Development and preclinical characterization of an MMAE-conjugated anti-EpCAM antibody–drug conjugate for colorectal cancer

Abstract

The epithelial cell adhesion molecule (EpCAM) is frequently overexpressed in colorectal cancer (CRC) and thus represents an attractive therapeutic target. However, the clinical efficacy of EpCAM-targeting antibodies has been limited by suboptimal binding properties and reliance on immune effector mechanisms. Here, we describe the development and characterization of anti-EpCAM #126, a fully human monoclonal antibody isolated from the OPAL phage-display library, and its corresponding antibody-drug conjugate (ADC). Anti-EpCAM #126 exhibited substantially higher binding affinity than adecatumumab, primarily owing to a markedly lower dissociation rate. High-density peptide microarray analysis and alanine-scanning mutagenesis identified NGSKLGRRAK (residues 74-83) within the EpCAM extracellular domain as the dominant epitope, with S76, R81, and K83 serving as key contact residues. Binding was conformation-dependent and markedly decreased following trypsin-mediated proteolytic cleavage of the epitope region. Anti-EpCAM #126 demonstrated robust staining of CRC xenograft and patient-derived tumor tissues and exhibited low cytokine release in human peripheral blood mononuclear cells and mouse models. Following the confirmation of efficient receptor-mediated internalization, anti-EpCAM #126 was conjugated to monomethyl auristatin E (MMAE) through a protease-cleavable valine-alanine linker, generating an ADC with a controlled average drug-to-antibody ratio of 4.31 while retaining nanomolar EpCAM-binding affinity. The resulting ADC exhibited potent cytotoxicity against EpCAM-expressing CRC cell lines and achieved 84.96% tumor growth inhibition in a patient-derived xenograft model at 10 mg/kg, whereas the unconjugated antibody showed no antitumor activity. These findings suggest that anti-EpCAM #126-MMAE is a high-affinity, epitope-specific ADC with promising preclinical efficacy against EpCAM-positive CRC.