Design, Synthesis, and Evaluation of [188Re]Organorhenium-Labeled Antibody Fragments with Renal Enzyme-Cleavable Linkage for Low Renal Radioactivity Levels
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- 作者：Tomoya Uehara ; Miho Koike ; Hideo Nakata ; Hiroshi Hanaoka ; Yasuhiko Iida ; Kazuyuki Hashimoto ; Hiromichi Akizawa ; Keigo Endo ; Yasushi Arano
- 刊名：Bioconjugate Chemistry
- 出版年：2007
- 出版时间：January 2007
- 年：2007
- 卷：18
- 期：1
- 页码：190 - 198
- 全文大小：138K
- 年卷期：v.18,no.1(January 2007)
- ISSN：1520-4812
文摘
Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy.We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl N
-maleoyl-lysine (HML) showed markedlylow renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuricacid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallicradionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated withN-tert-butoxycarbonyl-glycyl-lysine or N-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid.The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippurylN-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution ofradioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While[188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acidat a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes andnonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levelsthan did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higherthan those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fabsignificantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findingsindicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelateof high inertness and by designing a radiometabolite of high urinary excretion when released from antibodyfragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemicalstructure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysisreaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brushborder membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeledantibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitrosystem using BBMVs might be useful for selecting an appropriate peptide linkage.
-maleoyl-lysine (HML) showed markedlylow renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuricacid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallicradionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated withN-tert-butoxycarbonyl-glycyl-lysine or N-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid.The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippurylN-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution ofradioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While[188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acidat a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes andnonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levelsthan did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higherthan those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fabsignificantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findingsindicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelateof high inertness and by designing a radiometabolite of high urinary excretion when released from antibodyfragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemicalstructure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysisreaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brushborder membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeledantibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitrosystem using BBMVs might be useful for selecting an appropriate peptide linkage.