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J. Biol. Chem., Vol. 280, Issue 26, 24880-24887, July 1, 2005

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Fine Tuning of the Specificity of an Anti-progesterone Antibody by First and Second Sphere Residue Engineering^*

Olivier Dubreuil¶, Marc Bossus¶, Marc Graille, Maëlle Bilous, Alexandra Savatier¶, Michel Jolivet¶, André Ménez, Enrico Stura, and Frédéric Ducancel||

From the Unité Mixte Commissariat à l'Énergie Atomique, bioMérieux, Département d'Ingénierie et d'Études des Protéines, Commissariat à l'Énergie Atomique, Centre d'Études de Saclay, Gif-sur-Yvette Cedex 91191, and ^¶Département Recherches et Développements Immuno-essais et Protéomique, bioMérieux, Chemin de l'Orme, Marcy l'Étoile 69280, France

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5- or 5-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5- or 5-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

Received for publication, January 3, 2005 , and in revised form, March 14, 2005.

^* This work was supported by the society bioMérieux (France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

^|| To whom correspondence should be addressed: Dépt. d'Ingénierie et d'Études des Protéines, CEA de Saclay, Gif-sur-Yvette Cedex 91191, France. Tel.: 33-1-6908-8154; Fax: 33-1-6908-9071; E-mail: frederic.ducancel{at}cea.fr.

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