1-Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, Kooperativny Street, 5, Tomsk, 634009, Russian Federation; 2-National Research Tomsk Polytechnic University, Lenina Avenue, 30, Tomsk, 634050, Russian Federation; 3-Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, Moscow, 117997, Russian Federation; 4-Uppsala University, Sweden, Uppsala, Segerstedthuset, Dag Hammarskjölds väg 7 E-mail: [email protected]

Introduction. In recent years, alternative framework proteins are used for the targeted radionuclide imaging. Molecules of DARPin (Design Ankyrin Repeat Protein) are one of the representatives of scaffolds. Despite the continued increase in the number of molecular targets in oncological practice, the epidermal growth receptor Her2/neu is still of great interest, the overexpression of which is most often observed in patients with breast cancer and accounts for 15–20% of cases. However, the methods of the determination of Her2/neu have many significant drawbacks. The aim of the study. Assessment of the 99mTc-DARPin9_29 diagnostic efficacy for the diagnosis of breast cancer (BC) with Her2/neu overexpression. Methods. The study included 8 BC cancer patients (T1-4N0-2M0) who were not receiving systemic therapy at the time of the study: in 4 patients, Her2/neu overexpression was noted, in 4 patients – not detected. At the preclinical stage, all patients underwent morphological and immunohistochemical studies of the primary tumor biopsy material. Radiopharmaceutical 99mTc-DARPin9_29 was injected intravenously, WholeBody scintigraphy and SPECT were performed 2 hours after injection. Results. The distribution of radiopharmaceuticals in organs 2 hours after injection revealed the greatest accumulation in the liver and kidneys (26.28 and 6.68% of the administered dose, respectively). In studying tumor/background indices there were revealed values of the studied parameter in patients with overexpression of Her2 receptors to be more than by 2.8 times higher than the values in the subgroup of patients with negative expression of this marker. Conclusion. According to the results obtained at the initial stages of the study, it can be said that the 99mTc-DARPin9_29 preparation can be considered as a promising additional method for the diagnosis of BC with overexpression of the Her2 / neu receptor in the future.
breast cancer, DARPin9_29, target radionuclide diagnostics

Список литературы: 
  1. Bragina O.D., Chernov V.I., Zel'chan R.V., Sinilkin I.G., Medvedeva A.A., Lar'kina M.S. Al'ternativnye karkasnye belki v radionuklidnoj diagnostike zlokachestvennyh obrazovanij. Bjulleten' sibirskoj meditsiny. 2019; 18 (3): 125–33. [Bragina O.D., Chernov V.I., Zelchan R.V., Sinilkin I.G., Medvedeva A.A., Larkina M.S. Alternative scaffolds in radionuclide diagnosis of malignancies. Bulleten Sibirskoi Medicini. 2019; 18 (3): 125–33 (in Russian)]
  2. Chernov V.I., Bragina O.D., Zel'chan R.V., Medvedeva A.A., Sinilkin I.G., Lar'kina M.S., Stasjuk E.S., Nesterov E.A., Skuridin V.S. Mechenye analogi somatostatina v teranostike nejroendokrinnyh opuholej. Meditsinskaja radiologija i radiatsionnaja bezopasnost'. 2017; 62 (3): 42–9. [Chernov V.I., Bragina O.D., Zel’chan R.V., Medvedeva A.A., Sinilkin I.G., Larkina M.S., Stasyuk E.S., Nesterov E.A., Skuridin V.S. Labeled Somatostatin Analogues in Theranostics of Neuroendocrine Tumors. Medicinskaya radiologiya I radiacionnaya bezopasnost. 2017; 62 (3): 42–9 (in Russian)]
  3. Plückthun A. Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol. 2015; 55: 489–511.
  4. Garousi J., Honarvar H., Andersson K.G., Mitran B., Orlova A., Buijs J., Löfblom J., Frejd F.Y., Tolmachev V. Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX. Mol Pharm. 2016; 13 (11): 3676–87.
  5. Lindbo S., Garousi J., Mitran B., Altai M., Buijs J., Orlova A., Hober S., Tolmachev V. 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.
  6. Tolmachev V., Orlova A., Andersson K. Methods for radiolabelling of monoclonal antibodies. Methods Mol Biol. 2014; 1060: 309–30.
  7. 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.
  8. Stumpp M.T., Binz H.K., Amstutz P. DARPins: A new generation of protein therapeutics. Drug Discovery Today. 2008; 13 (15): 695–701.
  9. 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.
  10. Boersma Y.L., Pluckthun A. DARPins and other repeat protein scaffolds: advances in engineering and applications. Curr. Opin. Biotechnol. 2011; 22: 849–57.
  11. Binz H.K., Stumpp M.T., Forrer P., Amstutz P., Pluckthun A. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 2003; 332: 489–503.
  12. Goldstein R., Sosabowski J., Livanos M., Leyton J., Vigor K., Bhavsar G., Nagy-Davidescu G., Rashid M., Miranda E., Yeung J., Tolner B., Plückthun A., Mather S., Meyer T., Chester K.. Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging. Eur J Nucl Med Mol Imaging. 2015; 42 (2): 288–301.
