TW202400153A - Therapeutic uses of a krasg12c inhibitor - Google Patents

Abstract

The present invention provides a KRAS G12C inhibitor for use in a method of treatment of a cancer or solid tumor such as NSCLC which harbors a KRAS G12C mutation and a PD-L1 expression < 1% regardless of STK11 mutation status. The present invention also provides a KRAS G12C inhibitor for use in a method of treatment of a cancer or solid tumor such as NSCLC which harbors a KRAS G12C mutation and a PD-L1 expression < 1% regardless of STK11 mutation status or a cancer or solid tumor such as NSCLC which harbors a KRAS G12C mutation and a PD-L1 expression ≥ 1% and a STK11 co-mutation.

Description

Therapeutic uses of KRASG12C inhibitors

The present invention relates to KRAS G12C inhibitors (such as Compound A) or pharmaceutically acceptable salts thereof and their use in the treatment of cancer, especially cancers with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutations Status of cancers or solid tumors (eg, NSCLC), or cancers or solid tumors (eg, NSCLC) with KRAS G12C mutations and ≥ 1% PD-L1 expression and STK11 co-mutations. The present invention also relates to pharmaceutical compositions comprising a KRAS G12C inhibitor (such as Compound A) or a pharmaceutically acceptable salt thereof; and methods of using such compositions to treat or prevent cancer or solid tumors (such as NSCLC), the cancer or solid tumors (such as NSCLC) with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutation status, or with KRAS G12C mutations and ≥1% PD-L1 expression and STK11 co-mutations.

Cancer growth is driven by a variety of complex mechanisms. Some cancers inevitably become resistant to a given therapy. Inhibition of the MAPK pathway induces feedback mechanisms and pathway rewiring, leading to its subsequent reactivation. For example, one common mechanism is activation of receptor tyrosine kinases (RTKs).

Additionally, despite recent successes with targeted therapies and immunotherapies, some cancers, particularly metastatic cancers, remain largely incurable.

The KRAS oncoprotein is a GTPase with a critical role as a modulator of intracellular signaling pathways involved in proliferation, cell survival, and tumorigenesis, such as the MAPK, PI3K, and Ral pathways. Oncogene activation of KRAS occurs primarily 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 prevalent in approximately 13% of lung adenocarcinomas, 4% (3%-5%) of colon adenocarcinomas, and a smaller proportion of other cancer types.

In normal cells, KRAS alternates between an inactive GDP-bound state and an active GTP-bound state. Mutations in KRAS at codon 12 (eg, G12C) impair GTPase-activating protein (GAP)-stimulated GTP hydrolysis. So in this case, the conversion of KRAS G12C from GTP to GDP form will be very slow. As a result, KRAS G12C transitions into an active GTP-bound state, thereby driving oncogene signaling.

Lung cancer remains the most common type of cancer worldwide and the leading cause of cancer death 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 lung adenocarcinoma patients (Sequist et al. 2011). They are most commonly found at codon 12, with KRAS G12C mutations being the most common (40% overall) in both adenocarcinoma and squamous NSCLC (Liu et al. 2020). The presence of KRAS mutations predicts poor survival and is associated with reduced response to EGFR TKI therapy.

Immunotherapy for NSCLC using immune checkpoint inhibitors has shown promise, with some NSCLC patients experiencing durable disease control that lasts for years. However, such long-term nonprogression is uncommon, and treatment strategies that can increase the proportion of patients who respond to therapy and achieve durable remission with therapy are urgently needed.

The benefit of immunotherapy as monotherapy or in combination with platinum-based chemotherapy is generally related to the level of PD-L1 expression, with greater benefit observed in patients with advanced NSCLC and whose tumors exhibit high (≥50%) PD-L1 expression.

STK11 is a serine-threonine kinase that regulates cellular metabolism and cell cycle, and loss-of-function STK11 mutations promote an immunosuppressive microenvironment as they are associated with impaired T cell recruitment and activation and inhibition of neutrophil function. (Koyama et al. 2016). STK11 mutations are found in approximately 5% of patients with advanced NSCLC and are more common in approximately 15% of patients with KRAS G12C mutant NSCLC (Skoulidis et al. 2018; Scheffler et al. 2019; Shire et al. 2020; and Ricciuti et al. 2021) . Tumors with both KRAS G12C and STK11 mutations have gene expression profiles characterized by low expression of pro-inflammatory cytokines that promote an immunosuppressive tumor microenvironment, such as type I interferons, interferon Stimulator of genes (STING), DExD/H-Box helicase 58 (DDX58), toll-like receptor 4 (TLR4) and toll-like receptor 7 (TLR7) (Ricciuti et al. 2021).

Previous studies have shown that STK11 mutations are associated with worse prognosis and predict that patients with advanced NSCLC will benefit less from immunotherapy-based treatments (Ricciuti et al. 20201; Shire et al. 2020).

Approximately 40% to 50% of patients with advanced NSCLC are ineligible for subsequent treatment after discontinuation of first-line therapy, primarily due to clinical deterioration at disease progression (Davies et al. 2017). Therefore, more NSCLC patients may benefit from effective treatments administered earlier in the line.

Although the incorporation of immunotherapy into the first-line setting has significantly improved overall survival in patients with advanced NSCLC, previous studies have shown a benefit from immunotherapy in patients whose tumors have <1% PD-L1 manifestation or STK11 mutations. Lower magnitude: In a retrospective study including 1,261 patients with advanced lung adenocarcinoma treated with immunotherapy (42.5% and 20.6% had KRAS and STK11 mutations, respectively), Ricciuti et al. The presence of concurrent STK11 mutations was associated with worse PFS (HR = 2.04; 95% confidence interval [CI], 1.66-2.51; p < 0.0001) and OS (HR = 2.09; 95% CI, 1.68-2.61; p < 0.0001) (Ricciuti et al. 2021). In a meta-analysis involving a total of 10,074 patients and including 15 randomized controlled trials, Xu et al demonstrated that the magnitude of the OS benefit resulting from immunotherapy plus chemotherapy was related to the level of PD-L1 expression: for PD-L1 &lt; 1%, HR 0.60, 95% CI, 0.43-0.83; for PD-L1 1-49%, HR 0.56, 95% CI, 0.40-0.78; for PD-L1 ≥ 50%, HR 0.50, 95% CI, 0.35 -0.72 (Xu et al. 2019). Therefore, alternative approaches are needed to improve outcomes in these 2 subgroups of patients (PD-L1 expression <1% and STK11 mutations), who are presumed to benefit less from immunotherapy-based treatments.

Sotoraxib, a KRAS G12C inhibitor, recently received accelerated approval from the FDA for the treatment of patients with locally advanced or locally advanced KRAS G12C mutations as determined by an FDA-approved test who have received at least one prior systemic therapy. Adult patients with metastatic NSCLC. Sotorasiib received accelerated approval from the US FDA (Food and Drug Administration) in May 2021 and conditional labeling authorization from the European Commission (EC) in January 2022 for the treatment of locally advanced KRAS G12C mutant patients. or patients with metastatic non-small cell lung cancer (NSCLC). In this patient population, in a Phase 2 single-arm study of 126 patients, sotoraxib demonstrated an ORR of 37% (95% CI 28.6-46.2) and a median DOR of 11.1 months. PFS was 6.8 months, and median OS was 12.5 months (Skoulidis et al., N Engl J Med [New England Journal of Medicine]; 384:2371-81). Another KRAS G12C inhibitor, adagrasib, is also in clinical development for KRAS G12C mutant malignancies, with a preliminary ORR of 45% in patients with NSCLC (Janne et al., 2019, in October 2019 Published at the AACR-NCI-EORTC International Conference on Molecular Targets on the 28th).

However, the benefit of these targeted therapies in tumors with KRAS G12C mutations remains uncertain at this time, as not all patients respond, and in some cases, the reported duration of responses is short, possibly due to the development of Resistance is mediated, at least in part, by RAS gene mutations that disrupt inhibitor binding and reactivation of downstream pathways.

For example, of the 38 patients enrolled in the adagrasib study: 27 patients with non-small cell lung cancer, 10 patients with colorectal cancer, and 1 patient with appendiceal cancer, testing was performed in 17 patients (45% of the cohort) To the putative mechanisms of resistance to adagrasib, 7 (18% of the cohort) had multiple overlapping mechanisms. Acquired KRAS alterations include high-level amplification of the G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and KRASG12C alleles. Mechanisms of acquired bypass resistance include MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function in NF1 and PTEN mutation (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 affecting the switch-II pocket, to which adagrasib and other inactive KRAS G12C inhibitors bind, interfering with Key protein-drug interactions and confer resistance to these inhibitors in engineered and patient-derived KRASG12C cancer models.

Therefore, additional treatment options are needed to overcome resistance mechanisms that arise during treatment with KRAS inhibitors such as adagrasiib or sotorasib.

The efficacy of KRAS G12C inhibitors (such as adagrasiib or sotoracib) in the first-line setting remains unknown, as previous trials have focused on patients previously exposed to standard treatments (such as platinum-based chemotherapy and immunotherapy) Patients with advanced NSCLC.

Therefore, the medical need for new treatment options remains urgent for patients with lung cancer, including advanced and/or metastatic cancer, including NSCLC, especially when the cancer or solid tumor harbors a KRAS G12C mutation of. It will also be important to provide potentially beneficial novel therapies for incurable diseases, particularly for patients with KRAS G12C mutant tumors who have received and failed standard of care therapies for their indications, or Intolerance or ineligibility for approved therapies and therefore have limited treatment options.

The present invention provides new treatment options for patients with cancer, including advanced and/or metastatic cancer, and seeks to improve outcomes for patients with KRAS G12C- driven cancers. In particular, cancers to be treated by a KRAS G12C inhibitor (e.g., Compound A) or a pharmaceutically acceptable salt thereof are cancers or tumors with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutation status. cancers (such as NSCLC), or cancers or solid tumors (such as NSCLC) with KRAS G12C mutations and ≥ 1% PD-L1 expression and STK11 co-mutations.

Provided herein are compounds and methods thereof for the treatment of cancer or solid tumors (eg, NSCLC) with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutation status or in the treatment of patients with KRAS G12C mutations and ≥ 1 % of PD-L1 and STK11 co-mutated cancers or solid tumors such as NSCLC. The present invention also provides potentially beneficial novel therapies for incurable diseases, particularly for patients with KRAS G12C mutant tumors who are intolerant or ineligible for approved therapies and therefore have limited Treatment options. Such patients include patients with advanced NSCLC who have not received prior systemic therapy for advanced disease and whose tumors harbor KRAS G12C mutations and <1% PD-L1 expression or ≥1% PD-L1 expression and STK11 co-mutations.

In addition, the present invention also provides Compound A, alone or in combination with one or more additional therapeutic agents, for use in a method of treating cancer patients who have become resistant to other therapies, such as those previously treated with other KRAS Treatment with inhibitors (e.g., adagrasiib and sotoracib); preferably prior treatment with sotoracib.

