Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of its six hypervariable loops |
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Authors: | Laffly Emmanuelle Pelat Thibaut Cédrone Frédéric Blésa Stéphane Bedouelle Hugues Thullier Philippe |
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Affiliation: | 1 Groupe de Biotechnologie des Anticorps, Laboratoire d'Immunobiologie, Centre de Recherches du Service de Santé des Armées, 24 Avenue des Maquis du Grésivaudan, 38702 La Tronche, France 2 Biométhodes, Genavenir 8, 5 rue Henri Desbruères, 91030 Evry Cedex, France 3 Unit of Molecular Prevention and Therapy of Human Diseases (CNRS-URA 3012), Institut Pasteur, 28 rue Docteur Roux, 75724 Paris Cedex 15, France |
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Abstract: | The enhancement of antibody affinity by mutagenesis targeting only complementarity determining regions has the advantage of respecting the framework regions, which are important for tolerance if clinical use is envisaged. Here, starting from a Fab (antigen-binding fragment; 35PA83) capable of neutralizing the lethal toxin of anthrax and having an affinity of 3.4 nM for its antigen, a phage-displayed library of variants where all six complementarity determining regions (73 positions) were targeted for mutagenesis was built. This library contained 5 × 108 variants, and each variant carried four mutations on average. The library was first panned with adsorbed antigen and washes of increasing stringency. It was then screened in parallel with either small concentrations of soluble biotinylated antigen or adsorbed antigen and long elution times in the presence of soluble antigen. The stringencies of both selections were pushed as far as possible. Compared with 35PA83, the best selected clone had an affinity enhanced 19-fold, to 180 pM, and its 50% inhibitory concentration was decreased by 40%. The results of the two selection methods were compared, and the generality of these methods was considered. |
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Keywords: | Fab, antigen-binding fragment LT, lethal toxin PA, protective antigen CDR, complementarity determining region VH, heavy chain variable domain VL, light chain variable domain Kd, dissociation constant IC50, 50% inhibitory concentration koff, dissociation rate constant kon, association rate constant LF, lethal factor PBS, phosphate-buffered saline |
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