Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation
Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation
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DOI：
10.1093/protein/gzg093
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发表时间：
2003-10-01
期刊：
影响因子：
--
通讯作者：
Filpula, D
中科院分区：
文献类型：
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作者：
Yang, K;Basu, A;Filpula, D
如果这些最小抗原结合多肽的循环寿命可以延长和调节，则单链Fv蛋白作为治疗剂的效用将得以实现。我们已经开发了一种用于产生定制的单聚乙二醇化单链抗体的一般策略。游离半胱氨酸残基在抗TNF-α scFv中的C-末端或在两种scFv取向的接头区段（V-L-接头-V-H和V-H-接头-V-L）内被工程化。10个设计的变体scFv蛋白在毕赤酵母中的高水平表达允许快速纯化。实现了用5、20和40 kDa马来酰亚胺-PEG聚合物的位点特异性缀合物制备的优化，并比较了scFv蛋白及其PEG化对应物的结构和功能性质。肽图谱和MALDI-TOF质谱分析证实了每个PEG聚合物的独特附着位点。独立的生物化学和生物活性分析，包括结合亲和力和动力学、抗原性、流式细胞术分析和细胞毒性拯救，证明了10种设计的scFv缀合物的功能活性得以维持，而这些替代测定之间的scFv活性变化可以与缀合物和分析设计相关。PEG化scFv在小鼠中的药代动力学研究表明，在与临床抗体相当的范围内，循环寿命延长高达100倍。
The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V-L-linker-V-H and V-H-linker-V-L. High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Indepen dent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.