Non-Viral Engineering of Human iPSCs to Manufacture Rejuvenated TCR T Cells for Cancer Immunotherapy
Engineered, human induced pluripotent stem cells (iPSCs) offer a replenishable source to differentiate large numbers of T cells for cancer immunotherapy applications. However, the transduction efficiency of iPSCs by retroviral and lentiviral vectors is suboptimal, and there are safety concerns owing to the presence of viral gene products. Therefore, we explored using a non-viral, cGMP compliant, electroporation platform to transfect TCR-expressing piggyBac® transposons into human iPSCs. Three promoters were evaluated for optimal expression of a surface marker, and iPSCs were analyzed 14 days after electroporation by flow cytometry to determine the transposition efficiency. Remarkably, up to ~47% of cells expressed the surface marker protein without selection. Subsequently, iPSCs were electroporated to compare several TCR molecules. TCR-expressing iPSCs were differentiated into T cells and then evaluated for cytotoxic activity when co-cultured with peptide-pulsed target cells. Potent antigen-specific cytotoxicity was observed. Finally, iPSC-derived T cells expressing several TCR candidates were injected multiple times into mice engrafted with a luciferase-expressing liver cancer cell line. Significant tumor reduction was observed by Day 23 after the initial tumor cell introduction. These results demonstrate efficient TCR DNA transposition into iPSCs, followed by differentiation into T cells with functional activity both in vitro and in vivo.