Compound A is a selective covalent irreversible inhibitor of KRAS G12C and exhibits a novel binding mode by utilizing its unique interaction with KRASG12C. Notably, Compound A traps KRAS G12C in a GDP-bound inactive state while avoiding direct interaction with H95, a well-established resistance pathway (Awad-MM et al., New Engl J Med [New England Journal of Medicine] ] 2021;384:2382-2392). Compound A potently inhibits KRAS G12C H95Q, a double mutant that mediates resistance to adagrasib in clinical trials.

Compound A showed potent antitumor activity and favorable pharmacokinetic properties in preclinical models. Compound A was orally bioavailable, achieved exposure in the range predicted to confer antitumor activity, and was well tolerated.

Preliminary data (Phase Ib) from the KontRASt-01 study (NCT04699188) demonstrate that Compound A is a selective covalent and orally bioavailable inhibitor of KRASG12C, based on preliminary clinical trials in patients with KRAS G12C mutant solid tumors. Data, showing anti-tumor activity, high systemic exposure, and good safety profile at its recommended dose of 200 mg BID.

KRAS G12C inhibitors are specifically designed to inhibit KRAS G12C. However, many tumors harbor KRAS WT, HRAS, and NRAS proteins that are not inhibited by KRAS G12C inhibitors. For example, after KRAS G12C inhibitor treatment, reactivated RTKs can enter the MAPK pathway via these proteins, thereby counteracting anti-tumor activity. Likewise, many RTK and RAS proteins directly activate parallel pathways, such as the PI3K/AKT pathway.

In preclinical data, the presence of KRAS G12C mutations has been associated with an immunosuppressive tumor microenvironment. This effect can be caused by high levels of inhibitory interleukins such as nuclear factor kappa B (NF-κβ), signal transducer and activator of transcription 3 (STAT3), interleukin-6 (IL-6), interleukin 1-β (IL-1β)) and is mediated by myeloid-derived suppressor cells, regulatory T cells, and M2-differentiated tumor-associated macrophages in the tumor stroma (all of which impair anti-tumor immunity and promote tumor progression) mediated by the large presence of (Hamarsheh et al. 2020 and Cucurull et al. 2022). To further support this notion, KRAS G12C inhibitors stimulate the recruitment and activation of CD8+ T cells, dendritic cells, and M1-macrophages, thereby promoting a shift toward a more immunocompetent tumor microenvironment in NSCLC preclinical models ( Canon et al. 2019 and Briere et al. 2021). Compound A series compound 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -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, which has structure (Compound A).

It should be understood that reference herein to "Compound A" is intended to include the free base or a pharmaceutically acceptable salt or hydrate or solvate thereof. The terms "a compound of the invention" and "compounds of the invention" include Compound A in the free base form or a pharmaceutically acceptable salt or hydrate or solvate form thereof.

The present invention provides such compounds for use in the treatment of cancer as described herein.

The efficacy of the treatment methods and uses of the present invention can be determined by methods well known in the art, such as determining best overall response (BOR), overall response rate (ORR), duration of response (DOR), disease control according to RECIST v.1.1 rate (DCR), progression-free survival (PFS) and overall survival (OS). Accordingly, the invention provides pharmaceutical combinations of the invention that improve KRAS G12C inhibitor therapy, e.g., by best overall response (BOR), overall response rate (ORR), response according to RECIST v.1.1 Measured by an increase in one or more of duration of disease (DOR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS).

Methods to assess KRAS G12C and/or STK11 mutational status and PD-L1 expression are known in the art. For example, KRAS G12C and/or STK11 mutation status can be assessed in tumor tissue or in blood. KRAS G12C and/or STK11 mutations can be determined using molecular tests that detect mutations in DNA derived from tumor tissue or circulating tumor DNA (ctDNA) derived from plasma. Testing of KRAS G12C mutation status and/or STK11 mutation status can also be performed using next-generation sequencing (NGS) tests that detect DNA derived from formalin-fixed paraffin-embedded tumor tissue or ctDNA derived from plasma mutations in. PD-L1 Tumor Proportion Score (TPS) status can be assessed based on the PD-L1 immunohistochemistry (IHC) 22C3 or 28-8 pharmDx assay, or tumor cell (TC) membrane expression can be assessed based on the Ventana SP263 assay.

In another embodiment is a method for treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the invention.

In embodiments 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 lung squamous cell carcinoma, particularly when the cancer or tumor has a KRAS G12C mutation and KRAS G12C mutation and <1% PD-L1 expression regardless of STK11 mutation status.

In embodiments 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 lung squamous cell carcinoma, particularly when the cancer or solid tumor (such as NSCLC ) with KRAS G12C mutation and ≥ 1% PD-L1 manifestation and STK11 co-mutation.

In embodiments of the invention, the cancer or tumor to be treated is non-small cell lung cancer.

In an embodiment of the invention, the cancer or tumor to be treated is selected from the group consisting of colorectal cancer, cholangiocarcinoma, ovarian cancer, and pancreatic cancer, wherein the cancer has a KRAS G12C mutation and <1% PD-L1 expression, or wherein the cancer has KRAS G12C mutations and ≥ 1% of PD-L1 manifestations are co-mutated with STK11.

In additional embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients as described herein. KRAS G12C inhibitors

Examples of KRAS G12C inhibitors that can be used in the combinations and methods of the present invention include Compound A, sotoraxib (Amgen), adagrasiib (Mirati), D-1553 (Yifang) Biological (InventisBio)), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Eli 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 University) York University)), and RMC6291 (Revolution Pharmaceuticals, Inc.) or a pharmaceutically acceptable salt thereof.

KRAS G12C inhibitors also include the compounds detailed in A. "KRASG12C inhibitor" is a compound selected from the compounds detailed in the following documents: 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, WO 2017/058805, WO 2017015562, WO 2017058728, WO 2017058768, WO 2017058792, WO 2017058805, WO 2 017058807、WO 2017058902、WO 2017058915、WO 2017087528、WO 2017100546、WO 2017/ 201161、WO 2018/064510、WO 2018/068017、WO 2018/119183、WO 2018/217651、WO 2018/140512、WO 2018/140513、WO 2018/140514、WO 2018/140598、WO 20 18/140599、WO 2018/ 140600、WO 2018/143315、WO 2018/206539、WO 2018/218070、WO 2018/218071、WO 2019/051291、WO 2019/099524、WO 2019/110751、WO 2019/141250、WO 20 19/150305、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)pipien-1-yl)propan-2- En-1-one--methane (1/2) (compound 1); (S)-1-(4-(6-chloro-8-fluoro-7-(2-fluoro-6-hydroxyphenyl)quino oxazolin-4-yl)piperidine-1-yl)prop-2-en-1-one (compound 2); and 2-((S)-1-propenyl-4-(2-((( S)-1-methylpyrrolidin-2-yl)methoxy)-7-(naphth-1-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine- 4-yl)piperidine-2-yl)acetonitrile (compound 3). KRAS G12C inhibitor compound A

The preferred KRAS G12C inhibitor of the present invention is compound A, which is 1-{6-[(4 M )-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}propan -2-en-1-one or a pharmaceutically acceptable salt thereof. Compound A is also known as "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".

The synthesis of compound A is described in the following examples or in Example 1 of PCT application WO 2021/124222 published on June 24, 2021. The use of Compound A alone or in combination with additional therapeutic agents is described in PCT/CN 2021/139694 filed on December 20, 2021.

The structure of compound A is as follows: .

Alternatively, the structure of compound A can be drawn as follows: .

Compound A is a potent and selective small molecule inhibitor of KRAS G12C, which can covalently bind to mutant Cys12 and trap KRAS G12C in an inactive GDP-bound state. Compound A is structurally unique compared to sotorasiib or adagrasiib; its binding mode is a novel way to reach residue C12 and has no direct interaction with residue H95.

Preclinical data indicate that Compound A binds to KRAS G12C with low reversible binding affinity to the RAS SWII pocket, thereby inhibiting downstream cellular signaling and proliferation, particularly in KRAS G12C-driven cell lines but not in KRAS wild-type (WT ) or MEK Q56P mutant cell lines. Compound A demonstrated deep and sustained target occupancy in different KRAS G12C mutant xenograft models, resulting in anti-tumor activity. Cancers to be treated by the compounds and methods of the invention

Accordingly, the compounds of the present invention may be used to treat cancer and KRAS G12C mutated cancers or tumors. In particular, the compounds of the present invention can be used as cancers or solid tumors (such as NSCLC) with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutation status, or with KRAS G12C mutations and ≥ 1%. First-line treatment of cancers or solid tumors (such as NSCLC) that express PD-L1 and STK11 co-mutations.

The cancer or tumor to be treated may be selected from the group consisting of colorectal, cholangiocarcinoma, ovarian, and pancreatic cancer, wherein the particular cancer has a KRAS G12C mutation and <1% PD-L1 expression, or wherein the cancer has KRAS G12C mutations and ≥ 1% of PD-L1 manifestations are co-mutated with STK11.

The cancer can be early, intermediate, late or can be metastatic.

In some embodiments, the cancer is advanced cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is recurrent cancer. In some embodiments, the cancer is refractory to treatment. In some embodiments, the cancer is recurrent cancer. In some embodiments, the cancer is unresectable cancer.

Cancer can be early, intermediate, late or metastatic.

Compound A may also be used to treat solid malignancies characterized by additional RAS mutations.

The invention provides Compound A and combinations of the invention for use in the treatment of acquired KRAS alterations (selected from G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, Y96 D and KRASG12C for use in cancers or solid tumors characterized by high-level amplification of alleles) or by acquired bypass resistance mechanisms, including MET amplification; NRAS, BRAF, MAP2K1, and Activating mutations in RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN.

In another embodiment is a method of treating (e.g., alleviating, inhibiting, or delaying one or more of progression) a cancer or tumor in a subject, the method comprising administering to the subject in need thereof a compound comprising Compound A or its Pharmaceutical compositions of pharmaceutically acceptable salts.

Accordingly, the present invention provides methods of treating (e.g., reducing, inhibiting, or delaying one or more of progression) a cancer or tumor in a patient in need thereof, wherein the method comprises administering to the patient in need thereof a therapeutically active amount of The compound of the present invention, wherein the cancer is a cancer or solid tumor (such as NSCLC) with KRAS G12C mutation and <1% PD-L1 expression regardless of STK11 mutation status, or a KRAS G12C mutation and ≥1% PD - Cancers or solid tumors with L1 expression and STK11 co-mutations (such as NSCLC). The cancer may be selected from lung cancer (including lung adenocarcinoma and non-small cell lung cancer), colorectal cancer, cholangiocarcinoma, ovarian cancer, and pancreatic cancer.

The methods and combinations of the present invention may be used as first-line therapy (or as second-line or later-line therapy). For example, the patient may be treatment agnostic or a patient who has progressed and/or relapsed on prior therapy.

