A novel pretargeting method for measuring antibody internalization in tumor cells

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 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 is added to the wells of pretargeting group. The radioactivity of the direct targeting cells represents the sum of both internalized and cell surface bound antibodies while the radioactivity of the pretargeting cells is due only to surface bound antibodies since 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-PMSA antibody 3C6 in LNCaP cells was examined and the average cell surface residence time was determined as 2 h. 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.