound A is administered at a dose ranging from 50 to 1600 mg per day (e.g., 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, 1000, 1200 or 1600 mg) or 200 to 1600 mg per day (e.g., 200, 300, 400, 600, 800, 1000, 1200 or 1600 mg) and TNO155 is administered at a dose ranging from 10 to 80 mg per day (0, 15, 20, 30, 40, 60 or 80 mg), Compound A is administered on a continuous schedule and TNO is administered on either a 2 week on / 1 week off schedule or a continuous schedule.

[0126] In the combinations of the present invention, Compound A is administered at a dose ranging from 50 to 1600 mg (e.g., 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, 1000, 1200 or 1600 mg) per day on a continuous schedule or 200 to 1600 mg (e.g., 200, 300, 400, 600, 800, 1000, 1200 or 1600 mg) per day, and TNO155 is administered at a dose ranging from 10 to 80 mg (0, 15, 20, 30, 40, 60 or 80 mg) on ​​either a 2 week on / 1 week off schedule or a continuous schedule.

[0127] EGFR inhibitors, such as cetuximab, may be used in the combination therapy of the present invention, particularly when the cancer to be treated is colorectal cancer. When present, cetuximab is used as a concentrated injectable solution and is administered intravenously (IV). Cetuximab is administered at a dose of 400 mg / m 2 IV initial dose (typically administered as a 120-minute intravenous infusion), and 250 mg / m 2 / week, administered as a 60-minute infusion every other week. Alternatively, cetuximab may be administered at 500 mg / m once every two weeks. 2 The initial and subsequent doses may be administered once every two weeks. Typically, the total daily dose of compound A in the combination of the present invention may be selected from 100 mg to 400 mg, for example, 200 mg to 400 mg. The total daily dose may be administered once a day or twice a day (BID) continuously.

[0128] An example of a dosing regimen for the combination of Compound A and cetuximab is Compound A administered QD or BID continuously in combination with cetuximab administered once weekly (initial dose of 400 mg / m administered as a 120 minute intravenous infusion, followed by subsequent doses of 250 mg / m administered as a 60 minute infusion every week. Typically, the total exposure to cetuximab will not exceed an initial dose of 500 mg / m or 400 mg / m every 2 weeks followed by 250 mg / m once a week.

[0129] Typical dose levels for Compound A in combination with cetuximab may be as follows:

[0130] [Table 1]

[0131] MEK inhibitors, such as trametinib, may be used in the combination therapy of the present invention. Trametinib may be administered continuously (i.e., without a break) at a dose of 0.5 mg, 1 mg, or 2 mg once daily (QD). Based on clinical PK and PD data, a 1 mg QD dose of trametinib is considered to be potentially pharmacologically active. Compound A and / or trametinib may be administered with food. Typically, the total daily dose of Compound A in the combination of the present invention may be selected from 100 mg to 400 mg, for example, 200 mg to 400 mg. The total daily dose may be administered continuously once daily or twice daily (BID).

[0132] Typical dose levels for the combination of Compound A with trametinib may be as follows:

[0133] [Table 2]

[0134] CDK4 / 6 inhibitors, such as palbociclib or ribociclib, may be used in the combination therapy of the present invention. When ribociclib is used as the combination partner, it may be administered at a total daily dose of 100mg-600mg QD, 3 weeks on / 1 week off. For example, ribociclib may be administered once a day at a dose of 100mg, 200mg, 300mg, 400mg or 600mg. Typically, the total daily dose of compound A in the combination of the present invention may be selected from 100mg-400mg, for example, 200mg-400mg. The total daily dose may be administered once a day or twice a day (BID) continuously.

[0135] Typical dose levels for the combination of Compound A with ribociclib may be as follows:

[0136] [Table 3]

[0137] Pharmaceutical Compositions The KRAS G12C inhibitor (e.g., Compound A, or a pharma- ceutically acceptable salt thereof) may be administered simultaneously with, prior to, or subsequent to one or more (e.g., one or two) other therapeutically active agents. Compound A, or a pharma- ceutically acceptable salt thereof, may be administered separately by the same or different administration route, or together in the same pharmaceutical composition as the other therapeutically active agent.

[0138] In another aspect, the present invention provides pharma- ceutically acceptable compositions comprising a therapeutically effective amount of one or more (e.g., one or two) therapeutic agents selected from a KRAS G12C inhibitor (e.g., Compound A), a SHP2 inhibitor (e.g., TNO155), and optionally a third agent described herein, formulated together with one or more pharma- ceutically acceptable carriers (additives) and / or diluents.

[0139] In another aspect, the present invention provides a pharmaceutical composition comprising one, two or three compounds present in the combination of the present invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. In another aspect, the present invention provides a pharmaceutical composition comprising one or more (e.g., one or two) therapeutically active agents selected from a KRAS G12C inhibitor, e.g., Compound A, or a pharma- ceutically acceptable salt thereof, and a SHP2 inhibitor, e.g., TNO155, or a pharma- ceutically acceptable salt thereof, and a third therapeutically active agent. In a further embodiment, the composition comprises at least two pharma- ceutically acceptable carriers, e.g., those described herein. Preferably, the pharma- ceutical acceptable carriers are sterile. The pharmaceutical composition may be formulated for a particular route of administration, such as oral administration, parenteral administration, and rectal administration. Furthermore, the pharmaceutical composition of the present invention may be configured in a solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or in a liquid form (including, but not limited to, solutions, suspensions, or emulsions). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations, such as sterilization, and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifying agents, buffers, and the like.

[0140] Typically, the pharmaceutical composition is a tablet or gelatin capsule containing the active ingredient together with one or more of the following: a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; d) disintegrants, such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) Absorbents, colouring matters, flavouring matters and sweetening matters.

[0141] In one embodiment, the pharmaceutical composition is a capsule containing only the active ingredient.

[0142] Tablets may be film coated or enteric coated according to methods known in the art.

[0143] Compositions suitable for oral administration contain an effective amount of the compound in the combination of the present invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs, liquids or solid dispersions. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives to provide pharma-ceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharma-ceutically acceptable excipients suitable for the manufacture of tablets. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating or disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be used. Formulations for oral use can be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0144] Some injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or contain adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts for regulating the osmotic pressure and / or buffers. In addition, they may also contain other therapeutically useful substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75% of the active ingredient, or about 1-50% of the active ingredient.

[0145] The composition suitable for transdermal administration comprises an effective amount of the compound of the present invention and a suitable carrier.The carrier suitable for transdermal delivery comprises an absorbable pharmacologically acceptable solvent to support the passage through the host's skin.For example, the transdermal device is in the form of a dressing that comprises a backing member, a reservoir that contains the compound, optionally with a carrier, optionally a rate-controlling barrier for delivering the compound to the host's skin at a controlled and predetermined rate over a long period of time, and a means for fixing the device to the skin.

[0146] Compositions suitable for topical administration, such as administration to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations for delivery, such as by aerosol. Such topical delivery systems may be particularly suitable for skin applications, such as for treating skin cancer, and for preventative use, such as in sunscreen creams, lotions, and sprays. Thus, they are particularly suitable for topical use, including cosmetic formulations, well known in the art. Such compositions may contain solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives.

[0147] As used herein, topical application may also refer to inhalation or intranasal application. These may be conveniently delivered in the form of a dry powder from a dry powder inhaler (alone, in a mixture, e.g., as a dry blend with lactose, or as mixed component particles, e.g., with phospholipids), or in the form of an aerosol spray from a pressurized container, pump, spray, atomizer, or nebulizer, with or without the use of a suitable propellant.

[0148] In one embodiment, the present invention provides a product comprising compound A, or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition characterized by KRAS, HRAS or NRAS G12C mutation. The product provided as a combined preparation includes a composition comprising a compound of the present invention and one or more (e.g., one or two) therapeutically active agents selected from a SHP2 inhibitor (e.g., TNO155, or a pharmaceutically acceptable salt thereof), a KRAS inhibitor (e.g., compound A, or a pharmaceutically acceptable salt thereof), and the other therapeutic agent is in a separate form, e.g., in the form of a kit.

[0149] In one embodiment, the invention provides a pharmaceutical composition comprising a compound of the invention and another therapeutic agent. Optionally, the pharmaceutical composition may comprise a pharma- ceutically acceptable carrier as described above.

[0150] In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains Compound A, or a pharma- ceutically acceptable salt thereof; TNO155, or a pharma- ceutically acceptable salt thereof, and a third therapeutically active agent as described herein. In one embodiment, the kit comprises a means for separately holding the compositions, such as a container, a divided bottle, or a divided foil pouch. An example of such a kit is a blister pack, such as those typically used for packaging tablets, capsules, and the like.

[0151] The kits of the invention can be used to administer different dosage forms, e.g., oral and parenteral dosage forms, to administer the separate compositions at different dosage intervals, or to titrate the separate compositions relative to one another. To aid in compliance, the kits of the invention typically include administration instructions.

[0152] In the combination therapy of the present invention, the compound of the present invention and the other therapeutic agent can be manufactured and / or formulated by the same or different manufacturers. Furthermore, the compound of the present invention and the other therapeutic agent can be combined into a combination therapy (i) before delivery of the combination product to the physician (e.g., in the case of a kit containing the compound of the present invention and the other therapeutic agent); (ii) immediately before administration by the physician (or under the physician's guidance); (iii) by the patient himself, for example, during sequential administration of the compound of the present invention and the other therapeutic agent. The compound of the present invention can be administered simultaneously with, before or after one or more other therapeutic agents. The compound of the present invention can be administered separately by the same or different administration route, or together in the same pharmaceutical composition with the other agent.

[0153] In general, a suitable daily dose of the combination of the invention will be that amount of each compound that is the lowest dose effective to produce a therapeutic effect.

[0154] In another aspect, the present invention provides pharma- ceutically acceptable compositions comprising a therapeutically effective amount of one or more of the subject compounds described above, formulated together with one or more pharma- ceutically acceptable carriers (additives) and / or diluents.

[0155] definition The general terms used above and below preferably have the following meanings within the context of the present disclosure, unless otherwise stated; whenever used, more general terms may, independently of each other, be replaced by more specific definitions or remain as they are to define more specific embodiments of the invention.

[0156] In particular, when a dose or administration amount is mentioned, it is intended to include a range around the stated value of plus or minus 10%, or plus or minus 5%.

[0157] As is conventional in the art, dosage refers to the amount of therapeutic agent in free form. For example, if a dosage of 20 mg of TNO155 is referred to and TNO155 is used as its succinate salt, the amount of therapeutic agent used is equivalent to 20 mg of the free form of TNO155.

[0158] The term "subject" or "patient" as used herein is intended to include animals that may suffer from or be affected by cancer or any disorder in which cancer is directly or indirectly implicated. Examples of subjects include mammals, such as humans, apes, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In one embodiment, the subject is a human, e.g., a human that has, is at risk of, or may potentially have cancer.

[0159] The term "treat" or "treatment" as used herein includes treatment that alleviates, alleviates or relieves at least one symptom in a subject, or results in delay of progression of a disease. For example, treatment can be a reduction in one or several symptoms of a disorder, such as cancer, or partial or complete eradication of a disorder. Within the meaning of this disclosure, the term "treat" also refers to stopping a disease, delaying its onset (i.e., the period before clinical signs of the disease), and / or reducing the risk of developing or worsening a disease.

