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Designing Fcabs: well-expressed and stable high affinity antigen-binding Fc fragments

Wozniak-Knopp, Gordana and Stadlmayr, Gerhard and Perthold, Jan Walther and Stadlbauer, Katharina and Woisetschläger, Maximilian and Sun, Haijun and Rüker, Florian (2017) Designing Fcabs: well-expressed and stable high affinity antigen-binding Fc fragments. Protein engineering, design & selection : PEDS, 30 (9). pp. 657-671. ISSN 1741-0134

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 CH3 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 CH3 domain, while the overall structural integrity was retained. This allowed the Fcab to replace the Fc portion of a mAb, in which both the CH3 and Fab are capable of antigen engagement: a construct called mAb2 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.

Item Type: Article
Date Deposited: 14 Dec 2017 00:45
Last Modified: 14 Dec 2017 00:45
URI: https://oak.novartis.com/id/eprint/34915

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