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.
Yang
Karen
K
Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA.
Basu
Amartya
A
Wang
Maoliang
M
Chintala
Ramesh
R
Hsieh
Ming-Ching
MC
Liu
Sam
S
Hua
Jack
J
Zhang
Zhenfan
Z
Zhou
John
J
Li
Mark
M
Phyu
Hnin
H
Petti
Gerald
G
Mendez
Magda
M
Janjua
Haleema
H
Peng
Ping
P
Longley
Clifford
C
Borowski
Virna
V
Mehlig
Mary
M
Filpula
David
D
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