INTRODUTION. In the radioimmunotherapy (RIT), radiolabeled monoclonal antibodies (mAbs) are used for cancer therapy. This therapeutic modality improves the citotoxic effect of the monoclonal antibodies due to the coupling radioisotopes. RIT using anti-CD20 antibodies radiolabeled with 90Y and 131I has been used with success in the treatment of Non Hodking Linfoma (NHL). For labeling mAb with radioisotopes, conjugation was previously required in order to introduce a chelating group (DOTA or DTPA) in the protein chain. The conjugation and radiolabeling process involve special conditions of pH and temperature, long processes of manipulation and mixing. All this process can damage the antibody structure, compromise their receptor binding and compromises its clinical application. Therefore, the receptor binding ability is an important parameter to evaluate prior in vivo studies.

OBJECTIVE. This work describes the conjugation, radiolabeling and cell binding assays of DOTA-rituximab (anti-CD 20 Ab for specific binding to NHL cells) radiolabeled with lutetium-177 (177Lu), a ß- emitter with optimal physical characteristics for RIT of small tumors and metastases.

MATERIALS AND METHODS. Briefly, 10 mg of antibody (Rituximab, Mabthera-Roche®) previously purified by ultrafiltration device was conjugated with DOTA-NHS-ester (Macrocyclics®) in 50 fold molar excess. The reaction was conducted for 1 hour in phosphate buffer pH 8.0 and gently mixing at room temperature and remained for 24 hours under refrigeration. The immunoconjugated was purified by ultrafiltration device and radiolabeled with 37 MBq (1 mCi) of 177LuCl3 for 1 hour at 43 °C. The reaction was conducted in 0.4 M acetate buffer pH 5.5 and the radiochemical purity was determined using analysis by HPLC and TLC-SG plates. For specific cell binding assays, different amounts of lymphoma cells (Raji – Cell Bank of Rio de Janeiro.) was used in two groups: in the first, competitor was not added (non-radiolabeled antibody), whereas in the second group, a 100 fold excess of competitor was added.

RESULTS. Radiochemical purity above 95% was obtained when this radiolabeled antibody was purified in PD-10 column. The specific binding of the antibody to cells increases according to the number of cells, reaching 14% at the highest concentration.

CONCLUSION. The results suggests that the synthesis of 177Lu-DOTA-rituximab did not compromises its binding to CD20 positive tumor cells, and makes possible future studies in vivo.

INSTITUIÇÃO. Radiopharmacy Directory – Nuclear and Energy Research Institute – IPEN / CNEN – SãO Paulo – Brazil