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Infection and Immunity, June 1999, p. 2957-2963, Vol. 67, No. 6
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Disruption of Anthrax Toxin Binding with the Use of Human Antibodies and Competitive Inhibitors

Nick M. Cirino,1 Daniele Sblattero,2 David Allen,1 Scott R. Peterson,1 James D. Marks,3 Paul J. Jackson,1 Andrew Bradbury,2 and Bruce E. Lehnert1,*

Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 875451; International School for Advanced Studies (SISSA), Trieste, Italy 340132; and Department of Anesthesia and Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 941103

Received 28 December 1998/Returned for modification 5 February 1999/Accepted 1 March 1999

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.

* Corresponding author. Mailing address: MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 667-2753. Fax: (505) 665-3024. E-mail: lehnert{at}telomere.lanl.gov.

Infection and Immunity, June 1999, p. 2957-2963, Vol. 67, No. 6
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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