Fig 1: Functional blocking of OX40 receptor by Anticalin proteins in vitro. (A) OX40 antagonizing Anticalin proteins were added in titration to NF?B-Luc2/OX40 Jurkat reporter cells 30 min before stimulation with 3 nM OX40 ligand. The ligand concentration was previously adjusted to result in a NF?B-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.
Fig 2: Pharmacokinetics of Anticalin proteins in mice. (A, B) The serum concentrations after i.v. administration of 1.2–2.0 mg per kg bodyweight of Anticalin or Duocalin proteins with or without HLE module in CD-1 mice were analyzed by ELISA (n = 3, mean ± S.D.). (A) Comparison of serum concentrations over time for OX40 or NGAL Anticalin proteins. (B) The serum concentrations of corresponding Duocalin proteins were plotted against the PK of OX40 Ac-ABD (left) or OX40 Ac-IgBD (right). (C) The plasma concentrations after i.v. administration of 5 mg/kg Anticalin equivalent of OX40 Ac, OX40 Ac-ABD and OX40 Ac-Fc in hSA/hFcRn double humanized mice were analyzed by ECLA (n = 3, mean ± S.D.).
Fig 3: Binding of Anticalin proteins to OX40 receptor expressed on CHO cells. (A) OX40-specific Anticalin proteins or (B) Duocalin proteins specific for OX40 and a second TNFRSF receptor were analyzed for binding to Flp-In-CHO:huOX40 cells in flow cytometry in absence (left diagrams) or presence of 2% human serum (right diagrams). Normalization of data was done by setting the highest fluorescence value in each sample titration row to 100% and the fluorescence of the cell control to 0%. Data is represented as mean ± S.E.M. (standard error of the mean) of normalized datasets from four experiments.
Fig 4: Binding of Anticalin fusion proteins to OX40 receptor and serum proteins in SPR. The sensorgrams show the binding kinetics of exemplary OX40 Ac-ABD and OX40 Ac-IgBD analytes applied in serial dilutions to the immobilized OX40 receptor or serum protein ligands. Black lines represent a 1:1 model fit. (A) OX40 Ac-ABD (left) and OX40 Ac-IgBD (right) were analyzed for binding to recombinant huOX40-His coupled to a CM5 sensor chip. (B) OX40 Ac-ABD was analyzed for binding to biotinylated HSA (left) and biotinylated MSA (right) ligands immobilized on a CAP sensor chip. (C) OX40 Ac-IgBD was analyzed for binding to biotinylated trastuzumab (left) and biotinylated mouse IgG (right) ligands immobilized on a CAP sensor chip.
Fig 5: Anticalin proteins fused to serum half-life extension modules. (A) Schematic view of the Anticalin fusion proteins generated in this study. An Anticalin protein against OX40 (OX40 Ac), the control NGAL scaffold (NGAL Ac) or an Anticalin protein against another TNFRSF member (Rec2 Ac) were fused with ABD, IgBD or Fc in different Anticalin and Duocalin arrangements. Ig? L, immunoglobulin ? leader peptide; L1, (GGGGS)3; L2, GGSGGGGTGG; L3, (GGSGG)2; L4, AAAGGS. (B) Two µg each of Anticalin proteins (left) and Duocalin proteins (right) were analyzed by non-reducing and reducing SDS-PAGE and subsequent Coomassie staining. Anticalin proteins: (1) OX40 Ac-Fc, (2) OX40 Ac, (3) OX40 Ac-ABD, (4) ABD-OX40 Ac-ABD, (5) NGAL Ac-ABD, (6) OX40 Ac-IgBD, (7) IgBD-OX40 Ac-IgBD, (8) NGAL Ac-IgBD. Duocalin proteins: (1) OX40 Ac-Rec2 Ac, (2) OX40 Ac-Rec2 Ac-ABD, (3) OX40 Ac-Rec2 Ac-IgBD, (4) Rec2 Ac-OX40 Ac-ABD, (5) Rec2 Ac-OX40 Ac-IgBD.
Supplier Page from Sino Biological, Inc. for Human TNFRSF4 / OX40 / CD134 Protein (His Tag)