Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation.
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.
Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA.
Karen
K
Yang
Amartya
A
Basu
Maoliang
M
Wang
Ramesh
R
Chintala
Ming-Ching
MC
Hsieh
Sam
S
Liu
Jack
J
Hua
Zhenfan
Z
Zhang
John
J
Zhou
Mark
M
Li
Hnin
H
Phyu
Gerald
G
Petti
Magda
M
Mendez
Haleema
H
Janjua
Ping
P
Peng
Clifford
C
Longley
Virna
V
Borowski
Mary
M
Mehlig
David
D
Filpula
D000818:Animals; D015195:Drug Design; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D008301:Maleimides / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D015394:Molecular Structure; D008970:Molecular Weight; D010449:Peptide Mapping; D011092:Polyethylene Glycols / Q000737:chemistry*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000276:immunology / Q000633:toxicity
D007135:Immunoglobulin Variable Region; D008024:Ligands; D008301:Maleimides; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; C043592:maleimide; D011092:Polyethylene Glycols; D010450:Endopeptidases