Affinity Maturation Enables Dual Targeting of Cancer-Associated MUC1-Tn/STn and Drives Potent Tumor Control

A major challenge in solid tumor immunotherapy is identifying tumor-restricted antigens while overcoming intratumoral antigen heterogeneity. The truncated O-glycans Tn (GalNAcα-O-Ser/Thr) and STn (Neu5Acα2-6GalNAcα-O-Ser/Thr) are aberrantly displayed on the transmembrane glycoprotein MUC1 across a broad spectrum of epithelial malignancies, making MUC1-Tn/STn an attractive shared tumor-associated antigen class. However, heterogeneous intratumoral Tn/STn expression renders single-glycoform-specific antibodies vulnerable to antigen-driven escape. To address this challenge, we used structure-guided directed evolution and yeast surface display to affinity-mature the anti-MUC1-Tn antibody 5E5, generating variants with high-affinity dual recognition of MUC1-Tn and MUC1-STn. Co-crystal structures revealed that heavy-chain CDR loops cooperatively engage the GalNAc moiety primarily through hydrogen-bonding networks, in which HCDR3 residue 98 fine-tunes glycoform preference: Tyr98 favors MUC1-Tn recognition, whereas His98 enhances MUC1-STn engagement, while both support high-affinity recognition of both glycoforms. When reformatted as bispecific T-cell engagers, the affinity-matured variants mediated potent and durable cytolytic activity against cancer cells with heterogeneous glycan profiles, suppressed mixed Tn/STn tumors in vivo where the parental antibody failed, and showed enhanced activity in patient-derived tumor-like clusters. Together, these findings establish functional glycoform coverage as a design principle for antibody engineering, whereby a single paratope is optimized to recognize a defined spectrum of tumor-associated glycoforms while preserving antigen specificity, providing a strategy to overcome glycan heterogeneity in solid tumor immunotherapy.