Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels
Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels
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DOI：
10.1021/bc0602329
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发表时间：
2007-01-01
影响因子：
4.7
通讯作者：
Arano, Yasushi
中科院分区：
文献类型：
--
作者：
Uehara, Tomoya;Koike, Miho;Arano, Yasushi
放射性标记的抗体片段的肾定位构成靶向成像和放射治疗中的问题。我们曾报道，由于间-[I-131]碘马尿酸在刷状缘酶作用下快速选择性释放，用3 '-[I-131]碘马尿酸N-β-马来酰赖氨酸（HML）标记的Fab片段即使在注射后不久也显示出显著低的肾放射性水平。为了评估分子设计对金属放射性核素的适用性，将[Re-188]三羰基（环戊二烯基碳酸酯）双（[Re-188]CpTR-COOH）与N-叔丁氧羰基-甘氨酰-赖氨酸或N-顺丁烯二酰-甘氨酰-赖氨酸缀合以制备[Re-188]CpTR-GK-Boc或[Re-188]CpTR-GK。两种化合物的甘氨酰-赖氨酸键的裂解产生[Re-188]CpTR-COOH（[Re-188]CpTR-Gly）的甘氨酸缀合物，其具有与间碘马尿酸相似的体内行为。用大鼠肾刷状缘膜囊泡（BBMV）比较了[Re-188]CpTR-GK-Boc和3 ′-[I-125]碘马尿酰N-脱氨-Boc-赖氨酸（[I-125]HL-Boc）中肽键的水解速率。将[Re-188]CpTR-GK与巯基化Fab片段缀合以制备[Re-188]CpTR-GK-Fab。将注射[Re-188]CpTR-GK-Fab后放射性的生物分布与通过将[Re-188]CpTR-COOH的N-羟基琥珀酰亚胺酯与抗体片段偶联制备的[I-125]HML-Fab和[Re-188]CpTR-Fab进行比较。[Re-188]CpTR-GK-Boc在BBMV中释放[Re-188]CpTR-Gly，而[I-125]HL-Boc以更快的速率释放间-[I-125]碘马尿酸。此外，虽然[I-125]HL-Boc被金属酶和非金属酶水解，但金属酶负责[Re-188]CpTR-GK-Boc中的肽键的切割。在生物分布研究中，[Re-188]CpTR-GK-Fab的肾脏放射性水平显著低于[Re-188]CpTR-Fab。然而，[Re-188]CpTR-GK-Fab的肾脏放射性水平略高于[I-125]HML-Fab。对注射[Re-188]CpTR-GK-Fab后6 h收集的尿样进行分析，结果表明[Re-188]CpTR-Gly是主要的放射性代谢物。在荷瘤小鼠中，[Re-188]CpTR-GK-Fab显著降低肾脏放射性水平，而不损害肿瘤中的放射性水平。这些研究结果表明，HML的分子设计可以应用于金属放射性核素，通过使用放射性金属螯合物的高惰性，并通过设计高尿排泄的放射性代谢物时，从抗体片段的甘氨酰-赖氨酸键裂解后释放。这项研究还表明，化学结构的变化连接到甘氨酰-赖氨酸键显着影响参与水解反应的酶的放射性标记。由于有许多种酶可切割肾刷状缘膜上的各种肽键，因此选择对目标放射性金属螯合物最佳的肽键可提供放射性标记的抗体片段，其显示出与[I-131] HML标记的抗体片段相似的肾放射性水平。使用BBMV的体外系统可能有助于选择合适的肽连接。
Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[I-131]iodohippuryl N-epsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[I-131]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [Re-188]tricarbonyl(cyclopentadienylcarbonate)rhenium ([Re-188]CpTR-COOH) was conjugated with N-epsilon-tert-butoxycarbonyl-glycyl-lysine or N-epsilon-maleoyl-glycyl-lysine to prepare [Re-188]CpTR-GK-Boc or [Re-188]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [Re-188]CpTR-COOH ([Re-188]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [Re-188]CpTR-GK-Boc was compared with that in 3'-[I-125]iodohippuryl N-epsilon-Boc-lysine ([I-125]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [Re-188]CpTR-GK was conjugated to thiolated Fab fragments to prepare [Re-188]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [Re-188]CpTR-GK-Fab was compared with that of [I-125]HML-Fab and [Re-188]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [Re-188]CpTR-COOH with antibody fragments. While [Re-188]CpTR-GK-Boc liberated [Re-188]CpTR-Gly in BBMVs, [I-125]HL-Boc liberated m-[I-125]iodohippuric acid at a much faster rate. In addition, although [I-125]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [Re-188]CpTR-GK-Boc. In biodistribution studies, [Re-188]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [Re-188]CpTR-Fab. However, the renal radioactivity levels of [Re-188]CpTR-GK-Fab were slightly higher than those of [I-125]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [Re-188]CpTR-GK-Fab showed that [Re-188]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [Re-188]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [I-131]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.