For example, patients or subjects to be treated by the methods and combinations of the invention include patients with cancer, such as KRAS G12C mutant NSCLC (including advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), depending on Required in which the patient has received and progressed on prior therapy.

In embodiments of the invention, the subject or patient to be treated with Compound A and who may benefit from the treatment is selected from: - Patients with NSCLC whose tumors have KRAS G12C mutations and <1% PD-L1 expression; - Patients with NSCLC whose tumors harbor KRAS G12C mutations and <1% PD-L1 expression, regardless of STK11 mutation status; - Patients with locally advanced or metastatic NSCLC whose tumors harbor KRAS G12C mutations and <1% PD-L1 expression, regardless of STK11 mutation status; - Patients with NSCLC whose tumors harbor KRAS G12C mutations, ≥ 1% PD-L1 expression, and STK11 co-mutations; - Patients with locally advanced or metastatic NSCLC whose tumors harbor KRAS G12C mutations, ≥1% PD-L1 expression, and STK11 co-mutations.

In embodiments of the present invention, the patient has received standard of care therapy but failed it, or is intolerant to or ineligible for previously studied and/or approved therapy; Dosage and dosing regimen

In the methods and uses of the invention, Compound A may be administered in a total daily dose of between 100 mg and 600 mg or between 200 mg and 400 mg. The total daily dose of Compound A can be administered continuously on a QD (once daily) or BID (twice daily) schedule. For example, Compound A can be administered at a dose of 200 mg BID (total daily dose of 400 mg), 400 mg QD (total daily dose of 400 mg). Compound A may also be administered at a dose of 100 mg QD (total daily dose 100 mg) or 100 mg BID (total daily dose 200 mg) or at a dose of 200 mg QD (total daily dose 200 mg). Doses lower than the recommended dose of 400 mg (preferably administered twice daily) may be used particularly in cases of poor tolerability or high toxicity, while maintaining therapeutic efficacy.

Preclinical target occupancy models combined with PK patient data predict that BID programs may result in increased response in higher numbers of patients. Suitably, Compound A may therefore be administered at a dose of 100 mg BID or at a dose of 200 mg BID or at a dose of 300 mg BID. Typically, Compound A may be administered at a dose of 100 mg twice daily (for a total daily dose of 200 mg) or at a dose of 200 mg twice daily (for a total daily dose of 400 mg).

PK/PD modeling predicts sustained high levels of target occupancy at the recommended dose of 200 mg BID. The recommended total daily dose of Compound A is 400 mg, administered once daily (QD) or twice daily (BID), administered continuously (i.e., drug-free holidays).

Compound A is preferably administered with food, for example immediately after a meal (within 30 minutes).

In a preferred embodiment, the total daily dose of Compound A is 400 mg administered twice daily. This was determined based on clinical assessment of efficacy, safety, PK, PK-PD models, and a Bayesian hierarchical logistic regression model (BHLRM) model to assess the probability of DLT.

The 200 mg BID dose was evaluated and determined to be safe and tolerable. Among all dose levels tested, the highest AUC0-24h was obtained at 200 mg BID, which was >60% higher than the exposure at the 200 mg and 400 mg QD dose levels and higher than that obtained in the less sensitive xenograft model The effective exposure required for maximal efficacy was at least 3-fold higher and was highest for tumor regression in this xenograft model. At this dose level, an average KRAS G12C target occupancy of >90% was predicted based on the PK-PD model and was higher than the 70% average target occupancy required for tumor regression in various xenograft models.

The 200 mg BID dose level meets the Bayesian Hierarchical Logistic Regression Model (BHLRM) Escalation of Overdose Control (EWOC) criteria (i.e., <25% chance of true DLT rate greater than or equal to 33% during the DLT evaluation period), The posterior probability of excessive toxicity is less than 0.1%. No DLTs were observed at this dose level.

Therefore, it is expected that administration of a total dose of 400 mg of Compound A, preferably twice daily, would be particularly useful in the uses and methods of the present invention.

A total dose of 200 mg of Compound A, preferably administered daily (e.g., 100 mg BID), may also be useful in the uses and methods of the invention, particularly to reduce toxicity and/or improve the safety of the treatment and tolerance aspects. pharmaceutical composition

In another aspect, the invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a KRAS G12C inhibitor (eg, Compound A) in combination with one or more pharmaceutically acceptable Carriers (additives) and/or diluents are formulated together.

Preferably, the pharmaceutically acceptable carrier system is sterile. Pharmaceutical compositions may be formulated for specific routes of administration, such as oral administration, parenteral administration, rectal administration, and the like. In addition, the pharmaceutical composition of the present invention can be prepared in solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories) or in 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 auxiliary agents (such as preservatives, stabilizers, wetting agents, emulsifiers and buffer, etc.).

Typically, pharmaceutical compositions are tablets or gelatin capsules containing the active ingredient and 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 aluminum magnesium silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; d) disintegrating agents, such as starch, agar, alginic acid or sodium salts or effervescent mixtures thereof; and e) Adsorbents, colorants, flavorings and sweeteners.

In embodiments, the pharmaceutical composition contains only capsules of active ingredients.

Tablets may be film-coated or enteric-coated according to methods known in the art.

Suitable compositions for oral administration include effective amounts in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs, solutions or solids The compounds of the combinations of the invention are in the form of 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 sweetening agents, flavoring agents, coloring agents, and preservatives. The reagents are composed of groups of reagents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. Such excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin or Gum Arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over a longer period of time. For example, time delay materials such as glyceryl monostearate or glyceryl distearate may be used. Formulations for oral use may be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as hard gelatin capsules in which the active ingredient is mixed with an aqueous or oily vehicle (e.g., calcium carbonate, calcium phosphate, or kaolin). , peanut oil, liquid paraffin or olive oil) mixed soft gelatin capsules.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The composition may be sterile and/or contain adjuvants (eg, preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, salts for adjusting osmotic pressure and/or buffers). Additionally, they can contain other substances of therapeutic value. The composition is prepared according to conventional mixing, granulating or coating methods, and contains about 0.1%-75%, or about 1%-50% of active ingredients.

Suitable compositions for transdermal application include an effective amount of a compound of the invention and a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents that assist passage through the skin of the host. For example, a transdermal device is in the form of a bandage that includes a liner, a reservoir containing a compound and optionally a carrier, and a controller that delivers the compound to the skin of a host at a controlled and predetermined rate over an extended period of time. rapid barrier, and apparatus to secure the device to the skin.

Compositions suitable for topical administration (e.g., to the skin and eyes) include aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations, e.g., for delivery by aerosol or the like . Such topical delivery systems will be particularly suitable for dermal administration, eg, for the treatment of skin cancer, eg, for prophylactic use in sunscreens, lotions, sprays and the like. It is therefore particularly suitable for topical use, including cosmetics, formulations well known in the art. Such systems may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein, topical administration may also involve inhalation or intranasal administration. They are conveniently available in dry powder form (alone; as a mixture, for example with lactose; or as mixed component particles, for example with phospholipids) from a dry powder inhaler or as an aerosol spray from a pressurized container , delivered by pump, sprayer, atomizer or ejector, with or without suitable propellants.

In one embodiment, the present invention provides a product comprising Compound A, or a pharmaceutically acceptable salt thereof, for use in therapy. In one embodiment, the therapy is targeted to cancers or solid tumors (eg, NSCLC) with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutation status, or with KRAS G12C mutations and ≥1% PD - Cancers or solid tumors with L1 expression and STK11 co-mutations (such as NSCLC). Products provided as combination preparations include compositions containing the compounds of the invention in kit form.

In one embodiment, the kit includes means (eg, containers, separate bottles, or separate foil packets) for holding the compositions separately. An example of such a kit is a blister pack, as is typically used for the packaging of tablets, capsules, etc.

To aid compliance, the kits of the invention typically include instructions for administration.

definition

Unless stated otherwise, the general terms used above and below preferably have the following meanings in the context of the present disclosure, wherein the more general terms used in whatever context may be replaced by more specific definitions independently of each other. Or retain, thereby defining more detailed embodiments of the present invention:

In particular, where reference is made to dosage (dose or dosage), this is intended to include a range in the vicinity of ± 10% or ± 5% of the specified value.

According to common practice in the art, dosage refers to the amount of therapeutic agent in free form. For example, when a 200 mg dose of Compound A is referred to and Compound A is used in the form of its pharmaceutical salt, the amount of therapeutic agent used is equivalent to 20 mg of Compound A in its free form.

As used herein, the term "subject" or "patient" is intended to include animals susceptible to or suffering from cancer or any disorder involving, directly or indirectly, cancer. 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, such as a human who has, is at risk of, or may be susceptible to cancer.

The terms "treating" or "treatment" as used herein include treatment that relieves, alleviates, or relieves at least one symptom in a subject or achieves a delay in disease progression. For example, treatment may be to reduce the symptoms of one or several disorders, or to partially or completely eradicate a disorder (such as cancer). Within the meaning of this disclosure, the term "treatment" also means preventing, delaying the onset (i.e., the period prior to clinical manifestation of the disease) and/or reducing the risk of disease progression or disease progression.

"Treatment" may also be determined by efficacy and/or pharmacodynamic endpoints, and may be defined as improvement in one or more of safety, efficacy, and tolerability. The efficacy of monotherapy or combination therapy can be determined by determining the following according to RECIST v.1.1: best overall response (BOR), overall response rate (ORR), duration of response (DOR), disease control rate (DCR), Progression-free survival (PFS) and overall survival (OS).

The “best overall response” (BOR) rate was defined as the best response documented according to RECIST 1.1 from the start of treatment until disease progression/relapse.

“Overall response rate” (ORR) was defined as the proportion of patients with a BOR of CR or PR according to RECIST 1.1.

According to RECIST 1.1, “duration of response” (DOR) is the time between the first documented response (CR or PR) and the date of progression or death from any cause. Here, death from any cause is considered a conservative event and meets the PFS event definition.

“Disease control rate” (DCR) was defined as the proportion of patients with a BOR of CR, PR, or SD according to RECIST 1.1.

According to RECIST 1.1, “progression-free survival” (PFS) is defined as the time between the date of initiation of treatment and the date of first documented progression or death from any cause according to RECIST 1.1. If the patient does not have an event, PFS will be reviewed at the date of last adequate tumor assessment.

“Overall survival” (OS) was defined as the number of days from the date of initiation of study treatment to the date of death from any cause. If no death was reported before study termination or analysis cutoff, survival was censored on the last known patient survival date before/on the cutoff date. Survival time for patients without post-baseline survival information will be censored at the date of treatment initiation.

"Treatment" may also be defined as improvement in reducing the side effects of therapy with Compound A as described herein.

Unless otherwise indicated, the terms "comprising" and "including" are used herein in their open-ended and non-limiting sense.

Unless otherwise indicated herein or clearly contradicted by the context, the terms "a", "an" and "the" and similar designators shall be construed in the context of describing the invention (especially in the context of the following claims). To cover both the singular and the plural. When the plural form is used for compounds, salts, etc., this also means the singular compound, salt, etc.

