Site-Specific Antibody Functionalization Using Tetrazine–Styrene Cycloaddition

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,...

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Bibliographic Details
Published inBioconjugate chemistry Vol. 29; no. 5; pp. 1605 - 1613
Main Authors Umlauf, Benjamin J, Mix, Kalie A, Grosskopf, Vanessa A, Raines, Ronald T, Shusta, Eric V
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 16.05.2018
Subjects
Biological products
Cell Surface Display Techniques
Chemical bonds
Chemical reactions
Cycloaddition
Cycloaddition Reaction - methods
Heterocyclic Compounds, 1-Ring - chemical synthesis
Heterocyclic Compounds, 1-Ring - chemistry
Immunoconjugates - chemistry
Immunoglobulins
Proteins
Single-Chain Antibodies - chemistry
Styrene
Styrene - chemical synthesis
Styrene - chemistry
Styrenes
Sulfhydryl Compounds - chemical synthesis
Sulfhydryl Compounds - chemistry
Thiols
Yeast
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Summary: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.
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Present Address: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
ISSN:1043-1802
1520-4812
DOI:10.1021/acs.bioconjchem.8b00114