title: Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach.
abstract: In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive Phi angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ewert
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics; D020650:Combinatorial Chemistry Techniques; D016384:Consensus Sequence / Q000235:genetics; D006736:Horses; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library