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.

Keywords: Aggregation; IgG3; TLR4 agonist; hexamers; receptor clustering; tolerance.

我们之前已经证明，一种IgG3激动型TLR4/MD2抗体可以逆转小鼠急性1型糖尿病（T1D），诱导免疫耐受，并长期将TLR4/MD2内体隔绝。我们假设IgG3的Fc区对于小鼠TLR4抗体的激动活性至关重要，这得益于其独特的IgG3延长铰链区域以及形成更高阶寡聚结构的能力增强。在这里，我们通过使用板结合抗体和F(ab')2及F(ab)片段证明了Fc区的重要性，这些片段极大地减少了或消除了TLR4信号传导。重要的是，目前还没有关于人类的激动型TLR4抗体的描述。我们开发了四种新型的人源化IgG4激动型TLR4/MD2抗体作为治疗T1D的潜在候选药物。然而，人源化IgG4抗-TLR4抗体未能激活TLR4/MD2通路。将两种候选抗体从IgG4转换为IgG3后，则导致了强大的TLR4信号传导。使用IgG3二抗交联IgG4抗体也诱导了强大的TLR4信号传导。基于这一结果，表明增加的TLR4聚集可以增强信号传导，我们开发了四价IgG3和IgG4抗-TLR4抗体。然而，只有四价IgG3而非IgG4抗-TLR4抗体通过TLR4途径发出强大的信号。值得注意的是，用非IgG3二抗交联人类IgG3抗体会减少TLR4信号传导，与使用IgG3交联剂激活的IgG4同型异构体诱导的激活形成鲜明对比，这可能是通过干扰IgG3 Fc介导的寡聚化实现的。这些结果表明，IgG3 Fc区可以通过聚集TLR4受体来增强人类TLR4抗体的激动功能。从功能上看，人源化IgG3和四价IgG3抗体在原代人类单核细胞中诱导了免疫耐受，类似于小鼠抗体的作用。总之，我们开发出了新型的人类TLR4激动型抗体，证明了IgG3同种异构体和增强的多价性对于其TLR4信号传导是必要的，并且展示了它们治疗炎症性疾病时诱发免疫耐受的能力。

关键词：聚集；IgG3；TLR4激动剂；六聚体；受体聚集；耐受性。