Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation
| Autor: | Gerald Petti, Haleema Janjua, John Zhou, Jack Hua, Maoliang Wang, Mark Li, Magda Mendez, Clifford Longley, Ramesh Chintala, Zhenfan Zhang, Ming-Ching Hsieh, Hnin Phyu, Sam Liu, Ping Peng, Virna Borowski, David Filpula, Amartya Basu, Karen Yang, Mary Mehlig |
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| Rok vydání: | 2003 |
| Předmět: |
Antigenicity
Immunoglobulin Variable Region chemical and pharmacologic phenomena Bioengineering Ligands Protein Engineering Biochemistry Peptide Mapping Mass Spectrometry Receptors Tumor Necrosis Factor Pichia pastoris Polyethylene Glycols Substrate Specificity Maleimides Mice Structure-Activity Relationship Protein structure Endopeptidases Structure–activity relationship Animals Molecular Biology Mice Inbred ICR biology Molecular Structure Chemistry Tumor Necrosis Factor-alpha Protein engineering respiratory system biology.organism_classification Flow Cytometry Molecular biology Protein Structure Tertiary Molecular Weight Drug Design Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Female Linker Biotechnology Cysteine Conjugate |
| Zdroj: | Protein engineering. 16(10) |
| ISSN: | 0269-2139 |
| Popis: | 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. Independent 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. |
| Databáze: | OpenAIRE |
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