title: Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering.
abstract: 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 5beta- or 5alpha-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 5beta- or 5alpha-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.
authors.affiliation: Unité Mixte Commissariat à l'Energie Atomique, bioMérieux, Département d'Ingénierie et d'Etudes des Protéines, Commissariat à l'Energie Atomique, Centre d'Etudes de Saclay, Gif-sur-Yvette Cedex 91191, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Dubreuil
authors.forename: Marc
authors.initials: M
authors.lastname: Bossus
authors.forename: Marc
authors.initials: M
authors.lastname: Graille
authors.forename: Maëlle
authors.initials: M
authors.lastname: Bilous
authors.forename: Alexandra
authors.initials: A
authors.lastname: Savatier
authors.forename: Michel
authors.initials: M
authors.lastname: Jolivet
authors.forename: André
authors.initials: A
authors.lastname: Ménez
authors.forename: Enrico
authors.initials: E
authors.lastname: Stura
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ducancel
mesh_terms: D043582:5-alpha-Dihydroprogesterone / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques; D019921:Biotinylation; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006825:Hybridomas / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D019151:Peptide Library; D013256:Steroids; D011374:Progesterone; D043582:5-alpha-Dihydroprogesterone