Hofer Thomas, Thomas Joshua D, Burke Terrence R, Rader Christoph
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1203, USA.
Proc Natl Acad Sci U S A. 2008 Aug 26;105(34):12451-6. doi: 10.1073/pnas.0800800105. Epub 2008 Aug 21.
Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin alpha(4)beta(1). Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin alpha(4)beta(1) and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.
通过将终止密码子UGA重新编码为硒代半胱氨酸插入,硒代半胱氨酸在翻译过程中被共翻译插入到蛋白质中。硒代半胱氨酸的亲核硒醇基团赋予这种稀有氨基酸独特的化学反应性,使其在存在其他天然氨基酸的情况下能够进行区域特异性共价偶联。利用哺乳动物表达系统,我们生成了一种C末端带有硒代半胱氨酸的IgG1衍生Fc片段,其产量与传统单克隆抗体相当,并将其与一种拟肽的亲电衍生物偶联,该拟肽与整合素α(4)β(1)具有高亲和力和特异性结合。通过这种偶联,生物和化学成分都具有药理学优势。我们证明,虽然Fc蛋白将循环半衰期从几分钟延长至数天,并通过与新生儿Fc受体结合介导转胞吞作用,但拟肽引入了跨物种与细胞表面整合素α(4)β(1)的结合,并阻断其与血管细胞粘附分子-1的相互作用。与传统单克隆抗体相比,我们的技术通过将通用生物成分与可变化学成分相结合而在经济上受益。