A novel pretargeting method has been developed to quantitate antibody cellular internalization. In this study, the antibody was conjugated with a phosphorodiamidate morpholino oligomer (MORF) specific for the complementary MORF (cMORF) as an effector. Half the tumor cells were incubated with the MORF-antibody (pretargeting group) and the other half with the same MORF-antibody at the same concentration but radiolabeled (direct targeting group). After incubation, the same dosage of radiolabeled cMORF was added to the wells of the pretargeting group. The radioactivity of the direct targeting cells represented the sum of both internalized and cell-surface-bound antibodies, whereas the radioactivity of the pretargeting cells resulted only from the surface-bound antibodies, as the radiolabeled cMORF does not penetrate the cell surface. Therefore, the difference in radioactivity accumulation between pretargeting and direct targeting provides the internalized fraction. In this example, the internalization of a MORF conjugated anti-prostate-specific membrane antigen antibody, 3C6, in LNCaP cells was examined, and the average cell-surface residence time was determined as 2 hours. This method of measuring antibody internalization is directly applicable to pretargeting applications but can be a universal alternative to the conventional acid-wash method, with the advantage of leaving the cell membrane undamaged.

We are investigating a novel pretargeting approach involving an initial IV injection of antitumor antibody conjugated with a phosphorodiamidate morpholino oligomer (MORF, a DNA analog) and the subsequent IV injection of the radiolabeled complement oligomer (cMORF). In this paper, the cMORF was labeled with (188)Re using MAG(3) as chelator for therapeutic applications. Since (c)MORFs are unstable in acidic condition, an optimal labeling pH was first selected and the other labeling factors were then examined. A labeling efficiency of greater than 90% can be achieved even at a concentration of MAG(3)-cMORF as low as 0.8 microM. The labeled cMORF is stable and capable of hybridizing to its complement.