PhD: Investigations of monoclonal antibodies and peptides based therapeutic radiopharmaceuticals

Darinka Gjorgieva Ackova
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Macedonia
February, 2015


Recombinant monoclonal antibodies and peptides are currently rated as a significant and growing class of drugs with biological origin. According to the usage of the radioimmunoconjugates in therapy of non-Hodgkin's Lymphoma (NHL), we made investigations to newly prepared lyophilized formulations labeled with non-radioactive (Lu and Y) or radioactive isotopes (177Lu), of three different rituximab-bifunctional chelating agents (BFCA) conjugates, for assessment and evaluation of the structure. Three types of BFCA, p-Bn-SCN-DOTA, p-Bn-SCN-DTPA and 1B4M-DTPA were used. The results obtained by SDS-PAGE confirmed that conjugation and lyophilization steps and labeling of immunoconjugates with Lu and Y is successful, without significant changes in the molecules and forming of by-products (fragmentation of the antibody and/or presence of aggregates). Characterization of conjugates and determination of the average number of molecules of BFCA attached to each molecule of the antibody was made by MALDI-TOF MS where binding of an average 6.1 (DOTA), 8.8 (DTPA) and 8.3 (1B4M-DTPA) molecules per antibody were determined. Confirmation of the retention of secondary structure and possible modifications of the antibody after labeling, was carried out by means of vibrational spectroscopy. Based on the obtained frequencies for amide I, II and III bands, it was determined that the components of interest in this study, contain the highest percentage of β-conformation (parallel and antiparallel) in the structure, followed by α-helices. Several shifts of the characteristic bands of rituximab obtained in the recorded spectra of conjugates are dependent on the presence of a chelator and binded metal and indicate complex formation. Radioimmunoconjugates (labeled with 555 GBq/mg 177Lu) were further characterized by ITLC and SE-HPLC, with a high level of radiochemical purity and area under the peak more than 95% of the total area. Chemical and biological characterization of newly obtained immunoconjugates in in vitro and in vivo conditions for determination of important parameters for subsequent radiolabeling presents a basis for further qualification of this formulation for preclinical and clinical studies for diagnosis/treatment of lymphomas.