Importance of an alkyl group inserted into the DOTA backbone for reducing renal radioactivity levels of 111In-labeled antibody fragments.

INTRODUCTION: 1,4,7,10-Tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is a promising chelator for radiotheranostic applications. Recently, we developed a DOTA-based radiolabeled antibody fragment, [111In]In-DO3AiBu-Bn-FGK-Fab, which contains a cleavable linkage recognized by renal brush border membrane (BBM) enzymes. Molecular design of radiometabolites that rapidly escape from the coated vesicles in renal cells during the early stage of endocytosis is crucial for reducing renal radioactivity levels. In the present study, we prepared [111In]In-DO3AEt-Bn-FGK-Fab by replacing the isobutyl group of [111In]In-DO3AiBu-Bn-FGK-Fab with an ethyl group. We assessed the reduction effect of [111In]In-DO3AEt-Bn-FGK-Fab on renal radioactivity levels to investigate the importance of the choice of alkyl group inserted into the DOTA backbone.

METHODS: Density functional theory (DFT) calculations were performed for three In-complexes, In-DO3AEt-Bn, In-DO3AiBu-Bn, and In-DOTA-Bn. Enzymatic recognition of the cleavable FGK linkage was assessed by incubating a model low-molecular-weight (LMW) model substrate, [111In]In-DO3AEt-Bn-FGK(Boc) with brush border membrane vesicles. Then, DO3AEt-Bn-FGK-Fab was prepared by conjugating DO3AEt-Bn-FGK(Mal) to thiolated Fab and was radiolabeled with 111In. [111In]In-DO3AEt-Bn-FGK-Fab was administered to normal mice, and biodistribution studies and metabolic analyses of the kidney homogenate and urine samples were performed with comparison to an 111In-labeled Fab prepared by a conventional method ([111In]In-DOTA-Bn-SCN-Fab). Biodistribution studies and SPECT imaging were also performed for both [111In]In-DO3AEt-Bn-FGK-Fab and [111In]In-DOTA-Bn-SCN-Fab in tumor-bearing mice.

RESULTS: High similarity in the coordination environments was observed for the structures of In-DO3AEt-Bn and In-DO3AiBu-Bn predicted by DFT calculations. The release of [111In]In-DO3AEt-Bn-F was observed in an in vitro system using renal BBM vesicles and was inhibited by an angiotensin-converting enzyme inhibitor. [111In]In-DO3AEt-Bn-FGK-Fab showed a moderate reduction in renal radioactivity levels compared to the reference and higher renal radioactivity levels than [111In]In-DO3AiBu-Bn-FGK-Fab. Metabolic studies revealed that the release of [111In]In-DO3AEt-Bn-F also occurred in vivo. Contrary to the reduction in renal radioactivity levels, tumor accumulation was not impaired when [111In]In-DO3AEt-Bn-FGK-Fab was compared to the reference.

CONCLUSION: Since the difference in the radiometabolites liberated from [111In]In-DO3AEt-Bn-FGK-Fab and [111In]In-DO3AiBu-Bn-FGK-Fab was the alkyl (ethyl and isobutyl) group inserted into the DOTA structure, slight differences in the structure of the radiometabolites severely affected their renal residence time. These results provide a warning for further applications of the renal brush border strategy to other radiometal-chelator complexes. The radiometabolites would need to be carefully designed to ensure rapid elimination from renal cells.