Anti-CD20 and B-Cell Receptor-Mediated Growth Inhibition and Apoptosis: A Preclinical Study of Ibrutinib and Rituximab Combination Therapy in Mantle Cell Lymphoma in Vitro and in Vivo

Intruduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor outcome and therapeutic challenge. Oral single-agent ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, elicited a response rate of 68% in phase II clinical trial and has been approved by FDA for the treatment of MCL patients who received at least one prior therapy. To increase the response rate, the combination of ibrutinib with rituximab would be an ideal regimen because: (1) Both BTK and CD20 are effective therapeutic targets for MCL; (2) CD20-dependent apoptosis occurs associated with B-cell receptor (BCR) activity. Regardless rituximab-mediated immune response (ADCC and CDC) and ibrutinib-mediated ITK inhibition (Th1 polarization), an important potential effect of ibrutinib and rituximab may enhance the direct anti-MCL cytotoxicity of the combination.

Methods and Results We investigated the effects of ibrutinib and/or rituximab in 6 established MCL cell lines and 3 freshly isolated primary MCL cells in vitro. First, ibrutinib, as well as BTK-siRNA, inhibited the growth of MCL cells. Then we found that ibrutinib induced the growth inhibition and apoptosis of most MCL cell lines and primary MCL cells in a dose-dependent manner during 5-7 days of culture (IC50 range from 1.5 to 15 µM). On the other hand, rituximab alone induced 10-30% growth inhibition and apoptosis of both established and primary MCL cells in vitro. Importantly, our results showed that ibrutinib and rituximab combination synergistically induced apoptosis of MCL cells during 5-7 days of culture in vitro. Next, our animal study confirmed the compartmental shift phenomenon mediated by ibrutinib which was similar with the data from clinical study. Briefly, SCID mice were subcutaneously implanted with human fetal bone chips (SCID-hu). After 6 weeks of bone implantation, the freshly isolated MCL cells from patients were directly engrafted into the human fetal bone chips. The engrafted MCL cells produced measurable levels of human β₂-microglobulin (β₂M) in mouse serum. Once human β₂M had been detected in mouse serum, the primary MCL-bearing SCID-hu mice were treatment with 25 mg/kg ibrutinib oral gavage daily. A transient increase of human CD5⁺CD20⁺ cells in mouse peripheral blood was detected by flow cytometry from day 8 of treatment, representing a shift of human MCL cells from human fetal bone chip to mouse peripheral blood. At this time, 10 mg/kg rituximab was intravenously administrated every 3 days for total 7 doses. Our results demonstrated that rituximab and ibrutinib combination eradicated MCL cells in vivo and kept mice survival longer than either ibrutinib or rituximab alone.

Conclusions Based on our preclinical data of rituximab and ibrutinib combination in vitro and in primary MCL-bearing SCID-hu mice, ibrutinib plus rituximab could be an effective regimen in a clinical trial of relapsed or refractory MCL.