“nt-KO” indicates the control group of CAR-T cells electroporated with RNP complex containing nontargeting sgRNA, while “ITK-KO” indicates the group that received ITK-targeting sgRNA. (A) Schematic representation of the anti–human CD19–CAR molecule. CMV, cytomegalovirus promoter; CD8αSP, signal peptide of human CD8α; anti-CD19-scFv, single-chain fragment variable of anti–human CD19 antibody (clone: FMC63); CD8αTM, transmembrane domain of human CD8α; CD28, 4-1BB, and CD3ζ, signal transduction domains of human CD28, 4-1BB, and CD3ζ, respectively. (B) Generation of ITK-deficient CAR-T cells. Briefly, T cells were enriched from PBMCs and activated with anti-CD3/CD28 beads for 24 hours. Then, T cells were transduced with CAR-encoding lentivirus. Forty-eight hours after transduction, CAR-T cells were electroporated with RNP complex. (C) Gene editing efficiency of ITK locus by sgRNA1 targeting ITK (ITK-sgRNA1). CAR-T cells were collected for analysis 3 days after electroporation of RNP complex. (D) Validation of ITK deficiency at protein level by Western blotting. CAR-T cells were collected for Western blotting 5 days after electroporation. (E–G) In vitro killing assay against the indicated target tumor cells using control and ITK-KO CAR-T cells (n = 4). Luciferase-expressing MEC1, HG3, and Raji cells were mixed at the indicated ratios with CAR-T cells and analyzed 48 hours after coculture. (H) Representative flow cytometric plots of IFN-γ, TNF-α, and granzyme B expression in CAR-T cells stimulated as indicated. E, effector (CAR-T cells); T, target (MEC1 cells). (I) Summary of percentages of CAR-T cells expressing different cytokines in H (n = 4). Compiled data from 1 independent experiment for E–G and I. Technical replicates are shown in E–G and I. Data represent results of at least 2 independent experiments in C–I.