Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci

Abstract

Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self‐assembled, tumor‐targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor‐targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time‐deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24‐based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long‐term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

The Biological Potential Hidden in Inclusion Bodies

“…Many examples have been published during the last decade that prove that IBs are protein-based biomaterials with promising characteristics to be used for cell replacement therapies, for tissue engineering purposes, or even for cancer treatment [3][4][5][6][7][8][9][10][11]24], among other applications. However, their characteristics and their in vitro behavior have never been correlated with their in vivo performance compared with other standard formats.…”

Engineering Protein Venoms as Self‐Assembling CXCR4‐Targeted Cytotoxic Nanoparticles

“…[ 25 ] Such protein complexes, when formed by self‐assembling proteins, are able to release assembled nanoparticles to the media, for both local and systemic administration in different pathologies. [ 26,27 ] These released nanoparticles fully keep the targeting properties conferred by fused homing peptides displayed on such oligomers, as recently demonstrated. [ 26 ] In this particular format, that allows reaching high concentrations, toxins act already at 24 h of exposure ( Figure ), and melittin, at such high doses, is highly functional.…”

“…[ 26,27 ] These released nanoparticles fully keep the targeting properties conferred by fused homing peptides displayed on such oligomers, as recently demonstrated. [ 26 ] In this particular format, that allows reaching high concentrations, toxins act already at 24 h of exposure ( Figure ), and melittin, at such high doses, is highly functional. CLP71 is still, in this innovative format, the most cytotoxic agent among those tested (Figure 4).…”

Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

“…Therefore, this result has a direct application for biopharmaceutical and biotechnological applications through protein engineering. In fact, these protein nanoclusters have been described as a source of soluble active protein obtained upon incubation in non-denaturing conditions [14][15][16] and have also been administered as biocompatible depots for tumor targeting of therapeutic proteins [17][18][19][20][21]. Furthermore, protein aggregation seems to be a common mechanism described in most of the expression systems [47][48][49] that opens up the possibility of expanding this type of strategy to proteins that are di cult to produce in prokaryotes.…”

“…In bacteria, aggregates known as inclusion bodies (IB) are dynamic protein nanoclusters from which solubilized active protein conformers are released under physiological conditions [14][15][16]. In fact, recent experimental approaches have revealed the ability of active IBs to rescue enzymatic activities in cell cultures and to target cancer stem cells in cancer animal models [17][18][19][20][21]. Therefore, IBs are envisioned as depots of recombinant protein with the capacity to release the protein of interest from a complex and stable scaffold that protects the biological activity of the embed protein over time.…”