A Synthetic MUC1 Glycopeptide Bearing [beta]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 [beta]GalNAc-SerOBn and [beta]GalNAc-ThrOBn units, respectively, as mimetics of the natural Tn antigen ([alpha]GalNAc-Ser/Thr), along with the solid-phase assembly of the glycopeptides NHAcSer-Ala-Pro-Asp-Thr[[alpha]GalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (3-BSA) and NHAcSer-Ala-Pro-Asp-Thr[[beta]GalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (4-BSA), bearing [alpha]GalNAc-Thr or [beta]GalNAc-Thr units, respectively, as mimetics of MUC1 tumor mucin glycoproteins. According to ELISA tests, immunizations of mice with [beta]GalNAc-glycopeptide 4-BSA induced higher sera titers (1:320000) than immunizations with [alpha]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-[beta]GalNAc antibodies and [alpha]GalNAc-glycopeptide and vice versa was also verified. Lastly, molecular dynamics simulations and surface plasmon resonance (SPR) showed that [beta]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 [beta]GalNAc-Thr as Tn antigen isomer.