[0160] "Treatment" may also be determined by efficacy and / or pharmacodynamic endpoints and may be defined as an improvement in one or more of safety, efficacy and tolerability. Efficacy of monotherapy or combination therapy may be determined by determining best overall response (BOR) by RECIST v.1.1, overall response rate (ORR), duration of response (DOR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS).

[0161] "Best overall response" (BOR) rate is recorded from the start of treatment to disease progression / recurrence and is defined as the best response by RECIST 1.1.

[0162] "Overall response rate" (ORR) is defined as the proportion of patients with a BOR of CR or PR by RECIST 1.1.

[0163] "Duration of response" (DOR) by RECIST 1.1 is the time between the first reported response (CR or PR) and the date of progression or death from any cause, where death from any cause is considered a conservative event that meets the PFS event definition.

[0164] "Disease control rate" (DCR) by RECIST 1.1 is defined as the proportion of patients with a BOR of CR, PR, or SD by RECIST 1.1.

[0165] "Progression-free survival" (PFS) by RECIST 1.1 is defined as the time from the date of initiation of treatment to the date of first reported progression by RECIST 1.1 or death from any cause. If the patient was event-free, PFS will be censored at the date of the last adequate tumor assessment.

[0166] "Overall survival" (OS) is defined as the number of days between the date of initiation of study treatment and the date of death from any cause. If no death is reported before the end of the study or the analysis cutoff, survival will be censored at the date of the last known patient survival before / at the cutoff date. Survival times for patients without post-baseline survival information will be censored at the date of initiation of treatment.

[0167] "Treatment" may also be defined as an improvement in the reduction of adverse effects of monotherapy with Compound A or combination therapy as described herein.

[0168] The terms "comprising" and "including" are used herein in an open-ended, non-limiting sense, unless otherwise specified.

[0169] The terms "a," "an," and "the," and similar references in the context of describing the invention (particularly in the context of the claims which follow) should be construed to encompass both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. When the plural is used for compounds, salts, and the like, this is construed to mean a single compound, salt, or the like.

[0170] The term "combination therapy" or "in combination with" refers to the administration of two or more therapeutic agents to treat a condition or disorder (e.g., cancer) described in this disclosure. Such administration includes co-administration of these therapeutic agents substantially simultaneously, for example, in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration in multiple containers for each active ingredient, or in separate containers (e.g., capsules, powders, and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, such administration also includes the use of each type of therapeutic agent at about the same time or sequentially at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating the condition or disorder described herein.

[0171] Combination therapy can provide "synergistic effects" and be "synergistic", i.e., the effect achieved when active ingredients are used together is greater than the sum of the effects obtained from using the compounds separately. Synergistic effects can be obtained when active ingredients are (1) formulated and administered simultaneously or delivered simultaneously in a combined unit dose formulation; (2) delivered alternately or in parallel as separate formulations; or (3) delivered by some other regimen. When delivered in alternation therapy, synergistic effects can be obtained when compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., sequentially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. As used herein, synergy refers to the effect of two therapeutic agents, such as the compound TNO155 and compound A as SHP2 inhibitors, for example, in slowing the symptomatic progression of a proliferative disease, particularly cancer, or a condition thereof, which is greater than the simple addition of the effects of each drug administered by itself. Synergy can be calculated, for example, using a suitable method, such as the sigmoid Emax formula (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6:429-453 (1981)), the Loewe additive effect formula (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114:313-326 (1926)) and the median-effect formula (Chou, TC and Talalay, P., Adv. Enzyme Regul. 22:27-55 (1984)). Each of the equations referred to above can be applied to experimental data to generate corresponding graphs to aid in the evaluation of the effect of drug combinations. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, the isobologram curve, and the combination index curve, respectively.

[0172] The term "pharmaceutical combination" as used herein refers to either a fixed combination in one dosage unit form, or a non-fixed combination or kit-of-parts for combined administration, where two or more therapeutic agents can be administered simultaneously or separately and independently within a time interval, particularly such that these time intervals allow the combination partners to exhibit a synergistic effect, e.g. a synergistic effect.

[0173] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, or composition containing a compound of the invention effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0174] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio and without excessive toxicity, irritation, allergic response, other problem or complication.

[0175] As mentioned above, certain embodiments of the compounds may contain basic functional groups, such as amino or alkylamino groups, and thus may form pharma- ceutically acceptable salts with pharma-ceutically acceptable acids. The term "pharma-ceutically acceptable salts" in this respect refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts may be prepared in situ during the administration vehicle or dosage form manufacturing process, or by separately reacting the purified compounds of the present invention in free base form with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0176] Pharmaceutically acceptable salts of the subject compounds include conventional non-toxic salts or quaternary ammonium salts of the compounds, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids, such as hydrochloride, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids, such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. For example, a pharma-ceutically acceptable salt of TNO155 is the succinate salt.

[0177] In the combination of the present invention, Compound A, TNO155 and the third therapeutically active agent are also intended to represent unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have one or more atoms replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into TNO155 and the third therapeutically active agent include, where possible, isotopes of hydrogen, carbon, nitrogen, oxygen, and chlorine, e.g. 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 35 S, 36 The present invention includes isotopically labeled TNO155 and a third therapeutically active agent, including, for example, those that include a radioisotope, e.g. 3 H and 14 C, or non-radioactive isotopes, e.g. 2 H and 13 C. Isotopically labeled TNO155 and PD-1 inhibitors were used in metabolic studies ( 14 C), reaction kinetic studies (e.g. 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in radiotherapy of patients. Isotopically labeled compounds of the present invention may generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the accompanying examples using suitable isotopically labeled reagents.

[0178] In addition, the heavier isotopes, especially deuterium (i.e. 2Substitution with H or D) may result in certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improved therapeutic index. It is understood that deuterium is considered in this context as a substituent of either compound A, TNO155 or the third therapeutically active agent. The concentration of such heavier isotopes, specifically deuterium, can be defined by an isotopic enrichment factor. The term "isotopic enrichment factor" as used herein refers to the ratio between the abundance of an isotope and the natural abundance of a specified isotope. When a substituent in TNO155 or a third therapeutically active agent is designated as deuterium, such compounds have an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

[0179] In compound A, for example, the methyl group on the indazolyl ring can be deuterated or perdeuterated. EXAMPLES

[0180] Example 1: Preparation of 1-{6-[(4M)-4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one (Compound A) The synthesis of 1-{6-[(4M)-4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one (Compound A) is as described below.

[0181] Compound A is also known by the name "a(R)-1-(6-(4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptan-2-yl)prop-2-en-1-one."

[0182] General methods and conditions: Temperatures are given in ° C. Unless otherwise stated, all evaporations are carried out under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20 to 133 mbar).

[0183] The abbreviations used are conventional in the art.

[0184] Mass spectra were obtained on an LC-MS, SFC-MS, or GC-MS system using electrospray, chemical, and electron impact ionization methods with a Waters Acquity UPLC equipped with a Waters SQ detector, or mass spectra were obtained on an LCMS system using ESI methods with a Waters Acquity LCMS equipped with a PDA detector. [M+H] + refers to the protonated molecular ion of a chemical species.

[0185] NMR spectra were performed using Bruker Ultrashield™ 400 (400 MHz), Bruker Ultrashield™ 600 (600 MHz) and Bruker Ascend™ 400 (400 MHz) spectrometers with and without tetramethylsilane as an internal standard. Chemical shifts (δ values) are reported in ppm downfield from tetramethylsilane, and spectral splitting patterns are indicated as singlet (s), doublet (d), triplet (t), quartet (q), multiplet, unsplit or more overlapping signals (m), broad signal (br). Solvents are indicated in parentheses. Only proton signals observed and not overlapping with the solvent peak are reported.

[0186] Celite: Celite® (the Celite Corporation) = a diatomaceous earth-based filter aid Phase Separator: Biotage-Isolute Phase Separator - (Part Number: 120-1908-F for 70mL and Part Number: 120-1909-J for 150mL) SiliaMetS® Thiol: SiliCYCLE Thiol Metal Scavenger - (R51030B, particle size: 40-63 μm).

[0187] device Microwave: Unless otherwise stated, all microwave reactions were carried out in a Biotage Initiator operating at 0-400 W from a magnetron at 2.45 GHz with a Robot Eight / Robot Sixty throughput. UPLC-MS and MS analytical methods: A Waters Acquity UPLC equipped with a Waters SQ detector is used. UPLC-MS-1: Acquity HSS T3; particle size: 1.8 μm; column size: 2.1 × 50 mm; eluent A: HO + 0.05% HCOOH + 3.75 mM ammonium acetate; eluent B: CHCN + 0.04% HCOOH; gradient: 5 to 98% B in 1.40 min, then 98% B over 0.40 min; flow rate: 1 mL / min; column temperature: 60 °C. UPLC-MS-3: Acquity BEH C18; particle size: 1.7 μm; column size: 2.1 × 50 mm; eluent A: HO + 4.76% isopropanol + 0.05% HCOOH + 3.75 mM ammonium acetate; eluent B: isopropanol + 0.05% HCOOH; gradient: 1 to 98% B in 1.7 min, then 98% B over 0.1 min; flow rate: 0.6 mL / min; column temperature: 80 °C. UPLC-MS-4: Acquity BEH C18; particle size: 1.7 μm; column size: 2.1 × 100 mm; eluent A: HO + 4.76% isopropanol + 0.05% HCOOH + 3.75 mM ammonium acetate; eluent B: isopropanol + 0.05% HCOOH; gradient: 1–60% B in 8.4 min, then 60–98% B in 1 min; flow rate: 0.4 mL / min; column temperature: 80 °C. UPLC-MS-6: Acquity BEH C18; particle size: 1.7 μm; column size: 2.1 × 50 mm; eluent A: HO + 0.05% HCOOH + 3.75 mM ammonium acetate; eluent B: isopropanol + 0.05% HCOOH; gradient: 5 to 98% B in 1.7 min, then 98% B over 0.1 min; flow rate: 0.6 mL / min; column temperature: 80 °C.

[0188] Preparative method: Chiral SFC Method: C-SFC-1: Column: Amylose-C NEO 5 μm; 250 × 30 mm; Mobile phase; Flow rate: 80 mL / min; Column temperature: 40 °C; Back pressure: 120 bar. C-SFC-3: Column: Chiralpak AD-H 5μm; 100×4.6mm; Mobile phase; Flow rate: 3mL / min; Column temperature: 40°C; Back pressure: 1800psi.

[0189] [Table 4]

[0190] [Table 5]

[0191] [Table 6]

[0192] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts used in preparing the compounds of the invention are either commercially available or can be prepared by organic synthesis methods known to those skilled in the art. Additionally, the compounds of the invention can be prepared by organic synthesis methods known to those skilled in the art, as shown in the examples below.

[0193] The structures of all final products, intermediates and starting materials are confirmed by standard analytical spectroscopic properties, e.g., MS, IR, NMR. The absolute stereochemistry of the preferred (most active) atropisomer representatives has been determined by analysis of the X-ray crystal structure of the complex of each compound bound to the KRAS G12C mutant. In all other cases where X-ray structures are not available, the stereochemistry has been assigned by analogy, assuming that for each pair, the atropisomer showing the highest activity in the covalent competition assay has the same configuration as observed by X-ray crystallography for the representatives above. Absolute stereochemistry is assigned according to the Cahn-Ingold-Prelog rules.

