Demonstration of RQR8 activity when coexpressed with a CAR. (A) RQR8 is expressed with an anti-GD2 CAR in the SFG retroviral vector using the FMD-2A sequence. The retroviral vector expression is enhanced using a scaffold attachment region (SAR). (B) Primary human T cells were transduced with this retroviral vector. Following transduction, cells were stained with QBEnd10 and polyclonal goat anti-human Fc (the latter stains the CAR). Clear coexpression of RQR8 and CAR was observed. (C) T cells were sorted with QBEnd10 beads and mixed 1:1 with nontransduced T cells and exposed to either rituximab alone or rituximab and complement. Two hours later, anti-Fc and Annexin V staining was performed. Anti-Fc staining is shown gating on live T cells. Mean depletion was 97% of transduced cells. (D) This rituximab-depleted population, along with the undepleted T cells, and nontransduced T-cells were used as effectors in a chromium-release killing assay using GD2-positive target cells. This experiment was performed in 5 donors. LTR, long terminal repeat.