An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

A synthetic DNA template for fast manufacturing of versatile single epitope mRNA.

A flexible, affordable, and rapid vaccine platform is necessary to unlock the potential of personalized cancer vaccines in order to achieve full clinical efficiency. mRNA cancer vaccine manufacture relies on the rigid sequence design of multiepitope constructs produced by laborious bacterial cloning and time-consuming plasmid preparation. Here, we introduce a synthetic DNA template (SDT) assembly process, which allows cost- and time-efficient manufacturing of single (neo)epitope mRNA. We benchmarked SDT-derived mRNA against mRNA derived from a plasmid DNA template (PDT), showing that monocyte-derived dendritic cells (moDCs) electroporated with SDT-mRNA or PDT-mRNA, encoding HLA-I- or HLA-II-restricted (neo)epitopes, equally activated T cells that were modified to express the cognate T cell receptors. Furthermore, we validated the SDT-mRNA platform for neoepitope immunogenicity screening using the characterized HLA-A2-restricted neoepitope DHX40B and four new candidate HLA-A2-restricted melanoma neoepitopes. Finally, we compared SDT-mRNA with PDT-mRNA for vaccine development purposes. moDCs electroporated with mRNA encoding the HLA-A2-restricted, mutated Melan-A/Mart-1 epitope together with TriMix mRNA-generated high levels of functional Melan-A/Mart-1-specific CD8+ T cells. In conclusion, SDT single epitope mRNA can be manufactured in a more flexible, cost-efficient, and time-efficient way compared with PDT-mRNA, allowing prompt neoepitope immunogenicity screening, and might be exploited for the development of personalized cancer vaccines.