Ibrutinib impairs the phagocytosis of rituximab-coated leukemic cells from Chronic Lymphocytic Leukemia patients by human macrophages.

BORGE MERCEDES; ALMEJUN MARIA BELÉN; PODAZA ENRIQUE; COLADO ANA ; FERNANDEZ GRECCO HORACIO; CABREJO MARÍA; BEZARES RAIMUNDO FERNANDO; GIORDANO MIRTA; GAMBERALE ROMINA

HAEMATOLOGICA

FERRATA STORTI FOUNDATION

Año: 2015 vol. 100

0390-6078

Ibrutinib is an oral irreversible inhibitor of the Bruton Tyrosine Kinase (Btk) which hasshown promising efficacy and excellent tolerability in patients with Chronic LymphocyticLeukemia (CLL) and other B cell malignancies.1 Ibrutinib inhibits BCR signaling, impairschemokine-mediated adhesion and migration, and induces moderate levels of apoptosison CLL cells.2,3 It also reduces TCR downstream activation on Th2 cells by targetinganother kinase, the interleukin-2?inducible kinase (Itk).4 The combination of Ibrutinib withdifferent targeted therapies is now being explored. In a single arm study, the combinationof Ibrutinib and rituximab in patients with high-risk CLL was generally well-tolerated andresulted in an OR rate of 95% and a PFS of 78% at 18-months,5 the added effect ofrituximab to the Ibrutinb therapy is being tested in a randomized trial testing Ibrutinb versusIbrutinb and rituximab (NCT02007044). Given that macrophages are the most importanteffector cells in the anti-CD20-therapeutic effect in murine models6,7 and they probablyplay a key role in human anti-CD20 therapy8,9, we asked whether Ibrutinib could interferewith the capacity of human macrophages to mediate phagocytosis of rituximab-coated CLLcells. Our results showed that Ibrutinib impairs the phagocytosis of rituximab-opsonizedCLL cells by human macrophages.Macrophages differentiated from healthy peripheral blood monocytes were treatedwith or without Ibrutinib for 30 minutes and then cultured for 1, 2 or 3 hours with CFSElabeledCLL cells or rituximab-coated CFSE-labeled CLL cells. Then, cells were tripsinizedand the proportion of macrophages that have taken up CFSE-labeled CLL cells (CFSE+macrophages) were scored by flow cytometry and verified using confocal microscopy, aspreviously described.10 As expected, we found that the cultures with rituximab-coated CLLcells showed the highest percentage of CFSE+ macrophages, which increase in a timedependent manner (open circles in Figure 1A). Ibrutinib was able to reduce these values inall the times evaluated (solid circles in Figure 1A). Low percentages of CFSE+macrophages were obtained in cultures with uncoated CLL cells, which were not modifiedby Ibrutinib (open and solid squares, both at the bottom of the graph in Figure 1A). Inaddition, we found that Ibrutinib diminishes the percentage of CFSE+ macrophages in thecultures with rituximab-coated cells in a dose dependent manner (Figure 1B), which wasnot associated to a decreased viability of the macrophages (not shown). Moreover, theinhibitory effect of Ibrutinib was not limited to rituximab since comparable results wereobtained when campath-coated CFSE-labeled CLL cells were employed (Figure 1C).Similar results were found when macrophages from CLL patients were used: mean±SE ofthe % of CFSE+ macrophages: 26.8 ± 2.1 vs, 17.3 ± 2.7 vs 10.8 ± 0.7 for macrophagescultured with rituximab-coated CFSE-labeled CLL cells alone, with 0.5μM or 5μM ofIbrutinib (n= 6). Representative dot plots are shown in Figure 1D. The results obtained byflow cytometry analysis were validated by confocal microscopy quantifying the number ofmacrophages that engulfed at least one tumor target cell (Figure 1E). A representativeexperiment is shown in Figure 1F. In addition, by performing a binding assay at 4oC, weconfirmed that Ibrutinib did not reduce the binding of rituximab-coated CFSE-labeled CLLcells to macrophages (Figure 1G). It should be mentioned that although Ibrutinib 5 μMseems to increase the binding of CLL to macrophages, this difference was not statisticallysignificant.We next asked if macrophages could recover their phagocytic capacity onceIbrutinib was removed from the culture. To this aim macrophages were treated withIbrutinib for 2 hours and then either washed- or not washed- out, and immediately used forthe phagocytosis assay. Additionally, washed-out macrophages were cultured in Ibrutinibfreemedium for 7, 24 and 48hs before performing the phagocytosis assay. As it is shownin Figure 1H, the phagocytosis of rituximab-coated CLL cells by macrophages wasimpaired when Ibrutinib was present (T0 without wash-out) or immediately after Ibrutinibwas removed (T0 wash-out). Interestingly when Ibrutinib was washed out, macrophagesrecovered their phagocytic capacity after 7hs (T 7hs wash-out), 24hs (T 24hs wash-out)and 48 hours of culture (not shown) in drug-free medium (Figure 1H), showing a reversibleeffect on macrophages. In contrast, we confirmed a previous report by Honigberg, L. et alshowing that Ibrutinib was able to irreversibly inhibit BCR-mediated activation of CLL cells(not shown). Thus, while Ibrutinib binds to Btk in an irreversible manner on CLL cells, itsinhibitory effect on the phagocytosis of rituximab-coated CLL cells by macrophages wasreversible and needed the continuous presence of the drug, suggesting the existence of areversible-target, other than Btk. In line with this, Ibrutinib was shown to inhibit otherkinases11 including Hck, Fgr, and Lyn, which were described to be involved in Fc-mediatedphagocytosis on macrophages.12 So, the possibility exists that Ibrutinib impairment ofmacrophage phagocytosis involves the by binding to one or more of these kinases in areversible manner.Our results are in line with those recently reported by Kohrt, H.E. et al13 showingthat Ibrutinib prevented NK cell mediated cytotoxicity of antibody-coated CLL cells in vitro.They also found that the concurrent treatment with Ibrutinib and rituximab or trastuzumabreduces the therapeutic efficacy of both anti-CD20 antibodies in a mouse model, while thesequential treatment with Ibrutinib and rituximab restored its anti-lymphoma activity.Moreover, similar results showing Ibrutinib-inhibition of rituximab-dependent functions onmacrophages and neutrophils were published in this Journal while this manuscript wasunder evaluation.14 Although the combination of rituximab and Ibrutinib has shown to beactive and safe in high-risk CLL patients, 5 our results and those obtained by Kohrt et al13and by Da Roit et al, 14 suggest that the sequential administration of Ibrutinib followed byrituximab, and not the concurrent treatment with these agents, might enhance their antitumoractivity in vivo and improve CLL therapy.