Endolysosomal routing combined with immune stimulation enhances anti-tumor efficacy of an HPV16 mRNA therapeutic vaccine

Despite the widespread use of prophylactic vaccines, Human papillomavirus (HPV) related cancers persist as a substantial global health burden, highlighting the high demand for effective therapeutics. Signal peptide (SP) and major histocompatibility complex (MHC) class I trafficking domain (MITD) have been utilized in HPV messenger RNA (mRNA) therapeutic vaccines to improve MHC class I and II presentation. However, how antigen-processing modules (ubiquitin [Ub], SP-MITD, lysosome-associated membrane protein [LAMP]) and immune stimulators (granulocyte-macrophage colony-stimulating factor [GM-CSF], stimulator of interferon [IFN] genes [STING], CD4+ T cell epitopes) functionally interact within a single mRNA vaccine remains unexplored. Here, we identified an optimized structural configuration for HPV16 mRNA therapeutic vaccines that exhibited superior efficacy within our TC-1 murine models under the present conditions: SP-MITD-containing constructs were associated with greater anti-tumor efficacy than Ub-containing constructs; two distinct immune modulators—GM-CSF and STING—elicited comparable magnitudes of IFN-γ responses when integrated into the SP-MITD backbone; pan-HLA-DR binding epitope (PADRE) was selected among a panel of CD4+ T cell epitopes based on IFN-γ responses in PBMCs and previous clinical experiences, and showed a trend toward improved anti-tumor activity when added to the SP-MITD-GM-SCF construct. These findings guided the development of ABO-208, a lipid nanoparticle-encapsulated therapeutic mRNA vaccine targeting HPV16 E6-E7, incorporating SP-MITD, GM-CSF and PADRE. In preclinical models, ABO-208 elicited robust antigen-specific T cell responses and effectively inhibited both small (∼50 mm 3 ) and large (∼420 mm 3 ) tumors in TC-1 murine models. Furthermore, ABO-208 showed prophylactic potential, making it a promising candidate for HPV-related treatment and prevention under the current experimental setup. This potent efficacy highlights the potential value of the optimized structural integration evaluated in this study.