Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC–MS/MS Approach
Abstract
KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC–MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C -positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/μg of total protein has been achieved 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 level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.
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In a phase I trial of divarasib, among 57 evaluable patients with NSCLC, 26 were confirmed to be in partial remission, with a confirmed ORR of 46%.
“Of the 59 patients with NSCLC previously treated with divarasib monotherapy included, 57 patients had evaluable outcomes, 26 of whom were confirmed to be in partial remission (PR), with confirmed objective remission rate (ORR) of 46%.”
From Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials (2023) · cites this paper as [68]
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Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials
Xie, X., Yu, T., Li, X., Zhang, N., Foster, L. J., Peng, C., Huang, W., & He, G. (2023) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-023-01589-z · Cited by 396 · see citing works
- Authors
- Meng, L., Chan, E. W., Ng, C., Aimi, J., Tran, J. C., Oh, A. J., Merchant, M., Purkey, H. E., Heffron, T. P., Kaur, S., Xu, K., Shi, Z., & He, J.
- Affiliations
- Genentech (US)
- Corresponding author
- Shi, Zhen
- He, Jintang
- ORCID
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2022-09-09
- Volume
- 94
- Issue
- 37
- Pages
- 12927-12933
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- OpenAlex
- W4295038037
- Cited by
- 15
- References
- 14
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 6 · 2023: 3
- FWCI
- 1.06
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Lung Cancer Treatments and Mutations
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- KRAS G12C, target engagement, sotorasib, covalent inhibitors, immunoaffinity enrichment, 2D-LC-MS/MS, small molecule quantification, core needle biopsy, non-small cell lung cancer, pharmacodynamics, switch-II pocket
- MeSH terms
- Animals, Antineoplastic Agents, Biopsy, Carcinoma, Non-Small-Cell Lung, Chromatography, Liquid, Guanosine Triphosphate, Humans, Lung Neoplasms, Mutation, Proto-Oncogene Proteins p21(ras), Tandem Mass Spectrometry
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