Anti-Ly49C/I F(ab′)2increases anti-CD20 mAb-mediated NK-cell degranulation and cytotoxicity. (A) NK cells isolated and purified from C57BL/6 mice were analyzed for degranulation by CD107a mobilization after 24-hour culture with 5 conditions: medium alone; human CD20+ murine lymphoma cell line (EL4-huCD20) at a 1:1 ratio of tumor:NK cells; tumor and anti-CD20 mAb (CAT13; 10 µg/mL); tumor and anti-Ly49C/I F(ab′)2 (5E6; 10 µg/mL); or tumor, anti-CD20 mAb (10 µg/mL), and anti-Ly49C/I F(ab′)2 (10 µg/mL). *P = .0109. (B) Enzyme-linked immunosorbent assay (ELISA) results measuring IFN-γ following a 4-hour culture of purified murine NK cells in medium alone; EL4-huCD20 at a 1:1 ratio of tumor:NK cells; tumor and anti-CD20 mAb (CAT13; 10 µg/mL); tumor and anti-Ly49C/I F(ab′)2 (10 µg/mL); or tumor, anti-CD20 mAb (10 µg/mL), and anti-Ly49C/I F(ab′)2 (10 µg/mL). *P = .001. (C) Supernatant was harvested and analyzed by ELISA for IFN-γ. NK-cell cytotoxicity on EL4-huCD20 tumor cells was analyzed in a chromium-release assay. NK cells were purified before being incubated with 51Cr-labeled EL4-huCD20. (C) Percent lysis of target cells by chromium release at varying effector (purified murine NK cells):target (EL4-huCD20) cell ratios cultured with (●) medium alone, (▲) anti-Ly49C/I F(ab′)2 (10 µg/mL), (▼) anti-CD20 mAb (10 µg/mL), or (▪) the combination of both antibodies. *P = .019.
