title: Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.
abstract: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning.
  
  
  
  This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes).
  
  
  
  Therefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.
authors.affiliation: INSERM Center for Research in Transplantation and Immunology (CRTI) U1064; Université de Nantes; Centre Hospitalier Universitaire de Nantes Institut de Transplantation Urologie Néphrologie (ITUN), Nantes, F44000, France.|Transgenesis Rat ImmunoPhenomic Platform Structure Fédérative de Recherche François Bonamy Centre National de Recherche Scientifique UMS3556, Nantes, F44093, France.
authors.forename: Laure-Hélène
authors.initials: LH
authors.lastname: Ouisse
authors.affiliation: CRCNA UMR S892 INSERM 6299 CNRS Université de Nantes; Université de Nantes Faculté des Sciences et Techniques, Nantes, F44093, France.
authors.forename: Laetitia
authors.identifier: 0000-0003-0113-488X
authors.initials: L
authors.lastname: Gautreau-Rolland
authors.affiliation: CRCNA UMR S892 INSERM 6299 CNRS Université de Nantes; Centre Hospitalier Universitaire de Nantes, Nantes, F44093, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Devilder
authors.affiliation: Recombinant Antibody Technology Babraham Research Campus, Cambridge, CB22 3AT, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Osborn
authors.affiliation: CRCNA UMR S892 INSERM 6299 CNRS Université de Nantes; Centre Hospitalier Universitaire de Nantes, Nantes, F44093, France.
authors.forename: Melinda
authors.initials: M
authors.lastname: Moyon
authors.affiliation: Centre Hospitalier Universitaire de Bordeaux Laboratoire d'Immunologie et Immunogénétique Hôpital Pellegrin Bordeaux, Bordeaux, F33076, France.|Université de Bordeaux UMR CNRS 5164 , Talence, F33400, France.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Visentin
authors.affiliation: INSERM Center for Research in Transplantation and Immunology (CRTI) U1064; Université de Nantes; Centre Hospitalier Universitaire de Nantes Institut de Transplantation Urologie Néphrologie (ITUN), Nantes, F44000, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Halary
authors.affiliation: Recombinant Antibody Technology Babraham Research Campus, Cambridge, CB22 3AT, UK.
authors.forename: Marianne
authors.initials: M
authors.lastname: Bruggemann
authors.affiliation: Ligand Pharmaceuticals, San Diego, CA, USA.
authors.forename: Roland
authors.initials: R
authors.lastname: Buelow
authors.affiliation: INSERM Center for Research in Transplantation and Immunology (CRTI) U1064; Université de Nantes; Centre Hospitalier Universitaire de Nantes Institut de Transplantation Urologie Néphrologie (ITUN), Nantes, F44000, France. ianegon@nantes.inserm.fr.|Transgenesis Rat ImmunoPhenomic Platform Structure Fédérative de Recherche François Bonamy Centre National de Recherche Scientifique UMS3556, Nantes, F44093, France. ianegon@nantes.inserm.fr.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Anegon
authors.affiliation: CRCNA UMR S892 INSERM 6299 CNRS Université de Nantes; Université de Nantes Faculté des Sciences et Techniques, Nantes, F44093, France. xavier.saulquin@univ-nantes.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Saulquin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D002469:Cell Separation; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G
keywords: Cytofluorimetry; Human antibodies; Humanized rats; Tetramers; pMHC