title: A robust pipeline for rapid production of versatile nanobody repertoires.
abstract: Nanobodies are single-domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show promise as high-affinity reagents for research, diagnostics and therapeutics owing to their high specificity, small size (∼15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Our approach generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the subnanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultrahigh-affinity dimeric nanobodies with Kd values as low as ∼30 pM. The approach presented here is well suited for the routine production of high-affinity capture reagents for various biomedical applications.
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fridy
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA.
authors.forename: Yinyin
authors.initials: Y
authors.lastname: Li
authors.affiliation: Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Keegan
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Thompson
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Ilona
authors.initials: I
authors.lastname: Nudelman
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
authors.forename: Johannes F
authors.initials: JF
authors.lastname: Scheid
authors.affiliation: 1] Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada. [2] Département de Biochimie et Médecine Moléculaire, Faculté de Médecine, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Marlene
authors.initials: M
authors.lastname: Oeffinger
authors.affiliation: 1] Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA. [2] Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA.
authors.forename: Michel C
authors.initials: MC
authors.lastname: Nussenzweig
authors.affiliation: Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA.
authors.forename: David
authors.initials: D
authors.lastname: Fenyö
authors.affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
mesh_terms: D000818:Animals; D002161:Camelids, New World; D000939:Epitopes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000276:immunology* / Q000378:metabolism; D007114:Immunization; D008164:Luminescent Proteins / Q000276:immunology* / Q000378:metabolism; D008214:Lymphocytes / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000097573:Red Fluorescent Protein
chemical_list: D000939:Epitopes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D000097573:Red Fluorescent Protein