XmAb5574 stimulation of NK cells results in up-regulation of Erk1/2 phosphorylation. (A) Up-regulation of Erk1/2 phosphorylation in NK cells after stimulation by fixed XmAb5574 was detected by Western blot and compared with XmAb5603 and no antibody. Six-well flat-bottom plates were coated with 10 to 20 μg/mL of respective antibody and then plated with freshly isolated NK cells at 5 × 10⁶ cells/well. NK cells were harvested after 10-minute incubation and immediately subjected to lysis and protein extraction. (B) Specific MEK kinase inhibitor PD98059 (100μM) added to NK cells 30 minutes before their incubation with fixed XmAb5603 and XmAb5574 abrogates the enhancement of Erk1/2 phosphorylation by XmAb5574. Blots shown are representative of 3 individual experiments. Numbers denote densitometric ratios of p-Erk1/2 to total Erk1/2. (C) Ability of XmAb5574 to mediate ADCC against CLL cells through healthy donor NK cells was diminished but not significantly in the presence of media or PD98059 at 100μM (10.65% decrease with PD98059; 95% CI, 0.09-21.39; *P = .15; n = 27; compared with DMSO at E/T of 25:1). (D) Fixed XmAb5574 mediates minimal direct cytotoxicity against NK cells at 4 hours as shown by percentage of dead cells. Fixed antibody–mediated NK-cell cytotoxicity was determined by characterization of CD56⁺ and 7-AAD⁺ cells by FACS analysis on freshly isolated NK cells after 4-hour stimulation by 20 μg/mL of respective antibody coated on a 96-well flat-bottom plate. XmAb5574 did not mediate significantly higher NK-cell cytotoxicity than XmAb5603 (1% higher; 95% CI, −1.7% to 3.6%; *P = .4456), trastuzumab (1% higher; 95% CI, −1.7% to 3.6%; P = .446), or rituximab (0.8% higher; 95% CI, −1.8% to 3.5%; P = .518; n = 4). Percentage of dead cells was calculated as the number of 7-AAD⁺ cells in the CD56⁺ cell population, and all values were normalized to media control. Error bars represent SEMs.