Protein PEGylation decreases observed target association rates via a dual blocking mechanism.
نویسندگان
چکیده
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(HER-2) 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.
منابع مشابه
Mol 14910 1 Protein Pegylation Decreases Observed Target Association Rates via a Dual Blocking Mechanism
متن کامل
Deblocking Joint Photographic Experts Group Compressed Images via Self-learning Sparse Representation
JPEG is one of the most widely used image compression method, but it causes annoying blocking artifacts at low bit-rates. Sparse representation is an efficient technique which can solve many inverse problems in image processing applications such as denoising and deblocking. In this paper, a post-processing method is proposed for reducing JPEG blocking effects via sparse representation. In this ...
متن کاملPerformance-Based Plastic Design of Moment Frame-Steel Plate Shear Wall as a Dual System
Steel Plate Shear Wall (SPSW) is an emerging seismic load-resistant system that, compared to other systems, enjoys the advantages of stable ductile behavior, fewer detailing requirements, rapid constructability, and economy. American seismic provisions decree that a SPSW should be designed as a moment frame with a web infill plate. Specifically, in case of buildings taller than 160 ft, it decre...
متن کاملStudy on the fouling behavior of HDPE/PE-g-MA/EVA blend membrane fabricated via thermally induced phase separation method
In this study, neat HDPE and HDPE/PE-g-MA/EVA blend membranes were fabricated via thermally induced phase separation (TIPS) method and their fouling behaviors were examined using filtration of BSA protein. Membranes were characterized using FESEM, AFM, ATR-FTIR analyses and porosity measurement. Fouling behavior of membranes was analyzed using the resistance-in-series (RIS), classic and combine...
متن کاملProtein tetrazinylation via diazonium coupling for covalent and catalyst-free bioconjugation.
An efficient and bench-stable reagent was synthesized for direct and covalent introduction of tetrazines onto target protein or virus surfaces, which can be further modified based on tetrazine-ene ligation to achieve fluorescence labelling or PEGylation under mild conditions.
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Molecular pharmacology
دوره 68 5 شماره
صفحات -
تاریخ انتشار 2005