Fig 1: Functional blocking of OX40 receptor by Anticalin proteins in vitro. (A) OX40 antagonizing Anticalin proteins were added in titration to NFκB-Luc2/OX40 Jurkat reporter cells 30 min before stimulation with 3 nM OX40 ligand. The ligand concentration was previously adjusted to result in a NFκB-controlled luciferase activity approximately in the range of a half-maximal effect. After 5 h of ligand stimulation, luciferase substrate was added and resulting luminescence was measured. Data is represented as mean ± S.E.M. of triplicate values from one experiment, which is representative of three independent experiments. (B) Luciferase activity was monitored after addition of 1.5 µM HSA (upper graphs) or a mixture of equimolar amounts of human IgG1 IgG2 and IgG4 (lower graphs) in parallel with the Anticalin or Duocalin proteins. Column triplets represent three selected concentrations of Anticalin proteins (from left to right: 0.01, 1.95, 500 nM). Data is represented as mean ± S.E.M. of triplicate values. This data is a representation from three independent experiments. (C) The OX40 receptor blocking activity of a subset of Anticalin or Duocalin proteins was assessed in a co-culture assay setting. Therefore, panT cells from donor A or B were cultivated at a ratio 3:1 with mitomycin C treated Flp-In-CHO:vector cells to stimulate T cell alloreactivity. Four nM OX40 ligand was added to stimulate T cell activation in presence of anti-CD3 and anti-CD28 antibody. Dose-dependent inhibition of IL-2 release by OX40-specific Anticalin proteins is shown. Data is represented as mean ± S.E.M. of triplicate values from one experiment. Middle horizontal dotted lines represent the average of the background signal with anti-CD3 antibody, Flp-In-CHO:vector, panT cells and anti-CD28 antibody either in presence of 4 nM of OX40L (upper level of activation) or in absence of the ligand (lower background). Gray areas represent standard error of the mean of the average value for respective background levels.