title: Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.
abstract: In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK MedImmune Ltd, Milstein Building, Granta Park, Cambridge CB1 6GH, UK.
authors.forename: Gillian
authors.initials: G
authors.lastname: Houlihan
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Pietro
authors.initials: P
authors.lastname: Gatti-Lafranconi
authors.affiliation: MedImmune Ltd, Milstein Building, Granta Park, Cambridge CB1 6GH, UK.
authors.forename: David
authors.initials: D
authors.lastname: Lowe
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK fh111@cam.ac.uk.
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D017089:Ankyrin Repeat / Q000235:genetics; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics*; D019020:Directed Molecular Evolution; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic
chemical_list: D000906:Antibodies; D004247:DNA; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human
keywords: DARPin; SNAP display; alternative scaffold; antibody; directed evolution; in vitro compartmentalisation; trade-off