Dose-dependent effect of anti-CCR4 antibodies on CCL17-induced signaling using calcium flux assay.

<p>The results of four independent experiments on CCRF-CEM cells loaded with Fluo-4 using different concentration intervals are shown in panels (<b>a–d</b>), (<b>e–h</b>), (<b>i</b>) and (<b>j</b>). The cells were pre-incubated with human IgG1 variants 9E, 9E10J, 306, 406, 503 or with the control antibodies KM3060var and KW-0761var for 15 min before adding a CCR4-specific ligand CCL17 at a concentration of 10 ng/mL. The IgG concentrations were either 1 (<b>a</b>), 10 (<b>b</b>) and 100 ng/mL (<b>c</b>) or 0.1 (<b>e</b>), 1.0 (<b>f</b>) and 10 µg/mL (<b>g</b>) in the first and second set of experiments, respectively. The areas under the curves (AUC) were integrated using software PRISM (GraphPad) and plotted as percentage of AUC for maximal stimulation with CCL17 alone against antibody concentrations, as shown in panels (<b>d</b>) and (<b>h</b>) for the first and second set of experiments, respectively. The results of other two independent experiments using broader range of antibody concentrations are shown in panels (<b>i</b>) and (<b>j</b>).