Enhancing Cancer Vaccine Efficacy: Backbone Modification with β-Amino Acids Alters the Stability and Immunogenicity of MUC1-Derived Glycopeptide Formulations

Glycopeptides derived from the mucin-1 (MUC1) glycoprotein hold significant promise as cancer vaccine candidates, but their clinical utility is limited by proteolytic degradation and the poor bioavailability of L-α-amino acid-based peptides. In this study, we demonstrate that substitution of multiple α-amino acids with homologous β-amino acids (same side chain, but extended backbone) in <i>O</i>-glycosylated MUC1 derivatives significantly enhances their proteolytic stability. We further show that α-to-β substitutions within the most immunogenic epitope of MUC1 impede binding to an anti-MUC1 antibody, while substitutions outside the same epitope preserve antibody recognition. Structural investigations using circular dichroism, NMR spectroscopy, and molecular dynamics simulations reveal that the strongest α/β-peptide binders retain native-like conformations in the epitope region, both in their unbound state and when bound to the anti-MUC1 antibody. Conjugation of these high-affinity α/β-peptide analogs to gold nanoparticles induces robust immune responses in mice comparable to that of the native glycopeptide. Additionally, these α/β-analogs elicit elevated levels of the cytokine IFNγ, one of the key proteins for tumor cell elimination, surpassing levels produced by the native MUC1 glycopeptide. In contrast, a low-affinity α/β-analogue with lower proteolytic stability produces minimal cytokine responses, underscoring the critical role of these biochemical properties in vaccine efficacy. Collectively, our findings highlight that α-to-β modifications in the peptide backbone offer an effective strategy for developing biostable, highly immunogenic glycopeptide-based cancer vaccines, exemplifying the power of structure-based rational design in advancing next-generation vaccines.