Stability of a metabolizable ester bond in radioimmunoconjugates.

Ester bonds have been used as metabolizable linkages to reduce radioactivity levels in non-target tissues following the administration of antibodies labeled with metallic radionuclides. In this radiochemical design of antibodies, while the ester bonds should be cleaved rapidly in non-target tissues, high stability of the ester bonds in plasma is also required to preserve target radioactivity levels. To assess the structural requirements to stabilize the ester bond, a new benzyl-EDTA-derived bifunctional chelating agent with an ester bond, (1-[4-[4-(2- maleimidoethoxy)succinamido]benzyl]ethylenediamine-N,N,N',N' -tetraacetic acid; MESS-Bz-EDTA), was developed. MESS-Bz-EDTA was coupled with a thiolated monoclonal antibody (OST7, IgG1) prepared by reducing its disulfide bonds to introduce the ester bond close and proximal to the antibody molecule. For comparison, 1-[4-(5- maleimidopentyl)aminobenzyl]ethylenediamine-N,N,N',N'-tetraacetic acid (EMCS-Bz-EDTA) and meleimidoethyl 3-[131I]iodohippurate (MIH) was coupled to OST7 under the same conjunction chemistry. When incubated in 50% murine plasma or a buffered-solution of neutral pH, OST7-MESS-Bz-EDTA-111In rapidly released the radioactivity, and more than 95% of the initial radioactivity was liberated after a 24 h incubation in both solutions, due to a cleavage of the ester bond. On the other hand, only about 20% of the radioactivity was released from OST7-MIH-131I in both solutions during the same incubation period. In mice biodistribution studies, while a slightly faster radioactivity clearance from the blood with less radioactivity levels in the liver and kidneys was observed with OST7-MIH-131I than with OST7-EMCS-Bz-EDTA-111In, OST7-MESS-Bz-EDTA-111In indicated radioactivity clearance from the blood much faster than and almost comparable to that of OST7-MIH-131I and succinamidobenzyl-EDTA-111In, respectively. These findings as well as previous findings on radiolabeled antibodies with ester bonds suggested that while an introduction of an ester bond close to an antibody molecule stabilized the ester bond against esterase access, chemical structures of the linkages and radiolabels attached to the ester bonds play a significant role in the chemical stability of the ester bond. This may explain the different stability of the ester bonds in radioimmunoconjugates so far reported.

Radiolabeled metabolites of proteins play a critical role in radioactivity elimination from the liver.

We have recently reported that the behavior of radiolabeled metabolites in the liver appears to be responsible for the hepatic radioactivity levels after administration of protein radiopharmaceuticals. To better understand the role played by radiolabeled metabolites in hepatic radioactivity levels, two benzyl-EDTA derivatives rendering different radiolabeled metabolites, 1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) and 1-[p-(5-maleimidopentyl)aminobenzyl]ethylenediaminetetraacetic acid (ECMS-Bz-EDTA), were selected as bifunctional chelating agents (BCAs), and 111In labeling of galactosyl-neoglycoalbumin (NGA) and mannosyl-neoglycoalbumin (NMA) was performed. Biodistribution of radioactivity in mice and subcellular distribution of radioactivity in hepatocytes were then compared. After accumulation in hepatic parenchymal cells, NGA-EMCS-Bz-EDTA-111In rendered a faster elimination rate of radioactivity from the liver than NGA-SCN-Bz-EDTA-111In. Although each 111In-NMA exhibited a delayed elimination rate of radioactivity from the liver compared to the 111In-NGA counterpart, NMA-EMCS-Bz-EDTA-111In showed faster elimination rate of radioactivity than NMA-SCN-Bz-EDTA-111In. Analyses of radioactivity excreted in feces and urine and remaining in the liver indicated that both BCAs rendered mono-amino acid adducts as the major radiolabeled metabolites (cysteine-EMCS-Bz-EDTA-111In and lysine-SCN-Bz-EDTA-111In), which were generated in both cell types of the liver within 1 h postinjection. Subcellular distribution of radioactivity indicated that the radioactivity was copurified with lysosomes. These results demonstrate that although in vivo stability of radiometal chelates is essential, the biological properties of the radiolabeled metabolites generated after lysosomal proteolysis in hepatocytes play a critical role in radioactivity elimination from the liver.

