Functional characterization of RQR8. (A) Demonstration of typical cellular purification result achieved following CD34 magnetic bead selection. Primary human T cells were transduced with the bicistronic retroviral vector SFG.RQR8.IRES.eGFP and selected with Miltenyi QBEnd10 beads. (B) Efficiency of CDC in transduced sorted T cells. Primary human T cells were transduced with SFG.RQR8.IRES.eGFP, purified with QBEnd10 beads, and combined at equal concentration with nontransduced T cells. This mixed population was exposed to a 2-hour incubation with 100 μg/mL rituximab with or without 25% baby-rabbit complement. T cells were stained with Annexin/PI, and flow cytometric analysis of eGFP expression on the live population is shown. This is an example of an experiment repeated 6 times in different donors. More than 95% of the transduced population is deleted. (C) Time course and rituximab dose-titration assay. CDC-mediated deletion of targets was performed in primary human T cells with rituximab concentrations of 12.5, 25, 50, and 100 μg/mL analyzed at 1, 5, 10, 30, 60, and 120 minutes. Figure shows mean and standard deviation from 3 donors. CDC is highly effective at rituximab concentrations of ≥25 μg/mL, and killing occurs within 30 minutes. (D) Demonstration of ADCC-mediated sensitivity against T cells transduced with RQR8. Transduced T cells were incubated at 16:1, 8:1, 4:1, and 2:1 effector:target ratios of NK cell effectors derived from the same donor exposed to 100 μg/mL rituximab. Samples were stained with Annexin V/PI for live/dead exclusion with depletion assessed by flow cytometry analysis comparison of the ratio of eGFP/eBFP2 marker gene expression from the residual live population. Note: QR8 was not assessed for ADCC sensitivity.