Medical Radiology and Radiation Safety. 2020. Vol. 65. No. 1. P. 37–41

O.D. Bragina1, A.G.Vorobyeva5, V.M. Tolmachev5, A.M. Orlova5, V.I. Chernov1,2, S.M. Deyev2,4, G.N. Proshkina4, A.A. Shulga4, M.S. Larkina3, A.A. Medvedeva1, R.V. Zelchan1

In vitro and in vivo Evaluation of the Radiochemical Compound Based on
99mTechnetium Labelled DARPin9_29 for Molecular Visualization
of Malignancies Overexpressing Her2/neu

1. Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Science, Tomsk, Russia.
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2. National Research Tomsk Polytechnic University, Tomsk, Russia
3. Siberian State Medical University, Tomsk, Russia
4. Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
5. Uppsala University, Uppsala, Sweden


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.

Key words: malignancies, Her2/neu, radionuclide diagnostics, DARPin9_29

Для цитирования: Bragina OD, Vorobyeva AG, Tolmachev VM, Orlova AM, Chernov VI, Deyev SM, Proshkina GN, Shulga AA, Larkina MS, Medvedeva AA, Zelchan RV. In vitro and in vivo Evaluation of the Radiochemical Compound Based on 99mTechnetium Labelled DARPin9_29 for Molecular Visualization of Malignancies Overexpressing Her2/neu. Medical Radiology and Radiation Safety. 2020;65(1):37-41. (In Russ.).

DOI: 10.12737/1024-6177-2020-65-1-37-41

Список литературы / References

  1. Чернов ВИ, Медведева АА, Синилкин ИГ, и др. Ядерная медицина в диагностике и адресной терапии злокачественных новообразований. Бюллетень сибирской медицины. 2018;17(1):220-31. [Chernov VI, Medvedeva AA, Sinilkin IG, Zelchan RV, Bragina OD, Choynzonov EL. Nuclear medicine as a tool for diagnosis and targeted cancer therapy. Bulletin of Siberian Medicine. 2018;17(1):220-31. (In Russ.)].
  2. Чернов ВИ, Брагина ОД, Синилкин ИГ, и др. Радиоиммунотерапия: современное состояние проблемы. Вопросы онкологии. 2016;62(1):24-30. [Chernov VI, Bragina OD, Sinilkin IG, Medvedeva AA, Zel’chan RV Radioimmunotherapy: current state of the problem. Oncology Issues. 2016;62(1):24-30. (In Russ.)].
  3. Брагина ОД, Чернов ВИ, Зельчан РВ, и др. Альтернативные каркасные белки в радионуклидной диагностике злокачественных заболеваний. Бюллетень сибирской медицины. 2019;3(18):125-33. [Bragina OD, Chernov VI, Zelchan RV, Sinilkin IG, Medvedeva AA, Larkina MS. Alternative scaffolds in radionuclide diagnosis of malignancies. Bulletin of Siberian Medicine. 2019;18(3):125-33. (In Russ.)].
  4. Чернов ВИ, Брагина ОД, Синилкин ИГ, и др. Радиоиммунотерапия в лечении злокачественных образований. Сибирский онкологический журнал. 2016;15(2):101-6. [Chernov VI, Bragina OD, Sinilkin IG, Medvedeva AA, Zel’chan RV Radioimmunotherapy in the treatment of malignancies. Siberian journal of oncology. 2016;15(2):101-6. (In Russ.)].
  5. Tamaskovic R, Simon M, Stefan N, Schwill M, Plückthun A. Designed ankyrin repeat proteins (DARPins) from research to therapy. Methods Enzymol. 2012;503:101–34.
  6. Boersma YL, Pluckthun A. DARPins and other repeat protein scaffolds: advances in engineering and applications. Curr Opin Biotechnol. 2011;22:849-57.
  7. Брагина ОД, Ларькина МС, Стасюк ЕС, и др. Разработка высокоспецифичного радиохимического соединения на основе меченных 99mТс рекомбинантных адресных молекул для визуализации клеток с гиперэкспрессией Her-2/ neu. Бюллетень сибирской медицины. 2017;16(3):25-33. [Bragina OD, Larkina MS, Stasyuk ES, Chernov VI, Yusubov MS, Skuridin VS et al. Development of highly specific radiochemical compounds based on 99mTc-labeled recombinant molecules for targeted imaging of cells overexpressing Her-2/ neu. Bulletin of Siberian Medicine. 2017;16(3):25-33. (In Russ.)].
  8. Vorobyeva A, Bragina O, Altai M, Mitran B, Orlova A, Shulga A et al. Comparative Evaluation of Radioiodine and Technetium-Labeled DARPin 9_29 for Radionuclide Molecular Imaging of HER2 Expression in Malignant Tumors. Contrast Media & Molecular Imaging. 2018; 2018: 6930425.
  9. Goldstein R, Sosabowski J, Livanos M, Leyton J, Vigor K, Bhavsar G et al. Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging. Eur J Nucl Med Mol Imaging. 2015;42(2):288-301.
  10. Lindbo S, Garousi J, Mitran B, Altai M, Buijs J, Orlova A et al. Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label. J Nucl Med. 2018;59(1):93-9.
  11. Plückthun A Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol. 2015; 55:489-511.
  12. Garousi J, Honarvar H, Andersson KG, Mitran B, Orlova A, Buijs J et al. Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in vivo Expression of Carbonic Anhydrase IX. Mol Pharm. 2016 Nov 7;13(11):3676-87.
  13. Nicholes N, Date A, Beaujean P, Hauk P, Kanwar M, Ostermeier M. Modular protein switches derived from antibody mimetic proteins. Protein Engineering, Design and Selection. 2016; 29:77-85.
  14. Kramer L, Renko M, Završnik J, Turk D, Seeger MA, Vasiljeva O et al. Non-invasive in vivo imaging of tumour- associated cathepsin B by a highly selective inhibitory DARPin. Theranostics. 2017; 8:2806-21.
  15. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse a survival with amplification of the Her-2/neu oncogenes. Science. 1987; 235:177–82.
  16. Zavyalova M, Vtorushin S, Krakhmal N, Savelieva O, Tashireva L, Kaigorodova E et al. Clinicopathological features of nonspecific invasive breast cancer according to its molecular subtypes. Experimental Oncology. 2016;38(2):122-27.
  17. Vorobyeva A, Garousi J, Tolmachev V, Schulga A, Konovalova E, Deyev SM et al. Optimal composition and position of histidine-containing tags improves biodistribution of 99mTc-labeled DARPinG3. Scientific Reports. 2019; 9 (1): 9405.
  18. Чернов ВИ, Брагина ОД, Синилкин ИГ и др. Радионуклидная тераностика злокачественных образова- ний. Вестник рентгенологии и радиологии. 2016;97(5):306-13. [Chernov VI, Bragina OD, Sinilkin IG, Medvedeva AA, Zel’chan RV Radionuclide teranostic of malignancies. Journal of Radiology and Nuclear Medicine. 2016;97(5):306-13. (In Russ.)].

PDF (RUS) Full-text article (in Russian)

Conflict of interest. The authors declare no conflict of interest.

Financing. The research is carried out within the framework of the Federal target program «Development of the pharmaceutical and medical industry of the Russian Federation for the period up to 2020 and beyond» on the topic «Preclinical research of a radiopharmaceutical drug based on 99mTc-labeled recombinant targeted molecules for radionuclide diagnostics of cancer diseases with hyperexpression of Her-2/neu».

Contribution. Article was prepared with equal participation of the authors.

Article received: 28.05 2018. Accepted for publication: 11.12.2019.