title: Multitarget selection of catalytic antibodies with β-lactamase activity using phage display.
abstract: β-lactamase enzymes responsible for bacterial resistance to antibiotics are among the most important health threats to the human population today. Understanding the increasingly vast structural motifs responsible for the catalytic mechanism of β-lactamases will help improve the future design of new generation antibiotics and mechanism-based inhibitors of these enzymes. Here we report the construction of a large murine single chain fragment variable (scFv) phage display library of size 2.7 × 109with extended diversity by combining different mouse models. We have used two molecularly different inhibitors of the R-TEM β-lactamase as targets for selection of catalytic antibodies with β-lactamase activity. This novel methodology has led to the isolation of five antibody fragments, which are all capable of hydrolyzing the β-lactam ring. Structural modeling of the selected scFv has revealed the presence of different motifs in each of the antibody fragments potentially responsible for their catalytic activity. Our results confirm (a) the validity of using our two target inhibitors for the in vitro selection of catalytic antibodies endowed with β-lactamase activity, and (b) the plasticity of the β-lactamase active site responsible for the wide resistance of these enzymes to clinically available inhibitors and antibiotics.
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, France.|UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.
authors.forename: Melody A
authors.initials: MA
authors.lastname: Shahsavarian
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, France.|UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.
authors.forename: Nancy
authors.initials: N
authors.lastname: Chaaya
authors.affiliation: Service de Pharmacologie et d'Immuno-analyse (SPI), IBITECS, CEA, Université Paris-Saclay, Gif-sur-Yvette, France.
authors.forename: Narciso
authors.initials: N
authors.lastname: Costa
authors.affiliation: Service de Pharmacologie et d'Immuno-analyse (SPI), IBITECS, CEA, Université Paris-Saclay, Gif-sur-Yvette, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Boquet
authors.affiliation: Unité Fonctionnalité et Ingénierie des Protéines (UFIP), UMR CNRS 6286, Université de Nantes, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Atkinson
authors.affiliation: Unité Fonctionnalité et Ingénierie des Protéines (UFIP), UMR CNRS 6286, Université de Nantes, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Offmann
authors.affiliation: UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.|Institut National de la Santé et de la Recherche Médicale (INSERM) U 1138, Paris, France.|Equipe Immunopathology and Therapeutic Immunointervention, Centre de Recherche des Cordeliers, Paris, France.|International Associated Laboratory IMPACT, Institut National de la Santé et de la Recherche Médicale-France and Indian Council of Medical Research-India, National Institute of Immunohaemotology, Mumbai, India.
authors.forename: Srini V
authors.initials: SV
authors.lastname: Kaveri
authors.affiliation: UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.|Institut National de la Santé et de la Recherche Médicale (INSERM) U 1138, Paris, France.|Equipe Immunopathology and Therapeutic Immunointervention, Centre de Recherche des Cordeliers, Paris, France.|International Associated Laboratory IMPACT, Institut National de la Santé et de la Recherche Médicale-France and Indian Council of Medical Research-India, National Institute of Immunohaemotology, Mumbai, India.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lacroix-Desmazes
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, France.|UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Friboulet
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, France.|UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.
authors.forename: Bérangère
authors.initials: B
authors.lastname: Avalle
authors.affiliation: Génie Enzymatique et Cellulaire (GEC), FRE 3580 CNRS, Université de Technologie de Compiègne, France.|UMR S 1138, Sorbonne Universités, Université Pierre et Marie Curie - Paris VI, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Padiolleau-Lefèvre
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D007114:Immunization; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D010406:Penicillins / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology; D019151:Peptide Library; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D047090:beta-Lactams / Q000737:chemistry* / Q000378:metabolism
chemical_list: D017151:Antibodies, Catalytic; D010406:Penicillins; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D047090:beta-Lactams; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1
keywords: catalytic antibody; enzyme inhibitor; phage display; scFv library; β-lactamase