Abstract
We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.
Highlights
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We observed that Na citrate solution from vacutainer tubes, at the standard concentration used for anticoagulant activity (0.1 M), did not inhibit either complement activation induced by alemtuzumab in presence of 20% human serum (HS), measured as C3 and C9 deposition (Fig. 1A), or cell lysis (Fig. 1B)
We have set up novel whole blood assays to study the mechanism of action of therapeutic mAbs for circulating neoplastic B cells such as B-chronic lymphocytic leukemia (B-CLL) and B non-Hodgkin’s lymphoma (B-NHL), in conditions as physiological as possible
Summary
These data confirm using cytospins that complement is the major mechanism of lysis of B-CLL cells in presence of anti-C20 mAbs and serum and is dependent upon CD20 expression levels. These data suggest that lysis of B-CLL cells induced by 100 mg/ml rituximab or GA101 in whole blood is mostly due to complement activation and is dependent on CD20 expression levels.
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