Abstract
Optimizing co-stimulatory signaling to enhance T cell responses is central to effective antitumor immunity. In this study, we developed single-stranded RNAs (ssRNAs) utilizing the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) to express OX40L, 4-1BBL, and ICOSL and evaluated their efficacy. Co-culture of splenocytes with tumor cells transfected with these ssRNAs resulted in increased cytokine production and proliferation, along with altered T helper (Th) subsets. In vivo, intramuscular delivery of ssRNAs expressing co-stimulatory molecules expanded antigen-specific CD8(+) T cells. Furthermore, intratumoral delivery of these ssRNAs significantly suppressed tumor growth and induced complete tumor regression in a subset of melanoma-bearing mice. Mechanistically, ssRNAs expressing co-stimulatory molecules promoted immune cell infiltration into the tumor site and increased the cytotoxic CD8(+) T cells while reducing regulatory T cells (Tregs) in secondary lymphoid organs. These findings suggest that IRES-based ssRNAs expressing co-stimulatory molecules represent a promising platform for the development of effective cancer immunotherapies.