Abstract Purpose: The cardiac glycoside family of steroidal drugs has been shown to possess novel and potent antitumor activities in rodents. Yet most likely due to their cardiotoxic nature, these drugs have failed to show clinical efficacy at doses deemed safe to humans. In order to determine if this drug class could be made safer and more efficacious, we assessed the antitumor activity of a new antibody drug conjugate system in which these steroidal compounds are tethered to antibodies that direct them to protein complexes found on various types of cancer cells. Experimental design: The activity and safety profiles of these novel antibody drug conjugates were examined using multiple cancer cell lines, normal cells, xenograft models in immunodeficient mice and non-human primates. Results: We identified multiple monoclonal antibodies that when conjugated to cardiac glycosides through long stable linkers, could provide cell killing activity independent of effector functions. When tested in vitro on a number of cancer cell lines, these active antibody drug conjugates termed Extracellular Drug Conjugates or EDCs, displayed potent cytotoxic activities with half maximal effective concentration (EC50) in sub-nanomolar ranges via a pathway resembling apoptosis and/or necroptosis. These activities were dependent on the expression of the corresponding antibody, linker length and steroid chemistry. In vivo using various tumor xenograft models, the EDCs were able to partially or completely regress tumor growth when 1 to 10 mgs/kg were administered intravenously. The EDCs were also found to assist various clinically approved therapies regress tumor growth. Finally, because cynomolgus monkeys respond to cardiac glycosides is similar to humans, 5mgs/kg of EDC was slowly infused intravenously into a cynomolgus monkey with no adverse effects observed even though the relative blood concentration of the cardiac glycoside attached reached 1500 nanomolar. Conclusion: These results support efforts to further evaluate these unique targeting antibody drug conjugates for the treatment of human cancers. Citation Format: James R. Prudent, David Marshall, John Murphy, Chad Hall, Scott Harried. Antibody targeted steroids for the treatment of cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A125.