Renal brush border enzyme-cleavable linkages for low renal radioactivity levels of radiolabeled antibody fragments.

We previously demonstrated that Fab fragments labeled with 3'-[(131)I]iodohippuryl N(ε)-maleoyl-l-lysine ([(131)I]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[(131)I]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(ε)-Boc-l-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3'-[(125)I]iodohippuryl O-methyl-l-tyrosine (2c) liberated the highest amount of m-[(125)I]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'-[(125)I]iodohippuryl O-((2-maleimidoethyl)carbamoyl)methyl-l-tyrosine (HMT, 12c), was designed, synthesized, and subsequently conjugated to an Fab fragment. [(125)I]HMT-Fab exhibited renal radioactivity levels similar to and significantly lower than [(125)I]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 [(125)I]HMT-Fab showed that m-[(125)I]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.

In vitro system to estimate renal brush border enzyme-mediated cleavage of Peptide linkages for designing radiolabeled antibody fragments of low renal radioactivity levels.

Renal localization of radiolabeled antibody fragments presents a problem in targeted imaging and radiotherapy. We recently reported that Fab fragments labeled with 3'-[(131)I]iodohippuryl N(epsilon)-maleoyl-l-lysine (HML) demonstrated markedly low renal radioactivity levels from early postinjection in mice. Previous studies suggested that low renal radioactivity levels were attributable to cleavage of the glycyl-lysine sequence in HML by the action of renal brush border enzymes, followed by urinary excretion of the resulting m-iodohippuric acid. In this study, an in vitro system using brush border membrane vesicles (BBMVs) isolated from the rat kidney cortex was developed to estimate renal brush border enzyme(s)-mediated cleavage of the peptide linkage. Low molecular weight HML derivatives, 3'-[(125)I]iodohippuryl l-lysine (HL), 3'-[(125)I]iodohippuryl N(epsilon)-tert-butoxycarbonyl-l-lysine (HBL), and their d-amino acid counterparts, were synthesized and incubated in BBMVs. Both [(125)I]HL and [(125)I]HBL generated m-[(125)I]iodohippuric acid after incubation in BBMVs at 37 degrees C while the latter liberated significantly higher amounts of the metabolite. [(125)I]d-HL and [(125)I]d-HBL failed to release the metabolite under similar conditions. The liberation of m-[(125)I]iodohippric acid from [(125)I]HL was significantly facilitated or completely inhibited by the addition of an activator or an inhibitor for carboxypeptidase M. The release of m-[(125)I]iodohippuric acid from [(125)I]HBL increased by the addition of the activator, whereas the inhibitor partially inhibited the release of the metabolite from [(125)I]HBL. The BBMV-mediated release of m-[(125)I]iodohippuric acid from [(125)I]HBL was not impaired by the addition of inhibitors for neutral endopeptidase or renal dipeptidase. These findings showed that the glycyl-l-lysine sequence in HML would be recognized and cleaved by metalloenzymes and nonmetalloenzymes on the renal brush border even when iodine was incorporated into a benzene ring and the N(epsilon)-amine residue of lysine was chemically modified, which supported the hypothesis that low renal radioactivity levels of HML-conjugated Fab fragments would be attributed to the release of m-iodohippuric acid by renal brush border enzymes. This study suggested that this in vitro system using BBMVs would be useful to estimate radiolabeling reagents of antibody fragments or peptides designed to reduce renal radioactivity with a variety of radionuclides.