Related Experiment Video
Updated: Jan 11, 2026
Using High Content Imaging to Quantify Target Engagement in Adherent Cells
Published on: November 29, 2018
Quantitative Assessment of the Target Engagement of a KRAS G12C Inhibitor in Formalin-Fixed Paraffin-Embedded Tumor
Aiying Yu1, Jintang He1, Lingyao Meng1
1Genentech Inc., 1 DNA Way, South San Francisco 94080, California, United States.
Abstract:
Understanding the target engagement of covalent inhibitors, such as KRAS G12C inhibitors, is essential for evaluating their mechanisms of action and efficacy, particularly given the KRAS G12C mutation's significant role in cancer progression. We previously developed a method to directly evaluate KRAS G12C target engagement by measuring both free and drug-bound proteins in frozen tissues without the need for control samples. Since frozen tissues may not be readily available, we aimed to extend our method to formalin-fixed, paraffin-embedded (FFPE) tissues, which are the standard clinical specimens due to their superior preservation and long-term storage capabilities. This adaptation required addressing the analytical challenges associated with fixation-induced protein cross-linking and structural complexity. Here, we report an optimized workflow for quantification of both free and drug-bound KRAS G12C in FFPE tissues. The workflow integrates a specialized pretreatment process including deparaffinization, heat-induced antigen retrieval, and protein-level immunoaffinity enrichment with a targeted 2D-LC-MS/MS to enable a precise and reproducible determination of KRAS G12C target engagement in complex FFPE matrices. The method typically requires a minimum of 5 μg of total protein input, making it suitable for clinical applications with limited sample quantities such as core needle biopsies. A direct comparison of KRAS G12C target engagement measured in both FFPE and corresponding frozen tissues from a xenograft mouse study demonstrated a strong correlation between the two tissue formats. Overall, the reported workflow may serve as a sensitive and reliable tool for assessing the KRAS G12C engagement and potentially that of other covalent inhibitors.
Insights
This study adapted a method to measure KRAS G12C covalent inhibitor engagement in formalin-fixed, paraffin-embedded (FFPE) tissues. The optimized workflow enables precise target engagement assessment in standard clinical samples, crucial for cancer therapy evaluation.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- KRAS G12C mutations drive cancer progression, necessitating evaluation of covalent inhibitor efficacy.
- Assessing target engagement is vital for understanding drug mechanisms and patient response.
- Previous methods required frozen tissues, limiting clinical applicability.
Purpose of the Study:
- To adapt and optimize a method for quantifying KRAS G12C target engagement in formalin-fixed, paraffin-embedded (FFPE) tissues.
- To address analytical challenges posed by FFPE sample processing for covalent inhibitor assessment.
- To enable reliable target engagement measurement in standard clinical specimens.
Main Methods:
- Developed a workflow integrating deparaffinization, antigen retrieval, and immunoaffinity enrichment.
- Utilized targeted 2D-LC-MS/MS for precise quantification of free and bound KRAS G12C.
- Validated the method on FFPE tissues, requiring minimal protein input (5 μg).
Main Results:
- Successfully quantified KRAS G12C target engagement in FFPE tissues.
- Demonstrated a strong correlation between target engagement measurements in FFPE and frozen tissues.
- The method is suitable for limited clinical sample quantities, such as core biopsies.
Conclusions:
- The optimized workflow reliably assesses KRAS G12C covalent inhibitor engagement in FFPE tissues.
- This method extends target engagement analysis to the standard clinical specimen type.
- The workflow may be applicable to evaluating other covalent inhibitors in FFPE samples.
