Bioorthogonal reaction of o-quinone with furan-2(3H)-one for site-selective tyrosine conjugation and construction of bispecific antibody complexes

Abstract

Bioorthogonal reactions enable the chemical conjugation of functional moieties to native proteins and empower the development of new diagnostic tools and therapeutics. Through site-selective reactions, therapeutic molecules can be conjugated with antibodies in a stoichiometry- and site-controlled manner. Here, a one-pot chemoenzymatic reaction is reported that preferentially modifies a terminal tyrosine of recombinant proteins, or tyrosine 296 in the Fc domain of selected human antibodies. This reaction combines tyrosinase-catalyzed oxidation of phenol to o-quinone, the bioorthogonal addition reaction of o-quinone with an azide-containing furan-2(3H)-one (FuA) moiety, and the subsequent azide click reactions. To this surprise, experimental evidence indicates that the o-quinone−FuA reaction proceeds through nucleophilic addition instead of the cycloaddition pathway. This reaction enables site-selective modification of therapeutic human antibodies, including atezolizumab, trastuzumab, daratumumab, and cetuximab. Monofunctionalized antibody conjugates and DNA-templated bispecific antibody complexes (DNA-bsAbC) are thus constructed in a modular way. DNA-bsAbC acts as a bispecific engager to mediate the interaction between immune cells and cancer cells, resulting in antibody-dependent cellular cytotoxicity (ADCC) toward cancer cells. Taken together, here a bioorthogonal reaction is reported for site-selective tyrosine conjugation in recombinant proteins and human antibodies and showcase its application in constructing antibody conjugates for potential applications in immunotherapies.