Effect of crosslinking on dissociation of radiolabeled CD20 mAb.Comparison of the dissociation of radiolabeled CD20 mAb IgG (A) and the effect of cross-linking on dissociation (B-C). (A) ¹²⁵I-labeled CD20 mAbs (2 μg/mL) were added to DOHH cells, incubated for 2 hours at room temperature, and then pelleted and resuspended in 200 μL medium containing 1 mg/mL unlabeled mAbs. Cells were then cultured at room temperature and at the times indicated aliquots were taken to determine the level of cell-bound mAbs over time. Counts remaining at each time point were expressed as a percentage of the initial count. Symbols: rituximab, ▪; 7D8, ▴; and 2F2, ▾. This figure shows 1 of 3 experiments that gave similar results. (B-C) To determine the effect of cross-linking on mAb dissociation, DOHH cells were incubated with labeled rituximab (B) or 2F2 (C) as described in “Materials and methods,” pelleted, and the supernatant was removed. Cells were then resuspended and treated with sheep antihuman κ F(ab′)₂ fragment (open symbols) or not (filled symbols) for 30 minutes on ice. Excess mouse CD20 mAb, 1F5 (1 mg/mL), was added to act as a cold competitor that would not compete for binding to the sheep cross-linking Ab. At the times shown, aliquots of cells were taken and bound radiation was determined as for panel A. These results are representative of 2 experiments each showing the same outcome.