title: Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.
abstract: An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments.
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Julian
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Lee
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Tiller
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Lilia A
authors.initials: LA
authors.lastname: Rabia
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Evan K
authors.initials: EK
authors.lastname: Day
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Schick
authors.affiliation: Center for Biotechnology & Interdisciplinary Studies, Isermann Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA tessier@rpi.edu.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Tessier
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology*; D000915:Antibody Affinity; D019020:Directed Molecular Evolution / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000235:genetics; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D013205:Staphylococcal Protein A / Q000276:immunology
chemical_list: D016229:Amyloid beta-Peptides; D061905:Single-Domain Antibodies; D013205:Staphylococcal Protein A
keywords: CDR; VH; directed evolution; scFv; yeast surface display