title: Protection of the Furin Cleavage Site in Low-Toxicity Immunotoxins Based on Pseudomonas Exotoxin A.
abstract: Recombinant immunotoxins (RITs) are fusions of an Fv-based targeting moiety and a toxin. Pseudomonas exotoxin A (PE) has been used to make several immunotoxins that have been evaluated in clinical trials. Immunogenicity of the bacterial toxin and off-target toxicity have limited the efficacy of these immunotoxins. To address these issues, we have previously made RITs in which the Fv is connected to domain III (PE24) by a furin cleavage site (FCS), thereby removing unneeded sequences of domain II. However, the PE24 containing RITs do not contain the naturally occurring disulfide bond around the furin cleavage sequence, because it was removed when domain II was deleted. This could potentially allow PE24 containing immunotoxins to be cleaved and inactivated before internalization by cell surface furin or other proteases in the blood stream or tumor microenvironment. Here, we describe five new RITs in which a disulfide bond is engineered to protect the FCS. The most active of these, SS1-Fab-DS3-PE24, shows a longer serum half-life than an RIT without the disulfide bond and has the same anti-tumor activity, despite being less cytotoxic in vitro. These results have significance for the production of de-immunized, low toxicity, PE24-based immunotoxins with a longer serum half-life.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. gilad.kaplan@nih.gov.
authors.forename: Gilad
authors.initials: G
authors.lastname: Kaplan
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. fred.lee@nih.gov.
authors.forename: Fred
authors.initials: F
authors.lastname: Lee
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. OndaM@mail.nih.gov.
authors.forename: Masanori
authors.initials: M
authors.lastname: Onda
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. emily.kolyvas@nih.gov.
authors.forename: Emily
authors.initials: E
authors.lastname: Kolyvas
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Gauravb@uw.edu.|Institute for Protein Design, University of Washington, Seattle, WA 98195, USA. Gauravb@uw.edu.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Bhardwaj
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. dabaker@uw.edu.|Institute for Protein Design, University of Washington, Seattle, WA 98195, USA. dabaker@uw.edu.|Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. dabaker@uw.edu.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. pastani@mail.nih.gov.
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001427:Bacterial Toxins / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004220:Disulfides / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004355:Drug Stability; D005098:Exotoxins / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D045683:Furin / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D000922:Immunotoxins / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D000090204:Mesothelin; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy* / Q000473:pathology; D010084:Oxidation-Reduction; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D059016:Tumor Microenvironment; D037521:Virulence Factors / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D045683:Furin; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D000090204:Mesothelin; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C000715528:Msln protein, mouse
keywords: Pseudomonas exotoxin A; disulfide bond; mesothelin; recombinant immunotoxin