title: Expression and detection strategies for an scFv fragment retaining the same high affinity than Fab and whole antibody: Implications for therapeutic use in prion diseases.
abstract: Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.
authors.affiliation: Commissariat à l'Energie Atomique (CEA) Saclay, Service de Pharmacologie et d'Immunologie, Bat 136, 91191 Gif sur Yvette Cedex, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Padiolleau-Lefevre
authors.forename: Coralie
authors.initials: C
authors.lastname: Alexandrenne
authors.forename: Fatima
authors.initials: F
authors.lastname: Dkhissi
authors.forename: Gilles
authors.initials: G
authors.lastname: Clement
authors.forename: Sosthène
authors.initials: S
authors.lastname: Essono
authors.forename: Céline
authors.initials: C
authors.lastname: Blache
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Couraud
authors.forename: Anne
authors.initials: A
authors.lastname: Wijkhuisen
authors.forename: Didier
authors.initials: D
authors.lastname: Boquet
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D017096:Prion Diseases / Q000188:drug therapy / Q000276:immunology; D011328:Prions / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D011328:Prions; D011994:Recombinant Proteins