Analysis of active and inactive EGFR conformations

描述（由申请人提供）：表皮生长因子受体（EGFR）家族的酪氨酸激酶（RTKs）在许多人类癌症中很重要，并且是几种fda批准的治疗药物的靶点。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依赖性受体的激活。然而，现在很清楚，许多依赖EGFR的癌症是由EGFR的致癌突变引起的，这种突变促进了配体非依赖性信号传导——这可能不会被西妥昔单抗等抗体阻断。肺癌患者的致癌激活EGFR的研究是促进对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家族成员的细胞外区域发现了越来越多的突变。这为充分理解细胞外区域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受体激活模型并不能解释（或预测）这些体细胞突变中的哪一个将激活受体。在本提案中，我们首先询问EGFR细胞外区域的致癌突变如何促进受体的配体非依赖性激活，并完善我们对EGFR细胞外区域如何维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致癌激活的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靶向抗体和基于新型单链抗体的药物（VHH结构域）抑制癌症中发现的突变EGFR变异的组成型（不依赖配体）信号的能力。我们将结合体外细胞研究和EGFR细胞外区域的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细胞外EGFR的自抑制，以及它们如何通过配体结合和癌症患者中发现的体细胞突变被逆转。2：验证已知的EGFR致癌突变对EGFR靶向抗体和新型VHH结构域药物的抑制活性有不同影响的假设。总之，这些研究将为致癌突变导致EGFR和其他ErbB受体组成性激活的机制提供重要的新线索，同时也发现了独特的能够阻断EGFR突变体不依赖配体激活的新抗体。这类药物将是未来临床开发的重点。

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.