Molecularly defined antibody conjugation through a selenocysteine interface.
Molecularly defined antibody conjugation through a selenocysteine interface.
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DOI：
10.1021/bi901744t
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发表时间：
2009-12-22
期刊：
影响因子：
2.9
通讯作者：
Rader C
中科院分区：
文献类型：
--
作者：
Hofer T;Skeffington LR;Chapman CM;Rader C
抗体结合物在基础、临床前和临床应用中具有广泛的用途。通过赖氨酸胺基或半胱氨酸残基的硫醇基的常规抗体结合可产生化学计量比不确定的异质产物和相当大的批次之间的可变性。为了保持抗体分子的两个特点，精确度和可预测性，人们非常需要能够实现部位特异性抗体结合的方法。基于哺乳动物细胞表达系统，我们描述了利用第21种天然氨基酸硒半胱氨酸来产生具有独特亲核活性的免疫球蛋白和抗体分子，这种分子可以与生物素、荧光素和聚乙二醇的亲电衍生物进行位点特异性的结合。由此产生的抗体结合物被发现完全保留了它们的抗原结合能力，对于免疫球蛋白，还保留了介导效应器功能的能力。体外和体内实验均证实了其功能增强作用。虽然这些抗体结合物与各种蛋白质组、诊断和治疗应用相关，但它们也构成了产生分子定义的抗体-药物结合物和放射免疫结合物的原理证明。与其他位点特异性抗体偶联方法相比，硒半胱氨酸界面技术(I)只涉及C末端的微小修饰，不干扰二硫键，(Ii)不需要激活，(Iii)产生独特的生物和化学成分的1：1化学计量比。总的来说，我们的方法提供了高度明确的抗体结合物的生成，具有广泛的用途，从蛋白质组应用到治疗干预。
Antibody conjugates have broad utility in basic, preclinical, and clinical applications. Conventional antibody conjugation through the amine group of lysine or the thiol group of cysteine residues yields heterogeneous products of undefined stoichiometry and considerable batch-to-batch variability. To preserve the two hallmarks of the antibody molecule, precision and predictability, methods that enable site-specific antibody conjugation are in high demand. Based on a mammalian cell expression system, we describe the utilization of the 21st natural amino acid selenocysteine for the generation of IgG and Fab molecules with unique nucleophilic reactivity that affords site-specific conjugation to electrophilic derivatives of biotin, fluorescein, and poly(ethylene glycol). The resulting antibody conjugates were found to fully retain their antigen binding capability and, in case of IgG, the ability to mediate effector functions. Gain-of-function was demonstrated in vitro and in vivo. While these antibody conjugates are relevant for a variety of proteomic, diagnostic, and therapeutic applications, they also constitute a proof-of-principle for the generation of molecularly defined antibody-drug conjugates and radioimmunoconjugates. Compared to other site-specific antibody conjugation methods, selenocysteine interface technology (i) only involves a minor modification at the C-terminus that does not interfere with disulfide bridges, (ii) does not require activation, and (iii) generates unique 1:1 stoichiometries of biological and chemical component. Collectively, our method affords the generation of highly defined antibody conjugates with broad utility from proteomic applications to therapeutic intervention.
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影响因子：
2.9
作者：
Ernst, JA;Li, H;Vandlen, RL
通讯作者：
Vandlen, RL
DOI：
10.1073/pnas.0800800105
发表时间：
2008-08-26
影响因子：
11.1
作者：
Hofer, Thomas;Thomas, Joshua D.;Rader, Christoph
通讯作者：
Rader, Christoph
DOI：
10.1073/pnas.0610683104
发表时间：
2007-05-08
影响因子：
11.1
作者：
Novoselov, Sergey V.;Lobanov, Alexey V.;Gladyshev, Vadim N.
通讯作者：
Gladyshev, Vadim N.
影响因子：
2.9
作者：
HENRIKSON, KP;ALLEN, SHG;MALOY, WL
通讯作者：
MALOY, WL
影响因子：
2.4
作者：
Humphreys, David P.;Heywood, Sam P.;Brandt, Helen A.
通讯作者：
Brandt, Helen A.