Background/Purpose: Tocilizumab (TCZ) is a IL-6 receptor blockade, administered intravenously every 4 weeks and efficiently inhibits IL-6/STAT3 signaling pathway. We have previously reported the usefulness of adjusting the dosing interval based on the disease activity of rheumatoid arthritis (RA). We herein hypothesized that strength of IL-6/STAT3 signaling could differ in each patient. Accurate measurement strategy of inhibitory strength of IL-6/STAT3 signal in RA patients who administered TCZ, has not been currently established. So, we assessed IL-6 induced phosphorylated-STAT3 (pSTAT3) in RA patients treated with TCZ and achieved low disease activity.

Methods: Whole blood was collected from RA patients in low disease activity (LDA; CDAI <=�10) treated with intravenous TCZ (8mg/kg) every 3 weeks (3w group; n=10), 4 weeks (4w group; n=10), 5 weeks (5w group; n=10) or with methotrexate (mean dose: 9.0 mg/week, range: 4-14 mg/week) (control group, n=10). Recombinant human (rh) IL-6 (0, 0.1, 1, 10, 100 ng/ml) was exogenously added to whole blood and proportion of pSTAT3 positive CD4+ T cells (% in CD4+ T cell) was measured by Phosflow cytometric analysis (BD, USA). Serum IL-6 and soluble IL-6 receptor (sIL-6R) were measured by ELISA, expression of membrane IL-6R (mIL-6R, CD126) and gp130 (CD130) on CD4+T cell were analyzed by flow cytometry.

Results: Proportion of pSTAT3 positive CD4+ T cells increased in a dose dependent manner of exogenous rhIL-6 in each treatment group. Although, all patients were in LDA, %pSTAT3 was significantly increased in control group (mean: 58.6%) from the lowest concentration (0.1 ng/ml) of rhIL-6 compared to TCZ treated patients (mean: 0.0%, 0.0%, 12.2% in 3w, 4w, 5w group respectively). %pSTAT3 showed significant increase from low concentration (1 ng/ml) of rhIL-6 in 5w group (mean: 27.4%), while 4w group showed complete inhibition of pSTAT3 (mean: 0.0%). On the other hand, stimulation with 10, 100 ng/ml of rhIL-6 resulted in significantly suppressed pSTAT3 in 3w group (mean: 0.0%, 3.1%) compared to 4w group (mean: 5.6%, 29.2%). Serum IL-6 level was significantly higher in TCZ group than control group, and 3w and 4w group exhibited significantly higher IL-6 level compared to 5w group. Although sIL-6R concentration and mIL-6R expression were upregulated in TCZ group compared to control group, no significant difference were observed among 3w, 4w, 5w group. Meanwhile, gp130 level expression did not differ among each treatment group.

Conclusion: Our study demonstrated that IL-6 stimulated-pSTAT3 detection assay is a useful method to assess the inhibitory strength of IL-6/STAT3 signaling in RA patients treated with TCZ. Our results also suggested that further alteration of dosing interval of TCZ could be possible in certain RA patients, with our new method. Measurement of pSTAT3 in RA patients treated with TCZ could be a promising strategy to optimize treatment.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-new-bioassay-system-measuring-inhibitory-strength-of-il-6stat3-signal-under-tocilizumab-treatment-in-rheumatoid-arthritis-patients/