Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes.

The production of therapeutic recombinant toxins requires careful host cell selection. Bacteria, yeast, and mammalian cells are common choices, but no universal solution exists. Achieving the delicate balance in toxin production is crucial due to potential self-intoxication. Recombinant toxins from various sources find applications in antimicrobials, biotechnology, cancer drugs, and vaccines. "Toxin-based therapy" targets diseased cells using three strategies. Targeted cancer therapy, like antibody-toxin conjugates, fusion toxins, or "suicide gene therapy", can selectively eliminate cancer cells, leaving healthy cells unharmed. Notable toxins from various biological sources may be used as full-length toxins, as plant (saporin) or animal (melittin) toxins, or as isolated domains that are typical of bacterial toxins, including Pseudomonas Exotoxin A (PE) and diphtheria toxin (DT). This paper outlines toxin expression methods and system advantages and disadvantages, emphasizing host cell selection's critical role.

From Bacterial Toxin to Therapeutic Agent: The Unexpected Fate of Mycolactone.

"Recognizing a surprising fact is the first step towards discovery." This famous quote from Louis Pasteur is particularly appropriate to describe what led us to study mycolactone, a lipid toxin produced by the human pathogen Mycobacterium ulcerans. M. ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease manifesting as chronic, necrotic skin lesions with a "surprising" lack of inflammation and pain. Decades after its first description, mycolactone has become much more than a mycobacterial toxin. This uniquely potent inhibitor of the mammalian translocon (Sec61) helped reveal the central importance of Sec61 activity for immune cell functions, the spread of viral particles and, unexpectedly, the viability of certain cancer cells. We report in this review the main discoveries that marked our research into mycolactone, and the medical perspectives they opened up. The story of mycolactone is not over and the applications of Sec61 inhibition may go well beyond immunomodulation, viral infections, and oncology.

Study on Cellular Localization of Bin Toxin and its Apoptosis-inducing Effect on Human Nasopharyngeal Carcinoma Cells.

BACKGROUND: Bacterial pore-forming toxins, BinA and BinB together known as the binary toxin are potent insecticidal proteins, that share structural homology with antitumor bacterial parasporin-2 protein. The underlying molecular mechanism of Bin toxin-induced cancer cell cytotoxicity requires more knowledge to understand whether the toxin induced human cytotoxic effects occur in the same way as that of parasporin-2 or not. METHODS: In this study, anticancer properties of Lysinibacillus sphaericus derived Bin toxin on HK1 were evaluated through MTT assay, morphological analysis and lactate dehydrogenase efflux assay. Induction of apoptosis was determined from RT-qPCR, caspase activity and cytochrome c release assay. Internalization pattern of Bin toxin in HK1 cells was studied by confocal laser-scanning microscopic analysis. RESULTS: Activated Bin toxin had strong cytocidal activity to HK1 cancer cell line at 24 h postinoculation. Both BinA and BinB treated HK1 cells showed significant inhibition of cell viability at 12 µM. Induction of apoptotic mediators from RT-qPCR and caspase activity analyses indicated the activation of programmed cell death in HK1 cells in response to Bin toxin treatment. Internalization pattern of Bin toxin studied by using confocal microscopy indicated the localization of BinA on cell surface and internalization of BinB in the cytoplasm of cancer cells as well as colocalization of BinA with BinB. Evaluation of cytochrome c release also showed the association of BinB and BinA+BinB with mitochondria. CONCLUSION: Bin toxin is a cytotoxic protein that induces cytotoxic and apoptotic events in HK1 cells, and may have high therapeutic potential as an anti-cancer agent.

Future prospects of bacteria-mediated cancer therapies: Affliction or opportunity?

