Targeted drug conjugates which deliver highly cytotoxic payloads to malignant cells are a promising concept for cancer treatment. They enable the expansion of the tumor-targeting approach to antigens for which a simple occupation does not translate into an anti-tumor effect. One such antigen is the Epithelial Cell Adhesion Molecule (EpCAM), which is overexpressed on various carcinomas and a marker for cancer stem cells and disseminated tumor cells [1].
We have developed novel strategies to use Designed Ankyrin Repeat Proteins (DARPins) as innovative binding proteins which allow new chemical and engineering approaches to create EpCAM-targeted toxin moieties. DARPins are very stable and can be easily produced in bacteria in high yields [2].
The DARPin scaffold allows the site-specific incorporation of the non-natural amino acid azidohomoalanine (Aha) in E. coli at any desired position. Hence, the simultaneous, site-specific and stoichiometrically defined bioconjugation of a catalog of effector or half-life extension molecules using click chemistry reactions has become possible.
The off-target toxicity and short half-life of tumor targeting immunotoxins composed of Pseudomonas Exotoxin A (ETA) have largely limited the success of clinical trials in the past. Here, we report the engineering and preclinical testing of a novel functionally improved DARPin-ETA fusion protein with prodrug-like properties. Aha was introduced at defined positions in the fusion toxin and polyethylene glycol was coupled integrating a protease-cleavable linker. This inhibits the toxins activity and can be fully reverted by cleaving off the bulky polymer with the linker-specific protease [3].
Promising in vitro and in vivo data of this project, showing greatly extended therapeutic windows and significantly increased tolerability and circulation half-life will be presented, as well as work with other protease-cleavable half-life extension molecules.