Optimized Epitope-Flanking Sequences Enhance Antigen Presentation and T-Cell Responses Following mRNA Vaccination

mRNA vaccines can encode multiple tumor antigens whose presentation ultimately depends on adequate intracellular processing. To evaluate the potential impact of epitope-flanking sequences, we compared the immunogenicity of two mRNA vaccines in the MC38 mouse model. Both vaccines coded for the same tumor-specific antigens (TSAs). The only difference between the two vaccines was the identity of the epitope-flanking amino acids. The vaccine encoding TSAs bordered by optimized flanking sequences (TSA-Opti) cleared established tumors and elicited robust CD8⁺ T-cell responses. In contrast, the vaccine encoding the same TSAs surrounded by natural flanking sequences (TSA-Nat) induced weaker responses and incomplete protection against tumor growth. Quantitative immunopeptidomics showed that TSA-Opti increased antigen presentation levels, providing a mechanistic link between flanking-sequence design and the enhanced magnitude, clonal diversity, and functionality of TSA-specific CD8+ T cells. TSA-Opti further promoted durable memory, conferring long-term protection against tumor rechallenge. These results identify epitope-flanking sequences as tunable design elements that control peptide-MHC I density and thereby shape the magnitude, clonal diversity, tissue localization, and durability of antitumor CD8⁺ T-cell immunity.