Development of NOTA-based antibody-fragment labeling agent that reduces early renal radioactivity levels

Gallium-68 (⁶⁸Ga) and copper-64 (⁶⁴Cu) are radiometals used for nuclear medicine imaging and therapy. The compound 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms stable complexes with these radiometals. However, ⁶⁸Ga/⁶⁴Cu-labeled-low molecular weight proteins show high radioactivity levels in the kidneys. To address this problem, a NOTA-conjugated Met-Val-Lys (MVK)-maleimide (Mal) (NOTA-MVK-Mal), was recently designed to reduce renal radioactivity levels. The agent achieved this by releasing radiometal-NOTA-methionine as a radiometabolite via the action of renal brush border enzymes. As ⁶⁸Ga and ⁶⁴Cu have short half-lives, it is crucial to reduce renal radioactivity from the early post-injection-period. Based on this molecular strategy, this study designed and synthesized a new NOTA-based radiometal-labeling agent, designated NOTA-conjugated Phe-Gly-Lys (FGK)-Mal (NOTA-FGK-Mal), which reduced renal radioactivity in the earlier post-injection period.

Considering that the FGK sequence is a substrate of enzymes on the renal brush border, isothiocyanatobenzyl-NOTA was conjugated to an antibody Fab fragment through a FGK linkage to produce [⁶⁷Ga]Ga-NOTA-FGK-Fab. Enzymatic recognition of the linkage was evaluated using a low-molecular-weight [⁶⁷Ga]Ga-NOTA-FGK derivative. The biodistribution of radioactivity after the injection of [⁶⁷Ga]Ga-NOTA-FGK-Fab into mice was compared with that of [⁶⁷Ga]Ga-NOTA-MVK-Fab. The FGK linkage was recognized by brush border membrane enzymes to liberate [⁶⁷Ga]Ga-NOTA-Phe-Gly. This indicated that the recognition of the FGK sequence in the [⁶⁷Ga]Ga-NOTA-FGK derivative was the GK sequence, not the expected FG sequence. When injected into mice, [⁶⁷Ga]Ga-NOTA-FGK-Fab resulted in lower renal radioactivity levels than those induced by [⁶⁷Ga]Ga-NOTA-MVK-Fab without a reduction in tumor radioactivity levels.

These results suggest that NOTA-FGK(Mal) is a useful radiometal labeling agent for low-molecular-weight proteins that reduces renal radioactivity levels from as early as post-injection. This molecular design is not dependent on the type of antibody or its constructs, it is expected that NOTA-FGK(Mal) will also be applicable to a variety of target molecules.