title: Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies.
abstract: Despite their favorable pharmacokinetic properties, single-chain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 x 10(-9) to 1.5 x 10(-11) m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 x 10(-10) to <1.8 x 10(-12) m. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86 degrees C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.
authors.affiliation: ESBATech, ALCON Biomedical Research Unit, CH-8952 Schlieren, Switzerland.
authors.forename: Leo
authors.initials: L
authors.lastname: Borras
authors.forename: Tea
authors.initials: T
authors.lastname: Gunde
authors.forename: Julia
authors.initials: J
authors.lastname: Tietz
authors.forename: Ulrich
authors.initials: U
authors.lastname: Bauer
authors.forename: Valérie
authors.initials: V
authors.lastname: Hulmann-Cottier
authors.forename: John P A
authors.initials: JP
authors.lastname: Grimshaw
authors.forename: David M
authors.initials: DM
authors.lastname: Urech
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D022801:Complementarity Determining Regions / Q000737:chemistry; D042783:Endothelial Cells / Q000166:cytology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011817:Rabbits; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A