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Investigating the Relationship between Furin Cleavage and Cytotoxicity in a Recombinant Immunotoxin Based on Pseudomonas Exotoxin A
Author(s) -
Reifer Danielle,
Grossman Brian,
Weldon John E.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.472.8
Subject(s) - furin , immunotoxin , pseudomonas exotoxin , cleavage (geology) , recombinant dna , cytotoxicity , microbiology and biotechnology , exotoxin , endoplasmic reticulum , chemistry , diphtheria toxin , endocytosis , biology , toxin , biochemistry , receptor , in vitro , enzyme , gene , paleontology , fracture (geology)
Recombinant immunotoxins (RITs) are anti‐cancer therapeutic proteins that combine an antibody and a protein toxin. RITs based on the bacterial toxin Pseudomonas exotoxin A (PE) are currently in clinical trials, with one (moxetumomab pasudotox) recently approved by the FDA for the treatment of Hairy Cell Leukemia. PE‐based RITs are internalized by receptor‐mediated endocytosis and undergo retrograde trafficking to the endoplasmic reticulum, from which they translocate to the cytosol and catalytically inhibit protein synthesis. Early on during their intracellular trafficking route, PE‐based RITs are hydrolyzed and activated by furin, a serine protease ubiquitously expressed in mammalian cells. The furin cleavage site in PE, however, has a very slow cleavage rate compared to other targets of furin. Because the furin cleavage site of PE is so poor, it has been proposed that altering the cleavage site to enhance the rate would increase the cytotoxicity of PE‐based RITs. Previous research has shown that furin site mutations can modestly enhance RIT activity, but no correlation exists between cleavage efficiency and cytotoxicity across the full site. Interestingly, the correlation becomes significant if only mutations in the N‐terminal region of the cleavage site are considered (Spearman's r = 0.986; p = 0.0056). To investigate this relationship further, three different forms of an anti‐transferrin receptor PE‐based recombinant immunotoxin have been expressed and isolated: one with mutations on the N‐terminal region of the cleavage site, one with mutations on the C‐terminal region, and one with mutations over the entire cleavage site. Each toxin will be refolded, purified by column chromatography, and evaluated in cytotoxicity assays against HEK293 cells to determine how they impact cytotoxicity. It is expected that the RIT with optimizing mutations across the entire furin cleavage site will exhibit the greatest increase in cytotoxicity, followed closely by the N‐terminal mutations alone. These data will provide a better understanding of the relationship between cleavage efficiency and cytotoxicity, permitting better design of PE‐based RITs to produce more effective cancer treatments. It will also help to elucidate an incompletely understood PE intoxication pathway. Support or Funding Information This project was supported by funds from the Linda Sweeting Summer Research Fellowship and the Fisher College of Science and Mathematics. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .