Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab鈥?Fragments Improves Tumor MicroPET/CT Imaging in Mice

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• 作者：Haiming Ding ; Michelle M. Carlton ; Stephen P. Povoski ; Keisha Milum ; Krishan Kumar ; Shankaran Kothandaraman ; George H. Hinkle ; David Colcher ; Rich Brody ; Paul D. Davis ; Alex Pokora ; Mitchell Phelps ; Edward W. Martin ; Jr. ; Michael F. Tweedle
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：November 20, 2013
• 年：2013
• 卷：24
• 期：11
• 页码：1945-1954
• 全文大小：360K
• 年卷期：v.24,no.11(November 20, 2013)
• ISSN：1520-4812

文摘

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2鈥? kDa discrete, branched PEGylation reagents on mCC49 Fab鈥?(MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab鈥?(Fab鈥?NEM) was conjugated at a hinge region cysteine with maleimide-dPEG ₁₂-(dPEG₂₄COOH)₃ acid (Mal-dPEG-A), maleimide-dPEG₁₂-(dPEG₁₂COOH)₃ acid (Mal-dPEG-B), or maleimide-dPEG₁₂-(m-dPEG₂₄)₃ (Mal-dPEG-C), and then radiolabeled with iodine-124 (p>124p>I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab鈥?with Mal-dPEG-A (Fab鈥?A) reduced the binding affinity of the non PEGylated Fab鈥?by 30%; however, in vivo, Fab鈥?A significantly lengthened the blood retention vs Fab鈥?NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 卤 0.83 vs 3.83 卤 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab鈥?NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 卤 1.50 vs 1.97 卤 0.29%ID/g, p < 0.05) and 63% (3.83 卤 1.50 vs 1.42 卤 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.