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Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobody SS , a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. Monobody SS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobody SS -based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobody SS -PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ER SS /28-PE25, with a K D of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ER SS /28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Panton-Valentine leukocidin (PVL) of Staphylococcus aureus is associated with necrotizing infections. After binding to complement 5a receptor (C5aR/CD88) and CD45 it causes cytolysis in polymorphonuclear neutrophils (PMNs) as well as inflammasome activation in monocytes. The objective of this study was to test if (ant)agonists of C5aR and CD45 can attenuate the effect of PVL on PMNs and monocytes. We tested the effect of various concentrations of six C5aR (ant)agonists (avacopan, BM213, DF2593A, JPE-1375, PMX205 and W-54011) and one CD45 antagonist (NQ301) to attenuate the cytotoxic effect of PVL on human PMNs and monocytes in vitro. Shifts in the half-maximal effective concentration (EC 50 ) of PVL to achieve a cytotoxic effect on PMNs and modulation of inflammatory cytokine response from monocytes were determined by flow cytometry and IL-1β detection. Pre-treatment of PMNs with avacopan, PMX205 and W-54,011 resulted in 3.6- to 4.3-fold shifts in the EC 50 for PVL and were able to suppress IL-1β secretion by human monocytes in the presence of PVL. BM213, DF2593A and NQ301 were unable to change the susceptibility of PMNs towards PVL or reduce inflammasome activation in monocytes. Avacopan, PMX205 and W-54,011 showed protection against PVL-induced cytotoxicity and suppressed IL-1β secretion by monocytes. Clinical studies are needed to prove whether these substances can be used therapeutically as repurposed drugs., (© 2024. The Author(s).)

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers., (© 2024 Federation of American Societies for Experimental Biology.)

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli , achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC 50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP., (© 2023. The Author(s).)

Basophils are known to produce a large amount of IL-4 in response to stimuli and play a role in the initiation and propagation of type 2 inflammations. S. aureus secretes a series of pore-forming toxins: α-hemolysin, γ-hemolysins, and leukocidins. In this study, we examined the effects of α-hemolysin, γ-hemolysins (HlgAB and HlgCB), and leukocidins (LukAB, LukED, and Panton-Valentine leukocidin) on the function of basophils. All pore-forming toxins except for Panton-Valentine leukocidin bound to murine bone marrow-derived basophils (BMBs). HlgAB and LukED but not other toxins evoked the leakage of lactate dehydrogenase from BMBs at the concentration of 30 μg/ml γ-hemolysins, HlgAB and HlgCB, induced the secretion of IL-4 in BMBs at concentrations above 3.3 μg/ml. LukAB did not induce, and Hla and LukED induced only a small amount of IL-4. HlgBΔstem, the 5 amino acids deletion mutant of HlgB in the stem region, diminished IL-4 secretion by HlgAB and HlgCB in BMBs. These results suggest that the cell damage and the induction of IL-4 in basophils by HlgAB require pore formation. The induction of IL-4 by γ-hemolysins was also observed in fleshly isolated murine basophils. These results demonstrate a novel function of γ-hemolysins, the induction of IL-4 in basophils, in an IgE-independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Recently, cellulose nanocrystals (CNs) have attracted wide attention owing to their superior properties compared to their bulk materials. For example, they represent an outstanding model for fabricating green metallic/metal oxide nanoparticles (NPs). In this study, two CNs (carboxylated CNs and sulfated CNs) extracted from agro-wastes of palm sheath fibers were used as templates for the facile and green synthesis of ZnO NPs by employing the sono-co-precipitation method. The obtained nanomaterials were characterized using TEM, EDX, UV-visible, DLS, FT-IR, and XRD analysis. As a result, the size and concentration of synthesized ZnO NPs were inversely proportional to one another and were affected by the CNs utilized and the reaction temperature used. Contagious diseases incited by multifarious toxigenic bacteria present severe threats to human health. The fabricated bio-nanocomposites were evaluated in terms of their antimicrobial efficacy by agar well diffusion method and broth microdilution assay, showing that CN-ZnO bio-nanocomposites were effective against the tested Gram-negative (Escherichia coli and Salmonella) and Gram-positive (Listeria monocytogenes and Staphylococcus aureus) bacteria. The influence of the subinhibitory concentrations of these suspensions on the expression of the most critical virulence toxin genes of the tested strains was effective. Significant downregulation levels were observed through toxigenic operons to both fabricated CN-ZnO bio-nanocomposites with a fold change ranging from 0.004 to 0.510, revealing a decline in the capacity and virulence of microorganisms to pose infections. Therefore, these newly fabricated CNS-ZnO bio-nanocomposites could be employed rationally in food systems as a novel preservative to inhibit microbial growth and repress the synthesis of exotoxins., (© 2022. The Author(s).)

A. hydrophila is an important pathogen that mainly harms aquatic animals and has exhibited resistance to a variety of antibiotics. Here, to seek an effective alternative for antibiotics, the effects of umbelliferone (UM) at sub-MICs on A. hydrophila virulence factors and the quorum-sensing system were studied. Subsequently, RNA sequencing was employed to explore the potential mechanisms for the antivirulence activity of umbelliferone. Meanwhile, the protective effect of umbelliferone on grass carp infected with A. hydrophila was studied in vivo. Our results indicated that umbelliferone could significantly inhibit A. hydrophila virulence such as hemolysis, biofilm formation, swimming and swarming motility, and their quorum-sensing signals AHL and AI-2. Transcriptomic analysis showed that umbelliferone downregulated expression levels of genes related to exotoxin, the secretory system (T2SS and T6SS), iron uptake, etc. Animal studies demonstrated that umbelliferone could significantly improve the survival of grass carps infected with A. hydrophila , reduce the bacterial load in the various tissues, and ameliorate cardiac, splenic, and hepatopancreas injury. Collectively, umbelliferone can reduce the pathogenicity of A. hydrophila and is a potential drug for treating A. hydrophila infection.

