Background: Natalizumab-neutralizing antibodies (NABs) occur in 9% of natalizumab-treated multiple sclerosis (MS) patients. Loss of clinical and biological efficacy in patients with persisting NABs requires termination of natalizumab treatment. Because high-titer NABs are strongly associated with persistence of NABs, we investigated if determination of natalizumab saturation levels of alpha-4 integrins by flow cytometry has the potential to identify patients early with NABs. Methods: Cell-bound natalizumab and natalizumab saturation of alpha-4 integrins on T cells were detected by flow cytometry using a monoclonal anti-human IgG4 antibody. Peripheral blood mononuclear cells were enriched from venous blood collected at the start (baseline) and every 4 weeks immediately before subsequent infusions until up to 9 months from natalizumab-treated patients with NABs (n = 4) and at the start and after 1 (n = 15), 2 (n = 14), 3 (n = 9), 6 (n = 7), and 9 months (n = 3) from natalizumab-treated patients without NABs. Natalizumab saturation (in %) of T cells was determined by relating median fluorescence intensities (MFIs) of in vivo bound natalizumab to MFIs after in vitro incubation with saturating amounts of natalizumab. Determination of serum NABs was performed by ELISA. Results: In patients without NABs, the median natalizumab saturation of T cells over 9 months was 75% (confidence interval of 95%: 72-78%). In two of the four patients with NABs, natalizumab saturation of T cells only reached approximately 50% after the first infusion and further declined to baseline levels with the second infusion. Low-titer NABs were measured after the first infusion and development of persistent high-titer NABs led to termination of natalizumab treatment after 6 months. In another two patients, natalizumab saturation of T cells was 74% and 68% after the first infusion, temporarily decreased to approximately baseline levels and re-increased after approximately 6 months. Transient NABs were detected after 2 and 3 months, which resolved after 5 and 6 months. Conclusions: Monitoring natalizumab saturation on T cells is a fast and reliable method to identify patients with a reduced treatment effect due to NABs. Both high-and lowtiter NABs were equally effective in reducing cellular natalizumab saturation. We were able to show that natalizumab saturation, as detected by flow cytometry, is a sensitive method for detecting a prolonged NAB-mediated reduced treatment effect because NABs are apparently effective longer than suggested by the detection limit of ELISA.
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