GA101 induces Fc-independent PCD and HA. (A) A panel of CD20-positive, human B-lymphoma cell lines was treated with 10 μg/mL mAb and cell death was analyzed after 24 hours using the annexin V–FITC/PI assay. The anti-HER2 mAb trastuzumab was used as a human IgG1 isotype control. GA101 induced significantly higher cell death than rituximab in all the cell lines tested (for Raji and Granta 519, P < .001; Daudi, P < .01; SU-DHL4, P < .03). (B) Raji cells were treated with GA101 (10 μg/mL) for 4 hours and the viable cell population was isolated, rested overnight, then re-treated with GA101 for 4 hours. Subsequently, cell death was analyzed as in panel A. Mean and SEM of 2 independent experiments are shown. Cell death induced in viable isolated cells was equivalent to that induced in previously untreated cells. (C) The extent of HA induced by mAbs (10 μg/mL) was assessed by low-magnification light microscopy 4 hours after treatment, and HA in Daudi cells is shown as an example (scale bar, 500 μm). GA101 induced superior HA than rituximab. (D) Cell death induced by GA101 in Raji cells was directly compared with that induced by a non-glycoengineered, wild-type human IgG1-bearing derivative of GA101, GA101 (NG), and F(ab)′₂ fragments of GA101. All induced equivalent amounts of cell death confirming that GA101-induced cell death is Fc independent. (E) ⁵¹Cr-release cell death assay. Cells were prelabeled with ⁵¹Cr for 1 hour at 37°C before treatment with mAb and after 4 hours, ⁵¹Cr release was measured as described in “Cell death and cell viability assays.” Mean + SEM of quintuplicate samples representative of 2 independent experiments are shown. GA101 induced significantly greater ⁵¹Cr release than rituximab, and F(ab)′₂ fragments of GA101 were sufficient for the induction of ⁵¹Cr release.