Targeted tumor therapy is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) is overexpressed in tumor vasculature and plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. Immunotoxins due to their superior cell-killing ability and the relative simplicity of their preparation, have great potential in the clinical treatment of cancer. The aim of this study was to develop a novel immunotoxin against DLL4 as a cell cytotoxic agent and angiogenesis maturation inhibitor. In present study, an immunotoxin, named DLL4Nb-PE, in which a Nanobody as targeting moiety fused to the Pseudomonas exotoxin A (PE) was constructed, expressed and assessed by SDS-PAGE, western blotting, ELISA and flowcytometry. The functional assessment was carried out via MTT, apoptosis and chicken chorioallantoic membrane (CAM) assays. It was demonstrated DLL4Nb-PE specifically binds to DLL4 and recognizes DLL4-expressing MKN cells. The cytotoxicity assays showed that this molecule could induce apoptosis and kill DLL4 positive MKN cells. In addition, it inhibited neovascularization in the chicken chorioallantoic membrane. Our findings indicate designed anti-DLL4 immunotoxin has valuable potential for application to the treatment of tumors with high DLL4 expression., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is one of the major pathogens responsible for antimicrobial resistance-associated death. S. aureus can secrete various exotoxins, and staphylococcal biofilms play critical roles in antibiotic tolerance and the persistence of chronic infections. Here, we investigated the inhibitory effects of 18 hydroquinones on biofilm formation and virulence factor production by S. aureus. It was found that 2,5-bis(1,1,3,3-tetramethylbutyl) hydroquinone (TBHQ) at 1 µg/mL efficiently inhibits biofilm formation by two methicillin-sensitive and two methicillin-resistant S. aureus strains with MICs of 5 µg/mL, whereas the backbone compound hydroquinone did not (MIC > 400 µg/mL). In addition, 2,3-dimethylhydroquinone and tert-butylhydroquinone at 50 µg/mL also exhibited antibiofilm activity. TBHQ at 1 µg/mL significantly decreased the hemolytic effect and lipase production by S. aureus, and at 5−50 µg/mL was non-toxic to the nematode Caenorhabditis elegans and did not adversely affect Brassica rapa seed germination or growth. Transcriptional analyses showed that TBHQ suppressed the expression of RNAIII (effector of quorum sensing). These results suggest that hydroquinones, particularly TBHQ, are potentially useful for inhibiting S. aureus biofilm formation and virulence.

The leukotoxin (LtxA) of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a protein exotoxin belonging to the repeat-in-toxin family (RTX). Numerous studies have demonstrated that LtxA may play a critical role in the pathogenicity of A. actinomycetemcomitans since hyper-leukotoxic strains have been associated with severe disease. Accordingly, considerable effort has been made to elucidate the mechanisms by which LtxA interacts with host cells and induce their death. However, these attempts have been hampered by the unavailability of a tertiary structure of the toxin, which limits the understanding of its molecular properties and mechanisms. In this paper, we used homology and template free modeling algorithms to build the complete tertiary model of LtxA at atomic level in its calcium-bound Holo-state. The resulting model was refined by energy minimization, validated by Molprobity and ProSA tools, and subsequently subjected to a cumulative 600ns of all-atom classical molecular dynamics simulation to evaluate its structural aspects. The druggability of the proposed model was assessed using Fpocket and FTMap tools, resulting in the identification of four putative cavities and fifteen binding hotspots that could be targeted by rational drug design tools to find new ligands to inhibit LtxA activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

The cytotoxic necrotizing factors (CNFs) are a family of Rho GTPase-activating single-chain exotoxins that are produced by several Gram-negative pathogenic bacteria. Due to the pleiotropic activities of the targeted Rho GTPases, the CNFs trigger multiple signaling pathways and host cell processes with diverse functional consequences. They influence cytokinesis, tissue integrity, cell barriers, and cell death, as well as the induction of inflammatory and immune cell responses. This has an enormous influence on host-pathogen interactions and the severity of the infection. The present review provides a comprehensive insight into our current knowledge of the modular structure, cell entry mechanisms, and the mode of action of this class of toxins, and describes their influence on the cell, tissue/organ, and systems levels. In addition to their toxic functions, possibilities for their use as drug delivery tool and for therapeutic applications against important illnesses, including nervous system diseases and cancer, have also been identified and are discussed.

Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca 2+ , activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X 7 R antagonist. Similarly, blockage of P2X 7 Rs with oATP prevented the intracellular mobilization of Ca 2+ , an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X 7 R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death., (© 2021. The Author(s).)

Background/aim: Pseudomonas exotoxin (PE) is one of the most widely used toxins in the construction of therapeutic fusion proteins in pre-clinical studies followed by phase trials. In principle, PE acts by blocking protein synthesis through catalyzing the inactivation of elongation factor-2 (EF-2). The interleukin-13 fused PE (IL13-PE) cytotoxin was previously designed to target GBM cells. In this study, the cytotoxic effects of IL13-PE were evaluated in 5 different types of cancers and the therapeutic effects were further analyzed in a lung cancer cell line, NCI-H460. Conceptually, in another lung cancer cell line (A549), IL13Rα2 was overexpressed by lentiviruses (A549-IL13Rα2) and evaluated for cytotoxic efficacy of IL13-PE., Materials and Methods: The expression profile of IL13Rα2 in different cancer cell lines was determined by RT-PCR. Secretable toxin fusion was expressed in the toxin resistant HEK-293T cell line (293T-TxR) by using a plasmid coding for IL13-PE and IRES-GFP (LV-IL13-PE-IRES/GFP). Next, the cells were shown to produce and secrete functional IL13-PE by dot blot analysis, followed by cell viability assays and cell death analysis., Results: Upon treatment with IL13-PE, a significant decrease in cell viability was selectively demonstrated in cancer cells with cognate receptor expression. IL13-PE treatment increased the apoptotic/necrotic cell populations in the NCI-H460 cell line., Conclusion: Our results demonstrate that IL13-PE can be a therapeutic target for tumors bearing mostly IL13Rα2 positive cell populations. Our findings also suggest a cell-based delivery option for the recombinant toxins in the treatment of different cancers which can provide a solution for the clinical use of toxin therapy., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Recombinant immunotoxins are fusion proteins composed of a peptide toxin and a specific targeting domain through genetic recombination. They are engineered to recognize disease-specific target receptors and kill the cell upon internalization. Full-sized monoclonal antibodies, smaller antibody fragments and ligands, such as a cytokine or a growth factor, have been commonly used as the targeting domain, while bacterial Pseudomonas aeruginosa exotoxin (PE) is the usual toxin fusion partner, due to its natural cytotoxicity and other unique advantages. PE-based recombinant immunotoxins have shown remarkable efficacy in the treatment of tumors and autoimmune diseases. At the same time, efforts are underway to address major challenges, including immunogenicity, nonspecific cytotoxicity and poor penetration, which limit their clinical applications. Recent strategies for structural optimization of PE-based immunotoxins, combined with mutagenesis approaches, have reduced the immunogenicity and non-specific cytotoxicity, thus increasing both their safety and efficacy. This review highlights novel insights and design concepts that were used to advance immunotoxins for the treatment of hematological and solid tumors and also presents future development prospect of PE-based recombinant immunotoxins that are expected to play an important role in cancer therapy., (Copyright © 2021. Published by Elsevier B.V.)

