Engineering of combination QBEnd10/rituximab-binding constructs. (A) Cartoon of alternative epitope constructs illustrating alternative presentation formats of epitope constructs that were generated to enable comparison of sensitivity to CDC- and ADCC-mediated deletion. “Q” refers to the 16-amino-acid minimized QBEnd10 epitope; “R” refers to the CD20 circular mimotope; “8” refers to the CD8α stalk; and “Hinge-CH2CH3” refers to the hinge, CH2 and CH3, domains of human IgG1. (B) Binding of QBEnd10 and rituximab to constructs QR8 and RQ8 coexpressed with eGFP in primary human T cells is shown. Both antibodies could bind in either orientation. (C) Sensitivity to CDC depletion using primary human T cells transduced with constructs Q8, RCH2CH3, RQ8, RRQ8, RQR8, and full-length CD20 is shown. Following 4-hour incubation with 25% baby-rabbit complement and rituximab at 100 μg/mL, samples were stained with Annexin V/PI and the live population was assessed by flow cytometry analysis for the presence of the coexpressed eGFP marker gene. Results illustrate comparative deletion observed from 3 separate donors. (D) Similarly, sensitivity to ADCC-mediated depletion was assessed using primary human T-cell targets transduced with constructs were challenged by 16:1, 8:1, 4:1, and 2:1 effector:target ratios of NK cell effectors derived from the same donor. Following 48-hour incubation in the presence of 100 μg/mL rituximab, depletion was assessed by flow cytometry analysis. Samples were stained with Annexin V/PI for live/dead exclusion, with NK cells labeled with CellTRACE violet excluded from the live gate to identify the residual live population of targets cells identified by the presence of a coexpressed eGFP marker gene. Results illustrate comparative deletion by the 8:1 effector:target ratio observed from 3 separate donors. Construct RQR8 engenders equal CDC and ADCC to full-length CD20.