Analysis of active and inactive EGFR conformations

描述(申请人提供)：表皮生长因子受体(EGFR)家族的受体酪氨酸激酶(RTK)在许多人类癌症中是重要的，也是几种FDA批准的治疗药物的靶标。目前使用的EGFR靶向抗体疗法被专门选择来抑制EGF结合和EGF依赖的受体激活。然而，现在很清楚的是，许多依赖于EGFR的癌症是由EGFR的致癌突变引起的，这种突变促进了配体无关的信号转导--这种信号可能不会被西妥昔单抗等抗体阻断。对肺癌患者致癌激活的EGFR的研究一直是促进对EGFR激酶结构域如何调控的理解的关键。在胶质母细胞瘤和其他癌症中，在所有EGFR家族成员的胞外区域发现了越来越多的突变。这为充分了解细胞外区的调控提供了类似的机会-这是一个重要的目标，因为目前基于结构的EGFR受体激活模型不能解释(或预测)这些体细胞突变中的哪一个会激活受体。在这个建议中，我们首先询问EGFR胞外区的致癌突变如何促进受体的非配体依赖的激活，并加深我们对EGFR胞外区如何保持其非活性状态的理解。在第二个目标中，我们询问使用抗体疗法抑制肿瘤基因激活的EGFR的最有效策略是什么。单纯阻断配体结合的药物(如西妥昔单抗)可能不是由致癌突变激活的受体的有效抑制剂。我们将比较不同的EGFR靶向抗体和基于单链抗体的新型药物(VHH结构域)通过在癌症中发现的突变的EGFR变体来抑制构成(配体无关)信号的能力。我们将结合细胞研究和对EGFR胞外区的体外和结构分析来研究这些问题。我们的具体目标是：1：了解哪些分子内相互作用有助于EGFR的细胞外自抑制，以及它们如何被在癌症患者中发现的配体结合和体细胞突变逆转。2：验证已知的EGFR致癌突变不同地影响EGFR靶向抗体和新型VHH结构域药物的抑制活性的假设。总之，这些研究将对致癌突变导致EGFR和其他ErbB受体的结构性激活的机制提供重要的新线索，同时还将识别能够唯一地阻断EGFR突变体的配体非依赖性激活的新抗体。这类药物在未来的临床开发中将是高度优先的。 公共卫生相关性：目前用于癌症治疗的所有针对表皮生长因子的抗体--如西妥昔单抗--都以野生型受体为靶标，并阻断配体依赖的激活。然而，现在很清楚的是，许多由EGFR驱动的癌症是由EGFR(细胞内和细胞外)的致癌突变引起的，目前可用的抗体药物可能不适用于这些癌症。了解致癌的EGFR突变如何激活受体，以及它们如何影响一组新抗体的结合，将定义该小组中的哪些药物应优先开发用于临床应用。

DESCRIPTION (provided by applicant): The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTKs) is important in many human cancers, and is the target of several FDA-approved therapeutic agents. Currently-used EGFR-targeted antibody therapeutics were specifically selected to inhibit EGF binding and EGF-dependent activation of the receptor. However, it is now clear that many EGFR-dependent cancers arise from oncogenic mutations in EGFR that promote ligand-independent signaling - which may not be blocked by antibodies such as cetuximab. Studies of oncogenically activated EGFR from lung cancer patients have been key in advancing understanding of how the EGFR kinase domain is regulated. An increasing number of mutations are being identified in the extracellular region of all EGFR family members, in glioblastoma and other cancers. These open up a similar opportunity for fully understanding regulation of the extracellular region - an important goal since current structure-based models of EGFR receptor activation do not explain (or predict) which of these somatic mutations will activate the receptors. In this proposal we first ask how oncogenic mutations in the EGFR extracellular region can promote ligand-independent activation of the receptor, and also refine our understanding of how the extracellular region of EGFR is maintained in its inactive state. In a second aim, we ask what are the most effective strategies to inhibit oncogenically-activated EGFR using antibody therapeutics. Agents (such as cetuximab) that simply block ligand binding may not be effective inhibitors of receptors that are activated by oncogenic mutation. We will compare the ability of different EGFR-targeted antibodies and novel single chain antibody-based agents (VHH domains) to inhibit constitutive (ligand-independent) signaling by mutated EGFR variants found in cancers. We will combine cellular studies with in vitro and structural analyses of the EGFR extracellular region to investigate these questions. Our Specific Aims are: 1: To understand which intramolecular interactions contribute to extracellular autoinhibition of EGFR, and how they are reversed by ligand binding and somatic mutations found in cancer patients. 2: To test the hypothesis that known oncogenic mutations in EGFR differentially affect the inhibitory activity of EGFR-targeted antibodies and of novel VHH domain agents. In sum, these studies will shed important new light on the mechanisms through which oncogenic mutations cause constitutive activation of EGFR and other ErbB receptors, while also identifying new antibodies that are uniquely able to block ligand-independent activation of EGFR mutants. Such agents will be of high priority for future clinical development. PUBLIC HEALTH RELEVANCE: All antibodies against the epidermal growth factor that are currently in use for cancer therapy - such as cetuximab - target the wild-type receptor and block ligand-dependent activation. However, it is now clear that many EGFR-driven cancers arise from oncogenic mutations in EGFR (both intra- and extracellular), and currently available antibody drugs may not work for these cancers. Understanding how oncogenic EGFR mutations activate the receptor, and how they influence binding of a panel of novel antibodies, will define which agents in this panel should be developed for clinical application with the highest priority.