The phrase "therapeutically effective amount" as used herein refers to an amount of a compound, material or composition comprising a compound of the invention that results in a reasonable benefit in at least one subpopulation of cells in an animal suitable for use in any medical treatment/ The risk ratio is valid for producing some desired treatment effect.

As used herein, the phrase "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissue without producing undue toxicity, irritation, allergic reactions, or other problems or complications, Those compounds, materials, compositions, and/or dosage forms that also have a reasonable and proportionate benefit/risk ratio.

As noted above, certain embodiments of the compounds of the present invention may contain basic functional groups, such as amine or alkylamino groups, and are thereby capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this regard, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds of the invention. Such salts may be prepared in situ during manufacture of the administration vehicle or dosage form, or by separately reacting a purified free base form of the compound of the invention with a suitable organic or inorganic acid and isolating therefrom during subsequent purification. The salts formed are prepared. 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, glucose Heptonates, lactosurates and lauryl sulfonates, etc. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

In the methods and uses of the present invention, Compound A is also intended to represent unlabeled as well as isotopically labeled forms of the compound. Isotopically labeled compounds have one or more atoms replaced by atoms with a selected atomic mass or mass number. Examples of isotopes that may be ^incorporated include isotopes of hydrogen, carbon, nitrogen, oxygen and chlorine (where possible), such as ² H, ³ H, ^{11 C, 13} C, ¹⁴ C, ¹⁵ N, ³⁵ S, ³⁶ Cl. Isotopically labeled compounds can be used in metabolic studies (with ¹⁴ C), reaction kinetic studies (e.g. with 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 determination, or for radiation therapy in patients. Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically labeled reagents.

Furthermore, substitution with heavier isotopes, especially deuterium (i.e., ^H or D) may provide certain therapeutic advantages derived from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements or improved therapeutic index) . It should be understood that deuterium is considered a substituent of compound A in this context. The concentration of this heavier isotope (especially deuterium) can be defined by the isotope enrichment factor. As used herein, the term "isotopic enrichment factor" means the ratio between the abundance of an isotope and the natural abundance of a given isotope. If a substituent in a compound of the present invention is designated as deuterium, such compound has an isotope enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation per designated deuterium atom), at least 4000 (60% deuterium doped), at least 4500 (67.5% deuterium doped), at least 5000 (75% deuterium doped), at least 5500 (82.5% deuterium doped), at least 6000 (90% deuterium doped), at least 6333.3 (95% deuterium incorporated), at least 6466.7 (97% deuterium incorporated), at least 6600 (99% deuterium incorporated), or at least 6633.3 (99.5% deuterium incorporated).

In compound A, for example, the methyl group on the indazolyl ring can be deuterated or perdeuterated. Example Example 1: Compound A (JDQ443) shows antitumor activity in a KRAS G12C mutant CDX model driven by target occupancy

Single-agent antitumor activity of JDQ443 at daily oral doses of 10 mg/kg, 30 mg/kg, and 100 mg/kg in a panel of KRAS G12C mutant CDX models with different indications. The xenograft cell lines were: MIA PaCa-2 (PDAC); NCI-H2122, LU99, HCC-44, NCI-H2030 (NSCLC); and KYSE410 (esophageal cancer). JDQ443 inhibited growth in all models in a dose-dependent manner (Figure 1A), with model-specific differences in dose-response kinetics and maximal response patterns ranging from regression (MIA PaCa-2, LU99), to arrest (HCC44, NCI-H2122) , ranging from moderate tumor inhibition (NCI-H2030, KYSE410). The maximum dynamic range is observed in LU99. In contrast, JDQ443 showed no growth inhibition in a KRASG12V mutant xenograft model (NCI-H441; Figure 1B), confirming the specificity of KRASG12C and consistent with in vitro data. Efficacy was maintained when the same daily dose was administered once daily (QD) or twice daily (BID): 30 mg/kg QD versus 15 mg/kg BID in MIA PaCa-2 (Fig. 1C), or In NCI-H2122 and LU99, 100 mg/kg QD vs. 50 mg/kg BID (Figure 1D-1E). The efficacy of QD versus BID administration is closely related to the comparable daily area under the concentration-time curve (AUC) in the blood.

These findings demonstrate that the efficacy of JDQ443 is related to target occupancy (TO) and that effective AUC exposure can be achieved under both QD and BID dosing. To characterize whether AUC could serve as a surrogate for TO, the effects of continuous infusion versus oral administration in a LU99 xenograft model were studied. Once-daily oral administration of 30 mg/kg induced stasis followed by tumor progression for approximately one week, and 100 mg/kg induced tumor regression (Fig. 1F), with steady-state average concentrations (Cav) of approximately 0.3 µM and approximately 0.3 µM, respectively. 1 µM. To evaluate continuous dosing, JDQ443 was delivered intravenously via a programmable microinfusion pump to achieve target concentrations close to oral Cav. Continuous infusion and oral administration resulted in comparable antitumor responses (Figures 1F, 1G). PK/PD model simulations showed that efficacy was best correlated with TO and AUC of JDQ443 (Figures 1H, 1I), rather than other PK metrics. Example 2: Compound A potently inhibits KRAS G12C H95Q (a double mutant 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 (Kit) (Cat. No. 210518) Template: pcDNA3.1(+)EGFP-T2A-FLAG-KRAS G12C ) were produced and expressed in Cas9-containing Ba/F3 cells by stable transfection. Treat cells with a dose-response curve starting with 10 μM diluted 1/3 from 10 mM DMSO stock. Cell lines were treated with indicated compounds for 72 hours, and cell viability was measured using CellTiter-Glo. result:

Unlike MRTX-849 (adagrasib), JDQ443 (compound A) and AMG-510 (sotorracib) potently inhibit the cell viability of the KRASG12C H95Q double mutant. MRTX-849, AMG-510, or JDQ443 failed to inhibit KRASG12C Y96D or KRASG12C R68S double mutants at the indicated concentrations and conditions (Ba/F3 system, 3-day proliferation assay), and the double mutants were resistant to all three tests Resistance to KRASG12C inhibitors. KRAS G12C inhibitors GI ₅₀ [µM] +/- standard deviation (STDEV) KRAS G12C H95Q KRAS G12C Y96D KRAS G12C R68S NVP-JDQ443 (Compound A) 0.57 +/- 0.18 ＞10 ＞10 AMG-510 (Sotoracib) 0.26 +/- 0.06 ＞10 ＞10 MRTX-849 (adagrasib) ＞10 ＞10 ＞10 Conclusion:

In the case of KRASG12C H95Q, compound A may overcome resistance to adagrasib. Additionally, due to the unique binding interaction of Compound A with mutant KRAS G12C, when compared to sotoraxib and adagrasiib, Compound A alone or in combination with one or more therapeutic agents as described herein A, may be used to treat patients with cancer previously treated with other KRAS G12C inhibitors (such as sotoraxib or adagrasiib) or after the development of acquired KRAS resistance mutations during initial KRAS G12C inhibitor therapy Targeted resistance. Example 3: Compound A potently inhibits KRAS G12C double mutant

The effect of Compound A and other KRASG12C inhibitors on reported second site mutations conferring resistance to adagrasib was also studied as follows. Materials and Methods: Cell Lines and KRAS ^G12C Inhibitors:

Unless otherwise indicated, the Ba/F3 cell line is a mouse primary B cell line and was supplemented with 10% fetal bovine serum (FBS) (BioConcept, #2-01F30-I), 2 mM acetone Sodium phosphate (Bioconcepts, #5-60F00-H), 2 mM Stabilized Glutamine (Bioconcepts, #5-10K50-H), 10 mM HEPES (Bioconcepts, #5-31F00-H ) in RPMI 1640 (Bioconcepts, #1-41F01-I) at 37°C and 5% _CO . Parental Ba/F3 cells were cultured in the presence of 5 ng/ml recombinant murine IL-3 (Life Technologies, #PMC0035). Ba/F3 cells normally rely on IL-3 for survival and proliferation, however, by expressing oncogenes, they are able to switch their dependence from IL-3 to the expressed oncogene (Curr Opin Oncology, 2007 Jan;19(1):55-60. doi: 10.1097/CCO.0b013e328011a25f.) Single plastid mutagenesis and generation of Ba/F3 stable cell lines:

The QuikChange Lightning site-directed mutagenesis kit (Agilent; #210519) was used to generate resistance mutations on the pSG5_Flag-(codon optimized) KRAS ^G12C_puro plasmid template and by Sanger sequencing Confirm sequence. primer Primer sequence SEQ ID NO H95R_Forward 5'-gtcatttgaagatatccaccgttatcgcgagcagattaaga-3' 552 H95R_Reverse 5'-tcttaatctgctcgcgataacggtggatatcttcaaatgac-3' 553 H95Q_Forward 5'-tcatttgaagatatccaccagtatcgcgagcagattaagag-3' 554 H95Q_Reverse 5'-ctcttaatctgctcgcgatactggtggatatcttcaaatga-3' 555 H95D_Forward 5'-agtcatttgaagatatccacgattatcgcgagcagattaag-3' 556 H95D_reverse 5'-cttaatctgctcgcgataatcgtggatatcttcaaatgact-3' 557 MR68S_Forward 5'-gaagagtactccgcaatgagcgatcaatacatgaggacg-3' 558 R68S_Reverse 5'-cgtcctcatgtattgatcgctcattgcggagtactcttc-3' 559 Y96C_Forward 5'-cgaagtcatttgaagatatccaccattgtcgcgagcagatta-3' 560 Y96C_Reverse 5'-taatctgctcgcgacaatggtggatatcttcaaatgacttcg-3' 561 Y96D_Forward 5'-cgaagtcatttgaagatatccaccatgatcgcgagcagatt-3 562 Y96D_reverse 5'-aatctgctcgcgatcatggtggatatcttcaaatgacttcg-3' 563

The mutant plasmids were transfected into Ba/F3 WT cells by electroporation using NEON transfection kit (Invitrogen, #MPK10025). Therefore, two million Ba/F3 cells were electroporated with 10 μg pf plasmid using the NEON system (Invitrogen, #MPK5000) (using 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. IL-3 withdrawal ( withdrawal )

Ba/F3 cells normally rely on IL-3 for survival and proliferation, however, by expressing oncogenes, they are able to switch their dependence from IL-3 to the expressed oncogene. To evaluate whether KRAS ^G12C single and double mutants are able to sustain proliferation of Ba/F3 cells, engineered Ba/F3 cells expressing the mutant constructs were cultured in the absence of IL-3. Cell number and viability were measured every three days, and IL-3 withdrawal was completed after seven days. The expression of the mutant after IL-3 withdrawal was confirmed by Western blotting (data not shown, upshift of KRAS ^G12C/R68S was observed). Validation of drug response curves and resistance mutations to KRASG12C inhibitors:

1000 Ba/F3 cells/well were seeded into a 96-well plate (Greiner Bio-One, #655098). Treat on the same day with the Tecan D300e medication dispenser. CellTiter-Glo luminescent cell viability assay (Promega, #G7573) was used on a Tecan infinitiy M200 Pro reader (integration time 1000 ms), on the same day as the starting plate was processed (day 0) and detect viability three days after treatment (day 3).

