Ibrutinib antagonizes rituximab-dependent NK cell–mediated cytotoxicity

To the editor:

Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) with promising activity in CD20⁺ B-cell malignancies including recent US Food and Drug Administration approval in mantle cell lymphoma.¹  Given the homology between BTK and interleukin-2 inducible tyrosine kinase (ITK), we recently confirmed that ibrutinib irreversibly binds ITK.²  ITK expression in Fc rceptor (FcR)-stimulated natural killer (NK) cells leads to increased calcium mobilization, granule release, and cytotoxicity.³  As rituximab is a backbone of lymphoma therapy, with mechanisms of action including antibody-dependent cell-mediated cytotoxicity (ADCC), as well as direct induction of apoptosis and complement-dependent cytotoxicity⁴  and FcR stimulation is requisite for ADCC, we investigated if ibrutinib influenced rituximab’s anti-lymphoma activity in vitro by assessing NK cell interferon-γ secretion, degranulation by CD107a mobilization, and cytotoxicity by chromium release using CD20⁺ cell lines and autologous patient samples with chronic lymphocytic leukemia (CLL), as well as in xenotransplant lymphoma athymic, ν/ν mouse models, as previously described.⁵  Trastuzumab and human epidermal growth factor receptor 2⁺ (HER2⁺) breast cancer cell lines provided an ADCC control, and CGI-1746, lacking ITK inhibition, represented a BTK selective control.⁶ 

We found that FcR-stimulated NK cells following exposure to rituximab-coated lymphoma cells express high and moderate levels of ITK and BTK, respectively. Ibrutinib inhibited both rituximab- and trastuzumab-induced NK cell cytokine secretion in a dose-dependent manner at 0.1 and 1 μM of ibrutinib in vitro (Figure 1A; *P = .009, **P = .001, ***P < .001). Similarly, ibrutinib prevented FcR-stimulated NK cell degranulation by ∼60% and ∼90% at 0.1 and 1 μM, respectively (Figure 1B; *P = .013, **P = .017, ***P = .002, ****P = .001). Despite direct in vitro cytotoxicity due to ibrutinib independent of NK cells, NK cell–mediated cytotoxicity of both rituximab-coated, chromium-labeled lymphoma cells and trastuzumab-coated, chromium-labeled breast cancer cells was inhibited in an ibrutinib dose-dependent manner (Figure 1C; *P < .001, **P = .045, ***P = .036, ****P = .010). We hypothesize that a dose effect is seen in Figure 1C with trastuzumab and not with ibrutinib as a result of increasing apoptosis, which is a direct dose-dependent effect of ibrutinib monotherapy. Therefore, in vitro, as higher doses of ibrutinib are combined with rituximab, the direct effect of BTK inhibition outweighs the inhibition of NK cell’s ability to perform ADCC. In contrast, in vitro, CGI-1746 had no antagonistic effect on ADCC against rituximab-coated lymphoma cell lines or autologous CLL cells (Figure 1D; *P < .001). Abrogation of trastuzumab-dependent NK cell–mediated cytotoxicity was confirmed in vivo with concurrent ibrutinib daily dosing for 2 weeks during trastuzumab treatment (4 doses), as measured by tumor growth and survival (Figure 1E, *P < .001; Figure 1F, *P = .18). Concurrent ibrutinib daily dosing for 2 weeks during 4 doses of rituximab therapy similarly antagonized rituximab’s efficacy, with anti-lymphoma activity of the combination equivalent to ibrutinib monotherapy (Figure 1G, *P = .049, **P = .032; Figure 1H, *P = .29). Sequential ibrutinib for 1 week followed by 2 doses of rituximab or sequential rituximab (2 doses) followed by ibrutinib for 1 week resulted in restored anti-lymphoma activity superior to concurrent combination therapy of ibrutinib for 2 weeks and 4 doses of rituximab (Figure 1I, *P < .001; Figure 1J, *P < .001).

The combination of a promising novel agent and current standard of care for the treatment of B-cell lymphomas is currently being explored in multiple phase 2 and 3 trials. Surprisingly, our preclinical investigation of the combination of ibrutinib and rituximab results in antagonistic effects. We demonstrate that the abrogation of both rituximab’s and trastuzumab’s antitumor efficacy is a result of ibrutinib’s inhibition of FcR-stimulated NK cell function, specifically ADCC. Selective BTK inhibitors or alternative ibrutinib dosing schedules, sequential vs concurrent, may preserve the anti-lymphoma efficacy of both agents.