A dual-switch safety system enables safety controllability and high-efficiency enrichment for T cell immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has achieved durable and complete responses in a substantial proportion of patients with B cell malignancies. However, during CAR-T cell therapy, the occurrence of adverse events-including on-target/off-tumor toxicity against healthy tissues, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and the risk of T cell malignancies arising from random genomic integration of viral vectors-significantly threatens patient safety. In this study, we developed a novel truncated HER2 (tHER2)-herpes simplex virus thymidine kinase (HSVTK) dual-switch safety system and site-specifically integrated it into the PDCD1 locus using a Cas12a/adeno-associated virus (AAV)-mediated delivery platform. Importantly, this dual-switch system enables efficient magnetic bead-based enrichment of tHER2-HSVTK (tHER2-TK)-positive T cells, allows tunable modulation of engineered T cell persistence through Herceptin or the small-molecule drug ganciclovir (GCV), and also provides a highly effective purification method before infusion. Collectively, these features enhance safety without compromising therapeutic efficacy, facilitating the development of safer and more controllable engineered T cell immunotherapies.