Designing Fcabs: well-expressed and stable high affinity antigen-binding Fc fragments. Issue 9 (11th September 2017)

• Record Type: Journal Article
• Authors: Wozniak-Knopp, Gordana; Stadlmayr, Gerhard; Perthold, Jan Walther; Stadlbauer, Katharina; Woisetschläger, Maximilian; Sun, Haijun; Rüker, Florian
• Is Part Of: Protein engineering, design & selection. Volume 30:Issue 9(2017)
• Page Start: 657
• Page End: 671
• Publication Date: 2017-09-11
• Subjects: directed evolution -- Fcab -- molecular dynamics -- VEGF -- yeast display; Protein engineering -- Periodicals; 660.63
• DOI: 10.1093/protein/gzx042
• Languages: English
• ISSNs: 1741-0126

Abstract

Abstract: Fc fragment with antigen-binding (Fcab) is a novel construct which can be selected to recognize specifically a wide variety of target proteins. We describe the selection and affinity maturation of Fcab clones targeting VEGF, an important pro-angiogenesis factor. To investigate the extent of engineering permissible to Fcabs we applied targeted mutagenesis to all three C-terminal loop structures and the C-terminus of the CH 3 domain to isolate high-affinity binders by directed evolution and yeast display. The matured clone, CT6, binds to VEGF with low nanomolar affinity and inhibits VEGF-stimulated proliferation of human umbilical vein endothelial cells in vitro . Molecular dynamics simulations were performed to address flexibility of the molecular structure of CT6 and to approximate a structural ensemble in aqueous solution. Significantly higher RMSF levels of CT6 in comparison to wild-type Fc were limited to the elongated CD-loop in the CH 3 domain, while the overall structural integrity was retained. This allowed the Fcab to replace the Fc portion of a mAb, in which both the CH 3 and Fab are capable of antigen engagement: a construct called mAb 2 was assembled with CT6 and the Fab of bevacizumab. This bispecific molecule showed more potent antagonistic activity than bevacizumab in vitro . Further evaluation for the potential of the CT6 Fcab in targeted therapy is warranted due to the possibility of being combined with other therapeutically meaningful targets.