Department of Clinical Laboratory, The Affiliated Huai'an Hospital of Xuzhou Medical University, Huaian 223002, P. R. China.
Department of Clinical Laboratory, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, P. R. China.
J Bioinform Comput Biol. 2023 Apr;21(2):2350007. doi: 10.1142/S0219720023500075. Epub 2023 Apr 27.
Human epidermal growth factor receptor (EGFR) is strongly associated with malignant proliferation and has been established as an attractive therapeutic target of diverse cancers and used as a significant biomarker for tumor diagnosis. Over the past decades, a variety of monoclonal antibodies (mAbs) have been successfully developed to specifically recognize the third subdomain (TSD) of EGFR extracellular domain. Here, the complex crystal structures of EGFR TSD subdomain with its cognate mAbs were examined and compared systematically, revealing a consistent binding mode shared by these mAbs. The recognition site is located on the [Formula: see text]-sheet surface of TSD ladder architecture, from which several hotspot residues that significantly confer both stability and specificity to the recognition were identified, responsible for about half of the total binding potency of mAbs to TSD subdomain. A number of linear peptide mimotopes were rationally designed to mimic these TSD hotspot residues in different orientations and/or in different head-to-tail manners by using an orthogonal threading-through-strand (OTTS) strategy, which, however, are intrinsically disordered in Free State and thus cannot be maintained in a native hotspot-like conformation. A chemical stapling strategy was employed to constrain the free peptides into a double-stranded conformation by introducing a disulfide bond across two strand arms of the peptide mimotopes. Both empirical scoring and [Formula: see text]fluorescence assay reached an agreement that the stapling can effectively improve the interaction potency of OTTS-designed peptide mimotopes to different mAbs, with binding affinity increase by [Formula: see text]-fold. Conformational analysis revealed that the stapled cyclic peptide mimotopes can spontaneously fold into a double-stranded conformation that well threads through all the hotspot residues on TSD [Formula: see text]-sheet surface and exhibits a consistent binding mode with the TSD hotspot site to mAbs.
人表皮生长因子受体（EGFR）与恶性增殖密切相关，已被确立为多种癌症的有吸引力的治疗靶点，并被用作肿瘤诊断的重要生物标志物。在过去的几十年中，已经成功开发了多种单克隆抗体（mAbs）来特异性识别 EGFR 细胞外结构域的第三亚结构域（TSD）。在这里，系统地检查和比较了 EGFR TSD 亚结构域与其同源 mAbs 的复合物晶体结构，揭示了这些 mAbs 共享一致的结合模式。识别位点位于 TSD 梯状结构的 [Formula: see text]-片层表面上，从中鉴定出几个热点残基，这些残基显著赋予了识别的稳定性和特异性，负责 mAbs 对 TSD 亚结构域的总结合效力的一半左右。通过使用正交穿链（OTTS）策略，从多个线性肽模拟物中合理设计了模拟 TSD 热点残基的肽模拟物，这些模拟物以不同的取向和/或不同的头尾方式模拟这些热点残基，然而，在自由状态下它们本质上是无序的，因此不能保持在类似热点的天然构象中。采用化学订书钉策略通过在肽模拟物的两个链臂之间引入二硫键将自由肽约束成双链构象。经验评分和 [Formula: see text]荧光测定都达成了一致，即订书钉可以有效地提高 OTTS 设计的肽模拟物与不同 mAbs 的相互作用效力，结合亲和力增加了 [Formula: see text]倍。构象分析表明，订书钉环肽模拟物可以自发折叠成双链构象，该构象可以完全穿过 TSD [Formula: see text]-片层表面上的所有热点残基，并与 mAbs 上的 TSD 热点位点表现出一致的结合模式。
