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Chemical Design of Radiolabeled Antibody Fragments for Low Renal Radioactivity Levels¹

Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences [Y. A., Y. F., H. A., M. O., T. U., K. W., H. Saj.], and Department of Nuclear Medicine and Diagnostic Imaging, Graduate School of Medicine [H. Sak., J. K.], Kyoto University, Kyoto 606-8501; and Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 [M. N.], Japan

The renal uptake of radiolabeled antibody fragments presents a problem in targeted imaging and therapy. We hypothesized that the renal radioactivity levels of radiolabeled antibody fragments could be reduced if radiolabeled compounds of urinary excretion were released from glomerularly filtered antibody fragments before they were incorporated into renal cells by the action of brush border enzymes, present on the lumen of renal tubules. 3'-[¹³¹I]Iodohippuryl N-maleoyl-L-lysine ([¹³¹I]HML) was conjugated with a thiolated Fab fragment because the glycyl-lysine sequence in HML is a substrate for a brush border enzyme and meta-iodohippuric acid is released by cleavage of the linkage. Fab fragments were also radiolabeled by direct radioiodination (¹²⁵I-Fab) or by conjugation with meta-[¹²⁵I]-iodohippuric acid via an amide bond [N-(5-maleimidopentyl) 3'-iodohippuric acid amide ([¹²⁵I]MPH-Fab)] or an ester bond [maleimidoethy 3'-iodohippurate ([¹²⁵I]MIH-Fab)] by procedures similar to those used for [¹³¹I]HML-Fab.

In biodistribution experiments in mice, [¹³¹I]HML-Fab demonstrated markedly low renal radioactivity levels with kidney:blood ratios of radioactivity of 1 from 10 min to 1 h due to rapid release of meta-[¹³¹I]iodohippuric acid. [¹²⁵I]MIH-Fab and ¹²⁵I-Fab reached their peak ratios of 3.8 and 7.3 at 1 h, respectively, and [¹²⁵I]MPH-Fab showed the maximum ratio of 16.8 at 6 h. In subcellular distribution studies, both [¹²⁵I]MIH-Fab and ¹²⁵I-Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection, whereas the majority of the radioactivity was detected only in the membrane fraction after administration of [¹³¹I]HML-Fab at both time points. In nude mice, [¹³¹I]HML-Fab showed one-quarter of the renal radioactivity of simultaneously administered ¹²⁵I-Fab without impairing the target radioactivity levels 3 h after injection. These findings indicated that HML is a useful reagent for targeted imaging and therapy using antibody fragments as vehicles. These findings also suggested that the radiochemical design of radiolabeled antibody fragments that liberate radiometabolites of urinary excretion from antibody fragments by the action of brush border enzymes may constitute a new strategy for reducing the renal radioactivity levels of antibody fragments.

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