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The Influence of Furin and the KDEL Receptor on the Efficacy of Recombinant Immunotoxins based on Pseudomonas Exotoxin A
Author(s) -
Zhu Yuyi,
Weldon John
Publication year - 2021
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.2021.35.s1.04779
Subject(s) - kdel , furin , pseudomonas exotoxin , immunotoxin , receptor , endosome , hek 293 cells , microbiology and biotechnology , endoplasmic reticulum , recombinant dna , transfection , biology , chinese hamster ovary cell , cell culture , antibody , biochemistry , golgi apparatus , immunology , gene , monoclonal antibody , genetics , enzyme
Recombinant immunotoxins (RITs) are targeted immunotherapeutics for the treatment of cancers. Most RITs are composed of an antibody fragment joined to a bacterial toxin such as Pseudomonas exotoxin A (PE). PE‐based RITs undergo a complex intoxication pathway in order to kill cells. First, RITs are internalized by receptor‐mediated endocytosis upon engagement with their cognate receptor. Then, within the endolysosomal system, the protease furin cleaves RITs, separating PE from the antibody. Next, PE traffics from endosomes to the endoplasmic reticulum (ER) via retrograde transport. This is mediated by interaction of PE with the KDEL receptor. Finally, PE is exported from the ER into the cytosol, where PE covalently modifies translation elongation factor‐2 (EF2). This halts protein synthesis and induces apoptotic cell death. Previous research in our lab and others has demonstrated that replacement of the C‐terminal REDLK sequence of PE with the canonical KDEL sequence can enhance RIT efficacy. When we evaluated this improvement, however, the enhancement varied widely among RITs targeting different receptors and cell lines. I hypothesized that these differences could be due to variations in the expression of furin or the trafficking of different receptors. To investigate these possibilities, I have created HEK293 cell lines with and without furin that express uncommon receptors, CD22 or mesothelin. These HEK293 cells provide a uniform genetic background from which to study the roles of furin and the KDEL receptor. I plan to compare the relative sensitivity of the transgenic cell lines to different RIT constructs to determine the contribution of these factors to RIT trafficking and cytotoxicity. This information will be used to develop improved RIT‐based cancer therapies.