Abstract 1202: Target identification for a new type of ADC
Authors: James R. Prudent, Chad Hall, David J. Marshall, Scott Harried, John Murphy
Journal: Cancer Research (2016)

Abstract Purpose: To explore the mechanism of action of a potent new type of antibody drug conjugate (ADC) called Extracellular Drug Conjugates or EDCs. Experimental Design: EDCs are a new type of ADC that have similar activity and components, yet EDCs are different in the following ways; 1) EDCs do require internalization, 2) EDCs are not pro-drugs but instead require the antibody and payload to remain permanently linked and 3) EDCs target two proteins in unison, requiring long non-cleavable linkers to span distances between bound antibody to the payload binding site. To define the MOA of the EDCs, high definition phase contrast imaging and protein marker experiments were conducted. In order to verify the targets of a set of EDCs consisting of Na,K-ATPase specific payloads, siRNA knockdown experiments were designed and tested. Results: First, we observed that after 24 hour exposure to each of the EDCs (or the free payload), cells rounded up, partially detached from the substrate, swelled, and lost plasma membrane integrity - morphology resembling necrosis. These observations were consistent with no induction apoptosis or autophagy. Second, our siRNA studies determined that alpha 1 of the Na,K-ATPase was a lethal target. Consistent with this observation on all tested cells lines tested, the EC50 values of each EDC decreased with decrease in alpha 1 expression. Additionally, when the corresponding antibody target expression decreased, overall activity of each EDC went down. Alpha 1 expression was also found to change the effects of the free payload, indicating that the EDCs and the free payload share the same target. Yet unlike the EDCs, payload alone was not affected by the expression of any antibody target used in the study. We also observed that siRNA knockdown of the Na,K-ATPase beta 1 or beta 3 subunits alone did not affect EDC or payload activity, yet a significant decrease in activity could be observed when both subunits were knocked down simultaneously. These results are also consistent with microarray data analysis using siRNAs against all 8 alpha and beta subunits which show that only alpha-1, beta-1, and beta-3 are expressed in the cell types tested. Conclusions: These results show that when payloads targeting the Na,K-ATPase are attached to certain antibodies via long flexible linkers, the resulting EDC activity specifically target the alpha 1 subunit of the Na,K-ATPase, of which we determined to be a lethal target. These results also show that EDC's activity is dependent on the expression of the corresponding antibody target. In addition, the mechanisms of EDCs are similar to that of the free payload and appear to be necrosis like. These results support the continuing efforts to identify the detailed mechanism of the new ADCs which may lead to identifying patients most likely to respond to EDC based therapies. Citation Format: James R. Prudent, Chad Hall, David J. Marshall, Scott Harried, John Murphy. Target identification for a new type of ADC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1202.