WO2023199180A1

WO2023199180A1 - Therapeutic uses of a krasg12c inhibitor

Info

Publication number: WO2023199180A1
Application number: PCT/IB2023/053487
Authority: WO
Inventors: Saskia Maria Brachmann; Anna FARAGO; Andreas Weiss
Original assignee: Novartis Ag
Priority date: 2022-04-11
Filing date: 2023-04-05
Publication date: 2023-10-19
Also published as: TW202400153A

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 A KRASG12C INHIBITOR

FIELD OF THE INVENTION

The present invention relates to a KRAS G12C inhibitor such as Compound A, or a pharmaceutically acceptable salt thereof and its uses in treating cancer, particularly 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. 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 in the treatment or prevention 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.

BACKGROUND

Cancer growth is driven by many diverse complex mechanisms. Resistance to a given therapy inevitably occurs in some cancers. Inhibiting the MAPK pathway induces feedback mechanisms and pathway rewiring causing its subsequent reactivation. One common mechanism is for example the activation of Receptor Tyrosine Kinases (RTKs).

In addition, despite the recent successes of targeted therapies and immunotherapies, some cancers, in particular, metastatic cancers remain largely incurable.

The KRAS oncoprotein is a GTPase with an essential role as regulator of intracellular signaling pathways, such as the MAPK, PI3K and Rai pathways, which are involved in proliferation, cell survival and turn origenesis. Oncogenic activation of KRAS occurs predominantly through missense mutations in codon 12. KRAS gain-of-function mutations are found in approximately 30% of all human cancers. KRAS G12C mutation is a specific submutation, prevalent in approximately 13% of lung adenocarcinomas, 4% (3-5%) of colon adenocarcinomas and a smaller fraction of other cancer types.

In normal cells, KRAS alternates between inactive GDP -bound and active GTP -bound states. Mutations of KRAS at codon 12, such as G12C, impair GTPase-activating protein (GAP)- stimulated GTP hydrolysis. In that case, the conversion of the GTP to the GDP form of KRAS G12C is therefore very slow. Consequently, KRAS G12C shifts to the active, GTP -bound state, thus driving oncogenic signaling. Lung cancer remains the most common cancer type worldwide and the leading cause of cancer deaths in many counties, including the United States. NSCLC accounts for about 85% of all lung cancer diagnoses. KRAS mutations are detected in approximately 25% of patients with lung adenocarcinomas (Sequist et al 2011). They are most commonly seen at codon 12, with KRAS G12C mutations being most common (40% overall) in both adenocarcinoma and squamous NSCLC (Liu et al 2020). The presence of KRAS mutations is prognostic of poor survival and has been associated with reduced responsiveness to EGFR TKI treatment.

Immunotherapy for NSCLC with immune checkpoint inhibitors has demonstrated promise, with some NSCLC patients experiencing durable disease control for years. However, such long-term non-progressors are uncommon, and treatment strategies that can increase the proportion of patients responding to and achieving lasting remission with therapy are urgently needed.

The benefit of immunotherapy in monotherapy or in combination with platinum-based chemotherapy is often associated with PD-L1 expression levels, with a higher magnitude of benefit observed in those patients with advanced NSCLC and whose tumors present a high (>50%) PD-L1 expression.

STK1 l 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 around 5% of the patients with advanced NSCLC, being more frequent among those with KRAS G12C-mutant NSCLC - around 15% (Skoulidis et al 2018, Scheffler et al 2019, Shire et al 2020 and Ricciuti et al 2021). Tumors harboring both a KRAS G12C and a STK11 mutation have a gene expression profile characterized by a low expression of pro-inflammatory cytokines such as type I interferon, stimulator of interferon genes (STING), DExD/H-Box Helicase 58 (DDX58), toll-like receptor 4 (TLR4), and toll-like receptor 7 (TLR7), which promotes an immuno-suppressive tumor microenvironment (Ricciuti et al 2021).

Previous studies showed that STK11 mutations are associated with a worse prognosis and predict a low benefit from immunotherapy -based treatments in patients with advanced NSCLC (Ricciuti et al 20201, Shire et al 2020).

Approximately 40 to 50% of the patients with advanced NSCLC are not eligible to subsequent treatments after discontinuation of first-line therapy, mainly due to clinical deterioration at the moment of disease progression (Davies et al 2017. Hence, effective treatments administered at earlier lines have the potential to benefit more patients with NSCLC.

Although the incorporation of immunotherapy into the first-line setting has significantly improved the overall survival of patients with advanced NSCLC, previous studies have shown that a lower magnitude of benefit from immunotherapy is observed in patients whose tumors have a PD-L1 expression <1% or a STK11 mutation: in a retrospective study that included 1,261 patients (out of whom 42.5% and 20.6% had a KRAS and a STK11 mutation, respectively) with advanced lung adenocarcinoma treated with immunotherapy, Ricciuti et al have shown that the presence of a STK11 mutation co-occurring with a KRAS mutation 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 that included 15 randomized- controlled trials involving a total of 10,074 patients, Xu et al demonstrated that the magnitude of OS benefit yielded by immunotherapy added to chemotherapy was associated with PD-L1 expression levels: for PD-L1 <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, 0.35-0.72Xu et al 2019. Hence, alternatives to improve the outcomes of these 2 subgroups of patients (PD-L1 expression <1% and STK11 mutation) who presumably benefit less from immunotherapy -based treatment are needed.

Sotorasib, a KRAS G12C inhibitor, recently received accelerated approval from the FDA for the treatment of adult patients with KRAS G12C-mutant locally advanced or metastatic NSCLC, as determined by an FDA-approved test, who have received at least one prior systemic therapy. Sotorasib received accelerated approval by the US FDA (Food and Drug Administration) in May 2021 and conditional marking authorization by the European Commission (EC) in January 2022 in patients with KRAS G12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC). In this patient population in a phase 2 single arm study of 126 patients, sotorasib demonstrated an ORR of 37% (95% CI 28.6-46.2), median DOR of 11.1 months, median PFS of 6.8 months, and median OS of 12.5 months (Skoulidis et al, N Engl J Med; 384:2371-81). Adagrasib, another KRAS G12C inhibitor, is also in clinical development in KRAS G12C-mutated malignancies, with a preliminary ORR of 45% in patients with NSCLC (Janne et al 2019, Presented at AACR-NCI-EORTC International Conference on Molecular Targets, 28 October2019). However, the benefit of these targeted therapies for tumors harboring KRAS G12C mutations remains uncertain at present, as not all patients responded and in several instances, the duration of the reported responses were short, likely due to the emergence of resistance, mediated at least in part by RAS gene mutations that disrupt inhibitor binding and reactivation of downstream pathways.

For example, out of 38 patients included in a study with adagrasib: 27 with non-smallcell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer, putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAFI, and FGFR3; and loss-of-function mutations in NF1 and PTEN (Awad et al, Acquired Resistance to KRASG12C Inhibition in Cancer, N Engl J Med 2021;384:2382-93. Tanaka et al (Cancer Discov 2021 ; 11 : 1913-22) describe a novel KRAS Y 96D mutation affecting the switch-II pocket, to which adagrasib and other inactive-state KRAS G12C inhibitors bind, which interfered with key protein-drug interactions and conferred resistance to these inhibitors in engineered and patient-derived KRASG12C cancer models.

Additional treatment options to overcome resistance mechanisms that arise during treatment with KRAS inhibitors such as adagrasib or sotorasib are therefore needed.

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

There thus remains a high unmet medical need for new treatment options for patients suffering from lung cancer (including advanced and/or metastatic cancer including lung cancer including NSCLC, especially when the cancer or solid tumor harbors a KRAS G12C mutation. It is also important to provide a potentially beneficial novel therapy for incurable disease, especially for patients with KRAS G12C mutant tumors who have already received and failed standard of care therapy for their indication or are intolerant or ineligible to approved therapies and have therefore limited treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Compound A potently inhibited KRAS G12C cellular signaling and proliferation in a mutant- selective manner and demonstrated dose-dependent antitumor activity, with efficacy driven by daily AUC.

