Discovery and engineering of novel anti-IgE disruptive inhibitors

项目摘要 大多数过敏反应是由抗过敏原IgE抗体引起的，它能使过敏效应器致敏。 肥大细胞和嗜碱性粒细胞等细胞。与免疫球蛋白抗体不同，免疫球蛋白E以亚纳摩尔亲和力结合 高亲和力IgE受体(FceRI)在变态反应效应细胞上以受体结合的形式存在数月 即使在存在高亲和力的抗IgE抗体抑制剂的情况下，如奥马珠单抗。我们已经证明了 抗体和设计的Ankyrin重复蛋白(DARPins)都能够动力学地加速 IgE通过两种机制从FceRI解离：促进解离机制，允许 受体相邻抑制剂促进受体解离和变构机制将IgE限制为 构象与受体结合不相容。我们已经开发了一种酵母展示方法来选择 抗IgE抑制剂能够破坏受体复合体，并表明我们有能力选择更有效的 奥马珠单抗的抗IgE变异体。此外，我们还展示了工程设计更有效的途径 由干扰性抗IgE结构域和非竞争性IgE锚定组成的双价干扰性抑制剂 域。在这里，我们建议应用这些方法来询问抗IgE免疫库，以分离更多 有效的破坏性抑制剂，探索破坏性抑制剂的机理多样性并开发 与未来临床发展相适应的超强双价抗IgE抑制剂。

Project Summary The majority of allergic reactions are caused by anti-allergen IgE antibodies, which sensitize allergic effector cells such as mast cells and basophils. In contrast to IgG antibodies, IgE binds with subnanomolar affinity to the high affinity IgE receptor (FceRI) and persists in a receptor-bound form on allergic effector cells for months even in the presence of high affinity anti-IgE antibody inhibitors, such as omalizumab. We have shown that both antibodies and Designed Ankyrin Repeat Proteins (DARPins) are able to kinetically accelerate the dissociation of IgE from FceRI through two mechanisms: a facilitated dissociation mechanism that allows receptor-adjacent inhibitors to promote receptor dissociation and an allosteric mechanism that restricts IgE to a conformation incompatible with receptor binding. We have developed a yeast display approach to selecting anti-IgE inhibitors capable of disrupting receptor complexes and demonstrated our ability to select more potent anti-IgE variants of omalizumab. In addition, we have demonstrated pathways to engineering more potent bivalent disruptive inhibitors consisting of a disruptive anti-IgE domain and a non-competitive IgE anchoring domain. Here we propose to apply these approaches to interrogate an anti-IgE immune library, to isolate more potent disruptive inhibitors, to explore the mechanistic diversity of disruptive inhibitors and to develop ultrapotent bivalent anti-IgE inhibitors compatible with future clinical development.