[0194] Intermediate C1: Synthesis of tert-butyl 6-(3-bromo-4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate [ka] Step C.1: tert-Butyl 6-(tosyloxy)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C2) To a solution of tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate [1147557-97-8] (2.92 kg, 12.94 mmol) in DCM (16.5 L) was added DMAP (316.12 g, 2.59 mol) and TsCl (2.96 kg, 15.52 mol) at 20°C-25°C. To the reaction mixture was added Et3N (2.62 kg, 25.88 mol) dropwise at 10°C-20°C. The reaction mixture was stirred at 5°C-15°C for 0.5 h and then at 18°C-28°C for 1.5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. To the residue was added NaCl (5% in water, 23 L) followed by extraction with EtOAc (23 L). The combined aqueous layers were extracted with EtOAc (10 L x 2). The combined organic layers were washed with NaHCO3 (3% in water, 10 L x 2)) and concentrated under reduced pressure to give the title compound. 1 H NMR (400MHz, DMSO-d6)δ 7.81-7.70(m,2H), 7.53-7.36(m,2H), 4.79-4.62(m,1H), 3.84-3.68(m,4H ), 2.46-2.38(m,5H), 2.26-2.16(m,2H), 1.33(s,9H).UPLC-MS-1:Rt=1.18 min;MS m / z[M+H] + ;368.2.

[0195] Step C.2: 3,5-Dibromo-1H-pyrazole To a solution of 3,4,5-tribromo-1H-pyrazole [17635-44-8] (55.0 g, 182.2 mmol) in anhydrous THF (550 mL) was added n-BuLi (145.8 mL, 364.5 mmol) dropwise at -78°C over 20 min while maintaining the internal temperature at -78°C / -60°C. The RM was stirred at this temperature for 45 min. The reaction mixture was then carefully quenched with MeOH (109 mL) at -78°C and stirred at this temperature for 30 min. The mixture was allowed to reach 0°C and stirred for 1 h. The mixture was then diluted with EtOAc (750 mL) and HCl (0.5N, 300 mL) was added. The layers were concentrated under reduced pressure. The crude residue was dissolved in DCM (100 mL), cooled to -50°C and petroleum ether (400 mL) was added. The precipitated solid was filtered, washed with n-hexane (250 mL×2) and dried under reduced pressure to give the title compound. 1 H NMR (400MHz, DMSO-d6) δ 13.5(br s,1H), 6.58(s,1H).

[0196] Step C.3: tert-Butyl 6-(3,5-dibromo-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate To a solution of tert-butyl 6-(tosyloxy)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C2) (Step C.1, 900 g, 2.40 mol) in DMF (10.8 L) was added Cs2CO3 (1988 g, 6.10 mol) and 3,5-dibromo-1H-pyrazole (Step C.2, 606 g, 2.68 mol) at 15 °C. The reaction mixture was stirred at 90 °C for 16 h. The reaction mixture was poured into ice water / brine water (80 L) and extracted with EtOAc (20 L). The aqueous layer was re-extracted with EtOAc (10 L x 2). The combined organic layers were washed with brine (10 L), dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was triturated with dioxane (1.8 L) and dissolved at 60 °C. To the pale yellow solution, water (2.2 L) was added slowly and recrystallization started after the addition of 900 mL of water. The resulting suspension was cooled to 0° C., filtered and washed with cold water. The filter cake was triturated with n-heptane, filtered and then dried under reduced pressure at 40° C. to give the title compound. 1 H NMR(400MHz, DMSO-d6)δ 6.66(s,1H), 4.86-4.82(m,1H), 3.96-3.85(m,4H), 2.69-2.62(m,4H), 1.37(s,9H);UPLC-MS-3:Rt=1.19 min;MS m / z[M+H] + ;420.0 / 422.0 / 424.0.

[0197] Step C.4: Intermediate C3: tert-Butyl 6-(3-bromo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C3) To a solution of tert-butyl 6-(3,5-dibromo-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (step C.3, 960 g, 2.3 mol) in THF (9.6 L) was added dropwise at −80° C. under an inert atmosphere. The reaction mixture was stirred at −80° C. for 10 min. Then, iodomethane (1633 g, 11.5 mol) was added dropwise to the reaction mixture at −80° C. After stirring at −80° C. for 5 min, the reaction mixture was allowed to warm to 18° C. The reaction mixture was poured into saturated aqueous NH4Cl (4 L) and extracted with DCM (10 L). The separated aqueous layer was re-extracted with DCM (5 L) and the combined organic layers were concentrated under reduced pressure. The crude product was dissolved in 1,4-dioxane (4.8 L) at 60° C., then water (8.00 L) was added slowly dropwise. The resulting suspension was cooled to 17° C. and stirred for 30 minutes. The solid was filtered, washed with water and dried under reduced pressure to give the title compound. 1 H NMR (400MHz, DMSO-d6)δ 6.14(s,1H), 4.74-4.66(m,1H), 3.95-3.84(m,4H), 2.61-2.58(m,4H), 2.20(s,3H), 1.37(s,9H);UPLC-MS-1:Rt=1.18 min;MS m / z[M+H] + ;356.1 / 358.1.

[0198] Step C.5: tert-Butyl 6-(3-bromo-4-iodo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C4) To a solution of tert-butyl 6-(3-bromo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C3) (Step C.4, 350 g, 0.980 mol) in acetonitrile (3.5 L) was added NIS (332 g, 1.47 mol) at 15° C. The reaction mixture was stirred at 40° C. for 6 h. After completion of the reaction, the reaction mixture was diluted with EtOAc (3 L) and washed with water (5 L×2). The organic layer was washed with Na2SO3 (10% in water, 2 L), brine (2 L), dried (Na2SO4), filtered and concentrated under reduced pressure to give the title compound. 1 H NMR (400MHz, DMSO-d6)δ 4.81-4.77(m,1H), 3.94-3.83(m,4H), 2.61-5.59(m,4H), 2.26(s,3H), 1.37(s,9H);UPLC-MS-1:Rt=1.31 min;MS m / z[M+H] + ;482.0 / 484.0.

[0199] Step C.6: tert-Butyl 6-(3-bromo-4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C1) To a stirred suspension of tert-butyl 6-(3-bromo-4-iodo-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C4) (Step C.5, 136 g, 282 mmol) and 5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (Intermediate D1, 116 g, 310 mmol) in 1,4-dioxane (680 mL) was added aqueous KPO (2 M, 467 mL, 934 mmol), followed by RuPhos (13.1 g, 28.2 mmol) and RuPhos-Pd-G (14.1 g, 16.9 mmol). The reaction mixture was stirred at 80° C. for 1 h under inert atmosphere. After completion of the reaction, the reaction mixture was poured into 1M NaHCO3 aqueous solution (1 L) and extracted with EtOAc (1 L×3). The combined organic layers were washed with brine (1 L×3), dried (Na2SO4), filtered and concentrated under reduced pressure. The crude residue was purified by normal phase chromatography (eluent: 1 / 0 to 0 / 1 petroleum ether / EtOAc) to give a yellow oil. The oil was dissolved in petroleum ether (1 L) and MTBE (500 mL) and then concentrated under reduced pressure to give the title compound. 1 H NMR (400MHz, DMSO-d6) δ 7.81(s,1H), 7.66(s,1H), 5.94-5.81(m,1H), 4.90-4.78(m,1H), 3.99(br s,2H), 3.93-3.84(m,3H), 3.81-3.70(m,1H), 2.81-2.64(m,4H), 2.52(s,3H), 2.46-2.31(m,1 UPLC-MS-3:Rt=1.30 min;MS m / z[M+H] + ;604.1 / 606.1.

[0200] Intermediate D1: Synthesis of 5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole [ka] Step D.1: 1-Chloro-2,5-dimethyl-4-nitrobenzene To an ice-cold solution of 2-chloro-1,4-dimethylbenzene (3.40 kg, 24.2 mol) in AcOH (20.0 L) was added H2SO4 (4.74 kg, 48.4 mol, 2.58 L), followed by dropwise addition (dropping funnel) of a cold solution of HNO3 (3.41 kg, 36.3 mol, 2.44 L, 67.0% purity) in H2SO4 (19.0 kg, 193 mol, 10.3 L). The reaction mixture was then allowed to stir at 0-5 °C for 0.5 h. The reaction mixture was slowly poured into crushed ice (35.0 L) and a yellow solid precipitated. The suspension was filtered and the cake was washed with water (5.00 L x 5) to give a yellow solid, which was suspended in MTBE (2.00 L) for 1 h, filtered, and dried to give the title compound as a yellow solid. 1 H NMR (400MHz, CDCl3)δ 7.90(s,1H), 7.34(s,1H), 2.57(s,3H), 2.42(s,3H).

[0201] Step D.2: 3-Bromo-2-chloro-1,4-dimethyl-5-nitrobenzene To a cooled solution of 1-chloro-2,5-dimethyl-4-nitrobenzene (Step D.1, 2.00 kg, 10.8 mol) in TFA (10.5 L) was slowly added concentrated H2SO4 (4.23 kg, 43.1 mol, 2.30 L) and the reaction mixture was stirred at 20° C. NBS (1.92 kg, 10.8 mol) was added portionwise and the reaction mixture was heated at 55° C. for 2 h. The reaction mixture was cooled to 25° C. and then poured into a solution of crushed ice to give a pale white precipitate, which was filtered through vacuum, washed with cold water and dried under reduced pressure to give the title compound as a yellow solid, which was used in the next step without further purification. 1 H NMR (400MHz, CDCl3)δ 7.65(s,1H), 2.60(s,3H), 2.49(s,3H).

[0202] Step D.3: 3-Bromo-4-chloro-2,5-dimethylaniline To an ice-cold solution of 3-bromo-2-chloro-1,4-dimethyl-5-nitrobenzene (Step D.2, 2.75 kg, 10.4 mol) in THF (27.5 L) was added HCl (4M, 15.6 L) followed by Zn (2.72 kg, 41.6 mol) in portions. The reaction mixture was allowed to stir at 25° C. for 2 h. The reaction mixture was basified (to pH=8) by addition of saturated aqueous NaHCO3. The mixture was diluted with EtOAc (2.50 L), stirred vigorously for 10 min, and then filtered through a pad of Celite. The organic layer was separated and the aqueous layer was re-extracted with EtOAc (3.00 L×4). The combined organic layers were washed with brine (10.0 L), dried (Na2SO4), filtered, and concentrated under reduced pressure to give the title compound as a yellow solid, which was used in the next step without further purification. 1 H NMR (400MHz, DMSO-d6) δ 6.59(s,1H), 5.23(s,2H), 2.22(s,3H), 2.18(s,3H).

[0203] Step D.4: 3-Bromo-4-chloro-2,5-dimethylbenzenediazonium tetrafluoroborate BF3.Et2O (2.00 kg, 14.1 mol, 1.74 L) was dissolved in DCM (20.0 L) and cooled to -5 to -10 °C under nitrogen atmosphere. A solution of 3-bromo-4-chloro-2,5-dimethylaniline (step D.3, 2.20 kg, 9.38 mol) in DCM (5.00 L) was added to the above reaction mixture and stirred for 0.5 h. tert-Butylnitrile (1.16 kg, 11.3 mol, 1.34 L) was added dropwise and the reaction mixture was stirred at the same temperature for 1.5 h. TLC (petroleum ether: EtOAc = 5:1) showed no significant difference in the starting material (R f =0.45) was completely consumed. MTBE (3.00 L) was added to the reaction mixture to give a yellow precipitate, which was filtered through vacuum and washed with cold MTBE (1.50 L x 2) to give the title compound as a yellow solid, which was used in the next step without further purification.

