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Purpose: Evaluation of a radiopharmaceutical based on 99mTc-labeled targeted molecules DARPin9_29 for radionuclide diagnostics of malignancies with Her2/neu overexpression.

Material and methods: The DARPin9_29 sequence was amplified from the plasmid pET-DARP-6HIS for the DARPin9_29-His6 gene expression in E. coli cells. The eluent of 99mTcO4– (400–500 μl, 4 GBq) was added to the kit and incubated at a temperature of 100 °C for 20 minutes. After incubation, 40 μl of tricarbonyl technetium was added to 168 μg of DARPin9_29 in 100 μl of PBS (sodium phosphate buffer), followed by incubation at 40 °C for 60 minutes. The radiochemical yield and purity were determined by thin layer radiochromatography, the purification was performed using NAP-5 cleansing columns (GE Healthcare). Cell lines with different levels of Her2/neu expression were used: SKOV-3> BT474 >> DU-145 for the determination of the radiopharmaceutical specificity. Her2/neu expressing cell line SKOV-3 was used for in vitro study. The study was conducted 6 hours after the administration of the drug.

Results: The radiochemical yield was 72 ± 8 %, the radiochemical purity after purification was 98.7 ± 1.0 %. The stability in PBS (phosphate buffered saline) solution after 1 hour was 99.8 ± 0.2; after 3 hours – 98.2 ± 0.1. In vitro studies showed that the accumulation of explored compound was directly proportional to the level of Her2/neu expression in cells, while blocking the receptors with an excess of unlabeled protein showed a significant reduction in binding in the group of cells. Data on biodistribution and SPECT/CT in the body of the animal BALB/c nu/nu demonstrated rapid removal of the compound from the blood stream and high accumulation in the liver, kidney and bladder 6 hours after the introduction of the radiopharmaceutical.

Conclusion: The studies demonstrated high radiochemical yields and purity, as well as stability of the studied compound. The results of in vitro and in vivo analysis showed the specificity and affinity of the radiopharmaceutical to the Her2/neu receptor on the surface of tumor cells. The high accumulation of the drug in the liver and kidneys, detected in in vivo studies, is probably due to the lipophilicity of the 99mTc(CO)3-histidine tag and indicates the limitation of its further clinical use in assessing the condition of the above organs, which will require additional diagnostic methods, as well as possible modification chemical structure.