Engineering "off-the-shelf" TCR-T cells: A transient mRNA platform for balanced alloreactivity and functionality.

Adoptive T cell therapy using T cell receptor (TCR)-engineered T (TCR-T) cells shows promise for the treatment of cancer. Our lab employs mRNA electroporation to redirect T cell specificity toward HBV antigens expressed on HBV-related HCC, while exploiting mRNA's transient nature to limit toxicity. Here, we first demonstrated that peripheral blood mononuclear cells (PBMCs) from solid cancer patients are inferior manufacturing sources compared with healthy donors, showing reduced manufacturability and lower naive T cell frequencies. Since these limitations necessitate shifting to allogeneic "off-the-shelf" TCR-T products from healthy donors, we developed a non-gene-editing platform to produce such TCR-T cells. We addressed host-versus-graft rejection by combining transient tacrolimus-induced immunosuppression with immunosuppressive drug-resistant armored (IDRA) TCR-T cells. To reduce the risk of graft-versus-host disease, we optimized manufacturing using an IL-2/4/7 cytokine cocktail. The resulting TCR-T cells exhibited reduced alloreactivity, a less NK-like phenotype, and elevated IL-4/IL-10 secretion, while maintaining robust antigen-specific cytotoxicity and migratory capacity. This study provides an in vitro proof-of-concept for a feasible, multi-layered strategy to produce allogeneic TCR-T cell therapies, warranting further preclinical validation.