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 68 Ga‐labeled rituximab fragments, ( 68 Ga‐NOTA‐F (ab′)‐rituximab and 68 Ga‐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 68 Ga and characterized by SE‐HPLC. Intact rituximab was labeled with gallium‐68 for comparison. Specificity of 68 Ga‐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 68 Ga‐NOTA‐F (ab′) 2 ‐rituximab and 68 Ga‐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. 68 Ga‐labeled rituximab fragments were stable in saline and serum up to 2‐hour post preparation and exhibited specificity to CD20 antigen. Immunoreactivity of 68 Ga‐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 68 Ga‐ NOTA‐F (ab′) 2 ‐rituximab and 68 Ga‐NOTA‐F (ab′)‐rituximab as PET imaging agents for NHL.