Highly Potent, Anthracycline-based Antibody-Drug Conjugates Generated by Enzymatic, Site-specific Conjugation.

Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios.

Novel prodrug-like fusion toxin with protease-sensitive bioorthogonal PEGylation for tumor targeting.

Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients.

Increasing the antitumor effect of an EpCAM-targeting fusion toxin by facile click PEGylation.

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I-truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E.

Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy.

Introduction: The epithelial cell adhesion molecule (EpCAM) is abundantly expressed in epithelial tumors, on cancer stem cells and circulating tumor cells. Together with its role in oncogenic signaling, this has sparked interest in its potential for tumor targeting with antibodies and drug conjugates for safe and effective cancer therapy. Recent advances in protein engineering, linker design and drug formulations have provided a multitude of EpCAM-targeting anticancer agents, several of them with good perspectives for clinical development.

NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent.

The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al.

Designed ankyrin repeat proteins (DARPins) from research to therapy.

Designed ankyrin repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats.

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency.

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors.

A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity.

Purpose: Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)-specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A.

Experimental Design: DARPin Ec4 was genetically fused to a truncated form of Pseudomonas aeruginosa exotoxin A (ETA″) and expressed in Escherichia coli.