To determine growth, readings three days after treatment (Day 3) were normalized relative to the starting plate (Day 0). Percent viability was then calculated by normalizing treated wells relative to DMSO-treated control samples. Use XLfit to create a fitting curve (four-parameter curve) of the Sigmoidal dose-response model (Figure 2). The horizontal red dashed line represents the GI50 value. The tabular data is shown below. [Table]: Effect of compound A (JDQ443) on the proliferation of KRAS G12C/H95 double mutant. («STDEV» represents the standard deviation of the % growth value) % growth of treated cells relative to day 3 treatment Concentration [μM] 1 0.333333 0.111111 0.037037 0.012346 0.004115 0.001372 0.000457 G12C 4.279 20.252 44.204 72.785 89.361 93.832 97.516 95.501 G12C STDEV 0.961 0.345 3.567 3.058 1.072 0.770 3.921 3.639 H95R 0.321 5.323 23.425 52.178 66.971 83.996 92.517 103.118 H95R STDEV 0.943 0.276 0.779 1.034 0.897 3.344 3.811 7.044 H95Q 6.635 36.908 71.333 93.319 95.722 103.375 93.606 100.054 H95Q STDEV 2.025 0.910 11.656 4.209 0.919 6.685 3.996 2.621 H95D 28.569 68.199 88.358 102.308 97.783 90.379 91.567 96.429 H95D STDEV 1.055 4.247 2.997 6.409 3.644 0.087 2.074 7.212 R68S 69.159 93.111 103.721 108.276 104.608 100.329 103.390 100.931 R68S STDEV 7.892 4.607 9.627 6.839 0.781 4.288 2.838 0.996 Y96C 80.229 91.765 102.592 104.222 96.167 105.515 102.424 109.116 Y96C STDEV 1.667 0.222 2.981 1.230 0.896 4.837 5.863 4.380 Y96D 90.864 97.260 105.866 103.946 106.613 98.191 102.224 100.903 Y96D STDEV 0.852 0.105 4.943 6.090 3.050 1.435 5.305 3.229 [Table]: Effect of sotoracib (AMG510) on the proliferation of KRAS G12C/H95 double mutant («STDEV» represents the standard deviation of % growth value) % growth of treated cells relative to day 3 treatment Concentration [μM] 1 0.333333 0.111111 0.037037 0.012346 0.004115 0.001372 0.000457 G12C 24.244 46.199 71.315 77.193 92.241 95.211 94.786 100.303 G12C STDEV 5.785 4.703 0.727 0.776 2.500 1.603 2.016 5.046 H95R 3.485 8.354 23.503 53.471 71.098 83.029 90.450 93.527 H95R STDEV 1.825 1.731 0.971 4.408 1.615 6.613 10.693 5.561 H95Q 8.566 34.323 68.123 89.685 93.396 98.559 103.616 98.858 H95Q STDEV 2.203 1.572 6.416 5.515 0.970 1.603 4.610 1.842 H95D -1.171 33.638 74.059 91.299 91.484 99.189 93.064 103.216 H95D STDEV 0.374 1.962 1.683 0.716 2.837 6.975 2.489 5.237 R68S 79.748 94.396 102.811 98.842 99.936 100.994 94.166 95.566 R68S STDEV 3.473 7.672 2.021 3.308 0.455 0.885 1.904 1.680 Y96C 111.964 105.031 103.341 97.712 104.892 109.010 106.167 103.355 Y96C STDEV 3.326 0.058 2.472 2.258 0.105 2.374 0.266 3.889 Y96D 106.099 101.660 101.868 97.311 102.190 106.308 101.134 105.511 Y96D STDEV 7.943 8.231 5.850 1.065 8.679 8.652 10.362 3.819 [Table]: Effect of adagrasib (MRTX-849) on the proliferation of KRAS G12C/H95 double mutant («STDEV» represents the standard deviation of % growth value) % growth of treated cells relative to day 3 treatment Concentration [μM] 1 0.333333 0.111111 0.037037 0.012346 0.004115 0.001372 0.000457 G12C -0.266 16.329 51.013 72.820 87.908 90.538 97.358 99.591 G12C STDEV 0.026 0.281 1.081 5.419 4.200 3.922 1.669 7.272 H95R 64.097 87.126 83.910 90.693 96.751 85.570 95.027 89.422 H95R STDEV 6.732 10.191 5.867 1.326 1.261 8.143 11.049 7.126 H95Q 73.397 88.044 95.662 101.071 92.756 100.136 91.377 98.312 H95Q STDEV 6.148 0.323 0.148 0.289 1.657 1.053 5.052 1.475 H95D 82.356 91.214 103.587 100.461 103.684 89.514 98.169 92.404 H95D STDEV 6.938 2.123 4.434 4.185 5.798 3.766 1.029 2.848 R68S 26.823 84.668 94.008 101.452 105.903 100.837 99.142 97.443 R68S STDEV 2.129 0.233 1.666 0.085 7.640 1.368 3.217 1.746 Y96C 82.638 96.520 99.562 102.615 105.187 101.092 100.220 99.819 Y96C STDEV 9.540 4.002 2.065 3.987 4.264 2.673 6.930 10.013 Y96D 81.671 95.056 108.171 97.824 105.964 97.437 102.545 106.432 Y96D STDEV 3.884 2.842 0.058 4.085 8.415 7.575 0.349 6.625 Western inkblot technique

After treatment with different compounds at the indicated concentrations for the indicated times, cells were harvested, pelleted and snap-frozen at -80°C. Add 60 μL of lysis buffer (50 mM Tris HCl, 120 mM NaCl, 25 mM NaF, 40 mM β-glycerophosphate disodium pentahydrate, 1% NP40, 1 μM microcystin, 0.1 mM Na3VO3, 0.1 mM PMSF, 1 mM DTT, and 1 mM benzamidine, supplemented with 1 protease inhibitor cocktail tablet (Roche) per 10 mL of buffer. The 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 using a BCA protein assay kit (Pierce, 23225). After normalizing to the same total volume with lysis buffer, add NuPAGE™ LDS Sample Buffer 4X (Invitrogen, NP0007) and NuPAGE™ Sample Reducing Reagent 10X (Invitrogen, NP0009). Samples were heated at 70°C for 10 min and then loaded onto a 26-well NuPAGE™ Novex™ 4%-12% Bis-Tris Midi protein gel (Invitrogen, WG1403A). The gel was run at 200 V for 45 min (PowerPac HC, Biorad) in NuPAGE MES SDS running buffer (Invitrogen, NP0002). Proteins were transferred to Trans-Blot® Turbo™ Midi nitrocellulose transfer membrane (Bio-Rad, 1704159) at 135 mA per gel for 7 min using the Trans-Blot® Turbo™ System (Bio-Rad, 1704159), followed by Ponceau Red (Sigma, P7170) stained the membrane. Block membranes with TBST containing 5% milk at RT. Anti-RAS (Abcam, 108602) and anti-phospho-ERK 1/2 p44/42 MAPK (Cell Signaling, 4370) antibodies were incubated overnight at 4°C. Plakin (Sigma, V9131) antibody was incubated for 1 h at RT. The membrane was washed three times with TBST for 5 min, and anti-rabbit (Cell Communications, 7074) and anti-mouse (Cell Communications, 7076) secondary antibodies were incubated at RT for 1 h. Dilute all antibodies to 1/1000 in TBST, except anti-vinculin (1/3000). FusionCapt Advance FX7 software was used on Fusion FX (Vilber Lourmat), with WesternBright ECL (Advansta, K-12045-D20) for Ras and vinculin and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Fischer, 34096) to determine. (Figure 3). Results [Table]: Compound A (JDQ443) inhibited the proliferation of KRAS G12C/H95 double mutant. Treatment with JDQ443 (Compound A), AMG-510 (sotorasiib), and MRTX-849 (adagrasiib) (8-point dilutions starting at 1 mM) indicated FLAG-KRAS ^G12C single or double mutants of Ba/F3 cells for 3 days, and proliferation inhibition was assessed by Cell Titer Glo viability assay. The mean of GI ₅₀ ± standard deviation (St DV) from 4 independent experiments is shown. GI50 ± St DV [μM] JDQ443 AMG-510 MRTX-849 G12C 0.115 ± 0.060 0.389 ± 0.235 0.136 ± 0.071 G12C/H95R 0.024 ± 0.006 0.033 ± 0.008 ＞1 G12C/H95Q 0.284 ± 0.041 0.233 ± 0.022 ＞1 G12C/H95D 0.612 ± 0.151 0.262 ± 0.088 ＞1 G12C/R68S ＞1 ＞1 0.707 ± 0.165 G12C/Y96C ＞1 ＞1 ＞1 G12C/Y96D ＞1 ＞1 ＞1 Biophysical Data Materials and Methods: Preparation of Reagents: Selection, Performance, and Purification of RAS Protein Constructs

The E. coli expression constructs used in this study are based on the pET system and were generated using standard molecular selection techniques. cDNAs encoding KRAS, NRAS, and HRAS contained aa 1-169 after a cleavable N-terminal His affinity purification tag and were codon optimized and synthesized by GeneArt (Thermo Fisher Scientific) . Point mutations were introduced using the QuikChange Lightning site-directed mutagenesis kit (Agilent). All final expression constructs were sequence verified by Sanger sequencing.

Two liters of culture medium were inoculated with a preculture of E. coli BL21(DE3) freshly transformed with expression plasmids and incubated with 1 mM isopropyl-β-D-thiogalactopyranoside (Sigma) at 18°C. Protein expression was induced at C for 16 hours. Avi-tagged proteins were transformed into E. coli with compatible plasmids expressing the biotin ligase BirA, and the culture medium was supplemented with 135 µM d-biotin (Sigma).

Resuspend the cell pellet in buffer A (20 mM Tris, 500 mM NaCl, 5 mM imidazole, 2 mM TCEP, 10 % glycerol, pH 8.0). Cells were lysed three times by a homogenizer (Avestin) at 800-1000 bar, and the lysates were clarified by centrifugation at 40000 g for 40 min.

The lysate was loaded onto a HisTrap HP 5 ml column (Cytiva) installed on an ÄKTA Pure 25 chromatography system (Cytiva). Contaminating proteins were washed away with buffer A, and bound proteins were eluted with a linear gradient into buffer B (buffer A supplemented with 200 mM imidazole). During dialysis O/N, the N-terminal His affinity purification tags on untagged and avi-tagged proteins were cleaved by TEV or HRV3C protease, respectively. Reload the protein solution onto the HisTrap column and collect the stream containing the target protein.