A. Aggregated best tumor growth inhibition in six KRASG12C tumor models. JDQ443 efficacy was evaluated after oral dosing of 10, 30 and 100 mg/kg/day in six human KRAS G12C mutant CDX models in mice. In dark grey NSCLC cell line models are depicted, while in light grey PDAC (MIA Paca-2) and esophageal (KYSE-410) cancer cell line models are shown. Data are means from 2-11 independent in vivo studies. B-G. CDX-bearing mice with KRAS G12C- mutated (C-G) and non-KRAS G12C-mutated (NCI-441, KRASG12V; B) tumors were treated orally with JDQ443 at indicated doses and schedules. G. LU99 tumor-bearing mice were treated with JDQ443 by continuous intravenous infusion using a minipump. H-I. Simulated pop-PKPD metrics (H) daily AUC of JDQ443 in mouse blood and (I) average free KRASG12C levels in tumor at steady state, are correlated with the observed efficacy in LU99 (T/C or % regression). Points correspond to the mean and the error bars to ± 1 S.D of the simulated PK/PD metrics based on 100 simulations and observed efficacy metrics.

*p<0.05 vs vehicle, #p<0.05 vs each other, by one-way ANOVA.

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

Figure 3: Western blot analysis of ERK phosphorylation to assess the effect of Compound A (JDQ443), sotorasib (AMG510) and adagrasib (MRTX-849) on the signaling of KRAS G12C/H95 double mutants. Ba/F3 cells expressing the indicated FLAG-KRAS^G12C single or double mutants were treated with the indicated compound concentrations for 30 min and the inhibtion of the MAPK pathway was assessed by probing the cell lysates for reduction of pERK by westemblot.

Figure 4: PK and target occupancy profiles of JDQ443 RD 200 mg BID. Top panel shows the PK profile at steady state. Error bars indicate standard deviation for PK profile at each timepoint. The bottom panel shows the predicted target occupancy profile, where the line shows the simulated median and the shaded area shows the 5-95 percentile prediction interval. Figure 5: The top panel shows the best overall response across dose levels and indications for JDQ443 monotherapy. Waterfall plot: 37 (94.9%) patients with available change from baseline tumor assessments; data are plotted out of N=39 JDQ443 single-agent patients. Best overall responses are investigator assessed per RECIST vl. l. Three (7.7%) patients had a uPR, which contributed toward the ORR (confirmed and unconfirmed). uPR = unconfirmed PR pending confirmation, treatment ongoing with no PD. Intra-patient dose escalation, per protocol, occurred in four patients from 200 mg QD (administered once daily) to 200 mg BID (administered twice daily).

The bottom panel shows best overall responses across dose in all patients with NSCLC. Waterfall plot: 19 (95.0%) NSCLC patients with available change from baseline tumor assessments; data are plotted out of N=20 NSCLC patients in JDQ443 single-agent cohorts.

Figure 6: PET scans showing a substantial reduction in the 2-[fluorine-18]-fluoro-2- deoxy-d-glucose (18-F-FDG) avidity of the tumor mass after four cycles of treatment with Compound A administered at 200 mg BID to a patient with NSCLC. CT: computerized tomography; PET, positron emission tomography. Arrows indicate sites of tumor.

SUMMARY

The invention provides new treatment options for patients suffering from cancer (including advanced and/or metastatic cancer) and seeks to improve outcomes for patients with KRAS G12C-AAN&SX cancers. In particular, the cancer to be treated by a KRAS G12C inhibitor such as Compound A, or a pharmaceutically acceptable salt thereof, is 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.

Provided herein are compounds and their uses in methods of treating 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 in methods of treating a cancer or solid tumor such as NSCLC which harbors a KRAS G12C mutation and a PD-L1 expression >1% and a STK11 comutation. The present invention also provides a potentially beneficial novel therapy for incurable disease, especially for patients with KRAS G12C mutated tumors who are intolerant or ineligible to approved therapies and have therefore limited treatment options. Such patients include patients with advanced NSCLC with no previous systemic treatment for advanced disease whose tumors harbor a KRAS G12C mutation and either a PD-L1 expression <1% or a PD-L1 expression >1% and a STK11 co-mutation. 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 treatment for cancer patients who have developed resistance to other therapies, such as prior treatment with other KRAS inhibitors such as adagrasib and sotorasib; more preferably prior treatment with sotorasib.

Compound A is a selective covalent irreversible inhibitor of KRAS G12C which exhibits a novel binding mode, exploiting unique interactions with KRASG12C. Notably, Compound A traps KRAS G12C in a GDP -bound, inactive state while avoiding direct interaction with H95, a recognized route for resistance (Awad MM, et al. New Engl J Med 2021;384:2382-2392). Compound A potently inhibited KRAS G12C H95Q, a double mutant mediating resistance to adagrasib in clinical trials.

Compound A demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models. Compound A is orally bioavailable, achieves exposures in a range predicted to confer anti-tumor activity, and is well-tolerated.

Preliminary data (Phase lb) from the KontRASt-01 study (NCT04699188) showed that Compound A, a selective, covalent, and orally bioavailable KRASG12C inhibitor, demonstrated anti-tumor activity, high systemic exposure at its recommended dose of 200 mg BID and a favorable safety profile based on initial clinical data in patients with KRAS G12C-mutated solid tumors

KRAS G12C inhibitors are specifically designed to inhibit KRAS G12C. However, many tumors have KRAS WT, HRAS and NRAS proteins which are not inhibited by KRAS G12C inhibitors. Upon KRAS G12C inhibitor treatment, reactivated RTKs for instance can feed via these proteins into the MAPK pathway, thus counteracting anti-tumor activity. Likewise, many RTKs as well as RAS proteins directly activate parallel pathways, e.g. the PI3K/AKT pathway.

The presence of KRAS G12C mutations has been associated with an immunosuppressive tumor microenvironment in preclinical data. This effect can be mediated by high levels of inhibitory cytokines such as factor nuclear kappa B (NF-KP), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL-6), interleukin 1-P (IL-ip), as well as by a high presence of myeloid-derived suppressor cells, regulatory T cells, and M2-differentiated tumor- associated macrophages in the tumor stroma, all of which impair antitumor immunity and facilitate tumor progression (Hamarsheh et al 2020 and Cucurull et al 2022). Further supporting this concept, KRAS G12C inhibitors stimulate the recruitment and activation of CD8+ T cells, dendritic cells and Ml -macrophages, and thus promote a shift towards a more immune-activated tumor microenvironment in preclinical models of NSCLC (Canon et al 2019 and Briere et al 2021).

Compound A is the compound l-{6-[(4A/)-4-(5-Chloro-6-methyl-lJ/-indazol-4-yl)-5-methyl-3-(l-methyl- l/f-indazol-5-yl)- 1 //-py razol - 1 -yl]-2-azaspiro[3.3 ]heptan-2-yl }prop-2-en- 1 -one, having the structure

(Compound A).

It will 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 expressions “a compound of the invention” and “compounds of the invention” include Compound A, in free base form or in the form of a pharmaceutically acceptable salt or hydrate or solvate thereof.

The present invention provides these compounds for use in treating a cancer as described herein.

Efficacy of the therapeutic methods and uses of the invention may be determined by methods well known in the art, e.g. determining Best Overall Response (BOR), Overall Response Rate (ORR), Duration of Response (DOR), Disease Control Rate (DCR), Progression Free Survival, (PFS) and Overall Survival (OS) per RECIST v.1.1. The present invention therefore provides a pharmaceutical combination of the invention which improves KRAS G12C inhibitor therapy, e.g. as measured by an increase in one or more of Best Overall Response (BOR), Overall Response Rate (ORR), Duration of Response (DOR), Disease Control Rate (DCR), Progression Free Survival, (PFS) and Overall Survival (OS) per RECIST v.1.1.

Methods to assess, KRAS G12C and/or STK11 mutation status and PD-L1 expression are known in the art. For example, KRAS G12C and/or STK11 mutation status may be assessed in tumor tissue or in blood. KRAS G12C and/or STK11 mutations may be determined using a molecular test that detects mutations in DNA derived from tumor tissue or in circulating tumor DNA (ctDNA) derived from blood plasma. Testing for KRAS G12C mutation status and/or STK11 mutation status may also be carried out using Next Generation Sequencing (NGS) tests that detect mutations in DNA derived from formalin fixed, paraffin-embedded tumor tissue or ctDNA derived from blood plasma. PD-L1 tumor proportion score (TPS) status may be assessed according to PD- L1 immunohistochemistry (IHC) 22C3 or 28-8 pharmDx assay or tumor cell (TC) membrane expression according to the Ventana SP263 assay.

In another embodiment is a method for treating or preventing cancer in a subject in need thereof 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 squamous cell lung cancer), particularly when the cancer or tumor harbors a KRAS G12C mutation and a KRAS G12C mutation and a PD-L1 expression <1% 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 squamous cell lung cancer), particularly when the cancer or solid tumor such as NSCLC which harbors a KRAS G12C mutation and a PD-L1 expression >1% and a STK11 co-mutation.

