Institute of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.
Proteins. 2023 Sep;91(9):1316-1328. doi: 10.1002/prot.26509. Epub 2023 Jun 28.
In the last years, antibodies have emerged as a promising new class of therapeutics, due to their combination of high specificity with long serum half-life and low risk of side-effects. Diabodies are a popular novel antibody format, consisting of two F domains connected with short linkers. Like IgG antibodies, they simultaneously bind two target proteins. However, they offer altered properties, given their smaller size and higher rigidity. In this study, we conducted the-to our knowledge-first molecular dynamics (MD) simulations of diabodies and find a surprisingly high conformational flexibility in the relative orientation of the two F domains. We observe rigidifying effects through the introduction of disulfide bonds in the F -F interface and characterize the effect of different disulfide bond locations on the conformation. Additionally, we compare V -V orientations and paratope dynamics between diabodies and an antigen binding fragment (Fab) of the same sequence. We find mostly consistent structures and dynamics, indicating similar antigen binding properties. The most significant differences can be found within the CDR-H2 loop dynamics. Of all CDR loops, the CDR-H2 is located closest to the artificial F -F interface. All examined diabodies show similar V -V orientations, F -F packing and CDR loop conformations. However, the variant with a P14C-K64C disulfide bond differs most from the Fab in our measures, including the CDR-H3 loop conformational ensemble. This suggests altered antigen binding properties and underlines the need for careful validation of the disulfide bond locations in diabodies.
在过去的几年中，抗体作为一种有前途的新型治疗药物出现，这是由于它们具有高特异性、长血清半衰期和低副作用风险的结合。二价抗体是一种流行的新型抗体形式，由两个 F 结构域通过短接头连接而成。与 IgG 抗体一样，它们同时结合两个靶蛋白。然而，由于其体积较小且刚性较高，它们具有改变的特性。在这项研究中，我们进行了 - 据我们所知 - 首次对二价抗体的分子动力学 (MD) 模拟，发现两个 F 结构域相对取向具有惊人的高构象灵活性。我们通过在 F - F 界面引入二硫键观察到僵化效应，并表征了不同二硫键位置对构象的影响。此外，我们比较了二价抗体和相同序列的抗原结合片段 (Fab) 之间的 V - V 取向和抗原结合位动态。我们发现结构和动力学大多一致，表明具有相似的抗原结合特性。最显著的差异可以在 CDR-H2 环动力学中找到。在所有 CDR 环中，CDR-H2 最靠近人为的 F - F 界面。所有检查的二价抗体都显示出相似的 V - V 取向、F - F 包装和 CDR 环构象。然而，具有 P14C-K64C 二硫键的变体在我们的测量中与 Fab 的差异最大，包括 CDR-H3 环构象组合。这表明抗原结合特性发生了改变，并强调了在二价抗体中仔细验证二硫键位置的必要性。
