Chemoenzymatic Synthesis of Glycopeptide Library Decodes Sialylation‐Dependent Immunodominance to Enable a Potent Multicomponent Antitumor Vaccine

Aberrant sialylation is a hallmark of tumor glycosylation that promotes malignant progression and immune evasion. While sialylated carbohydrates represent attractive targets for anticancer vaccines, structural and antigenic insights into glycopeptides are still hampered by limited access to well-defined structures, rendering their immunogenicity unclear. Herein, we describe an efficient chemoenzymatic platform for facile synthesis of a glycopeptide library comprising 18 structures with diverse O-glycans at different glycosylation sites. Glycopeptide microarray-based serological screening revealed a consistent preference of cancer patient sera for sialylated glycopeptides over their non-sialylated counterparts. Motivated by this finding, we performed systematic immunological evaluations of sialylated and non-sialylated glycopeptide-tetanus toxoid (TT) conjugates, demonstrating that sialylated glycopeptide-TT conjugates elicit stronger antibody responses, enhanced tumor cell recognition, and more potent tumor growth inhibition through synergistic activation of antigen-presenting cells and T cell subsets. Furthermore, we developed a multicomponent cocktail vaccine by incorporating four sialylated glycopeptides, which elicited broader and more potent immune activation than single-antigen formulations. Antisera from the cocktail vaccine exhibited strong binding to human tumor tissues, underscoring the clinical relevance of our findings. This work establishes sialylation as a key determinant of glycopeptide immunogenicity and provides a rational strategy for developing next-generation glycopeptide-based cancer vaccines.