A ROR1-specific CAR expressed in human CD8⁺ T cells confers specific recognition of ROR1-positive tumor cells. (A) Design of the transgene encoding the ROR1-CAR. The CAR-cassette contains a truncated extracellular EGFR transduction marker, separated by a cleavable T2A element to identify transduced T cells. (B) Phenotype and transgene expression of a ROR1-CAR transduced and a GFP-transduced CD8⁺ T-cell clone obtained from a healthy donor. To identify CAR-transduced T cells, primary staining was either performed with biotinylated anti-EGFR mAb or biotinylated recombinant Fc-ROR1 fusion protein that contained the extracellular domain of ROR1 and binds directly to the scFv of the CAR, and compared with staining with Fc-protein alone. Secondary staining was performed with streptavidin-conjugated PE. (C) Specific cytotoxicity of ROR1-CAR transduced CD8⁺ T cells against primary B-CLL and ROR1-transfected K562 cells by chromium release assay. Cytotoxicity data are presented as mean values of triplicate wells. The standard deviation of each triplicate was ≤ 3% in all cases (C-F). (D) ROR1-CAR modified CD8⁺ T cells but not T cells transduced with a GFP-encoding control vector specifically recognize primary B-CLL and MCL samples from multiple patients and a panel of ROR1-positive tumor cell lines. Cytotoxicity was analyzed by chromium release assay at an E/T ratio of 20:1. (E) Chromium release assay comparing the cytotoxicity of ROR1-CAR and CD20-CAR transduced CD8⁺ T cells obtained from the same donor against primary B-CLL cells and autologous resting and EBV-transformed B cells. (F) Recognition of autologous B cells that had been activated by BCR-crosslinking, with PMA/ionomycin, and stimulation through CD40 by ROR1-CAR, CD20-CAR, and control GFP-transduced CD8⁺ T cells by chromium release assay at an E/T ratio of 20:1.