Protein PEGylation Decreases Observed Target Association Rates via a Dual Blocking Mechanism

Abstract

PEGylation is an attractive strategy to enhance the therapeutic efficacy of proteins with a short serum half-life. It can be used to extend the serum persistence and to reduce the immunogenicity of proteins. However, PEGylation can also lead to a decrease in the functional activity of the molecule to which it is applied. We constructed site-specifically PEGylated variants of anti-p185 HERϪ2 antibody fragments in the format of a monovalent single-chain variable fragment and a divalent miniantibody and characterized the antigen binding properties in detail. Mass-transport limited BIAcore measurements and binding assays on HER-2-overexpressing cells demonstrated that the immunoreactivity of the antibody fragments is fully maintained after PEGylation. Nevertheless, we found that the attachment of a 20-kDa polyethylene glycol (PEG) moiety led to a reduction in apparent affinity of approximately 5-fold, although in both formats, the attachment site was most distal to the antigen binding regions. This decrease in affinity was observed in kinetic BIAcore measurements as well as in equilibrium binding assays on whole cells. By analysis of the binding kinetics, we could pinpoint this reduction exclusively to slower apparent on rates. Through both experimental and computational analyses, we demonstrate that these reduced on-rates do not arise from diffusion limitations. We show that a mathematical model accounting for both intramolecular and intermolecular blocking mechanisms of the PEG moiety can robustly explain the observed binding kinetics. The results suggest that PEGylation can significantly alter the binding-competent fraction of both ligands and receptors and may help to explain some of the beneficial effects of PEGylation in vivo.

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

“…We could confirm the theoretical molecular mass of about 50 kDa at least for the monomer-PEG20 construct by multi-angle static light scattering (MALS), performed online during the gel filtration runs (55). This behavior of PEG in gel filtration is in agreement with the findings of other groups (49,50,68,69).…”

“…For the multivalent species only apparent values of functional affinities or avidities (valid for the particular immobilization density used) can be deduced. However, because of the very slow dissociation rate, deviations from a 1:1 model are not apparent from the quality of the fit of the multivalent species (55).…”

“…By attachment of a 20-kDa polyethylene glycol moiety, a highly flexible, bulky residue with a long reach was added to the protein. This PEG-tail can dynamically interfere with the antigen-antibody binding interaction, and in addition, upon binding, block the binding to a neighboring antigen (55).…”

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

“…S4 and Table S1). After PEGylation, however, the association rate constant of PEG 20kDa -Ec1-ETA 00 was 2-fold lower than the non-PEGylated fusion toxin, whereas no difference was found for k d. This resulted in an overall reduction in K D by a factor of two (139 pmol/L before and 290 pmol/L after PEGylation), possibly due to intramolecular blocking effects (37). In addition, the maximal response on the chip (which is proportional to the number of fusion toxin molecules able to interact with the surface) showed a reduction for the PEGylated fusion toxin, compared with its non-PEGylated counterpart, probably due to intermolecular blocking effects resulting from steric hindrance by the PEG 20kDa polymer ( Supplementary Fig.…”

Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles

“…The decoration of AuNPs with thiolated poly(ethylene glycol) (PEG-SH) via the formation of stable, covalent gold-thiol linkages (bond energy = 30-40 kJ/mol) (19) can reduce nonspecific interactions (20), allowing the targeting ligands immobilized on the particle surface to engage cell surface receptors with high specificity. PEGylation also prolongs the circulation time of nanoparticles (21), giving sufficient time for particles to localize in different organs.…”