title: Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries.
abstract: We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.
authors.affiliation: Dyax s a, Boulevard du Rectorat 27B, Sart Tilman, 4000 Liège 1, Belgium.
authors.forename: Twan
authors.initials: T
authors.lastname: van den Beucken
authors.forename: Henk
authors.initials: H
authors.lastname: Pieters
authors.forename: Mieke
authors.initials: M
authors.lastname: Steukers
authors.forename: Marcel
authors.initials: M
authors.lastname: van der Vaart
authors.forename: Robert Charles
authors.initials: RC
authors.lastname: Ladner
authors.forename: Hennie R
authors.initials: HR
authors.lastname: Hoogenboom
authors.forename: Simon E
authors.initials: SE
authors.lastname: Hufton
mesh_terms: D000915:Antibody Affinity; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification*; D019809:Streptavidin / Q000378:metabolism
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D019809:Streptavidin