Cancer, as a disease characterized by uncontrolled growth of cells, is recognized as one of the significant challenges in the field of health and medicine. There are various treatments for cancer like surgery, hormone therapy, chemotherapy, etc., but they have negative effects on the patient's lifestyle. Numerous side effects, and recently the emergence of drug resistance to these methods are weaknesses of these treatments. The utilization of bacteria as a treatment for cancer has attracted scientists' attention in the last decade. There are various methods of using bacteria to treat cancer, including the use of live, attenuated, or genetically engineered microbes, the use of bacterial toxins as an immunotoxin or conjugated to tumor antigens, bacteria-based cancer immunotherapy, bacterial vectors for gene-directed enzyme prodrug, and also the undeniable role of probiotics in treatment, are the cases that today are used for treatment. Bacterial therapy has shown a greater promise in cancer treatment due to its ability to lyse the tumor cells and deliver therapeutic products. However, the potential cytotoxicity of bacteria for healthy tissues, their inability to entirely lyse cancerous cells, and the possibility of mutations in their genomes are among the challenges of bacteriotherapy for cancer. Herein, we summarize the mechanism of bacteria, their potential benefits and harms, and the future of research in this field.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Comparison of Ameliorative Effects between Probiotic and Biodegradable Bacillus subtilis on Zearalenone Toxicosis in Gilts.

This study was conducted to compare the potential ameliorative effects between probiotic Bacillus subtilis and biodegradable Bacillus subtilis on zearalenone (ZEN) toxicosis in gilts. Thirty-six Landrace×Yorkshire gilts (average BW = 64 kg) were randomly divided into four groups: (1) Normal control diet group (NC) fed the basal diet containing few ZEN (17.5 µg/kg); (2) ZEN contaminated group (ZC) fed the contaminated diet containing an exceeded limit dose of ZEN (about 300 µg/kg); (3) Probiotic agent group (PB) fed the ZC diet with added 5 × 109 CFU/kg of probiotic Bacillus subtilis ANSB010; (4) Biodegradable agent group (DA) fed the ZC diet with added 5 × 109 CFU/kg of biodegradable Bacillus subtilis ANSB01G. Results showed that Bacillus subtilis ANSB010 and ANSB01G isolated from broiler intestinal chyme had similar inhibitory activities against common pathogenic bacteria. In addition, the feed conversion ratio and the vulva size in DA group were significantly lower than ZC group (p < 0.05). The levels of IgG, IgM, IL-2 and TNFα in the ZC group were significantly higher than PB and DA groups (p < 0.05). The levels of estradiol and prolactin in the ZC group was significantly higher than those of the NC and DA groups (p < 0.05). Additionally, the residual ZEN in the feces of the ZC and PB groups were higher than those of the NC and DA groups (p < 0.05). In summary, the ZEN-contaminated diet had a damaging impact on growth performance, plasma immune function and hormone secretion of gilts. Although probiotic and biodegradable Bacillus subtilis have similar antimicrobial capacities, only biodegradable Bacillus subtilis could eliminate these negative effects through its biodegradable property to ZEN.

Moxetumomab Pasudotox in Hairy Cell Leukaemia: A Profile of Its Use.

Moxetumomab pasudotox (Lumoxiti®), an anti-CD22 recombinant immunotoxin, is an important treatment option that is approved in adults with relapsed or refractory hairy cell leukaemia (HCL) who have received at least two prior lines of treatment with systemic therapies including purine nucleoside analogues. In a pivotal phase III trial, treatment with moxetumomab pasudotox resulted in approximately one third of patients achieving durable complete response lasting more than 6 months, as well as improvements in other haematological parameters and disease-related symptoms. Moxetumomab pasudotox had a generally manageable tolerability profile; the most common treatment-related adverse events (AEs) included nausea, peripheral oedema, headache and pyrexia. AEs of special interest (including haemolytic uraemic syndrome and capillary leak syndrome) were generally manageable and reversible with monitoring and supportive care.

HCL is a rare form of leukaemia (≈ 2% of all leukaemia cases) that may cause symptoms such as anaemia, easy bruising and recurrent infection due to the low production of normal blood cells. Although treatment options are available, options are limited in patients with HCL who have relapsed (disease has reappeared after remission) or are refractory (does not respond) to treatment. Moxetumomab pasudotox (Lumoxiti^®) binds to a specific protein that is overexpressed on the surface of malignant B cells, and is approved to treat adults with relapsed or refractory HCL who have been treated at least twice with systemic therapies, including a purine nucleoside analogue, for HCL. In approximately one third of patients treated with moxetumomab pasudotox, no HCL cells were found in blood or bone marrow for at least 6 months; disease-related symptoms were also improved. Moxetumomab pasudotox had a generally manageable adverse event profile. While infrequent, serious adverse events such as haemolytic uraemic syndrome and capillary leak syndrome can occur, and are generally manageable and reversible with monitoring and supportive medical care (e.g. adequate oral hydration). Thus, moxetumomab pasudotox is an important treatment option in patients with relapsed or refractory HCL who have received at least two previous treatments for HCL.

