American Association for Cancer Research
10780432ccr151304-sup-150526_1_supp_3082603_nsbs9s.pptx (551.07 kB)

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

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posted on 2023-03-31, 19:04 authored by Martin Skarzynski, Carsten U. Niemann, Yuh Shan Lee, Sabrina Martyr, Irina Maric, Dalia Salem, Maryalice Stetler-Stevenson, Gerald E. Marti, Katherine R. Calvo, Constance Yuan, Janet Valdez, Susan Soto, Mohammed Z.H. Farooqui, Sarah E.M. Herman, Adrian Wiestner

Supplementary Figure 1. Uniform CD20 loss from the surface of primary CLL cells after in vivo exposure to ibrutinib. Supplementary Figure 2. Loss of CD20 on ibrutinib and basal CD20 expression levels do not correlate with high risk prognostic factors. Supplementary Figure 3. Selective NF-κB inhibitor diminishes CD20 cell surface expression. Supplementary Figure 4. Complement regulatory proteins membrane cofactor protein and protectin are not consistently inhibited by ibrutinib.



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. Clin Cancer Res; 22(1); 86–95. ©2015 AACR.