Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition

Bioconjug Chem. 2018 May 16;29(5):1605-1613. doi: 10.1021/acs.bioconjchem.8b00114. Epub 2018 May 3.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Surface Display Techniques
  • Cycloaddition Reaction / methods*
  • Heterocyclic Compounds, 1-Ring / chemical synthesis
  • Heterocyclic Compounds, 1-Ring / chemistry*
  • Immunoconjugates / chemistry*
  • Single-Chain Antibodies / chemistry*
  • Styrene / chemical synthesis
  • Styrene / chemistry*
  • Sulfhydryl Compounds / chemical synthesis
  • Sulfhydryl Compounds / chemistry


  • Heterocyclic Compounds, 1-Ring
  • Immunoconjugates
  • Single-Chain Antibodies
  • Sulfhydryl Compounds
  • Styrene