Interactions between Ibrutinib and Anti-CD20 Antibodies: Competing Effects on the Outcome of Combination Therapy

Abstract

Purpose: Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox.

Experimental design: Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs.

Results: We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P < 0.001) compared with baseline. Concomitantly, CD20 mRNA was decreased concurrent with reduced NF-κB signaling. An NF-κB binding site in the promoter of MS4A1 (encoding CD20) and downregulation of CD20 by NF-κB inhibitors support a direct transcriptional effect. Ex vivo, tumor cells from patients on ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P < 0.001); however, opsonization by the complement protein C3d, which targets cells for phagocytosis, was relatively maintained. Expression of decay-accelerating factor (CD55) decreased on ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy.

Conclusions: Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation.

Figure 1

In vivo ibrutinib treatment decreases CD20 expression on circulating CLL cells. A-B, flow cytometric quantification of the change in CD20 expression on leukemic (CD19+, CD5+) cells in the peripheral blood of CLL patients (A) after a single dose (day 2) of ibrutinib (n=12) and (B) on days 28 and 56 (n=22) compared to pre-treatment. Lines depict median values. C, CD20 antibody binding capacity (ABC) values of circulating CLL cells from patients before ibrutinib treatment and at 6 months (n=27). D, Percent change in CD20 ABC at 6 months (n=27). ABC measurements were made on fresh samples using QuantiBrite beads. P values were calculated using the matched-pairs Wilcoxon signed rank test.

Figure 2

In vivo ibrutinib treatment reduces CD20 expression on bone marrow-resident CLL cells. A, bone marrow immunohistochemistry staining for CD20 and CD79a in specimens taken from the same patient pre-ibrutinib, at 2 months (day 56) and 6 months on treatment. CD79a staining is shown for comparison of CLL infiltration in matched areas of the bone marrow. B, semi-quantitative assessment of CD79a and CD20 staining intensity in bone marrow specimens during treatment (n=11). C, CD20 antibody binding capacity (ABC) values at 2 months (left panel) and 6 months (right panel) in bone marrow of patients treated with ibrutinib (n=13). ABC measurements were made on fresh samples using QuantiBrite beads. P values were calculated using the matched-pairs Wilcoxon signed rank test. The whiskers on all box plots depict the range (min and max values).

Figure 3

Inhibition of CD20 mRNA expression by ibrutinib correlates with reduced NF-κB activity. A, expression of CD20 mRNA was quantified by RT-PCR in CD19+ PBMCs on day 2 (n=8) and after 1 month (d28, n=12) on ibrutinib. The % change on treatment compared to baseline is shown. B, correlation between CD20 mRNA (MS4A1 gene) expression and CD20 cell surface expression on CLL cells on day 28, n=8. C, correlation between NF-κB signature score and CD20 gene expression. LN core biopsies (day 2, n=11) are shown as triangles while day 28 peripheral blood samples are represented as circles (n=12). Panels B and C are shown on a log2 scale. P values for all comparisons were calculated using the matched-pairs Wilcoxon signed rank test. Correlations were determined using Pearson's test. The whiskers on all box plots depict the range (min and max values).

Figure 4

Ibrutinib decreases ofatumumab-mediated complement-dependent cytotoxicity. A, flow cytometric analysis of complement-dependent cytotoxicity (CDC) mediated by ofatumumab in 33% normal human serum (NHS) against CLL cells obtained from 12 patients before treatment (Pre), the day after treatment began (d2), and at 2 and 6 months. B, correlation of ibrutinib's effect on CD20 expression (x axis) and CDC (y axis) in the patients shown in panel a, n=12. C, CLL samples from the ten patients with >20% decrease in CD20 expression and CDC were reassessed for CD20 expression and susceptibility to CDC after 1-4 days of culture in AIM V media. Symbols represent mean values. P values for all comparisons were calculated using the two-tailed student's paired t test. Correlations were determined using Pearson's test. The whiskers on all box plots depict the range (min and max values).

Figure 5

Inhibition of CD55 expression by ibrutinib rescues ofatumumab-mediated complement opsonization. A, correlation between cell lysis and the degree of C3d opsonization of CLL cells exposed to ofatumumab and normal human serum ex vivo. CLL cells were obtained from patients (n=16) pre-treatment and at 2 months on ibrutinib. B, the % change in ofatumumab-mediated CDC and C3d opsonization on ibrutinib compared to baseline (n=16). C, the mean fluorescence intensity of CD55 on the surface of CLL cells obtained from patients before ibrutinib (pre) and on day 56. (n=20). D, correlation between the % change in CD55 expression and C3d opsonization (n=16). P values for all comparisons were calculated using the two-tailed student's paired t test. Correlations were determined using Pearson's test. The whiskers on all box plots depict the range (min and max values).

Figure 6

Ibrutinib reduces ofatumumab-induced loss of CD20 by inhibiting trogocytosis. A, representative flow plots showing transfer of ofatumumab-AlexaFluor 594-bound CD20 complexes to THP1 monocytes pre-treated with ibrutinib or vehicle. B, quantification of the effect of ibrutinib and PMA pre-treatment on the trogocytic loss of cell surface CD20, obtained from three independent experiments (n=9). P values were calculated using the two-tailed student's paired t test. The whiskers on all box plots depict the range (min and max values).