Conformation and dynamics of interchain cysteine-linked antibody-drug conjugates as revealed by hydrogen/deuterium exchange mass spectrometry.
Conformation and dynamics of interchain cysteine-linked antibody-drug conjugates as revealed by hydrogen/deuterium exchange mass spectrometry.
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通过氢/氘交换质谱法揭示链间半胱氨酸连接的抗体-药物缀合物的构象和动力学。
DOI：
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发表时间：
2014
影响因子：
7.4
通讯作者：
John F. Valliere
中科院分区：
文献类型：
--
作者：
L. Pan;O. Salas‐Solano;John F. Valliere
抗体-药物结合物(ADC)是将靶向特异性单抗(MAb)与药物分子偶联的蛋白质疗法。ADC的制造涉及额外的偶联步骤，这些步骤是在亲本单抗上进行的，评估药物偶联过程如何影响单抗的构象和动力学是很重要的。在这里，我们提出了链间半胱氨酸连接的IgG1ADC和相应的单抗的氢/氢交换质谱学(HDX-MS)的比较研究。我们发现，ADC与单甲基金黄色素E或F(vcMMAE/mcMMAF)偶联的∼一级序列的90%表现出与单抗相同的HDX动力学，表明ADC和单抗在溶液中具有非常相似的构象和动力学。相对于mAb，ADDC中的两个Fc区域的HDX动力学速率略有增加，这表明这两个区域在ADDC中变得更具结构动力学和/或更容易获得溶剂。这些发现导致了随后的调查，即局部构象变化是由于链间半胱氨酸残基上存在药物，还是由于缺乏完整的链间二硫键，或者两者兼而有之。为了解决这个问题，对ADC、mAbs、还原mAbs(包含8个还原的半胱氨酸硫醇)和部分还原的mAbs(结合过程中间体)进行了并排的HDX比较。我们的结果表明，在ADC的这两个区域检测到的构象动力学的轻微增加是由于缺乏完整的链间二硫键，而不是由于烷基化的链间半胱氨酸残基上存在vcMMAE或mcMMAF。这些结果突出了HDX-MS在询问ADC和其他蛋白质疗法的高阶结构方面的实用价值。
Antibody-drug conjugates (ADCs) are protein therapeutics in which a target specific monoclonal antibody (mAb) is conjugated with drug molecules. The manufacturing of ADCs involves additional conjugation steps, which are carried out on the parent mAbs, and it is important to evaluate how the drug conjugation process impacts the conformation and dynamics of the mAb. Here, we present a comparative study of interchain cysteine linked IgG1 ADCs and the corresponding mAb by hydrogen/deuterium exchange mass spectrometry (HDX-MS). We found that ∼90% of the primary sequence of the ADC conjugated with either monomethyl auristatin E or F (vcMMAE/mcMMAF) displayed the same HDX kinetics as the mAb, indicating the ADCs and mAbs share very similar conformation and dynamics in solution. Minor increases in HDX kinetic rates were observed in two Fc regions in the ADCs relative to the mAb which indicated that both regions become more structurally dynamic and/or more solvent-accessible in the ADCs. The findings led to a subsequent inquiry into whether the local conformational changes were due to the presence of drugs on the interchain cysteine residues or the absence of intact interchain disulfides or both. To address this question, a side-by-side HDX comparison of ADCs, mAbs, reduced mAbs (containing 8 reduced interchain cysteine thiols), and partially reduced mAbs (conjugation process intermediate) was performed. Our results indicated that the slight increase in conformational dynamics detected at the two regions in the ADCs was due to the absence of intact interchain disulfide bonds and not the presence of vcMMAE or mcMMAF on the alkylated interchain cysteine residues. These results highlight the utility of HDX-MS for interrogating the higher-order structure of ADCs and other protein therapeutics.
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影响因子：
2.8
作者：
Bobst CE;Kaltashov IA
通讯作者：
Kaltashov IA
影响因子：
7.4
作者：
Bobst, Cedric E.;Abzalimov, Rinat R.;Houde, Damian;Kloczewiak, Marek;Mhatre, Rohin;Berkowitz, Steven A.;Kaltashov, Igor A.
通讯作者：
Kaltashov, Igor A.
影响因子：
7.4
作者：
Kaltashov, Igor A.;Bobst, Cedric E.;Abzalimov, Rinat R.
通讯作者：
Abzalimov, Rinat R.
影响因子：
16
作者：
Kaltashov, Igor A.;Bobst, Cedric E.;Abzalimov, Rinat R.;Wang, Guanbo;Baykal, Burcu;Wang, Shunhai
通讯作者：
Wang, Shunhai