CD63-Mediated SARS-CoV-2 RBD Fusion Neoantigen DNA Vaccine Enhances Antitumor Immune Response in a Mouse Panc02 Model via EV-Targeted Delivery.

BACKGROUND: Although DNA vaccines offer a flexible platform for tumor immunotherapy, their weak immunogenicity remains a key limitation. This study aimed to improve the immunogenicity of DNA vaccines by enhancing the efficiency of tumor neoantigen delivery through extracellular vesicles (EVs), thereby promoting stronger dendritic cell (DC) activation and antitumor responses. METHODS: A novel DNA vaccine (pCSP) was engineered by fusing tumor-specific neoantigens to the EV-associated protein CD63 and incorporating a SARS-CoV-2 receptor-binding domain (RBD) fragment to facilitate EV uptake by DCs. The resulting EVs were expected to carry neoantigens into the immunoproteasome for major histocompatibility complex I (MHC-I) presentation. The immunological and antitumor effects of pCSP were assessed through in vitro functional assays and in vivo experiments in a murine pancreatic cancer model. Safety was evaluated through histological and biochemical analyses. RESULTS: In vitro, pCSP significantly promoted EV internalization by DCs by approximately twofold and enhanced their immune activation, as evidenced by elevated cytokine production. In vivo, pCSP markedly suppressed tumor growth with a decrease in volume by over 70% relative to controls, boosted CD8+ T cell responses, and increased immune infiltration into the tumor microenvironment. Safety assessments revealed that while liver/kidney function markers were within physiological ranges, mild inflammatory infiltrates were consistently observed in the lungs, indicating a localized safety concern that warrants further monitoring. CONCLUSIONS: The pCSP vaccine enhances the immunogenicity of neoantigen DNA vaccines by improving EV uptake and immune activation in DCs. These findings provide a potential strategy for improving DNA vaccine efficacy in the context of cancer immunotherapy while maintaining acceptable safety.