Guanosine 5'-diphosphate sodium salt (GDP, Sigma) or GppNHp-tetralithium salt (Jena Biosciences) was added to a molar excess of 24-32 times the protein. Add EDTA (pH adjusted to 8) to a final concentration of 25 mM. After 1 hour 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 (Stofan Co., Ltd.). GDP (for KRAS G12C resistance mutants H95Q/D/R, Y96D/C and R68S) or GppNHp was added to the eluted protein to a molar amount exceeding the protein by 24-32-fold. 40 U of shrimp alkaline phosphatase (New England Biolabs) was added to GppNHp containing sample only. Samples were then incubated at 5°C for 1 hour. Finally, add MgCl2 to a concentration of approximately 30 mM.

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

The purity and concentration of the protein were determined by RP-HPLC and its identity was confirmed by LC-MS. The present nucleotide was determined by ion pair chromatography [Eberth et al., 2009]. Covalent rate constant determination and curve fitting by RapidFire MS

Serial dilutions of test compounds (50 µM, ½ dilution) were prepared in 384-well plates and incubated with 1 µM KRAS G12C (with/without additional mutants) in 20 mM Tris (pH 7.5), 150 Incubate in mM NaCl, 100 µM MgCl ₂ , and 1% DMSO. The reaction was stopped at various time points by adding 1% formic acid. MS measurements were performed using an Agilent 6530 quadrupole time-of-flight (QToF) MS system coupled to an Agilent RapidFire autosampler RF360 device to derive the % modification value for each well. At the same time, compound solubility is assessed by turbidimetry, and compound concentrations that result in measurable turbidity are excluded from curve fitting.

Plot % modification versus time to extract k _obs values for different compound concentrations. In a second step, the obtained k _obs values are plotted against compound concentration. The rate constant (i.e. k _inact /K _I ) is derived from the initial linear part of the resulting curve. MS measurement

Injections were performed using the RapidFire autosampler RF 360. Solvent was delivered by Agilent 1200 pump. C18 solid phase extraction (SPE) cartridges were used for all experiments.

Aspirate a volume of 30 µL from each well of the 384-well plate. At a flow rate of 1.5 mL/min (H2O, 0.1% formic acid), sample load/wash time is 3000 ms; elution time is 3000 ms (acetonitrile, 0.1% formic acid); at 1.25 mL/min (H2O, 0.1% formic acid) ), the rebalancing time is 500 ms.

Mass spectrometry (MS) data were acquired on an Agilent 6530 quadrupole time-of-flight (QToF) MS system connected to a double electrospray (AJS) ion source (in positive mode). The instrument parameters are as follows: gas temperature 350°C, dry gas 10 L/min, atomizer 45 psi, shielding gas 350°C, shielding gas flow rate 11 L/min, capillary 4000 V, nozzle 1000 V, fragmentation voltage 250 V , skimmer 65 V, eight-pole RF 750 V. Acquire data at 6 spectra/s. Mass calibration was performed in the range 300-3200 m/z.

All data processing was performed using a combination of Agilent MassHunter Qualitative Analysis, Agilent Rapid-Fire control software, and Agilent DA Reprocessor Offline Utilities. The maximum entropy algorithm produces a zero-charge spectrum in a separate bin for each injection. Batch processing generates a single file that combines all mass spectra in text format into x,y coordinates. Use this profile to calculate the percentage of protein modification in each well. result

Second-order rate constants for modifications for specified constructs (all GDP-loaded) were quantified using kinetic MS experiments, measuring % modification over a range of compound concentrations at different time points. K _inact /K _I is derived from the initial slope of the k _obs versus compound concentration curve. The activity relative to KRAS G12D:GDP was set to 1 and the relative activity of the resistant mutants is given. The table below gives the average of n = 4 experiments for KRAS G12C, n = 3 for G12C_Y96D, and n = 2 for other mutants. [Table]: Fold change of the second-order rate constant (K _inact /K _I ) of the resistant mutant relative to KRAS G12C KRAS mutant (GDP) G12C G12C_H95R G12C_H95Q G12C_H95D G12C_R68S G12C_Y96C G12C_Y96D Compound A (JDQ443) 1 1.04 0.40 0.20 0.14 0.03 0.004 sothorasib 1 2.39 1.67 1.45 0.31 ＜0.002 < 0.001 Adaghrasib 1 ＜0.05 ＜0.05 ＜0.05 0.38 ＜0.002 ＜0.001

Second-order rate constants for modifications for specified constructs (all GDP-loaded) were quantified using kinetic MS experiments, measuring % modification over a range of compound concentrations at different time points. K _inact /KI is derived from the initial slope of the K _obs versus compound concentration curve. Averages are given for n = 4 experiments for KRAS G12C, n = 3 for G12C_Y96D, and n = 2 for other mutants. [Table]: Second-order rate constants (K _inact /KI [mM-1*s-1]) of compound A (JDQ443), sotorasib and adagrasiib relative to the resistant mutant KRAS mutant (GDP) G12C G12C_H95R G12C_H95Q G12C_H95D G12C_R68S G12C_Y96C G12C_Y96D Compound A (JDQ443) 24.3 25 9.5 4.85 3.45 0.65 0.09 sothorasib 9 21.5 15 13.05 2.75 ＜0.02 ＜0.01 Adaghrasib 26 < 1 < 1 < 1 9.9 ＜0.04 ＜0.01 Conclusion

First-generation KRAS G12C inhibitors have shown efficacy in clinical trials. However, the emergence of mutations that disrupt inhibitor binding and reactivation of downstream pathways limits response duration. Second site mutants conferring resistance to adagrasib in clinical trials will be reported (Reference: N Engl J Med. [New England Journal of Medicine] 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. April 6, 2021 Electronic Publication. 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 the binding pattern, Compound A inhibited proliferation and signaling of the KRAS G12C H95 double mutant. Compound A potently inhibited the proliferation of G12C/H95R and G12C/H95Q (GI ₅₀ = 0.024 ± 0.006 mM, GI ₅₀ = 0.284 ± 0.041 mM, respectively), while the performance of G12C/R68S, G12C/Y96C and G12C/Y96D conferred Resistance to Compound A (all GI ₅₀ > 1 mM).

Surprisingly, although compound A does not directly interact with histidine 95, the behavior of G12C/H95D resulted in reduced sensitivity to compound A (GI = _0.612 ± 0.151 mM) compared to H95R or Q. Western blot analysis of pERK after Compound A treatment and rate constant analysis of Compound A in a biophysical environment toward the SWII pocket mutations observed clinically ( Biophysical Data, above ) are consistent with the cell growth inhibition data (see surface).

The difference between H95D and H95R or Q may be due to the negative charge of aspartic acid, which can 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 to this mutant. Another possible explanation is that the H95D mutation may affect the kinetics of KRAS, making it more difficult to obtain a conformation that allows compound A to bind.

Taken together, the data show that compound A should overcome adagrasib-induced resistance in the context of G12C/Q95R or G12C/H95Q. Compound A treatment, particularly in the methods of the invention, may still be useful in the context of G12C/H95Q where it has shown activity. Example 7: Clinical Efficacy of Compound A

Compound A (JDQ443) alone and Phase Ib/II open-label, multicenter, dose-escalation study of its combination with specific agents. This study was conducted to evaluate the anti-tumor efficacy, safety and tolerability of JDQ443 as a single agent and in combination with other agents.

Patients to be treated include patients with advanced KRAS G12C-mutant solid tumors who have received standard of care therapies, are intolerant to, or are ineligible for approved therapies; or who have Eastern Cooperative Oncology Group performance status (Eastern Cooperative Oncology Group performance status). Cooperative Oncology Group Performance Status, ECOG PS 0-1); or have not been previously treated with a KRAS ^G12C inhibitor. The main exclusion criteria for the JDQ443 monotherapy arm were active brain metastases and/or prior KRASG12C inhibitor therapy.

Patients with NSCLC include those previously treated with platinum-based chemotherapy regimens and immune checkpoint inhibitors in combination or sequentially, unless otherwise ineligible for such therapies.

Patients with CRC included those who had previously received standard of care therapy, which included fluoropyrimidine, oxaliplatin, and irinotecan-based chemotherapy, unless they were ineligible for such therapy.

Preliminary data from the monotherapy dose-escalation arm study are as follows.

As of January 5, 2022, 39 patients were treated with Compound A at 200 mg QD, 400 mg QD, 200 mg BID, or 300 mg BID. Compound A is administered with food.

Patients had a median of 3 prior lines of antineoplastic therapy. The recommended dose for monotherapy is 200 mg Compound A orally twice daily (BID). Efficacy data from the pooled Phase Ib JDQ443 single-agent cohort (n = 39) (as of January 05, 2022) show: • In NSCLC, 57% (4/7) confirmed overall response rate (ORR) at 200 mg BID • In NSCLC, 45% (9/20) confirmed and unconfirmed ORR across doses • In NSCLC, 35% (7/20) confirmed ORR across doses • PD/PK modeling predicts sustained high levels of target occupancy at the recommended dose of 200 mg BID

Compound A treatment was generally well tolerated. Most treatment-related adverse events (TRAEs) were grade 1-2 (Gr). There are no Level 4-5 TRAEs. Four Grade 3 TRAEs occurred in 4 separate patients. The most common TRAEs are fatigue, nausea, edema, diarrhea and vomiting. There was one DLT (Grade 3 fatigue) and one treatment-related serious AE (Grade 3 photosensitivity reaction) in separate patients each treated with 300 mg BID.

At the recommended dose of 200 mg BID, there is prolonged absorption, with a median time to reach maximum plasma concentration (Tmax) of 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 area under the steady-state curve (AUCss) was more than three times greater than the exposure required for maximal efficacy in the less sensitive KRAS G12C xenograft model. Figure 9 shows the PK curve at steady state. T _max ( hr ), median (minimum - maximum) C _max ,ss ( ng/mL ), geometric mean ( CV% ) AUC _0-12 , ss ( ng*hr/mL ), geometric mean ( CV% ) 3.2 (2.0-7.8) 5950 (35.0) 49,400 (39.0)

The predicted target occupancy curve is shown in Figure 4. Patient PK and preclinical target occupancy models were integrated to predict patient target occupancy >90% in >82% of patients. These models assume that JDQ443 binding and target (KRAS) turnover are identical in mice and humans (KRAS has a half-life of approximately 25 hr) and that only free drug can bind to the target.

The best overall response across dose levels and indications is shown in the upper part of Figure 5 and in the table below. Best overall response as assessed by investigator based on Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) All patients, N = 39, n (%) Partial Response (PR) (Confirmed) 8(20.5) stable disease (SD) 24 (61.5) Progressive disease (PD) 5(12.8) Not Evaluable (NE) 2 (5.1) Overall response rate (ORR) (confirmed and unconfirmed) 11 (28.2) ORR (confirmed) 8(20.5)

The bottom half of Figure 5 and the table below show the best overall response at each dose level for all patients with NSCLC. All patients with a partial response or an unconfirmed partial response continued treatment at the time of data cutoff. Investigator-assessed best overall response according to RECIST v1.1 All patients with NSCLC, n = 20, n (%) PR (confirmed) 7(35.0) SD 11 (55.0) PD 0 NE 2 (10.0) ORR (confirmed and unconfirmed) 9 (45.0) ORR (confirmed) 7(35.0)

NE, not evaluable; NSCLC, non-small cell lung cancer; ORR, overall response rate; PD, progressive disease; PR, partial response; QD, once daily.

