A new bioassay for measuring the strength of IL-6/STAT3 signal inhibition by tocilizumab in patients with rheumatoid arthritis

Background: Interleukin-6 (IL-6) transduces signals via phosphorylation of STAT3 (pSTAT3). Tocilizumab (TCZ) is an IL-6 receptor blocker, which, when administered intravenously every 4 weeks, efficiently ameliorates rheumatoid arthritis (RA). Since IL-6 signal strength varies among patients with RA, the intensity necessary for appropriate IL-6 signal inhibition by TCZ might vary between individuals. In a previous study, we have examined the clinical utility of increasing (dosing interval shortened to 3 weeks) and decreasing (interval extended to 5 weeks) the dose frequency of TCZ. However, there is currently no established method for accurately measuring the strength of IL-6 signal inhibition by TCZ among individual patients. We therefore sought to develop such an assay. Methods: Whole blood samples were collected from RA patients with low disease activity (clinical disease activity index (CDAI) <= 10) who were treated with TCZ at dosing intervals of 3 weeks (3-week group, n = 10), 4 weeks (4-week group, n = 10) or 5 weeks (5-week group, n = 10), or with methotrexate (control group, n = 10). Recombinant human IL-6 (0, 0.1, 1, 10, 100 ng/ml) was exogenously added to whole blood and the proportion of pSTAT3-positive CD4+ T cells (% pSTAT3+/CD4+) was measured by Phosflow cytometric analysis. Results: The addition of exogenous IL-6 increased the proportion of pSTAT3-positive CD4+ T cells in a dose-dependent manner in each group. Inhibition of IL-6 signaling was strongest in the 3-week dosing group, followed by the 4-week, 5-week and control group. Significant differences in % pSTAT3+/CD4+ cells were observed between dose interval groups when stimulated with 10 ng/ml and 100 ng/ml of IL-6. Conclusion: Assessment of the proportion of pSTAT3-positive CD4+ T cells under IL-6 stimulation is a highly sensitive and useful method for determining differences in the strength of IL-6 signal inhibition in patients treated with TCZ. It is suggested that different TCZ treatment intervals were necessary to lower disease activity in each group of patients, and these findings also indicate that the IL-6 signaling pathway may differ in each RA patient. Our assay may support strategies for optimizing TCZ treatment in RA patients.