51: Exploiting receptor clustering enhances inhibitory effects of a therapeutic monoclonal antibody to TLR4 revealing a novel mechanism of action driving the pathology of rheumatoid arthritis

Abstract

Aims Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking TLR4 activation induced by multiple ligands on inflammatory leukocytes represents an elegant strategy when targeting the underlying causes of human diseases. A ligand-independent anti-TLR4 neutralizing antibody, which utilizes a novel mechanism of action to enhance its inhibitory effects on inflammatory leukocytes, is evaluated as a strategy for TLR4 neutralization in rheumatoid arthritis (RA). Methods and results An anti-human TLR4 mAb, NI-0101, binds to human TLR4 in a region involved in receptor dimerization. Using exogenous, endogenous or chemical ligands, NI-0101 was shown to block TLR4 signaling regardless of activator. The mechanism of action of the antibody was further explored using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis. The results revealed that NI-0101 harnesses the clustering of TLR4 and Fc gamma receptors (Fc γ R) in lipid rafts to achieve increased potency on inflammatory cells. The advantage of the novel mechanism of action was tested with a newly reported TLR4 ligand in RA, immune complexes containing citrullinated fibrinogen (cFb-IC), and synovial fluid samples from RA patients. The results demonstrated that NI-0101 is well suited to block TLR4-dependent pro-inflammatory cytokine production (e.g., IL-6, TNF). Conclusions Our data demonstrate that NI-0101 is superior in blocking multiple ligand-induced TLR4 activation on Fc γ R + inflammatory cells. The results, thus, suggest that this novel mechanism of action is well-suited to block TLR4 activation in RA patients where immune complexes are thought to drive disease.