Johns Terrance G, Adams Timothy E, Cochran Jennifer R, Hall Nathan E, Hoyne Peter A, Olsen Mark J, Kim Yong-Sung, Rothacker Julie, Nice Edouard C, Walker Francesca, Ritter Gerd, Jungbluth Achim A, Old Lloyd J, Ward Colin W, Burgess Antony W, Wittrup K Dane, Scott Andrew M
Tumour Targeting Program, Ludwig Institute for Cancer Research, Austin Hospital, Heidelberg 3084, Australia.
J Biol Chem. 2004 Jul 16;279(29):30375-84. doi: 10.1074/jbc.M401218200. Epub 2004 Apr 9.
The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, an observation often correlated with poor clinical outcome. Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. Monoclonal antibody (mAb) 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild type (wt) EGFR when overexpressed but does not bind the wt EGFR expressed in normal tissues. Despite only binding to a low proportion of the wt EGFR expressed in A431 tumor cells (approximately 10%), mAb 806 displays robust antitumor activity against A431 xenografts grown in nude mice. To elucidate the mechanism leading to its unique specificity and mode of antitumor activity, we have determined the EGFR binding epitope of mAb 806. Analysis of mAb 806 binding to EGFR fragments expressed either on the surface of yeast or in an immunoblot format identified a disulfide-bonded loop (amino acids 287-302) that contains the mAb 806 epitope. Indeed, mAb 806 binds with apparent high affinity (approximately 30 nm) to a synthetic EGFR peptide corresponding to these amino acids. Analysis of EGFR structures indicates that the epitope is fully exposed only in the transitional form of the receptor that occurs because EGFR changes from the inactive tethered conformation to a ligand-bound active form. It would seem that mAb 806 binds this small proportion of transient receptors, preventing their activation, which in turn generates a strong antitumor effect. Finally, our observations suggest that the generation of antibodies to transitional forms of growth factor receptors may represent a novel way of reducing normal tissue targeting yet retaining antitumor activity.
表皮生长因子受体（EGFR）在许多上皮癌中过度表达，这一现象通常与不良临床预后相关。EGFR的过度表达通常由EGFR基因扩增引起，有时与细胞外结构域存在内部缺失的变异型EGFR（de2-7 EGFR或EGFRvIII）的表达有关。单克隆抗体（mAb）806是一种具有显著抗肿瘤活性的新型EGFR抗体，当EGFR过度表达时，它既能识别de2-7 EGFR，也能识别野生型（wt）EGFR的一个亚群，但不与正常组织中表达的wt EGFR结合。尽管mAb 806仅与A431肿瘤细胞中表达的低比例wt EGFR（约10%）结合，但它对裸鼠体内生长的A431异种移植瘤显示出强大的抗肿瘤活性。为了阐明导致其独特特异性和抗肿瘤活性模式的机制，我们确定了mAb 806的EGFR结合表位。对mAb 806与在酵母表面表达或以免疫印迹形式表达的EGFR片段的结合分析，确定了一个包含mAb 806表位的二硫键连接环（氨基酸287 - 302）。实际上，mAb 806与对应于这些氨基酸的合成EGFR肽以明显高亲和力（约30 nM）结合。EGFR结构分析表明，该表位仅在受体的过渡形式中完全暴露，这种过渡形式是由于EGFR从无活性的束缚构象转变为配体结合的活性形式而产生的。似乎mAb 806结合了这一小部分瞬时受体，阻止它们的激活，进而产生强大的抗肿瘤作用。最后，我们的观察结果表明，针对生长因子受体过渡形式产生抗体可能代表了一种减少正常组织靶向但保留抗肿瘤活性的新方法。
