Despite the success in infectious diseases, mRNA vaccines have inherent limitations in stability and immunogenicity. Non-small cell lung cancer remains the leading cause of cancer-related death worldwide, with poor prognosis in advanced stages, underscoring the urgent need for novel therapeutic strategies. Previously, we have developed a scarless circular RNA synthesis platform (NeoAna) that exhibited enhanced stability and long-lasting antigen expression with minimal cytotoxicity compared to mRNA. In this study, we construct a circRNA vaccine using NY-ESO-1 as the antigen and deliver circRNA vaccine with lipid nanoparticles. The circRNA vaccine elicited robust innate and adaptive immune responses both in vitro and in vivo. It demonstrated superior antitumor efficacy relative to mRNA vaccine, achieving 93.1% tumor inhibition when combined with immune checkpoint blockade. Prophylactic vaccination prevented tumor establishment, while adjuvant therapy conferred protection against tumor re-challenge, highlighting durable immune memory. Mechanistically, circRNA vaccine promoted dendritic cell maturation and enhanced tumor infiltration of T cells, and mediated antitumor immunity in a CD8+ T cell-dependent manner. Additionally, single-nucleus RNA sequencing revealed that it remodeled the tumor microenvironment toward a pro-inflammatory and antitumor phenotype. Following circRNA vaccination, the proportions of cytotoxic T cells, pro-inflammatory macrophages, and antigen-presenting cancer-associated fibroblasts were all increased within the tumor microenvironment, accompanied by activation of immunity-related signaling pathways. In conclusion, our findings demonstrate the immunogenicity and therapeutic potential of the NeoAna circRNA-based therapeutic vaccine, supporting its further evaluation in clinical trials.
