NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximabinduced complement fixation, natural killer (NK)-cell activation, and NK cellmediated ADCC. Down-modulation of NKcell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heatinactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell-mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC. IntroductionMonoclonal antibodies (mAbs) are now a mainstay of therapy for a number of cancers. Rituximab was the first chimeric mAb to be approved for clinical use and remains the most extensively used mAb in cancer therapy. Rituximab binding of CD20 has been shown to signal apoptosis in a subset of lymphoma cell lines. 1 However, there is little evidence that signaling plays an important role in clinical responses to rituximab. Growing evidence suggests multiple interacting mechanisms, including complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC), play a role in the antitumor response of rituximab and other mAbs.Evidence is conflicting related to the role CMC plays in mediating the antitumor effects of rituximab. Van Meerten et al used target cells that express varying amounts of CD20 on their surface, and concluded that rituximab-mediated CMC depends on CD20 expression level and acts in a complementary manner to ADCC. 2 Target cell expression of the complement inhibitory proteins CD55 and CD59 correlates with the ability of rituximab to induce CMC in vitro, 3 and CMC is enhanced when these proteins are blocked. 4 However, no correlation was found between CD55/ CD59 expression by lymphoma cells and clinical response to therapy. 5 In mouse models using murine lymphomas expressing human CD20, Golay et al found that complement plays a key role in mediating rituximab's antitumor effects. 6,7 Cragg and Glennie reached similar conclusions from studies of human B-cell lines in severe combined immunodeficiency (SCID) mice. 8 Clinically, depletion of complement and evidence for complement fixation on target cells can be seen following rituximab...

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