Mutations In The Furin Cleavage Site To Improve Cytotoxicity In Anti‐Mesothelin Recombinant Immunotoxins Based On Pseudomonas Exotoxin A

Abstract

A recombinant immunotoxin (RIT) is a fusion protein composed of an antibody and a toxin. The antibody binds to receptors on target cells and promotes internalization. Once internalized, the toxin kills the cell. RITs based on Pseudomonas exotoxin A (PE) have been created to target and kill cancer cells. In order to do this, internalized PE must reach the cytosol through a retrograde trafficking route, during which it encounters and is cleaved by the protease furin. Furin has been established as an integral part of PE‐based RIT intoxication pathways. Mutations in the PE furin cleavage site have been shown to influence cleavage efficiency and RIT cytotoxicity. In order to explore the role of furin in RIT intoxication further, we have constructed anti‐mesothelin/PE‐based RITs with mutations on either side of the furin cleavage site. Based on previous results, we initially hypothesized that mutations N‐terminal to the cleavage site would increase both cleavage efficiency and cytotoxicity in these RITs, while mutations C‐terminal to the cleavage site would increase cleavage efficiency without significant improvements to cytotoxicity. We expressed, purified, and tested the cytotoxicity of these constructs in tissue culture and found that mutations N‐terminal to the cleavage site decreased cytotoxicity relative to the wild‐type RIT, while mutations C‐terminal to the cleavage site had minimal effect. We next plan to explore cleavage efficiency of the mutant RITs.Support or Funding InformationTowson University Department of Biological Sciences