Abstract

A number of monoclonal antibodies (mAb) are presently in development or approved for CD20-directed immunotherapy. Ofatumumab, a human IgG1 anti-CD20 mAb, is currently being evaluated in phase III clinical trials for B-CLL and FL, and in phase II clinical trials for rheumatoid arthritis (RA). Rituximab, a chimeric IgG1 anti-CD20 mAb, has been approved for 1st line treatment of CD20-positive B cell lymphomas alone or in combination with chemotherapy, and in RA. Virtually all rituximab-treated patients relapse after single-agent treatment. The clinical efficacy of rituximab might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising anti-lymphoma effects. We studied the influence of statins on ofatumumab- and rituximab-mediated cytotoxicity. Surprisingly, B cells incubated with statins showed decreased rather than increased CD20 mAb-mediated complement-dependent cytotoxicity (CDC) in cell viability assays. However, cell lysis of statin-treated B cells remained higher when using ofatumumab in comparison to rituximab. Statins decreased CD20 immunostaining in flow cytometry but did not affect total cellular CD20 levels when compared to non-treated cells. Incubation of B cells with other cholesterol depleting agents (methyl-β-cyclodextrin (MβCD) and berberine) established that the presence of plasma membrane cholesterol and not lipid rafts may be required for CD20 mAb-mediated CDC. Cholesterol restitution reversed ofatumumab- and rituximab-mediated CDC and CD20 mAb staining. Statin incubation resulted in conformational changes of CD20 and impaired binding of CD20 mAb to the CD20 molecule as observed by atomic force microscopy. Freshly isolated cells of 4 B cell lymphoma patients were treated with the cholesterol-depleting agent MβCD, and CD20 mAb (B9E9) binding and rituximab-mediated CDC were significantly decreased (P<0.05 t-test, versus control) In addition, 5 hypercholesterolemia patients were treated with atorvastatin. Ofatumumab binding to freshly isolated B cells was found decreased in all patients upon statin treatment (P<0.01, paired t-test). Based on these data, statins may interfere with CD20 detection and anti-lymphoma activity of CD20 mAb and may have significant clinical implications as impaired mAb binding to CD20 conformational epitopes elicited by statins may delay diagnosis, postpone treatment or impair anti-lymphoma activity of CD20 mAb. MAb which are less sensitive to such changes may minimize possible effects of statins on mAb-mediated immunotherapy in these patients.

Author notes

Disclosure:Employment: Patrick Engelberts, Wendy Mackus and Paul Parren are employed by Genmab BV. Ownership Interests:; Patrick Engelberts, Wendy Mackus and Paul Parrren may have ownershiph interest (stock options) in Genmab. Research Funding: Funding for this research will be provided by Genmab in near future.