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Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition

Author(s)
Mix, Kalie; Raines, Ronald T
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Abstract
Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.
Date issued
2018-05
URI
https://hdl.handle.net/1721.1/125600
Department
Massachusetts Institute of Technology. Department of Chemistry
Journal
Bioconjugate chemistry
Publisher
American Chemical Society (ACS)
Citation
Umlauf, Benjamin J. et al. “Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition.” Bioconjugate chemistry 29 (2018): 1605-1613 © 2018 The Author(s)
Version: Author's final manuscript
ISSN
1043-1802

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