Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes
Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes
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DOI：
10.1073/pnas.1019266108
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发表时间：
2011-05-03
影响因子：
11.1
通讯作者：
Wakarchuk, Warren W.
中科院分区：
文献类型：
--
作者：
Lindhout, Theresa;Iqbal, Umar;Wakarchuk, Warren W.
用末端唾液酸对治疗性蛋白质进行翻译后修饰是改善其循环半衰期从而改善其效率的一种手段。我们已经开发了糖蛋白的两步体外酶促修饰，其先前仅通过化学手段实现[Gregoriadis G，Jain S，Papaioannou I，Laing P（2005）Int J Pharm 300：125-130）。该两步程序使用空肠弯曲杆菌Cst-II α 2，8-唾液酸转移酶提供N-连接聚糖上的引物，然后使用脑膜炎奈瑟菌α 2，8-聚唾液酸转移酶进行聚唾液酸化。在这里，我们已经证明了该系统的能力，以修改三个糖蛋白与不同的N-连接的聚糖组合物：人类治疗蛋白α-1-抗胰蛋白酶（A1 AT）和因子IX，以及牛胎球蛋白。通过控制反应条件优化了聚唾液酸加成的链长。在证明了该系统修饰多种蛋白质的能力后，检查了聚唾液酸化对A1 AT的活性和血清半衰期的影响。A1 AT的聚唾液酸化没有不利地影响其对人中性粒细胞弹性蛋白酶的体外抑制活性。当将修饰的蛋白质注射到CD-1小鼠中时，A1 AT的聚唾液酸化导致显著改善的药代动力学特征。总之，这些结果表明，聚唾液酸化A1 AT可能有助于改善这种蛋白质缺乏患者的增强治疗，并且这种修饰可以应用于其他治疗蛋白质。
The posttranslational modification of therapeutic proteins with terminal sialic acids is one means of improving their circulating half-life, thereby improving their efficiency. We have developed a two-step in vitro enzymatic modification of glycoproteins, which has previously only been achieved by chemical means [Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Int J Pharm 300:125-130). This two-step procedure uses the Campylobacter jejuni Cst-II alpha 2,8-sialyltransferase to provide a primer on N-linked glycans, followed by polysialylation using the Neisseria meningitidis alpha 2,8-polysialyltransferase. Here, we have demonstrated the ability of this system to modify three glycoproteins with varying N-linked glycan compositions: the human therapeutic proteins alpha-1-antitrypsin (A1AT) and factor IX, as well as bovine fetuin. The chain length of the polysialic acid addition was optimized by controlling reaction conditions. After demonstrating the ability of this system to modify a variety of proteins, the effect of polysialylation on the activity and serum half-life of A1AT was examined. The polysialylation of A1AT did not adversely affect its in vitro inhibition activity against human neutrophil elastase. The polysialylation of A1AT resulted in a significantly improved pharmacokinetic profile when the modified proteins were injected into CD-1 mice. Together, these results suggest that polysialylated A1AT may be useful for improved augmentation therapy for patients with a deficiency in this protein and that this modification may be applied to other therapeutic proteins.