[0204] Step D.5: 4-Bromo-5-chloro-6-methyl-1H-indazole To 18-crown-6 ether (744 g, 2.82 mol) in chloroform (20.0 L) was added KOAc (1.29 kg, 13.2 mol) and the reaction mixture was cooled to 20° C. Then, 3-bromo-4-chloro-2,5-dimethylbenzenediazonium tetrafluoroborate (Step D.4, 3.13 kg, 9.39 mol) was added slowly. Then, the reaction mixture was allowed to stir at 25° C. for 5 hours. After completion of the reaction, the reaction mixture was poured into ice-cold water (10.0 L) and the aqueous layer was extracted with DCM (5.00 L×3). The combined organic layers were washed with saturated aqueous NaHCO3 (5.00 L), brine (5.00 L), dried (Na2SO4), filtered and concentrated under reduced pressure to give the title compound as a yellow solid. 1 H NMR (600MHz, CDCl3)δ 10.42(br s,1H), 8.04(s,1H), 7.35(s,1H), 2.58(s,3H).UPLC-MS-1:Rt=1.02 min;MS m / z[M+H] + ;243 / 245 / 247.

[0205] Step D.6: 4-Bromo-5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole To a solution of PTSA (89.8 g, 521 mmol) and 4-bromo-5-chloro-6-methyl-1H-indazole (step D.5, 1.28 kg, 5.21 mol) in DCM (12.0 L) was added DHP (658 g, 7.82 mol, 715 mL) dropwise at 25 °C. The mixture was stirred at 25 °C for 1 h. After completion of the reaction, the reaction mixture was diluted with water (5.00 L) and the organic layer was separated. The aqueous layer was re-extracted with DCM (2.00 L). The combined organic layers were washed with saturated aqueous NaHCO3 (1.50 L), brine (1.50 L), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by normal phase chromatography (eluent: petroleum ether / EtOAc from 100 / 1 to 10 / 1) to give the title compound as a yellow solid. 1H NMR (600MHz, DMSO-d6)δ 8.04(s,1H), 7.81(s,1H), 5.88-5.79(m,1H), 3.92-3.83(m,1H), 3.80-3.68(m,1H), 2.53(s,3H), 2.40-2.3 2(m,1H), 2.06-1.99(m,1H), 1.99-1.93(m,1H), 1.77-1.69(m,1H), 1.60-1.56(m,2H).UPLC-MS-6:Rt=1.32 min;MS m / z[M+H] + ;329.0 / 331.0 / 333.0

[0206] Step D.7: 5-Chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (Intermediate D.1) A suspension of 4-bromo-5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (Step D.6, 450 g, 1.37 mol), KOAc (401 g, 4.10 mol) and B2Pin2 (520 g, 2.05 mol) in 1,4-dioxane (3.60 L) was degassed with nitrogen for 0.5 h. Pd(dppf)Cl2.CH2Cl2 (55.7 g, 68.3 mmol) was added and the reaction mixture was stirred at 90 °C for 6 h. The reaction mixture was filtered through diatomaceous earth and the filter cake was washed with EtOAc (1.50 L x 3). The mixture was concentrated under reduced pressure to give a black oil, which was purified by normal phase chromatography (eluent: petroleum ether / EtOAc from 100 / 1 to 10 / 1) to give the desired product as a brown oil. The residue was suspended in petroleum ether (250 mL) for 1 h to give a white precipitate. The suspension was filtered and dried under reduced pressure to give the title compound as a white solid. 1H NMR (400MHz, CDCl3)δ 8.17(d,1H), 7.52(s,1H), 5.69-5.66(m,1H), 3.99-3.96(m,1H), 3.75-3.70(m,1H), 2.51(d,4H) ), 2.21-2.10(m,1H), 2.09-1.99(m,1H), 1.84-1.61(m,3H), 1.44(s,12H);UPLC-MS-6:Rt=1.29 min;MS m / z[M+H] + ;377.1 / 379.

[0207] Synthesis of Compound A [ka] Step 1: tert-Butyl 6-(4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate In a 500 mL flask, tert-butyl 6-(3-bromo-4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-5-methyl-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Intermediate C1, 10 g, 16.5 mmol), (1-methyl-1H-indazol-5-yl)boronic acid (6.12 g, 33.1 mmol), RuPhos (1.16 g, 2.48 mmol), and RuPhos-Pd-G3 (1.66 g, 1.98 mmol) were suspended in toluene (165 mL) under argon. K3PO4 (2M, 24.8 mL, 49.6 mmol) was added and the reaction mixture was placed in a preheated oil bath (95 °C) and stirred for 45 min. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc (x3). The combined organic layers were washed with saturated aqueous NaHCO3, dried (phase separator) and concentrated under reduced pressure. The crude residue was diluted with THF (50 mL) and SiliaMetS® thiol (15.9 mmol) was added and the mixture was rotated at 40 °C for 1 h. The mixture was filtered, the filtrate was concentrated and the crude residue was purified by normal phase chromatography (eluent: MeOH in CH2Cl2 0-2%) and the purified fraction was purified again by normal phase chromatography (eluent: MeOH in CH2Cl2 0-2%) to give the title compound as a light brown foam. UPLC-MS-3: Rt = 1.23 min; MS m / z [M+H] + ;656.3 / 658.3.

[0208] Step 2: 5-chloro-6-methyl-4-(5-methyl-3-(1-methyl-1H-indazol-5-yl)-1-(2-azaspiro[3.3]heptan-6-yl)-1H-pyrazol-4-yl)-1H-indazole TFA (19.4 mL, 251 mmol) was added to a solution of tert-butyl 6-(4-(5-chloro-6-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptane-2-carboxylate (Step 1, 7.17 g, 10.0 mmol) in CHCl (33 mL). The reaction mixture was stirred at room temperature under nitrogen for 1.5 h. The reaction mixture was concentrated under reduced pressure to give the title compound as the trifluoroacetate salt, which was used in the next step without purification. UPLC-MS-3: Rt=0.74 min; MS m / z[M+H] + ;472.3 / 474.3.

[0209] Step 3: 1-(6-(4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptan-2-yl)prop-2-en-1-one A mixture of acrylic acid (0.69 mL, 10.1 mmol), propylphosphonic anhydride (50% in EtOAc, 5.94 mL, 7.53 mmol) and DIPEA (21.6 mL, 126 mmol) in CHCl (80 mL) was stirred at room temperature for 20 min and then added (dropping funnel) to an ice-cold solution of 5-chloro-6-methyl-4-(5-methyl-3-(1-methyl-1H-indazol-5-yl)-1-(2-azaspiro[3.3]heptan-6-yl)-1H-pyrazol-4-yl)-1H-indazole trifluoroacetate (step 2, 6.30 mmol) in CHCl (40 mL). The reaction mixture was stirred at room temperature under nitrogen for 15 min. The reaction mixture was poured into saturated aqueous NaHCO and extracted with CHCl (×3). The combined organic layers were dried (phase separator) and concentrated. The crude residue was diluted with THF (60 mL) and LiOH (2N, 15.7 mL, 31.5 mmol) was added. The mixture was stirred at room temperature for 30 min until disappearance of the by-product resulting from the reaction of acryloyl chloride with the free NH group of the indazole (UPLC), then poured into saturated aqueous NaHCO3 and extracted with CHCl2 (3x). The combined organic layers were dried (phase separator) and concentrated. The crude residue was purified by normal phase chromatography (eluent: MeOH in CHCl2 0-5%) to give the title compound. The isomers were separated by chiral SFC (C-SFC-1; mobile phase: CO2 / [IPA+0.1%Et3N]: 69 / 31) to give compound A, i.e., a(R)-1-(6-(4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptan-2-yl)prop-2-en-1-one), as the second eluting peak (white powder): 1H NMR(600MHz,DMSO-d6)δ 13.1(s,1H),7.89(s,1H),7.59(s,1H),7.55(s,1H),7.42(m,2H),7.30 (d,1H),6.33(m,1H),6.12(m,1H),5.68(m,1H),4.91(m,1H),4.40(s,1 UPLC-MS-4:Rt=4.22 min;MS m / z[M+H] + 526.3 / 528.3; C-SFC-3 (mobile phase: CO2 / [IPA+0.1%Et3N]: 67 / 33): Rt=2.23 min. The compound of Example 1 is also referred to as "Compound A".

[0210] The atropisomer of compound A, a(S)-1-(6-(4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl)-2-azaspiro[3.3]heptan-2-yl)prop-2-en-1-one, was obtained as the first eluting peak: C-SFC-3 (mobile phase: CO2 / [IPA+0.1%Et3N]:67 / 33): Rt=1.55 min.

[0211] Example 2: Compound A (JDQ443) exhibits antitumor activity in the KRAS G12C mutant CDX model driven by target occupancy Single-agent antitumor activity of JDQ443 at 10 mg / kg, 30 mg / kg, and 100 mg / kg once-daily oral doses in a panel of KRAS G12C mutant CDX models across different indications. Cell lines for xenografting were MIA PaCa-2 (PDAC); NCI-H2122, LU99, HCC-44, NCI-H2030 (NSCLC); and KYSE410 (esophageal cancer). JDQ443 dose-dependently inhibited the growth of all models (Figure 8A), with dose-response kinetics differing model-specifically, with maximal response patterns ranging from regression (MIA PaCa-2, LU99) to quiescence (HCC44, NCI-H2122) to moderate tumor inhibition (NCI-H2030, KYSE410). The greatest dynamic range was observed in LU99. In contrast, JDQ443 showed no growth inhibition in a KRASG12V mutant xenograft model (NCI-H441; FIG. 8B), supporting KRAS G12C specificity and consistent with the in vitro data. Efficacy was maintained over the same daily doses of once (QD) or twice (BID) administration: 30 mg / kg QD vs. 15 mg / kg BID in MIA PaCa-2 (FIG. 8C), or 100 mg / kg QD vs. 50 mg / kg BID in NCI-H2122 and LU99 (FIGS. 8D-E). Efficacy of QD vs. BID administration correlated well with comparable daily area under the curve (AUC).

[0212] These findings suggested that JDQ443 efficacy was related to target occupancy (TO) and that effective AUC exposure could be achieved under both QD and BID dosing. To characterize whether AUC could act as a surrogate for TO, the effect of continuous infusion versus oral dosing in the LU99 xenograft model was investigated. Once-daily oral dosing at 30 mg / kg induced approximately 1 week of stasis followed by tumor progression, and 100 mg / kg induced tumor regression (Figure 8F), with approximate steady-state average concentrations (Cav) of 0.3 μM and approximately 1 μM, respectively. To evaluate continuous dosing, JDQ443 was delivered intravenously via a programmable microinfusion pump to achieve target concentrations approximating oral Cav. Continuous infusion and oral dosing resulted in comparable antitumor responses (Figure 8F,G). PK / PD model simulations showed that efficacy correlated best with TO and AUC of JDQ443 rather than other PK metrics (Figure 8H,I).

[0213] Example 3: Compound A potently inhibits the double mutant KRAS G12C H95Q that mediates resistance to adagrasib in clinical trials GFP-tagged KRASG12C H95Q, KRASG12C Y96D or KRASG12C R68S double mutations were generated by site-directed mutagenesis (QuikChange Lightning Site-Directed Mutagenesis Kit (Cat# 210518) Template: pcDNA3.1(+)EGFP-T2A-FLAG-KRAS G12C) and expressed by stable transfection in Cas9-containing Ba / F3 cells. Cells were treated with a dose-response curve starting at 10 μM in a 1 / 3 dilution from a 10 mM DMSO stock solution. Cell lines were treated with the indicated compounds for 72 hours and cell viability was measured by CellTiter-Glo.

