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High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

Thibaut Pelat,^1, Michael Hust,^2, Emmanuelle Laffly,¹ Florence Condemine,¹ Chantal Bottex,¹ Dominique Vidal,¹ Marie-Paule Lefranc,³ Stefan Dübel,² and Philippe Thullier^1*

Groupe de Biotechnologie des Anticorps, Département de Biologie des Agents Transmissibles, Centre de Recherche du Service de Santé des Armées, La Tronche,¹ Laboratoire d'ImmunoGénétique Moléculaire (LIGM), Université Montpellier II, UPR CNRS 1142, Institut de Génétique Humaine (IGH), Montpellier, and Institut Universitaire de France, Paris, France,³ Institut für Biochemie und Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany²

Received 5 December 2006/ Returned for modification 18 February 2007/ Accepted 10 May 2007

The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 x 10⁸ clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ± 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

Published ahead of print on 21 May 2007.

T.P. and M.H. contributed equally to this work.

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