The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a Tc chelate of an isonicotinic acid derivative of 2-picolylglycine (Tc-IPG). Tc-IPG-glycine was liberated fromTc-IPG-GFK by the enzymes, while Tc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice,Tc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of Tc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m ( 99m Tc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG 3 )-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG 3 -GFK-suc-TFP, was designed, synthesized, and evaluated. 99m Tc-labeled Fab was obtained by reacting MAG 3 -GFK-Fab conjugate with 99m Tc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 ( 111 In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [ 99m Tc]Tc-MAG 3 -GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [ 99m Tc]Tc-MAG 3 -Gly. However, [ 99m Tc]Tc-MAG 3 -GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111 In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [ 99m Tc]Tc-MAG 3 -Gly to the intestine. The retrospective comparison of [ 99m Tc]Tc-MAG 3 -GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [ 99m Tc]Tc-MAG 3 -Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG 3 -GFK-suc-TFP provided 99m Tc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the postlabeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.