title: Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.
abstract: The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.
authors.affiliation: Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
authors.forename: N M
authors.initials: NM
authors.lastname: Cirino
authors.forename: D
authors.initials: D
authors.lastname: Sblattero
authors.forename: D
authors.initials: D
authors.lastname: Allen
authors.forename: S R
authors.initials: SR
authors.lastname: Peterson
authors.forename: J D
authors.initials: JD
authors.lastname: Marks
authors.forename: P J
authors.initials: PJ
authors.lastname: Jackson
authors.forename: A
authors.initials: A
authors.lastname: Bradbury
authors.forename: B E
authors.initials: BE
authors.lastname: Lehnert
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000915:Antibody Affinity; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D018000:Receptors, Peptide / Q000276:immunology; D012967:Sodium Dodecyl Sulfate; D013997:Time Factors
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007128:Immunoglobulin Fragments; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors; C055966:immunoglobulin Fv; D012967:Sodium Dodecyl Sulfate