Optimization of TCR/CD3-CAR NK-92 cell–mediated purification and comparison with MACS-mediated CD3-depletion. (A) Experimental setup for lentiviral (LV) or virus-free TCR-to-CAR replacement and subsequent NK-92 cell–mediated depletion of residual TCR-unedited cells in 1 or 2 coculture cycles. n = 4, TRAC-edited CAR T-cell lines derived from 2 donors (per donor: 1 × LV CAR gene transfer + TRAC-KO and 1 × TRAC-CAR knockin). (B) From left to right: representative flow cytometry dot plots of virus-free TRAC-integrated CAR T cells when cultured alone, after 2 coculture cycles with unedited NK-92 cells, after MACS-mediated CD3-depletion (independent experiment), or after 1, or 2 coculture cycles with TCR/CD3-specific (CAR4) NK-92 cells. (C) Summary of purity, impurity (in terms of the residual TCR/CD3⁺ cells), and cleaning efficiency (relative reduction of the CD3⁺ fraction) after 0, 1, or 2 coculture cycles. (D) Impurity and cleaning efficiency after MACS-mediated CD3-depletion (left, independent experiment; n = 12) or coculture with TCR/CD3-specific (CAR4) NK-92 cells. Data point shapes: triangular, LV gene transfer; round, virus-free gene transfer. (E) Ratio of successfully edited (TCR/CD3^-) cells per residual unedited (TCR/CD3⁺) wild-type cell.