title: REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.
abstract: We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.
authors.affiliation: Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Darmstadt, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Rhiel
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Krah
authors.affiliation: Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Darmstadt, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Günther
authors.affiliation: Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Darmstadt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
authors.affiliation: Protein Engineering and Antibody Technologies, Merck Serono, Merck KGaA, Darmstadt, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Hock
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D061505:Cell Surface Display Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D057166:High-Throughput Screening Assays / Q000379:methods; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D019809:Streptavidin
chemical_list: D000906:Antibodies; D017931:DNA Primers; D007074:Immunoglobulin G; D019809:Streptavidin