Conformational epitope engagement by anti-MSLN ADC RC88 confers resistance to soluble mesothelin

Abstract Background Mesothelin (MSLN) is a therapeutic target for antibody-drug conjugates (ADCs) due to its tumor selective overexpression. However, soluble MSLN (sMSLN) in circulation compromises efficacy by acting as a decoy. RC88 is a clinical stage ADC composed of a humanized anti-MSLN antibody conjugated to a monomethyl auristatin E (MMAE) payload. This study elucidates the unique binding mechanism that allows RC88 to maintain superior efficacy despite sMSLN interference. Methods We characterized the RC88-MSLN interaction via structural analysis and mutagenesis. The impact of sMSLN on binding was assessed using competitive cellular assays. In vitro internalization kinetics and cytotoxicity were evaluated to determine the therapeutic potential. Results RC88 targets a high affinity N-terminal epitope overlapping the MUC16/CA125 binding site, competitively inhibiting this interaction. Notably, due to the structural flexibility of MSLN, RC88 also engages a secondary, low affinity juxtamembrane epitope at the C-terminus. This conformational binding confers resistance to sMSLN interference and enhanced tumor cell retention. In vitro, RC88-ADC maintained near complete cytotoxic potency even in the presence of physiologically relevant sMSLN concentrations. In vivo, RC88 suppressed tumor growth in ovarian cancer xenografts. Conclusion Our findings demonstrate that RC88 utilizes a novel conformational binding strategy to overcome the limitations of soluble antigen interference. These findings provide a mechanistic rationale for the encouraging clinical activity of RC88 and suggest that conformational epitope targeting represents a promising strategy for overcoming sMSLN interference in MSLN-positive cancers.