Novel long-lasting interferon alpha derivatives designed by glycoengineering
Novel long-lasting interferon alpha derivatives designed by glycoengineering
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DOI：
10.1016/j.biochi.2007.10.013
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发表时间：
2008-03-01
期刊：
影响因子：
3.9
通讯作者：
Oggero, A.
中科院分区：
文献类型：
--
作者：
Ceaglio, N.;Etcheverrigaray, M.;Oggero, A.
人α干扰素（hIFN-α）包括一个密切相关的蛋白质家族，其阻断病毒感染、抑制细胞增殖和调节细胞分化。重组hIFN-α 2已被证明可用于治疗多种人类病毒性疾病和癌症。然而，这种细胞因子的临床使用由于其短的循环半衰期而受到限制，这使得需要在延长的时间内频繁给药。为了避免这个问题，使用定点诱变进行糖工程化策略。通过将一个N-糖基化共有序列插入细胞因子的不同位置构建了14个突变体。突变集中在被认为对蛋白质的结构或功能不重要的氨基酸位置。考虑到保留的特异性体外生物活性和较高的碳水化合物含量，选择5个N-糖基化位置引入分子中。在每次添加功能性共有序列后观察到分子量的连续增加，导致具有4个和5个N-连接的碳水化合物（4 N-和5 N-IFN）的类似物具有增加的大小和电荷，这是降低蛋白质的肾清除率的因素。药代动力学实验显示4 N-和5 N-IFN变体的相似行为，与皮下给药大鼠后的非糖基化rhIFN-α 2相比，消除半衰期增加25倍，全身清除率降低20倍。此外，与非糖基化细胞因子相比，4 N类似物的分布和消除半衰期均更长，确定静脉内接种后曲线下面积增加10倍。因此，本文中我们首次描述了具有显著改善的药代动力学性质的高度糖基化的IFN类似物，这使得我们能够设计出将联合收割机较低的给药频率与增强的治疗功效相结合的药物。(c)2007年，Elsevier Masson SAS。All rights reserved.
Human alpha interferons (hIFN-alpha) comprise a family of closely related proteins that block viral infection, inhibit cell proliferation and modulate cell differentiation. Recombinant hIFN-alpha 2 has proved useful for the treatment of a variety of human viral diseases and cancers. However, the clinical use of this cytokine has been restricted due to its short circulating half-life, which makes frequent dosing over an extended period necessary. To circumvent this problem, a glycoengineering strategy was carried out using site-directed mutagenesis. Fourteen mutants were constructed by the insertion of one N-glycosylation consensus sequence into different positions of the cytokine. Mutations were focused on amino acid positions that were believed not to be critical for the protein's structure or function. Taking into account the retained specific in vitro bioactivity and the higher carbohydrate content, five N-glycosylation positions were selected to be introduced into the molecule. Successive increases in molecular weight were observed after each addition of a functional consensus sequence, resulting in analogs with 4 and 5 N-linked carbohydrates (4N- and 5N-IFN) with increased size and charge, factors that reduce renal clearance of proteins. Pharmacokinetic experiments showed a similar behavior of 4N- and 5N-IFN variants, with a 25-fold increase in the elimination half-life and a 20-fold decrease in the systemic clearance rate compared with the non-glycosylated rhIFN-alpha 2 following subcutaneous administration to rats. Besides, both distribution and elimination half-lives of the 4N analog were longer in comparison with the non-glycosylated cytokine, determining a 10-fold increase in the area under the curve after intravenous inoculation. Thus, herein we describe for the first time heavily glycosylated IFN analogs with a remarkable improvement in pharmacokinetic properties, which allow us to project drugs that combine less frequency of administration with enhanced therapeutic efficacy. (c) 2007 Elsevier Masson SAS. All rights reserved.