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

In embodiments of the invention the cancer or tumor to be treated is selected from colorectal cancer, bile duct cancer, ovarian cancer, and pancreatic cancer where the cancer harbors a KRAS G12C mutation and a PD-L1 expression <1% or where the cancer harbors a KRAS G12C mutation and a PD-L1 expression >1% and a STK11 co-mutation

In a further embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients as described herein.

KRAS G12C inhibitors

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

A KRAS G12C inhibitor also includes a compound detailed in A “KRASG12C inhibitor” is a compound selected from the compounds detailed in WO2013/155223, WO2014/143659, WO2014/152588, W02014/160200, WO2015/054572, WO2016/044772, WO20 16/049524, WO2016164675, WO2016168540, W02017/058805, WO2017015562,

WO2017058728, WO2017058768, WO2017058792, W02017058805, W02017058807, W02017058902, WO2017058915, WO2017087528, W02017100546, W02017/201161, WO20 18/064510, WO2018/068017, WO2018/119183, WO2018/217651, W02018/140512, W02018/140513, W02018/140514, WO2018/140598, WO2018/140599, W02018/140600,

WO2018/143315, WO2018/206539, WO2018/218070, WO2018/218071, WO2019/051291,

WO20 19/099524, WO2019/110751, W02019/141250, W02019/150305, WO2019/155399,

WO2019/213516 WO2019/213526 WO2019/217307 and WQ2019/217691. Examples are: 1-

(4-(6-chloro-8-fluoro-7-(3-hydroxy-5-vinylphenyl)quinazolin-4-yl)piperazin-l-yl)prop-2-en-l- one— methane (1/2) (compound 1); (S)-l-(4-(6-chloro-8-fluoro-7-(2-fluoro-6- hydroxyphenyl)quinazolin-4-yl)piperazin-l-yl)prop-2-en-l-one (compound 2); and 2-((S)-l- acryloyl-4-(2-(((S)-l-methylpyrrolidin-2-yl)methoxy)-7-(naphthalen-l-yl)-5, 6,7,8- tetrahydropyrido[3,4-d]pyrimidin-4-yl)piperazin-2-yl)acetonitrile (compound 3).

KRAS G12C inhibitor Compound A

A preferred KRAS G12C inhibitor of the present invention is Compound A is l-{6- [(4A )-4-(5-Chloro-6-methyl-U/-indazol-4-yl)-5-methyl-3-(l -methyl- U/-indazol-5-yl)- 1H- pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, or a pharmaceutically acceptable salt thereof. Compound A is also known by the name “a(A)-l-(6-(4-(5-chloro-6-methyl-lH-indazol- 4-yl)-5-methyl-3-(l-methyl-lH-indazol-5-yl)-lH-pyrazol-l-yl)-2-azaspiro[3.3]heptan-2-yl)prop- 2-en-l-one”.

The synthesis of Compound A is described in the Examples below or in Example 1 of PCT application WO2021/124222, published 24 June 2021. Uses of Compound A, alone or in combination with an additional therapeutic agent are described in PCT/CN2021/139694, filed on December 20, 2021.

The structure of Compound A is as follows:

Alternatively, the structure of Compound A may be drawn as follows:

Compound A is a potent and selective KRAS G12C small molecule inhibitor that covalently binds to mutant Cysl2, trapping KRAS G12C in the inactive GDP -bound state. Compound A is structurally unique compared with sotorasib or adagrasib; 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, inhibiting downstream cellular signaling and proliferation specifically in KRAS G12C-driven cell lines but not KRAS wild-type (WT) or MEK Q56P mutant cell lines. Compound A showed deep and sustained target occupancy resulting in anti-tumor activity in different KRAS G12C mutant xenograft models.

Cancers to be treated by the compounds and methods of the invention

The compounds of the invention may thus be useful in the treatment of cancer and in cancers or tumors which are KRAS G12C mutated. In particular, the compounds of the invention may be useful as first-line treatment for 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.

The cancer or tumor to be treated may be selected from the group consisting of colorectal cancer, bile duct cancer, ovarian cancer, and pancreatic cancer where the specific cancer harbors a KRAS G12C mutation and a PD-L1 expression <1% or where the cancer harbors harbors a KRAS G12C mutation and a PD-L1 expression >1% and a STK11 comutation.

The cancer may be at an early, intermediate, late stage or may be metastatic cancer. In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer is a refractory cancer. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer is an unresectable cancer.

The cancer may be at an early, intermediate, late stage or metastatic cancer.

Compound A may also be useful in the treatment of solid malignancies characterized by additional mutations of RAS.

The present invention provides Compound A and combinations of the invention for use in the treatment of a cancer or solid tumor characterized by an acquired KRAS alteration which is selected from Gl 2D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, Y96 D and high-level amplification of the KRASG12C allele, or characterized by an acquired bypass mechanisms of resistance, These bypass mechanisms of resistance include MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAFI, and FGFR3; and loss-of-function mutations in NF1 and PTEN.

In another embodiment is method of treating (e.g., one or more of reducing, inhibiting, or delaying progression) a cancer or a tumor in a subject comprising administering to a subject in need thereof a pharmaceutical composition comprising Compound A, or pharmaceutically acceptable salt thereof.

The present invention therefore provides a method of treating (e.g., one or more of reducing, inhibiting, or delaying progression) 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 invention, wherein the cancer is 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. The cancer may be selected from lung cancer (including lung adenocarcinoma and non-small cell lung cancer), colorectal cancer, bile duct cancer, ovarian cancer, and pancreatic cancer.

The methods and combinations of the invention may be useful as first line therapy (or as second or more advanced lines of therapy). For example, the patient may be a treatment agnostic patient or a patient who has progressed and/or relapsed on previous therapy.

For example, the patient or subject to be treated by the methods and combinations of the invention include a patient suffering from cancer, e.g. KRAS G12C mutant NSCLC (including advanced (metastatic or unresectable) KRAS G12C mutant NSCLC), optionally wherein the patient has received and progressed on previous therapy.

In embodiments of the invention, the subject or patient to be treated and likely to benefit from treatment with Compound A is selected from

-a patient suffering from NSCLC whose tumors harbor a KRAS G12C mutation and a PD-L1 expression <1%:

- a patient suffering from NSCLC whose tumors harbor a KRAS G12C mutation and a PD-L1 expression <1%, regardless of STK11 mutation status;

-a patient suffering from locally advanced or metastatic NSCLC whose tumors harbor a KRAS G12C mutation and a PD-L1 expression <1%, regardless of STK11 mutation status;

- a patient suffering from NSCLC whose tumors harbor a KRAS G12C mutation, a PD-L1 expression >1% and a STK11 co-mutation;

- a patient suffering from locally advanced or metastatic NSCLC whose tumors harbor a KRAS G12C mutation, a PD-L1 expression >1% and a STK11 co-mutation.

In embodiments of the invention, the patient has received and failed standard of care therapy or is intolerant or ineligible to previous investigative and/or approved therapies;

Dosages and dosing regimens

In the methods and uses of the present invention, the total daily dose of Compound A may be administered between 100 and 600, or between 200 and 400 mg. The total daily dose of Compound A may be administered continuously, on a QD (once a day) or BID (twice a day) regimen. For example, Compound A may be administered at a dose of 200 mg BID (total daily dose of 400 mg), 400 mg QD (total daily dose 400 mg). Compound A may also be administered at a dose of 100 mg QD (total daily dose 100 mg), or at 100 mg BID (total daily dose of 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 in particular be used in cases of poor tolerability or higher toxicity whilst retaining efficacy of the treatment.

Preclinical target occupancy models coupled with PK patient data predict that a BID scheduling may lead to an increased response in a larger number of patients. Suitably, Compound A may thus 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 administered twice a day (total daily dose of 200 mg) or at a dose of 200 mg administered twice a day (total daily dose of 400 mg).

PK/PD modelling predicts sustained, high-level target occupancy at the recommended dose of 200 mg BID. The total daily recommended dose of Compound A is 400 mg, given once daily (QD) or twice daily (BID), given continuously (i.e. with no drug holiday).

Compound A is preferably administered with food, e.g. taken immediately (within 30 minutes) following a meal.

