title: A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs.
abstract: Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs.
authors.affiliation: a Miltenyi Biotec GmbH, Friedrich-Ebert-Straße , Bergisch Gladbach , Germany.
authors.forename: Sarah-Jane
authors.initials: SJ
authors.lastname: Kellmann
authors.affiliation: b Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics , Braunschweig , Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.affiliation: a Miltenyi Biotec GmbH, Friedrich-Ebert-Straße , Bergisch Gladbach , Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Thie
mesh_terms: D000494:Allosteric Regulation; D000915:Antibody Affinity / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology; D002147:Calmodulin / Q000737:chemistry*; D002846:Chromatography, Affinity; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D015202:Protein Engineering / Q000379:methods*; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology
chemical_list: D002147:Calmodulin; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies
keywords: Affinity modulation; antibody engineering; antibody fragments; calmodulin/calmodulin-binding peptide interaction; circular permutation; conformational change; scFv linker