Induction of programmed cell death in ErbB2/HER2-expressing cancer cells by targeted delivery of apoptosis-inducing factor

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with NADH oxidase activity that has a vital function in healthy cells but is also an important mediator of caspase-independent programmed cell death in stressed and damaged cells. Here, we generated a truncated AIF derivative (AIFΔ100) that lacks the mitochondrial import signal of the protein. Bacterially expressed AIFΔ100 was functionally active and induced cell death on microinjection into Vero cells accompanied by clear signs of apoptosis. For specific targeting to tumor cells, AIFΔ100 was genetically fused to the scFv(FRP5) antibody fragment that recognizes the ErbB2 (HER2) receptor tyrosine kinase frequently overexpressed in many human cancers. Recombinant scFv(FRP5)-AIFΔ100 (5-AIFΔ100) protein and a similar scFv(FRP5)-ETA252-366-AIFΔ100 (5-E-AIFΔ100) molecule harboring in addition the nontoxic translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed binding to ErbB2-expressing cells followed by protein internalization and accumulation in intracellular vesicles. In the presence of the endosomolytic reagent chloroquine 5-E-AIFΔ100 but not the similar 5-AIFΔ100 protein displayed potent cell killing activity, which was strictly dependent on the expression of ErbB2 on the target cell surface. Our results show that recombinant AIF specifically targeted to human cancer cells and delivered into the cytosol has potent cell killing activity, suggesting this molecule as an effector function suitable for the development of humanized immunotoxin-like molecules. [Mol Cancer Ther 2009;8(6):1526–35]