Alemtuzumab Induces Cell Death in Human Chronic Lymphocytic Leukemia Cells through a Lipid Raft-Dependent Mechanism.

Abstract Campath-1H is a humanized IgG1 kappa antibody directed against the cell surface antigen CD52, a GPI linked protein of unknown function that is present on B and T lymphocytes as well as CLL cells. Campath-1H is approved for fludarabine refractory CLL and ongoing studies will continue to define its role in the treatment of CLL. Despite its clinical use, little is known about the in vivo mechanism of its cytotoxicity. Mechanisms invoking complement, effector cells and direct antibody- induced cytotoxicity have been proposed. Here we show that campath-1H is directly toxic to human CLL cells in vitro in the absence of complement or effector cells. Furthermore, we observed that normal T-cells isolated from random blood donors are less sensitive to direct campath-1H induced cytotoxicity than CLL cells. Using annexin V-FITC/propidium iodide (PI) staining with FACS analysis, we found that treatment of CLL samples for 4 hrs with campath-1H (10μg/ml) or campath-1H with a cross-linking anti-Fc IgG (10μg/ml) leads to positive staining in 9.5 % and 29% of the cell respectively (n=11). Parallel study using the MTT assay showed a 7.9 % and 20.4 % loss of viability with campath-1H or campath-1H + anti-Fc IgG (n=5). Antibody directed cellular cytoxicity, using the chromium release assay, showed 20 % killing at 4 hrs (effector to target cell ratio 25:1, n = 4). After treating primary CLL cells with campath-1H for 4 hrs in the presence of human complement, 67 % of the cells stained positive with annexin V-FITC/PI (n=5). Direct campath-1H induced killing of normal T-cells was reduced compared to CLL cells. After a 4 hr treatment with campath-1H + anti-Fc IgG, 6.7 % of the cells stained positive with annexin V-FITC/PI (n=5). The direct campath-1H cytoxicity we observed in CLL cells is caspase-independent. Immunoblot analysis showed no evidence of caspase-3, 8, or 9 activation or PARP cleavage, nor was there a reduction in cytotoxicity with the addition of Z-VAD-fmk. Using minimal detergent solubilization of CLL cells followed by density-gradient centrifugation and immunoblot analysis, we found that CD52 colocalizes with the glycosphingolipid GM1. Fluorescence microscopy analysis revealed that upon cross-linking CD52 with campath-1H + anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM1 formed on the plasma membrane of primary CLL cells. Cholesterol depletion of lymphocyte membranes with methyl-β-cyclodextran diminished the formation of these CD52 and GM1 enriched patches and caps. Inhibition of actin polymerization with cytochalasin B also reduced the formation of campath-1H + anti-Fc IgG induced caps. The cytotoxic effect of cross-linked campath-1H upon CLL cells was reduced by methyl-β-cyclodextran or cytochalasin B pretreatment as measured by annexin V-FITC/PI staining and MTT assay analysis. In conclusion, we found that campath-1H treatment of CLL cells leads to rapid cell death through a caspase-independent mechanism in the absence of complement or effector cells. Experiments using methyl-β-cyclodextran or cytochalasin B treated cells, suggest that the direct cytotoxicity of campath-1H is dependent on formation of large antigen/lipid raft aggregates upon the cell membrane, with underlying cytoskeletal rearrangement. The difference in direct killing observed between CLL cells and normal T-cells suggests that death mechanisms may differ significantly and thus provide the opportunity to refine CD52 directed antibodies with the aim of reducing the suppression of cellular immunity.