Mutational and biophysical robustness in a prestabilized monobody

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dc.contributor.authorChandler, Peter G
dc.contributor.authorTan, Li Lynn
dc.contributor.authorPorebski, Benjamin T
dc.contributor.authorGreen, James S
dc.contributor.authorRiley, Blake
dc.contributor.authorBroendum, Sebastian
dc.contributor.authorHoke, David E
dc.contributor.authorFalconer, Robert J
dc.contributor.authorMunro, Trent P
dc.contributor.authorBuckle, Malcolm
dc.contributor.authorJackson, Colin
dc.contributor.authorBuckle, Ashley
dc.date.accessioned2024-03-04T23:10:24Z
dc.date.available2024-03-04T23:10:24Z
dc.date.issued2021
dc.date.updated2022-10-16T07:26:18Z
dc.description.abstractThe fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability–function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.en_AU
dc.format.mimetypeapplication/pdfen_AU
dc.identifier.issn0021-9258en_AU
dc.identifier.urihttp://hdl.handle.net/1885/315698
dc.language.isoen_AUen_AU
dc.provenanceThis is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).en_AU
dc.publisherAmerican Society for Biochemistry and Molecular Biology Incen_AU
dc.rights© 2021 The Author(s). Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.en_AU
dc.rights.licenseCreative Commons Attribution-NonCommercial-NoDerivs Licenseen_AU
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/en_AU
dc.sourceJournal of Biological Chemistryen_AU
dc.titleMutational and biophysical robustness in a prestabilized monobodyen_AU
dc.typeJournal articleen_AU
dcterms.accessRightsOpen Accessen_AU
local.bibliographicCitation.lastpage10en_AU
local.bibliographicCitation.startpage1en_AU
local.contributor.affiliationChandler, Peter G, Monash Universityen_AU
local.contributor.affiliationTan, Li Lynn, College of Science, ANUen_AU
local.contributor.affiliationPorebski, Benjamin T, Medical Research Council Laboratory of Molecular Biologyen_AU
local.contributor.affiliationGreen, James S, Monash Universityen_AU
local.contributor.affiliationRiley, Blake, Monash Universityen_AU
local.contributor.affiliationBroendum, Sebastian, Monash Universityen_AU
local.contributor.affiliationHoke, David E, Monash Universityen_AU
local.contributor.affiliationFalconer, Robert J, University of Adelaideen_AU
local.contributor.affiliationMunro, Trent P, University of Queenslanden_AU
local.contributor.affiliationBuckle, Malcolm, Universite Paris-Saclayen_AU
local.contributor.affiliationJackson, Colin, College of Science, ANUen_AU
local.contributor.affiliationBuckle, Ashley, Monash Universityen_AU
local.contributor.authoremailu4040768@anu.edu.auen_AU
local.contributor.authoruidTan, Li Lynn, u1039977en_AU
local.contributor.authoruidJackson, Colin, u4040768en_AU
local.description.notesImported from ARIESen_AU
local.identifier.absfor340402 - Biomolecular modelling and designen_AU
local.identifier.ariespublicationa383154xPUB19114en_AU
local.identifier.citationvolume296en_AU
local.identifier.doi10.1016/j.jbc.2021.100447en_AU
local.identifier.scopusID2-s2.0-85103618605
local.identifier.thomsonIDWOS:000672866400421
local.identifier.uidSubmittedBya383154en_AU
local.publisher.urlhttps://www.elsevier.com/en-auen_AU
local.type.statusPublished Versionen_AU

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