Maleimidoethyl 3-(tri-n-butylstannyl)hippurate: a useful radioiodination reagent for protein radiopharmaceuticals to enhance target selective radioactivity localization.

In pursuit of radiolabeled monoclonal antibodies (mAbs) with rapid urinary excretion of radioactivity from nontarget tissues, radioiodinated mAbs releasing a m-iodohippuric acid from the mAbs in nontarget tissues were designed. A novel reagent, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was synthesized by reacting N-(hydroxyethyl)maleimide with N-Boc-glycine before coupling with N-succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE). MIH possessed a maleimide group for mAb conjugation and a butylstannyl moiety for high-yield and site-specific radioiodination, and the two functional groups were linked via an ester bond to release m-iodohippuric acid. To investigate the fate of radiolabels after lysosomal proteolysis, hepatic parenchymal cells were used as a model nontarget tissue and 131I-labeled MIH was conjugated with galactosyl-neoglycoalbumin (NGA). Further conjugation of [131I]MIH with a mAb against osteogenic sarcoma (OST7) after reduction of its disulfide bonds was followed up. In murine biodistribution studies, [131I]MIH-NGA exhibited rapid accumulation in the liver followed by radioactivity elimination from the liver at a rate that was identical to and faster than those of 131I-labeled NGA via direct iodination ([131I]NGA) and [131I]ATE-labeled NGA, respectively. While [131I]NGA indicated high radioactivity levels in the murine neck, stomach, and blood, such increases in the radioactivity count were not detectable by the administration of either [131I]MIH-NGA or [131I]ATE-NGA. At 6 h postinjection of [131I]MIH-NGA, 80% of the injected radioactivity was recovered in the urine. Analyses of urine samples indicated that m-iodohippuric acid was the sole radiolabeled metabolite. In biodistribution studies using [131I]-MIH-OST7 and [131I]ATE-OST7, while both 131I-labeled OST7s registered almost identical radioactivity levels in the blood up to 6 h postinjection, the former demonstrated a lower radioactivity level than [131I]ATE-OST7 in nontarget tissues throughout the experiment. Such chemical and biological characteristics of MIH would enable high target/nontarget ratios in diagnostic and therapeutic nuclear medicine using mAbs and other polypeptides.

A biological method to evaluate bifunctional chelating agents to label antibodies with metallic radionuclides.

UNLABELLED: Since the lysosome is a common organelle for protein digestion, pursuing the fate of radiolabeled metabolites after lysosomal proteolysis in liver cells is ideal to evaluate bifunctional chelating agents (BCAs). METHODS: We used galactosyl-neoglycoalbumin (NGA) and mannosyl-neoglycoalbumin (NMA) as carrier proteins for hepatic parenchymal and nonparenchymal cells, respectively. These proteins were labeled with 111In using 1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) as a model. RESULTS: NGA-SCN-Bz-EDTA-111In exhibited rapid accumulation in the hepatic parenchymal cells, followed by hepatobiliary excretion of the metabolites with an elimination rate that was faster and much slower than that of NGA-DTPA-111In and NGA-131I, respectively. This metabolite represented all the radioactivity registered in the liver at 1 hr postinjection. Subcellular distribution studies indicated the metabolites were located only in the lysosome fraction, and the difference in elimination rates of the metabolites from the lysosome fraction was responsible for the variations in radioactivity clearance from the cells. CONCLUSION: The biological characteristics of radiolabeled metabolites play a critical role in eliminating the radiolabel from liver cells. The present method portrays a highly useful model to pursue the fate of radiolabeled metabolites in the liver.