Therapeutic Uses of Bacterial Subunit Toxins.

The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb3) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin-receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb3 (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb3 is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb3 is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb3 levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb3 is upregulated in the neovasculature of many tumours, irrespective of tumour Gb3 status. Gb3 is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.

Pseudomonas Exotoxin A-Based Immunotherapy Targeting CCK2R-Expressing Colorectal Malignancies: An In Vitro and In Vivo Evaluation.

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 IC50 values of GD9P in vitro ranged from 1.61 to 4.55 nM. Pharmacokinetic studies were conducted in mice with a T1/2 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.

Effects of trans-resveratrol on type 1 diabetes-induced up-regulation of apoptosis and mitogen-activated protein kinase signaling in retinal pigment epithelium of Dark Agouti rats.

Microvascular changes and retinal degeneration precede diabetic retinopathy. Oxidative stress alters several intracellular signaling pathways, which form the basis of diabetic retinopathy. Many antioxidants have been investigated as possible preventive and therapeutic remedies for diabetic retinopathy. The current study investigated the modulatory effects of trans-resveratrol on streptozotocin-induced type 1 diabetes mediated changes in the transcription and levels of apoptosis-related proteins and mitogen-activated protein kinases (MAPKs) in the retinal pigment epithelium (RPE) of adult male dark Agouti rats. In control rats, 5 mg/kg/d trans-resveratrol administration for 30 days increased gene expressions of tumor suppressor protein 53, Bcl2-associated X protein, B-cell lymphoma-2 (Bcl2), Caspase-3 (CASP3), CASP8 and CASP9, p38αMAPK, c-Jun N-terminal kinase-1 (JNK1), and extracellular signal-regulated kinase-1 (ERK1). On the other hand, diabetes decreased gene expressions of CASP3, CASP8, p38αMAPK, JNK, and ERK1. Trans-resveratrol reversed the inhibited gene expressions of CASP8, p38αMAPK, JNK, and ERK1 to normal control levels in diabetic rats. Trans-resveratrol normalized diabetes-induced upregulation of CASP3 and -9, cytochrome-c, Bcl-2, and ERK1 proteins. In conclusion, Trans-resveratrol-induced alterations in gene expressions do not seem to affect RPE functions as they do not reflect as altered protein functions. Trans-resveratrol imparts its protective effects by normalizing apoptosis-related proteins and ERK1 but does not affect JNK proteins. Trans-resveratrol causes cytostasis in RPE of normal rats by upregulating Bcl2 protein and apoptotic proteins.

[Hairy cell leukemia: What are the best treatment options for relapsed or refractory patients?] / Leucémie à tricholeucocytes : quelles sont les meilleures options thérapeutiques pour les patients en rechute ou réfractaires ?

Hairy cell leukemia is a rare form of leukemia: three hundred new cases are diagnosed each year in France. The diagnosis is based on: (1) morphological examination of the blood and bone marrow smear, (2) analysis by flow cytometry of hairy cells, which express three or the four following markers: CD11c, CD25, CD103 and CD123, (3) identification of the BRAFV600E mutation, a true molecular marker of the disease. The management of treatment has evolved considerably in recent years. As of today, the purine analogues remain the standard treatment in the first line. Relapses are however observed in about 40% of cases. In the event of a first relapse, the preferred option is treatment with immunochemotherapy i.e. a combination of cladribine plus rituximab. Subsequent relapses are treated with moxetumomab pasudotox or BRAF inhibitors which provide indisputable benefits if third-line treatment is required. We will discuss in patients with relapsed/refractory hairy cell leukemia the needs for personalized medicine and the advantages and disadvantages of each treatment modality. The good prognosis for LT requires treatments that are not immunosuppressive, non-myelotoxic, and do not increase the risk of secondary cancers.

