Effects of Ibrutinib on Rituximab and GA-101 Induced Antibody-Dependent Cell Cytotoxicity (ADCC) in Lymphoma Cells in Vitro

Introduction: Ibrutinib is a first-in-class, oral, once-daily, small molecule that covalently binds to and inhibits Bruton's tyrosine kinase (BTK), an essential enzyme in the B-cell receptor signaling pathway. Ibrutinib has shown single-agent efficacy and good tolerability in patients with various B-cell malignancies, and has been approved for treatment of previously treated chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, ibrutinib is also being evaluated in combination with several other anticancer agents, including the anti-CD20 monoclonal antibodies rituximab and ofatumumab, as well as obinutumumab (GA-101), a next-generation anti-CD20 antibody recently approved to treat CLL. A previous report suggested that the combination of ibrutinib and rituximab under certain conditions results in antagonistic effects on ADCC, a possible mechanism of action of rituximab (Kohrt HE, et al. Blood. 2014;123:1957-1960). However, clinical data in high-risk CLL in combination with rituximab showed high response rates compared with available data on the single agents (Burger JA, et al. Blood. 2012;120 (21):187). As the variance could be due to experimental conditions that do not replicate well the unique pharmacokinetics (PK; such as short half-life and lower Cmax; Advani RH, et al. JCO. 2013;31:88-94) and selectivity profile of ibrutinib under clinical dosing conditions, we modified the assay to better mimic the physiological PK profile, and further, to test these possible effects of ibrutinib in combination with the newer anti-CD20 antibody obinutumumab.

Methods:Peripheral blood mononuclear cells (PBMC) isolated from different donors were used as effector cells in this study, in addition to the purified natural killer (NK) cells used previously, and were treated as follows: (1) no treatment for 24 hrs (control), (2) pretreatment with 200nM ibrutinib for 2hrs (2 hr T), (3) pretreatment with 200nM ibrutinib for 2 hours with a 24-hour washout period (2hr T/24 hr WO) which mimics the physiological PK profile of ibrutinib, (4) pretreatment with 200nM ibrutinib for 24 hours (24 hr C). ADCC assays were then conducted on target lymphoma cell lines (Ramos and Daudi) with rituximab (10 µg/ml) or obinutuzumab (10 µg/ml) alone or in combination with 200nM ibrutinib for 3 hours. Percent-specific lysis was measured using the Calcein AM assay. BTK occupancy was measured as described previously (Honigberg LA, et al. PNAS. 2010;107:13075-13080). Cytokine levels were measured using the 10-plex Proinflammatory Panel 1 from Meso Scale Discovery per recommended procedures.

Results: Continuous exposure to ibrutinib reduced rituximab-mediated ADCC in the studied lymphoma cell lines. However, under physiologically-relevant conditions where cells were exposed to ibrutinib for 2 hrs and then washed off, the antagonistic effect on rituximab-mediated ADCC was minimized. Even with a 2 hr exposure, BTK was still fully occupied at 24 hrs after washoff, suggesting that the chronic effect may be due at least in part to other targets of ibrutinib (Honigberg LA, et al. PNAS. 2010;107:13075-13080) not continuously inhibited under physiological conditions. Obinutuzumab induced a higher level of ADCC than seen with rituximab in the same cell lines, and ibrutinib pretreatment had minimal effects on obinutuzumab-mediated ADCC, especially at 2 hr exposure with or without ibrutinib washout. Figure 1 shows typical data in Daudi cells, derived from a Burkitt's patient and not appreciably killed by ibrutinib at 200nM in 24 hrs. Cytokine measurements showed that interferon-gamma was reduced by chronic exposure to ibrutinib but this effect was also mitigated by 2 hr ibrutinib exposure followed by 24 hr washoff.

Figure 1.

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ADCC Induced by Rituximab (Left) and Obinutuzumab (GA-101, Right) in Daudi Cells. 2 hr T and 24 hr C Represent Continuous Pre-Exposure to Ibrutinib (200 nm), While 2hr T/24 hr WO Represents the 2 hr Exposure to Ibrutinib Followed by 24 hr Washout Period

Figure 1.

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ADCC Induced by Rituximab (Left) and Obinutuzumab (GA-101, Right) in Daudi Cells. 2 hr T and 24 hr C Represent Continuous Pre-Exposure to Ibrutinib (200 nm), While 2hr T/24 hr WO Represents the 2 hr Exposure to Ibrutinib Followed by 24 hr Washout Period

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Conclusions: These results demonstrate that, when administered under physiologically-relevant conditions in vitro, the effects of ibrutinib on anti-CD20 antibody-induced ADCC are substantially mitigated, particularly with obinutuzumab (GA-101).