Glioblastomas (GBMs) are refractory to current treatments and novel therapeutic approaches need to be explored. Pro‑apoptotic tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) is tumor‑specific and has been shown to induce apoptosis and subsequently kill GBM cells. However, approximately 50% of GBM cells are resistant to TRAIL and a combination of TRAIL with other therapeutics is necessary to induce mechanism‑based cell death in TRAIL‑resistant GBMs. The present study examined the ability of the tumor cell surface receptor, interleukin (IL)‑13 receptor α2 (IL13Rα2)‑ and epidermal growth factor receptor (EGFR)‑targeted pseudomonas exotoxin (PE) to sensitize TRAIL‑resistant GBM cells and assessed the dual effects of interleukin 13‑PE (IL13‑PE) or EGFR nanobody‑PE (ENb‑PE) and TRAIL for the treatment of a broad range of brain tumors with a distinct TRAIL therapeutic response. Receptor targeted toxins upregulated TRAIL death receptors (DR4 and DR5) and suppressed the expression of anti‑apoptotic FLICE‑inhibitory protein (FLIP) and X‑linked inhibitor of apoptosis protein (XIAP). This also led to the induction of the cleavage of caspase‑8 and caspase‑9 and resulted in the sensitization of highly resistant established GBM and patient‑derived GBM stem cell (GSC) lines to TRAIL‑mediated apoptosis. These findings provide a mechanism‑based strategy that may provide options for the cell‑mediated delivery of bi‑functional therapeutics to target a wide spectrum of TRAIL‑resistant GBMs.

Cholecystokinin-2 receptor (CCK2R) has been proven to be a specific biomarker for colorectal malignancies. Immunotoxins are a valuable class of immunotherapy agents consisting of a targeting element and a bacterial or plant toxin. Previous work demonstrated that targeting CCK2R is a good therapeutic strategy for the treatment of colorectal cancer (CRC). In the present study, we developed a new version of CCK2R-targeting immunotoxin GD9P using a targeted peptide, GD9, as the binding motif and a truncated Pseudomonas exotoxin A (PE38) as the cytokiller. BALB/c nude mice were treated with different doses of GD9P, and pharmacodynamics, pharmacokinetic, and toxicological data were obtained throughout this study. Compared to the parental immunotoxin rCCK8PE38, GD9P exhibited about 1.5-fold yield, higher fluorescence intensity, and increased antitumor activity against human CRC in vitro and in vivo. The IC 50 values of GD9P in vitro ranged from 1.61 to 4.55 nM. Pharmacokinetic studies were conducted in mice with a T 1/2 of 69.315 min. When tumor-bearing nude mice were treated with GD9P at doses ≥2 mg/kg for five doses, a rapid shrinkage in tumor volume and, in some cases, complete remission was observed. A preliminary safety evaluation demonstrated a good safety profile of GD9P as a Pseudomonas exotoxin A-based immunotherapy. The therapy in combination with oxaliplatin can increase the antitumor efficacy and reduce the toxic side effects caused by chemotherapy. In conclusion, the data support the use of GD9P as a promising immunotherapy targeting CCK2R-expressing colorectal malignancies.

Purpose: Endophthalmitis models have reported the virulent role of Panton-Valentine leucocidin (PVL) secreted by Staphylococcus aureus on the retina. PVL targets retinal ganglion cells (RGCs), expressing PVL membrane receptor C5aR. Interactions between PVL and retinal cells lead to glial activation, retinal inflammation, and apoptosis. In this study, we explored oxidative stress and retinal neurotransmitters in a rabbit retinal explant model incubated with PVL., Methods: Reactive oxygen species (ROS) production in RGCs has been assessed with fluorescent probes and immunohistochemistry. Nuclear magnetic resonance (NMR) spectroscopy quantified retinal concentrations of antioxidant molecules and neurotransmitters, and concentrations of neurotransmitters released in the culture medium. Quantifying the expression of some pro-inflammatory and anti-inflammatory factors was performed using RT-qPCR., Results: PVL induced a mitochondrial ROS production in RGCs after four hours' incubation with the toxin. Enzymatic sources of ROS, involving nicotinamide adenine dinucleotide phosphate-oxidase and xanthine oxidase, were also activated after four hours in PVL-treated retinal explants. Retinal antioxidants defenses, that is, glutathione, ascorbate and taurine, decreased after two hours' incubation with PVL. Glutamate retinal concentrations and glutamate release in the culture medium remained unaltered in PVL-treated retinas. GABA, glycine, and acetylcholine (Ach) retinal concentrations decreased after PVL treatment. Glycine release in the culture medium decreased, whereas Ach release increased after PVL treatment. Expression of proinflammatory and anti-inflammatory cytokines remained unchanged in PVL-treated explants., Conclusions: PVL activates oxidative pathways and alters neurotransmitter retinal concentrations and release, supporting the hypothesis that PVL could induce a neurogenic inflammation in the retina.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC 50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.

