Pseudomonas Exotoxin-Based Immunotoxins: Over Three Decades of Efforts on Targeting Cancer Cells With the Toxin

Cancer is one of the prominent causes of death worldwide. Despite the existence of various modalities for cancer treatment, many types of cancer remain uncured or develop resistance to therapeutic strategies. Furthermore, almost all chemotherapeutics cause a range of side effects because they affect normal cells in addition to malignant cells. Therefore, the development of novel therapeutic agents that are targeted specifically toward cancer cells is indispensable. Immunotoxins (ITs) are a class of tumor cell-targeted fusion proteins consisting of both a targeting moiety and a toxic moiety. The targeting moiety is usually an antibody/antibody fragment or a ligand of the immune system that can bind an antigen or receptor that is only expressed or overexpressed by cancer cells but not normal cells. The toxic moiety is usually a protein toxin (or derivative) of animal, plant, insect, or bacterial origin. To date, three ITs have gained Food and Drug Administration (FDA) approval for human use, including denileukin diftitox (FDA approval: 1999), tagraxofusp (FDA approval: 2018), and moxetumomab pasudotox (FDA approval: 2018). All of these ITs take advantage of bacterial protein toxins. The toxic moiety of the first two ITs is a truncated form of diphtheria toxin, and the third is a derivative of Pseudomonas exotoxin (PE). There is a growing list of ITs using PE, or its derivatives, being evaluated preclinically or clinically. Here, we will review these ITs to highlight the advances in PE-based anticancer strategies, as well as review the targeting moieties that are used to reduce the non-specific destruction of non-cancerous cells. Although we tried to be as comprehensive as possible, we have limited our review to those ITs that have proceeded to clinical trials and are still under active clinical evaluation.

Pseudomonas Exotoxin A Based Toxins Targeting Epidermal Growth Factor Receptor for the Treatment of Prostate Cancer

The epidermal growth factor receptor (EGFR) was found to be a valuable target on prostate cancer (PCa) cells. However, EGFR inhibitors mostly failed in clinical studies with patients suffering from PCa. We therefore tested the targeted toxins EGF-PE40 and EGF-PE24mut consisting of the natural ligand EGF as binding domain and PE40, the natural toxin domain of Pseudomonas Exotoxin A, or PE24mut, the de-immunized variant thereof, as toxin domains. Both targeted toxins were expressed in the periplasm of E.coli and evoked an inhibition of protein biosynthesis in EGFR-expressing PCa cells. Concentration- and time-dependent killing of PCa cells was found with IC50 values after 48 and 72 h in the low nanomolar or picomolar range based on the induction of apoptosis. EGF-PE24mut was found to be about 11- to 120-fold less toxic than EGF-PE40. Both targeted toxins were more than 600 to 140,000-fold more cytotoxic than the EGFR inhibitor erlotinib. Due to their high and specific cytotoxicity, the EGF-based targeted toxins EGF-PE40 and EGF-PE24mut represent promising candidates for the future treatment of PCa.

Targeting c-Myc with a novel Peptide Nuclear Delivery Device

Biologics such as peptides and antibodies are a well-established class of therapeutics. However, their intracellular delivery remains problematic. In particular, methods to efficiently inhibit intra-nuclear targets are lacking. We previously described that Pseudomonas Exotoxin A reaches the nucleoplasm via the endosomes-to-nucleus trafficking pathway. Here, we show that a non-toxic truncated form of PE can be coupled to peptides and efficiently reach the nucleoplasm. It can be used as a Peptide Nuclear Delivery Device (PNDD) to deliver polypeptidic cargos as large as Glutathione- S-transferase (GST) to the nucleus. PNDD1 is a fusion of PNDD to the c-myc inhibitor peptide H1. PNDD1 is able to inhibit c-Myc dependent transcription at nanomolar concentration. In contrast, H1 fused to various cell-penetrating peptides are active only in the micromolar range. PNDD1 attenuates cell proliferation and induces cell death in various tumor cell lines. In particular, several patient-derived Diffuse Large B-Cell Lymphomas cell lines die after exposure to PNDD1, while normal B-cells survive. Altogether, our data indicate that PNDD is a powerful tool to bring active cargo to the nucleus and PNDD1 could be the basis of a new therapy against lymphoma.