Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.