Figure 5.

Inducible caspase-9 (iCasp9) as a safety switch for CAR-engineered NK-92 cells. (A) NK-92 cells transduced with a lentiviral vector that encodes iCasp9 and ErbB2-specific CAR 5.28.z separated by a Thosea asigna virus self-cleaving peptide (T2A) (NK-92/iCasp9_T2A_5.28.z) were incubated in the presence of 10 nM of the homodimerizer AP20187 for iCasp9 activation. Lysates of cells collected after 10, 20, 30, or 60 min of exposure to AP20187 were subjected to SDS-PAGE and subsequent immunoblotting with a caspase-9-specific antibody. Lysates of NK-92/iCasp9_T2A_5.28.z cells kept without dimerizer and parental NK-92 cells incubated in the absence or presence of AP20187 served as controls. (B) Cytotoxicity of NK-92/iCasp9_T2A_5.28.z cells against ErbB2-overexpressing MDA-MB453 breast carcinoma cells was investigated in flow cytometry-based cytotoxicity assays after co-incubation of NK cells and tumor cells at different effector to target ratios (E/T) for 2 h in the absence (filled circles) or presence of AP20187 (open circles). Parental NK-92 cells were included for comparison (gray boxes). Mean values ± SEM are shown; n = 2.