Renal Brush Border Enzyme-Cleavable Linkages for Low Renal Radioactivity Levels of Radiolabeled Antibody Fragments
Renal Brush Border Enzyme-Cleavable Linkages for Low Renal Radioactivity Levels of Radiolabeled Antibody Fragments
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DOI：
10.1021/bc300428b
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发表时间：
2013-02-01
影响因子：
4.7
通讯作者：
Arano, Yasushi
中科院分区：
文献类型：
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作者：
Akizawa, Hiromichi;Imajima, Mitsuo;Arano, Yasushi
我们之前证明，用 3'-[I-131] 碘马嘌呤 N-ε-马来酰基-L-赖氨酸 ([I-131]HML) 标记的 Fab 片段在注射后早期就表现出较低的肾放射性，这是由于肾刷状缘酶的作用释放了间-[I-131]碘马尿酸。由于肾刷状缘膜上有大量酶，因此将甘氨酰-L-赖氨酸以外的肽键评估为可裂解键以探索化学设计。在本研究中，我们评估了四种肽键，通式为间碘苯甲酰基-甘氨酰-X（X：L-酪氨酸O-甲基、L-天冬酰胺、L-谷氨酰胺和N-ε-Boc-L-赖氨酸）。使用肾刷状缘膜囊泡 (BBMV) 的体外研究表明，4 种底物中，3'-[I-125]碘马尿基 O-甲基-L-酪氨酸 (2c) 释放最高量的间-[I-125]碘马尿酸，并且连接结构的变化改变了负责水解反应的酶种类。为了进一步评估该连接的适用性，设计、合成了含有甘氨酰-酪氨酸连接 3'-[I-125]碘马嘌呤 O((2-马来酰亚胺乙基)氨基甲酰基)甲基-L-酪氨酸 (HMT, 12c) 的放射性碘化试剂，随后将其与 Fab 片段缀合。 [I-125]HMT-Fab 的肾放射性水平与 [I-125]HML-Fab 和直接放射性碘化的 Fab 相似且显着较低，而三者的血液清除率相似。 [I-125]HMT-Fab 注射后 24 小时的尿液分析表明，间-[I-125]碘马尿酸作为主要放射性代谢物被排出。研究结果表明，由于肾刷状缘膜上存在的酶的作用释放了间碘马尿酸，甘氨酰-酪氨酸连接也可降低放射性碘标记的 Fab 片段的肾放射性水平。这些发现表明，肽键的适当选择将允许从共价缀合的多肽中释放设计的放射性标记的化合物，以制备低肾放射性水平的放射性标记的多肽。为了选择最合适的肽键，使用 BBMV 的体外系统将有助于将候选肽缩小到少数。
We previously demonstrated that Fab fragments labeled with 3'-[I-131]iodohippuryl N-epsilon-maleoyl-L-lysine ([I-131]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[I-131]iodohippuric acid by the action of renal brush border enzymes. Since there are lots of enzymes on renal brush border membrane, peptide linkages other than the glycyl-L-lysine were evaluated as the cleavable linkages to explore the chemical design. In this study, we evaluated four peptide linkages with a general formula of m-iodobenzoyl-glycyl-X (X: L-tyosine O-methyl, L-asparagine, L-glutamine, and N-epsilon-Boc-L-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3'-[I-125]iodohippuryl O-methyl-L-tyrosine (2c) liberated the highest amount of m-[I-125]iodohippuric acid among the four substrates and the change in the linkage structure altered enzyme species responsible for the hydrolysis reaction. To further assess the applicability of the linkage, a radioiodination reagent containing a glycyl-tyrosine linkage, 3'-[I-125]iodohippuryl O((2-maleimidoethyl)carbamoyl)methyl-L-tyrosine (HMT, 12c), was designed, synthesized, and subsequently conjugated to an Fab fragment. [I-125]HMT-Fab exhibited renal radioactivity levels similar to and significantly lower than [I-125]HML-Fab and directly radioiodinated Fab, while the blood clearance rates of the three were similar. The analyses of urine for 24 h postinjection of [I-125]HMT-Fab showed that m-[I-125]iodohippuric acid was excreted as the major radiometabolite. The findings indicated that glycyl-tyrosine linkage is also available to reduce renal radioactivity levels of radioiodinated Fab fragments, due to liberation of m-iodohippuric acid by the action of enzymes present on renal brush border membrane. These findings suggest that an appropriate selection of peptide linkages would allow the liberation of a designed radiolabeled compound from covalently conjugated polypeptides to prepare radiolabeled polypeptides of low renal radioactivity levels. For the selection of the most appropriate peptide linkage, the in vitro system using BBMVs would be useful to narrow the candidates to just a few.