Single-Pot Enzymatic Synthesis Of Cancer-Associated Muc16 O-Glycopeptide Libraries And Multivalent Protein Glycoconjugates: A Step Towards Cancer Glycovaccines

Cancer cells often overexpress and/or express de novo glycoproteins modified with short-chain sialylated O-glycans, sialyl-Tn (STn), sialyl-3-T (S3T) and sialyl-6-T (S6T) antigens, that hold potential for carbohydrate-based vaccines. However, the generation of glycopeptide libraries composed by structurally defined standards remains a critical step for carbohydrate metrology and vaccine development. A simple and fast multi-enzymatic single-pot method was applied for generating a wide array of STn, S3T and S6T glycopeptides. A 20-mer tandem repeat peptide from the cancer-associated glycoprotein MUC16 was used as a scaffold towards this end. Different glycosyltransferases and nucleotide sugars were combined in a stepwise manner to generate the glycopeptide libraries. TiO2 enrichment was further used to isolate sialoglycopeptides, and the glycopeptide libraries were characterized by nanoLC-MS. Multiple glycopeptides were conjugated with protein immunogens, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), foreseeing multivalent glycoepitopes that may pave the way for immunotherapy. We have synthesized MUC16-Tn antigens containing up to 5 glycosites and mapped the main occupied glycosites. We have also successfully synthesized MUC16-STn, S3T and S6T glycopeptides and hybrid MUC16-Tn/STn, MUC16-Tn/S3T, MUC16-T/S6T and MUC16-Tn/T/S6T glycoproteoforms. As proof-of-concept, we have coupled MUC16-Tn/STn glycopeptides to immunogenic proteins. This sets the molecular background for the future development of multivalent glycan-based vaccines exploiting MUC16 glycoepitopes.