Production and evaluation of [Ga]-DTPA-Rituximab

Rituximab is a chimeric mouse-human monoclonal antibody. Rituximab, selectively, binds with high affinity to the CD20 antigen (human B-lymphocyte-restricted differentiation antigen, Bp35), a hydrophobic transmembrane protein, which is expressed on B-lymphocytes and on more than 90% of B cell non-Hodgkin's lymphomas. This antigen regulates the early step(s) in the activation process for cell cycle initiation and differentiation (1). CD20 is not shed from the cell surface and does not internalize upon antibody binding. Free CD20 antigen is not found in the circulation. Rituximab is thought to deplete CD20-positive cells via antibody-dependent cell-cytotoxicity and complement mediated cell lysis. Several studies of radiolabeled anti-CD20 monoclonal antibodies at nonmyeloablative doses in treating B-cell NHL have been reported, and several others are in progress. The agents for which most data are available are Itositumomab (Bexxar) (2) and yttrium Yibritumomab tiuxetan (Zevalin) (3). These studies have reported response rates of 25% to 40% with median response duration of 6 to 18 months in most studies and some very durable responses of more than 5 years (4-7). In some studies rituximab has been labeled for metabolism and localization of CD20 antigens throughout body and/or penetration of the antibody to specific organs (8). In order to obtain an anti-CD20 conjugate to be used in future therapeutic studies using therapeutic radioisotopes, Ga-labeled antibody was prepared as a model of metal chelated immunoconjugate for preliminary dosimetric and biodistribution studies. Based on our recent experiences on the preparation of radiometal-labeled antibodies in our group (9), we were interested in the preparation of cyclotron-derived antiCD-20 immunoconjugate. A precise labeling strategy was employed Background: In order to obtain an anti-CD20 conjugate to be used in future therapeutic studies with therapeutic radioisotopes, Ga-labeled antibody was prepared as a model of metal chelated immunoconjugate for preliminary dosimetric and biodistribution studies. Materials and Methods: Rituximab was labeled with [Ga]-gallium chloride after residulation with freshly prepared cyclic DTPAdianhydride. The best results of the conjugation were obtained by the addition of 1 ml of a rituximab pharmaceutical solution (5 mg/ml, in phosphate buffer, pH=8) to a glass tube pre-coated with DTPAdianhydride (0.01 mg) at 25|o|C with continuous mild stirring for 30 min. The final isotonic Ga-DTPArituximab complex was checked by gel electrophoresis for radiolysis/chemolysis control. Radio-TLC was performed to ensure the formation of only one species. Preliminary in vivo studies in normal rat model were performed to determine the biodistribution of the radioimmunoconjugate up to 6 hours. Results: Radio-thin layer chromatography showed an overall radiochemical purity of 96-99% at optimized conditions (specific activity =300-500 MBq/mg, labeling efficiency 77%). Gel electrophoresis showed no protein cleavage after radiolabeling. Conclusion: Preliminary in vivo studies in normal rat model showed [Ga]-DTPA-rituximab is a good probe for bio-dosimetry of therapeutic rituximab conjugates.