Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

umen of intraluminal vesicles or to the cytoplasm; 7. Following transportation to late endosome (LE), back fusion of intraluminal vesicles with the limiting membrane delivers the "trapped" toxic factors to the cytoplasm; 8. In the cytoplasm, LF functions as a zinc metalloproteinase that cleaves the N termini of MKK/MEK proteins, blocking their signaling activity. EF acts as a Ca 2+ /calmodulin activated adenylate cyclase that dramatically elevates cytoplasmic cAMP level and consequently disrupts normal cellular activities. [421] were the first to introduce and apply a new concept in which replacing the natural furin cleavage site in anthrax PA protein (such cleavage must occur on the cell surface in order to achieve binding and internalization of a catalytic LF and EF) with a sequence recognized by a tumor-associated protease may confer the recombinant molecule with the ability of targeting the protease overexpressing cells. To this end, in two mutated PA proteins, PA-L1(named also PrAg-L1) and PA-L2, the natural furin recognition site was replaced by sequences susceptible to cleavage by MMP-2 (gelatinase A) and MMP-9 (gelatinase B) that are reported to be related to invasion and metastasis in various human cancers [473] [474] [475] [476] . The toxic catalytic polypeptide that has been used in this study was a fusion between the ADP-ribosylation domain of Pseudomonas exotoxin A and amino acids 1-254 of LF (LF N ), which contains the PA binding domain that proved sufficient to achieve translocation of a fused "passenger" polypeptide to the cytosol of cells in a PA-dependent process [477] [478] [479] . By combining the re-engineered PA with the fusion toxic polypeptide (which was denoted FP59), the researchers have demonstrated selective killing of MMP-overexpressing human tumor cell lines while sparing nontumorigenic normal cells. Protection against challenge from PA-L1/L2 plus FP59 by MMP inhibitors further demonstrated that cell killing is highly dependent on the MMPs activity expressed by the tumor cells. Furthermore, specific eradication of MMP overexpressing tumor cells in a co-culture model indicated that PA activation occurred on the tumor cell surface and not in the culture supernatant. Activation of MMPs on the cell membrane by plasmin and/or membrane-anchored MMP, together with binding to cell surface receptors, were proposed as factors that may contribute to the retention of soluble active MMPs on the surface of tumor cells [421] .