In a preferred embodiment, the total daily dose of Compound A is 400 mg, administered twice daily. This was determined based on the clinical assessment of efficacy, safety, PK, PK-PD modeling as well as the Bayesian hierarchical logistic regression model (BHLRM) model assessing the probability of DLTs.

The 200 mg BID dose was evaluated and determined to be safe and tolerable. The highest AUC0-24h was obtained at 200 mg BID among all dose levels tested, >60% higher than the exposure for 200 mg and 400 mg QD dose levels, and was at least 3-fold above the efficacious exposure required for maximum efficacy in less sensitive xenograft models which had the highest exposure requirement for tumor regression. At this dose level, a >90% average KRAS G12C target occupancy was predicted based on PK-PD modeling and was higher than the 70% average target occupancy required for tumor regression in various xenograft models. The 200 mg BID dose level satisfied the escalation with overdose control (EWOC) criteria (i.e. <25% chance that the true DLT rate was greater than or equal to 33% during DLT evaluation period) by Bayesian hierarchical logistic regression model (BHLRM) with posterior probability of excessive toxicity less than 0.1%. No DLTs were observed at this dose level.

It is therefore expected that administration of a total dose of 400 mg of Compound A, preferably administered twice daily, will be particularly useful in the uses and methods of the invention.

A total dose of 200 mg of Compound A, preferably administered daily (for example 100 mg BID) may also be useful in the uses and methods of the invention, specially in reducing toxicity and/or improving safety and tolerability of the treatment.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically effective amount of KRAS G12C inhibitor (e.g. Compound A), formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.

Preferably, pharmaceutically acceptable carriers are sterile. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.

Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of: a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) absorbents, colorants, flavors and sweeteners.

In an embodiment, the pharmaceutical compositions are capsules comprising the active ingredient only.

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

Suitable compositions for oral administration include an effective amount of a compound in a combination of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs, solutions or solid dispersion. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75%, or contain about 1-50%, of the active ingredient. Suitable compositions for transdermal application include an effective amount of a compound of the invention with a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

Suitable compositions for topical application, 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 in particular be appropriate for dermal application, e.g., for the treatment of skin cancer, e.g., for prophylactic use in sun creams, lotions, sprays and the like. They are thus particularly suited for use in topical, including cosmetic, formulations well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein, a topical application may also pertain to an inhalation or to an intranasal application. They may be conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray, atomizer or nebulizer, with or without the use of a suitable propellant.

In one embodiment, the invention provides a product comprising Compound A, or a pharmaceutically acceptable salt thereof, for use in therapy. In one embodiment, the therapy is 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. Products provided as a combined preparation include a composition comprising the compound of the present invention in the form of a kit.

In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like. To assist compliance, the kit of the invention typically comprises directions for administration.

Definitions

The general terms used hereinbefore and hereinafter preferably have within the context of this disclosure the following meanings, unless otherwise indicated, where more general terms whereever used may, independently of each other, be replaced by more specific definitions or remain, thus defining more detailed embodiments of the invention:

In particular, where a dose or dosage is mentioned, it is intended to include a range around the specified value of plus or minus 10%, or plus or minus 5%.

As is customary in the art, dosages refer to the amount of the therapeutic agent in its free form. For example, when a dosage of 200 mg of Compound A is referred to, and Compound A is used as a pharmaceutical salt thereof, the amount of the therapeutic agent used is equivalent to 20 mg of the free form of Compound A.

The term “subject” or “patient” as used herein is intended to include animals, which are capable of suffering from or afflicted with a cancer or any disorder involving, directly or indirectly, a cancer. Examples of subjects include mammals, e.g., humans, apes, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In an embodiment, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancers.

The terms “treating”, or “treatment”, as used herein comprise a treatment relieving, reducing or alleviating at least one symptom in a subject or effecting a delay of progression of a disease. For example, treatment can be the diminishment of one or several symptoms of a disorder or partial or complete eradication of a disorder, such as cancer. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.

“Treatment” may also be determined by efficacy and/or pharmacodynamic endpoints and may be defined as an improvement in one or more of safety, efficacy and tolerability. Efficacy of the monotherapy or the combination therapy may be determined by determining Best Overall Response (BOR), Overall Response Rate (ORR), Duration of Response (DOR), Disease Control Rate (DCR), Progression Free Survival, (PFS) and Overall Survival (OS) per RECIST v.1.1. “Best overall response” (BOR) rate is defined as the best response recorded from the start of the treatment until disease progression/recurrence and according to RECIST 1.1.

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

“Duration of Response” (DOR) per RECIST 1.1 is the time between the first documented response (CR or PR) and the date of progression or death due to any cause. Here, death due to any cause is considered as an event to be conservative and align with PFS event definition.

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

“Progression Free Survival” (PFS) per RECIST 1.1 is defined as the time from the date of start of treatment to the date of the first documented progression according to RECIST 1.1, or death due to any cause. If a patient has not had an event, PFS will be censored at the date of last adequate tumor assessment.

“Overall survival” (OS) is defined as the number of days between the date of start of study treatment to the date of death due to any cause. If no death is reported prior to study termination or analysis cut off, survival will be censored at the date of last known date patient alive prior to/on the cut-off date. Survival time for patients with no post-baseline survival information will be censored at the date of start of treatment.

“Treatment” may also be defined as an improvement in a reduction of adverse effects of the therapy with Compound A, as described herein.

The terms “comprising” and “including” are used herein in their open-ended and nonlimiting sense unless otherwise noted.

The terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like.

The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

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

In the methods and uses of the invention, Compound A, is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have one or more atoms replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated include isotopes, where possible, of hydrogen, carbon, nitrogen, oxygen, and chlorine, for example, ²H, ³H, ^UC, ¹³C, ¹⁴C, ¹⁵N, ³⁵ S, ³⁶C1. Isotopically labelled compounds are useful in metabolic studies (with ¹⁴C), reaction kinetic studies (with, for example ²H or ³H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents.

Further, substitution with heavier isotopes, particularly deuterium (i.e., ²H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent of Compound A. The concentration of such a heavier isotope, specifically deuterium, may be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in the compounds of the present invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

In Compound A, a methyl group, e.g. on the indazolyl ring, may be deuterated or perdeuterated.

EXAMPLES

Example 1 : Compound A (JDQ443) shows anti-tumor activity in KRAS G12C-mutated CDX models, 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-mutated CDX models across different indications. Cell lines for xenografting were: MIA PaCa-2 (PDAC); NCI-H2122, LU99, HCC-44, NCLH2030 (NSCLC); and KYSE410 (esophageal cancer). JDQ443 inhibited the growth of all models in a dose-dependent manner (Fig. 1 A), with model-specific differences in dose-response dynamics and maximal response patterns that ranged from regression (MIA PaCa-2, LU99), to stasis (HCC44, NCI-H2122), to moderate tumor inhibition (NCLH2030, KYSE410). The largest dynamic range was observed in LU99. In contrast, JDQ443 showed no growth inhibition in a KRASG12V-mutated xenograft model (NCI-H441; Fig. IB), confirming KRASG12C specificity and consistent with the in vitro data. Efficacy was maintained across once- (QD) or twice-daily (BID) administration of the same daily dose: 30 mg/kg QD versus 15 mg/kg BID in MIA PaCa-2 (Fig. 1C), or 100 mg/kg QD versus 50 mg/kg BID in NCLH2122 and LU99 (Fig. 1D-E). The efficacy of QD vs BID dosing correlated well with comparable daily area under the concentration-time curve (AUC) in blood. These findings suggested that JDQ443 efficacy is related to target occupancy (TO), and that efficacious AUC exposures can be achieved under both QD and BID dosing. To characterize whether AUC can act as a surrogate for TO, the effect of continuous infusion versus oral dosing in the LU99 xenograft model was investigated. Once-daily oral dosing at 30 mg/kg induced stasis for about one week followed by tumor progression, and 100 mg/kg induced tumor regression (Fig. IF), with approximate steady-state average concentrations (Cav) of 0.3 pM and ~1 pM, respectively. To assess continuous dosing, JDQ443 was delivered intravenously via programmable microinfusion pumps to achieve target concentrations approximating the oral Cav. Continuous infusion and oral dosing resulted in comparable antitumor responses (Fig. 1F,G). PK/PD model simulation showed that efficacy correlates best with TO and the AUC of JDQ443 (Fig. 1H, I), rather than other PK metrics.

