title: High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.
abstract: Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.
authors.affiliation: a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
authors.forename: Valencio
authors.initials: V
authors.lastname: Salema
authors.affiliation: a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Mañas
authors.affiliation: a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
authors.forename: Lidia
authors.initials: L
authors.lastname: Cerdán
authors.affiliation: a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Piñero-Lambea
authors.affiliation: a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
authors.forename: Elvira
authors.initials: E
authors.lastname: Marín
authors.affiliation: b Cell Biology, Department of Biology, Science Faculty, Utrecht University , Utrecht , The Netherlands.
authors.forename: Rob C
authors.initials: RC
authors.lastname: Roovers
authors.affiliation: b Cell Biology, Department of Biology, Science Faculty, Utrecht University , Utrecht , The Netherlands.
authors.forename: Paul M P
authors.initials: PM
authors.lastname: Van Bergen En Henegouwen
authors.affiliation: a Department of Microbial Biotechnology , Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus UAM Cantoblanco , Madrid , Spain.
authors.forename: Luis Ángel
authors.initials: LÁ
authors.lastname: Fernández
mesh_terms: D000818:Animals; D061505:Cell Surface Display Techniques / Q000379:methods*; D066246:ErbB Receptors / Q000276:immunology*; D004926:Escherichia coli; D006801:Humans; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000302:isolation & purification*
chemical_list: D019151:Peptide Library; D061905:Single-Domain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Antibody display; E. coli; EGFR; bacterial display; cell surface antigen; nanobodies; on-cell selection; single-domain antibodies; tumor-associated antigen