Site-specific antibody conjugations using bacterial transglutaminase and the Diels-Alder cycloaddition reaction

Antibody-drug conjugates (ADCs) have emerged as a formidable class of targeted biologics. Whereas all 11 Food and Drug Adminis-tration-approved ADCs are based on random conjugation, accumu-lating evidence suggests a potential for improved therapeutic index with site-specific ADCs. Here, we present a two-step sequential conjugation approach that marries the site specificity of the bacte-rial transglutaminase (TG) reaction with the robustness of Diels-Alder (DA) chemistry to generate stable antibody conjugates with favorable physiochemical properties. Further, we identify diene-based linkers with different reaction kinetics for DA coupling, and we use these linker derivatives to conjugate antibodies to two distinct small molecules in tandem. In addition, we show strong potency of antibody conjugates, prepared via TG-DA chemistry, in both cell-based assays and a mouse model. Overall our conjugation approach is site specific, versatile, and can potentially be used to generate different classes of antibody conjugates for multiple therapeutic areas.