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

Abstract

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.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

“…As shown in Supplementary Fig. S3, non-PEGylated Ec1-ETA 00 eluted with the expected molecular weight of approximately 60 kDa, whereas mono-PEGylated Ec1-ETA 00 eluted at smaller volumes, which is comparable with a molecular weight of more than 250 kDa typical for a mono-PEGylated protein (36).…”

111In-LLP2A-DOTA Polyethylene Glycol–Targeting α4β1 Integrin: Comparative Pharmacokinetics for Imaging and Therapy of Lymphoid Malignancies

“…The observed extended half-life in circulation of (LLP2A-DOTA) 4 -PEG 10,000 suggests that the branched PEG may expand the Stoke radius of a PEG peptidomimetic conjugate. Because the nature of PEG already expanded this size beyond its actual mass (32), only the Stoke radius of the (LLP2A-DOTA) 4 -PEG 10,000 molecule was likely to have been above the effective size-exclusion limit for first-pass glomerular filtration. Tumor uptake of the tetramer was similar to (and retention better than) that in the LLP2A 1-nM study, despite the calculations that the 4 units/molecule increased the ligand moles injected by a factor of 4, making it 4.4 nmol of LLP2A binding units, compared with the 1.1 molecular nanomoles.…”

Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region

“…The availability of maleimide-PEG has allowed conjugation to antibodies through reaction with free native or engineered cysteines (12). This chemistry has proven to be suitable not only for Fab but also for single-chain variable fragments (scFvs) (1,37,54), immunoliposomes (38), and other conjugates (19).…”