IgG-Paraoxonase-1 Fusion Protein for Targeted Drug Delivery across the Human Blood-Brain Barrier
IgG-Paraoxonase-1 Fusion Protein for Targeted Drug Delivery across the Human Blood-Brain Barrier
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DOI：
10.1021/mp800113g
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发表时间：
2008-11-01
影响因子：
4.9
通讯作者：
Pardridge, William M.
中科院分区：
文献类型：
--
作者：
Boado, Ruben J.;Zhang, Yun;Pardridge, William M.
对氧磷酶(PON)-1是人类抗有机磷（OP）毒素最有效的有机磷酶活性蛋白。OP化合物很容易穿过血脑屏障（131313），并在脑内具有致命的作用机制。通过将人类胰岛素受体（HIR）的单克隆抗体（mAb）重组为融合蛋白，可以产生穿过131313的人类脑穿透型PON1。HIRMAb通过内源性胰岛素受体穿过131313，并作为分子特洛伊木马将POW运送到大脑。人POW被融合到嵌合HIRMAb重链的羧基端。用重链基因和轻链基因双转染COS细胞，用蛋白A层析法亲和纯化了HIRMAb-PON1融合蛋白。人IgG或人PON1抗体的Western blotting显示，HIRMAb-PON1融合蛋白的重链比嵌合HIRMAb的重链大40 kDa。嵌合HIRMAb和HIRMAb- pon1融合蛋白与HIR胞外结构域结合的ED50分别为0.55 +/- 0.07 nM和1.1 +/- 0.1 nM。根据荧光酶测定，融合蛋白的POW酶活性约为人血浆酶活性的25%。总之，人类POW已被重新设计为一种igg -有机磷酸酶融合蛋白，可以穿透人血脑屏障。
Paraoxonase (PON)-1 is the most potent human protein with organophosphatase activity against organophosphate (OP) toxins. OP compounds readily cross the blood-brain barrier (131313) and have lethal mechanisms of action within the brain. The production of a brain penetrating form of human PON1, which crosses the 131313, is possible with the re-engineering of the enzyme as a fusion protein with a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb crosses the 131313 via the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the POW into brain. The human POW was fused to the carboxyl terminus of the heavy chain of the chimeric HIRMAb. COS cells were dual transfected with the heavy chain gene and the light chain gene, and the HIRMAb-PON1 fusion protein was affinity purified with protein A chromatography. Western blotting with antibodies to human IgG or human PON1 showed the heavy chain of the HIRMAb-PON1 fusion protein was 40 kDa larger than the heavy chain of the chimeric HIRMAb. The ED50 of binding to the HIR extracellular domain was 0.55 +/- 0.07 nM and 1.1 +/- 0.1 nM, respectively, for the chimeric HIRMAb and the HIRMAb-PON1 fusion protein. The POW enzyme activity of the fusion protein was approximately 25% of the enzyme activity in human plasma, based on a fluorometric enzymatic assay. In conclusion, human POW has been re-engineered as an IgG-organophosphatase fusion protein that penetrates the human BBB.