Abstract B168: Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations
Authors: Pagliarini Raymond A , Milgram Benjamin C , Borrelli Deanna R , O'Hearn Erin , Huff Michael R. , Ladd Brendon , Brooijmans Natasja , Wang Weixue , Kuzmic Petr , Guzman-Perez Angel , Stuart Darrin D 
Published: December 2023

Abstract
Background: EGFR mutations are well validated clinical targets in non-small cell lung cancer (NSCLC). Osimertinib, a highly-selective EGFR mutation-targeting covalent drug, is increasingly used in the first line setting for patients with NSCLC bearing EGFR L858R mutation or exon 19 deletions (ex19del). In a subset of these patients, co-occurring L858R/C797x or ex19del/C797x mutations (“double mutants”) are emerging as an on-target resistance mechanism, necessitating the need for new therapies, particularly in patients with CNS metastases.
Materials and methods: STX-241 was tested across a panel of in vitro biochemical, cell signaling, and proliferation assays for potency against EGFR L858R and ex19del single mutants and the corresponding C797S double mutants. Additionally, STX-241 was tested for in vivo activity in mice bearing human NSCLC cell line xenografts NCI-H3255 (L858R) and PC-9 (ex19del), and in a PC-9-derived ex19del/C797S double mutant knock-in xenograft. Free CNS penetration (Kp,uu) was determined in non-tumor bearing mice. Osimertinib and gefitinib (an approved reversible EGFR inhibitor) were used as benchmark molecules across assays.
Results: STX-241, an ATP-competitive reversible EGFR inhibitor representing novel chemical matter, demonstrated potent and selective inhibition of recombinant EGFR L858R/C797S mutant protein relative to wild-type, with increased mutant residence time relative to gefitinib. Potent (high picomolar to low nanomolar) inhibition of L858R, ex19del, L858R/C797S, and ex19del/C797S mutants was observed in proliferation assays using engineered Ba/F3 and human NSCLC cell lines. STX-241 demonstrated &gt;150x selectivity for all tested EGFR mutants relative to wild-type EGFR in engineered Ba/F3 cells and human cancer cells. In these assays, STX-241 selectivity exceeded that of the benchmark reversible EGFR inhibitor gefitinib. Strong potency and double mutant selectivity was also observed for STX-241 in pharmacodynamic assays measuring EGFR pathway activation (pEGFR). STX-241 was well tolerated at doses of 15 mg/kg BID or 50 mg/kg QD in mice, where regression of EGFR exon 19 or 21 mutant NSCLC xenografts was observed, concomitant with EGFR pathway suppression. Notably, using an isogenic pair of PC-9 (EGFR ex19del) NSCLC xenografts that differ only by the presence of a C797S mutation, STX-241 demonstrates no drop off in antitumor activity in the presence of C797S. In mouse pharmacokinetic studies measuring free CNS penetration, Kp,uu measurements were comparable to Osimertinib, run in parallel as a benchmark.
Conclusions: STX-241 demonstrates strong potency and selectivity against EGFR L858R/C797S and ex19del/C797S double mutants, as well as robust CNS penetrance. These data warrant further exploration of this potential best-in-class inhibitor in the clinic for patients progressing on Osimertinib via C797x mutation.
Citation Format: Raymond A Pagliarini, Benjamin C Milgram, Deanna R Borrelli, Erin O'Hearn, Michael R. Huff, Brendon Ladd, Natasja Brooijmans, Weixue Wang, Petr Kuzmic, Angel Guzman-Perez, Darrin D Stuart. Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B168.