Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach
KRAS is one of the most commonly mutated oncogenes, and the KRAS G12C mutation has recently emerged as a viable target for small molecule inhibitors. GDC-6036 is an investigational KRAS G12C inhibitor that works by irreversibly binding to the switch II pocket of KRAS G12C in its inactive GDP-bound state, thereby preventing GTP binding and activation. Evaluating target engagement is crucial in clinical drug development, as it helps demonstrate the drug’s mechanistic activity, inform dose selection, understand pharmacodynamics in relation to clinical response, and explore resistance mechanisms.
We have developed an ultra-sensitive method for assessing KRAS G12C engagement. This approach combines immunoaffinity enrichment using a commercially available anti-RAS antibody with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. We applied this method to a KRAS G12C-positive non-small cell lung cancer xenograft model treated with GDC-6036, demonstrating the assay’s capability to analyze small core needle biopsies. As anticipated, we observed dose-dependent KRAS G12C engagement. Currently, the assay achieves a sensitivity of 0.08 fmol/μg of total protein for both free and GDC-6036-bound KRAS G12C, with as little as 4 μg of total protein extracted from human tumor samples. This sub-fmol/μg sensitivity offers a powerful tool for evaluating covalent inhibitor target engagement at the site of action using core needle tumor biopsies in clinical studies.