SpyLigase-Catalyzed Modification of Antibodies

: Immunoconjugates are essential tools in diagnostics for the detection and quantification of proteins and in cell biology for the characterization of different cell populations as well as for tracking intracellular pathways. In recent years, antibody-drug conjugates (ADCs) have emerged as promising therapeutics to treat cancer and have moved into the focus of interest of the pharmaceutical industry. Here we describe a conjugation method for the generation of antibody conjugates that relies on the formation of a spontaneous isopeptide bond between two peptide tags referred to as SpyTag and KTag. This reaction is catalyzed by SpyLigase, an engineered cell surface protein obtained from Streptococcus pyogenes. We describe the preparation of SpyLigase by expression from E. coli cells, chemical solid-phase synthesis of the KTag peptide and its coupling to reporter molecules and cytotoxins as well as the transient expression from mammalian cells to produce Spy-tagged antibodies. Furthermore, we describe the purification and analytics of the formed conjugates.