Abstract

Glypican-3 (GPC3) is a sought-after immunotherapeutic target for hepatocellular carcinoma (HCC) due to its tumor-specific expression. However, GPC3-derived peptide vaccines suffer from limited epitope coverage and weak immunogenicity. Herein, we developed a dual-adjuvanted GPC3 mRNA vaccine delivered by local-retention lipid nanoparticles (LNPs). Through rational ionizable lipids screening, we engineered L1-based lipid nanoparticles (L1-LNPs) that achieved 4-fold greater local expression than ALC-0315-LNPs with reduced hepatic accumulation. A fusion mRNA construct (HGPC3C) encoding GPC3 flanked by two molecular adjuvants of HSP70 peptide and complement C3d was further designed. Mechanistic analyses suggested that HSP70 was associated with enhanced cellular immune responses, including elevated IFN-γ and IL-12p70, whereas C3d was associated with enhanced humoral immune responses, including increased IL-6 and anti-GPC3 antibody titers, together supporting coordinated activation of both immune arms. In prophylactic models, L1-LNPs/HGPC3C mRNA achieved 80.4% tumor suppression. In therapeutic settings, L1-LNPs/HGPC3C (59.9%) significantly outperformed ALC-0315-LNPs and non-adjuvanted formulations, validating the coordinated advantages of local delivery and dual-adjuvant design. Its combination with PD-L1 blockade further increased suppression to 78.3%, with enhanced CD8 + T cell infiltration and antibody production. Collectively, these findings establish L1-LNPs/HGPC3C mRNA as a promising immunotherapeutic approach for HCC treatment and its combination with immune checkpoint blockade holds clinical translational potential.