Intestinal Transcytosis of a Protein Cargo and Nanoparticles Mediated by a Non-Toxic Form of Pseudomonas aeruginosa Exotoxin A

The low permeability of nanoparticles (NPs) across the intestinal epithelium remains a major challenge for their application of delivering macromolecular therapeutic agents via the oral route. Previous studies have demonstrated the epithelial transcytosis capacity of a non-toxic version of Pseudomonas aeruginosa exotoxin A (ntPE). Here, we show that ntPE can be used to deliver the protein cargo green fluorescent protein (GFP) or human growth hormone (hGH), as genetic fusions, across intact rat jejunum in a model where the material is administered by direct intra-luminal injection (ILI) in vivo in a transcytosis process that required less than 15 min. Next, ntPE chemically coupled onto biodegradable alginate/chitosan condensate nanoparticles (AC NPs-ntPE) were shown to transport similarly to ntPE-GFP and ntPE-hGH across rat jejunum. Finally, AC NPs-ntPE loaded with GFP as a model cargo were demonstrated to undergo a similar transcytosis process that resulted in GFP being colocalized with CD11c+ cells in the lamina propria after 30 min. Control NP preparations, not decorated with ntPE, were not observed within polarized epithelial cells or within the cells of the lamina propria. These studies demonstrate the capacity of ntPE to facilitate the transcytosis of a covalently associated protein cargo as well as a biodegradable NP that can undergo transcytosis across the intestinal epithelium to deliver a noncovalently associated protein cargo. In sum, these studies support the potential applications of ntPE to facilitate the oral delivery of macromolecular therapeutics under conditions of covalent or non-covalent association.

Dual Targeting of Cancer Cells with DARPin-Based Toxins for Overcoming Tumor Escape

We report here a combined anti-cancer therapy directed toward HER2 and EpCAM, common tumor-associated antigens of breast cancer cells. The combined therapeutic effect is achieved owing to two highly toxic proteins—a low immunogenic variant of Pseudomonas aeruginosa exotoxin A and ribonuclease Barnase from Bacillus amyloliquefaciens. The delivery of toxins to cancer cells was carried out by targeting designed ankyrin repeat proteins (DARPins). We have shown that both target agents efficiently accumulate in the tumor. Simultaneous treatment of breast carcinoma-bearing mice with anti-EpCAM fusion toxin based on LoPE and HER2-specific liposomes loaded with Barnase leads to concurrent elimination of primary tumor and metastases. Monotherapy with anti-HER2- or anti-EpCAM-toxins did not produce a comparable effect on metastases. The proposed approach can be considered as a promising strategy for significant improvement of cancer therapy.

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A–both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.

HER2-Specific Targeted Toxin DARPin-LoPE: Immunogenicity and Antitumor Effect on Intraperitoneal Ovarian Cancer Xenograft Model

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.