Next-generation RNA vaccine modalities enable high-fidelity antigen discovery and T-cell immunogenicity screening

RNA-based cancer vaccines provide exceptional flexibility for encoding tumour-associated antigens (TAAs) or patient-specific neoantigens and recent clinical data show promising efficacy over traditional approaches, yet comparative functional data across RNA formats remain limited. We developed an integrated evaluation workflow to benchmark linear mRNA, circular RNA (circRNA), self-amplifying RNA (saRNA), and 27-mer peptide vaccines for their ability to drive antigen expression and prime functional antigen-specific T-cell responses. Using GFP reporter constructs, expression levels and persistence were assessed across selected RNA formats in primary antigen-presenting cells. Linear mRNA and circRNA robustly produced higher and more durable antigen expression than peptide pulsing, consistent with emerging insights into RNA vaccine potency. Vaccine candidates were subsequently screened using an antigen-specific T-cell expansion workflow, which expands rare tumour-reactive CD8+ T cells via a mature dendritic cell (DC) co-culture system. This platform addresses challenges of low precursor frequency and HLA diversity, enabling de novo priming of T cells against TAAs such as MART-1 or unique neoantigens. In the majority of donors, RNA-encoded antigens elicited stronger T-cell responses than 27-mer peptides, reflected by greater expansion of antigen-specific CD8+ T cells and increased IFNgamma release following restimulation. Functional killing was confirmed using peptide-loaded tumour targets and tumour cell lines endogenously expressing relevant-antigen. RNA-encoded antigens produced T cells with higher cytotoxic potential, evidenced by enhanced tumour apoptosis in killing assays. Crucially, the strongest response seen was with a circRNA neoantigen vaccine candidate relative to the same neoantigen encoded in linear mRNA or peptide formats. To support personalised vaccine programs, putative neoantigens were first validated using TAP-deficient MHC-loading assays to confirm direct presentation, then screened in the DC-T-cell co-cultures to generate donor-resolved immunogenicity hierarchies. This enabled rapid identification of high-value epitopes suitable for inclusion in bespoke mRNA or circRNA vaccine constructs. Collectively, these data demonstrate that RNA vaccine modalities can outperform peptide-based vaccination in generating functional tumour-reactive T cells. The integrated platform provides a modality-agnostic, human-relevant workflow spanning antigen selection, RNA vaccine characterisation, and comprehensive functional T-cell assessment, supporting rapid advancement of next-generation cancer vaccine pipelines.