INVESTIGATIONS OF THE 125I-LABELLED TARGET MOLECULES DARPIN9_29 FOR RADIONUCLIDE DIAGNOSTICS OF MALIGNANT TUMORS WITH HER2/NEU OVEREXPRESSION

DOI: https://doi.org/10.29296/24999490-2018-06-07

O.D. Bragina(1), A.G. Vorobyeva(5), V.M. Tolmachev(5), A.M. Orlova(5), V.I. Chernov(1, 2), S.M. Deev(2, 4), G.N. Proshkina(4), A.A. Shulga(4), M.S. Larkina(3), R.V. Zelchan(1), I.G. Sinilkin(1), A.A. Medvedeva(1) 1-Tomsk National Research Medical Center, Kooperativny Street, 5, Tomsk, 634009, Russian Federation; 2-National Research Tomsk Polytechnic University, Lenina Avenue, 30, Tomsk, 634050, Russian Federation; 3-Siberian State Medical University, Moskovsky trakt, 2, Tomsk, 634055, Russian Federation; 4-Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya str., 16/10, Moscow, 117997, Russian Federation; 5-Uppsala University, Dag Hammarskjölds väg 7, Segerstedthuset, Uppsala, Sweden E-mail: [email protected]

Introduction. Her2/neu is one of the most studied molecular target the overexpression of which is associated with poor prognosis and aggressive course of the disease. The use of modern non-invasive radionuclide techniques with the application of highly affinity directed molecules acquires vogue the in molecular imaging. Recently, a new class of binding proteins has been actively studied and called «alternative skeletal proteins». DARPin (Design Ankyrin Repeat Protein) meets all necessary requirements for the creation of effective radiopharmaceuticals. The aim of the study. To evaluate a radiochemical compound based on 125I-labeled targeted molecules DARPin9_29 for radionuclide diagnostics of malignancies with HER2/neu overexpression. Methods. A modified genetic structure was used with a sequence of encoding recombinant address DARPin9_29 molecules. The tagging was performed according to the standard method, radiochemical yield and purity were determined using thin-layer chromatography (ITLC), purification was carried out using NAP-5 purification columns (GE Healthcare). The stability test was performed by incubation of labeled protein in PBS (Phosphate-buffered saline) solution at room temperature for 1 and 3 hours, the analysis was performed on thin-layer chromatography. 3 cell lines overexpressing Her2/neu were used in in vitro studies: SKOV-3, BT474, and DU145. Specificity of the compound was determined by blocking receptors. SKOV-3 cells were studied in vivo and injected subcutaneously into the right hind limb of BALB/C nu/nu mice. Evaluation of results of the biodistribution and tumor imaging was performed 6 hours after injection. Results. Radiochemical yield amounted to 96,2±0,7%, radiochemical purity – 99,7±0,5%. Stability in PBS solution after 1 hour accounted for 99,7±0,4; 3 hours later – 97,9±1,0. The association of compound accumulation with the level of Her2/neu receptor expression in the cell lines was demonstrated in studies in vitro. Blocking the receptor with the excess of unlabeled protein showed a significant reduction in binding. The main characteristics of biodistribution were rapid removal from the bloodstream, as well as relatively low capture rates of the studied compound in the lungs, liver and spleen, kidneys and bones. Results of SPECT/CT for laboratory animals showed clearly visualization of Her2/neu-tumors in all cases and the radiotracer accumulation in the bladder. Conclusion. The completed studies have demonstrated a new stable compound with high radiochemical yield and purity. Results of in vitro and in vivo studies showed the high sensitivity and specificity to the Her2/neu receptor
Keywords: 
Her2/neu, molecular imaging, radionuclide diagnostics, malignancies, DARPins
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