# IRES-based RNAs expressing co-stimulatory molecules: Promising candidates for cancer immunotherapy

## TL;DR

This study shows that RNA molecules using IRES can boost T cell responses and shrink tumors, offering a new approach for cancer immunotherapy.

## Contribution

The novel use of IRES-based ssRNAs to express co-stimulatory molecules for cancer immunotherapy is introduced.

## Key findings

- ssRNAs expressing co-stimulatory molecules increased cytokine production and T cell proliferation in co-culture experiments.
- Intratumoral delivery of these ssRNAs suppressed tumor growth and induced complete regression in some melanoma models.
- ssRNAs promoted immune cell infiltration and increased cytotoxic CD8+ T cells while reducing Tregs in lymphoid organs.

## 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.

IRES-based RNAs encoding 4-1BB, OX40L, and ICOSL enhanced T cell immunity and suppressed tumor progression by promoting immune infiltration and attenuating T cell exhaustion and regulatory T cell expansion in lymphoid organs, supporting their potential as RNA-based platforms for rationally designed cancer immunotherapy.