FIGURE 7.

NI-1201 targets the same epitope on the human protein as 25F10 does for the mouse receptor but inhibits both IL-6 cis- and trans-signaling. A, PEAK cells were transiently transfected with wild-type or mutated hIL-6R as indicated in each panel. NI-1201 or isotype control was added to the cells, and mAbs bound to the surface were detected with anti-human IgG-APC and analyzed by flow cytometry (NI-1201, black line; isotype control, filled gray). Results are representative of at least two independent experiments. B and E, PEAK cells were transfected with pSIEM-STAT3-luciferase vector and incubated with serial dilutions of mAbs or isotype control as indicated and with 100 ng/ml hIL-6Rc WT or hIL-6Rc-T264E. Firefly luciferase activity was monitored 16 h later. C and F, PEAK cells were transfected with a vector containing hIL-6R or hIL-6R-T264E and 1 day later with pSIEM-STAT3-luciferase vector. They were then co-incubated with serial dilutions of mAbs or isotype control as indicated and with 10 ng/ml hIL-6. Data are expressed as the mean ± S.E. (error bars) and are representative of four independent experiments. D, anti-human Fc was immobilized on a CM5 chip. Shgp130-hFc, hIL-6Rc WT, and NI-1201 Fab were sequentially injected at 10, 10, and 50 μg/ml, respectively. Arrows indicate the start of the injection. Results are representative of at least two independent experiments. G, anti-human Fc was immobilized on a CM5 chip. Shgp130-hFc, mutated hIL-6Rc-T264E, and 25F10 were sequentially injected at 10, 10, and 5 μg/ml, respectively. Arrows indicate the start of an injection. Results are representative of at least two independent experiments. RLU, relative light units; RU, relative units.