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

BORGE MERCEDES; ALMEJÚN MARÍA BELÉN; PODAZA ENRIQUE; COLADO ANA; RISNIK DENISE; FERNANDEZ GRECCO HORACIO; CABREJO MARÍA; 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 inBCR signaling have been developed and entered the clinical stage showing promisingefficacy and excellent tolerability. The Btk inhibitor, ibrutinib, has been approved in USAlast year for CLL treatment and the combination of ibrutinib and other kinases inhibitors withtargeted therapies such as the anti-CD20 antibody rituximab, is now being explored inclinical trials. Anti-CD20 antibodies are thought to act through different mechanisms butthere is strong evidence suggesting that macrophages, acting through Fcγ-receptors, are thekey immune effector cell for anti-CD20 therapeutic effect in vivo [1,2]. Given that kinaseinhibitors might affect Fcγ-receptor signaling, the aims of our study were: i) to evaluate theeffect 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 ifAVL-292 and GS9973 modulate CD20 expression on leukemic cells. Human monocytesderivedmacrophages were treated with or without ibrutinib for 30 minutes and then culturedfor 1 hour with CFSE-labeled CLL cells coated or not with rituximab. Then, the proportion ofmacrophages that have taken up CFSE-labeled CLL cells (CFSE+ macrophages) were scoredby flow cytometry and confocal microscopy. As shown in Figure 1A and 1B we observedthat ibrutinib inhibited macrophage-mediated phagocytosis of rituximab-coated CLL cells at0.5 and 5M (n=17, p0.01). We confirmed that ibrutinib impairs the phagocytosis but not thebinding of rituximab-coated CLL cells to macrophages by performing the same experiment at4oC (Figure 1C). Interestingly we found that the inhibition of phagocytosis was no longerpresent when ibrutinib was washed out and macrophages were cultured in absence of the drugfor 7 or 24 hours (Figure 1D). Given that ibrutinib binds to Btk in an irreversible manner thisobservation suggests the existence of a reversible-target of ibrutinib in macrophages otherthan Btk [3]. Our next aim was to evaluate if AVL-292 or GS9973, two kinase inhibitorscurrently being tested in clinical trials for CLL, could also affect macrophage phagocytosis.We found that GS9973, a Syk inhibitor, impaired phagocytosis at 0.5M while the Btkinhibitor, AVL-292, only impaired the phagocytosis at 5M (Figure 1E). Finally, we foundthat the modulation of CD20 expression on CLL cells by ibrutinib, AVL-292 and GS9973was heterogeneous among our cohort of patients, and that while CD20 expression wasdownregulated by ibrutinib in accordance with a previous report [4], it was not significantlymodify by AVL-292 and GS9973 (Figure 1F). Our results show that kinases inhibitorsinterfere with the macrophage-mediated anti-tumour activity of rituximab suggesting that thesequential administration of kinases inhibitors and rituximab, and not the concurrenttreatment 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 M) didnot significantly impair macrophage-phagocytosis suggesting that this inhibitor might haveless interference with the anti-tumor effect of rituximab in vivo.