INTERNALIZATION OF AN INTACT DOXORUBICIN IMMUNOCONJUGATE

An immunoconjugate between doxorubicin and anti-(carcinoembryonic anti gen) (CEA) was prepared by using aminodextran (Mr = 40000) as the inte rmediate carrier, and the carbohydrate moiety of the antibody as the l inking site. The resulting immunoconjugate was subjected to an in vitr o evaluation for the internalization on the target cells (LoVo), and c ompared to that of unconjugated antibody, as well as the cellular upta ke of unconjugated doxorubicin. The internalization was evaluated micr oscopically by following the translocation of the red fluorescence of doxorubicin and the green fluorescence of the fluorescein-isothiocyana te-labeled goat anti-(mouse Ig) antibody, which visualizes the locatio n of the primary mouse antibody. Anti-CEA monoclonal antibody (NP-4) w as found to internalize into LoVo cells. The immunoconjugate made with this antibody was similarly internalized, and the doxorubicin was fou nd to distribute with the primary antibody. The cell surface and cytop lasm were the major compartments of their distribution. These results indicate that the drug molecules were indeed delivered into the cells by the antibody as an intact conjugate. Unconjugated doxorubicin, on t he contrary, was quickly absorbed by the cells and concentrated in the nucleus within 30 min, and never showed a distribution in the cytopla sm or cell membrane as in the nucleus by this procedure. The intermedi ate drug conjugate, doxorubicin-dextran, did not show internalization. The internalization of NP-4 antibody (or the doxorubicin conjugate) w as also confirmed by studying the intracellular catabolism of the cell -bound antibody (or conjugate). The release of the degraded antibody b y the cells, as differentiated by trichloroacetic acid precipitation t echniques, was considered an indication of internalization. Lysosomes were involved in the degradation, since the process was markedly inhib ited in the presence of the lysosomal enzyme inhibitor, ammonium chlor ide.