Document Detail


Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation.
MedLine Citation:
PMID:  14600206     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Karen Yang; Amartya Basu; Maoliang Wang; Ramesh Chintala; Ming-Ching Hsieh; Sam Liu; Jack Hua; Zhenfan Zhang; John Zhou; Mark Li; Hnin Phyu; Gerald Petti; Magda Mendez; Haleema Janjua; Ping Peng; Clifford Longley; Virna Borowski; Mary Mehlig; David Filpula
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Protein engineering     Volume:  16     ISSN:  0269-2139     ISO Abbreviation:  Protein Eng.     Publication Date:  2003 Oct 
Date Detail:
Created Date:  2003-11-05     Completed Date:  2004-07-02     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8801484     Medline TA:  Protein Eng     Country:  England    
Other Details:
Languages:  eng     Pagination:  761-70     Citation Subset:  IM    
Affiliation:
Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Drug Design
Endopeptidases / metabolism
Female
Flow Cytometry
Immunoglobulin Variable Region / chemistry*,  genetics,  immunology,  metabolism*
Ligands
Maleimides / chemistry
Mass Spectrometry
Mice
Mice, Inbred ICR
Molecular Structure
Molecular Weight
Peptide Mapping
Polyethylene Glycols / chemistry*
Protein Engineering*
Protein Structure, Tertiary
Receptors, Tumor Necrosis Factor / antagonists & inhibitors,  metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Structure-Activity Relationship
Substrate Specificity
Tumor Necrosis Factor-alpha / antagonists & inhibitors,  immunology,  toxicity
Chemical
Reg. No./Substance:
0/Immunoglobulin Variable Region; 0/Ligands; 0/Maleimides; 0/Polyethylene Glycols; 0/Receptors, Tumor Necrosis Factor; 0/Tumor Necrosis Factor-alpha; 541-59-3/maleimide; EC 3.4.-/Endopeptidases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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