177-Lu-Anti-CD20 monoclonal antibody: A potential radiopharmaceutical for treatment of non-Hodgkin's lymphoma

P. AUDICIO; M. TASSANO; E. REZZANO; M. FERNÁNDEZ; A. ROBLES; G. CASTELLANO; P. CABRAL; H. BALTER; P. OLIVER

Cartagena

Congreso; 3rd International Conference on Radiopharmaceutical Therapy (ICRT-2008); 2009

World Radiopharmaceutical Therapy Council

Anti-CD20 monoclonal antibody generated specifically against the surface antigen CD20 (transmembrane) of human B-lymphocytes is used for the treatment of non-Hodgkin´s lymphoma as immunotherapeutic agent, alone or associated with chemotherapy. The labelling of this anti-CD20 antibody with ß-emitters increases the therapeutic effectiveness due to radiological and cytotoxic effects of ionizing radiation. Lutetium-177 (177Lu, T1/2 6.7 d), is a ß- (497 keV) and α emitter (150 keV) and has a range of tissue penetration up to 2 mm; these properties give the possibility to obtain images of the biodistribution, to do dosimetric calculations and produce the desired therapeutic effect specific to this ß- emission. Our objective was the optimization of the labelling, the evaluation of the biological behavior and dosimetric studies of 177-Lu-anti-CD20 as radiopharmaceutical for treatment of non-Hodgkin´s lymphoma. Methodology: Before labelling, mab-anti- CD20 (Mabthera ®) was purified by gel filtration and conjugated with 235 μl (3 μmol) of DOTA-Ossu, at pH 7.5 and incubated 18 hours at 4°C, with subsequent purification by gel-permeation with Sephadex G-25 (PD-10 columns from Pharmacia). Fractions of conjugated antibody were stored at 4 and -20°C for further stability evaluation. The labelling of this conjugate was performed through the addition of 55.5 – 259 MBq of 177-LuCl3 and 100µl of gentisic acid (10 mg/mL) to 100µL (500 μg) of anti CD20- DOTA-OSSu, incubating 30 minutes at 37°C and purifying by PD-10 column. Stability of 177Lu-anti-CD20 in human serum and in saline was analyzed by using 2 chromatographic systems: ITLC-SG and saturated ITLC-SG strips (BSA 5%) as carrier, and Sodium acetate 14% and EtOH-NH4OH-H2O (2:1:5) as solvents, respectively. Biodistribution studies were carried out in CD-1 normal mice in triplicate at 4, 16 and 24 hours post injection of 22.6 - 40.7 MBq of anti-CD20-DOTA-177Lu. In order to study the effect of previous administration of unlabelled anti- CD20 in the uptake by critical organs, biodistribution studies were also done at the same conditions using 150 mg/m² and 250 mg/m² of unlabelled anti-CD20. Dosimetric studies were accomplished using Monte Carlo Simulation method, subroutines Penelope, considering 177Lu-anti-CD20 uniformly distributed in a spheroid as a model of tumor mass. Results The Mab labelling with 177- Lu gave yields ranging from 60% to 75% and a radiochemical purity higher than 97% after purification for up to 24 hours elapsed time, both in human serum and in saline. Also, there was no significant difference in the performance and stability studies, between antibody stored at 4ºC and at -20ºC. The biological distributions without previous administration of unlabelled anti-CD20 showed significant uptake by liver up to 40% and urinary elimination up to 30% at 24 hours. Biodistributions studies with blocking by previous administration of unlabelled anti-CD20 showed a significant dose-dependent decrease, reaching half the initial values in the uptake of 177-Lu-anti-CD20 by liver tissue, while in blood there is not significant change. Dosimetric studies using Monte Carlo Simulation showed that 83% of the total dose was deposited at the tumor mass, while only the remaining 17% affects the surrounding non- pathological tissue. Conclusions: Optimization of the labelling of 177-Lu-anti-CD20 was achieved giving reliable results with a radiochemical purity consistent with its potential clinical application as a therapeutic radiopharmaceutical for treatment of non- Hodgkin´s lymphoma. Biodistributions studies with blocking by previous administration of unlabelled anti- CD20 showed the importance of this previous administration in order to decrease the uptake by critical organs to achieve a protective effect, optimizing the therapeutic desired effect in terms of the administered dose. Dosimetric studies using the simulation method determined that 177-Lu is a radionuclide suitable for treatment of tumor masses of small and medium size.