Genetic Code Expanded T Cell for Controllable Immunotherapy.

Chimeric antigen receptor (CAR)-T cell therapy has demonstrated curative potential against hematologic malignancies, but its clinical application remains constrained by the risk of uncontrolled immune activation. To address this, we engineered a translational control system for CAR expression based on Genetic Code Expansion (GCE), enabling tight, dose-dependent, and function-preserving regulation through nonsense codon suppression via noncanonical amino acids (ncAAs). By introducing amber stop codons into CAR constructs and engineered aminoacyl-tRNA synthetase and tRNA pair, we developed a leak-free regulatory module applicable in both Jurkat and primary human T cells. NcAA-treated GCE-CAR-T cells exhibited antigen-specific cytotoxicity and cytokine secretion comparable to wild-type CAR-T cells. In a xenograft mouse model, tumor-specific immune responses were observed only upon ncAA administration, with untreated controls showing no therapeutic effect. This work establishes a stringent, fast-acting translational switch that enables precise modulation of CAR-T cell function without compromising efficacy, offering a promising platform for next-generation programmable cell therapies.