Example 2: Compound A potently inhibits KRAS G12C H95Q, a double mutant mediating 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 (Catalog # 210518) Template: pcDNA3.1(+)EGFP-T2A-FLAG-KRAS G12C and expressed in Cas9 containing Ba/F3 cells by stable transfection. Cells were treated with a dose response curve starting at lOpM with 1/3 dilution from a lOmM DMSO stock. Cell lines were treated with indicated compounds for 72 hours and the viabilities of the cells were measured with CellTiter-Glo.

Results:

In contrast to MRTX-849 (adagrasib), JDQ443 (Compound A) and AMG-510 (sotorasib) are potently inhibiting the cellular viability of the KRASG12C H95Q double mutant. KRASG12C Y96D or KRASG12C R68S double mutant are not inhibited by MRTX-849, AMG- 510 or JDQ443 at the indicated concentrations and in the described setting (Ba/F3 system, 3-day proliferation assay) and confer resistance to all three tested KRASG12C inhibitors.

Conclusion:

Compound A might overcome resistance towards adagrasib in the KRASG12C H95Q setting. In addition, since Compound A has unique binding interactions with mutated KRAS G12C, when compared with sotorasib and adagrasib, Compound A, alone or in combination with one or more therapeutic agent as described herein, may be useful to treat patients suffering from cancer who have previously been treated with other KRAS G12C inhibitors such as sotorasib or adagrasib, or to target resistance after an acquired KRAS resistance mutation emerges on the initial KRAS G12C inhibitor treatment.

Example 3: Compound A potently inhibits KRAS G12C double mutants

The effect of Compound A and other KRASG12C inhibitors on second-site mutations reported to confer resistance to adagrasib was also investigated as follows.

Materials and Methods:

Cell lines and KRAS^G12C Inhibitors:

The Ba/F3 cell line is a murine pro-B-cell line and is cultured in RPMI 1640 (Bioconcept, #1 - 1F01 -I) supplemented with 10 % Fetal Bovine Serum (FBS) (BioConcept, #2- 01F30-I), 2 mM Sodium pyurvate (BioConcept, # 5-60F00-H), 2 mM stable Glutamine (BioConcept, # 5-10K50-H), 10 mM HEPES (BioConcept, # 5-31F00-H) and at 37 °C with 5 % CO2, except as otherwise indicated. The parental Ba/F3 cells were cultured in the presence of 5 ng/ml of recombinant murine IL-3 (Life Technologies, #PMC0035). Ba/F3 cells are normally dependent on IL-3 to survive and proliferate, however, by expressing oncogenes they are able to switch their dependency from IL-3 to the expressed oncogene (Curr Opin Oncology, 2007 Jan;19(l):55-60. doi: 10.1097/CCO.0b013e328011a25f.)

Individual plasmid mutagenesis and generation of Ba/F3 stable cell lines:

QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent; # 210519) was used to generate the resistant mutations on the pSG5_Flag-(codon optimized) KRAS^G12C_puro plasmid template and sequences were confirmed by Sanger sequencing.

The mutant plasmids were transfected into the Ba/F3 WT cells by electroporation with the NEON transfection kit (Invitrogen, #MPK10025). Therefore, two million Ba/F3 cells have been electroporated with 10 pg pf plasmids with the NEON System (Invitrogen, #MPK5000), using following conditions Voltage (V) 1635, Width (ms) 20, Pulses 1. After 72 h of electroporation, puromycin selection was performed at 1 pg / ml to generate stable cell lines.

IL-3 withdrawal

Ba/F3 cells are normally dependent on IL-3 to survive and proliferate, however, by expressing oncogenes they are able to switch their dependency from IL-3 to the expressed oncogene. To assess whether the KRAS^G12C single and double mutants are able to sustain the proliferation of Ba/F3 cells, the engineered Ba/F3 cells expressing the mutant constructs were cultured in absence of IL-3. Cell number and viability was measured every three days and after seven days the IL-3 withdrawal was completed. The expression of the mutants after the IL-3 withdrawal were confirmed by Western Blot (data not shown, an upwards shift was observed for KRAS^G12C/R68S).

Drug response curves for KRASG12C inhibitors and validation of resistance mutations:

1000 Ba/F3 cells/well were seeded at in 96-well plates (Greiner Bio-One, #655098). Treatment was performed on the same day with a Tecan D300e drug dispenser. Viability was detected on the same day of treatment for the start plate (Day 0) and three days post-treatment (Day 3) using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, #G7573) on a Tecan infinitiy M200 Pro reader (Intergration Time 1000ms). To determine the growth, the three days post-treatment (Day 3) readout was normalized to start plate (Day 0). The percentage viability was then calculated by normalizing treated wells to DMSO treated control samples. XLfit was used to make the fitted curve with a Sigmoidal Dose-Response Model (four-parameter curve) (Figure 2). The horizontal red dotted line represents the GI50 value. Tabular data are shown below.

Table: Effect of Compound A (JDQ443) on the the proliferation of KRAS G12C/H95 double mutants. («STDEV» indicates the standard deviation for the % growth value)

Table: Effect of sotorasib (AMG510) on the the proliferation of KRAS G12C/H95 double mutants («STDEV» indicates the standard deviation for the % growth value)

Table: Effect of adagrasib (MRTX-849) on the the proliferation of KRAS G12C/H95 double mutants («STDEV» indicates the standard deviation for the % growth value)

Western blot

After treatment with the different compounds at the indicated concentrations and for the indicated time, the cells were collected, pelleted and snap frozen at - 80 °C. Sixty pL of lysis buffer (50 mM Tris HC1, 120 mM NaCl, 25 mM NaF, 40 mM P-glycerol phosphate disodium salt pentahydrate, 1% NP4O, 1 pM microcystin, 0.1 mM Na3VO3, 0.1 mM PMSF, 1 mM DTT and 1 mM benzamidine, supplemented with 1 protease inhibitor cocktail tablet (Roche) for 10 mL of buffer) was added to each sample. The samples were then vortexed, incubated on ice for 10 min, vortexed again and centrifuged at 14000 rpm at 4 °C for 10 min. Protein concentration was determined with the BCA Protein Assay kit (Pierce, 23225). After normalization to the same total volume with lysis buffer, NuPAGE™ LDS Sample buffer 4 X (Invitrogen, NP0007) and NuPAGE™ Sample reducing agent 10 X (Invitrogen, NP0009) was added. The samples were heated at 70 °C for 10 min before loading on a NuPAGE™ Novex™ 4 - 12 % Bis-Tris Midi Protein Gel, 26 - wells (Invitrogen, WG1403A). Gels were run for 45 min at 200 V (PowerPac HC, Biorad) in NuPAGE MES SDS running buffer (Invitrogen, NP0002). The proteins were transferred for 7 min at 135 mA per gel on a Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs membrane (Biorad, 1704159) using the Trans-Blot® Turbo™ system (Biorad) before staining the membrane with Ponceau red (Sigma, P7170). The membranes were blocked with TBST with 5 % of milk at RT. Anti-RAS (Abeam, 108602) and anti-phospho-ERK 1/2 p44/42 MAPK (Cell Signaling, 4370) antibodies were incubated overnight at 4 °C, the anti-vinculin (Sigma, V9131) antibody was incubated for 1 h at RT. Membranes were washed 3 X for 5 min with TBST and the anti-rabbit (Cell Signaling, 7074) and anti-mouse (Cell Signaling, 7076) secondary antibodies were incubated for 1 h at RT. All antibodies were diluted in TBST to 1/1000, except of anti-vinculin (1/3000). Revelation was performed with WesternBright ECL (Advansta, K-12045-D20) for Ras and vinculin and with SuperSignal West Femto maximum sensitivity substrate (Thermo Fischer, 34096), on a Fusion FX (Vilber Lourmat) using the FusionCapt Advance FX7 software. (Figure 3).

Results

Table: Compound A (JDQ443) inhibits the proliferation of KRAS G12C/H95 double mutants.

Ba/F3 cells expressing the indicated FLAG-KRAS^G12C single or double mutants were treated with JDQ443 (Compound A, AMG-510 (sotorasib) and MRTX-849 (adagrasib) (8-point dilution starting at 1 mM) for 3 days and the inhibtion of proliferation was assessed by Cell titer gio viability assay. The average of GIso ± standard deviation (St DV) of 4 independent experiments are shown.

Biophysical data Material and methods:

Preparation of reagents:

Cloning, expression and purification of RAS protein constructs

The E. coll expression constructs used in this study were based on the pET system and generated using standard molecular cloning techniques. Following the cleavable N-terminal his affinity purification tag the cDNA encoding KRAS, NRAS, and HRAS comprised aa 1-169 and was codon-optimized and synthesized by GeneArt (Thermo Fisher Scientific). Point mutations were introduced with the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent). All final expression constructs were sequence verified by Sanger sequencing.

