Evaluation of the cytotoxic potencies of the major maytansinoid metabolites of antibody maytansinoid conjugates detected in vitro and in preclinical mouse models

2150 Antibody-maytansinoid conjugates (AMCs) are anticancer agents composed of three components-a monoclonal antibody that binds specifically to a target antigen, a maytansinoid cell-killing agent (e.g., DM1 or DM4), and a linker that stably connects the two. The choice of the linker impacts both the activity and tolerability of AMCs in preclinical models. Three AMCs are currently in phase II clinical trials for the treatment of cancer -IMGN242 (huC242-DM4), IMGN901 (huN901-DM1), and trastuzumab-DM1 (T-DM1). All three conjugates consist of an average of 3-4 maytansinoid (DMx) molecules tethered to a humanized monoclonal antibody (huMab) via a stable linker. IMGN901 and IMGN242 both contain disulfide links, while T-DM1 contains a thioether link. We have isolated and identified by independent synthesis and mass spectrometry the major maytansinoid metabolites produced in COLO 205 tumor-bearing SCID mice and non-tumor bearing CD-1 mice following administration of huMab-[3H]DMx conjugates utilizing the same three linkers employed by IMGN242, IMGN901, and T-DM1. We found that the nature of the linker determines the metabolism in both target tumor tissue as well as liver tissue of mice. Surprisingly, no detectable metabolism of the macrocyclic core of the maytansinoid is observed. Instead, metabolism appears to be restricted to the linker arm of the maytansinoids. In this study, we synthesized all of the major observed metabolites and compare their cytotoxic potencies against a panel of human carcinoma cell lines to a reference maytansine standard. Two of the metabolites formed from the disulfide-linked conjugates, S-methyl-DM1 and S-methyl-DM4, were more potent than maytansine towards all cell lines tested, while the other metabolites displayed considerably less activity. The cytotoxic potencies of the different maytansinoid metabolites appear to be related to their uptake into cells.