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.