Analysis of active and inactive EGFR conformations

描述(申请人提供)：表皮生长因子(EGF)受体或受体酪氨酸激酶(RTK)ErbB家族与许多人类癌症有关。已经证实，针对EGF受体(EGFR)胞外区的抗体可以阻止配体结合和/或受体信号转导，从而抑制体内肿瘤的生长。至少有5种这样的抗EGFR抗体正在进行临床试验；1种是西妥昔单抗/Erbitux，于2004年2月被FDA批准用于治疗晚期结肠癌。最近对EGFR家族胞外区的结构研究发现了一种新的配体诱导受体二聚化的机制。未连接的受体以一种特有的“自抑制”构型被发现，在这种构型中，受体二聚界面被分子内的“系绳”完全封闭。配体结合诱导EGFR发生显著的构象变化，暴露这个正常埋在细胞外的二聚化位点，促进二聚化和受体激活。我们将分析针对EGFR胞外区(SEGFR)的特定抗体如何产生显著不同的效果。某些抗体抑制EGFR的激活，而其他抗体改变(或偏向)细胞表面受体的亲和力状态，有时允许信号继续进行。还有一些特别地削弱了一个配体的结合而不影响另一个配体的结合。我们假设这些不同的效应反映了由特定抗体稳定的不同的细胞外构象。我们将分析抗体与已定义构象改变的sEGFR突变体的结合，并使用X射线结晶学来确定Fab：sEGFR复合体的结构。最后，为了利用最近的结构进展，我们将筛选单链Fv(ScFv)分子的噬菌体展示文库，这些分子与sEGFR中的关键表位结合，我们假设将以新的方式抑制EGFR。我们希望对细胞外构象与细胞表面EGFR的活性、亲和力和特异性之间的关系有更深入的了解。其具体目的是：1.通过分析已知对体内受体功能有影响的结合抗体的结构后果，探讨EGFR反应性与胞外区构象之间的关系。2.使用选择性抑制一组EGFR激动剂结合的抗体，探索EGFR激活中的配基特异性。

DESCRIPTION (provided by applicant): The Epidermal Growth Factor (EGF) receptor or ErbB family of receptor tyrosine kinases (RTKs) have been implicated in many human cancers. It is well established that antibodies to the extracellular region of the EGF receptor (EGFR) that prevent ligand binding and/or receptor signaling can inhibit tumor growth in vivo. At least 5 such anti-EGFR antibodies are in clinical trials; 1, cetuximab/Erbitux, was FDA-approved to treat advanced colon cancer in February 2004. Recent structural studies of EGFR family extracellular regions have identified a novel mechanism for ligand induced receptor dimerization. The unliganded receptor is found in a characteristic "autoinhibited" configuration in which the receptor dimerization interface is completely occluded in an intramolecular "tether". Ligand binding induces a dramatic conformational change in EGFR that exposes this normally buried extracellular dimerization site, promoting dimerization and receptor activation. We will analyze how particular antibodies against the EGFR extracellular region (sEGFR) can have dramatically different effects. Certain antibodies inhibit EGFR activation, while others alter (or bias) the affinity state of the receptor at the cell surface, sometimes allowing signaling to proceed. Still others specifically impair binding of 1 ligand without affecting binding of another. We hypothesize that these different effects reflect the distinct extracellular conformations stabilized by the particular antibodies. We will analyze antibody binding to sEGFR mutants with defined altered conformations, and use X-ray crystallography to determine structures of Fab:sEGFR complexes. Finally, in an effort to exploit recent structural advances, we will screen phage display libraries for single-chain Fv (scFv) molecules that bind to key epitopes in sEGFR and that we hypothesize will inhibit EGFR in novel ways. We hope to gain structural insight into relationships between extracellular conformation and activity, affinity and specificity of EGFR at the cell surface. The specific aims are: 1. To investigate relationships between EGFR responsiveness and extracellular domain conformation by analyzing the structural consequences of binding antibodies with known effects on in vivo receptor function. 2. To probe ligand specificity in EGFR activation using an antibody that selectively inhibits binding of a subset of EGFR agonists.