A general approach to site-specific antibody drug conjugates

Significance Here we demonstrate the ability to genetically incorporate nonnative amino acids into proteins in mammalian cells using both transient and stable platform expression systems that provide yields and fidelities compatible with commercial applications. To illustrate the utility of this methodology we have generated chemically homogeneous antibody drug conjugates (NDCs) with precise control over the site and stoichiometry of drug conjugation. In rodent xenograft models these NDCs display improved properties, including half-life, efficacy and safety, relative to conventional heterogeneous ADCs. These advances allow the generation of therapeutic antibody drug conjugates with medicinal chemistry like control over structure, which should greatly facilitate the optimization of their pharmacological activities. Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low–antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.