USE OF SITE-DIRECTED MUTAGENESIS TO ENHANCE THE EPITOPE-SHIELDING EFFECT OF COVALENT MODIFICATION OF PROTEINS WITH POLYETHYLENE-GLYCOL
USE OF SITE-DIRECTED MUTAGENESIS TO ENHANCE THE EPITOPE-SHIELDING EFFECT OF COVALENT MODIFICATION OF PROTEINS WITH POLYETHYLENE-GLYCOL
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DOI：
10.1073/pnas.88.16.7185
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发表时间：
1991-08-01
影响因子：
11.1
通讯作者：
SHORT, SA
中科院分区：
文献类型：
--
作者：
HERSHFIELD, MS;CHAFFEE, S;SHORT, SA
聚乙二醇共价结合修饰可以降低蛋白质的免疫原性，延长蛋白质的循环寿命，但这种方法对任何蛋白质的应用都受到聚乙二醇结合位点(例如赖氨酸残基的epsilon-氨基)的数量和分布的限制。我们开发了一种策略，通过使用定点突变来选择性地用赖氨酸密码子取代精氨酸，并用来自大肠杆菌的嘌呤核苷磷酸化酶(PNP)进行测试，PNP是一种非常稳定但具有免疫原性的酶，可能用于治疗PNP的遗传性缺陷。构建了一个具有3个Arg-->；Lys取代的三重突变体RK3，使每个PNP亚基的赖氨酸数量从14个增加到17个，为每个六聚体酶分子提供了额外的18个潜在的聚乙二醇结合位点。野生型和RK3酶具有相似的催化活性、抗原性和免疫原性。经聚乙二醇化修饰后，两种酶都保持了催化活性，在小鼠体内的血浆半衰期从几乎相等的4小时增加到4天，并且两种酶与未修饰酶的抗血清的结合作用明显减弱。然而，针对野生型聚乙二醇单核苷酸的抗体不能结合聚乙二醇RK3酶。PEG-RK3 PNP的免疫原性也明显低于野生型的PEG-PNP。在12只接受PEG-RK3 PNP治疗的小鼠中，有2只发生了加速抗体介导的清除PEG-PNP的作用，而在16只接受改良的野生型酶治疗的小鼠中，有10只发生了加速的清除。这种定向突变和聚乙二醇化修饰的组合使用旨在允许最广泛的蛋白质选择，包括遗传和化学“工程”的产品，用于治疗遗传性和获得性疾病。
Modification by covalent attachment of polyethylene glycol (PEG) can reduce the immunogenicity and prolong the circulating life of proteins, but the utility of this approach for any protein is restricted by the number and distribution of PEG attachment sites (e.g., epsilon-amino groups of lysine residues). We have developed a strategy for introducing additional sites for PEG attachment by using site-directed mutagenesis to selectively replace arginine with lysine codons and tested it with purine nucleoside phosphorylase (PNP) from Escherichia coli, an extremely stable but immunogenic enzyme, that could potentially be used to treat an inherited deficiency of PNP. A triple mutant, RK3, possessing three Arg --> Lys substitutions was constructed that increased the number of lysines per PNP subunit from 14 to 17, providing an additional 18 potential PEG attachment sites per hexameric enzyme molecule. The wild-type and RK3 enzymes had similar catalytic activity, antigenicity, and immunogenicity. After PEG modification, both enzymes retained catalytic activity, the plasma half-life of both enzymes in mice increased from almost-equal-to 4 hr to 4 days, and the binding of both enzymes by antisera raised against each unmodified enzyme was markedly diminished. However, antibody raised against wild-type PEG-PNP did not bind the PEG-RK3 enzyme. PEG-RK3 PNP was also substantially less immunogenic than wild-type PEG-PNP. Accelerated antibody-mediated clearance of PEG-PNP occurred in 2 of 12 mice treated with PEG-RK3 PNP, compared with 10 of 16 mice treated with the modified wild-type enzyme. This combined use of directed mutagenesis and PEG modification is aimed at permitting the widest choice of proteins, including products of genetic and chemical "engineering," to be used for therapy of inherited and acquired disorders.