Preclinical characterization of D3S-001, a highly potent, selective, and differentiated covalent inhibitor of KRAS G12C.

e15102 Background: KRAS G12C mutation is an oncogenic driver most frequently found in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and other tumor types. Covalent small molecule inhibitors such as sotorasib, recently FDA-approved, and others in clinical trials that target G12C-altered protein by irreversibly binding to the GDP-bound inactive form of KRAS are clinically active in NSCLC patients carrying this somatic mutation. However, in in vitro assays we have developed to evaluate cellular GTP-bound KRAS G12C level, these molecules did not achieve maximal KRAS G12C target inhibition at the minimum concentration (C min ) of their respective clinically doses. We therefore hypothesize that the potency of KRAS G12C inhibitors may be further improved through medicinal chemistry optimization. Methods: A series of in vitro and in vivo studies were conducted to assess the preclinical pharmacological activity of D3S-001. The biochemical potency of D3S-001 was evaluated by SPR binding analysis. The kinetics of KRAS G12C target engagement was measured by ELISA method. The inhibitor effect of D3S-001 on phospho-ERK1/2 was determined by HTRF assay, and the anti-proliferation effect on cancer cell lines was evaluated by CTG luminescence assay. For in vivo efficacy studies, several human cancer xenograft models and a mouse syngeneic model were employed. Results: D3S-001 demonstrated an exceptionally high Kinact/KI value of 1.58^10 6 M -1 s -1 by SPR binding analysis. In target engagement assays, D3S-001 achieved rapid and near complete (> 95%) KRAS G12C inhibition within 2 hours at 5 nM. This rapid and effective target engagement correlated with its substantially enhanced cellular potency in both in vitro and in vivo efficacy studies. D3S-001 demonstrated single-digit nanomolar IC 50 values in inhibiting cellular phospho-ERK1/2 and cell proliferation in a panel of cancer cell lines harboring the KRAS G12C mutation. D3S-001 was also high selective with no anti-proliferation effects on non-KRAS G12C mutant cell lines. In in vivo studies, D3S-001 demonstrated robust anti-tumor effects as monotherapy in KRAS G12C xenograft models with tumor regression observed at 10 mg/kg and a 30% durable complete remission (CR) rate in CT26, a KRAS G12C syngeneic model, at 30 mg/kg. When combined with an anti-PD-1 antibody, a 70% durable CR rate was achieved with no tumor re-growth observed in CR mice after rechallenging with CT26 tumor, suggesting a memory T cell-induced response. Conclusions: D3S-001 is a highly potent, selective KRAS G12C inhibitor that is differentiated by its rapid and effective target engagement. Human PK modeling predicts > 95% constant target blockage using a 200 mg QD dosing regimen, which may be important and relevant to the induction of deeper and more durable clinical response. D3S-001 will enter a first-in-human Phase 1 clinical trial in 2022.