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Research Articles: Therapeutics, Targets, and Development

A novel, potent, and specific ephrinA1-based cytotoxin against EphA2 receptor–expressing tumor cells

Departments of ¹ Neurosurgery, and ² Radiation Oncology and Cancer Biology, Brain Tumor Center of Excellence, Wake Forest University School of Medicine Comprehensive Cancer Center, Winston-Salem, North Carolina

Requests for reprints: Waldemar Debinski, Departments of Neurosurgery, Radiation Oncology, and Cancer Biology, Brain Tumor Center of Excellence, Comprehensive Cancer Center of Wake Forest University, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: 336-716-9712; Fax: 336-713-7639. E-mail: debinski{at}wfubmc.edu

Abstract

We have previously shown that the EphA2 receptor tyrosine kinase is overexpressed in glioblastoma multiforme (GBM) and represents a novel, attractive therapeutic target for the treatment of brain tumors. Here, we have developed an EphA2-targeted agent, ephrinA1-PE38QQR, a novel cytotoxin composed of ephrinA1, a ligand for EphA2, and PE38QQR, a mutated form of Pseudomonas aeruginosa exotoxin A. EphrinA1-PE38QQR showed potent and dose-dependent killing of GBM cells overexpressing the EphA2 receptor in cell viability and clonogenic survival assays, with an average IC₅₀ of 10^–11 mol/L. The conjugate was also highly effective in killing breast and prostate cancer cells overexpressing EphA2. The cytotoxic effect of ephrinA1-PE38QQR was specific, as it was neutralized by an excess of EphA2 ligands. Moreover, normal human endothelial cells and breast cancer cells that do not overexpress EphA2, as well as GBM cells that have down-regulated EphA2, were not susceptible to the cytotoxin. EphrinA1-PE38QQR–mediated cytotoxicity induced caspase-dependent apoptosis, which was, however, not responsible for cell death in response to the conjugate. In addition, the conjugate elicited no changes in the activity of survival pathways such as phosphoinositide 3-kinase, measured by AKT phosphorylation. This is the first attempt to create a cytotoxic therapy using any of the ephrin ligands of either class (A or B) conjugated to a bacterial toxin. EphrinA1-PE38QQR is very potent and specific, produces cell death that is caspase independent, and forms the basis for the further development of clinically applicable EphA2-targeted cytotoxins. [Mol Cancer Ther 2007;6(12):3208–18]

Grant support: F31 NSO55533-01 (J. Wykosky), and RO1 CA 74145 and Brain Tumor Center of Excellence (W. Debinski). NIH grant CA-12197 to the Cell and Virus Vector Core Laboratory of the Comprehensive Cancer Center of Wake Forest University.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁶ J. Wykosky, D.M. Gibo, W. Debinsky, unpublished data.

Received 3/21/07; revised 9/18/07; accepted 10/17/07.