Antibody-maytansinoid conjugates are potent anti-cancer agents that are activated in the lysosome and arrest cancer cells in G 2 /M phase of the cell cycle in tissue culture and in xenograft tumor models
Authors: Peter U. Park, Anna Skaletskaya, Linda Hylander, Kate Connors, Michele Mayo, Thomas Chittenden, Victor S. Goldmacher
Journal: Cancer Research (2006)

1175   The recent approvals of monoclonal antibodies in oncology have given clinicians new tools for cancer treatment and renewed interest in other monoclonal antibody-based anti-cancer therapies, such as antibody-cytotoxic drug conjugates. Antibody-drug conjugates are composed of a monoclonal antibody that targets an antigen expressed on the surface of cancer cells and a small cytotoxic drug covalently linked to the antibody. While a number of antibody-drug conjugates have been shown to have anti-tumor activities, both in pre-clinical tumor models and in clinical settings, the mechanisms of action of these agents at the cellular level are not well-understood. A generally assumed notion is that upon binding to its target antigen, an antibody-drug conjugate is internalized  via  antigen-mediated endocytosis, which is followed by the intracellular release of the drug from the antibody. Release mechanisms such as proteolytic degradation of the antibody in the lysosomes or cleavage of the linker connecting the antibody and the drug by specific intracellular processes have been proposed. In this study we examined conjugates of huC242, a humanized monoclonal antibody that binds to the tumor-associated antigen, MUC1, and maytansinoid drugs (DM drugs), which are potent anti-mitotic drugs that inhibit microtubule polymerization. As expected, huC242-DM conjugates arrested antigen-positive COLO 205 colon cancer cells at the G 2 /M phase of the cell cycle  in vitro . By performing  in situ  immunohistochemistry using MPM-2, a monoclonal antibody that binds to mitotic phosphoproteins, we could also demonstrate that a major fraction of cells in subcutaneous COLO 205 xenograft tumors in mice underwent mitotic arrest following the treatment of mice with huC242-DM conjugates.  In vitro , cells were arrested as quickly as within 10 hr of the exposure to the conjugate, which indicates a fast process for the conversion of the conjugated DM moiety into an active drug species and delivery to its molecular target in the cytoplasm of the cell. Bafilomycin A1 and chloroquine are compounds that neutralize the pH in lysosomes and endosomes by two distinct mechanisms and as a result, block trafficking between late endosomes and lysosomes. The presence of either of these lysosomal inhibitors abolished the G 2 /M arrest induced by huC242-DM conjugates, but not by the free DM drug. This result indicates that lysosomal activation is an essential step in the process of cell killing by these conjugates. We extended these studies to other antibodies targeting different antigens on different cancer cell lines and to other cytotoxic drugs, which are unrelated to DM, and observed a similar requirement of lysosomal activation. Thus, lysosomal processing is likely to be a general activation mechanism for antibody-drug conjugates.