Gan Hui K, Walker Francesca, Burgess Antony W, Rigopoulos Angela, Scott Andrew M, Johns Terrance G
Oncogenic Signalling Laboratory, Ludwig Institute of Cancer Research, Austin Hospital, Level 6, Harold Stokes Building, Studley Road, Heidelberg, Victoria 3084, Australia.
J Biol Chem. 2007 Feb 2;282(5):2840-50. doi: 10.1074/jbc.M605136200. Epub 2006 Nov 8.
The epidermal growth factor receptor (EGFR) has at least two fundamental conformations: an inactive tethered conformation and an active untethered, ligand-bound "back-to-back" dimer, which may be part of an oligomeric complex. Monoclonal antibody (mAb) 806 is an EGFR-specific antibody that only binds a transitional form of the receptor after it untethers but before forming the back-to-back, ligated, active oligomer. We have shown that AG1478, a tyrosine kinase inhibitor of the EGFR, synergistically inhibits the growth of tumors overexpressing EGFR when used in combination with mAb 806 but the mechanism for this was not elucidated (Johns, T. G., Luwor, R. B., Murone, C., Walker, F., Weinstock, J., Vitali, A. A., Perera, R. M., Jungbluth, A. A., Stockert, E., Old, L. J., Nice, E. C., Burgess, A. W., and Scott, A. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15871-15876). We now show that AG1478 increases binding of mAb 806 to the cell surface through two distinct mechanisms: an immediate effect on the conformation of EGFR and a longer term increase in cell surface under-glycosylated EGFR, an event known to increase mAb 806 reactivity. Cross-linking studies demonstrated the presence of spontaneously occurring mAb 806-reactive dimers on the surface of cells overexpressing EGFR, which are rapidly increased by AG1478. Because they react with mAb 806, these dimers must exist in a conformation distinct from the ligated back-to-back dimer. Indeed, we detected similar dimers in 293T cells expressing the EGFR lacking the small dimerization/activation arm essential to the formation of the back-to-back dimer. Thus, some of the EGFR on the cell surface of cancer cells must exist as an untethered dimer that adopts a previously unreported conformation that is inactive. This information was used to optimize the therapeutic synergy between mAb 806 and AG1478 in a xenograft model.
表皮生长因子受体（EGFR）至少有两种基本构象：一种无活性的束缚构象和一种有活性的非束缚、配体结合的“背靠背”二聚体，后者可能是寡聚复合物的一部分。单克隆抗体（mAb）806是一种EGFR特异性抗体，它仅在受体解开束缚但在形成背靠背、连接的活性寡聚体之前结合受体的一种过渡形式。我们已经表明，EGFR的酪氨酸激酶抑制剂AG1478与mAb 806联合使用时，能协同抑制过表达EGFR的肿瘤生长，但其中的机制尚未阐明（约翰斯，T.G.，卢沃尔，R.B.，穆罗内，C.，沃克，F.，温斯托克，J.，维塔利，A.A.，佩雷拉，R.M.，容布卢特，A.A.，斯托克特，E.，奥尔德，L.J.，尼斯，E.C.，伯吉斯，A.W.，以及斯科特，A.M.（2003年）《美国国家科学院院刊》100，15871 - 15876）。我们现在表明，AG1478通过两种不同机制增加mAb 806与细胞表面的结合：对EGFR构象的即时影响以及细胞表面糖基化不足的EGFR的长期增加，已知这一事件会增加mAb 806的反应性。交联研究证明，在过表达EGFR的细胞表面存在自发形成的mAb 806反应性二聚体，AG1478能使其迅速增加。因为它们与mAb 806反应，所以这些二聚体必定以一种不同于连接的背靠背二聚体的构象存在。实际上，我们在表达缺乏形成背靠背二聚体所必需的小二聚化/激活臂的EGFR的293T细胞中检测到了类似的二聚体。因此，癌细胞表面的一些EGFR必定以一种未报道过的无活性构象的非束缚二聚体形式存在。这些信息被用于优化异种移植模型中mAb 806与AG1478之间的治疗协同作用。