Massive neutrophil infiltration is an early key event in infectious inflammation, accompanied by chemotactic leukotriene (LT)B 4 generation. LTB 4 biosynthesis is mediated by 5-lipoxygenase (5-LOX), but which pathogenic factors cause 5-LOX activation during bacterial infections is elusive. Here, we reveal staphylococcal exotoxins as 5-LOX activators. Conditioned medium of wild-type Staphylococcus aureus but not of exotoxin-deficient strains induced 5-LOX activation in transfected HEK293 cells. Two different staphylococcal exotoxins mimicked the effects of S. aureus-conditioned medium: (1) the pore-forming toxin α-hemolysin and (2) amphipathic α-helical phenol-soluble modulin (PSM) peptides. Interestingly, in human neutrophils, 5-LOX activation was exclusively evoked by PSMs, which was prevented by the selective FPR2/ALX receptor antagonist WRW4. 5-LOX activation by PSMs was confirmed in vivo as LT formation in infected paws of mice was impaired in response to PSM-deficient S. aureus. Conclusively, exotoxins from S. aureus are potent pathogenic factors that activate 5-LOX and induce LT formation in neutrophils.

Enhancing the sensitivity of hepatocellular carcinoma (HCC) cells to sorafenib (SFN) is an essential clinical bottleneck to be solved. Here we report that the expression of CD47 negatively correlated with HCC sensitivity to SFN. The microbiota-derived Staphylococcal superantigen-like protein 6 (SSL6) inhibited CD47 and promoted SFN-induced apoptosis of HCC cells Huh-7 and MHCC97H. Mechanistically, the sensitivity of HCC cells to SFN was inhibited by elevated Warburg effect (glycolysis), and SSL6 down-regulated PI3K/Akt-mediated glycolysis by blocking CD47. Knockdown of CD47 also dampened glycolysis and sensitized HCC cells to SFN. Moreover, SFN-resistant HCC cells exhibited enhanced glycolysis and CD47 expression. SSL6 significantly re-sensitized the resistant HCC cells to SFN. More importantly, we identified the anti-tumor effect of SSL6 in combination with SFN in HCC-bearing mice. Our results clarify the mechanism by which SSL6 enhances SFN sensitivity in HCC cells, providing a molecular basis for combination targeted therapy with microbiota-derived SSL6 to treat HCC., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Immunotoxins are a class of targeted cancer therapeutics in which a toxin such as Pseudomonas exotoxin A (PE) is linked to an antibody or cytokine to direct the toxin to a target on cancer cells. While a variety of PE-based immunotoxins have been developed and a few have demonstrated promising clinical and preclinical results, cancer cells frequently have or develop resistance to these immunotoxins. This review presents our current understanding of the mechanism of action of PE-based immunotoxins and discusses cellular mechanisms of resistance that interfere with various steps of the pathway. These steps include binding of the immunotoxin to the target antigen, internalization, intracellular processing and trafficking to reach the cytosol, inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (EF2), and induction of apoptosis. Combination therapies that increase immunotoxin action and overcome specific mechanisms of resistance are also reviewed., Competing Interests: The authors declare no conflicts of interest.

Background and Aims: Treatment of hepatocellular carcinomas using our glypican-3 (GPC3)-targeting human nanobody (HN3) immunotoxins causes potent tumor regression by blocking protein synthesis and down-regulating the Wnt signaling pathway. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic., Approach and Results: To address these concerns, we engineered HN3-based immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3-T20, which was modified to remove T-cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B-cell deimmunized immunotoxin (HN3-mPE24) and our original HN3-immunotoxin with a wild-type PE domain (HN3-PE38). All of our immunotoxins displayed high affinity to human GPC3, with HN3-T20 having a K D value of 7.4 nM. HN3-T20 retained 73% enzymatic activity when compared with the wild-type immunotoxin in an adenosine diphosphate-ribosylation assay. Interestingly, a real-time cell growth inhibition assay demonstrated that a single dose of HN3-T20 at 62.5 ng/mL (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20's serum retention, we tested the effect of adding a streptococcal albumin-binding domain (ABD) and a llama single-domain antibody fragment specific for mouse and human serum albumin. For the detection of immunotoxin in mouse serum, we developed a highly sensitive enzyme-linked immunosorbent assay and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20 (326 minutes vs. 7.3 minutes); consequently, addition of an ABD resulted in HN3-ABD-T20-mediated tumor regression at 1 mg/kg., Conclusion: These data indicate that ABD-containing deimmunized HN3-T20 immunotoxins are high-potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer., (© 2019 by the American Association for the Study of Liver Diseases.)

Bacterial toxins have received a great deal of attention in the development of antitumor agents. Currently, these protein toxins were used in the immunotoxins as a cancer therapy strategy. Despite the successful use of immunotoxins, immunotherapy strategies are still expensive and limited to hematologic malignancies. In the current study, for the first time, a nano-toxin comprised of truncated pseudomonas exotoxin (PE38) loaded silver nanoparticles (AgNPs) were prepared and their cytotoxicity effect was investigated on human breast cancer cells. The PE38 protein was cloned into pET28a and expressed in Escherichia coli, BL21 (DE3), and purified using metal affinity chromatography and was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AgNPs were biologically prepared using cell-free supernatant of E. Coli K12 strain. Nanoparticle formation was characterized by energy dispersive spectroscopy, transmission electron microscopy, and dynamic light scattering. The PE38 protein was loaded on AgNPs and prepared the PE38-AgNPs nano-toxin. Additionally, in vitro release indicated a partial slow release of toxin in about 100 hr. The nano-toxin exhibited dose-dependent cytotoxicity on MCF-7 cells. Also, real-time polymerase chain reaction results demonstrated the ability of nano-toxin to upregulate Bax/Bcl-2 ratio and caspase-3, -8, -9, and P53 apoptotic genes in the MCF-7 tumor cells. Apoptosis induction was determined by Annexin-V/propidium flow cytometry and caspases activity assay after treatment of cancer cells with the nano-toxin. In general, in the current study, the nano-toxin exhibit an inhibitory effect on the viability of breast cancer cells through apoptosis, which suggests that AgNPs could be used as a delivery system for targeting of toxins to cancer cells., (© 2019 Wiley Periodicals, Inc.)

