CONTROL AND ACTIVATION OF THE TUMOR NECROSIS FACTOR RECEPTORS

项目总结 这项提议的目标是确定控制肿瘤受体激活的规则。 肿瘤坏死因子受体超家族(TNFRSF)，用于辅助开发治疗性抗体 以这些受体为目标。确实需要深入了解通过什么机制 这些受体被激活，因为它们中的许多都是基于抗体的免疫治疗的靶标。而受体 伴随受体聚集的激活是观察到的TNFRSF受体的普遍现象， 现有的开发激动型/拮抗型抗体的方法在很大程度上依赖于反复试验，而没有 已经观察到抗体亲和力和功能性之间的相关性。拟议的研究是基于 我们最近发现，对于TNFRSF的少数成员来说，仅受体跨膜螺旋(TMH)就可以 介导高阶受体聚集以驱动下行信号，并认为 受体胞外结构域在缺乏配体的情况下阻止了TMH介导的受体激活。因此， 开发专门调节胞外结构域自身抑制状态的抗体将产生更多 选择性有效的受体激活剂或抑制剂。要调查此方法的一般适用性，请执行以下操作 机制，我们将对受体TMH齐聚和前配体的结构和功能进行研究 TNFRSF其他成员协会。基于我们对受体机制的理解 自我抑制，我们将开发高通量技术来发现激活或抑制抗体 TNFRSF成员以特定于构象的方式。具体地说，在目标1中，我们将执行彻底的 TMH受体聚集法鉴定TNFRSF成员与TMH形成高亲和力 订单交互网络和驱动信令。在目标2中，我们将描述配基前的缔合 几个具有代表性的TNFRSF成员的结构以了解受体胞外结构域是如何物理地 抑制TMH聚集。在目标3中，我们将广泛地概述受体激活的基本要求，以测试我们的 提出了受体激活的3-2法则。在目标4中，我们将利用自抑制概念来实现 酵母菌展示技术筛选特定破坏或稳定前配体的纳米体/纤维 胞外区结合，分别作为激活或抑制受体的手段。建议数 研究将极大地促进我们对TNFRSF受体激活的机制的理解 潜在地发现几个TNFRSF成员的特定激动剂或拮抗剂，例如DR5， TNFR1、TNFR2、OX40、4-1BB、CD40和GITR，这些都是重要的免疫治疗靶点。

PROJECT SUMMARY The goal of this proposal is to determine the rules governing the activation of the receptors in the tumor necrosis factor receptors super family (TNFRSF), for aiding the development of therapeutic antibodies targeting these receptors. There are genuine needs for in-depth understanding of the mechanism by which these receptors are activated, as many of them are targets for antibody-based immunotherapy. While receptor activation accompanying receptor clustering is a general phenomenon observed for receptors of the TNFRSF, existing approaches of developing agonistic/antagonistic antibodies rely largely on trial and error, and no correlation between antibody affinity and functionality has been observed. The proposed research is based on our recent finding that, for a few members of the TNFRSF, the receptor transmembrane helix (TMH) alone can mediate higher-order receptor clustering to drive downstream signaling and that an important role of the receptor ectodomain in the absence of ligand is preventing the TMH-mediated receptor activation. Thus developing antibodies that specifically modulate the auto-inhibitory state of the ectodomain would yield more selective and efficient activators or inhibitors of the receptors. To investigate the general applicability of this mechanism, we will perform structural and functional studies of receptor TMH oligomerization and pre-ligand association for other members of the TNFRSF. Based on our understanding of the mechanism of receptor autoinhibition, we will develop high-throughput technology for discovering antibodies that activate or inhibit members of the TNFRSF in conformation-specific manner. Specifically, in Aim 1, we will perform a thorough survey of receptor TMH clustering to identify TNFRSF members with TMHs that are capable of forming higher- order interaction network and driving signaling. In Aim 2, we will characterize the pre-ligand association structures for a few representative members of the TNFRSF to understand how receptor ectodomain physically inhibit TMH clustering. In Aim 3, we will broadly survey the basic requirements of receptor activation to test our proposed 3-2 rule of receptor activation. In Aim 4, we will exploit the autoinhibition concept to implement a yeast display technology to screen for nanobodies/Fabs that specifically break or stabilize the pre-ligand ectodomain association, as means of activating or inhibiting the receptors, respectively. The proposed research will significantly advance our mechanistic understanding of receptor activation for the TNFRSF while potentially discovering specific agonistic or antagonistic agents for several TNFRSF members such as DR5, TNFR1, TNFR2, OX40, 4-1BB, CD40, and GITR, all of which are important immunotherapy targets.