Response was assessed by investigators according to RECIST v1.1. Two (10.0%) patients had uPR, which contributed to ORR (confirmed and unconfirmed).

uPR = unconfirmed PR pending confirmation, on treatment, no PD. After data cutoff, one of the two patients with uPR had a confirmed PR.

Figure 6 shows PET scans showing 2-[fluoro-18]-fluoro-2-deoxy-d-glucose ( 18-F-FDG) affinity is significantly reduced. The patient had received pemetrexed/pembrolizumab, docetaxel, tegafur/gemelapyrimidine/octilizumab, and carboplatin/paclitaxel/atezolizumab. Post-cycle 2 scans showed a 30.4% reduction in the sum of the longest diameters of target lesions compared with baseline. PR confirmed on follow-up scan

A 57-year-old man with metastatic KRAS G12C mutant NSCLC. Local molecular testing using next-generation sequencing (NGS) identified no mutations in TP53. The mutational status of STK11, KEAP1 and NRF2 is unknown. The patient had received prior carboplatin/pemetrexed/pembrolizumab, docetaxel, tegafur-gemetrexed-octilizumab, and carboplatin/paclitaxel/atezolizumab. He was enrolled in the JDQ443 monotherapy dose-escalation portion of the study at a dose of JDQ443 200 mg BID given continuously in 21-day cycles. Disease assessment after 2 treatment cycles demonstrated a RECIST 1.1 partial response, in which the sum of the longest diameters of target lesions changed by -30.4% compared to baseline. The partial response was confirmed on subsequent scans (Figure 6), and the patient continued treatment. Positron emission tomography imaging at baseline and after 4 treatment cycles also showed a substantial decrease in 2-[fluoro-18]-fluoro-2-deoxy-d-glucose avidity of the tumor mass. treatment T _max (hr) C _max (ng/mL) AUC _0-24h (h ^x ng/mL ⁾ Compound A 200 mg BID continuous 7.8 5920 75930 Example 8: JDQ443 single agent as first-line treatment in patients with locally advanced or metastatic KRAS G12C mutant non-small cell lung cancer with <1% PD-L1 manifestation or ≥1% PD-L1 manifestation and STK11 co-mutations Efficacy and safety studies

Clinical studies demonstrating the therapeutic use of Compound A (JDQ443) can be conducted as follows.

This study was designed to evaluate the antitumor activity and safety of JDQ443 as a single agent as first-line therapy in participants with locally advanced or metastatic non-small cell lung cancer (NSCLC) whose tumors harbor a KRAS G12C mutation and < 1% PD-L1 manifestation regardless of STK11 mutation status (cohort A) or ≥ 1% PD-L1 manifestation and STK11 co-mutation (cohort B).

Tests to determine PD-L1 expression status, KRAS G12C mutation and STK11 mutation status, for example in tumor tissue or blood samples, are known in the art. Goals and end points: Target end point main To evaluate the antitumor activity of JDQ443 single agent as first-line therapy in participants with locally advanced or metastatic NSCLC whose tumors harbor KRAS G12C mutations <1% PD-L1 expression regardless of STK11 mutation status (group Group A). Overall response rate (ORR), defined as a complete response (CR) or partial response (PR) confirmed by a blinded independent review committee (BIRC) according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) as best overall response (BOR) ) of participants. Key and secondary To evaluate the antitumor activity of JDQ443 single agent as first-line therapy in participants with locally advanced or metastatic NSCLC whose tumors harbor KRAS G12C mutations, ≥1% PD-L1 expression, and STK11 co-mutations (cohort B). ORR determined by BIRC according to RECIST 1.1. Duration of response (DOR) was assessed in both cohorts. DOR is defined as the time from the first occurrence of PR or CR as determined by BIRC according to RECIST 1.1 to disease progression or death from any cause. secondary Progression-free survival (PFS) was assessed in both cohorts. PFS, defined as the time from the date of enrollment to the date of first documented disease progression or death from any cause as determined by BIRC according to RECIST 1.1. Overall survival (OS) was assessed in both cohorts. OS, defined as the time from the date of enrollment to death from any cause. The anti-tumor activity of JDQ443 was evaluated in two cohorts. Disease control rate (DCR), defined as the proportion of participants with BOR of CR, PR, and stable disease (SD) confirmed by BIRC according to RECIST 1.1. Time to response (TTR) was defined as the time from the date of enrollment to the first recorded CR or PR response by BIRC according to RECIST 1.1. The anti-tumor activity of JDQ443 was evaluated in both cohorts based on local radiological assessment. ORR, DOR, DCR, TTR and PFS determined according to RECIST 1.1 by regional radiological assessment. To evaluate the antitumor activity of JDQ443 single agent as first-line treatment in participants whose tumors harbor STK11 mutations regardless of PD-L1 performance status (pooled from both cohorts). ORR, DOR, DCR, TTR and PFS determined by BIRC and by local radiological assessment according to RECIST 1.1. OS. Characterize the security characteristics of JDQ443. Type, frequency and severity of adverse events, changes in laboratory values, vital signs, electrocardiogram (ECG). Characterizing the pharmacokinetics of JDQ443. Concentration of JDQ443 in plasma. To assess the impact of JDQ443 on patient-reported lung cancer symptoms, health-related quality of life, and health status. Time to final worsening of pain, cough, and dyspnea from NSCLC SAQ, and general health status and QoL items from EORTC QLQ-C30 (primary PRO variables of interest) NSCLC-SAQ, Patient Global Impression of Symptom Severity (PGI- S), changes from baseline in physical and role functional domains of EORTC-QLQ C30 and EQ-5D-5L

The study will have 2 non-comparison cohorts that will recruit participants in parallel based on the following characteristics: Cohort A : Participants whose tumors have a KRAS G12C mutation and <1% PD-L1 expression regardless of STK11 mutation status (N = 90). • Cohort B : Participants whose tumors had KRAS G12C mutations, ≥1% PD-L1 expression, and STK11 co-mutations (N = 30).

Compound A (JDQ443) was administered to all subjects as study treatment: JDQ443, 200 mg orally (PO) twice daily, continuously (i.e., no drug holidays). Key Inclusion Criteria • Participants with histologically confirmed locally advanced (stage IIIb/IIIc not eligible for clear chemoradiotherapy or surgery) or metastatic (stage IV) NSCLC with no prior systemic therapy for metastatic disease. Receipt of prior (neo)adjuvant therapy with chemotherapy and/or immunotherapy for localized disease or locally advanced disease, or in combination with chemotherapy and/or immunotherapy if the time between completion of therapy and enrollment is >12 months Previous radiation therapy given sequentially or concurrently. • Presence of KRAS G12C mutation (all patients) and: • Cohort A : < 1% PD-L1 manifestation regardless of STK11 mutation status • Cohort B : ≥ 1% PD-L1 manifestation and STK11 co-mutation • At least one measurable lesion according to RECIST 1.1. • ECOG performance status ≤ 1. • Participants with brain metastases were allowed if clinically stable. • Participants who are able to swallow study medication. Key Exclusion Criteria • Participants with EGFR activating mutations or ALK rearrangements are not eligible. Participants diagnosed with other targetable mutations based on local testing will be excluded if required by local guidelines. • Prior use of a KRAS G12C inhibitor or prior systemic therapy for metastatic NSCLC. • A medical condition that causes increased photosensitivity (ie, solar urticaria, lupus erythematosus, etc.). • Participants who are taking prohibited medications (strong CYP3A inducers) that cannot be discontinued for at least seven days before the first dose of study treatment and for the duration of the study

Therefore, the following implementation is now provided:

Embodiment 1. A treatment for a cancer or solid tumor with a KRAS G12C mutation and <1% PD-L1 expression regardless of STK11 mutation status or with a KRAS G12C mutation and ≥1% PD-L1 expression in a subject in need thereof A method for cancer or solid tumors such as NSCLC that expresses L1 and is co-mutated with STK11, wherein the method includes administering to the subject a therapeutically effective amount of a KRAS G12C inhibitor or a pharmaceutically acceptable salt thereof.

Embodiment 2. The method of embodiment 1, wherein the KRAS G12C inhibitor is selected from 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -indazole- 4-yl)-5-methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptane -2-yl}prop-2-en-1-one (Compound A), sotorasibu (Amgen), adagrasibu (Mirati), D-1553 (Yifang Biological), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Eli Lilly), BI1823911 (Boehringer), AS KRAS G12C ( Ascent Pharmaceuticals), SF KRAS G12C (Sanofi), RMC032 (Revolution Pharmaceuticals), JAB-21822 (Jax Pharmaceuticals), AST-KRAS G12C (Alix Pharmaceuticals), AZ KRAS G12C (AstraZeneca), NYU-12VC1 (New York University), and RMC6291 (Revolutionary Medicines) or their pharmaceutically acceptable salts.

Embodiment 3. The method of embodiment 2, wherein the KRAS G12C inhibitor is selected from 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -indazole- 4-yl)-5-methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptane -2-yl}prop-2-en-1-one (Compound A), sotorasiib, adagrasiib, D-1553, and GDC6036 or pharmaceutically acceptable salts thereof.

Embodiment 4. The method of embodiment 2, wherein the KRAS G12C inhibitor is 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -indazole-4 -yl)-5-methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptane- 2-yl}prop-2-en-1-one (Compound A) or a pharmaceutically acceptable salt thereof.

Embodiment 5. The method of embodiment 2, wherein the KRAS G12C inhibitor is 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -indazole-4 -yl)-5-methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptane- 2-yl}prop-2-en-1-one (Compound A).

Embodiment 6. The method of any one of the preceding embodiments, wherein the cancer or tumor is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer, and lung squamous cell carcinoma), colorectal cancer, and cholangiocarcinoma. , ovarian cancer, pancreatic cancer, duodenal papilla cancer and a group of cancers or tumors composed of solid tumors.

Embodiment 7. The method of any one of the preceding embodiments, wherein the cancer is non-small cell lung cancer.

Embodiment 8. The method of any one of the preceding embodiments, wherein each therapeutic agent is administered to the subject in need thereof in an amount effective to treat the cancer or tumor.

Embodiment 9. The method of any one of the preceding embodiments, wherein Compound A or a pharmaceutically acceptable dose thereof is administered at a therapeutically effective dose ranging from 100 to 600 mg/day, such as from 200 to 400 mg/day. of salt.

Embodiment 10. The method of any one of the preceding embodiments, wherein Compound A or a pharmaceutically acceptable dose thereof is administered at a therapeutically effective dose selected from the group consisting of 100, 150, 200, 250, 300, 350 and 400 mg/day. Take that with a grain of salt.

