A Synthetic MUC1 Glycopeptide Bearing GalNAc-Thr as a Tn Antigen Isomer Induces the Production of Antibodies against Tumor Cells

This study presents the synthesis of the novel protected O-glycosylated amino acid derivatives 1 and 2, containing GalNAc-SerOBn and GalNAc-ThrOBn units, respectively, as mimetics of the natural Tn antigen (GalNAc-Ser/Thr), along with the solid-phase assembly of the glycopeptides NHAcSer-Ala-Pro-Asp-Thr[GalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (3-BSA) and NHAcSer-Ala-Pro-Asp-Thr[GalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (4-BSA), bearing GalNAc-Thr or GalNAc-Thr units, respectively, as mimetics of MUC1 tumor mucin glycoproteins. According to ELISA tests, immunizations of mice with GalNAc-glycopeptide 4-BSA induced higher sera titers (1:320000) than immunizations with GalNAc-glycopeptide 3-BSA (1:40000). Likewise, flow cytometry assays showed higher capacity of the obtained anti-glycopeptide 4-BSA antibodies to recognize MCF-7 tumor cells. Cross-recognition between immunopurified anti-GalNAc antibodies and GalNAc-glycopeptide and vice versa was also verified. Lastly, molecular dynamics simulations and surface plasmon resonance (SPR) showed that GalNAc-glycopeptide 4 can interact with a model antitumor monoclonal antibody (SM3). Taken together, these data highlight the improved immunogenicity of the unnatural glycopeptide 4-BSA, bearing GalNAc-Thr as Tn antigen isomer.