The synthesis began with an OSu-activation of BCN-CH2OH, after which it was exposed to PEG3bisamine, resulting in the formation of the desired bis-BCN linker L1 (Scheme 2B) (see ESI for detailed synthesis schemes). After deglycosylation of Asn300 of Tras using the enzyme peptide-N-glycosidase F (PNGase F) (Scheme 2C), an exposed proximal tyrosine residue in the Fc region becomes available for SPOCQ conjugation. Specifically, mushroom tyrosinase was applied to oxidize a tyrosine into a reactive o-quinone, which, in the presence of a strained alkyne, i.e., L1 at 4 °C for 16 h, led to an inverse electron-demand Diels-Alder cycloaddition. Using L1, each heavy chain was equipped with a free BCN handle. Having successfully attached the click handles, tras-BCN2 was treated with one of the different azide-functionalized payloads (1-4) at 4 °C for 16 h, affording the various target ADCs (5-8) at a drug-to-antibody ratio (DAR) of 2. RP-HPLC-MS and SDS-PAGE confirmed the formation of the desired products (Scheme 2C, Figures ). Using the same method, a cetuximab (Cetux) conjugate of Dp44mT-O 1 was synthesized for use as a negative control (Figure ). The ADCs correspondingly displayed trastuzumab-dependent baseline activity at low concentrations in SK-BR-3 cells, which was absent in MCF-7 cells (Figures and). This supports the notion that the antibody remained conjugated to the payload for the incubation time.