title: Biophysical properties of human antibody variable domains.
abstract: There are great demands on the stability, expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V(H)) and light (V(L)) germline family consensus domains and then a systematic series of V(H)-V(L) combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V(H) and seven V(L) domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL). The isolated V(H) and V(L) domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V(lambda) domains show high levels of stability, even though V(lambda) domains are surprisingly unstable by themselves. This is due to a strong interaction with the V(H) domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstr 190, CH-8057 Zürich, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ewert
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Annemarie
authors.initials: A
authors.lastname: Honegger
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D022801:Complementarity Determining Regions; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019151:Peptide Library; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library; D019791:Guanidine