Integrating molecular dynamics simulation with small- and wide-angle X-ray scattering to unravel the flexibility, antigen-blocking, and protease-restoring functions in a hindrance-based pro-antibody.

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein Science Pub Date : 2024-09-01 DOI:10.1002/pro.5124
Jun Min Liao, Shih-Ting Hong, Yeng-Tseng Wang, Yi-An Cheng, Kai-Wen Ho, Shu-Ing Toh, Orion Shih, U-Ser Jeng, Ping-Chiang Lyu, I-Chen Hu, Ming-Yii Huang, Chin-Yuan Chang, Tian-Lu Cheng
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Abstract

Spatial hindrance-based pro-antibodies (pro-Abs) are engineered antibodies to reduce monoclonal antibodies' (mAbs) on-target toxicity using universal designed blocking segments that mask mAb antigen-binding sites through spatial hindrance. By linking through protease substrates and linkers, these blocking segments can be removed site-specifically. Although many types of blocking segments have been developed, such as coiled-coil and hinge-based Ab locks, the molecular structure of the pro-Ab, particularly the region showing how the blocking fragment blocks the mAb, has not been elucidated by X-ray crystallography or cryo-EM. To achieve maximal effect, a pro-Ab must have high antigen-blocking and protease-restoring efficiencies, but the unclear structure limits its further optimization. Here, we utilized molecular dynamics (MD) simulations to study the dynamic structures of a hinge-based Ab lock pro-Ab, pro-Nivolumab, and validated the simulated structures with small- and wide-angle X-ray scattering (SWAXS). The MD results were closely consistent with SWAXS data (χ2 best-fit = 1.845, χ2 allMD = 3.080). The further analysis shows a pronounced flexibility of the Ab lock (root-mean-square deviation = 10.90 Å), yet it still masks the important antigen-binding residues by 57.3%-88.4%, explaining its 250-folded antigen-blocking efficiency. The introduced protease accessible surface area method affirmed better protease efficiency for light chain (33.03 Å2) over heavy chain (5.06 Å2), which aligns with the experiments. Overall, we developed MD-SWAXS validation method to study the dynamics of flexible blocking segments and introduced methodologies to estimate their antigen-blocking and protease-restoring efficiencies, which would potentially be advancing the clinical applications of any spatial hindrance-based pro-Ab.

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将分子动力学模拟与小角和广角 X 射线散射相结合,揭示基于阻碍的原抗体的灵活性、抗原阻断和蛋白酶恢复功能。
基于空间阻碍的原抗体(pro-Abs)是一种工程抗体,利用通用设计的阻断段,通过空间阻碍掩盖 mAb 抗原结合位点,从而降低单克隆抗体(mAbs)的靶向毒性。通过蛋白酶底物和连接体的连接,这些阻断段可针对特定位点去除。虽然目前已开发出多种类型的阻断片段,如基于线圈和铰链的抗体锁,但原抗体的分子结构,尤其是显示阻断片段如何阻断 mAb 的区域,尚未通过 X 射线晶体学或低温电子显微镜阐明。为了达到最佳效果,原 Ab 必须具有较高的抗原阻断和蛋白酶恢复效率,但结构不清限制了其进一步优化。在此,我们利用分子动力学(MD)模拟研究了一种基于铰链的抗体锁定原-抗体(pro-Nivolumab)的动态结构,并用小角和广角 X 射线散射(SWAXS)验证了模拟结构。MD 结果与 SWAXS 数据密切吻合(χ2 best-fit = 1.845,χ2 allMD = 3.080)。进一步的分析表明,Ab 锁链具有明显的灵活性(均方根偏差 = 10.90 Å),但它仍然掩盖了 57.3%-88.4% 的重要抗原结合残基,因此其抗原阻断效率提高了 250 倍。引入的蛋白酶可及表面积方法证实,轻链(33.03 Å2 )的蛋白酶效率高于重链(5.06 Å2 ),这与实验结果一致。总之,我们开发了 MD-SWAXS 验证方法来研究柔性阻断片段的动力学,并引入了估算其抗原阻断和蛋白酶恢复效率的方法,这将有可能推动任何基于空间阻碍的原抗体的临床应用。
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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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