SummaryThe effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.

2365 Poster Board II-342 Introduction: Monoclonal antibodies against CD20 have great potential as therapeutics for the treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL), since CD20 is expressed on all mature B-cells except stem or plasma cells. Therefore treatment with anti-CD20 antibodies leads to both, direct cell death induction (DCD) by receptor engagement and to cell killing by Fc-mediated mechanisms, namely antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In a pre-clinical in vitro assessment we compared the effects of rituximab, which was approved for treatment of non-Hodgkin lymphoma more than a decade ago, with GA101, a third-generation glyco-engineered anti-CD20-antibody recognizing a type II epitope. Methods: CLL cells were isolated from blood samples by Ficoll gradient centrifugation after treatment with a RosetteSep antibody cocktail. The effects of antibody treatment on membrane integrity were assessed by annexin V binding. Antibody-mediated B cell depletion in whole blood samples was determined flow cytometrically by enumerating CD19- and CD3-positive B and T lymphocytes and by relating the B/T cell ratios after antibody treatment with those of the corresponding untreated samples. Whole blood samples from 25 patients were investigated. Significance thresholds were determined by unpaired Student's T-test and linear correlation analysis. Results: The average decreases in viability of freshly isolated CLL cells due to treatment with 10 μg/ml of anti-CD20 antibodies were 21 % for GA101 (n=8) and 6 % for rituximab (n=6), while the mean B cell depletion in whole blood samples from CLL patients was 32 % for GA101 (n=18) and 11 % for rituximab (n=17). Thus the whole blood assay indicated the antibody effects with greater sensitivity than analysis of annexin V binding in isolated cells. In both assays GA101 performed better than rituximab as evidenced by superior DCD (p=0.021) and antibody-dependent B cell depletion (p=0.001). In about two thirds of whole blood samples from CLL patients treatment with 1 μg/ml of GA101 led to more than 20 % antibody-mediated B-cell depletion. The maximum B-cell depletion observed among the investigated CLL samples after treatment with 10 μg/ml of antibody was 85 % for GA101 and 25 % for rituximab. The B cell depletion from whole blood samples mediated by GA101 and rituximab correlated with the CD20 surface expression on CLL cells from the same patients (r=0.643, n=15, p<0.01 and r=0.610, n=14, p<0.05). In contrast, CLL cell depletion in six whole blood samples did not correlate with the increases in annexin V binding of the corresponding isolated CLL cells. Conclusions: The size of GA101 effects observed in the present B cell depletion assay in a majority of whole blood samples encourages attempts to dissect antibody-mediated killing mechanisms in individual patient samples by specifically inhibiting ADCC or CDC to determine their contribution to B cell depletion. As compared to rituximab, GA101 had stronger effects on isolated CLL cells and, to an even higher degree, led to superior B cell depletion in whole blood samples. These pre-clinical in vitro data obtained with CLL cells indicate that many patients might benefit from this promising therapeutic agent. Disclosures: Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Hallek:Roche: Honoraria, Research Funding. Krause:Roche: Research Funding.