INVESTIGATION OF p-SCN-Bn-DOTA-TRASTUZUMAB LABELED WITH RADIOACTIVE AND NON-RADIOACTIVE LUTETIUM AND YTTRIUM: A CRUCIAL STEP FOR FUTURE APPLICATIONS

Marija Arev; Marija Mirković; Magdalena  Radović; Katarina Davalieva; Petre Makreski; Paulina Apostolova; Sanja Vranješ-Djurić; Aleksandar Dimovski; Predrag Džodić; Emilija Janevik-Ivanovska

Marija Arev University "Goce Delčev" – Faculty of Medical Sciences
Marija Mirković University in Belgrade – Vinča Institute of Nuclear Sciences
Magdalena Radović University in Belgrade – Vinča Institute of Nuclear Sciences
Katarina Davalieva Macedonian Academy of Sciences and Arts, Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”
Petre Makreski Ss. Cyril and Methodius University, Faculty of Natural Sciences and Mathematics, Institute of Chemistry
Paulina Apostolova University "Goce Delčev" – Faculty of Medical Sciences
Sanja Vranješ-Djurić University in Belgrade – Vinča Institute of Nuclear Sciences
Aleksandar Dimovski Macedonian Academy of Sciences and Arts, Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”
Predrag Džodić University of Niš – Faculty of Medicine, Department of Pharmacy
Emilija Janevik-Ivanovska University "Goce Delčev" – Faculty of Medical Sciences

Abstract

The significance lies in preparing stable, trastuzumab-immunoconjugate through the utilization of non-radioactive LuCl₃ and YCl₃, via p-SCN-Bn-DOTA. This approach is crucial to determine potential physicochemical alterations in immunoconjugate structure following metal binding. Post-conjugation, employing a 1:20 molar ratio, freeze-drying was performed to obtain stable immunoconjugates for subsequent analysis. Several chemical methods were employed to characterize antibody stability and retained immunoreactivity within the formulated immunoconjugates. Proof of protein integrity came from SDS-PAGE electrophoresis, with uniform fragment intensities (25 kDa for light chain, 50 kDa for heavy chain) indicating antibody non-degradation (1). IR and Raman spectroscopy verified secondary structural changes, with the presence of characteristic amide bands in both spectra indicating the retention of native secondary structure (2). Employing MALDI-TOF-MS, 4.9 p-SCN-Bn-DOTA molecules were determined per antibody molecule. The promising outcomes from non-radioactive labeling provide an opportunity for potential labeling with radioactive lutetium-177 and yttrium-90, each with a specific activity of 200 µCi/mL. Radioisotopes were incubated with p-SCN-Bn-DOTA-trastuzumab for an hour at 40 ºC. Evaluation of radiochemical purity and stability was conducted using the ITLC-SG system. Optimal mobile phases, specifically 0.4 M methanol:sodium acetate (1:1) for yttrium-90 and 0.9% NaCl for lutetium-177, facilitated thorough examination. Remarkable radiolabeling efficiency was achieved, >96% for yttrium-90 and >99% for lutetium-177. Stability assessments after 72 hours demonstrated greater stability in ¹⁷⁷Lu-p-SCN-Bn-DOTA-trastuzumab (<1.5% lutetium-177 release) compared to the ⁹⁰Y-labeled counterpart (<17% yttrium-90 release). This study demonstrates the successful development of radioimmunoconjugates, positioning this agent for potential application in vivo investigations.

References

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2. Sterjova M, Džodić P, Makreski P, Duatti A, Risteski M, Janevik-Ivanovska E. Vibrational spectroscopy as a tool for examination to the secondary structure of metal-labeled trastuzumab immunoconjugates. J. Radioanal. Nucl. Chem. 2019; 320(1): 209-218.