Preparation and preliminary bioevaluation studies of 68Ga-NOTA-rituximab fragments as radioimmunoscintigraphic agents for non-Hodgkin lymphoma

Rituximab is used for the treatment of non-Hodgkin lymphoma (NHL). This study focuses on development of Ga-68-labeled rituximab fragments, (Ga-68-NOTA-F (ab ')-rituximab and Ga-68-NOTA-F (ab ')(2)-rituximab, as PET-imaging agents for NHL. Rituximab was digested with immobilized pepsin and papain to yield F (ab ')(2) and Fab fragments respectively that were characterized by size exclusion HPLC (SE-HPLC) and SDS-PAGE. They were conjugated with p-SCN-Bn-NOTA, labeled with Ga-68 and characterized by SE-HPLC. Intact rituximab was labeled with gallium-68 for comparison. Specificity of Ga-68-labeled immunoconjugates was ascertained by immunoreactivity and cell binding studies in Raji cells, while biodistribution studies were performed in normal Swiss mice. Gradient SDS-PAGE under nonreducing condition showed molecular weights of F (ab ')(2)-rituximab and F (ab ')-rituximab as approximately 100 and 40 Kd, respectively. Radiochemical purity (RCP) of Ga-68-NOTA-F (ab ')(2)-rituximab and Ga-68-NOTA-F (ab ')-rituximab were 98.2 +/- 0.5% and 98.8 +/- 0.2% respectively with retention times of 17.1 +/- 0.1 min and 19.3 +/- 0.1 min in SE-HPLC. Ga-68-labeled rituximab fragments were stable in saline and serum up to 2-hour post preparation and exhibited specificity to CD20 antigen. Immunoreactivity of Ga-68-labeled immunoconjugates was greater than 80%. Clearance of the fragmented radioimmunoconjugates was predominantly through renal route. Preliminary results from this study demonstrate the potential of Ga-68- NOTA-F (ab ')(2)-rituximab and Ga-68-NOTA-F (ab ')-rituximab as PET imaging agents for NHL.