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 IC(50) of approximately 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.