Two liters of culture medium were inoculated with a pre-culture of E. coli BL21(DE3) freshly transformed with the expression plasmid and protein expression induced with 1 mM isopropyl-P-D-thiogalactopyranoside (Sigma) for 16 hours at 18 °C. Proteins with an avi-tag were transformed into E. coli harboring a compatible plasmid expressing the biotin ligase BirA and the culture medium was supplemented with 135 pM d-biotin (Sigma).

Cell pellets were resuspended in buffer A (20 mM Tris, 500 mM NaCl, 5 mM imidazole, 2 mM TCEP, 10 % glycerol, pH 8.0) supplemented with Turbonuclease (Merck) and cOmplete protease inhibitor tablets (Roche). The cells were lysed by three passages through a homogenizer (Avestin) at 800-1000 bar and the lysate clarified by centrifugation at 40000 g for 40 min. The lysate was loaded onto a HisTrap HP 5 ml column (Cytiva) mounted on an AKTA Pure 25 chromatography system (Cytiva). Contaminating proteins were washed away with buffer A and bound protein was eluted with a linear gradient to buffer B (buffer A supplemented with 200 mM imidazole). During dialysis O/N the N-terminal His affinity purification tags on the nontagged and avi-tagged proteins were cleaved off by TEV or HRV3C protease, respectively. The protein solution was re-loaded onto a HisTrap column and the flow through containing the target protein collected.

Guanosine 5 ’-diphosphate sodium salt (GDP, Sigma) or GppNHp-Tetralithium salt (Jena Bioscience) was added to a 24-32x molar excess over protein. EDTA (pH adjusted to 8) was added to a final concentration of 25 mM. After 1 hour at room temperature the buffer was exchanged on a PD-10 desalting column (Cytiva) against 40 mM Tris, 200 mM (NH4)2SO4, 0.1 mM ZnC12, pH 8.0. GDP (for KRAS G12C resistance mutants H95Q/D/R, Y96D/C and R68S) or GppNHp was added to a 24-32x molar excess over protein to the eluted protein. 40 LT Shrimp Alkaline Phosphatase (New England Biolabs) was added to GppNHp containing samples only. The sample was then incubated for 1 hour at 5 °C. Finally, MgC12 was added to a concentration of about 30 mM. The protein was then further purified over a HiLoad 16/600 Superdex 200 pg column (Cytiva) pre-equilibrated with 20 mM HEPES, 150 mM NaCl, 5 mM MgC12, 2 mM TCEP, pH 7.5. The purity and concentration of the protein was determined by RP-HPLC, its identity was confirmed by LC-MS. Present nucleotide was determined by ion-pairing chromatography [Eberth et al, 2009],

Determination of covalent rate constants by RapidFire MS

Assay and curve fitting

Serial dilutions of the test compounds (50 pM, /i dilutions) were prepared in 384well plates and incubated with 1 pM KRAS G12C (with/without additional mutants) in 20mM Tris pH7.5, 150mM NaCl, 100 pM MgCh, 1% DMSO at room temperature. Reactions were stopped at different time points by addition of formic acid to 1%. MS measurements were carried out using a Agilent 6530 quadrupole time-of-flight (QToF) MS system coupled to an Agilent RapidFire autosampler RF360 device, resulting in % modification values for each well. In parallel, compound solubility was assessed by nephelometry and compound concentrations resulting in measurable turbidity were excluded from curve fitting.

Plotting the % modification vs. time allowed for extraction of k_Obs values for the different compound concentrations. In a second step, the obtained k_Obs values were plotted against the compound concentrations. Rate constants (i.e. kinact/ i) were derived from the initial linear part of the resulting curves.

MS measurements

The RapidFire autosampler RF 360 was used to perform the injections. Solvents were delivered by Agilent 1200 pumps. A C18 Solid Phase Extraction (SPE) cartridge was used for all experiments.

A volume of 30 pL was aspirated from each well of a 384-well plate. The sample load/wash time was 3000 ms at a flow rate of 1.5 mL/min (H2O, 0.1% formic acid); elution time was 3000 ms (acetonitrile, 0.1% formic acid); reequilibration time was 500 ms at a flow rate of 1.25 mL/min (H2O, 0.1% formic acid).

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

All data processing was performed using a combination of Agilent MassHunter Qualitative Analysis, Agilent Rapid-Fire control software, and the Agilent DA Reprocessor Offline Utilities. A Maximum Entropy algorithm produced zero-charge spectra in separate files per injection. A batch processing generated a single file incorporating all mass spectra in a text format as x,y coordinates. This file was used to calculate the % of protein modification in each well.

Results

Quantification of the second order rate constants for modification for the indicated constructs (all GDP -loaded) was carried out using kinetic MS experiments, measuring %modification at different time points for a range of compound concentrations. Kinact/Ki was extrapolated from the initial slope of the k_Obs vs. compound concentration plot. Activities against KRAS G12D:GDP were set to 1 and relative activities for the resistance mutants are given. Average values of n=4 experiments for KRAS G12C, n=3 for G12C Y96D and n=2 for other mutants are given in the Table below.

Table: Fold change of second order rate constant for resistance mutants relative to

KRAS G12C

Quantification of the second order rate constants for modification for the indicated constructs (all GDP -loaded) was carried out using kinetic MS experiments, measuring %modification at different time points for a range of compound concentrations. Kinact/KI was extrapolated from the initial slope of the K_Obs vs. compound concentration plot. Average values of n=4 experiments for KRAS G12C, n=3 for G12C Y96D and n=2 for other mutants are given.

Table: Second order rate constants (Kjnact/KI lmM-l*s-11) for Compound A (JDQ443), sotorasib and adagrasib against resistance mutants

Conclusions

First generation KRAS G12C inhibitors have shown efficacy in clinical trials. However, the emergence of mutations that disrupt inhibitor binding and reactivation in downstream pathways, limits the duration of response. Second-site mutants reported to confer resistance to adagrasib in clinical trials (ref: N Engl J Med. 2021 Jun 24;384(25):2382-2393. doi: 10.1056/NEJMoa2105281., Cancer Discov. 2021 Aug; 11(8): 1913-1922. doi: 10.1158/2159- 8290. CD-21-0365. Epub 2021 Apr 6.PMID: 33824136.) were expressed in Ba/F3 cells and analyzed for their sensitivity towards Compound A (JDQ443) in comparison to KRAS G12C (GEo = 0.115 ± 0.060 mM). As expected from the binding mode, Compound A inhibited proliferation and signaling of KRAS G12C H95 double mutants. Compound A potently inhibited the proliferation of G12C/H95R and G12C/H95Q (GI50 = 0.024 ± 0.006 mM, GI50 = 0.284 ± 0.041 mM, respectively), while expression of G12C/R68S, G12C/Y96C and G12C/Y96D conferred resistance to Compound A (GI50 >1 mM, all).

Surprisingly, expression of G12C/H95D resulted in reduced sensitivity to Compound A (GI50 = 0.612 ± 0.151 mM) compared to H95R or Q although Compound A is not directly interacting with Histidine 95. Western blot analysis of pERK upon Compound A treatment as well as the analysis of the rate constants of Compound A (biophysical data, above) towards these clinically observed SWII pocket mutations in biophysical settings were in agreement with the cellular growth inhibition data (see table).

The difference between H95D compared to H95R or Q could be due the negative charge of the aspartate, which could further increase the negative electrostatic potential of the KRAS G12C surface. This might affect ligand recognition and therefore decrease the specific reactivity and cellular activity of Compound A for this mutant. Another possible explanation is that the H95D mutation could affect KRAS dynamic so that the conformation allowing Compound A binding becomes less accessible. In conclusion, the data show Compound A should overcome adagrasib induced resistance in G12C/Q95R or G12C/H95Q settings. Compound A treatment, particularly in methods of the invention, may still be useful in the G12C/H95Q setting where it has shown activity.

Example 7: Clinical efficacy of Compound A

A phase Ib/II open-label, multi-center, dose escalation study of Compound A (JDQ443) alone and in combination with specific agents is conducted in patients with advanced solid tumors harboring the KRAS G12C mutation, including KRAS G12C-mutated NSCLC and KRAS G12C-mutated colorectal cancer (KontRASt-01 (NCT04699188)). The study is conducted to evaluate the antitumor efficacy, safety and tolerability of JDQ443 as a single agent and JDQ443 in combination with other agents.

