Bispecific and biparatopic antibodies targeting tumor-specific Tn-glycoepitopes on MUC1 and MUC4 enable broad, selective tumor coverage in epithelial cancers

Altered O-glycosylation in epithelial cancers generates tumor-specific Tn-glycoepitopes that are largely absent from normal tissues. Using advanced O-glycoproteomics, we have leveraged these neoepitopes to create a large suite of highly cancer-specific, humanized monoclonal antibodies targeting Tn-glycoepitopes in tumor-associated proteins. Here, we combine select Tn-targeting mAbs to develop two next-generation antibody-drug conjugates (ADCs): Tn-MUC1 biparatopic (M100A×M100B) and MUC1/MUC4 bispecific (M400×M100B) that broaden tumor coverage, increase avidity, while still maintaining the clean target profile with no reactivity to healthy tissue. Both designs were engineered as site-specific ADCs using an mc-vc-PAB-MMAE linker to generate homogeneous DAR2 conjugates. Biophysical analyses confirmed dual-epitope engagement and enhanced binding avidity: M100A×M100B bound two distinct Tn-MUC1 epitopes with sub-nanomolar affinity, mitigating epitope heterogeneity within MUC1, while M400 × M100B simultaneously recognized MUC1-Tn and MUC4-Tn, improving cross-mucin coverage in heterogeneous tumors. Immunohistochemistry demonstrated broad reactivity across breast, lung, ovarian, pancreatic, and gastrointestinal cancers with negligible binding to normal tissue. In vitro, both ADCs exhibited potent cytotoxicity against dual-positive tumor cell lines and sustained activity in mixed A+/B+ heterogeneity models. In vivo, M400 × M100B-vedotin and M100A × M100B-vedotin achieved tumor regressions comparable or superior to combinations of parental ADCs, while maintaining favorable tolerability in preclinical models. These data establish MUC1 and MUC4-targeted bispecific and biparatopic designs as powerful next-generation therapeutics for overcoming tumor heterogeneity and improving selectivity in solid tumors.