30. May the antibody mechanism of target binding be more critical than its affinity for the target?

CANZIANI GA, BAKER A, LACY E, DUFFY K, LAMB R, MBOW L, CUNNINGHAM M, BRIGHAM-BURKE M.

Baltimore, MD, USA

Simposio; 19th Biophysical Society Symposium; 2007

The Biophysical Society, USA

There are ten identified human Toll-Like cellular receptors (TLR) that specifically recognize a remarkably wide variety of pathogen-derived molecular signatures. The activation of TLR by pathogen-derived molecules sets in motion an immune inflammatory process that controls the innate and adaptive immune responses against invading pathogens. Among the TLR family, TLR3 specifically recognizes double stranded viral RNA (dsRNA) and endogenous ligands from necrotic cells. TLR3 is expressed both intracellularly and extracellulary in many cell types including fibroblasts, epithelial and dendritic cells. TLR3 receptor ectodomain (ECD) has the characteristic concave structure observed in proteins with leucine-rich repeats (LRRs). The recent resolution of the crystal structure of TLR3 and the molecular characterization of the putative dsRNA-binding domain support a mechanism for TLR3 oligomerization that triggers TLR3 signaling and IFN I up-regulation. We generated monoclonal antibodies against TLR3 and ranked them by their ability to interfere with Poly(I:C)-induced NF-kB activation and cytokine production. However, the mechanism through which neutralizing anti-TLR3 MAbs may inhibit TLR3 function remains elusive since TLR3 neutralizing antibodies and Fab block TLR3 signaling and cytokine production. We characterized the anti-TLR3 neutralizing and non-neutralizing monoclonal antibody complexes with TLR3 by their kinetic and equilibrium binding constants. Since these mAb binding mechanisms appeared to be distinct, we studied the properties of reagents and complexes using SLS-SEC. From these results we propose mechanisms of neutralizing and non-neutralizing binding. By comparing these mechanisms, we discovered that the antibody-TLR3 complex formation and the stoichiometry of binding, rather than the affinity for TLR3, are relevant in the development of an effective TLR3 antagonist.