title: Directed evolution of Her2/neu-binding IgG1-Fc for improved stability and resistance to aggregation by using yeast surface display.
abstract: An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.
authors.affiliation: Christian Doppler Laboratory for Antibody Engineering, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Traxlmayr
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Lobner
authors.forename: Bernhard
authors.initials: B
authors.lastname: Antes
authors.forename: Manuela
authors.initials: M
authors.lastname: Kainer
authors.forename: Susanne
authors.initials: S
authors.lastname: Wiederkum
authors.forename: Christoph
authors.initials: C
authors.lastname: Hasenhindl
authors.forename: Gerhard
authors.initials: G
authors.lastname: Stadlmayr
authors.forename: Florian
authors.initials: F
authors.lastname: Rüker
authors.forename: Max
authors.initials: M
authors.lastname: Woisetschläger
authors.forename: Kevin
authors.initials: K
authors.lastname: Moulder
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000737:chemistry; D019020:Directed Molecular Evolution; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D010843:Pichia; D011485:Protein Binding; D011487:Protein Conformation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human