Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.
Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.
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DOI：
10.4161/21645515.2014.990856
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发表时间：
2015
影响因子：
4.8
通讯作者：
Norman AB
中科院分区：
文献类型：
--
作者：
Kirley TL;Norman AB
翻译后修饰的变化对于治疗性抗体的稳定性和体内行为是重要的。重组人源化抗可卡因单克隆抗体（h2 E2）的特征在于N-连接糖基化和二硫键的异质性。此外，还检查了部分由于重链上存在或不存在C-末端赖氨酸所致的电荷异质性。对于可卡因过量治疗，Fab片段可以是治疗性的，因此，设计了通过内切蛋白酶Lys-C消化产生的Fab片段的产生、纯化和表征的简化方法。完整的h2 E2抗体和纯化的Fab片段都分析了它们对可卡因及其2种代谢产物苯甲酰芽子碱和可卡因乙烯的亲和力，通过这些药物结合产生的固有抗体酪氨酸和色氨酸荧光的荧光猝灭。从荧光猝灭测量得到的结合常数与最近发表的放射性配体和ELISA结合测定一致。测定的h2 E2单克隆抗体及其Fab片段的解离常数分别约为1、5和20 nM（可卡因、可卡因和苯甲酰芽子碱）。在酪氨酸和色氨酸的激发（280 nm）或色氨酸单独的选择性激发（295 nm）之后测量色氨酸荧光猝灭（在330 nm处发射）。使用色氨酸选择性激发在295 nm处获得更准确的结合常数，这可能是由于可卡因和代谢物在280 nm处的干扰吸收。这些淬灭结果与先前发表的该抗体可变区的3-D模型中可卡因的预测结合位置中或附近的多个色氨酸和酪氨酸残基一致。
Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.