First-in-human validation of enzymolysis clearance strategy for decreasing renal radioactivity using modified [ 68 Ga]Ga-HER2 Affibody

Purpose Enzymolysis clearance strategy, characterized by releasing the non-reabsorbable radioactive fragment under the specific cleavage of enzymes, is confirmed to be a safe and effective way to reduce the renal radioactivity accumulation in mice. However, the effectiveness of this strategy in humans remains unknown. Human epidermal growth factor receptor 2 (HER2) is overexpressed in various types of tumors, and radiolabeled HER2 Affibody is believed to be an attractive tool for HER2-targeted theranostics. However, its wide application is limited by the high and persistent renal uptake. In this study, we intend to validate the effectiveness of enzymolysis clearance strategy in reducing renal accumulation by using a modified HER2 Affibody. Materials and methods A new HER2 Affibody ligand, NOTA-MVK-Z HER2:2891 , containing a cleavable Met-Val-Lys (MVK) linker was synthesized and labeled with 68 Ga. The microPET imaging study was performed in SKOV-3 tumor mice to assess the uptakes of the control ligand and the MVK one in tumors and kidneys. Seven healthy volunteers were included for biodistribution and dosimetric studies with both the control and MVK ligands performed 1 week apart. Urine and blood samples from healthy volunteers were collected for in vivo metabolism study of the two ligands. Four HER2-positive and two HER2-negative patients were recruited for [ 68 Ga]Ga-NOTA-MVK-Z HER2:2891 PET/CT imaging at 2 and 4 h post-injection (p.i.). Results [ 68 Ga]Ga-NOTA-MVK-Z HER2:2891 was stable both in PBS and in mouse serum. MicroPET images showed that the tumor uptake of [ 68 Ga]Ga-NOTA-MVK-Z HER2:2891 was comparable to that of [ 68 Ga]Ga-NOTA-Z HER2:2891 at all the time points, while the kidney uptake was significantly reduced 40 min p.i. ( P < 0.05). The biodistribution study in healthy volunteers showed that the kidney uptake of MVK ligand was significantly lower than that of the control ligand at 1 h p.i. ( P < 0.05), with the SUV mean of 34.3 and 45.8, respectively, while the uptakes of the two ligands in the other organs showed negligible difference. The effective doses of the MVK ligand and the control one were 26.1 and 28.7 µSv/MBq, respectively. The enzymolysis fragment of [ 68 Ga]Ga-NOTA-Met-OH was observed in the urine samples of healthy volunteers injected with the MVK ligand, indicating that the enzymolysis clearance strategy worked in humans. The PET/CT study of patients showed that the range of SUV max of HER2-positive lesions was 9.4–21, while that of HER2-negative lesions was 2.7–6.2, which suggested that the MVK modification did not affect the ability of Z HER2:2891 structure to bind with HER2. Conclusion We for the first time demonstrated that enzymolysis clearance strategy can effectively reduce renal radioactivity accumulation in humans. This strategy is expected to decrease renal radiation dose of peptide and small protein-based radiotracers, especially in the field of radionuclide therapy.