CD16xCD33 BiKE enhances NK cell cytotoxicity and cytokine production against MDSC targets. (A) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled NK cells were cultured for 5 days in basal medium or medium supplemented with 10 ng/mL IL-15 in the presence or absence (E:T ratio = 1:2) of cytokine-derived MDSCs from normal PBMCs, and proliferation was evaluated via FACS analysis. Histogram plots represent 1 of 6 normal donors. (B) PBMCs from normal donors were coated with 10 μg/mL of CD16xCD33 BiKE or scFv CD16 control and cocultured with cytokine-derived MDSC or HL-60 targets, and NK cell-mediated cytotoxicity was evaluated via a ⁵¹Cr-release assay (****P < .0001; n = 4). (C-E) PBMCs from normal donors were coated with 10 μg/mL of CD16xCD33 BiKE or scFv CD16 control and cocultured with cytokine-derived MDSC or HL-60 targets, and NK cell CD107a expression and intracellular TNF-α and IFN-γ production were evaluated via FACS analysis (**P < .01, ****P < .0001; n = 6). (F-H) Mononuclear cells from paired PB and BM samples were isolated from normal donors (n = 4) and MDS patients (n = 10), coated with 10 μg/mL of CD16xCD33 BiKE or scFv CD16 control, and cocultured with cytokine-derived MDSC, and NK cell (F) CD107a expression and intracellular (G) TNF-α and (H) IFN-γ production were evaluated via FACS analysis (*P < .05, **P < .01, ***P < .01, ****P < .0001).