Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery

Abstract

Background: To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. Results: Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. Conclusion: We believe that this reengineered system is an important step forward to addressing efficient cellspecific delivery of proteins to the cytosol.

Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin

“…Cloning of most constructs used in this study has been described before [ 6 , 9 ]. DARPin cargoes were cloned into the SpeI/AgeI-restricted pQIq-LF N -cargo-avi-HA backbone for protein delivery.…”

“…The modular structure of anthrax toxin enabled us and others to generate engineered PA variants with altered cell specificity and the capability to translocate alternative LF cargo molecules [ 4 , 5 , 6 , 7 , 8 , 9 ]. Our group and others [ 6 , 10 , 11 , 12 , 13 ] showed that cytosolic translocation by pore-forming toxins depends on the ability of the molecule to fit through this channel.…”

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy

“…As a starting point, it is useful to consider that approaches that report cytosolic protein delivery in 2D systems in vitro often report values of cytosolic concentrations for the delivered protein that are comparatively high (mid- or high-nanomolar range) [ 18 , 24 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ], which is well over the average level of molecules of a specific protein in a cell, which is around 2000–8000 molecules/cell (~2–10 nM) [ 27 ]. Hence, addressing cytosolic targets in vitro is already possible for some applications.…”

Harnessing the Membrane Translocation Properties of AB Toxins for Therapeutic Applications

“…Similarly, Becker et al used designed ankyrin repeat proteins (DARPins) fused to a PA-CMG2-based construct to specifically target transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM) at the surface of cells. These engineered constructs were shown to target EpCAM-expressing cells with a high specificity and to deliver LF N -based constructs to the cytosol [ 58 ]. Overall, these engineered proteins show that both the A and B subunits of anthrax toxin have strong potential as a protein delivery system, and they open many new routes for investigating the development of therapeutics.…”

“…Targeting to non-native receptors using Affibodies, DARPins or receptors ligand for the cytosolic delivery of non-native cargos [ 55 , 56 , 57 , 58 ]…”