[0214] result: In contrast to MRTX-849 (adagrasib), JDQ443 (compound A) and AMG-510 (sotrasib) potently inhibit cell viability of the KRASG12C H95Q double mutant. The KRASG12C Y96D or KRASG12C R68S double mutants are not inhibited by MRTX-849, AMG-510, or JDQ443 at the concentrations and settings shown (Ba / F3 line, 3-day proliferation assay), conferring resistance to all three tested KRASG12C inhibitors.

[0215] [Table 7]

[0216] Conclusion: Compound A can overcome resistance to adagrasib in the KRASG12C H95Q setting. Furthermore, because compound A has a unique binding interaction with mutant KRAS G12C compared to sotorasib and adagrasib, compound A alone or in combination with one or more of the therapeutic agents described herein can be useful for treating patients with cancers that have already been treated with other KRAS G12C inhibitors, such as sotorasib or adagrasib, or for targeting resistance after acquired KRAS resistance mutations emerge during initial KRAS G12C inhibitor treatment.

[0217] Example 4: Compound A potently inhibits KRAS G12C double mutants The effects of Compound A and other KRASG12C inhibitors on second-site mutations that have been reported to confer resistance to adagrasib were also investigated as follows.

[0218] Materials and Methods: Cell lines and KRAS G12C Inhibitors: The Ba / F3 cell line is a murine pro-B cell line, which is cultured at 37°C and 5% CO2 in RPMI 1640 (BioConcept, #1-41F01-I) supplemented with 10% fetal bovine serum (FBS) (BioConcept, #2-01F30-I), 2 mM sodium pyruvate (BioConcept, #5-60F00-H), 2 mM stable glutamine (BioConcept, #5-10K50-H), and 10 mM HEPES (BioConcept, #5-31F00-H) unless otherwise indicated. Parental Ba / F3 cells were cultured in the presence of 5 ng / ml recombinant mouse IL-3 (Life Technologies, #PMC0035). Ba / F3 cells are normally dependent on IL-3 for survival and proliferation, but by expressing oncogenes they can switch their dependence from IL-3 to the expressed oncogene (Curr Opin Oncology, 2007 Jan;19(1):55-60. doi:10.1097 / CCO.0b013e328011a25f.).

[0219] Individual plasmid mutagenesis and generation of Ba / F3 stable cell lines: pSG5_Flag-(codon-optimized)KRAS was mutagenesised using the QuikChange Lightning site-directed mutagenesis kit (Agilent; #210519). G12C Resistance mutations were generated on the _puro plasmid template and the sequence was confirmed by Sanger sequencing.

[0220] [Table 8]

[0221] The mutated plasmids were transfected into Ba / F3 WT cells by electroporation with the NEON transfection kit (Invitrogen, #MPK10025). Thus, 2 million Ba / F3 cells were electroporated with 10 μg of pf plasmid using the NEON system (Invitrogen, #MPK5000) under the following conditions: voltage (V) 1635, width (ms) 20, pulse 1. 72 hours after electroporation, puromycin selection was performed at 1 μg / ml to generate stable cell lines.

[0222] IL-3 removal Ba / F3 cells are normally dependent on IL-3 for survival and proliferation, but by expressing an oncogene, they can switch their dependence from IL-3 to the expressed oncogene. G12C To assess whether the single and double mutants could sustain Ba / F3 cell proliferation, engineered Ba / F3 cells expressing the mutant constructs were cultured in the absence of IL-3. Cell numbers and viability were measured every 3 days, and IL-3 withdrawal was completed after 7 days. Expression of the mutants after IL-3 withdrawal was confirmed by Western blot (data not shown; an upshift indicates KRAS G12C / R68S was observed).

[0223] Drug response curves and validation of resistance mutations for KRASG12C inhibitors: 1000 Ba / F3 cells / well were seeded in 96-well plates (Greiner Bio-One, #655098). Treatments were performed on the same day with a Tecan D300e drug dispenser. Viability was detected on the same day of treatment (day 0) and 3 days after treatment (day 3) for the starting plates using the CellTiter-Glo fluorescent cell viability assay (Promega, #G7573) on a Tecan infinitiy M200 Pro reader (integration time 1000 ms).

[0224] To determine growth, readouts after 3 days of treatment (day 3) were normalized to the starting plate (day 0). Viability was then calculated by normalizing treated wells to the DMSO-treated control samples. Curves were fitted with a sigmoidal dose-response model (4-parameter curve) using XLfit (Figure 9). The horizontal red dotted line represents the GI50 value. The tabulated data is shown below.

[0225] [Table 9]

[0226] [Table 10]

[0227] [Table 11]

[0228] [Table 12]

[0229] Western blot After treatment with different compounds at the indicated concentrations and times, cells were harvested, pelleted and snap frozen at -80°C. 60 μL of lysis buffer (50 mM Tris HCl, 120 mM NaCl, 25 mM NaF, 40 mM β-glycerol phosphate disodium salt pentahydrate, 1% NP40, 1 μM microcystin, 0.1 mM Na3VO3, 0.1 mM PMSF, 1 mM DTT and 1 mM benzamidine, supplemented with one protease inhibitor cocktail tablet (Roche) per 10 mL of buffer) was added to each sample. Samples were then vortexed, incubated on ice for 10 min, vortexed again and centrifuged at 14000 rpm for 10 min at 4°C. Protein concentration was determined by BCA protein assay kit (Pierce, 23225). After normalization to the same total volume with lysis buffer, NuPAGE™ LDS Sample Buffer 4x (Invitrogen, NP0007) and NuPAGE™ Sample Reducing Agent 10x (Invitrogen, NP0009) were added. Samples were heated at 70°C for 10 min before loading onto NuPAGE™ Novex™ 4-12% Bis-Tris Midi Protein Gels, 26 well (Invitrogen, WG1403A). Gels were run in NuPAGE MES SDS running buffer (Invitrogen, NP0002) at 200V (PowerPac HC, Biorad) for 45 min. Proteins were transferred into Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs membranes (Biorad, 1704159) at 135 mA per gel using the Trans-Blot® Turbo™ system (Biorad) for 7 min and then the membranes were stained with Ponceau Red (Sigma, P7170). Membranes were blocked with TBST with 5% milk at RT. Anti-RAS (Abcam, 108602) and anti-phospho-ERK1 / 2p44 / 42 MAPK (Cell Signaling, 4370) antibodies were incubated overnight at 4° C. and anti-vinculin (Sigma, V9131) antibody was incubated for 1 h at RT.Membranes were washed 3 times for 5 min with TBST and incubated with anti-rabbit (Cell Signaling, 7074) and anti-mouse (Cell Signaling, 7076) secondary antibodies for 1 h at RT. All antibodies were diluted 1 / 1000 in TBST except for anti-vinculin (1 / 3000). Visualization was performed with WesternBright ECL (Advansta, K-12045-D20) for Ras and vinculin, and with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer, 34096) on a Fusion FX (Vilber Lourmat) using FusionCapt Advance FX7 software (Figure 10).

[0230] result

[0231] [Table 13]

[0232] Biophysical data Materials and Methods: Preparation of reagents: Cloning, expression and purification of RAS protein constructs The E. coli expression constructs used in this study were based on the pET system and were generated using standard molecular cloning techniques. cDNAs encoding KRAS, NRAS, and HRAS, including aa 1-169, followed by a cleavable N-terminal his affinity purification tag, were codon-optimized and synthesized by GeneArt (Thermo Fisher Scientific). Point mutations were introduced with the QuikChange Lightning site-directed mutagenesis kit (Agilent). All final expression constructs were sequence-verified by Sanger sequencing.

[0233] Two liters of culture medium were inoculated with a preculture of E. coli BL21(DE3) freshly transformed with the expression plasmids, and protein expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (Sigma) for 16 h at 18° C. Proteins with avi tags were transformed into E. coli harboring a compatible plasmid expressing biotin ligase BirA, and the culture medium was supplemented with 135 μM d-biotin (Sigma).

[0234] The cell pellet was resuspended in buffer A (20 mM Tris, 500 mM NaCl, 5 mM imidazole, 2 mM TCEP, 10% glycerol, pH 8.0) supplemented with Turbonuclease (Merck) and cOmplete protease inhibitor tablets (Roche). Cells were lysed at 800-1000 bar via homogenizer (Avestin) for 3 passages and the lysate was cleared by centrifugation at 40000g for 40 min.

[0235] The lysate was loaded onto a HisTrap HP 5 ml column (Cytiva) mounted on an AeKTA Pure 25 chromatography system (Cytiva). Contaminating proteins were washed away with buffer A and bound proteins were eluted with a linear gradient to buffer B (buffer A supplemented with 200 mM imidazole). During overnight dialysis, the N-terminal His affinity purification tags on the non-tagged and avi-tagged proteins were cleaved off by TEV or HRV3C protease, respectively. The protein solution was reloaded onto the HisTrap column and the flow-through containing the target protein was collected.

[0236] Guanosine 5'-diphosphate sodium salt (GDP, Sigma) or GppNHp tetralithium salt (Jena Bioscience) was added to a 24-32 fold molar excess over protein. EDTA (adjusted pH to 8) was added to a final concentration of 25 mM. After 1 h at room temperature, the buffer was exchanged with 40 mM Tris, 200 mM (NH4)2SO4, 0.1 mM ZnCl2, pH 8.0 on a PD-10 desalting column (Cytiva). GDP (for KRAS G12C resistant mutants H95Q / D / R, Y96D / C and R68S) or GppNHp was added to the eluted protein to a 24-32 fold molar excess over protein. 40 U of shrimp alkaline phosphatase (New England Biolabs) was added to the GppNHp containing sample only. The samples were then incubated at 5 °C for 1 h. Finally, MgCl2 was added to a concentration of approximately 30 mM.

[0237] The protein was then further purified on a HiLoad 16 / 600 Superdex 200 pg column (Cytiva) pre-equilibrated with 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 2 mM TCEP, pH 7.5.

[0238] Protein purity and concentration were determined by RP-HPLC and its identity was confirmed by LC-MS. The nucleotide sequence was determined by ion-pairing chromatography [Eberth et al, 2009].

[0239] Determination of Covalent Binding Rate Constants by RapidFire MS Assay and curve fitting Serial dilutions of test compounds (50 μM, 1 / 2 dilutions) were prepared in 384-well plates and incubated with 1 μM KRAS G12C (with / without additional mutations) in 20 mM Tris pH 7.5, 150 mM NaCl, 100 μM MgCl2, 1% DMSO at room temperature. Reactions were stopped at different time points by the addition of formic acid up to 1%. MS measurements were performed using an Agilent 6530 quadrupole time-of-flight (QToF) MS system coupled to an Agilent RapidFire autosampler RF360 device, resulting in a % modified value for each well. In parallel, compound solubility was assessed by turbidimetry and compound concentrations resulting in measurable turbidity were excluded from the curve fitting.

[0240] The plot of % modification versus time shows the k for different compound concentrations. obs In the second step, the obtained k obs The values ​​were plotted versus compound concentration. The rate constant (i.e., k inact / K I ) was derived from the initial linear portion of the resulting curve.

[0241] MS measurement Injections were performed using a RapidFire autosampler RF360. Solvents were delivered by an Agilent 1200 pump. C18 solid phase extraction (SPE) cartridges were used for all experiments.

