CONTROL AND ACTIVATION OF THE TUMOR NECROSIS FACTOR RECEPTORS

项目摘要 这项提案的目的是确定肿瘤中受体激活的规则 坏死因子受体超家族（TNFRSF），用于帮助开发治疗性抗体 针对这些受体。确实需要深入了解 这些受体被激活，因为它们中的许多是基于抗体的免疫疗法的靶。While受体 伴随受体聚集的活化是TNFRSF受体观察到的普遍现象， 现有的开发激动/拮抗抗体的方法主要依赖于试验和错误， 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的一些成员，受体跨膜螺旋（TMH）单独可以 介导高阶受体聚集以驱动下游信号传导， 受体胞外域在缺乏配体的情况下阻止TMH介导的受体活化。因此 开发特异性调节胞外域自身抑制状态的抗体将产生更多的 受体的选择性和有效的激活剂或抑制剂。为了研究这种方法的普遍适用性， 机制，我们将进行受体TMH寡聚化和前配体的结构和功能研究 该协会为TNFRSF的其他成员。基于我们对受体作用机制的理解， 自抑制，我们将开发高通量技术，发现抗体，激活或抑制 TNFRSF成员的构象特异性的方式。具体而言，在目标1中，我们将执行一个彻底的 受体TMH聚类的调查，以鉴定具有TMH的TNFRSF成员，其能够形成更高的 命令交互网络和驱动信令。在目标2中，我们将表征前配体缔合 TNFRSF的一些代表性成员的结构，以了解受体胞外域如何物理 抑制TMH聚类。在目标3中，我们将广泛调查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一个 酵母展示技术，以筛选特异性破坏或稳定前配体的纳米抗体/Fab 胞外域结合，分别作为激活或抑制受体的手段。拟议 这项研究将大大推进我们对TNFRSF受体激活机制的理解， 潜在地发现了几种TNFRSF成员如DR 5的特异性激动剂或拮抗剂， TNFR 1、TNFR 2、OX 40、4-1BB、CD 40和GITR，所有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.