Sequential CD2/CD3/CD28 activation improves nonviral large-payload chimeric antigen receptor knock-in at the T cell receptor α constant locus.

CRISPR/Cas9-mediated nonviral targeted integration via homology-directed repair (HDR) offers a strategy for generating uniform, next-generation chimeric antigen receptor (CAR) T cells while avoiding risks associated with viral vectors. However, efficient delivery of large therapeutic payloads into primary T cells is limited by low HDR efficiency and electroporation-induced toxicity. Here, we show that sequential CD2/CD3/CD28 activation improves HDR-mediated large-payload knock-in at the T cell receptor α constant (TRAC) locus and increases viable CAR-T cell yield under an AZD7648-supported nonviral editing framework. Transcriptomic profiling and mitochondrial membrane potential analysis suggest that this condition is associated with cell-cycle- and metabolism-related programs, which may contribute to improved tolerance to electroporation stress and processing of large DNA templates. Functionally, engineered nonviral TRAC-CD19.CAR-T cells exhibit antigen-specific cytotoxicity in vitro and suppress leukemia progression in an NSG xenograft model, with antitumor activity that approached that of a lentiviral CAR-T reference in this proof-of-function xenograft model. This work establishes an optimized sequential activation strategy to improve nonviral, large-payload TRAC-targeted CAR-T cell engineering and may inform future development of precision cellular immunotherapy manufacturing workflows.