[0242] A volume of 30 μL was aspirated from each well of a 384-well plate. Sample load / wash time was 3000 ms at a flow rate of 1.5 mL / min (HO, 0.1% formic acid); elution time was 3000 ms (acetonitrile, 0.1% formic acid); re-equilibration time was 500 ms at a flow rate of 1.25 mL / min (HO, 0.1% formic acid).

[0243] Mass spectrometry (MS) data were acquired on an Agilent 6530 quadrupole time-of-flight (QToF) MS system coupled to a dual electrospray (AJS) ion source in positive mode. Instrument parameters were as follows: gas temperature 350°C, drying gas 10 L / min, nebulizer 45 psi, sheath gas 350°C, sheath gas flow 11 L / min, capillary 4000V, nozzle 1000V, fragmentor 250V, skimmer 65V, octapole RF 750V. Data were acquired at a rate of 6 spectra / s. Mass equilibration was performed over the 300-3200 m / z range.

[0244] All data processing was performed using a combination of Agilent MassHunter Qualitative Analysis, Agilent Rapid-Fire control software, and the Agilent DA Reprocessor offline utility. The Maximum Entropy algorithm generated zero-charge spectra in separate files for each injection. Batch processing generated a single file capturing all mass spectra in text format as x,y coordinates. This file was used to calculate the % protein modification in each well.

[0245] result Quantification of second order rate constants for modification for the indicated constructs (all GDP loaded) was performed using kinetic MS experiments to measure % modification at different time points for a range of compound concentrations. inact / K I k obs Extrapolated from the initial slope of the plot versus compound concentration. KRAS G12D: Activity against GDP was set to 1, giving the relative activity for the resistant mutants. The average values ​​of n=4 experiments for KRAS G12C, n=3 for G12C_Y96D, and n=2 for the other mutants are given in the table below.

[0246] [Table 14]

[0247] Quantification of second order rate constants for modification for the indicated constructs (all GDP loaded) was performed using kinetic MS experiments to measure % modification at different time points for a range of compound concentrations. inact / KI to k obs Extrapolated from the initial slope of the compound vs. concentration plot. Mean values ​​are given for n=4 experiments for KRAS G12C, n=3 for G12C_Y96D, and n=2 for the other mutants.

[0248] [Table 15]

[0249] conclusion First-generation KRAS G12C inhibitors have shown efficacy in clinical trials. However, the emergence of mutations that disrupt inhibitor binding and reactivation in downstream pathways limits the duration of response. Second-site mutants reported to confer resistance to adagrasib in clinical trials (references: N Engl J Med. 2021 Jun 24;384(25):2382-2393. doi:10.1056 / NEJMoa2105281., Cancer Discov. 2021 Aug;11(8):1913-1922. doi:10.1158 / 2159-8290. CD-21-0365. Epub 2021 Apr 6. PMID:33824136.) were expressed in Ba / F3 cells and analyzed for their sensitivity to compound A (JDQ443) compared to KRAS G12C (GI 50 =0.115±0.060 mM). As expected from its binding mode, compound A inhibited the proliferation and signaling of KRAS G12C H95 double mutants. Compound A potently inhibited the proliferation of G12C / H95R and G12C / H95Q (GI 50 =0.024±0.006mM, G.I. 50 =0.284±0.041 mM), whereas expression of G12C / R68S, G12C / Y96C, and G12C / Y96D conferred resistance to compound A (all GI 50 >1mM).

[0250] Surprisingly, expression of G12C / H95D resulted in a decreased sensitivity to compound A (GI 163166) compared to H95R or Q, even though compound A does not directly interact with histidine 95. 50 = 0.612 ± 0.151 mM). Western blot analysis of pERK upon Compound A treatment and analysis of the kinetic constants of Compound A against clinically observed SWII pocket mutations in a biophysical setting (biophysical data above) were consistent with the cell growth inhibition data (see table).

[0251] The difference of H95D compared to H95R or Q may be due to the negative charge of aspartic acid, which may further increase the negative electrostatic potential of the KRAS G12C surface. This may affect ligand recognition and thus reduce the specific reactivity and cellular activity of Compound A for this mutant. Another possible explanation is that the H95D mutation may affect KRAS dynamics, resulting in a lower conformational accessibility that allows Compound A binding.

[0252] Taken together, the data indicate that Compound A should overcome adagrasib-induced resistance in the G12C / Q95R or G12C / H95Q setting. Compound A treatment, especially in the combination of the present invention, may still be useful in the G12C / H95Q setting in which it has shown activity.

[0253] Example 5: In vivo JDQ443 antitumor efficacy is enhanced in combination with inhibitors of RAS upstream and downstream signaling The antitumor efficacy of JDQ443± inhibitors of RAS upstream or downstream signaling was evaluated in a panel of human KRAS G12C mutant NSCLC and CRC PDXs.

[0254] Patient-derived xenograft (PDX) models of human NSCLC and CRC were established by direct subcutaneous implantation of patient NSCLC or CRC tumor tissues into nude mice. PDX models were maintained via in vivo serial passaging.

[0255] Cohorts of mice were implanted subcutaneously with tumor fragments from each PDX model (typically passages 4-9). Ten NSCLC and nine CRC PDX models were used. For identification and tracking purposes, each model is designated with a code, e.g., 30580-HX, 30581-HX, etc. Individual mice were transplanted at 10-30 days after their tumor volume reached 200-250 mm. 3 Animals were assigned to treatment or control groups when they reached T = 0 on the x-axis of the spider plot. One animal per PDX model was assigned to each treatment arm. Upon enrollment in a treatment arm, tumor volumes were measured twice weekly using calipers and calculated using the formula: length x width. 2 / 2 to mm 3 Tumor volumes were estimated by the 10-day time point. The end of the study for each model was determined as a minimum of 28 days of treatment or when untreated tumors reached 1500 mm 3 The time to tumor doubling was defined as the duration to reach 0.01 or the duration of doubling of untreated tumors, whichever was later.

[0256] Mice were orally treated with a KRAS G12C inhibitor (Compound A at 100 mg / kg QD) alone or in combination with the combination partners listed in the table below. For example, Compound A was administered at 100 mg / kg once daily (QD) in combination with LXH254 (napolafenib) at 50 mg / kg twice daily (BID).

[0257] [Table 16]

[0258] [Table 17]

[0259] Compound A and TNO155 were formulated as suspensions in 0.1% Tween 80 and 0.5% methylcellulose in water. The Raf inhibitor (LXH254 (napolafenib)) was formulated as a suspension. The MEK inhibitor (trametinib) was formulated as a suspension in 0.2% Tween 80, 0.5% hydroxypropyl methylcellulose (HPMC) pH adjusted to approximately pH 8. The ERK inhibitor (LTT462 (linetelqib)) was formulated as a suspension in 0.5% hydroxypropylcellulose (HPC) / 0.5% Pluronic in pH 7.4 phosphate buffered saline (PBS) buffer, pH 4. The CDK4 / 6 inhibitor (LEE011) was formulated as a suspension in 0.5% methylcellulose. The PI3K inhibitor (BYL719) was formulated as a suspension in 0.5% Tween 80 and 1% carboxymethylcellulose in water. The mTOR inhibitor (RAD001) was formulated in 5% glucose.

[0260] The control group received no treatment.

[0261] result: Tumor volume improvements and objective antitumor responses were greater than JDQ443 monotherapy for all combination treatments in both NSCLC and CRC models (Figures 1-6). Similarly, benefits of combination treatments were observed for time to tumor volume doubling in both models (Figure 7).

[0262] In CRC models, Compound A treatment alone caused a moderate antitumor response in some models. Combination of Compound A with each of its combination partners improved the antitumor response. Triple combinations appeared to further improve the response (Figures 1 and 2).

[0263] In NSCLC models, Compound A treatment alone induced no to moderate antitumor responses in half of the models and no good antitumor responses in the other half of the models. Combination of Compound A with each of its combination partners improved the antitumor response (Figures 3, 4 and 5).

[0264] Example 6: PI3K inhibitors show maximal synergy scores in a 3-day proliferation assay in combination with a KRAS G12C inhibitor alone or in the presence of an SHP2 inhibitor. Matrix combination proliferation assay (treatment time 3 days, cell titer growth assay) was performed using KRAS G12C Inhibitors (labeled "KRAS" in Figure 11) G12C 11 ) as single agents or in combination with 10 μM SHP099, an SHP2 inhibitor (denoted as "SHP2i" in FIG. 11 ), in the presence of the upstream receptor kinase inhibitor BGJ398, an FGFR inhibitor (denoted as "FGFRi" in FIG. 11 ), and either erlotinib, an EGFR inhibitor (denoted as "EGFRi" in FIG. 11 ), or trametinib, a MEK inhibitor (denoted as "MEKi" in FIG. 11 ), or the PI3K effector arm inhibitors alpelisib (denoted as "PI3Kαi" in FIG. 11 ), and GDC0941, a pan-PI3K inhibitor (denoted as "panPI3Ki" in FIG. 11 ).

[0265] Synergy scores (SS) were calculated by the Loewe index and are shown above each grid as "SS" values. Values ​​in the grid are % growth inhibition values: values ​​higher than 100% indicate cell death. % growth inhibition: 0-99=proliferation retardation, 100=growth arrest / stasis, 101-200=reduction in cell number / cell death.

[0266] The values ​​on the x-axis of each grid indicate the concentration (μM) of the KRAS G12c inhibitor used. The values ​​on the y-axis of each grid indicate the concentration (μM) of the second agent (i.e., FGFR inhibitor, EGFR inhibitor, MEK inhibitor, PI3αK inhibitor and pan-PI3K inhibitor, respectively).

[0267] As shown in Figures 11A and 11B, the addition of an SHP2 inhibitor to a dual combination of a KRAS G12C inhibitor and a second agent selected from an FGFR inhibitor, an EGFR inhibitor, a MEK inhibitor, and a PI3K inhibitor increases the synergy score, for example, the synergy score increases from 1.522 for the dual combination of a KRAS G12C inhibitor and an EGFR inhibitor to 3.533 for the triple combination of a KRAS G12C inhibitor, an EGFR inhibitor, and an SHP2 inhibitor.

[0268] The highest synergy scores were obtained in the presence of a combination of a PI3K inhibitor with a KRAS G12C inhibitor alone or in the presence of an SHP2 inhibitor (FIGS. 11A and 11B).

[0269] Example 7: Dose response of JDQ443 in combination with erlotinib or cetuximab in NSCLC cell lines. Beneficial effect of the combination of Compound A and ribociclib on NSCLC xenograft models. The combination test of compound A and ribociclib was carried out in mouse KRAS G12C and CDKN2A mutant LU99 xenograft model. Compound A alone induces tumor regression for approximately two and a half weeks, and then induces tumor recurrence while treatment is still continued. Ribociclib alone does not have any effect on tumor growth. The combination significantly improves the durability of response and time to recurrence seen with compound A as a single agent.

[0270] Example 8: Combination of Compound A with a SHP2 inhibitor, a PI3K inhibitor or a CDK4 / 6 inhibitor delays time to progression (TPP) compared to single agent treatment with Compound A in NSCLC xenograft models. In vivo efficacy studies of compound A (JDQ443) as a single agent or its combinations (double, triple, quadruple) with TNO155 (SHP2 inhibitor), BYL719 (alpelisib, PI3K inhibitor) and LEE011 (ribociclib, CDK4 / 6 inhibitor) were carried out in mouse KRAS G12C, PIK3CA and CDKN2A mutant LU99 xenograft models. Daily administration of 100 mg / kg JDQ443 induced profound tumor regression for approximately two and a half weeks, followed by tumor recurrence while treatment was still ongoing. TNO155 given at 7.5 mg / kg daily did not have any effect on tumor growth compared to the vehicle group.

