The type II anti-CD20 antibody GA101 mediates superior direct cell death induction in normal and malignant B cells. (A) GA101 induces PS exposure and cell death in the annexin V/PI FACS assay at levels superior to those induced by the type II anti-CD20 antibody B1 and rituximab. Z138 NHL cells were seeded and treated with 10 μg/mL GA101, nonglycoengineered GA101 (wt GA101), B1, rituximab, or camptothecin as control for 24 hours. The graph shows the mean percentage of total annexin V–positive cells, that is, annexin V/PI double-positive (AxV⁺/PI⁺) and annexin V–positive, PI-negative cells (AxV⁺/PI⁻; n = 3). (B) NHL cells were seeded and either left untreated (□) or treated with 10 μg/mL GA101 (■) or rituximab (▩), respectively, for 72 hours. The graph shows the percentage of total annexin V–positive cells for a cell line panel of 3 Burkitt lymphoma, 4 DLBCL, and 1 MCL cell lines from 1 representative experiment. (C) GA101 (black curve) induces increased cell death compared with rituximab in purified human nonmalignant B cells isolated from 2 healthy donors (gray curve), as measured by annexin V/PI double-positive staining at 36 hours. All measurements were performed in duplicate; the mean of replicate samples from multiple donors (n = 2) and SD are shown. (D) Murine purified B cells from human CD20 transgenic mice were treated as indicated for 36 hours ex vivo with GA101 or rituximab either with or without prior mitogenic stimulation by IgM or CD40L, and cell death induction was measured by annexin V/PI staining as described in “Annexin V/PI FACS assay.” Two animals were used per stimulation. All measurements were performed in duplicate; the mean of replicate samples from multiple animals (n = 2) and SD are shown.