RAS is the most frequently mutated oncogene in cancer. KRAS G12C mutations are most prevalent in lung adenocarcinoma (~13%) and colorectal adenocarcinoma (~4%), and occur less commonly in other solid tumor malignancies. First generation KRAS^G12C inhibitors show anti-tumor activity in early phase clinical trials. However, the emergence of resistance, mediated at least in part by RAS gene mutations that disrupt inhibitor binding and reactivation of downstream pathways, limit the duration of response. Here we report the identification of JDQ443 (NVP-JDQ443), a novel KRAS^G12C inhibitor which binds under the switch II loop with a novel binding mode, exploiting unique interactions with the KRAS^G12C protein compared to sotorasib and adagrasib. JDQ443 potently inhibits KRAS^G12C cellular signaling and proliferation in a mutant selective manner by irreversibly trapping the GDP-bound state of KRAS^G12C through formation of a covalent bond with cysteine at position 12. Consistent with its mechanism as an irreversible inhibitor, JDQ443 shows sustained target occupancy (TO) in vivo (KRAS^G12C TO t1/2 ~ 66 h in the MiaPaCa2 model) despite a blood half-life of ~ 2 hours, and exhibits a linear PK/PD relationship. JDQ443 has dose-dependent anti-tumor activity in mice bearing KRAS G12C mutated tumor xenografts comparable to sotorasib and adagrasib. In mouse, rat, and dog, JDQ443 is orally bioavailable, achieves exposures in a range predicted to confer anti-tumor activity, and is well-tolerated. Continuous delivery of JDQ443 using mini-pump administration demonstrates that area under the curve (AUC), rather than maximal concentration (Cmax), is the driver of efficacy. Combination of JDQ443 with the SHP2 inhibitor TNO155 further increases KRAS G12C target occupancy in vivo, enhanced pre-clinical anti-tumor activity, and delayed the emergence of resistance in xenografts. A genome-wide CRISPR screen in 5 KRAS G12C mutated lung cancer cell lines identifies novel mechanisms of resistance to the KRAS/SHP2 drug combination. Furthermore, the characterization of JDQ443 alone and in combination with TNO155 in BaF/3 pools addicted to KRAS alleles that have previously been shown to mediate resistance to adagrasib in clinical samples will be discussed. Collectively, these data show that JDQ443 is a potent, mutant-selective, covalent irreversible KRAS^G12C inhibitor with favorable pharmaceutical properties. A phase Ib/II clinical trial of JDQ443 alone and in combination with TNO155 in patients with advanced solid tumors harboring the KRAS G12C mutation is ongoing (NCT04699188). Citation Format: Saskia M. Brachmann, Andreas Weiss, Daniel A. Guthy, Kim Beyer, Johannes Voshol, Michel Maira, Anirudh Prahallad, Diana Graus Porta, Christian Schnell, Nils Ostermann, Andrea Vaupel, Marc Gerspacher, Catherine Leblanc, Dirk Erdmann, Dario Sterker, Grainne Kerr, Giovannoni Jerome, Victoria Head, Rowan Stringer, Ruben De Kanter, Kearns Jeff, Danielle Roman, Toni Widmer, Peter Wessels, Eloisa Jimenez Nunez, Richard Sedrani, Frederic Zecri, Francesco Hofmann, Jeff Engleman, Edwige Lorthiois, Simona Cotesta. JDQ443, a covalent irreversible inhibitor of KRAS G12C, exhibits a novel binding mode and demonstrates potent anti-tumor activity and favorable pharmacokinetic properties in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P124.