1. 17th European Symphosium on Radiopharmacy and Radiopharmaceuticals, April 24-27, Pamplona, Spain In vivo and in vitro study of 177 Lu and 90 Y labeled DOTA(SCN)-Rituximab conjugate Wioletta Wojdowska, Urszula Karczmarczyk, Michał Maurin, Agnieszka Sawicka, Ewa Laszuk, Piotr Garnuszek, Renata Mikołajczak National Centre for Nuclear Research, Radioisotope Centre POLATOM, Otwock, Poland Immunoreactivity Biodistribution of 177 Lu-DOTA-rituximab in the tumour-bearing Rj:NMRI-Foxn1 nu /Foxn1 nu mice (n=3) Literature: 1.U. Karczmarczyk, W. Wojdowska, M. Maurin, E. Byszewska-Szpocinska, P. Garnuszek, R. Mikołajczak: Post-conjugation purification of DOTA-anti-CD20 (Rituximabâ)and influence on 177Lu-labeling yield. Eur J Nucl Med Mol Imaging (2013) 40 (Suppl 2):Str 293 2.Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA Jr (1984) Determination of the immunoreactive fraction of radiolabelled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods 72: 77-89 Monoclonal antibodies (mAb) are attractive molecules in the field of molecular imaging agent development. In 2011 the IAEA initiated a Coordinated Research Project (CRP) on the “Development and preclinical evaluations of therapeutic radiopharmaceuticals based on 177 Lu and 90 Y labelled monoclonal antibodies and peptides”. The aim of this study was evaluation of the 177 Lu and 90 Y radiolabeled DOTA-Rituximab conjugates as a potential therapeutics for radioimmunotherapy (RIT) of tumors overexpressing CD20 antigens. Introduction DOTA(SCN)-rituximab and DOTA(NHS)-rituximab immunoconjugates were prepared in the form of freeze-dried kits for 177 Lu and 90 Y labelling. 177 Lu-DOTA-rituximab of specific activity up to 1.5 GBg/mg was obtained with high radiochemical purity (> 94.7%). The radioimmunoconjugate was stable up to 72 h in HSA at RT. 90 Y-DOTA-rituximab with radiochemical purity (> 92%) and specific activity of 0.7 GBg/mg was obtained. The radioimmunoconjugate was stable up to 48 h in HSA at RT. The in vitro assay in Raji cells showed high immunoreactivity of both 177 Lu-DOTA-rituximab and 90 Y-DOTA-rituximab (immunoreactive fractions ca. 90%). Biodistribution investigations revealed typical for antibodies slow pharmacokinetics and hepatobiliary excretion route. In vivo imaging studies confirmed accumulation and retention of the radioimmunoconjugate in tumour. Conclusions The assay for determination of the fraction of immunoreactive antibody was adapted with slight modifications of the method described by Lindmo et al. [2]. The immunoreactivity of the labelled coumpounds was performed using constant concentrations of 177 Lu- / 90 Y-DOTA-rituximab and serial dilutions of Raji cells. The presented data ( 177 Lu-DOTA(SCN)-rituximab) demonstrates a very close linear relationship of ‘total applied/ specific binding’ as a function of the inverse cell concentration. Physiological distribution of 177 Lu-DOTA-rituximab was determined in normal Balb/c mice and in the tumour-bearing mice. The radioactive preparations were diluted with normal saline to injection dose of 0.01 mg of the antibody and radioactivity of 9-12 MBq in 0.1 mL. At the fixed time points (4, 24, 48 and 72 h p.i.v.) the animals were sacrificed, then blood samples were collected and the selected organs were dissected and weighted. The radioactivity in all organs was expressed as percentage of injected dose per gram of tissue (% ID/g). The tumour model was prepared in immunodeficient Rj:NMRI- Foxn1 nu /Foxn1 nu (male, weight 18-25 g) mice, which were subcutaneously xenografted with suspension of Raji cells (2 mln in 0.3 mL) in presence of MatrixGel. The tumors were allowed to grow for 2-3 weeks. All animal experiments were performed after approval by The IVth Local Animal Ethics Committee in Warsaw (authorization number 34/2013) and were carried out in accordance with the principles of good laboratory practice (GLP). Chelator used Immunoreactivity [%] 177 Lu-DOTA-Rituximab 90 Y-DOTA-Rituximab p-SCN-Bn-DOTA89,2 ± 4,8 (n=3) 88,0 ± 6,6 (n=2) DOTA-NHS-ester90,0 ± 4,6 (n=3) 95,7 ± 0,7 (n=2) Biodistribution of 177 Lu-DOTA-rituximab in normal Balb/c mice (n=5) Both 177 Lu-DOTA-rituximab and 90 Y-DOTA-rituximab revealed slow blood clearance (ca. 40, 20 and 16 %ID/g after 1, 24 and 72 h, respectively). The highest radioactivity uptake was observed in the liver and spleen, confirming the hepatobiliary excretion route. The biodistriobution of 177 Lu-DOTA-rituximab in the tumour- bearing mice revealed faster clearance of radioactivity from blood and excretion with urine than observed in normal mice. The uptake (%ID/g) of 177 Lu-DOTA-rituximab in tumor was increasing from 1.8 and 2.6 at 4 h p.i.v. to 9.3 and 6.9 after 72 h p.i.v. for the 177 Lu conjugates with DOTA(SCN) and DOTA(NHS), respectlively. Advantageous and increasing with time post injection, the tumor/muscle (T/NT) ratios were noted (see figure below). Biodistribution Radiolabelling Freeze-drying: The commercially available mAb anti-CD20 (Rituximab, Mabthera, Roche) was conjugated with bifunctional chelator DOTA: p-SCN-Bn-DOTA and DOTA-NHS-ester. After purification by ultrafiltration (Amicon Ultra, MWCO 30 000, Millipore), the DOTA-rituximab conjugate (2.0 mg) was prepared in the freeze-dried kit form by lyophilization with mannitol (5 mg), chlorid (4.5 mg) and ammonium acetate (9.62 mg). Labelling: The rituximab kits were reconstituted with 0.5 mL of water and then mixed at RT for 5 min. The activities of 177 Lu (LutaPOL) or 90 Y (ItraPOL) used were 500 - 1500 MBq per kit. The reaction mixtures were incubated at 40 0 C for 1 hour. Size exclusion HPLC using BioSepSEC 3000 column (Phenomenex) was applied for quality control of the radiolabelled antibody. Using the developed freeze-dried kit, 177 Lu-DOTA-Rituximab and 90 Y-DOTA-Rituximab radioimmunoconjugates were obtained with high radiolabelling yield (RCP > 98%) and specific activities up to 1.5 GBq/mg and 0.7 GBq/mg, respectively [1]. No release of free 177 Lu and 90 Y were detected up to 24 hours. Tendency towards aggregation was more pronounced in case of the 177 Lu-labelled conjugate. PhotonIMAGER TM System (Biospace LAB) was applied for in vivo imaging of 90 Y-DOTA-rituximab in the tumour-bearing Rj:NMRI-Foxn1 nu /Foxn1 nu (Raji s.c.) mice. The mice were intravenously injected with 5 µg of 90 Y-DOTA- rituximab (0.7 GBq/mg). Cerenkov imaging of the animals anesthetized by izoflurane inhalation was performed 2, 24 and 48 h p.i. 2 h p.i. 24 h p.i. 48 h p.i. Optical imaging 177 Lu-DOTA-rituximab 90 Y-DOTA-rituximab Cerenkov imaging of 90 Y-DOTA-rituximab