Embodiment 11. The method of any one of the preceding embodiments, wherein the total daily dose of Compound A is administered once daily or twice daily.

Embodiment 12. The method of any one of the preceding embodiments, wherein Compound A is administered at a dose of 100 mg twice daily or 200 mg twice daily.

Embodiment 13. The method of any one of the preceding embodiments, wherein Compound A is administered with food.

Embodiment 14. Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer or solid tumors with KRAS G12C mutations and <1% PD-L1 expression regardless of STK11 mutation status or with KRAS G12C mutations and Used in ≥ 1% of cancers that express PD-L1 and STK11 co-mutations or solid tumors such as NSCLC.

Embodiment 15. The compound of embodiment 14, wherein the cancer or solid tumor is selected from lung cancer (including lung adenocarcinoma, non-small cell lung cancer and lung squamous cell carcinoma), colorectal cancer, cholangiocarcinoma, ovarian cancer , pancreatic cancer and duodenal papilla cancer as well as solid tumors.

Embodiment 16. The compound of embodiment 15, wherein the cancer or solid tumor is non-small cell lung cancer.

Embodiment 17. A compound, the compound is 1-{6-[(4 M )-4-(5-chloro-6-methyl- 1H -indazol-4-yl)-5-methyl-3 -(1-Methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-ene- 1-one (Compound A) or a pharmaceutically acceptable salt thereof, for use in the method of treating cancer or tumors as described in any one of Embodiments 1 to 13.

Embodiment 18. A method or compound for use as described in any one of the preceding embodiments, wherein the treatment is for first-line treatment.

Embodiment 19. A compound for use in the method of treating cancer or solid tumors as described in any embodiment, or a compound for use in the method of treating cancer or solid tumors as described in any embodiment A combination for use in, or a method of treating cancer or solid tumors according to any embodiment, wherein the cancer or solid tumor is present in a patient who has previously been treated with a KRAS G12C inhibitor or a KRAS G12C inhibitor-naïve patient (i.e., patients who have not previously received KRAS G12C inhibitor therapy).

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

References in this specification to "the present invention" are intended to reflect the embodiments of the several inventions disclosed in this specification and should not be construed as unnecessarily limiting the claimed subject matter.

It should be understood that the examples and implementations described herein are for illustrative purposes only, and various modifications or changes thereof will be apparent to those skilled in the art and are included within the spirit and scope of the present application and the appended claims. within the range.

While specific embodiments of the invention have been discussed, the foregoing description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the invention should be determined by reference to the full scope of the patent claims and their equivalents, and the specification, along with such changes.

without

[Figure 1]: Compound A potently inhibited KRAS G12C cell signaling and proliferation in a mutation-selective manner and showed dose-dependent anti-tumor activity, with efficacy driven by daily AUC.

A. Comprehensive optimal tumor growth inhibition among six KRASG12C tumor models. The efficacy of JDQ443 was evaluated following oral administration of 10, 30, and 100 mg/kg/day in six human KRAS G12C mutant CDX models in mice. NSCLC cell line models are depicted in dark gray, while PDAC (MIA Paca-2) and esophageal cancer (KYSE-410) cell line models are shown in light gray. Data are averages from 2-11 independent in vivo studies. B-G. CDX tumor-bearing mice with KRAS G12C mutant (C-G) and non-KRAS G12C mutant (NCI-441, KRASG12V; B) tumors were treated orally with JDQ443 at the indicated doses and schedules. G. Treatment of LU99 tumor-bearing mice 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 KRASG12C levels in steady-state tumors correlate with efficacy (T/C or % regression) observed in LU99. Points correspond to mean and error bars ± 1 S.D of simulated PK/PD metrics based on 100 simulations and observed efficacy metrics.

By one-way ANOVA, * p < 0.05 compared to vehicle; # p < 0.05 compared to each other.

[Figure 2]: Effects of Compound A (JDQ443), sotorasib (AMG510) and adagrasiib (MRTX-849) on the proliferation of KRAS G12C/H95 double mutant . Ba/F3 cells expressing the indicated FLAG-KRAS ^G12C single or double mutants were treated with the indicated compound concentrations for 3 days, and proliferation inhibition was assessed by Cell titer glo viability assay. The y-axis shows the % growth of treated cells relative to day 3 treatment, and the x-axis shows the logarithmic concentration of KRASG12C inhibitor (μM).

[Figure 3]: Western blot analysis of ERK phosphorylation to evaluate signaling of KRAS G12C/H95 double mutant by Compound A (JDQ443), sotorasiib (AMG510), and adagrasiib (MRTX-849) influence. Ba/F3 cells expressing the specified FLAG-KRAS ^G12C single or double mutants were treated with the indicated compound concentrations for 30 min, and inhibition of the MAPK pathway was assessed by detecting the decrease in pERK in cell lysates by Western blotting.

[Figure 4]: PK and target occupancy curve of JDQ443 RD 200 mg BID. The top panel shows the PK curve at steady state. Error bars represent the standard deviation of the PK curve at each time point. The bottom panel shows the predicted target occupancy curve, where the line shows the simulated median and the shaded area shows the 5%-95% prediction interval.

[Figure 5]: The top panel illustrates the best overall response of JDQ443 monotherapy across dose levels and indications. Waterfall plot: 37 (94.9%) patients had measurable changes compared to baseline tumor assessment; data from N = 39 JDQ443 single-agent patients plotted. Investigators assessed best overall response according to RECIST v1.1. Three (7.7%) patients had uPR, which contributed to ORR (confirmed and unconfirmed). uPR = unconfirmed PR pending confirmation, on treatment, no PD. Per protocol, intrapatient dose escalation from 200 mg QD (administered once daily) to 200 mg BID (administered twice daily) occurred in four patients.

The bottom panel shows the best overall response at different doses for all patients with NSCLC. Waterfall plot: 19 (95.0%) NSCLC patients had measurable changes compared to baseline tumor assessment; data plotted for N = 20 NSCLC patients in the JDQ443 single-agent cohort.

[Figure 6]: PET scan showing 2-[fluoro-18]-fluoro-2-deoxy-d-glucose in tumor masses after four cycles of treatment with 200 mg BID of Compound A in patients with NSCLC. (18-F-FDG) affinity decreased significantly. CT: computed tomography; PET, positron emission tomography. Arrows indicate tumor sites.

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Claims (19)

A cancer or solid tumor with a KRAS G12C mutation and ≥ 1% PD-L1 expression regardless of STK11 mutation status or a KRAS G12C mutation and ≥ 1% PD-L1 expression and STK11 in a subject in need of treatment A method of co-mutating cancer or solid tumors such as NSCLC, wherein the method includes administering to the subject a therapeutically effective amount of a KRAS G12C inhibitor or a pharmaceutically acceptable salt thereof. The method of claim 1, wherein the KRAS G12C inhibitor is selected from 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -indazol-4-yl) -5-Methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl }Propan-2-en-1-one (Compound A), sotorasibu (Amgen), adagrasiib (Mirati), D-1553 (Yifang Biological), BI1701963 (Bolin GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Eli Lilly), BI1823911 (Boehringer), AS KRAS G12C (Ascent Pharmaceuticals) Company), SF KRAS G12C (Sanofi), RMC032 (Revolutionary Pharmaceuticals), JAB-21822 (Jax Pharmaceuticals), AST-KRAS G12C (Alix Pharmaceuticals), AZ KRAS G12C (AstraZeneca) Company), NYU-12VC1 (New York University), and RMC6291 (Revolutionary Pharmaceuticals, Inc.) or their pharmaceutically acceptable salts. The method of claim 2, wherein the KRAS G12C inhibitor is selected from 1-{6-[(4 M )-4-(5-chloro-6-methyl-1 H -indazol-4-yl) -5-Methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl }Propan-2-en-1-one (Compound A), sotoraxib, adagrasiib, D-1553, and GDC6036 or pharmaceutically acceptable salts thereof. The method of claim 2, wherein the KRAS G12C inhibitor is 1-{6-[(4 M )-4-(5-chloro-6-methyl- 1H -indazol-4-yl)- 5-Methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl} Propan-2-en-1-one (Compound A) or a pharmaceutically acceptable salt thereof. The method of claim 2, wherein the KRAS G12C inhibitor is 1-{6-[(4 M )-4-(5-chloro-6-methyl- 1H -indazol-4-yl)- 5-Methyl-3-(1-methyl-1 H -indazol-5-yl)-1 H -pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl} Propan-2-en-1-one (Compound A). The method according to any one of the preceding claims, wherein the cancer or tumor is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and lung squamous cell carcinoma), colorectal cancer, cholangiocarcinoma, ovarian cancer, A group of cancers or tumors consisting of pancreatic cancer, duodenal papilla cancer, and solid tumors. The method of any one of the preceding claims, wherein the cancer is non-small cell lung cancer. The method of any one of the preceding claims, wherein each therapeutic agent is administered to the subject in need thereof in an amount effective to treat the cancer or tumor. The method of any one of the preceding claims, wherein Compound A or a pharmaceutically acceptable salt thereof is administered in a therapeutically effective dose ranging from 100 to 600 mg/day, for example from 200 to 400 mg/day. The method of any one of the preceding claims, wherein Compound A or a pharmaceutically acceptable salt thereof is administered at a therapeutically effective dose selected from the group consisting of 100, 150, 200, 250, 300, 350 and 400 mg/day. A method as claimed in any one of the preceding claims, wherein the total daily dose of Compound A is administered once daily or twice daily. The method of any one of the preceding claims, wherein Compound A is administered at a dose of 100 mg twice daily or 200 mg twice daily. A method as claimed in any one of the preceding claims, wherein Compound A is administered with food. Compound A, or a pharmaceutically acceptable salt thereof, for the treatment of cancer or solid tumors with KRAS G12C mutations and < 1% PD-L1 expression regardless of STK11 mutation status or with KRAS G12C mutations and ≥ 1% This method is used in cancers or solid tumors such as NSCLC that express PD-L1 and STK11 co-mutations. The compound of claim 14, wherein the cancer or solid tumor is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and lung squamous cell carcinoma), colorectal cancer, cholangiocarcinoma, ovarian cancer, and pancreatic cancer and duodenal papilla carcinoma and solid tumors. The compound of claim 15, wherein the cancer or solid tumor is non-small cell lung cancer. A compound, the compound is 1-{6-[(4 M )-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) or a pharmaceutically acceptable salt thereof, for use in the method of treating cancer or tumors as described in any one of claims 1 to 13. A method or compound for use as claimed in any one of the preceding claims, wherein the treatment is for first-line treatment. A compound for use in the method of treating cancer or solid tumors as described in any one of the claims, or a compound for use in the method of treating cancer or solid tumors as described in any of the claims A combination for use in, or a method of treating cancer or solid tumors as described in any one of the claims, wherein the cancer or solid tumors are present in patients who have previously been treated with a KRAS G12C inhibitor or who are new to a KRAS G12C inhibitor. in patients who have not previously received KRAS G12C inhibitor therapy.