Okazaki Kyo, Kobashigawa Yoshihiro, Morita Hikari, Yamauchi Soichiro, Fukuda Natsuki, Liu Chenjiang, Toyota Yuya, Sato Takashi, Morioka Hiroshi
Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
ACS Omega. 2023 Jun 9;8(25):22945-22954. doi: 10.1021/acsomega.3c01948. eCollection 2023 Jun 27.
Antibody drugs are denatured under physical stress, e.g., friction, heat, and freezing, which triggers formation of aggregates and resultant allergic reactions. Design of a stable antibody is thus critical for the development of antibody drugs. Here, we obtained a thermostable single-chain Fv (scFv) antibody clone by rigidifying the flexible region. We first conducted a short molecular dynamics (MD) simulation (3 runs of 50 ns) to search for weak spots in the scFv antibody, i.e., flexible regions located outside the CDR (complementarity determining region) and the interface between the heavy-chain and light-chain variable regions. We then designed a thermostable mutant and evaluated it by means of a short MD simulation (3 runs of 50 ns) based on reductions in the root-mean-square fluctuation (RMSF) values and formation of new hydrophilic interactions around the weak spot. Finally, we designed the VL-R66G mutant by applying our strategy to scFv derived from trastuzumab. Trastuzumab scFv variants were prepared by using an expression system, and the melting temperature-measured as a thermostability index-was 5 °C higher than that of the wild-type trastuzumab scFv, while the antigen-binding affinity was unchanged. Our strategy required few computational resources, and would be applicable to antibody drug discovery.
抗体药物在物理应激（如摩擦、加热和冷冻）下会发生变性，这会引发聚集体的形成并导致过敏反应。因此，设计稳定的抗体对于抗体药物的开发至关重要。在此，我们通过使柔性区域刚性化获得了一种热稳定的单链Fv（scFv）抗体克隆。我们首先进行了短时间的分子动力学（MD）模拟（3次，每次50纳秒），以寻找scFv抗体中的薄弱点，即位于互补决定区（CDR）之外的柔性区域以及重链和轻链可变区之间的界面。然后，我们设计了一个热稳定突变体，并基于均方根波动（RMSF）值的降低以及薄弱点周围新的亲水相互作用的形成，通过短时间的MD模拟（3次，每次50纳秒）对其进行评估。最后，我们将我们的策略应用于源自曲妥珠单抗的scFv，设计了VL-R66G突变体。使用表达系统制备了曲妥珠单抗scFv变体，作为热稳定性指标测量的熔解温度比野生型曲妥珠单抗scFv高5℃，而抗原结合亲和力未发生变化。我们的策略所需的计算资源很少，并且将适用于抗体药物发现。