Hepatocellular carcinoma (HCC) is a complicated and poor prognosis cancer, necessitating the development of a potential treatment strategy. In this study, we initially revealed that LukS-PV belonged to leukocidin family performs an anti-HCC action. Then, we used liquid chromatography-mass spectrometry (LC/MS) to compare protein expression profiles of the LukS-PV-treated human HCC cell lines HepG2 and the control cells. GO annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis were carried out of differential expression followed by protein-protein interactome, to explore the underlying cancer suppressor mechanisms of LukS-PV for human HCC. A total of 88 upregulated proteins and 46 downregulated proteins were identified. The top 10 proteins identified by the MCC method are FN1, APP, TIMP1, nucleobindin-1, GOLM1, APLP2, CYR61, CD63, ENG, and CD9. Our observation on protein expression indicated that LukS-PV produces a signature affecting central carbon metabolism in cancer, galactose metabolism, and fructose and mannose metabolism pathways. The results give a functional effects and molecular mechanism insight, following LukS-PV treatment.

Background & Aims: Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease., Methods: C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin., Results: The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the β-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis., Conclusions: Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis., Lay Summary: Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the β-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease., (Published by Elsevier B.V.)

Five peptide ligands of four different cell surface receptors (nucleolin, CXCR1, CMKLR1, and CD44v6) have been evaluated as targeting moieties for triple-negative human breast cancers. Among them, the peptide F3, derived from phage display, promotes the fast and efficient internalization of a genetically fused green fluorescent protein (GFP) inside MDA-MB-231 cancer stem cells in a specific receptor-dependent fashion. The further engineering of this protein into the modular construct F3-RK-GFP-H6 and the subsequent construct F3-RK-PE24-H6 resulted in self-assembling polypeptides that organize as discrete and regular nanoparticles. These materials, 15-20 nm in size, show enhanced nucleolin-dependent cell penetrability. We show that the F3-RK-PE24-H6, based on the Pseudomonas aeruginosa exotoxin A (PE24) as a core functional domain, is highly cytotoxic over target cells. The combination of F3, the cationic peptide (RK) n , and the toxin domain PE24 in such unusual presentation appears as a promising approach to cell-targeted drug carriers in breast cancers and addresses selective drug delivery in otherwise difficult-to-treat triple-negative breast cancers.

Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of lung cancer cases. LukS-PV, one of the two components of Panton-Valentine leucocidin (PVL), is produced by Staphylococcus aureus. The present study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) lines (THP-1 and HL-60). However, the role of LukS-PV in NSCLC is unclear. In this study, we treated NSCLC cell lines A549 and H460 and a normal lung cell line, 16HBE, with LukS-PV and investigated the biological roles of LukS-PV in NSCLC. Cells were treated with varying concentrations of LukS-PV and cell viability was evaluated by CCK8 and EdU assay. Flow cytometry was used to detect cell apoptosis and analyze the cell cycle, and the expression of apoptosis and cell cycle-associated proteins and genes were identified by western blotting analysis and qRT-polymerase chain reaction, respectively. We found that LukS-PV inhibited the proliferation of NSCLC cells but had little cytotoxicity in normal lung cells. LukS-PV induced NSCLC cell apoptosis and increased the BAX/BCL-2 ratio, triggering S-phase arrest in A549 and H460 cells while increasing P21 expression and decreasing CDK2, cyclin D1, and cyclin A2 expression. We also observed increased P-p38 and P-ERK in NSCLC cells treated with LukS-PV. Treatment of NSCLC with LukS-PV combined with p38 and ERK inhibitors reversed the pro-apoptotic and pro-cell cycle arrest effects of LukS-PV. Overall, these findings indicate that LukS-PV has anti-tumor effects in NSCLC and may contribute to the development of anti-cancer agents., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: One of the approaches to cancer gene therapy relies on tumor transfection with DNA encoding toxins under the control of tumor-specific promoters., Methods: Here, we used DNA plasmids encoding very potent anti-ERBB2 targeted toxin, driven by the human telomerase promoter or by the ubiquitous CAG promoter (pTERT-ETA and pCAG-ETA) and linear polyethylenimine to target cancer cells., Results: We showed that the selectivity of cancer cell killing by the pTERT-ETA plasmid is highly dependent upon the method of preparation of DNA-polyethylenimine complexes. After adjustment of complex preparation protocol, cell lines with high activity of telomerase promoter can be selectively killed by transfection with the pTERT-ETA plasmid. We also showed that cells transfected with pTERT-ETA and pCAG-ETA plasmids do not exert any detectable bystander effect in vitro., Conclusion: Despite this, three intratumoral injections of a plasmid-polyethylenimine complex resulted in substantial growth retardation of a poorly transfectable D2F2/E2 tumor in mice. There were no significant differences in anti-tumor properties between DNA constructs with telomerase or CAG promoters in vivo., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that has been strongly associated with localized aggressive periodontitis. The capacity of A. actinomycetemcomitans to produce a leukotoxin (LtxA) that activates pyroptosis in macrophages and induces the release of endogenous danger signals is thought to play a key role in the disease process. The aim of the present study was to investigate the effects of cranberry proanthocyanidins (PACs) on gene expression and cytotoxic activities of LtxA. We showed that cranberry PACs dose-dependently attenuate the expression of genes making up the leukotoxin operon, including ltxB and ltxC , in the two strains of A. actinomycetemcomitans tested. Cranberry PACs (≥62.5 µg/mL) protected macrophages against the cytotoxic effect of purified LtxA. Moreover, cranberry PACs reduced caspase-1 activation in LtxA-treated macrophages and consequently decreased the release of both IL-1β and IL-18, which are known as damage-associated molecular patterns (DAMPs) and contribute to the progression of periodontitis by increasing cell migration and osteoclastogenesis. In addition, cranberry PACs reduced the expression of genes encoding the P2X7 receptor and NALP3 (NACHT, LRR and PYD domains-containing protein 3), which play key roles in pore formation and cell death. Lastly, cranberry PACs blocked the binding of LtxA to macrophages and consequently reduced the LtxA-mediated cytotoxicity. In summary, the present study showed that cranberry PACs reduced LtxA gene expression in A. actinomycetemcomitans and neutralized the cytolytic and pro-inflammatory responses of human macrophages treated with LtxA. Given these properties, cranberry PACs may represent promising molecules for prevention and treatment of the aggressive form of periodontitis caused by A. actinomycetemcomitans .

