Messenger (m)RNA vaccines are currently being developed for a range of diseases including cancer. Tumour-targeting mRNA vaccines that express tumour antigens need to activate antigen-presenting cells (APCs) to stimulate a tumour-killing immune response. However the tumour microenvironment is immunosuppressive inhibiting this. Toll-like receptor 2/6 (TLR2/6) agonists such as Pam2CSK4 have been shown to enhance antibody titres and T cell responses in viral vaccine models, and TLR2/6-stimulating drugs enhance anti-tumour immunity making them promising candidates for boosting mRNA cancer vaccine potency.
This project investigates whether co-delivery of a TLR2/6 agonist can enhance mRNA-expressed antigen uptake and expression. We are using a lipid nanoparticles (LNPs) to deliver GFP mRNA with (or without) Pam2CSK4 to test the hypothesis that co-formulation of Pam2CSK4 in LNPs does not compromise mRNA expression whilst boosting APC activation.
To determine whether TLR2/6 stimulation can enhance antigen presentation, dose-response experiments were conducted on a human monocytic cell line (THP-1) and a mouse macrophage cell line (RAW 264.7) treated with Pam2CSK4. In parallel, optimisation of GFP-LNPs with various ionisable lipids was completed with SM-102-based LNPs achieving good mRNA uptake and expression. GFP-mRNA LNPs formulated with and without Pam2CSK4 showed comparable physicochemical properties (Z-average 194nm Vs. 189nm; PDI 0.19 Vs. 0.18) and high mRNA encapsulation efficiency (>90%), indicating that agonist incorporation does not compromise LNP mRNA formulation. Flow cytometry confirmed successful mRNA delivery to RAW 264.7 cells regardless of formulation with Pam2CSK4, with 70–80% GFP-positive cells and comparable GMFI, demonstrating that Pam2CSK4 incorporation during LNP mRNA formulation does not impair mRNA delivery or expression; LNP uptake and GFP expression were similarly confirmed in both cell types by fluorescence microscopy. Pam2CSK4-containing LNPs also induced expression of IL-6 and TNF-α by both cell lines, indicating cross-species innate immune activation. TLR2/6 stimulation also upregulated CD80 and HLA-DR, consistent with enhanced APC activation .
Future work will assess Pam2CSK4-induced dendritic cell maturation and evaluate antigen-specific T cell responses following in vivo immunisation, with the goal of testing this platform in a tumour challenge model. Together, these findings support TLR2/6 stimulation as a promising strategy for enhancing the potency of mRNA cancer vaccines.