Real-time analysis of the detailed sequence of cellular events in mAb-mediated complement-dependent cytotoxicity of B-cell lines and of chronic lymphocytic leukemia B-cells

Complement-dependent cytotoxicity is an important mechanism of action of certain mAbs used in cancer immunotherapy, including ofatumumab and rituximab. However, the detailed sequence of cellular changes that occur in nucleated cells attacked by mAb and complement has not been delineated. Recently developed CD20 mAbs, engineered to form hexamers on binding to cells, react with B-cells in serum, chelate C1q, and then activate complement and promote cell killing considerably more effectively than their wild-type precursors. We used these engineered mAbs as a model to investigate the sequence of events that occur when mAbs bind to B-cell lines and to primary cells from patients with chronic lymphocytic leukemia and then activate complement. Based on four-color confocal microscopy real-time movies and high resolution digital imaging, we find that after CD20 mAb binding and C1q uptake, C3b deposits on cells, followed by Ca²⁺ influx, revealed by bright green signals generated on cells labeled with FLUO-4, a Ca²⁺ indicator. The bright FLUO-4/Ca²⁺ signal fades, replaced by punctate green signals in mitochondria, indicating Ca²⁺ localization. This step leads to mitochondrial poisoning followed by cell death. The entire sequence is completed in <2min for hexamerization-enhanced CD20 mAb-mediated killing. To our knowledge this is the first time the entire process has been characterized in detail in real time. By identifying multiple discrete steps in the cytotoxic pathway for nucleated cells our findings may inform future development and more effective application of complement-fixing mAbs to cancer treatment.