Moxetumomab pasudotox (MP) is an immunotoxin that recently received US Food and Drug Administration (FDA) approval for the treatment of hairy cell leukemia (HCL) that has failed at least 2 prior lines of therapy, including a purine analog. MP is a recombinant immunotoxin that consists of an anti-CD22 immunoglobulin variable domain genetically joined to Pseudomonas exotoxin (PE38). Unlike most antibody-drug conjugates, which use a chemical linker, recombinant DNA techniques are used to produce MP. MP and its predecessor, BL22, were initially developed to treat non-Hodgkin lymphoma, acute lymphoblastic leukemia, and HCL. However, MP was found to be particularly effective in HCL due to the high level of CD22 cell-surface expression. The recent pivotal phase 3 trial of MP in relapsed/refractory HCL demonstrated a durable complete remission rate of 30%, and 85% of complete responders achieved minimal residual disease negativity, which is associated with improved disease-free survival outcomes in HCL. In addition to an exceptional depth of response, MP appears to be less immunosuppressive than purine analogs. MP is generally well tolerated but has unique toxicities, including capillary leak syndrome and hemolytic uremic syndrome, which are poorly understood. This review will encompass the preclinical and clinical development of MP, with particular attention to its current indication in HCL., (© 2019 by The American Society of Hematology.)

Introduction : Hairy cell leukemia is a rare indolent B-cell malignancy, characterized by pancytopenia, recurrent infections, and splenomegaly. After initial therapy with purine nucleoside analogs, up to 50% of patients relapse after several years of remission. The number of relapsed patients is increasing and, until recently, there was no approved therapy with durable responses for hairy cell leukemia patients in the relapsed setting, thus the need for new non-chemotherapy approach with significant efficacy and less myelosuppression. Areas covered : Moxetumomab pasudotox is a recombinant immunotoxin containing a Fv fragment of an anti-CD22 monoclonal antibody and truncated Pseudomonas exotoxin (PE38). The authors reviewed pre-clinical and clinical studies that led to the FDA approval of the drug in patients with relapsed and/or refractory hairy cell leukemia, who received at least two prior therapies, including at least one purine nucleoside analog. Expert opinion : Moxetumomab pasudotox demonstrated a durable complete remission rate of 30% in heavily pretreated patients with hairy cell leukemia, and MRD eradication in 85% of responding patients. Moxetumomab pasudotox got a global FDA approval in September 2018. The US prescribing information carries boxed warnings regarding the risk of capillary leak syndrome and hemolytic uremic syndrome. Long-term follow-up of the pivotal study is ongoing (NCT01829711).

Aggregatibacter actinomycetemcomitans is an oral pathogen that produces the RTX toxin, leukotoxin (LtxA; Leukothera ® ). A. actinomycetemcomitans is strongly associated with the development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans to subvert the host immune response by binding to the β 2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs), causing cell death. In this paper, we reviewed the state of knowledge on LtxA interaction with WBCs and the subsequent mechanisms of induced cell death. Finally, we touched on the potential therapeutic applications of LtxA (trade name Leukothera ® ) toxin therapy for the treatment of hematological malignancies and immune-mediated diseases.

Purpose: Hairy cell leukemia (HCL) is a rare mature B cell leukemia. Purine analogs are the mainstay of treatment of HCL, but relapse after purine analog therapy is common. Outcomes of treatment of relapsed/refractory HCL typically diminish with each successive line of therapy. Moxetumomab pasudotox-tdfk is a novel recombinant immunotoxin approved for the treatment of patients with relapsed/refractory HCL who have received at least two prior therapies, including a purine analog. This article reviews HCL treatment, focusing on moxetumomab pasudotox-tdfk, its place in therapy, considerations for preparation and administration, and strategies for prevention and management of toxicities., Methods: A literature search was conducted in the PubMed database from inception to January 2019, using the following terms: moxetumomab, hairy cell leukemia, relapsed/refractory hairy cell leukemia, immunotoxin, and CD22. The package insert and available posters and abstracts were also reviewed., Results: FDA approval of moxetumomab pasudotox-tdfk was based on a phase III single-arm, open-label trial in 80 patients. Treatment with moxetumomab pasudotox-tdfk yielded a durable complete response rate of 30% with a median duration of response that had not yet been reached at a median follow-up of 16.7 months. The objective response rate was 75% based on blinded independent central review. The most common adverse reactions were infusion-related reactions, edema, nausea, fatigue, headache, pyrexia and anemia. Serious adverse events include capillary leak syndrome and hemolytic uremic syndrome., Conclusions: Clinicians providing care for patients receiving moxetumomab pasudotox-tdfk should be aware of the strategies required for safe administration, including the management of serious adverse events.

Beta haemolytic Group A streptococcus (GAS) or Streptococcus pyogenes are strict human pathogens responsible for mild to severe fatal invasive infections. Even with enormous number of reports exploring the role of S. pyogenes exotoxins in its pathogenesis, inadequate knowledge on the biofilm process and the potential role of exotoxins in bacterial dissemination from matured biofilms has been a hindrance in development of effective and targeted treatments. Therefore, the present study was aimed in investigating the uncharted role of these exotoxins in biofilm process. Through our study the putative role of ciaRH in the SpeA dependent ablation of biofilm formation could be speculated and thus helping in bacterial dissemination. The seed-dispersal effect of SpeA was time and concentration dependent and seen to be consistent within various streptococcal species. Transcriptome analysis of SpeA treated S. pyogenes biofilms revealed the involvement of many transcriptional regulators (ciaRH) and response genes (luxS, shr, shp, SPy_0572), hinting towards specific mechanisms underlying the dispersal effect by SpeA. This finding opens up a discussion towards understanding a new mechanism involved in the pathogenesis of Streptococcus pyogenes and might help in understanding the bacterial infections in a better way.

