Hexamerization-enhanced CD20 antibody mediates complement-dependent cytotoxicity in serum genetically deficient in C9

Publication date: Available online 31 May 2017
Source:Clinical Immunology
Author(s): Ronald P. Taylor, Margaret A. Lindorfer, Erika M. Cook, Frank J. Beurskens, Janine Schuurman, Paul W.H.I. Parren, Clive S. Zent, Karl R. VanDerMeid, Richard Burack, Masashi Mizuno, B. Paul Morgan
We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca²⁺ during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca²⁺ to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca²⁺ signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca²⁺ influx represents the most proximate mediator of cell death by CDC.



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