Understanding the determinants of antibody specificity is one of the challenging tasks in antibody development. Monospecific antibodies are still dominant in approved antibody therapeutics but there is a significant body of work to show that multispecific antibodies can increase the overall therapeutic effect. Dual‐specific or “Two‐in‐One” antibodies can bind to two different antigens separately with the same antigen‐binding site as opposed to bispecifics, which simultaneously bind to two different antigens through separate antigen‐binding units. These nonstandard dual‐specific antibodies were recently shown to be promising for new antibody‐based therapeutics. Here, we physicochemically and structurally analyzed six different antibodies of which two are monospecific and four are dual‐specific antibodies derived from monospecific templates to gain insight about dual‐specificity determinants. These dual‐specific antibodies can target both human epidermal growth factor receptor 2 and vascular endothelial growth factor at different binding affinities. We showed that a particular region of clustered Vernier zone residues might play key roles in gaining dual specificity. While there are minimal intramolecular interactions between a certain Vernier zone region, namely LV4 and LCDR1 of monospecific template, there is a significant structural change and consequently close contact formation between LV4‐LCDR1 loops of derived dual‐specific antibodies. Although Vernier zone residues were previously shown to be important for humanization applications, they are mostly underestimated in the literature. Here, we also aim to resurrect Vernier zone residues for antibody engineering efforts.

中文翻译：

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游标区区域的构象变化：对抗体双重特异性的影响。

了解抗体特异性的决定因素是抗体开发中的挑战性任务之一。单特异性抗体在批准的抗体治疗剂中仍然占主导地位，但是有大量工作表明多特异性抗体可以提高总体治疗效果。双特异性或“二合一”抗体可以通过相同的抗原结合位点分别与两种不同的抗原结合，而双特异性抗体则通过单独的抗原结合单元同时与两种不同的抗原结合。这些非标准双特异性抗体最近被证明对基于抗体的新疗法有希望。这里，我们从理化和结构上分析了六种不同的抗体，其中两种是单特异性抗体，四种是从单特异性模板衍生而来的双特异性抗体，以了解双特异性决定簇。这些双特异性抗体可以以不同的结合亲和力靶向人类表皮生长因子受体2和血管内皮生长因子。我们表明，聚集的游标区残基的特定区域可能在获得双重特异性中起关键作用。尽管某个游标区区域（即单特异性模板的LV4和LCDR1）之间的分子内相互作用极小，但衍生的双特异性抗体的LV4-LCDR1环之间存在显着的结构变化，因此紧密接触形成。尽管先前显示出游标区残留物对人源化应用很重要，但在文献中它们大多被低估了。在这里，我们还旨在复活游标区残基以进行抗体工程。