Characterization of a human toll-like receptor 3 antibody designed to block ligand binding and ligand mediated signaling (180.16)

Abstract

Abstract Toll-like receptors (TLRs) play key roles during pathogen invasion to initiate innate immune responses. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (microbial and endogenous) and transmits signals to activate NF-kappa B. TLR3 is restricted to acidic pH+ intracellular compartments of innate immune cells (viz immature DC). Ligand binding and formation of multimeric signal complexes, to activate down-stream signaling, are facilitated by acidic pH in these compartments. Mutational and co-crystallization studies determined the interaction of TLR3 & ligand binding at the molecular level and also individual residues important in ligand recognition and signal transduction. Besides, neutralizing antibodies were utilized as tools for better understanding of ligand binding and TLR3 signaling. In the present study, we report results of a TLR3 antibody which blocks ligand (poly IC) binding and its' effect on functional activity. We tested multiple TLR3 antibodies, generated through use of synthetic peptides designed to include various regions of protein and also the residues playing important role in ligand binding. The binding and blocking (ligand) activities were analyzed by flow cytometry using TLR3 transfectant cells in presence/absence of poly IC. We also tested blocking activity in a functional assay by measuring secreted alkaline phosphatase (SEAP) in cultures of TLR3-NF-kB-SEAP co-transfectants stimulated wih poly IC in the presence of antibody.