Thermostability detection and optimization of glycoengineered antibodies and antibody-drug conjugates based on differential scanning flouremitry analysis.
Thermostability detection and optimization of glycoengineered antibodies and antibody-drug conjugates based on differential scanning flouremitry analysis.
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DOI：
10.1016/j.bioorg.2019.103391
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发表时间：
2019-11
影响因子：
5.1
通讯作者：
Ken Qin;Weiwei Shi;Lei Zhao;Mingjie Li;Yubo Tang;Faridoon;Bofeng Jiang;Feng Tang;Wei Huang
中科院分区：
文献类型：
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作者：
Ken Qin;Weiwei Shi;Lei Zhao;Mingjie Li;Yubo Tang;Faridoon;Bofeng Jiang;Feng Tang;Wei Huang
单抗和抗体-药物结合物的热稳定性作为生物治疗的一个重要性质，对它们的物理化学过程、药效学和药代动力学具有重要意义。单抗的Fc糖基化在包括热稳定性在内的抗体功能中起着至关重要的作用，然而，由于缺乏均一的糖基化来比较，糖形式对单抗和ADC热稳定性的确切影响仍然是具有挑战性的。在本文中，我们利用差示扫描荧光技术(DSF)研究了Fc结构域、糖工程mAbs和ADC的热稳定性，并将其与N-糖链结构进行了比较。结果表明，与复合型N-聚糖相比，高甘露糖型N-葡聚糖显著降低了Fc的Tm值。我们还发现，核岩藻糖有助于单抗的热稳定性，而对二天线N-糖链非还原末端的非自然修饰可以弥补单抗的稳定性下降，并保持增强抗体依赖的细胞介导的细胞毒(ADCC)的优势。对赖氨酸连接的ADC和糖基化ADC的DSF分析表明，糖链连接的ADC的热稳定性降低，但使用优化的连接可以提高热稳定性。本工作对具有均一糖型的单抗和ADC的热稳定性进行了深入的分析，并提出了利用非天然糖链和稳定的连接来优化糖工程单抗和糖位特异性ADC的新策略。
Thermostability of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), as a critical property of biotherapeutics, is important for their physicochemical processes, pharmacodynamics, and pharmacokinetics. Fc glycosylation of mAbs plays a crucial role in antibody functions including thermostability, however, due to the lack of homogeneous glycosylation for comparison, the precise impact of glycoforms on thermostability of mAbs and ADCs remains challenging to elucidate. In this paper, we employed the technique of differential scanning fluorimetry (DSF) to investigate the thermostability of Fc domains, glycoengineered mAbs, and ADCs, carrying well-defined N-glycan structures for comparison. The results revealed that high-mannose-type N-glycans dramatically reduce the Tmvalue of Fc, compared to complex-type N-glycans. We also found that core-fucose contributes to the thermostability of mAbs, and the unnatural modification on non-reducing end of biantennary N-glycan can compensate the reduced stability of afucosylated mAbs and maintain the advantage of enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). DSF analysis of lysine-linked and glycosite-specific ADCs indicated that thermostability of glycan-linked ADCs is reduced, but it could be improved by using an optimized linkage. This work provides an in-depth analysis on thermostability of mAbs and ADCs with homogeneous glycoforms, and also proposes new strategies for optimizing glycoengineered mAbs and glycosite-specific ADCs using unnatural glycan and stabilized linkage.