Binding and redistribution properties of different anti-CD20 antibodies.

(A) Binding of ¹²⁵I-labeled F(ab′)₂ fragments of 1F5, Ritux, and B1 anti-CD20 mAbs to EHRB and Daudi cells.¹²⁵I-labeled F(ab′)₂ fragments of anti-CD20 or control (anti-CD3) mAbs were incubated with Daudi or EHRB cells for 2 hours at 37°C. The exception to this is the curve for IF5 binding to Daudi cells, for which ¹²⁵I-labeled whole IgG was used. The cell-bound and free ¹²⁵I-labeled mAbs were then separated by centrifugation through phthalate oils and the cell pellets together with bound antibody counted for radioactivity. (B) 1F5 and Ritux, but not B1, induce clustering of YFP-HuCD20 in BCL₁-3B3–transfected cells upon binding to CD20. BCL₁-3B3 cells transfected with YFP-HuCD20 were incubated with various anti-CD20 mAbs for 20 minutes at room temperature. Cells were then cytospun onto slides and fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. YFP-HuCD20 clustering was visualized using confocal microscopy. Representative images from 3 separate experiments are shown (original magnification, × 100). During this investigation we performed microscopic examination of anti-CD20 mAbs binding to cells using FITC-mAbs. These experiments revealed clear differences in the distribution of CD20.