TLR reporter cell lines for screening TLR agonists and antagonists (136.20)

Abstract We have developed novel TLR/NF-κB SEAPorter™ HEK 293 cell lines each expressing an intact Toll-like Receptor (TLR) as well as a secreted alkaline phosphatase (SEAP) reporter whose expression is under control of an NF-κB response element. These cell lines are suitable for TLR-dependent functional assays and are an ideal fit for high-throughput screening of TLR agonists or antagonists. TLR expression of each cell line has been validated by Flow Cytometry. Using a 96-well plate format, functional activity has been evaluated by criteria including cell number titration, ligand dose response, cell stimulation time and DMSO tolerance. Validation results yield an average signal-to-background (S/B) ratio of 16.9±0.7 as well as Z’ value of 0.75±0.05 for ligand dose-response tests. Assay validation is based on the 96-well plate format screening assay, but are also suitable for 384- or 1536-well plate format screening assays. For TLR agonist screening, each compound in a library can be directly applied to cells and induced SEAP in culture media can be measured without additional steps. TLR antagonist screening can be performed as agonist screening except the addition of TLR ligands is done before or after treatment with library compounds. Consequently, the TLR-specific cell-based high-throughput feature of our novel TLR/NF-κB SEAPorter™ HEK 293 cell lines will provide a fast, flexible and convenient means for lead compound screening in the pharmaceutical drug development fields.