title: Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin ZHER2:2891-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain.
abstract: Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (KD=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (KD=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.
authors.affiliation: School of Biotechnology, Division of Protein Technology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.affiliation: School of Biotechnology, Division of Protein Technology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Seijsing
authors.affiliation: Affibody AB, Solna, Sweden.
authors.forename: Fredrik Y
authors.initials: FY
authors.lastname: Frejd
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.affiliation: School of Biotechnology, Division of Protein Technology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Torbjörn
authors.initials: T
authors.lastname: Gräslund
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000494:pharmacology*; D000418:Albumins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000494:pharmacology*; D015374:Biosensing Techniques; D002417:Cattle; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005098:Exotoxins / Q000235:genetics / Q000494:pharmacology*; D006207:Half-Life; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000494:pharmacology*; D051379:Mice; D009369:Neoplasms / Q000378:metabolism* / Q000628:therapy; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D037521:Virulence Factors / Q000235:genetics / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000418:Albumins; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C508053:ERBB2 protein, human