Abstract
Background: Although survival rates in Mantle cell lymphoma (MCL) have improved, by addition of the anti-CD20-monoclonal antibody (anti-CD20-mAb) rituximab, the disease is still regarded incurable and novel combinations are required to improve outcome. Ibrutinib, a BTK-inhibitor, has shown activity in MCL and is currently combined with anti-CD20 therapy in clinical trials. However, preclinical data on CLL has shown reduced effect of anti-CD20-mAb when combined with ibrutinib in vitro, possibly due to reduced activation of NK cells. (Da Roit et al., 2015) Consequently, we investigated how ibrutinib affects the cytotoxic effect of anti-CD20-mAb on MCL cell lines in vitro. We also investigated if the addition of lenalidomide, a potential sensitizer of anti-CD20-ab would overcome an inhibitory effect of ibrutinib.
Methods: PBMC from healthy donors were pretreated with ibrutinib 0/0.01/0.05/0.1/1/5 µM, 1h, 37°C before incubation with anti-CD20-opsonized (rituximab/obinutuzumab, 1µM, 20 min, 37°C), CFSE-labelled MCL cell lines (Jeko-1 and REC), o/n, 37°C. Ratio eff: target 100:1. On day 2, samples were stained with 7-AAD and analyzed in flow cytometry (iQue screener plus®). Rate of cell death was calculated from rate of 7-AAD out of CFSE-positive cells compared to control (PBMC + anti-CD20-mAb+target cells). In secondary experiments, PBMC was incubated with lenalidomide (0/0.01/0.05/1 µM, 20 min, 37°C) before the addition of ibrutinib 1 µM and subsequent proceeding according to protocol as described above.
Results: A significant lower rate of cell death compared to control could be observed in Jeko-1 with rituximab and ibrutinib (figure 1a) at 0.5µM: (25 ±5.56%, p=0.0227), 1 µM (20±3.03%, p=0.0241) and 5 µM (21±1.65% , p=0.0123) and with obinutuzumab and ibrutinib (figure 2) at 0.1 µM (48±0.26%,p=0.0032), 1 µM (17±0.58%, p=0,0045) and 5 µM (11±1.09%, p=0.0078). Further, obinutuzumab was associated with enhanced cell death compared to rituximab in Jeko-1, (149±100% vs 31±25.4%, p=0.0296). For REC, a significant lower cell death was observed at 5µM ibrutinib(27±2.7%), p=00012) in series with rituximab. In series with obinutuzumab, cell death was significant lower at 0.5 µM (86 ±0.99%, p= 0.0437) ibrutinbi but not for higher concentrations. Lenalidomide did not affect the rate of cell death (data not shown).
Conclusion: Our study shows that pretreatment of PBMC with ibrutinib negatively affected the cytotoxic effect of anti-CD20-mAb rituximab and obinutuzumab on MCL in vitro. Further, obinutuzumab was associated with enhanced cytotoxicity compared to rituximab. The addition of lenalidomide, a potential synergizer of anti-CD20-ab did not overcome the inhibitory effect of ibrutinib. Further studies, i.e. of sequential administration, may reveal how to optimize the combination of ibrutinib with anti-CD20-ab to improve outcome in MCL patients.
No relevant conflicts of interest to declare.
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