Structure-function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation
Structure-function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation
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DOI：
10.1021/bc050322y
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发表时间：
2006-05-17
影响因子：
4.7
通讯作者：
Filpula, David
中科院分区：
文献类型：
--
作者：
Basu, Amartya;Yang, Karen;Filpula, David
重组干扰素-β-1b（IFN-β-1b）在临床上用于治疗多发性硬化症。与许多生物配体一样，IFN-β-1b表现出相对较短的血清半衰期，中和抗体可能会进一步降低生物利用度。虽然聚乙二醇化是通常用于增加蛋白质治疗剂的血液停留时间的方法，但是分子工程方法还需要解决治疗性蛋白质的稳定性、溶解性、聚集、免疫原性和体内暴露。我们在超过20个位点选择性单聚乙二醇化或多聚乙二醇化IFN-β-1b生物缀合物中研究了重组人IFN-β-1b的这五个参数。伯胺通过聚（乙二醇）的单个或多个附件进行修饰，或者在N-末端位点特异性地，或者随机地在11个赖氨酸上。在两种替代方法中，定点诱变独立地用于构建设计的IFN-β-1b变体，所述变体含有用于位点特异性PEG化的单个游离半胱氨酸或赖氨酸。实现了用12 kDa、20 kDa、30 kDa和40 kDa胺选择性PEG聚合物的缀合物制备的优化，并进行了IFN-β-1b蛋白及其PEG化对应物的结构和功能性质的比较。肽图谱和MALDI-TOF质谱分析证实了PEG聚合物的附着位点。独立的生物化学和生物活性分析，包括抗病毒和抗增殖生物测定，圆二色谱，毛细管电泳，流式细胞仪分析，反相和尺寸排阻HPLC，和免疫分析表明，设计的IFN-β-1b缀合物的功能活性得到保持，而IFN-β-1b的可溶性或不溶性聚集体的形成得到改善。在小鼠和大鼠中对所选聚乙二醇化IFN-β-1b化合物进行的免疫原性和药代动力学研究表明，与未修饰的蛋白质相比，IgG应答降低，AUC暴露增加100倍以上。结果表明，这种大分子工程策略的能力，以解决药理学和制剂的高度疏水性，易于聚集的蛋白质的挑战。在制剂优化和生物学研究中进一步研究了先导单聚乙二醇化候选物40 kDa PEG 2-IFN-β-1b的性质。
Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly( ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead monoPEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies.