Higher order receptor clustering due to the IgG3 subclass is necessary for TLR4 signaling and tolerance induction by novel human anti-TLR4 antibodies.

We have previously demonstrated that an IgG3 agonistic TLR4/MD2 antibody reversed acute murine Type 1 diabetes (T1D), induced immune tolerance, and induced long-term endosomal sequestration of TLR4/MD2. We hypothesized that the IgG3 Fc was critical for agonist activity of the mouse TLR4 antibodies, due to the unique IgG3 extended hinge region and enhanced ability to form higher-order oligomeric structures. Here, we prove the essential role of the Fc region using plate-bound antibody and F(ab')2 and F(ab) fragments, which greatly reduced or eliminated TLR4 signaling. Importantly, no agonistic TLR4 antibodies have been described for humans. We developed four novel IgG4 human agonistic TLR4/MD2 antibodies as potential therapeutic candidates for T1D. The human IgG4 anti-TLR4 antibodies failed to activate the TLR4/MD2 pathway. Switching two candidate antibodies from IgG4 to IgG3, however, resulted in robust TLR4 signaling. Cross-linking the IgG4 antibody with an IgG3 secondary antibody also induced robust TLR4 signaling. Based on this result, which suggested that increased TLR4 clustering could increase signaling, we developed tetravalent IgG3 and IgG4 anti-TLR4 antibodies. Tetravalent IgG3, but not IgG4, anti-TLR4 antibodies robustly signaled via the TLR4 pathway. Notably, however, cross-linking human IgG3 antibodies with non-IgG3 secondaries reduced TLR4 signaling, in marked contrast to activation induced by IgG4 isotypes with IgG3 crosslinker, potentially through interference with IgG3 Fc-mediated oligomerization. These results suggest that the IgG3 Fc enhances agonist function of human TLR4 antibodies via aggregation of the TLR4 receptor. Functionally, human IgG3 and IgG3 tetravalent antibodies induced tolerance in primary human monocytes, analogous to the mouse antibody. In conclusion, we developed novel human TLR4 agonistic antibodies, demonstrated that the IgG3 isotype and enhanced multivalency are necessary for their TLR4 signaling, and demonstrated their tolerogenic potential for treating inflammatory diseases.