No abstract

Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcγRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-γ/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-γ had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.

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 (r2 = 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.

No abstract

Ofatumumab (OFA) is a human anti-CD20 Ab approved for treatment of fludarabine-refractory B chronic lymphocytic leukemia (B-CLL). The efficacy of different immunotherapeutic strategies is best investigated in conditions that are as physiologic as possible. We have therefore compared the activity OFA and rituximab (RTX), alone or in combination with chemotherapeutic agents in unmanipulated whole blood assays, using flow cytometry. OFA (10–100 μg/ml) lysed B-CLL targets in whole blood more efficiently and with faster kinetics than RTX, with a mean 56% lysis at 24 h compared with 16%. This activity of OFA was fully complement dependent, as shown by >99% inhibition by anti-C5 Ab eculizumab and a lack of NK cell activation in whole blood. OFA-mediated NK cell activation was blocked by complement. OFA-mediated lysis could be increased an additional 15% by blocking CD55 and CD59 complement inhibitors. Interestingly, OFA-mediated lysis correlated significantly with CD20 expression levels (r2 = 0.79). OFA showed overlapping dose response curves similar to those for RTX in phagocytosis assays using either human macrophages or neutrophils. However, phagocytosis was inhibited in the presence of serum or whole blood. Finally, combined treatment with mafosfamide and fludarabine showed that these therapeutic drugs are synergistic in B-CLL whole blood assays and show superior activity when combined with OFA compared with RTX. These results confirm in B-CLL samples and in physiologic conditions the superior complement mediated cytotoxicity induced by OFA alone compared with RTX, the lack of NK cell activation, and phagocytosis in these conditions and suggest effective chemoimmunotherapy strategies using this new generation anti-CD20 Ab.