Il 6rα

We used an ELISA-based assay [71 (link)] to determine if MH can bind to recombinant IL-6Rα, IL-8R, and IL-11Rα proteins (all purchased from R&D Systems; Minneapolis, MN, USA). IL-6Rα (2 μg/mL), IL-8R (0.5 μg/mL), or IL-11Rα (1 μg/mL) proteins were coated on 96-well plates by overnight incubation at 4 °C. The plates were then washed with PBS + 0.05% tween 20 and blocked with PBS containing 1% BSA for 1 h at room temperature. For competition assay, various concentrations of MH (0.03–3%) were added to recombinant receptor-coated plates and incubated for 1 h at room temperature. After thorough washing, recombinant IL-6, IL-8, or IL-11 cytokines were added (50 ng/mL) and incubated for 1 h at 37 °C. The plates were then thoroughly washed and biotin-conjugated mAbs to IL-6 (0.25 µg/mL), IL-8 (0.5 µg/mL) or IL-11 (0.5 µg/mL) were added for 1 h at room temperature. After another round of washing, HRP-conjugated streptavidin was added for 30 min, washed, and developed by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for 10 min. Additionally, the potential effect of flavonoid compounds (luteolin, quercetin, galangin, and chrysin; dose range 0.5–50 μM) on binding to IL-6Rα was investigated in the same assay. The dose-response was modeled based on mass-action kinetic drug-response interactions.

DARPins have been described previously [19 (link), 21 ]. Two rounds of cell-based affinity selections were each performed on 10⁷ activated expanded Treg cells originating from seven donors using a DARPin phage display library containing up to 10⁹ binding members, as described previously [17 (link), 18 (link)]. The following recombinant human proteins were included as de-selection antigens: TCR α and β chains, CD5 (Cambridge Biosciences); CD69, CD3ε, and ITGB2 (Sino Biological); CD2 (Abcam); CD132, CD122, CD39, IL-10Rα, sCD4, CD109, IL-1R, IL-6Rα, IL-14Rα, IFN-γR1, CD45, CD38, TNFRI, 4-1BB, CD30, GITR, PD-1, B7-H1, CD44, CD25, CD27, and CD28 (R&DSystems). DARPins isolated from two rounds of selections were reformatted as either human IgG₁ Fc domain fusions or mouse IgG_2a Fc domain fusions and expressed in HEK293 cells (ATCC).