Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.