title: Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability.
abstract: The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer's Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability.
authors.affiliation: Center for Biotechnology &Interdisciplinary Studies, Isermann Dept. 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 Dept. of Chemical &Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Li
authors.affiliation: Center for Biotechnology &Interdisciplinary Studies, Isermann Dept. of Chemical &Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Shekhar
authors.initials: S
authors.lastname: Garde
authors.affiliation: Center for Biotechnology &Interdisciplinary Studies, Isermann Dept. of Chemical &Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Wilen
authors.affiliation: Center for Biotechnology &Interdisciplinary Studies, Isermann Dept. of Chemical &Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Tessier
mesh_terms: D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity / Q000502:physiology*; D000937:Antigen-Antibody Reactions; D022801:Complementarity Determining Regions / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D016296:Mutagenesis; D010446:Peptide Fragments / Q000276:immunology; D055550:Protein Stability; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D016229:Amyloid beta-Peptides; D000906:Antibodies; D022801:Complementarity Determining Regions; D010446:Peptide Fragments; D057127:Single-Chain Antibodies; C075222:amyloid beta-protein (1-42)