S. Kubetzko†, C.A. Sarkar†, and A. Plückthun. "Protein PEGylation decreases observed target association rates via a dual blocking mechanism." Molecular Pharmacology, 68:1439-1454 (2005). †Contributed equally.

    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 also 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-p185HER-2 antibody fragments in the format of a monovalent single-chain Fv 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 PEG moiety led to about a 5-fold reduction in apparent affinity, 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 ligands and may help to explain some of the beneficial effects of PEGylation in vivo.

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