Comparison of Cytotoxic Mechanisms of Anti-CD20 Antibodies GA101 and Rituximab Against Fresh Chronic Lymphocytic Leukemia Cells

Abstract 2467

GA101, a novel glycoengineered type II IgG1 antibody against CD20, has shown a direct and immune effector cell-mediated cytotoxicity in numerous B-cell disorders. Chronic Lymphocytic Leukemia (CLL) is the most common hematologic malignancy in the western world. Since circulating mature B-CLL cells express high levels of antiapoptotic proteins that are implicated in the survival mechanism, we investigated whether the effects of GA101 compared to rituximab, induces apoptosis in these cells and what mechanism underlies GA101-mediated cytotoxicity. CLL cells were isolated from peripheral blood samples by density gradient centrifugation and B lymphocytes were purified by a negative selection method using the EasySep® B Cell Enrichment Cocktail. Cell viability was measured flow cytometrically by annexinV binding. We assessed the mitochondrial transmembrane potential (ΔΨm) by staining with 3,3-dihexyloxacarbocyanine iodide (DiOC6[3]), the generation of reactive oxygen species by staining with Dihydroethidine (DHE) as well as cytochrome c release. Moreover, the expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak and Bad) and the activation of the caspase cascade were evaluated by immunoblotting on 34 fresh peripheral blood B-CLL specimens. We showed that GA101 initiates an early extensive cell death. The average decrease of viability of freshly isolated and purified CLL cells 24 hours post-treatment with 10μg/ml of anti CD20 antibodies were 37.6% for GA101 (n=11) and 28.8% for Rituximab (n=11). The GA101-induced cell death was paralleled by a rapid loss of mitochondrial membrane potential accompanied with the production of ROS and cytochrome c release that occurred significantly as early as 3 hours post-treatment. However, rituximab was unable to initiate a loss of ΔΨm and the production of ROS. The use of antioxidants such as N-acetyl cysteine and L-ascorbic acid were unable to circumvent either the GA101-induced cell death or the loss of ΔΨm. However, the preincubation of CLL cells with Z-VAD.fmk (N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone), a broad caspase inhibitor, abolished the exposure of phosphatidylserine residues, the generation of reactive oxygen species and reversed the loss of ΔΨm. Furthermore no change was observed in the expression level of Bcl2 pro-survival family members, while GA101 induced the pro-apoptotic proteins such as Bax and Bak and caused cleavage of the active form of caspase 9 and 3 and the proteolytic cleavage of PARP, in 5 out of 9 patients studied. Altogether, these data show that GA101 induced-cell death in B-CLL cells, unlike what has been observed in cell lines, is mediated by a caspase-dependent mechanism involving the loss of ΔΨm and the generation of ROS. Ongoing studies aim to analyze the role, the conformational changes and the cellular redistribution of Bax and Bak in response to GA101 and the modifications of other apoptosis-related proteins in CLL cells.