Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

Abstract

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

Ibrutinib does not synergize with anti-CD20 antibodies in inducing cell death. (A) The MEC-1 and BJAB cell lines were treated with 1, 3 or 10 μM ibrutinib. The percentage viable cells was analyzed after 72 h using Alamar vital dye. (B) The MEC-1 and BJAB cell lines were treated with 0.1, 1 or 10 μM ibrutinib. The percentage of dead cells was analyzed by 7-AAD staining and flow cytometry at 72 h. (C, D) The MEC-1 cell line (C) or peripheral blood mononuclear cells from CLL patients (D) were treated for 48 h with the indicated concentrations of ibrutinib, in the presence or absence of 10 μg/mL rituximab (RTX), ofatumumab (OFA) or obinutuzumab (OBZ). The Alamar blue vital dye was then added and the percentage of viable cells was measured after overnight incubation. The results are the mean percentages and standard deviations of viable cells compared to untreated control, from three independent experiments. For all conditions in the presence of ibrutinib, the statistical significance indicated refers to ibrutinib treated versus equivalent samples in the absence of ibrutinib. For samples not treated with ibrutinib (0, panels C and D), statistical significance is shown for samples treated with anti-CD20 monoclonal antibodies versus untreated samples. *P<0.05; **P<0.01; ***P<0.001.

Figure 2.

Exposure to ibrutinib does not significantly affect CDC induced by anti-CD20 antibodies. (A) BJAB cells were incubated with increasing concentrations of ibrutinib for 1 h, after which 1 or 3 μg/mL ofatumumab (OFA) and 20% human serum were added. 7-AAD was added after 1 h and cell death (%7-AAD+) was measured by flow cytometry. (B) Ibrutinib was added to whole blood from normal donors (ND) and after 1 h at 37°C, CLL cells opsonized with 3 μg/mL OFA were added. The percentage of CD19+/7-AAD− viable CLL cells was measured by flow cytometry after 24 h. (C) Ibrutinib was added to whole blood from CLL patients and after 1 h at 37°C, 3 μg/mL OFA were added. The percentage of CD19+/7-AAD− viable CLL cells was measured by flow cytometry after 24 h. (D) Ibrutinib was added to whole blood from normal donors and after 1 h at 37°C, CLL cells opsonized with 3 μg/mL OFA were added; C3 deposition was measured after 1 h. Unless otherwise indicated all results are the means and standard deviations for three independent experiments. Statistical significance is indicated (*P<0.05; **P<0.01; ***P<0.001) and refers to the presence versus absence of OFA. Differences between ibrutinib-treated versus untreated samples were not statistically significant. *P<0.05; **P<0.01; ***P<0.001.

Figure 3.

Ibrutinib strongly inhibits antibody-mediated NK cell-degranulation and ADCC. (A,B): Peripheral blood mononuclear cells (PBMC) (A) or whole blood (B) from CLL patients were treated with 0.1 to 10 μM ibrutinib for 1 h, after which the indicated anti-CD20 monoclonal antibodies were added at 1 μg/mL. NK-cell degranulation was analyzed after a further 4 h of incubation, by double staining with anti-CD56-APC and anti-CD107a-PE and flow cytometry. (C,D) ADCC assays were performed by 51Cr release assays, using the DOHH-2 (C) or MEC-1 (D) cell lines as targets, in the absence or presence of the indicated anti-CD20 monoclonal antibodies at 1 μg/mL and/or increasing concentrations of ibrutinib. The results are the means and standard deviations for three independent experiments. The statistical significance (*P<0.05; **P<0.01; ***P<0.001) indicated above each bar refers to CD20 monoclonal antibody-treated versus untreated control sample, for values obtained in the absence of ibrutinib (0). Statistical significance in the presence of different doses of ibrutinib (1–10 μM) was calculated with respect to the equivalent controls in the absence of the drug. In the absence of ibrutinib, statistical significance refers to the comparison with versus without anti-CD20 antibodies. TRZ: trastuzumab; RTX: rituximab; OFA: ofatumumab; OBZ: obinutuzumab.

Figure 4.

Antibody-mediated NK-cell activation ex vivo is inhibited following in vivo ibrutinib treatment. (A) Blood samples from three patients with B-NHL were collected before the initiation of treatment (PRE), or 4 h after administration of the first 560 mg ibrutinib tablet (POST 4h). Peripheral blood mononuclear cells were purified from all samples and co-cultured with the BJAB cell line opsonized with 1 μg/mL anti-CD20 monoclonal antibodies and degranulation was analyzed 2 h later by double staining with anti-CD56-APC and anti-CD107a-PE and flow cytometry. (B) In one case, blood samples were also collected at day 21 of continuous treatment, before (POST 21 days) and 4 hour after administration of ibrutinib on day 21 (POST 21 days + 4h). The degranulation capacity of peripheral blood mononuclear cells from this patient was then measured as in (A). CTRL: no antibody; TRZ: trastuzumab; RTX: rituximab; OFA: ofatumumab; OBZ: obinutuzumab.

Figure 5.

Ibrutinib inhibits antibody-dependent phagocytosis mediated by both macrophages and PMN. (A) In vitro differentiated macrophages were pretreated with ibrutinib for 1 h, after which CFSE-labeled CLL cells either untreated (CTRL), or opsonized with 10 μg/mL anti-CD20 or control trastuzumab (TRZ) were added. After 2 h the cells were stained with CD19-APC and CD11b-PE and phagocytosis was measured by flow cytometry and triple fluorescence analysis. Phagocytosis was defined as the percentage of CD11b+ cells that were CFSE+/CD19−. (B) Whole blood from normal donors was incubated with ibrutinib for 1 h, after which CLL cells, either untreated (CTRL), or opsonized with 10 μg/mL obinutuzumab (OBZ) or control TRZ monoclonal antibody were added. PMN activation after 6 h was measured by staining with CD11b-PE and flow cytometry. (C) Whole blood from normal donors was incubated with ibrutinib for 1 h, after which PKH26-labeled-CLL cells opsonized with 10 μg/mL OBZ or control TRZ monoclonal antibody were added. Phagocytosis was measured after 20 h by staining with anti-CD15-FITC and anti-CD19-APC and flow cytometry. All results are the means and standard deviations for three independent experiments. The statistical significance was calculated as defined in the legend to Figure 1. *P<0.05; **P<0.01; ***P<0.001.

Figure 6.

Idelalisib (IDE) also inhibits cell-mediated activities of CD20 monoclonal antibodies (mAb), but more weakly than ibrutinib (IBRU). The following immune-mediated mechanisms of anti-CD20 monoclonal antibodies were studied in the absence or presence of 10 μM IBRU or 10 μM IDE: (A) Ofatumumab (OFA)-dependent CDC of CLL cells in normal donor whole blood, (B) NK-cell degranulation in peripheral blood mononuclear cells induced by obinutuzumab (OBZ)-opsonized CLL cells, (C) OFA- and OBZ-dependent ADCC of the MEC-1 cell line, (D) PMN activation in whole blood in response to OBZ-opsonized CLL and (E) phagocytosis by macrophages of OFA-opsonized CLL. All results are the means and standard deviations for three independent experiments. *P<0.05; **P<0.01; ***P<0.001.