Orthogonal Assembly of a Designed Ankyrin Repeat Protein–Cytotoxin Conjugate with a Clickable Serum Albumin Module for Half-Life Extension

Abstract

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.

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

“…DARPins contain no cysteines and most importantly only a single N-terminal methionine at position 1 (ATG) in addition to another Met in the backbone, which can be conveniently replaced by leucine without loss of biophysical performance (8,9,10). The anti-EpCAM DARPin Ec1 contains even only the methionine at position 1, which we replaced by Aha to enable bioorthogonal strain-promoted cycloaddition of a DBCO-activated PEG 20kDa (9,10).…”

“…The anti-EpCAM DARPin Ec1 contains even only the methionine at position 1, which we replaced by Aha to enable bioorthogonal strain-promoted cycloaddition of a DBCO-activated PEG 20kDa (9,10). We demonstrate that this strategy is perfectly suited to modify DARPin-ETA 00 fusion toxins beyond the classical way of protein engineering by genetic alterations.…”

“…The data were analyzed using Excel (Microsoft) and Prism (v 5.04, GraphPad Software Inc.). Dose-response curves with variable slope were fitted to all data points (10).…”

“…Recently, we reported the use of bioorthogonal click chemistry as a further strategy of DARPin functionalization (8,9). The DARPin scaffold was found to well tolerate the replacement of its N-terminal methionine by a nonnatural clickable L-azidohomoalanine (Aha; and, if necessary, the replacement of any other Met residues), resulting in a fully functional binder, which can be sitespecifically conjugated with polyethylene glycol (PEG) or other conjugation partners in a one-step reaction (8,9,10).…”

Enzymatic Prenylation and Oxime Ligation for the Synthesis of Stable and Homogeneous Protein–Drug Conjugates for Targeted Therapy

“…[4] Thehomogeneity and stability of protein-drug conjugates (PDCs) have ap rofound effect on their pharmacological properties,s uch as the plasma half-life,t herapeutic efficacy, and off-target toxicity. [5] However,c oupling reactions of amines with NHS-ester or thiols with maleimide,w hich have been widely employed for the synthesis of drug conjugates,h ave mostly resulted in heterogeneous products with different stoichiometries,l eading to unwanted toxicity and increased clearance.F urthermore,t hiol-maleimide linkages are prone to hydrolysis and undergo ar eversible exchange reaction with serum protein thiols or free cysteine through ar etro-Michael reaction, causing premature drug release and inefficient drug delivery to the target site.…”