Generation of TCR/CD3-specific CAR NK-92 cell lines for the removal of residual TCR⁺ T-cells from TCR-deleted CAR T-cell product. (A) Outline of the cell-mediated purification strategy. Gamma-irradiated NK-92 cells equipped with a TCR/CD3-specific CAR are added to expanding TCR^– CAR T-cells. This coculture then leads to the removal of residual TCR-wild-type T-cells from the TCR^– CAR T-cell product. (B) Generation of TCR/CD3-specific CAR NK-92 cell lines by site-specific integration of 4 CAR transgenes (termed CAR1-CAR4), with specificity for either the TCRα/β dimer or CD3ε in the human AAVS1 safe-harbor locus. (C) CAR expression after knockin (left) and MACS-enrichment. (D,E) Experimental setup and results of flow cytometric VITAL assays used to test target-specific cytotoxicity of the 4 TCR/CD3-CAR NK-92 cell lines. The VITAL assays were set up as cocultures of unlabeled effector (E) TCR/CD3-CAR NK-92 cells, DDAO-labeled C cells TRAC-KO (TCR-negative) T-cells, and CFSE-labeled target (T) wild-type T-cells. Relative killing was calculated from T:C ratios at 4 and 16 hours after the start of the coculture.