Cardiac glycosides (CG) are potent inhibitors of Na+/K+-ATPase (NKA), inducing apoptosis through the inhibition of ion current. We developed a synthetic CG, CEN09-106, with enhanced cytotoxic potential over conventional CGs such as oubain and others. For specific delivery to the tumor, CEN09-106 was conjugated through a stable chemical linker to antibodies targeted against cell surface proteins that form complexes with NKA. Due to this molecular design CEN09-106 is locally presented to the tumor and inhibits NKA activity. Internalization of CEN09-106 is not required for its cytotoxicity, and hence we refer to this family of antibody-CG complexes as extracellular drug conjugates (EDC). Ovarian tumors overexpress FXYD5, a modulator of NKA activity. We have developed EDC1 to target FXYD5. Here, we demonstrate the ability of EDC1 to target ovarian tumors and confirm its mechanism of action. First, we compared the activity of oubain, CEN09-106, and EDC1 in cell viability assays. While the potency of oubain was approximately 1-10 mM, CEN09-106 and EDC1 both had IC50 between 1-2.5 nM when tested against OVCAR-5, OV2008, and SKOV3 cells. Expression of FXYD5 in these cell lines was confirmed by flow cytometry. The inhibition of cell viability was attributed to apoptosis induced by CEN09-106 and EDC1 as measured by increase in cleaved caspase3 and annexin V binding and decrease in Bcl-2 expression. CEN09-106 and EDC1 also inhibited viability of the cisplatin-resistant C13 (IC50 ~2.5 nM) cells. Whole-cell patch-clamp recording of cancer cells revealed NKA ionic current in SKOV-3 and OV2008 cells after inhibition of majority of the cell surface ion transporters and channels. Outward ion current detected in SKOV3 and OV2008 prior to treatment with CEN09-106 and EDC1 was in the range of 500-1000 pA. Upon treatment with CEN09-106 and EDC1 (2.5 nM, each), the current in these two cell lines decreased to only 200-400 pA, a 60-75% decrease. In vivo administration of EDC1 in nonhuman primates established its low general toxicity profile. In vivo studies in xenograft mouse models are currently under way and are showing promising results with significant inhibition of SKOV3 and OV2008 tumors when CEN09-106 and EDC1 were administered intraperitoneally. In ongoing work, we are screening additional platinum-sensitive and -resistant ovarian cancer lines for their responses to EDC1. These basic and translational studies will serve as the foundation to demonstrate the clinical value of EDC1 in the treatment of patients with ovarian tumors.
Citation Format: Yousef Alharbi, Arvinder Kapur, Jim Prudent, David Marshall, Mildred felder, Bikash Pattnaik, Manish Patankar. Antibody-conjugated cardiac glycosides: Potent agents for treatment of ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B11.