Herein, we report the development of a novel bivalent neoantigen vaccine against hepatocellular carcinoma (HCC) that integrates the technologies of lipid nanoparticles (LNPs) and messenger RNA (mRNA) vaccines. A spleen-homing LNP formulation, CL15H6 LNPs, was used to deliver mRNAs encoding for the epitopes of HCC-specific neoantigens; Alpha fetoprotein (AFP) and Glypican 3 (GPC3), to splenic dendritic cells (DCs) via intravenous administration. The mRNA delivery efficiency and subsequent cellular immune responses were assessed post-administration. The prophylactic and therapeutic activities were then evaluated against HCC challenges in mice, and the biosafety of the developed vaccine was evaluated through histo-pathological, serological, and cytokine examinations. CL15H6 LNPs demonstrated functional mRNA delivery efficiency, antigen presentation efficiency, and expression of co-stimulatory molecules on splenic DCs that proved superior to that of clinically relevant formulations, and also elicited a higher proportion of antigen-specific cytotoxic T lymphocytes (CTLs). Pre-immunization with two doses of the developed bivalent vaccine completely protected mice from HCC challenge, unlike the corresponding monovalent vaccines. Moreover, treatment of HCC-bearing mice with two doses of the bivalent vaccine successfully eliminated HCC. Characterization of the immune microenvironment in the spleen post-treatment revealed a high upregulation of CTL-related genes, immunostimulatory cytokines, and memory function markers. Furthermore, repeated administrations of the developed vaccine induced less pro-inflammatory cytokines than a clinically relevant counterpart, and neither damaged tissues nor impaired organ functions. The novel neoantigen vaccine and the versatile mRNA delivery platform reported herein are promising for use in clinical applications.