Introduction : Cladribine and pentostatin are the drugs of choice in the treatment of hairy cell leukemia (HCL). Recently, immunotoxin moxetumomab pasudotox has been introduced to improve the prognosis in relapsed and refractory HCL. Areas covered : This review discusses the mechanism of action, safety, and efficacy of moxetumomab pasudotox in HCL patients. A literature review of the MEDLINE database for articles in English concerning immunotoxins, moxetumomab pasudotox, and hairy cell leukemia was conducted via PubMed. Publications from 2000 through December 2018 were scrutinized. The search terms used were immunotoxins and moxetumomab pasudotox in conjunction with hairy cell leukemia . Conference proceedings from the previous five years of the American Society of Hematology, European Hematology Association and American Society of Clinical Oncology were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles. Results/conclusion : Moxetumomab pasudotox, a novel recombinant anti-CD22 immunotoxin, was well tolerated and active in the previous phase 1 and 3 studies in patients with HCL. The drug has been approved in 2018 by the FDA for the treatment of patients with relapsed/refractory HCL who had at least two prior systemic therapies including at least one purine nucleoside analog. Expert opinion : The use of moxetumomab pasudotox is a promising new strategy for the treatment of HCL.

Objective: Recombinant immunotoxins (RITs) are antibody-toxin fusion proteins that can selectively eliminate populations of cells expressing specific surface receptors. They are in evaluation as therapeutic agents for cancer. RITs based on Pseudomonas exotoxin A (PE) are in use clinically for the treatment of hairy cell leukemia, and under trial for the treatment of other cancers. In an effort to improve the efficacy of PE-based RITs, we evaluated the potential of combination therapy with several common antibiotics (tetracycline, chloramphenicol, streptomycin, linezolid, fusidic acid, and kanamycin) on human cell lines HEK293, OVCAR8, and CA46. Antibiotics were selected based on their potential to inhibit mitochondrial protein synthesis and disrupt energy metabolism in cancer cells., Results: Tetracycline, chloramphenicol, linezolid, and fusidic acid alone killed cultured human cells at high concentrations. At high but nontoxic concentrations of each antibiotic, only chloramphenicol treatment of the Burkitt's lymphoma cell line CA46 showed enhanced cytotoxicity when paired with an anti-transferrin receptor/PE RIT. This result, however, could not be replicated in additional Burkitt's lymphoma cell lines Ramos and Raji. Although the six antibiotics we tested are not promising candidates for RIT combination therapy, we suggest that fusidic acid could be considered independently as a potential cancer therapeutic.

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.

LukS-PV is one of the two components of Panton-Valentine leucocidin (PVL). Our previous study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) THP-1 and HL-60 cells. C5aR (C5a receptor) is the receptor for PVL, but whether C5aR plays a key role in LukS-PV induced apoptosis is unclear. The aim of this study was to establish whether C5aR plays a physiological role in apoptosis of leukemia cells induced by LukS-PV. We investigated the role of C5aR in leukemia cell apoptosis induced by LukS-PV by pretreatment of THP-1 and HL-60 cells with C5aR antagonist and transfection to knockdown C5aR in THP-1 cells or overexpress C5aR in Jurkat cells before treatment with LukS-PV. Cell apoptosis was analyzed by staining with Annexin V/propidium iodide or Annexin V-PE/7-AAD. Mitochondrial membrane potential (MMP) was determined using JC-1 dye. The expression of apoptosis-associated genes and proteins was identified by qRT-polymerase chain reaction and Western blotting analysis, respectively. As the C5aR antagonist concentration increased, the rate of apoptosis induced by LukS-PV decreased, the MMP increased, and expression of pro-apoptotic Bax and Bak genes and proteins was downregulated while that of anti-apoptotic Bcl-2 and Bcl-x genes and proteins was upregulated. Knockdown of C5aR also decreased LukS-PV-induced THP-1 cell apoptosis. LukS-PV did not induce apoptosis of Jurkat cells, which have no endogenous C5aR expression; however, LukS-PV did induce apoptosis in Jurkat cells after overexpression of C5aR. Correspondingly, the MMP decreased and Bax and Bak were upregulated while Bcl-2 and Bcl-x were downregulated. LukS-PV can induce apoptosis in AML cells by targeting C5aR. C5aR may be a potential therapeutic target for AML and LukS-PV is a candidate targeted drug for the treatment of AML., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Standard treatment, unfortunately, yields a poor prognosis for patients with primary or metastatic cancers in the central nervous system, indicating a necessity for novel therapeutic agents. Immunotoxins (ITs) are a class of promising therapeutic candidates produced by fusing antibody fragments with toxin moieties. In this study, we investigated if inherent resistance to IT cytotoxicity can be overcome by rational combination with pro-apoptotic enhancers. Therefore, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting chondroitin sulfate proteoglycan 4 (CSPG4) with a panel of Bcl-2 family inhibitors (ABT-737, ABT-263, ABT-199 [Venetoclax], A-1155463, and S63845) against patient-derived glioblastoma, melanoma, and breast cancer cells/cell lines. In vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized the different tumors to IT treatment, and improved the IC50 values of 9.2.27-PE38KDEL up to >1,000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that increased levels of intracellular IT, processed (active) exotoxin, and PARP cleavage correlated with the enhanced sensitivity to the combination treatment. Furthermore, we confirmed the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy in orthotopic GBM xenograft and cerebral melanoma metastasis models in nude mice. Our study defines strategies for overcoming IT resistance and enhancing specific antitumor cytotoxicity in primary and metastatic brain tumors., Competing Interests: The authors have declared that no competing interests exist.

Immunotoxin targeted therapy is a promising way of cancer therapy that is made from a toxin attached to an antibody which target a specific protein presented on cancer cells. In this study, we introduce immunotoxins comprising of truncated pseudomonas exotoxin A (PEA) and diphtheria toxin (DT) conjugated to trastuzumab. The effectiveness of 20 and 30 μg/ml immunotoxins and trastuzumab were studied on SK-BR-3 and BT-474 HER2/neu positive breast cancer cell lines by a cell death assay test. The produced immunotoxins have the potential to reduce the therapeutic dose of the trastuzumab and in the same time achieve higher efficiency.

