Effect of the Btk inhibitors, ibruitinb and AVL-292, and the Syk inhibitor, GS9973, on macrophage-mediated anti-tumor activity of rituximab

BORGE MERCEDES; COLADO ANA; PODAZA ENRIQUE; AMEJÚN MARÍA BELÉN; BEZARES FERNANDO RAIMUNDO; BEZARES FERNANDO RAIMUNDO; GIORDANO MIRTA; GAMBERALE ROMINA

Sydney

Workshop; Young Investigator Meeting. XVI International Workshop on Chronic Lymphocytic Leukaemia; 2015

In the past few years several orally administrated inhibitors targeting kinases involved in BCR signaling have been developed and entered the clinical stage showing promising efficacy and excellent tolerability. The Btk inhibitor, ibrutinib, has been approved in USA last year for CLL treatment and the combination of ibrutinib and other kinases inhibitors with targeted therapies such as the anti-CD20 antibody rituximab, is now being explored in clinical trials. Anti-CD20 antibodies are thought to act through different mechanisms but there is strong evidence suggesting that macrophages, acting through Fc-receptors, are the key immune effector cell for anti-CD20 therapeutic effect in vivo (1,2). Given that kinase inhibitors might affect Fc-receptor signaling, the aims of our study were: i) to evaluate the effect of ibrutinib on macrophage-mediated phagocytosis of rituximab-coated leukemic cells, ii) to assess the effect AVL-292 and GS9973 on macrophage phagocytosis, and iii) to study if AVL-292 and GS9973 modulate CD20 expression on leukemic cells.Human monocytes-derived macrophages were treated with or without ibrutinib for 30 minutes and then cultured for 1 hour with CFSE-labeled CLL cells coated or not with rituximab. Then, the proportion of macrophages that have taken up CFSE-labeled CLL cells (CFSE+ macrophages) were scored by flow cytometry and confocal microscopy. As shown in Figure 1A and 1B we observed that ibrutinib inhibited macrophage-mediated phagocytosis of rituximab-coated CLL cells at 0.5 and 5µM (n=17, p˂0.01). We confirmed that ibrutinib impairs the phagocytosis but not the binding of rituximab-coated CLL cells to macrophages by performing the same experiment at 4oC (Figure 1C). Interestingly we found that the inhibition of phagocytosis was no longer present when ibrutinib was washed out and macrophages were cultured in absence of the drug for 7 or 24 hours (Figure 1D). Given that ibrutinib binds to Btk in an irreversible manner this observation suggests the existence of a reversible-target of ibrutinib in macrophages other than Btk(3).Our next aim was to evaluate if AVL-292 or GS9973, two kinase inhibitors currently being tested in clinical trials for CLL, could also affect macrophage phagocytosis. We found that GS9973, a Syk inhibitor, impaired phagocytosis at 0.5µM while the Btk inhibitor, AVL-292, only impaired the phagocytosis at 5µM (Figure 1E). Finally, we found that the modulation of CD20 expression on CLL cells by ibrutinib, AVL-292 and GS9973 was heterogeneous among our cohort of patients, and that while CD20 expression was downregulated by ibrutinib in accordance with a previous report (4), it was not significantly modify by AVL-292 and GS9973 (Figure 1F).Our results show that kinases inhibitors interfere with the macrophage-mediated anti-tumour activity of rituximab suggesting that the sequential administration of kinases inhibitors and rituximab, and not the concurrent treatment with these agents, might enhance their anti-tumor activity in CLL patients. Interestingly we found that the Btk inhibitor AVL-292 at clinical relevant doses (0.5 µM) did not significantly impair macrophage-phagocytosis suggesting that this inhibitor might have less interference with the anti-tumor effect of rituximab in vivo.  1.J. Uchida et al., J Exp Med199, 1659 (Jun 21, 2004).2.M. Taskinen, M. L. Karjalainen-Lindsberg, H. Nyman, L. M. Eerola, S. Leppa, Clin Cancer Res13, 5784 (Oct 1, 2007).3.M. Borge et al., Haematologica2015 Jan 23. [Epub ahead of print].4.K. Bojarczuk et al., Leukemia28, 1163 (May, 2014).