Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates
Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates
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定量碰撞诱导的解折叠区分模型抗体药物缀合物
DOI：
10.1002/pro.3560
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发表时间：
2018
期刊：
影响因子：
8
通讯作者：
Ruotolo, Brandon T.
中科院分区：
文献类型：
--
作者：
Tian, Yuwei;Lippens, Jennifer L.;Netirojjanakul, Chawita;Campuzano, Iain D. G.;Ruotolo, Brandon T.
抗体-药物缀合物（ADC）是基于抗体的治疗剂，已被证明是高效的癌症治疗平台。它们由单克隆抗体通过化学接头与高效药物缀合组成。与半胱氨酸靶向化学相比，天然赖氨酸残基的缀合可导致更高程度的结构异质性，因此评价缀合对抗体构象的影响很重要。在这里，我们提出了一个工作流程，涉及天然离子迁移率（IM）-MS和气相去折叠赖氨酸连接的单克隆抗体（mAb）-生物素缀合物的结构表征。在通过变性液相色谱-质谱（LC-MS）测量确定结合状态后，我们进行了分子排阻色谱（SEC）和天然IM-MS测量，以比较生物素化和未修饰IgG 1分子的结构。由于这些抗体-生物素偶联物的柔性结构和有限的仪器分辨率，流体动力学半径（Rh）和碰撞截面（CCS）值不足以区分其构象变化。相比之下，碰撞诱导的解折叠（CIU）分析能够检测生物素缀合后mAb中的细微结构和稳定性差异，表现出对mAb缀合的灵敏度超过单独的天然MS分析。通过CIU和差示扫描量热法（DSC）数据检测到mAb-生物素缀合物的不稳定性，表明这两种测量工具之间存在先前未知的相关性。最后，我们讨论了IM‐MS和CIU技术对ADC开发管道未来的影响。
Antibody–drug conjugates (ADCs) are antibody‐based therapeutics that have proven to be highly effective cancer treatment platforms. They are composed of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine‐targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)‐MS and gas‐phase unfolding for the structural characterization of lysine‐linked monoclonal antibody (mAb)–biotin conjugates. Following the determination of conjugation states via denaturing Liquid Chromatography‐Mass Spectrometry (LC–MS) measurements, we performed both size exclusion chromatography (SEC) and native IM‐MS measurements in order to compare the structures of biotinylated and unmodified IgG1 molecules. Hydrodynamic radii (Rh) and collision cross‐sectional (CCS) values were insufficient to distinguish the conformational changes in these antibody–biotin conjugates owing to their flexible structures and limited instrument resolution. In contrast, collision induced unfolding (CIU) analyses were able to detect subtle structural and stability differences in the mAb upon biotin conjugation, exhibiting a sensitivity to mAb conjugation that exceeds native MS analysis alone. Destabilization of mAb–biotin conjugates was detected by both CIU and differential scanning calorimetry (DSC) data, suggesting a previously unknown correlation between the two measurement tools. We conclude by discussing the impact of IM‐MS and CIU technologies on the future of ADC development pipelines.
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影响因子：
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DOI：
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发表时间：
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影响因子：
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