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII-specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-ETA ex vivo. SUMMARY: Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII (FVIII) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII, the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A (ETA) from Pseudomonas aeruginosa for selective elimination of FVIII-specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full-length FVIII protein and the resulting human C2 domain-ETA fusion protein (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested ex vivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-ETA and controls. FVIII-specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody-secreting cells (ASC) and detected by an enzyme-linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC2-ETA reduced the number of C2-specific ASC in a dose-dependent fashion, indicating specific and efficient elimination of C2-specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII-specific B cells in patients with hemophilia A and persisting inhibitors., (© 2018 International Society on Thrombosis and Haemostasis.)

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple‑targeted therapies have been developed, with a recent clinical application of the antibody‑mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N‑terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF‑ETA), which has ADP‑ribosylation activity and induces apoptosis. The EGF‑ETA protein was expressed in E. coli as a His‑tagged fusion. Our results showed that EGF‑ETA significantly inhibited the proliferation of EGFR‑positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549‑IC50 1,000 ng/ml; MCF‑7‑IC50 >10,000 ng/ml). Compared to cetuximab, EGF‑ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF‑ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild‑type HT29 colon cancer cells. Finally, co‑incubation of EGF‑ETA with an anti‑EGF antibody abrogated its effect on the EGFR‑positive A431 cells. Our results show that the chimeric EGF‑ETA toxin is extremely effective against EGFR‑positive cancers and raises the potential to further develop this chimera for use in targeting EGFR‑positive tumours resistant to monoclonal antibodies.

Cell targeting protein toxins have gained increasing interest for cancer therapy aimed at increasing the therapeutic window and reducing systemic toxicity. Because recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli ( E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. Here, we describe the production of full-length antibody immunotoxins using self-splicing split inteins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of trastuzumab, a human epidermal growth factor receptor 2 (HER2) receptor targeting antibody and to a nanobody-Fc fusion targeting the HER1 receptor, respectively. Both antibodies were produced in Expi293F cells. The longer C-terminal counterpart of the intein was genetically fused to the protein toxins gelonin or Pseudomonas Exotoxin A, respectively, and expressed in E. coli via fusion to maltose binding protein. Using optimized in vitro splicing conditions, we were able to generate a set of specific and potent immunotoxins with IC 50 values in the mid- to subpicomolar range.

Anti-CD22 moxetumomab pasudotox achieved 46% complete remissions (CRs) in previously reported phase 1 testing in relapsed/refractory hairy cell leukemia (HCL; n = 28). The importance of minimal residual disease (MRD) after CR in HCL is unknown. A 21-patient extension cohort received 50 µg/kg every other day for 3 doses in 4-week cycles. These patients plus 12 previously reported at this upper dose level received 143 cycles without dose-limiting toxicity. The combined 33-patient cohort achieved 64% CR and 88% overall response rates, with median CR duration of 42.4 months. Of 32 50-µg/kg patients evaluable for MRD by bone marrow aspirate flow cytometry (most stringent assessment), median CR duration was 13.5 (4.9-42.4) months in 9 MRD-positive CRs vs 42.1 (24.0-69.2) months in 11 MRD-negative CRs ( P < .001). Among MRD-negative CRs, 10 patients had ongoing CR, 9 without MRD, at end of study. To our knowledge, moxetumomab pasudotox is the only nonchemotherapy regimen that can eliminate MRD in a significant percentage of HCL patients, to enhance CR duration. Repeated dosing, despite early neutralizing antibodies, increased active drug levels without detectable toxicity from immunogenicity. The activity and safety profiles of moxetumomab pasudotox support ongoing phase 3 testing in HCL. This trial was registered at www.clinicaltrials.gov as #NCT00586924.

Key Message: This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7 µg per 100 mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.

Recombinant immunotoxins (RITs) are chimeric proteins consisting of a Fv that binds to a cancer cell and a portion of a protein toxin. One of these, Moxetumomab pasudotox, was shown to be effective in treating patients with some leukemias, where the cells are readily accessible to the RIT. However, their short half-life limits their efficacy in solid tumors, because penetration into the tumors is slow. Albumin and agents bound to albumin have a long half-life in the circulation. To increase the time tumor cells are exposed to RITs, we have produced and evaluated variants that contain either an albumin-binding domain (ABD) from Streptococcus or single-domain antibodies from Llama. We have inserted these ABDs into RITs targeting mesothelin, between the Fv and the furin cleavage site. We find that these proteins can be produced in large amounts, are very cytotoxic to mesothelin-expressing cancer cell lines, and have a high affinity for human or mouse serum albumin. In mice, the RIT containing an ABD from Streptococcus has a longer half-life and higher antitumor activity than the other two. Its half-life in the circulation of mice ranges from 113 to 194 min compared with 13 min for an RIT with no ABD. Cell uptake studies show the RIT enters the target cell bound to serum albumin. We conclude that RITs with improved half-lives and antitumor activity should be evaluated for the treatment of cancer in humans., Competing Interests: Conflict of interest statement: I.P. is an inventor on several patents on immunotoxins that have all been assigned to the NIH. All other authors declare no potential conflicts of interest.

Most human listeriosis outbreaks are caused by Listeria monocytogenes evolutionary lineage I strains which possess four exotoxins: a phosphatidylinositol-specific phospholipase C (PlcA), a broad-range phospholipase C (PlcB), listeriolysin O (LLO) and listeriolysin S (LLS). The simultaneous contribution of these molecules to virulence has never been explored. Here, the importance of these four exotoxins of an epidemic lineage I L. monocytogenes strain (F2365) in virulence was assessed in chicken embryos infected in the allantoic cavity. We show that LLS does not play a role in virulence while LLO is required to infect and kill chicken embryos both in wild type transcriptional regulator of virulence PrfA (PrfA WT ) and constitutively active PrfA (PrfA*) backgrounds. We demonstrate that PlcA, a toxin previously considered as a minor virulence factor, played a major role in virulence in a PrfA* background. Interestingly, GFP transcriptional fusions show that the plcA promoter is less active than the hly promoter in vitro, explaining why the contribution of PlcA to virulence could be observed more importantly in a PrfA* background. Together, our results suggest that PlcA might play a more important role in the infectious lifecycle of L. monocytogenes than previously thought, explaining why all the strains of L. monocytogenes have conserved an intact copy of plcA in their genomes.