Abstract B167: Preclinical characterization of THE-349, a mutant-selective, CNS-active, fourth-generation EGFR inhibitor to overcome T790M- and C797S-mediated resistance in NSCLC
Authors: Zhang Sen , Nadworny Sara , Huang Wei-Sheng , Talbot Adam , Ye Emily , Narasimhan Narayana , Shakespeare William C , Rivera Victor M 
Published: December 2023

Background: EGFR activating mutations are observed in 10-50% of NSCLC patients. Although the common mutations (L858R [L] and exon 19 deletions [D]) are initially sensitive to first-, second-, and third-generation EGFR inhibitors (e.g., erlotinib [1G], afatinib [2G], and osimertinib [3G], respectively), on-target resistance is observed in a substantial percentage of patients, with T790M (T) and C797S (C) observed most frequently (post-1G/2G and post-3G, respectively).  Furthermore, EGFR mutational heterogeneity can increase during treatment with existing inhibitors, inhibition of wild type (WT) EGFR leads to dose-limiting toxicities, and tumors commonly metastasize to the brain. We have designed THE-349 to be a fourth-generation EGFR inhibitor with potent activity against all 8 major single- (L, D), double- (LT, DT, LC, DC), and triple- (LTC, DTC) mutant variants, selectivity over WT EGFR, and activity in the CNS.

Methods: Kinome selectivity was evaluated biochemically against a panel of 412 kinases. In vitro potency was determined by assessing effects (IC50s) on human and BaF3 cell lines expressing WT or mutant forms of EGFR. Efficacy studies were conducted using engineered BaF3 cell lines, a luciferase-tagged human NSCLC cell line, and a patient-derived xenograft (PDX) model dosed orally, once-daily at levels that did not exceed the maximum tolerated dose (40 mg/kg THE-349 and 25 mg/kg osimertinib).

Results:THE-349 potently inhibited the biochemical activity of all major EGFR mutant variants (IC50s &lt;2 nM) with a high degree of kinome selectivity. In engineered BaF3 cells in vitro, THE-349 potently inhibited viability of all 8 major EGFR mutant variants (L, LT, LC, LTC, D, DT, DC, and DTC), with IC50s &lt;6 nM and with ≥10-fold selectivity over WT EGFR. Similarly, THE-349 demonstrated robust anti-tumor activity against the same 8 EGFR variants in vivo, in all cases inducing deep tumor regressions (&gt;80% TR) in mice implanted with engineered BaF3 cells. In a PDX model from an osimertinib-resistant tumor containing D and DTC variants, THE-349 induced deep and sustained regressions (99% TR) through 56 days of dosing, and tumor regrowth that occurred during a subsequent 5-week dosing holiday was reversed (88% TR) upon redosing with THE-349. In an intracranial tumor model using a triple-mutant (LTC) NSCLC cell line, THE-349 significantly prolonged median survival (from 42.5 to 71.5 days), while osimertinib had no effect. THE-349 has a favorable ADME profile and promising safety profile in IND-enabling toxicology studies.

Conclusion:  THE-349 is a fourth-generation, CNS-active, mutant-selective EGFR inhibitor that has potent, single-agent activity against all major classes of EGFR activating and resistance mutations. A phase 1 clinical trial of THE-349 is planned.

Citation Format: Sen Zhang, Sara Nadworny, Wei-Sheng Huang, Adam Talbot, Emily Ye, Narayana Narasimhan, William C Shakespeare, Victor M Rivera. Preclinical characterization of THE-349, a mutant-selective, CNS-active, fourth-generation EGFR inhibitor to overcome T790M- and C797S-mediated resistance in NSCLC [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 B167.