Structure-guided phage display discovery of antibodies for (S)Tn-glycans in protein context

Developing high-affinity monoclonal antibodies (mAbs) against tumor-associated carbohydrate antigens such as Tn and STn on carrier proteins remains a major challenge in cancer therapy. These antigens, expressed as glycan–peptide epitopes (combotopes), require precise recognition for high specificity. Through structural studies, we found that V_H domains of certain antibodies primarily recognize glycans, whereas V_L domains bind peptide sequences. Using these insights, we developed a V_H-focused and V_L-diverse phage display library to discover mAbs with combotope-binding characteristics. Notably, structural analysis enabled us to convert Tn-specific mAbs into STn-specific mAbs through modification of V_H complementarity-determining region 3, demonstrating the versatility of this approach. Our hypothesis was validated with glycoprotein targets MUC1 and CD43, yielding antibodies with high specificity and affinity. Furthermore, internalization studies using the parental antibody scaffold show efficient uptake by tumor cells, supporting its use in antibody–drug conjugates. This platform addresses the challenge of generating glycoform-specific antibodies for cancer therapy.