Structure-guided phage display discovery of antibodies for (S)Tn-glycans in protein context
Ramon Hurtado-Guerrero*, Spyridon Gatos, Irene Ginés-Alcober, Javier Macías-León, Andrés Manuel González-Ramírez, Ilknur Kasapoglu, Billy Veloz, Ismael Compañón, Mattia Ghirardello, Pedro Merino, Francisco Corzana & Ola Blixt*. Nature Chemical Biology, 2025. DOI; 10.1038/s41589-025-01971-8. * joint corresponding authorship
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 VH domains of certain antibodies primarily recognize glycans, whereas VL domains bind peptide sequences. Using these insights, we developed a VH-focused and VL-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 VH 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.
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 VH domains of certain antibodies primarily recognize glycans, whereas VL domains bind peptide sequences. Using these insights, we developed a VH-focused and VL-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 VH 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.