Tumor cell-selective cytotoxicity of matrix metalloproteinase-activated anthrax toxin

Matrix metalloproteinases (MMPs) are overexpressed in a variety of tumor ti ssues and cell lines, and their expression is highly correlated to tumor in vasion and metastasis. To exploit these characteristics in the design of tu mor cell-selective cytotoxins, we constructed two mutated anthrax toxin pro tective antigen (PA) proteins in which the furin protease cleavage site is replaced by sequences selectively cleaved by MMPs. These MMP-targeted PA pr oteins were activated rapidly and selectively on the surface of MMP-overexp ressing tumor cells. The activated PA proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor resi dues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A. The toxicity of the mutated PA proteins for MMP-overexpressing cells was bl ocked by hydroxamate inhibitors of MMPs, including BB94, and by a tissue in hibitor of matrix metalloproteinases (TIMP-2). The mutated PA proteins kill ed MMP-overexpressing tumor cells while sparing nontumorigenic normal cells when these were grown together in a coculture model, indicating that PA ac tivation occurred on the tumor cell surface and not in the supernatant, Thi s method of achieving cell-type specificity is conceptually distinct from, and potentially synergistic with, the more common strategy of retargeting a protein toxin by fusion to a growth factor, cytokine, or antibody.