[0271] Dual combinations of JDQ443 with TNO155, BYL719, or LEE011, triple combinations of JDQ443 and TNO155 with BYL719 or LEE011, and quadruple combinations of JDQ443 with TNO155, BYL719, and LEE011 improved the durability of response and time to progression seen with JDQ443 as a single agent in the following order: single agent < dual combination < triple combination < quadruple combination (Figure 12).

[0272] Example 9: Dose response of Compound A (JDQ443) in combination with EGFR inhibitors in NSCLC and CRC cell lines The combination of cetuximab and Compound A provides additive benefit to Compound A and cetuximab treatment in a CRC cell line (SW1463) (Figure 13, top panel).

[0273] The % growth inhibition was also increased for the combination of erlotinib or cetuximab with Compound A in NSCLC (NCI-H358 and NCI-H2122) cell lines (Figure 13, middle and bottom panels).

[0274] Example 10: Effect of Compound A, SOS-inhibitor BI-3406 and the combination of Compound A, SOS-inhibitor BI-3406 on NSCLC and CRC cell lines. Matrix combination proliferation assays were performed as follows: For each of the cell lines, cells were dispensed into tissue culture treated 384-well plates (Greiner #781098) in a final volume of 25 μL per well. Cells were allowed to attach and initiate growth for 24 hours. On plates, counts were made before treatment (=day 1) and other plates were treated with compound or DMSO using a HP D300 digital dispenser. After 72 hours, medium was refreshed by supplementing with 25 μl culture medium per well containing the corresponding compound or DMSO. All treatments were performed in triplicate.

[0275] Seven days after treatment initiation, cell growth was determined using CellTiter-Glo® (Promega #G7573), which measures the amount of ATP in the wells. Plates were equilibrated to room temperature for approximately 30 minutes, and a volume of CellTiter-Glo® reagent equal to the volume of cell culture medium was added. Cell lysis was induced for 2 minutes on an orbital shaker, plates were incubated at room temperature for 10 minutes, and luminescence was recorded.

[0276] Cells were treated with compounds at the indicated final concentrations. Dose-response curves were derived using XLfit dose response one site, model 205. Percentage of growth inhibition relative to DMSO (percentage GI) is reported after subtraction of the day 1 readings.

[0277] Low growth inhibition was observed with single agent treatment with the SOS-inhibitor BI-3406. A combination benefit was observed with the addition of a KRAS G12C inhibitor (Figure 14).

[0278] Example 11: Clinical efficacy of Compound A as monotherapy and combination therapy A Phase Ib / II open-label, multicenter, dose-escalation study of Compound A (JDQ443) alone and in combination with defined agents is conducted in patients with advanced solid tumors with KRAS G12C mutations, including KRAS G12C mutant NSCLC and KRAS G12C mutant colorectal cancer (KontRASt-01 (NCT04699188)). This study is conducted to evaluate the antitumor efficacy, safety, and tolerability of JDQ443 as a single agent and in combination with other agents. JDQ443 + TNO155 and JDQ443 + PD1-inhibitors, such as tislelizumab, can be used to treat patients with KRAS G12C mutant solid tumors.

[0279] Patients to be treated are those who have received standard of care therapy or who are intolerant or ineligible for approved therapy; Eastern Cooperative Oncology Group Performance Status (ECOG PS 0-1); or KRAS G12C Patients with advanced KRAS G12C mutated solid tumors who had no prior treatment with an inhibitor are included. The main exclusion criteria for the JDQ443 monotherapy arm are active brain metastases and / or prior KRAS G12C inhibitor treatment.

[0280] Patients with NSCLC include those who have already been treated with a platinum-based chemotherapy regimen and an immune checkpoint inhibitor, either in combination or sequentially, unless ineligible to receive such therapy.

[0281] Patients with CRC include those who have previously received standard of care therapy, including fluoropyrimidine, oxaliplatin, and irinotecan-based chemotherapy, unless they are ineligible to receive such therapy.

[0282] Preliminary data from the monotherapy dose escalation arm study include:

[0283] At the cutoff date of January 5, 2022, 39 patients were treated with 200 mg QD, 400 mg QD, 200 mg BID, or 300 mg BID Compound A. Compound A was administered with food.

[0284] Patients had a median of three prior lines of antineoplastic therapy. The recommended dose for monotherapy is 200 mg of Compound A taken orally twice daily (BID). Efficacy data from the pooled Phase Ib JDQ443 single agent cohort (n=39) (cutoff January 5, 2022) showed the following: Confirmed overall response rate (ORR) of 57% (4 / 7 patients) at 200 mg BID in NSCLC Confirmed and unconfirmed ORR of 45% (9 / 20) across doses in NSCLC Confirmed ORR of 35% (7 / 20) across doses in NSCLC PD / PK modeling predicted sustained high levels of target occupancy at the recommended dose of 200mg BID

[0285] Compound A treatment was generally well tolerated. Most treatment-related adverse events (TRAEs) were grade (Gr) 1-2. There were no grade 4-5 TRAEs. Four grade 3 TRAEs occurred in four separate patients; the most common TRAEs were fatigue, nausea, edema, diarrhea, and vomiting. There was one DLT (grade 3 fatigue) and one treatment-related severe AE (grade 3 photosensitivity reaction) in each separate patient treated with 300 mg BID.

[0286] [Table 18]

[0287] At the recommended dose of 200 mg BID, absorption was prolonged, with the median time to maximum plasma concentration (Tmax) being 3-4 hours after administration with food. No significant accumulation was observed at steady state, and there was no evidence of autoinduction. The half-life was approximately 7 hours, and the steady-state area under the curve (AUCss) exceeded the exposure required for maximum efficacy 3-fold in the insensitive KRAS G12C xenograft model. Figure 15 shows the PK profile at steady state.

[0288] [Table 19]

[0289] The predicted target occupancy profile is shown in Figure 15. Patient PK and preclinical target occupancy models were integrated to predict target occupancy in patients at >90% in >82% of patients. The model assumes that JDQ443 binding and target (KRAS) turnover rates are the same in mice and humans (half-life of approximately 25 hours for KRAS) and that only free drug can bind to the target.

[0290] The best overall efficacy across dose levels and indications is shown in the top half of Figure 16 and in the table below.

[0291] [Table 20]

[0292] The best overall response across dose levels in all patients with NSCLC is shown in the bottom half of Figure 16 and in the table below. All patients with a partial response or unconfirmed partial response were continuing treatment at the time of data cutoff.

[0293] [Table 21]

[0294] Response will be assessed by the investigator according to RECIST v1.1. Two (10.0%) patients had uPR contributing to ORR (confirmed and unconfirmed). uPR = unconfirmed PR pending confirmation with treatment continuing without PD. One of the two patients with uPR had a confirmed PR after data cutoff.

[0295] - Figure 17 shows PET scans showing a significant reduction in 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (18-F-FDG) avidity of the tumor mass after 4 cycles of treatment with Compound A administered at 200 mg BID in a patient with NSCLC. The patient was receiving pemetrexed / pembrolizumab, docetaxel, tegafur / gimeracil / oteracil, and carboplatin / paclitaxel / atezolizumab. The scan after cycle 2 showed a 30.4% reduction in the sum of the longest diameters of the target lesions compared to baseline. PR was confirmed on subsequent scans.

[0296] The combination of Compound A and an SHP2 inhibitor, such as TNO155, also showed clinical efficacy. Figure 18 shows post-cycle 2 scans from a patient with KRAS G12C mutation duodenal papillary carcinoma, previously treated with cisplatin / gemcitabine and tegafur, each with best response of progression. The patient was treated with JDQ443 200mg QD continuously and TNO155 20mg QD 2 weeks on / 1 week off. Post-cycle 2 scans showed a 44.2% reduction in the sum of the longest diameters of target lesions compared to baseline.

[0297] Two patients treated in a first-in-human clinical trial are provided here to illustrate the clinical antitumor activity of JDQ443 alone or together with TNO155 (FIGS. 17 and 18).

[0298] Example 1: A 57-year-old male with metastatic KRAS G12C-mutated NSCLC. Local molecular testing using next-generation sequencing (NGS) did not identify a mutation in TP53. STK11, KEAP1, and NRF2 mutation status was unknown. The patient had received prior carboplatin / pemetrexed / pembrolizumab, docetaxel, tegafur-gimeracil-oteracil, and carboplatin / paclitaxel / atezolizumab. The male was enrolled in the JDQ443 monotherapy dose escalation part of the study at a dose of JDQ443 200 mg BID given in consecutive 21-day cycles. Disease assessment after two cycles of treatment demonstrated a RECIST 1.1 partial response, with a -30.4% change in the sum of the longest diameter of the target lesions compared to baseline. The partial response was confirmed on subsequent scans (Figure 17), and the patient continued treatment. Positron emission tomography imaging at baseline and after four cycles of treatment also demonstrated a significant decrease in 2-[fluorine-18]-fluoro-2-deoxy-d-glucose avidity of the tumor mass.

[0299] Example 2: A 58-year-old female with liver metastatic KRAS G12C mutant papillary duodenal carcinoma. An R175H mutation in TP53 was observed by NGS (Foundation One panel). The patient had received prior treatment with cisplatin / gemcitabine and tegafur, both with best response of progression. The woman was enrolled in the dose escalation portion of the JDQ443+TNO155 arm of the study, where the patient received continuous 200mg QD JDQ443 and 20mg QD TNO155 2 weeks on / 2 weeks off. Disease assessment after 2 cycles of treatment demonstrated a RECIST 1.1 partial response, with a -44.2% change in the sum of the longest diameter of the target lesions compared to baseline (Figure 18). The partial response was confirmed on subsequent scans, and the patient continued treatment.

[0300] [Table 22]

[0301] Example 12: Clinical trial investigating Compound A versus docetaxel in patients with previously treated locally advanced or metastatic KRAS G12C mutated NSCLC An open-label study designed to compare Compound A as monotherapy with docetaxel in subjects with advanced non-small cell lung cancer (NSCLC) harboring a KRAS G12C mutation who have been previously treated sequentially or in combination with platinum-based chemotherapy and immune checkpoint inhibitor therapy can be conducted.

[0302] The exam consists of two parts: - The randomized part will evaluate the efficacy and safety of Compound A as monotherapy compared with docetaxel. - The expansion part will be open after the final progression-free survival (PFS) analysis (if the primary endpoint meets statistical significance) for subjects randomized to docetaxel treatment to cross over to receive compound A treatment.

[0303] The study population includes adult subjects with locally advanced or metastatic (stage IIIB / IIIC or IV) KRAS G12C mutant non-small cell lung cancer who received prior platinum-based chemotherapy and prior immune checkpoint inhibitor therapy administered sequentially or as a combination therapy.

[0304] Subjects will be treated with Compound A or docetaxel according to local guidelines as per standard of care and product labeling (docetaxel concentrated injectable solution, administered intravenously).

[0305] Primary outcome measures included: Progression-free survival (PFS) PFS is the time from the date of randomization / treatment initiation to the date of the first documented event defined as progression or death from any cause. PFS is based on central assessment and uses RECIST 1.1 criteria.

