Generation of TCR KO CAR T cells. (A) Schematic overview of the time points for retroviral transduction, CRISPR/Cas9-mediated TCR KO, duration of cultivation, and purification of the final CAR–T-cell product. (B) Structure of the CAR construct: the second-generation CAR consists of the anti-CD19 scFv region, followed by the myc tag, CD8 extracellular (EC), transmembrane (TM), and intracellular (IC) domain, as well as a costimulatory 4-1BB domain and T-cell–activating CD3ζ chain. (C) A mean CAR transduction rate of >35% could be reached for the _RV19BB_TCR⁺, _RV19BB_CRTCR⁺, and _RV19BB_CRTRBC⁻ CARs as assessed by flow cytometry (3 independent experiments). (D) The mean TCR KO rate within CD4⁺CD8⁺ cells was 78.2%, and (E) the proportion of cells expressing the CAR and lacking the TCR reached around 32.2% (3 independent experiments). (F) Exemplary flow cytometry plots to determine transduction rate and TCR KO efficacy, and (G) correlation of CD3 and TCR expression in TCR⁺ and TCR⁻ T cells within _RV19BB_CRTRBC⁻ CARs is shown in this histogram. (H) Fold expansion of the untransduced T cells, _RV19BB_TCR⁺, _RV19BB_CRTCR⁺, and _RV19BB_CRTRBC⁻ CARs after T-cell isolation and activation, transduction, and electroporation (n ≥ 4). PB, peripheral blood.