A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

Background Human papillomavirus (HPV) infection is strongly associated with multiple malignancies, primarily driven by the viral oncoproteins E6 and E7, which play a central role in HPV-induced malignant transformation. Although current prophylactic HPV vaccines have shown remarkable efficacy in preventing initial infections, there remains an urgent need for therapeutic vaccines targeting pre-existing HPV infections and HPV-associated malignancies. Methods In this study, we developed TriStim-E6/E7, a novel HPV mRNA vaccine that combines E6/E7 antigens with three T cell co-stimulatory molecules (CD80, 4-1BBL, and CD70). The TriStim-E6/E7 mRNA was encapsulated in lipid nanoparticles (LNPs) and administered intramuscularly to mice, followed by analysis of cellular immunogenicity. The anti-tumor efficacy of TriStim-E6/E7 mRNA was evaluated using an HPV-positive TC-1 tumor model. In vitro T cell activation and proliferation assays were conducted to investigate the mechanistic underpinnings of the vaccine. Additionally, safety was assessed in a PBMC-reconstituted mouse model. Results Administration of TriStim-E6/E7 mRNA elicited robust antigen-specific cellular immune responses and achieved complete regression of HPV-positive TC-1 tumors in a syngeneic mouse model. The vaccine also demonstrated durable immunoprotection, preventing tumor recurrence upon rechallenge. Through targeted T cell depletion experiments, we established that CD8 + T cells are indispensable for the vaccine’s anti-tumor activity, whereas CD4 + T cell depletion had no significant impact on therapeutic outcomes. Treatment with TriStim-E6/E7 mRNA significantly induced the proliferation and tumor infiltration of E7 antigen-specific T cells. Mechanistic investigations revealed that TriStim-E6/E7 mRNA significantly enhanced T cell activation and proliferation in vitro compared to control mRNAs lacking co-stimulatory elements. Furthermore, combination therapy with anti-PD-L1 antibody synergistically enhanced the anti-tumor efficacy of TriStim-E6/E7 mRNA. To evaluate clinical translatability, we developed a humanized version (hTriStim-E6/E7) encoding human CD80, 4-1BBL, and CD70 co-stimulatory molecules together with HPV E6/E7 fusion proteins. This humanized construct effectively induced antigen-specific cellular immune responses and suppressed tumor growth in CD28/4-1BB/CD27 triple-humanized mice. Additionally, a “cytokine release storm” experiment using PBMC-reconstituted mice confirmed a favorable safety profile of TriStim-E6/E7 mRNA. Conclusions These findings collectively demonstrate the promising therapeutic potential of TriStim−E6/E7 mRNA for clinical translation against HPV−associated malignancies.