1 research outputs found
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, 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