Moxetumomab pasudotox in heavily pre-treated patients with relapsed/refractory hairy cell leukemia (HCL): long-term follow-up from the pivotal trial.

BACKGROUND: Moxetumomab pasudotox is a recombinant CD22-targeting immunotoxin. Here, we present the long-term follow-up analysis of the pivotal, multicenter, open-label trial (NCT01829711) of moxetumomab pasudotox in patients with relapsed/refractory (R/R) hairy cell leukemia (HCL). METHODS: Eligible patients had received ≥ 2 prior systemic therapies, including ≥ 2 purine nucleoside analogs (PNAs), or ≥ 1 PNA followed by rituximab or a BRAF inhibitor. Patients received 40 µg/kg moxetumomab pasudotox intravenously on Days 1, 3, and 5 of each 28-day cycle for up to six cycles. Disease response and minimal residual disease (MRD) status were determined by blinded independent central review. The primary endpoint was durable complete response (CR), defined as achieving CR with hematologic remission (HR, blood counts for CR) lasting > 180 days. RESULTS: Eighty adult patients were treated with moxetumomab pasudotox and 63% completed six cycles. Patients had received a median of three lines of prior systemic therapy; 49% were PNA-refractory, and 38% were unfit for PNA retreatment. At a median follow-up of 24.6 months, the durable CR rate (CR with HR > 180 days) was 36% (29 patients; 95% confidence interval: 26-48%); CR with HR ≥ 360 days was 33%, and overall CR was 41%. Twenty-seven complete responders (82%) were MRD-negative (34% of all patients). CR lasting ≥ 60 months was 61%, and the median progression-free survival without the loss of HR was 71.7 months. Hemolytic uremic and capillary leak syndromes were each reported in ≤ 10% of patients, and ≤ 5% had grade 3-4 events; these events were generally reversible. No treatment-related deaths were reported. CONCLUSIONS: Moxetumomab pasudotox resulted in a high rate of durable responses and MRD negativity in heavily pre-treated patients with HCL, with a manageable safety profile. Thus, it represents a new and viable treatment option for patients with R/R HCL, who currently lack adequate therapy. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01829711; first submitted: April 9, 2013. https://clinicaltrials.gov/ct2/show/NCT01829711.

Botulinum Toxin: An Update on Pharmacology and Newer Products in Development.

Since its introduction as a treatment for strabismus, botulinum toxin (BoNT) has had a phenomenal journey and is now recommended as first-line treatment for focal dystonia, despite short-term clinical benefits and the risks of adverse effects. To cater for the high demand across various medical specialties, at least six US Food and Drug Administration (FDA)-approved formulations of BoNT are currently available for diverse labelled indications. The toxo-pharmacological properties of these formulations are not uniform and thus should not be used interchangeably. Synthetic BoNTs and BoNTs from non-clostridial sources are not far from clinical use. Moreover, the study of mutations in naturally occurring toxins has led to modulation in the toxo-pharmacokinetic properties of BoNTs, including the duration and potency. We present an overview of the toxo-pharmacology of conventional and novel BoNT preparations, including those awaiting imminent translation from the laboratory to the clinic.

Anti-cancer Immunotoxins, Challenges, and Approaches.

The development of recombinant immunotoxins (RITs) as a novel therapeutic strategy has made a revolution in the treatment of cancer. RITs result from the fusion of antibodies to toxin proteins for targeting and eliminating cancerous cells by inhibiting protein synthesis. Despite indisputable outcomes of RITs regarding inhibition of multiple cancer types, high immunogenicity has been known as the main obstacle in the clinical use of RITs. Various strategies have been proposed to overcome these limitations, including immunosuppressive therapy, humanization of the antibody fragment moiety, generation of immunotoxins originated from endogenous human cytotoxic enzymes, and modification of the toxin moiety to escape the immune system. This paper is devoted to review recent advances in the design of immunotoxins with lower immunogenicity.

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.

Lectin drug conjugate therapy for colorectal cancer.

Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT-29, LoVo, LS174T, and DLD-1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN-38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line-derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD-1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN-PE38 group was significantly reduced compared with that using control treatment alone. However, the HT-29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN-PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans.