The generation of
drug conjugates for safe and effective
tumor targeting requires
binding proteins tolerant to functionalization by rational engineering. Here, we show that
Designed Ankyrin Repeat Proteins (
DARPins), a novel class of
binding proteins not derived from
antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for
tumor targeting with tailored properties.
DARPin Ec1, which targets the
Epithelial Cell Adhesion Molecule (
EpCAM), was genetically modified with a C-terminal
cysteine for conjugation of the small molecule
cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural
amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse
serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable
drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including
EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the
DARPin increased receptor-specific uptake of the
drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice,
albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the
DARPin format for facile modular assembly of
drug conjugates with improved pharmacokinetic performance for
tumor targeting.