Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation

被引：100

作者：

Yang, K ^{[1
]}

Basu, A ^{[1
]}

Wang, ML ^{[1
]}

Chintala, R ^{[1
]}

Hsieh, MC ^{[1
]}

Liu, S ^{[1
]}

Hua, J ^{[1
]}

Zhang, ZF ^{[1
]}

Zhou, J ^{[1
]}

Li, M ^{[1
]}

Phyu, H ^{[1
]}

Petti, G ^{[1
]}

Mendez, M ^{[1
]}

Janjua, H ^{[1
]}

Peng, P ^{[1
]}

Longley, C ^{[1
]}

Borowski, V ^{[1
]}

Mehlig, M ^{[1
]}

Filpula, D ^{[1
]}

机构：

[1] Enzon Pharmaceut, Piscataway, NJ 08854 USA

来源：

PROTEIN ENGINEERING | 2003年 / 16卷 / 10期

关键词：

Pichia; polyethylene glycol; therapeutic antibodies; TNF-alpha;

D O I：

10.1093/protein/gzg093

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：761 / 770

页数：10

共 32 条

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