Reducing renal radioactivity of radiolabeled tetravalent RGD peptides via cleavable linkers for targeted radionuclide therapy.

INTRODUCTION: In targeted radionuclide therapy, high renal radioactivity raises concerns that nephrotoxicity may limit therapeutic utility. Our recently developed 211At-labeled tetravalent RGD peptide ([211At]At-(RGD)4) also faces this challenge, despite achieving high and persistent tumor accumulation. Here, we evaluated whether introducing renal brush-border enzyme-cleavable linkers (renal brush-border strategy) could reduce renal radioactivity and improve the tumor-to-kidney ratio of radiolabeled tetravalent RGD peptides. 125I-labeled analogues were used due to limited availability of 211At. METHODS: Gly-Lys (GK) or Gly-Asp (GD) linkers were inserted between the radiolabeling moiety and the tetravalent RGD peptide. The biodistribution of [125I]GK-(RGD)4 and [125I]GD-(RGD)4 was evaluated in nude mice bearing U87MG human glioblastoma. Metabolite analysis was performed using urine, kidney, and tumor samples. U87MG cells were incubated with [125I]I-(RGD)4 or [125I]GD-(RGD)4 in the presence or absence of lysosomal enzyme inhibitors. Fluorescently labeled tetravalent RGD peptide (FL-(RGD)4) was used to evaluate cellular internalization. RESULTS: Both [125I]GK-(RGD)4 and [125I]GD-(RGD)4 showed reduced renal retention compared with [125I]I-(RGD)4 but also exhibited decreased tumor retention, resulting in an unchanged tumor-to-kidney ratio. [125I]iodohippuric acid was detected in the tumor, kidney, and urine samples, confirming in vivo linker cleavage. Lysosomal enzyme inhibition restored the cellular accumulation of [125I]GD-(RGD)4 to levels comparable to [125I]I-(RGD)4. Fluorescence microscopy visualized internalization of FL-(RGD)4 followed by trafficking to lysosomal compartments. CONCLUSION: GK and GD linkers reduced renal radioactivity but impaired tumor retention, likely due to internalization and lysosomal linker cleavage. These findings clarify both the utility and limitations of the renal brush-border strategy for improving tumor-to-kidney ratios.