mRNA vaccine encoding neoantigen for cancer immunotherapy using mouse melanoma as a model

Abstract

Cancer is the leading cause of death globally with an urgent need to find more effective approaches for prevention and treatment. mRNA vaccine is a promising vaccine platform over conventional vaccines in terms of safety, ease of large-scale production and effectiveness in inducing both cellular and humoral immune responses. Breakthrough in mRNA technology allowed novel strategies to improve mRNA stability and efficacy including nucleoside modifications. Modification of mRNA with N1-methylpseudouridine (m1Ψ) was reported to enhance protein expression and decrease innate immunogenicity due to low binding affinity to pattern recognition receptors, and low type I interferon (IFN-I) secretion. Therefore, this study aimed to evaluate the immunogenicity and anti-tumor responses of different degrees of m1Ψ replacement in mRNA vaccine following intramuscular administration in B16 melnamoma mouse model. Moreover, non-dominant neoantigens, fusion of Pbk and Actn4 somatic mutations of B16F10 tumor were selected for investigation as neoantigens in mRNA vaccine. First, the translation efficiency of mRNA vaccines depended on the degree of nucleoside modification and inversely correlated with induction of IFN-I secretion and antigen-presenting cell maturation. Immunization of ovalbumin encoding mRNA formulated in lipid nanoparticles (LNPs) (OVA-LNP) with unmodified nucleosides stimulated both Th1 and Th2 responses, whereas modified mRNA induced mainly Th1 response. Both conditions however equally activated OVA-specific cytotoxic effector T cells. Second, unmodified mRNA OVA-LNP showed potent antitumor efficacy and lung metastasis inhibition, accompanied by a significant increase in the influx of mature migratory cDC1 to the tumor. In addition, while both unmodified and modified mRNA showed a significant expansion of OVA-specific splenic CD8 effector T cells, modified mRNA showed a significant increase in PD-1+ CD8+ T cells. Third, the role of IFN-I signaling in anti-tumor response induced by mRNA vaccine administration was investigated by IFN-I receptor blockade. Intact IFN-I signaling resulted in a significant tumor growth suppression and increase in OVA specific IFN-γ-producing T cells with decreased PD-1 expression and less tumor-infiltrating M2-like macrophages. Lastly, LNPs containing neoantigens (Pbk-Actn4) encoding mRNA was immunogenic especially in unmodified mRNA. This neoantigen mRNA-LNP effectively controlled B16F10 tumor growth. Taken together, this study showed that intramuscular administration of unmodified nucleoside mRNA vaccine exhibits higher therapeutic antitumor effects in type I IFN-dependent manner. Presently, lipid nanoparticles (LNPs) are often used as nucleic acid carriers due to greater payload and design flexibility. It facilitates rapid uptake and mRNA escapes to the cytoplasm. In this study, we formulated two different formulas of LNPs and complexed with plasmid DNA (pDNA) at varied N/P ratio (1:1, 1:3, 1:5, 1:7, 1:9, 3:1, 5:1, 7:1, 9:1). The first formula LNP consisted of cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1:1 mole ratio. The second formula LNP consisted of DOTAP, DOPE and Lipid-anchored polyethyleneglycol at 50:49.25:0.75 mole ratio. The LNP composition and the N/P ratio had an effect on particle size and charge. Both formulas showed non-toxic effects on RAW264.7 cell line and facilitated antigen transfection and protein translation in RAW264.7 cell line.