Ibrutinib antagonizes antibody-dependent NK cell–mediated cytotoxicity. To evaluate NK cell function, purified NK cells were isolated from healthy peripheral blood mononuclear cells and cultured with 0.1 or 1 μM of ibrutinib for 4 hours together with rituximab-coated (10 µg/mL) lymphoma cells, DHL4, or trastuzumab-coated (10 µg/mL) HER2⁺ breast cancer cells, HER18, and (A) supernatant was harvested and analyzed by enzyme-linked immunosorbent assay for interferon-γ, and (B) NK cells isolated and analyzed for degranulation by flow cytometry for CD107a mobilization. (C-D) NK cell cytotoxicity as percent lysis of DHL4 or HER18 tumor cells was analyzed in chromium release assays with purified NK cells incubated with (C) chromium-labeled DHL4 or HER18 cells for 4 hours at variable effector:target ratios, rituximab (10 µg/mL), and ibrutinib (0.1 or 1 μM) or (D) chromium-labeled Raji or autologous CLL cells for 4 hours at variable rituximab concentrations at a constant effector:target ratio of 25:1 and ibrutinib (1 μM) or CGI-1746 (1 μM). All in vitro experiments were performed in triplicate. To evaluate NK cell function, in vivo athymic ν/ν mouse models (10 mice per group) were xenotransplated with HER18 or DHL4 tumor cells (1 × 10⁶) subcutaneously along the flank on day 0 and monitored for (E,G,I) tumor growth and (F,H,J) survival with experiments performed in duplicate. (E-F) In vivo therapy of the HER18 tumor model included intraperitoneal (ip) immunoglobulin G (IgG) control on days 4, 8, 12, and 16; ip trastuzumab (200 μg) on days 4, 8, 12, and 16; ibrutinib (25 mg/kg/dose) twice daily on days 4 to 18 by oral gavage (og); or the combination. (G-H) In vivo concurrent therapy of the DHL4 lymphoma model included ip IgG control on days 14, 18, 22, and 26; ip rituximab (200 μg) on days 14, 18, 22, and 26, ibrutinib og (25 mg/kg/dose) twice daily on days 14 to 28; or the combination. (I-J) In vivo sequential versus concurrent therapy of the DHL4 lymphoma model included sequential ibrutinib og (25 mg/kg/dose) twice daily on days 14 to 21 and ip rituximab (200 μg) on days 22 and 26; or sequential ip rituximab (200 μg) on days 14 and 18 and ibrutinib og (25 mg/kg/dose) twice daily on days 21 to 28; or concurrent ip rituximab (200 μg) on days 14, 18, 22, and 26 and ibrutinib og (25 mg/kg/dose) twice daily on days 14 to 28. BID, twice daily.