XmAb5574 induces modest MDM-mediated ADCP against CLL cells. (A) CD14 selected monocytes isolated from healthy donor whole blood was used to derive macrophages under monocyte-colony stimulating factor and used in ADCP assays against primary patient CLL cells. Macrophages were harvested and labeled with PKH-26 membrane dye, whereas CLL cells were labeled with PKH-67 and incubated on ice for 1 hour with no antibody, trastuzumab, XmAb5603, or XmAb5574 (10 μg/mL each). Macrophages and CLL cells were subsequently coincubated for 2.5 hours at 37°C and analyzed by FACS. XmAb5574 was unable to significantly enhance the MDM-induced ADCP against CLL cells compared with XmAb5603 (12.37% vs 10.51%; estimated ratio, 1.18; 95% CI, 0.61-2.27; *P = .58) or rituximab (11.82% phagocytosis; estimated ratio, 1.05; 95% CI, 0.54-2.02; P = .87 compared with XmAb5574). XmAb5574 did, however, show a statistically significant increase in ADCP compared with our negative control trastuzumab (12.37% vs 1.18%; estimated ratio, 10.47; 95% CI, 5.43-20.17; P = < .001; n = 6). An E/T ratio of 1:2 was used for all experiments. Percentage of phagocytosis was calculated as events positive for both PKH-67 and PKH-26 dyes. Error bars represent SEMs. (B-C) XmAb5574 induces potent ADCC by NK cells against both allogeneic and autologous B-CLL cells. XmAb5574 induced NK cell–mediated ADCC against allogeneic (B) and autologous (C) CLL cells. Ability of rituximab, trastuzumab, XmAb5603, or XmAb5574 to mediate ADCC was evaluated with the use of either fresh allogeneic or autologous human NK cells as effector cells and B-CLL cells as target cells at the indicated E/T ratios by standard 4-hour ⁵¹Cr-release assay. ⁵¹Cr-labeled target cells (5 × 10⁴ CLL cells) were placed in 96-well plates after incubating them with 10 μg/mL of various antibodies for 30 minutes and subsequently coincubating them with NK cells (from healthy donors or patients with CLL) at indicated ratios. Specific inhibitors were added to NK cells 30 minutes before coincubation. After 4-hour incubation, supernatants were removed and counted in a γ counter, and the percentage of specific cell lysis was determined by the following formula: percentage of lysis = 100 × (ER − SR)/(MR − SR), where ER, SR, and MR represent experimental, spontaneous, and maximum release, respectively. (B) XmAb5574 induces progressively higher ADCC by allogeneic NK cells derived from healthy donors with increasing E/T ratios (24.1% increase; 95% CI, 12.9%-35.3%; P for trend < .001) and significantly higher ADCC compared with XmAb5603 (26.9% increase; 95% CI, 14.5%-39.2%; *P ≤ .001, at the highest E/T ratio used of 25:1) or rituximab (33.5% increase; 95% CI, 20.8%-46.2%; P ≤ .001; at E/T of 25:1; n = 11). (C) Similarly, with the use of autologous NK cells derived from the same patients with CLL there was a 23.9% progressive increase in ADCC with increasing E/T ratios (95% CI, 9.4%-38.3%; P = .006). XmAb5574 was also able to significantly increase ADCC mediated by autologous NK cells compared with XmAb5603 (23.6% increase; 95% CI, 6.0%-41.2%; *P = .01; at E/T of 25:1) or rituximab (27.1% increase; 95% CI, 9.9%-44.3%; P = .0026; at E/T of 25:1; n = 5).