GA101 exhibits characteristics typical of a type II anti-CD20 antibody. (A) Fluorescence intensity at the EC50 value (half-maximal binding) of GA101 compared with rituximab. Titration of a panel of NHL cell lines with Cy-5–labeled GA101 (■) shows that only half the amount of antibody is bound to CD20 on the cells, compared with Cy-5 labeled rituximab (▩) at the EC50 concentration. (B) GA101 does not mobilize CD20 into lipid rafts. On binding of GA101 to CD20 in Ramos cells, CD20 is mainly found in the Triton X-100–soluble fraction (S) and not in the Triton X-100–insoluble pellet (P) representing lipid rafts (top row). In contrast, binding of rituximab resulted in the distribution of CD20 into the Triton X-100–insoluble pellet fraction (S; top row). The distribution of Lyn as a typical lipid raft marker and CD71 as a nonlipid raft marker is not affected (bottom 2 rows). (C) Confocal microscopy: Ramos cells were stained for 30 minutes at 37°C with Cy-3–labeled rituximab or GA101 (red fluorescence, middle panel) and costained with the Alexa 488-labeled lipid raft marker cholera toxin subunit B (CTB-Alexa 488, green fluorescence, left panel), which binds to the membrane ganglioside GMI in lipid rafts. The overlay (right panel) confirms that rituximab binding to CD20 results in accumulation of CD20 clusters in lipid rafts as shown by colocalization with CTB-Alexa 488 (yellow fluorescence), whereas CD20 molecules do not redistribute to lipid rafts on binding of GA101 and do not colocalize with CTB-Alexa 488. Pictures were captured with a Leica TCS SP2 confocal microscope with an HCX PL APO CS 63.0×/1.32 OIL UV objective (numeric aperture 1.32) in glycerol and aquired with Leica Confocal Software Version 2.61. Image manipulation was performed with Metamorph Version 7.0r3 and Jasc Paint Shop Pro. (D) CDC assays (LDH release) with Z138 mantle cell lymphoma cells. In the presence of physiologic concentrations of human unspecific IgG (10 mg/mL RedImmune), the type II anti-CD20 antibody GA101 (black) mediates greatly reduced CDC induction compared with rituximab (gray; n = 3). (E) GA101 induces rapid and pronounced homotypic aggregation of SU-DHL4 cells, whereas rituximab induces only a weak aggregation. Pictures were taken 24 hours after the addition of antibody. Pictures were captured with a Zeiss Axiovert 135 microscope with a Zeiss Fluar 5×/0.25 objective (numeric aperture 0.25) in RPMI 1640, 10% FCS, 2mM L-Glutamin on non fixed Ramos cells and a Cool SNAP K4 camera (Visitron Systems GmbH). Image acquisition and manipulation were performed with Metamorph Version 7.0r3 and Jasc Paint Shop Pro.
