A modular synthetic DNA template platform for rapid generation of TCR-encoding mRNA and streamlined validation of cognate TCR–epitope reactivity

The interaction between T-cell receptors (TCRs) and their cognate ligands plays an important role in the regulation of the adaptive immune system as this is key in inducing immune responses in cancer, infectious and autoimmune diseases. As identification of a cognate TCR relies on laborious experimental validation, we propose the use of a synthetic DNA template assembly approach for the rapid generation of TCR encoding in vitro transcribed (IVT) mRNA, enabling mRNA-based screening. We show antigen-specific T-cell activation of human CD8 + T cells after delivery of TCR encoding mRNA and coculture with antigen presenting cells, presenting the cognate antigen/human leukocyte antigen (HLA) complex. Additionally, we demonstrated functionality of CD8 + T cells expressing an mRNA delivered, MAGEA1/HLA-A*02:01 cognate TCR, showing in vitro killing of MAGEA1 + HLA-A*02:01 + non-small cell lung cancer cells. This work showcases a fast platform for TCR-mRNA generation, facilitating TCR functionality and reactivity validation in personalized in vitro screening assays.