Abstract
BACKGROUND: Dendritic cell-derived exosomes (DEXs) are emerging as effective platforms for cancer vaccines due to their capacity to present tumor antigens and regulate immune responses. Here, we developed a multifunctional DEX vaccine (DEX(AGNP)) that integrates liver cancer epitope presentation, innate immune activation, and checkpoint modulation. Human DEX(AGNP) was generated by loading HLA-A*02:01-restricted hAFP(158-166) and hGPC3(144-152) peptides, and murine DEX(AGNP) by loading mAFP(212-219) and mGPC3(127-136) peptides. The N-terminal functional domain of high-mobility group nucleosome-binding protein 1 (N1ND) was membrane-anchored via CHOL-PEG2000, and programmed death ligand 1 (PD-L1) of DEXs surface was blocked with anti-PD-L1 antibodies to mitigate immunosuppression. RESULTS: DEX(AGNP) vaccine enhanced antigen-specific CD8⁺ T-cell responses, as supported by peptide-MHC tetramer staining, and promoted cytokine-associated effector activity. In vitro, DEX(AGNP)-primed T cells mediated cytotoxicity against both human and murine liver cancer cell lines. In vivo, DEX(AGNP) suppressed tumor growth in an immunocompetent subcutaneous H22 model and a humanized HepG2 xenograft model, and demonstrated antitumor activity in an orthotopic HCC model. In addition, DEX(AGNP) induced measurable killing in ex vivo assays using clinical HCC specimens and, in the prophylactic setting, was associated with enhanced memory-like T-cell responses. CONCLUSION: DEX(AGNP) is a modular exosome vaccine that combines epitope-specific antigen presentation with immune activation and PD-L1 blockade, enabling robust antitumor T-cell responses across complementary liver cancer models. This engineered DEX platform provides a practical blueprint for developing next-generation exosome-based cancer vaccines.