  13. Hausammann S., Vogel M., Kremer J.A. Designed Ankyrin Repeat Proteins: A New Approach to Mimic Complex Antigens for Diagnostic Purposes? PLoS One. 2013; 8: 1–9.
  14. Moody P., Chudasama V., Nathani R. I., Maruani A., Martin S., Smith M.B., Caddick S. A rapid, site-selective and efficient route to the dual modification of DARPins. Chem Commun (Camb). 2014: 50 (38): 4898–900.
  15. Kramer L., Renko M., Završnik J., Turk D., Seeger M.A., Vasiljeva O., Grütter M.G., Turk V., Turk B. .Non-invasive in vivo imaging of tumour-associated cathepsin B by a highly selective inhibitory DARPin. Theranostics. 2017; 8: 2806–21.
  16. Houlihan G., Gatti-Lafranconi P., Lowe D., Hollfelder F. Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process. Protein Eng Des Sel. 2015; 28 (9): 269–79.
  17. Hanenberg M., McAfoose J., Kulic L. Amyloid-β peptide-specific DARPins as a novel class of potential therapeutics for Alzheimer disease. J. Biol. Chem. 2014; 26: 27080–9.
  18. Romond E.H., Perez E.A., Bryant J., Suman V.J., Geyer C.E. Jr., Davidson N.E., Tan-Chiu E., Martino S., Paik S., Kaufman P.A., Swain S.M., Pisansky T.M., Fehrenbacher L., Kutteh L.A., Vogel V.G., Visscher D.W., Yothers G., Jenkins R.B., Brown A.M., Dakhil S.R., Mamounas E.P., Lingle W.L., Klein P.M., Ingle J.N., Wolmark N. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N. Engl. J. Med. 2005; 353: 1673–84.
  19. Zahid M., Khan S., Khan R. Detection of Her2/neu gene amplification by fluoroscence in situ hybridization technique. Pathology. 2016; 48 (1): 163–70.
  20. Orlando L., Viale G., Bria E. Lutrino E.S., Sperduti I., Carbognin L., Schiavone P., Quaranta A., Fedele P., Caliolo C., Calvani N., Criscuolo M., Cinieri S. Discordance in pathology report after central pathology review: Implications for breast cancer adjuvant treatment. Breast. 2016; 30: 151–5.
  21. Telugu R.B., Chowhan A.K., Rukmangadha N., Patnayak R., Phaneendra B.V., Prasad B.C., Reddy M.K. Human epidermal growth factor receptor 2/neu protein expression in meningiomas: An immunohistochemical study. J. Neurosci Rural Pract. 2016; 7 (4): 526–31.
  22. Bragina O.D., Lar'kina M.S., Stasjuk E.S., Chernov V.I., Jusubov M.S., Skuridin V.S., Deev S.M., Zel'chan R.V., Buldakov M.A., Podrezova E.V., Belousov M.V. Razrabotka vysokospetsifichnogo radiohimicheskogo soedinenija na osnove mechenyh 99mTc rekombinantnyh adresnyh molekul dlja vizualizatsii kletok s giperekspressiej Her2/neu. Bjulleten' sibirskoj meditsiny. 2017; 16 (3): 25–33. [Bragina O.D., Larkina M.S., Stasyuk E.S., Chernov V.I., Yusubov M.S., Skuridin V.S., Deyev S.M., Zel’chan R.V., Buldakov M.A., Podrezova E.V., Belousov M.V. The development of a highly specific radiochemical compound based on labeled 99mtc recombinant molecules for targeted imaging of cells with the overexpression of Her-2 / neu. Bulleten Sibirskoi Medicini. 2017; 16 (3): 25–33 (In Russian)]
  23. Vorobyeva A., Garousi J., Tolmachev V., Schulga A., Konovalova E., Deyev S.M., Güler R., Löfblom J., Sandström M., Chernov V., Bragina O., Orlova A. Optimal composition and position of histidine-containing tags improves biodistribution of 99mTc-labeled DARPinG3. Scientific Reports. 2019: 9 (1); 9405.
  24. Vorobyeva A., Bragina O., Altai M., Mitran B., Orlova A., Shulga A., Proshkina G., Chernov V., Tolmachev V., S. Deyev. 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; 6930425.
  25. Bragina O.D., Vorob'eva A.G., Tolmachev V.M., Orlova A.M., Chernov V.M., Deev S.M., Proshkina G.M., Shul'ga A.A., Lar'kina M.S., Zel'chan R.V., Sinilkin I.G., Medvedeva A.A. Doklinicheskie issledovanija mechennyh 125I targetnyh molekul DARPin9_29 dlja radionuklidnoj diagnostiki zlokachestvennyh opuholej s giperekspressiej Her2/neu. Molekuljarnaja meditsina. 2018; 16 (6): 41–5. [Bragina O.D., Vorobyeva A.G., Tolmachev V.M., Orlova A.M., Chernov V.I., Deev S.M., Proshkina G.N., Shulga A.A., Larkina M.S., Zelchan R.V., Sinilkin I.G., Medvedeva A.A. Investigations of the 125I labelled target molecules DARPIN9_29 for radionuclide diagnostics of malignant tumors with Her2/neu overexpression. Moleculyarnaya medicina. 2018; 16 (6): 41–5 (In Russian)]