Patients to be treated include patients with advanced, KRAS G12C-mutated solid tumors who have received standard-of-care therapy, or who are intolerant of or ineligible for approved therapies; or , Eastern Cooperative Oncology Group Performance Status (ECOG PS 0-1); or had no prior treatment with KRAS^G12C inhibitors. Key exclusion criteria for the JDQ443 monotherapy arm are: active brain metastases and/or prior KRASG12C inhibitor treatment.

Patients with NSCLC include patients previously treated with a platinum-based chemotherapy regimen and an immune checkpoint inhibitor, either in combination or in sequence, unless ineligible to receive such therapy.

Patients with CRC include patients who have previously received standard-of-care therapy, including fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, unless ineligible to receive such therapy.

The preliminary data from the monotherapy dose escalation arm study are as follows.

At a cut-off date of January 5, 2022, 39 patients were treated with 200 mg QD, 400 mg QD, 200 mg BID or 300 mg BID of Compound A. Compound A was administered with food.

Patients had a median of 3 prior lines of anti -neoplastic therapy. The recommended dose for the monotherapy is a dose of 200 mg of Compound A taken orally twice daily (BID). Efficacy data (cutoff of 05 Jan 2022) from the pooled Phase lb JDQ443 single agent cohort (n=39) showed:

• 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 in NSCLC

• PD/PK modeling predicted sustained, high-level 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 (Gr) 1-2. There were no Grade 4-5 TRAEs. Four Grade 3 TRAEs occurred in 4 separate pts;. The most common TRAEs were fatigue, nausea, edema, diarrhea, and vomiting. There was one DLT (Grade 3 fatigue) and one treatment-related serious AE (Grade 3 photosensitivity reaction), each in separate patients treated at 300 mg BID.

At the recommended dose of 200 mg BID, there was prolonged absorption, with a median time to maximum plasma concentration (Tmax) of 3-4 hrs following administration with food. No significant accumulation was observed at steady state, and there was no evidence of auto-induction. The half-life was about 7 hours, and steady-state area under the curve (AUCss) was more than threefold above the exposure required for maximum efficacy in less-sensitive KRAS G12C xenograft models. Figure 9 shows the PK profile at steady state.

The predicted target occupancy profile is shown in Figure 4. Patient PK and preclinical target occupancy models were integrated to predict target occupancy in patients at >90% in >82% patients. The models assume that JDQ443 binding and target (KRAS) turn-over rates are the same in mice and humans (~25 hr half-life for KRAS) and that only free drug can bind the target.

The best overall response across dose levels and indications is shown in the top half of Figure 5 and in the Table below.

The best overall response across dose levels in all patients with NSCLC is shown in the bottom half of Figure 5 and in the Table below. All patients with a Partial Response or unconfirmed Partial Response were ongoing treatment at the data cut-off.

NE, not evaluable; NSCLC, non-small cell lung cancer; ORR, overall response rate;

PD, progressive disease; PR, partial response; QD, once daily.

Responses are investigator assessed per RECIST vl.l. Two (10.0%) patients had a uPR, which contributed toward the ORR (confirmed and unconfirmed). uPR = unconfirmed PR pending confirmation, treatment ongoing with no PD. One of two patients with a uPR had confirmed PR after the data cut-off.

Figure 6 shows PET scans showing a substantial reduction in the 2-[fluorine-18]-fluoro-2- deoxy-d-glucose (18-F-FDG) avidity of the tumor mass after four cycles of treatment with Compound A administered at 200 mg BID to a patient with NSCLC. The patient had received pemetrexed/pembrolizumab, docetaxel, tegafur/gimeracil/oteracil, and carboplatin/ paclitaxel/atezolizumab. Post-Cycle 2 scan showed a 30.4% reduction in the sum of the longest diameters of target lesions compared with baseline. PR was confirmed on subsequent scans

A 57 year old male with metastatic KRAS G12C-mutated NSCLC. Local molecular testing using next generation sequencing (NGS) identified no mutations in TP53. Mutation status of STK11, KEAP1 and NRF2 were unknown. The patient had received prior carboplatin/pemetrexed/pembrolizumab, docetaxel, tegafur-gimeracil-oteracil, and carboplatin/paclitaxel/atezolizumab. He was enrolled to the JDQ443 monotherapy dose escalation part of the study at a dose of JDQ443 200 mg BID given continuously on a 21 -day cycle. Disease assessment after 2 cycles of treatment demonstrated a RECIST 1.1 partial response, with a -30.4% change in the sum of the longest diameters of target lesions compared with baseline. Partial response was confirmed on subsequent scans (Fig. 6) and the patient continued on treatment. Positron emission tomography imaging at baseline and after 4 cycles of treatment also showed substantial reduction in 2-[fluorine-18]-fluoro-2-deoxy-d-glucose avidity of the tumor mass.

Example 8: Study of efficacy and safety of JDQ443 single-agent as first-line treatment for patients with locally advanced or metastatic KRAS G12C-mutant non-small cell lung cancer with a PD-L1 expression <1% or a PD-L1 expression >1% and a STK11 co-mutation

A clinical study demonstrating the therapeutic use of Compound A (JDQ443) may be carried out as follows.

This study aims to evaluate the antitumor activity and safety of JDQ443 single-agent as first-line treatment for participants with locally advanced or metastatic non-small cell lung cancer (NSCLC) whose tumors harbor a KRAS G12C mutation and have a PD-L1 expression <1%, % regardless of STK 11 mutation status, (cohort A) or a PD-L 1 expression > 1 % and STK 11 co-mutations (cohort B).

Tests to determine PD-L1 expression status, KRAS G12C mutation and STK11 mutation status, e.g. in tumor tissue or blood samples, are known in the art. Objectives and Endpoints:

The study will have 2 non-comparative cohorts that will recruit participants in parallel according to the following characteristics:

• Cohort A: participants whose tumors harbor a KRAS G12C mutation and a PD-L1 expression <1%, regardless of STK11 mutation status (N=90).

• Cohort B: participants whose tumors harbor a KRAS G12C mutation, a PD-L1 expression >1% and a STK11 co-mutation (N=30).

Compound A (JDQ443) administered to all subjects as study treatment: • JDQ443 per os (PO) 200 mg twice a day continuously (i.e. with no drug holiday).

Key Inclusion criteria

• Histologically confirmed locally advanced (stage IHb/IIIc ineligible for definitive chemoradiation or surgery) or metastatic (stage IV) NSCLC participants without previous systemic treatment for metastatic disease. Prior (neo)adjuvant treatment with chemotherapy and/or immunotherapy, or prior radiotherapy administered sequentially or concomitantly with chemotherapy and/or immunotherapy for localized or locally advanced disease are accepted if the time between therapy completion and enrollment is > 12 months.

• Presence of a KRAS G12C mutation (all patients) and:

• Cohort A: PD-L1 expression <1%, regardless of STK11 mutation status

• Cohort B: PD-L1 expression >1% and a STK11 co-mutation

• At least one measurable lesion per RECIST 1.1.

• ECOG performance status < 1.

• Participants with brain metastases are allowed if clinically stable.

• Participants capable of swallowing study medication.

Key Exclusion criteria

• Participants with EGFR activating mutations or ALK rearrangements are not eligible. Participants with other targetable mutations diagnosed per local tests will be excluded if required by local guidelines.

• Previous use of a KRAS G12C inhibitor or previous systemic treatment for metastatic NSCLC.

• A medical condition that results in increased photosensitivity (i.e. solar urticaria, lupus erythematosus, etc).

• Participants who are taking a prohibited medication (strong CYP3A inducers) that cannot be discontinued at least seven days prior to the first dose of study treatment and for the duration of the study

Accordingly, there is now provided the following embodiments:

Embodiment 1. A method of treating a cancer or solid tumor 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, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a KRAS G12C inhibitor, or a pharmaceutically acceptable salt thereof.

Embodiment 2. The method according to Embodiment 1, wherein the KRAS G12C inhibitor is selected from l-{6-[(4A/)-4-(5-Chloro-6-methyl-lZ/-indazol-4-yl)-5-methyl-3-(l- methyl-l//-indazol-5-yl)-lZ/-pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A), sotorasib (Amgen), adagrasib (Mirati), D-1553 (InventisBio), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Lilly), BI1823911 (Boehringer), AS KRAS G12C (Ascentage Pharma), SF KRAS G12C (Sanofi), RMC032 (Revolution Medicine), JAB-21822 (Jacobio Pharmaceuticals), AST-KRAS G12C (Allist Pharmaceuticals), AZ KRAS G12C (Astra Zeneca), NYU-12VC1 (New York University), and RMC6291 (Revolution Medicines), or a pharmaceutically acceptable salt thereof.

