Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

Abstract

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

Keywords: ADCC; ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP; ADCP, antibody-dependent cellular phagocytosis; NHL, non-Hodgkin's lymphoma; NK, natural killer; ibrutinib; idelalisib; kinase inhibitors; monoclonal antibodies; obinutuzumab; rituximab; trastuzumab.

Figure 1.

Effect of kinase inhibitors ibrutinib, idelalisib, NVP-BEZ235, and LY294002 on the ADCC effect of trastuzumab (A), rituximab (B) and obinutuzumab (C). ADCC was performed using NK-92-CD16 cells as effectors and BT474 cells (trastuzumab) or RL cells (rituximab and obinutuzumab) as target cells, with the corresponding antibody at 1 μg/mL final. The effector : target (E:T) ratio = 5:1 for trastuzumab or 2:1 for rituximab and obinutuzumab. Ibrutinib, idelalisib, NVP-BEZ235 or LY294002 were added simultaneously at 10 μM final. Means ± SD of 3 independent experiments are shown. *P < 0.05; **P < 0,01.

Figure 2.

Dose response effect of ibrutinib on ADCC effect of trastuzumab (A), rituximab (B) and obinutuzumab (C). ADCC was performed using NK-92-CD16 cells as effectors and BT474 cells (trastuzumab) or RL cells (rituximab and obinutuzumab) as target cells, with the corresponding antibody at 1 μg/mL final, in the presence of indicated concentrations of ibrutinib. E:T = 5:1 for trastuzumab and E:T = 2:1 for rituximab and obinutuzumab. A representative experiment is shown.

Figure 3.

Effect of tyrosine kinase inhibitors ibrutinib, idelalisib, NVP-BEZ235, and LY294002 on the ADCP effect of trastuzumab (A), rituximab (B) and obinutuzumab (C). ADCP was performed using neutrophils as effectors and BT474 cells (trastuzumab) or RL cells (rituximab and obinutuzumab) as target cells, with the corresponding antibody at 1 μg/mL final. The effector : target (E:T) ratio = 5:1 for trastuzumab or 2:1 for rituximab and obinutuzumab. Ibrutinib, idelalisib, NVP-BEZ235 or LY294002 were used at 10 μM final.

Figure 4.

Effect of tyrosine kinase inhibitors ibrutinib, idelalisib, NVP-BEZ235, and LY294002 on phagocytic activity of normal human neutrophils. Phagocytic activity was evaluated using the FagoFlowEx® Kit after the stimulation of neutrophils with E. coli bacteria, in the presence of 10 μM of ibrutinib, idelalisib, NVP-BEZ235 or LY294002. Phorbol 12-myristate 13-acetate (PMA) was used as positive control. Median fluorescence intensity (MFI) is reported. Means ± SD of 2 independent experiments are shown.

Figure 5.

In vivo effect of combinations of antibodies with ibrutinib: (A) combination of rituximab and obinutuzumab with ibrutinib; (B) combination of trastuzumab with ibrutinib. The human follicular line RL (Fig. 5A) and the human breast cancer line BT474 (Fig. 5B) were grown as subcutaneous xenografts in SCID mice. Treatment with antibodies (rituximab 30 mg/kg/week, obinutuzumab 30 mg/kg/week, trastuzumab 25 mg/kg/week) and ibrutinib (25 mg/kg/day, 5 d a week) was initiated when tumor volume reached 100 mm³. Medians ± SEM are shown (n = 10 mice/group). *P < 0.05; **P < 0.01.