Antibody-drug conjugates targeting EGFR ligand Epiregulin inhibit colorectal tumor growth irrespective of RAS mutational status

Purpose: The EGFR ligand, epiregulin (EREG), is highly expressed in colorectal cancers (CRCs) and a biomarker for predicting benefit in RAS wildtype patients receiving EGFR-targeted therapies. Here, we report the development and preclinical evaluation of EREG-targeted antibody-drug conjugates (ADCs) incorporating diverse chemical linkers and duocarmycin DM (DuoDM) payload in CRC models of different mutational status and subtypes. Experimental Design: RNA-seq datasets from different patient cohorts were analyzed for EREG expression. EREG monoclonal antibody, H231, was characterized for specificity, affinity, internalization, and biodistribution and tumor uptake using 89Zr-immunoPET. H231 was conjugated to a series of cleavable dipeptide and tripeptide linker-DuoDM payloads and cytotoxicity of EREG ADCs were assessed in a panel of CRC cell lines. Safety, antitumor efficacy, and survival was evaluated in vivo. Results: EREG was high in both RAS mutant and wildtype tumors and inversely associated with microsatellite instability. 89Zr-immunoPET showed significant tumor uptake of H231 with minimal uptake in normal tissues. EREG ADCs incorporating tripeptide linkers demonstrated highest potency in EREG-expressing CRC cells (IC50s = 0.01-0.50 nmol/L). EREG ADCs were safe and well-tolerated at doses tested and showed significant tumor growth inhibition with increased survival in patient-derived tumor xenograft models. Conclusions: EREG is a promising target for the development of ADCs for treating colorectal and other tumor types that express high levels of EREG. Importantly, EREG ADCs demonstrated significant therapeutic efficacy in both RAS mutant and wildtype CRC cell lines and tumors, suggesting their potential as an alternative to EGFR-targeted therapy to benefit a broader patient population. ### Competing Interest Statement Y.A. and K.T. are named inventors on all or some of the patent applications (WO2018218004A1, US11629122B2, EP3630189A4 and WO2023122587A3) relating to the linker technologies described in this article. K.T. is a co-founder of and holds equity in CrossBridge Bio.