Comparative Binding of Disulfide-Bridged PEG-Fabs

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• 作者：Hanieh Khalili ; Antony Godwin ; Ji-won Choi ; Rebecca Lever ; Steve Brocchini
• 刊名：Bioconjugate Chemistry
• 出版年：2012
• 出版时间：November 21, 2012
• 年：2012
• 卷：23
• 期：11
• 页码：2262-2277
• 全文大小：664K
• 年卷期：v.23,no.11(November 21, 2012)
• ISSN：1520-4812

文摘

Protein PEGylation is the most clinically validated method to improve the efficacy of protein-based medicines. Antibody fragments such as Fabs display rapid clearance from blood circulation and therefore are good candidates for PEGylation. We have developed PEG-bis-sulfone reagents 1 that can selectively alkylate both sulfurs derived from a native disulfide. Using PEG-bis-sulfone reagents 1, conjugation of PEG specifically targets the disulfide distal to the binding region of the Fab (Scheme 2). PEG-bis-sulfone reagents 1 (10鈥?0 kDa) were used to generate the corresponding PEG-mono-sulfones 2 that underwent essentially quantitative conjugation to give the PEG-Fab product 4. Four Fabs were PEGylated: Fab_beva, Fab鈥?sub>beva, Fab_rani, and Fab_trast. Proteolytic digestion of bevacizumab with papain gave Fab_beva, while digestion of bevacizumab with IdeS gave F(ab鈥?_2-beva, which after reaction with DTT and PEG-mono-sulfone 2 gave PEG₂-Fab鈥?sub>beva. Ranibizumab, which is a clinically used Fab, was directly PEGylated to give PEG-Fab_rani. Trastuzumab was proteolytically digested with papain, and its corresponding Fab was PEGylated to give PEG-Fab_trast. Purification of the PEGylated Fabs was accomplished by a single ion exchange chromatography step to give pure PEG-Fab products as determined by silver-stained SDS-PAGE. No loss of PEG was detected post conjugation. A comparative binding study by SPR using Biacore with low ligand immobilization density was conducted using (i) VEGF₁₆₅ for the bevacizumab and ranibizumab derived products or (ii) HER2 for the trastuzumab derived products. VEGF₁₆₅ is a dimeric ligand with two binding sites for bevacizumab. HER2 has one domain for the binding of trastuzumab. Binding studies with PEG-Fab_beva indicated that the apparent affinity was 2-fold less compared to the unPEGylated Fab_beva. Binding properties of the PEG-Fab_beva products appeared to be independent of PEG molecular weight. Site-specific conjugation of two PEG molecules gave PEG_2脳20-Fab鈥?sub>beva, whose apparent binding affinity was similar to that observed for PEG-Fab_beva derivatives. The k_d values were similar to those of the unPEGylated Fab_beva; hence, once bound, PEG-Fab_beva remained bound to the same degree as Fab_beva. Biacore analysis indicated that both Fab_rani and PEG₂₀-Fab_rani did not dissociate from the immobilized VEGF at 25 掳C, but ELISA using immobilized VEGF showed 2-fold less apparent binding affinity for PEG₂₀-Fab_rani compared to the unPEGylated Fab_rani. Additionally, the apparent binding affinities for trastuzumab and Fab_trast were comparable by both Biacore and ELISA. Biacore results suggested that trastuzumab had a slower association rate compared to Fab_trast; however, both molecules displayed the same apparent binding affinity. This could have been due to enhanced rebinding effects of trastuzumab, as it is a bivalent molecule. Analogous to PEG-Fab_beva products, PEG₂₀-Fab_trast displayed 2-fold lower binding compared to Fab_trast when evaluated by ELISA. The variations in the apparent affinity for the PEGylated Fab variants were all related to the differences in the association rates (k_a) rather than the dissociation rates (k_d). We have shown that (i) Fabs are well-matched for site-specific PEGylation with our bis-alkylation PEG reagents, (ii) PEGylated Fabs display only a 2-fold reduction in apparent affinity without any change in the dissociation rate, and (iii) the apparent binding rates and affinities remain constant as the PEG molecular weight is varied.