[0306] Secondary outcome measures included: ·Overall survival (OS) OS is defined as the time from the date of randomization to the date of death from any cause. Overall response rate (ORR) ORR is defined as the proportion of patients with a best overall response of complete response (CR) or partial response (PR) based on central and local investigator assessment by RECIST 1.1. Disease control rate (DCR) DCR is defined as the proportion of subjects with a complete response (CR), partial response (PR), stable disease (SD) or best overall response (BOR) of non-CR / non-PD. Time to recurrence (TTR) TTR is defined as the time from the date of randomization to the date of the first documented response (CR or PR, which must be subsequently confirmed). Duration of response (DOR) DOR is calculated as the time from the date of first documented response (complete response (CR) or partial response (PR)) to the date of first documented progression or death from cancer of any cause. Progression-free survival (PFS2) after next-line therapy PFS2 (based on local investigator assessment) is defined as the time from the date of randomization to the date of first documented progression on next-line therapy or death from any cause, whichever occurs first. Plasma concentrations of Compound A and its metabolites To characterize the pharmacokinetics of compound A and its metabolite HZC320 Time to definite deterioration in Eastern Cooperative Oncology Group (ECOG) performance status Deterioration of Eastern Cooperative Oncology Group (ECOG) performance status (PS) Time to a definite 10-point worsening in symptom scores for chest pain, cough, and dyspnea on the QLQ-LC13 · EORTC QLQ LC13 is a 13-item lung cancer-specific questionnaire module that includes both multi-item and single-item measures of lung cancer-related symptoms (i.e. cough, hemoptysis, dyspnea and pain) and side effects from conventional chemo- and radiotherapy (i.e. alopecia, neuropathy, oral pain and dysphagia). Time to definite 10-point worsening is defined as the time from the date of randomization to the date of the event, defined as an absolute increase (worsening) of at least 10 points from baseline without subsequent change below the threshold or death from any cause. Time to significant deterioration in general health status / QoL, shortness of breath and pain according to QLQ-C30 The EORTC QLQ-C30 is a questionnaire developed to assess the health-related quality of life in cancer subjects. The questionnaire contains 30 items and consists of both multi-item and single-item scales based on the subject's experiences over the past week. These include five domains (physical, role, emotional, cognitive and social functioning), three symptom scales (fatigue, nausea / vomiting and pain), six single items (dyspnea, insomnia, anorexia, constipation, diarrhea and financial difficulties) and a general health status / HRQoL scale. Time to definite 10-point deterioration is defined as the time from the date of randomization to the date of the event, defined as an absolute increase (worsening) of at least 10 points from baseline in the corresponding scale score without a subsequent change below the threshold or death from any cause. Change from baseline in EORTC-QLQ-C30 The EORTC QLQ-C30 is a questionnaire developed to assess the health-related quality of life in cancer subjects. The questionnaire contains 30 items and consists of both multi-item and single-item scales based on the subjects' experiences over the past week. These include five domains (physical, role, emotional, cognitive and social functioning), three symptom scales (fatigue, nausea / vomiting and pain), six single items (dyspnea, insomnia, loss of appetite, constipation, diarrhea and financial difficulties) and a general health status / HRQoL scale. Higher scores indicate greater presence of symptoms. Change from baseline in EORTC-QLQ-LC13 The EORTC QLQ LC13 is a 13-item lung cancer-specific questionnaire module that includes both multi-item and single-item measures of lung cancer-related symptoms (i.e. cough, hemoptysis, dyspnea, and pain) and side effects from conventional chemo- and radiotherapy (i.e. alopecia, neuropathy, oral pain, and dysphagia). Higher scores indicate greater presence of symptoms. Change from baseline in EORTC-EQ-5D-5L The EQ-5D-5L is a general instrument for describing and assessing health. It is based on a descriptive system that defines health in five dimensions: mobility, self-care, activities of daily living, pain / discomfort, and anxiety / depression. Change from baseline in NSCLC-SAQ The Non-Small Cell Lung Cancer Symptom Assessment Questionnaire (NSCLC-SAQ) is a 7-item patient-reported outcome measure assessing patient-reported symptoms associated with advanced NSCLC. It contains 5 domains and subitems identified as symptoms of NSCLC: cough (1 item), pain (2 items), dyspnea (1 item), fatigue (2 items), and appetite (1 item). PFS based on KRAS G12C mutation status in plasma To compare clinical outcomes for Compound A versus docetaxel based on KRAS G12C mutation status in plasma OS based on KRAS G12C mutation status in plasma To compare clinical outcomes for Compound A versus docetaxel based on KRAS G12C mutation status in plasma ORR based on KRAS G12C mutation status in plasma To compare clinical outcomes for Compound A versus docetaxel based on KRAS G12C mutation status in plasma

[0307] Example 13: Clinical trial of JDQ443 with selected combinations in patients with advanced solid tumors harboring KRAS G12C mutations A Phase Ib / II multicenter, open-label platform study of JDQ443 in select combinations in patients with advanced solid tumors harboring KRAS G12C mutations will be conducted. The study aims to characterize the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of JDQ443 in combination with select therapies in adult patients with solid tumors harboring KRAS G12C mutations.

[0308] This study focuses on a single-molecule subset of patients whose tumors harbor the KRAS G12C mutation and have shown only modest response to single-agent KRAS G12C inhibition or are predicted to do so based on historical data. Combining JDQ443 with selected targeted therapy or other anti-neoplastic therapy may prevent or overcome this resistance in KRAS G12C mutant tumors and enable deeper, more durable responses than previously seen with KRAS G12C inhibitor monotherapy in similar patient populations.

[0309] Each treatment arm will include a dose escalation part (Phase Ib) and a Phase II part. Dose escalation will be performed in KRAS G12C mutant solid tumors to establish safety / efficacy and determine the maximum tolerated dose (MTD) and / or recommended dose (RD) (JDQ443 + cetuximab may be explored in CRC).

[0310] The Phase II part of the study will further explore RD in select indications (e.g., NSCLC and CRC) in combination with select therapies. The objective of Phase II is to evaluate the antitumor efficacy of JDQ443 in combination with select therapies in RD and to further explore its safety and tolerability.

[0311] [Table 23]

[0312] [Table 24]

[0313] All publications, patents, and accession numbers mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0314] References herein to the "present invention" are intended to reflect certain inventive embodiments disclosed herein and should not be construed as unnecessarily limiting the claimed subject matter.

[0315] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims.

[0316] While specific embodiments of the invention have been disclosed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon reference to this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification and such variations.

Claims

1. A combined medicament comprising a KRAS G12C inhibitor or a pharmaceutically acceptable salt thereof, and a therapeutically active agent selected from an EGFR inhibitor, a MEK inhibitor, a CDK4 / 6 inhibitor, and combinations thereof.

2. The combined medicament according to claim 1, wherein the KRAS G12C inhibitor is selected from 1-{6-[(4M)-4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one (Compound A), sotorasib, adagrasib, D-1553, and GDC6036, or a pharmaceutically acceptable salt thereof, and the therapeutically active agent is selected from an EGFR inhibitor, a MEK inhibitor, a CDK4 / 6 inhibitor, and combinations thereof.

3. The combined medicament according to claim 2, wherein the KRAS G12C inhibitor is 1-{6-[(4M)-4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methyl-1H-indazol-5-yl)-1H-pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-1-one (Compound A).

4. The combined medicament according to claim 1, wherein the therapeutically active agent is an EGFR inhibitor selected from cetuximab, panitumumab, afatinib, lapatinib, erlotinib, gefitinib, osimertinib, and nazartinib, (ii) a MEK inhibitor selected from pimasertib, PD-0325901, selumetinib, trametinib, binimetinib, and cobimetinib, and a CDK4 / 6 inhibitor selected from ribociclib, palbociclib, and abemaciclib, or a pharmaceutically acceptable salt thereof.

5. The combined medicament according to claim 4, wherein the therapeutically active agent is selected from cetuximab, trametinib, and ribociclib, or a pharmaceutically acceptable salt thereof.

6. The combined medicament according to claim 5, wherein the therapeutically active agent is trametinib or a pharmaceutically acceptable salt thereof.

7. The combined medicament according to claim 5, wherein the therapeutically active agent is ribociclib or a pharmaceutically acceptable salt thereof.

8. The combined medicament according to claim 5, wherein the therapeutically active agent is cetuximab or a pharmaceutically acceptable salt thereof.

9. The combined medicament according to any one of claims 1 to 8, for use in a method of treating cancer or a tumor in a subject in need thereof.

10. The combined medicament according to claim 9, wherein the cancer or tumor is a KRAS G12C mutant cancer or tumor.

11. The combined medicament according to claim 9, wherein the cancer or tumor is selected from lung cancer, colorectal cancer, pancreatic cancer, uterine cancer, rectal cancer, appendiceal cancer, small intestine cancer, esophageal cancer, hepatobiliary tract cancer, bladder cancer, ovarian cancer, duodenal papilla cancer, and solid tumors.

12. The combined medicament according to claim 9, wherein the cancer or tumor is selected from lung cancer selected from non-small cell lung cancer, colorectal cancer, pancreatic cancer, and solid tumors.

13. The combined medicament according to claim 9, wherein the therapeutic agent in the combination therapy is administered simultaneously, separately, or over a period of time.

14. The combined medicament according to claim 9, wherein compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose selected from 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, and 600 mg per day.

15. The combined medicament according to claim 14, wherein compound A is administered at a dose of 100 mg or 200 mg twice a day.

16. The combined medicament according to claim 9, wherein the trametinib or a pharmaceutically acceptable salt thereof is administered once a day at a dose of 0.5 mg.

17. The combined medicament according to claim 9, wherein the trametinib or a pharmaceutically acceptable salt thereof is administered once a day at a dose of 1 mg.

18. The combined medicament according to claim 9, wherein the trametinib or a pharmaceutically acceptable salt thereof is administered once a day at a dose of 2 mg.

19. The combined medicament according to claim 9, wherein the ribociclib or a pharmaceutically acceptable salt thereof is administered once a day at a dose of 200 mg.

20. The combined medicament according to claim 9, wherein the ribociclib or a pharmaceutically acceptable salt thereof is administered once a day at a dose of 400 mg.

21. The combined medicament according to claim 19, wherein the ribociclib or a pharmaceutically acceptable salt thereof is administered once a day for 3 weeks and then discontinued for 1 week.

22. The combined medicament according to claim 9, wherein cetuximab or a pharmaceutically acceptable salt thereof is administered once every 2 weeks at a dose of 500 mg / m ２ 2.

23. The combined medicament according to claim 9, wherein cetuximab or a pharmaceutically acceptable salt thereof is administered once every 2 weeks at a dose of 400 mg / m ２ 2.

24. The combined medicament according to claim 9, wherein cetuximab or a pharmaceutically acceptable salt thereof is administered once every 2 weeks at a dose of 300 mg / m ２ 2.

25. The subject or patient to be treated is - A patient suffering from a KRAS G12C mutant solid tumor (e.g., advanced (metastatic or unresectable) KRAS G12C mutant solid tumor), optionally having received standard of care therapy and having failed or for whom approved therapies are intolerant or ineligible; - A patient suffering from KRAS G12C mutant NSCLC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), optionally having received platinum-based chemotherapy regimen and immune checkpoint inhibitor therapy in combination or sequentially and having failed; - A patient suffering from KRAS G12C mutant NSCLC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), optionally having already been treated with a KRAS G12C inhibitor (e.g., sotorasib, adagrasib, GDC6036 or D-1553); and - A patient suffering from KRAS G12C mutant CRC (e.g., advanced (metastatic or unresectable) KRAS G12C mutant CRC), optionally having received standard of care therapy including fluoropyrimidine, oxaliplatin, and / or irinotecan-based chemotherapy and having failed The combination medicament according to claim 9, selected from

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