Fig. 1. 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 2^nd generation CAR consists out of the anti-CD19 scFv region, followed by the myc tag, CD8 extra-, trans- and intracellular domain, as well as a costimulatory 4-1BB domain and T-cell activating CD3zeta chain. (C) A mean CAR transduction rate of more than 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 78.20 % 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). PBMC=peripheral blood mononuclear cell, PB=peripheral blood, KO=knockout, TRBC=constant T-cell receptor β-chain, SSC=side scatter, _RV19BB_TCR⁺=conventional CAR-T cells without electroporation, _RV19BB_CRTCR⁺=CAR-T cells electroporated with nonsense gRNA, _RV19BB_CRTRBC^-=CAR-T cells electroporated with TRBC-targeting gRNA.