Embodiment 3. The method according to Embodiment 2, wherein the KRAS G12C inhibitor is selected from l-{6-[(4A -4-(5-Chloro-6-methyl-l//-indazol-4-yl)-5-methyl-3-(l -methyl- 1/7- indazol-5-yl)-U/-pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A), sotorasib, adagrasib, D-1553, and GDC6036), or a pharmaceutically acceptable salt thereof.

Embodiment 4. The method according to Embodiment 2, wherein the KRAS G12C inhibitor is 1- {6-[(4A/)-4-(5-Chloro-6-methyl-l//-indazol-4-yl)-5-methyl-3-(l-methyl-l//-indazol-5-yl)-l//- pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A), or a pharmaceutically acceptable salt thereof.

Embodiment 5. The method according to Embodiment 2, wherein the KRAS G12C inhibitor is 1- {6-[(4A/)-4-(5-Chloro-6-methyl-l//-indazol-4-yl)-5-methyl-3-(l-methyl-l//-indazol-5-yl)-l//- pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A).

Embodiment 6. The method according to any one of the previous Embodiments, wherein the cancer or tumor is a cancer or tumor which is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer, bile duct cancer, ovarian cancer, pancreatic cancer and duodenal papillary cancer and a solid tumor.

Embodiment 7. The method according to any one of the previous Embodiments, wherein the cancer is non-small cell lung cancer.

Embodiment 8. The method according to any one of the previous Embodiments, wherein the amount of each therapeutic agent is administered to the subject in need thereof is effective to treat the cancer or tumor. Embodiment 9. The method according to any one of the previous Embodiments, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose ranging from 100 to 600 mg per day, e.g. from 200 to 400 mg per day.

Embodiment 10. The method according to any one of the previous Embodiments, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose which is selected from 100, 150, 200, 250, 300, 350 and 400 mg per day.

Embodiment 11. The method according to any one of the previous Embodiments, wherein the total daily dose of Compound A is administered once daily or twice daily.

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

Embodiment 13. The method according to any one of the previous Embodiments, wherein Compound A is administered with food.

Embodiment 14. Compound A, or a pharmaceutically acceptable salt thereof, for use in a method of treating a cancer or solid tumor 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.

Embodiment 15. The compound according to Embodiment 14, wherein the cancer or solid tumor is selected from lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer, bile duct cancer, ovarian cancer, pancreatic cancer and duodenal papillary cancer and a solid tumor.

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

Embodiment 17. A compound which is l-{6-[(4A/)-4-(5-Chloro-6-methyl-lJ/-indazol-4- yl)-5-methyl-3-(l-methyl-17/-indazol-5-yl)- 177-pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop- 2-en-l-one (Compound A), or a pharmaceutically acceptable salt thereof, for use in a method of treating a cancer or a tumor, according to any one of Embodiments 1 to 13. Embodiment 18. A method or a compound for use according to any one of the previous Embodiments, wherein the treatment is for first-line treatment.

Embodiment 19. A compound for use in a method of treating a cancer or a solid tumor, or a combination for use in in a method of treating a cancer or a solid tumor, or a method of treating a cancer or a solid tumor according to any one of the Embodiments, wherein the cancer or a solid tumor is present in a patient who has previously received KRAS G12C inhibitor treatment or who is a KRAS G12C inhibitor naive patient (i.e. has not previously received KRAS G12C inhibitor treatment).

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

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

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

While specific embodiments of the subject invention have been discussed, the above specification 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 claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

What is claimed is:

1. A method of treating a cancer or solid tumor 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 comutation, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a KRAS G12C inhibitor, or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein the KRAS G12C inhibitor is selected from 1- {6-[(4A/)-4-(5-Chloro-6-methyl-U/-indazol-4-yl)-5-methyl-3-(l-methyl-U/-indazol-5-yl)-UT- pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A), sotorasib (Amgen), adagrasib (Mirati), D-1553 (InventisBio), BI1701963 (Boehringer), GDC6036 (Roche), JNJ74699157 (J&J), X-Chem KRAS (X-Chem), LY3537982 (Lilly), BI1823911 (Boehringer), AS KRAS G12C (Ascentage Pharma), SF KRAS G12C (Sanofi), RMC032 (Revolution Medicine), JAB-21822 (Jacobio Pharmaceuticals), AST-KRAS G12C (Allist Pharmaceuticals), AZ KRAS G12C (Astra Zeneca), NYU- 12 VC 1 (New York University), and RMC6291 (Revolution Medicines), or a pharmaceutically acceptable salt thereof.

3. The method according to claim 2, wherein the KRAS G12C inhibitor is selected from 1- {6-[(4A/)-4-(5-Chloro-6-methyl-U/-indazol-4-yl)-5-methyl-3-(l-methyl-U/-indazol-5-yl)-UT- pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A), sotorasib, adagrasib, D-1553, and GDC6036), or a pharmaceutically acceptable salt thereof.

4. The method according to claim 2, wherein the KRAS G12C inhibitor is l-{6-[(4A/)-4-(5- Chloro-6-methyl-U/-indazol-4-yl)-5-methyl-3-(l-methyl-U/-indazol-5-yl)-U/-pyrazol-l-yl]-2- azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A), or a pharmaceutically acceptable salt thereof.

5. The method according to claim 2, wherein the KRAS G12C inhibitor is l-{6-[(4A/)-4-(5- Chloro-6-methyl-U/-indazol-4-yl)-5-methyl-3-(l-methyl-U/-indazol-5-yl)-U/-pyrazol-l-yl]-2- azaspiro[3.3]heptan-2-yl}prop-2-en-l-one, (Compound A).

6. The method according to any one of the previous claims, wherein the cancer or tumor is a cancer or tumor which is selected from the group consisting of lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer, bile duct cancer, ovarian cancer, pancreatic cancer and duodenal papillary cancer and a solid tumor.

7. The method according to any one of the previous claims, wherein the cancer is non-small cell lung cancer.

8. The method according to any one of the previous claims, wherein the amount of each therapeutic agent is administered to the subject in need thereof is effective to treat the cancer or tumor.

9. The method according to any one of the previous claims, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose ranging from 100 to 600 mg per day, e.g. from 200 to 400 mg per day.

10. The method according to any one of the previous claims, wherein Compound A, or a pharmaceutically acceptable salt thereof, is administered at a therapeutically effective dose which is selected from 100, 150, 200, 250, 300, 350 and 400 mg per day.

11. The method according to any one of the previous claims, wherein the total daily dose of Compound A is administered once daily or twice daily.

12. The method according to any one of the previous claims, wherein Compound A is administered at a dose of 100 mg twice daily or 200 mg twice daily.

13. The method according to any one of the previous claims, wherein Compound A is administered with food.

14. Compound A, or a pharmaceutically acceptable salt thereof, for use in a method of treating a cancer or solid tumor 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.

15. The compound according to claim 14, wherein the cancer or solid tumor is selected from lung cancer (including lung adenocarcinoma, non-small cell lung cancer and squamous cell lung cancer), colorectal cancer, bile duct cancer, ovarian cancer, pancreatic cancer and duodenal papillary cancer and a solid tumor.

16. The compound according to claim 15, wherein the cancer or solid tumor is non-small cell lung cancer.

17. A compound which is l-{6-[(4A/)-4-(5-Chloro-6-methyl-lJ/-indazol-4-yl)-5-methyl-3-(l- methyl-lJT-indazol-5-yl)- 17/-pyrazol-l-yl]-2-azaspiro[3.3]heptan-2-yl}prop-2-en-l-one (Compound A), or a pharmaceutically acceptable salt thereof, for use in a method of treating a cancer or a tumor, according to any one of claims 1 to 13.

18. A method or a compound for use according to any one of the previous claims, wherein the treatment is for first-line treatment.

19. A compound for use in a method of treating a cancer or a solid tumor, or a combination for use in in a method of treating a cancer or a solid tumor, or a method of treating a cancer or a solid tumor according to any one of the claims, wherein the cancer or a solid tumor is present in a patient who has previously received KRAS G12C inhibitor treatment or who is a KRAS G12C inhibitor naive patient (i.e. has not previously received KRAS G12C inhibitor treatment).