Background: Multiple ADCs are in clinical trials for CRC and the optimal strategy for selecting patients who may benefit from the treatment is evolving. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also for markers of resistance or response to the payload. We have built a multiplexed ADC biomarker panel in FFPE tumor tissue that simultaneously quantifies the protein levels of the antibody targets and also the payload markers. Methods: FFPE tumor tissues from 363 CRC patients were microdissected and solubilized for mass spectrometry-based targeted proteomic analysis in our CLIA certified laboratory. We quantified protein levels of EGFR, HER2, HER3, Axl, Mesothelin, FRalpha, Trop2 (antibody targets), tubulin-beta3 and TOPO1 (payload resistance and response markers, respectively) simultaneously. The multiplexed assay also quantified additional 22 clinically relevant proteins. Results: Expression of EGFR(83%), HER2(52%), HER3(21.5%), Axl(3.7%), Mesothelin(26.5%), FRalpha(3.7%), and Trop2(59.8%) may indicate likely response to ADCs. Expression of TUBB3(+) and TOPO1 (>1350amol/µg) in antibody target-positive subset may suggest resistance or response to payloads, such as taxanes and irinotecan, respectively (Table). Previously we identified that HER2 expression >750amol/µg correlated with HER2 positivity. Accordingly, 1.4% (5/355) of CRC patients were HER2 positive, of which 40% (2/5) had TOPO1 expression >1350amol/µg (75th percentile) suggesting that these 2 patients may receive benefit from a HER2/TOPO1 ADC. (+) indicates expression ≥LOQ; (-) indicates expression <LOQ. Conclusions: In patients with CRC, quantitative proteomics identified both antibody targets and markers of resistance or response to the payloads for multiple approved and investigational ADC therapies.

BiomarkersN of CRC testedPrevalence (potential response rate)HER2(+), TOPO1(>1350)35527.0%HER2(+), TUBB3(-)29818.8%HER3(+), TOPO1(>1350)35512.7%HER3(+), TUBB3(-)29810.1%EGFR(+), TUBB3(-)29632.1%Axl(+), TUBB3(-)2980.3%Mesothelin(+), TUBB3(-)2988.4%FRalpha(+), TUBB3(-)2981.7%Trop2(+), TOPO1(>1350)10219.6%