CD40 Stimulation Sensitizes CLL Cells to CD20-Triggered Cell Death by Rituximab and GA101 Via a Different Mechanism

Abstract 3979 Although treatment results for Chronic Lymphocytic Leukemia (CLL) have improved considerably over the last decade, unfortunately a curative treatment is still not available. In part this might be due to the interaction of CLL cells with their micro-environment in lymph nodes, spleen and bone marrow. Micro-environment derived signals are not only capable of driving proliferation of CLL cells, but can also induce resistance of CLL to cytotoxic drugs. Previously we have shown that in vitro CD40 stimulation of peripheral blood derived CLL cells can to a certain extent mimic the lymph node microenvironment and result in resistance to cytotoxic drugs ( Kater AP et al. Br J Haematol 2004;127:404; Smit LA et al. Blood 2007;109:1660.; Hallaert DY et al. Blood 2008;112:5141). At present it is not known whether sensitivity of CLL cells to CD20 monoclonal antibodies (mAbs) is modulated by micro environmental stimuli. Therefore in the present study we investigated anti-CD20 mediated cell death of CD40-stimulated CLL cells from 17 CLL patients. We observed that in sharp contrast with the response towards cytotoxic drugs, CD40 stimulation sensitizes CLL cells to cell death mediated by anti-CD20 mAbs. In CD40-stimulated CLL cells, cell death induced by both Rituximab (a type I anti-CD20 mAb) and GA101 (a novel type II anti-CD20 mAb) ( Moessner E et al. Blood, 2010;127:404, 115(22):4393-402 ) is increased. Both anti-CD20 mAbs induce a non-apoptotic, caspase- and p53-independent rapid cell death, but interestingly the mechanism of Rituximab and GA101-induced cell death appears to be different. Rituximab-induced cell death is dependent on extracellular Ca 2+ and ROS production and CD40 stimulation sensitizes CLL cells by increasing basal ROS production. In contrast, GA101 induces cell death via a lysosome-dependent mechanism and CD40 stimulation sensitizes CLL cells by increasing the lysosomal volume of the cell. Moreover, in contrast to Rituximab, GA101 induces cell death in the absence of a secondary crosslinking mAb. Combination of GA101 with fludarabine, chlorambucil, bortezomib or bendamustine shows additive effects and results in strong cell death of CD40-stimulated CLL cells, even in p53 dysfunctional CLL cells. Our findings not only provide a rationale for combining cytotoxic drugs and anti-CD20 monoclonal antibodies, but also show that GA101 is a potent promising new anti-CD20 mAb for the treatment of CLL. Disclosures: Klein: Roche: Employment, Equity Ownership, Patents & Royalties.