Arev, Marija and Dzodic, Predrag and Makreski, Petre and Vanjes Duric, Sanja and Jankovic, Drina and Mirkovic, Marija and Radovic, Magdalena and Janevik-Ivanovska, Emilija (2019) Preparation and examination of 177Lu- and 90Y-labeled immunoconjugates of Trastuzumab. In: ISTR.
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Abstract
Trastuzumab is a monoclonal antibody for treatment of HER2 positive breast cancer. Labelled antibody with lutetium-177 and yttrium-90 has been investigated as potential radiopharmaceutical agent for use in radioimmunotherapy. In this study, the labeling was done via DOTA, DTPA and 1B4M-DTPA as a chelator in molar ratios of 1:20. A several chemical techniques have been used to characterize the stability and retained immunoreactivity of the antibody in the formulated immunoconjugates. A protein integrity and purity were proved by applying of reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Vibrational spectroscopy (Infra-red (IR) and Raman spectroscopy) provide molecular structure information and are convenient for verification of changes in the secondary structure. The number of chelating groups per one trastuzumab molecule was calculated by MALDI-TOF-MS. After conjugation, the freeze-drying process was performed in order to obtain stable immunoconjugates for further labeling. Quality control and stability were examined with ITLC using a three different mobile phases (0.9% saline solution, 0.4 M methanol:sodium acetate (1:1) and 0.1M acetic buffer).
The same intensity of the fragments (25 kDa for light chain and 50 kDa for heavy chain) of lyophilized immunoconjugates and pure trastuzumab was indicated that there is no degradation of the antibody. The presence of characteristic amide bands in IR spectra (amide I (1700-1600 cm-1), amide II (1480-1575 cm-1) and amide III bands (1255-1244 cm-1) and Raman spectra (amide I band at ~1670 cm-1 and amide III band (1230-1300 cm-1) also have indicated that all samples have retained native secondary structure. An average of 3.92 p-SCN-Bn-DTPA, 3.69 p-SCN-Bn-DOTA and 4.43 1B4M-DTPA groups could be randomly conjugated to an antibody molecule, which is a good result for successful labeling. The immunoconjugates were labeled with 177Lu and 90Y with specific activity of 200 µCi/mL. The ITLC studies have shown stability of the radioimmunoconjugates in 0.9% NaCl after 72 h. The 0.4 M methanol:sodium acetate (1:1) was the most appropriate mobile phase for examination of 90Y labeled conjugates and the 0.9% NaCl for 177 Lu labeled. The stability studies after 72 h have shown that 177Lu-labeled trastuzumab is more stable (< 10% of the released 177Lu) than 90Y-labeled one (< 25% of released 90Y). Our study shows successful formulation of stable radioimmunoconjugates which makes this agent as potential use for in vivo investigations.
| Item Type: | Conference or Workshop Item (Poster) |
|---|---|
| Subjects: | Medical and Health Sciences > Basic medicine |
| Divisions: | Faculty of Medical Science |
| Depositing User: | Marija Arev |
| Date Deposited: | 26 Nov 2019 13:56 |
| Last Modified: | 18 May 2023 08:21 |
| URI: | https://eprints.ugd.edu.mk/id/eprint/22967 |
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