Spatiotemporally controlled Pseudomonas exotoxin transgene system combined with multifunctional nanoparticles for breast cancer antimetastatic therapy.

The tumor microenvironment is a barrier to breast cancer therapy. Cancer-associated fibroblast cells (CAFs) can support tumor proliferation, metastasis, and drug resistance by secreting various cytokines and growth factors. Abnormal angiogenesis provides sufficient nutrients for tumor proliferation. Considering that CAFs express the sigma receptor (which recognizes anisamide, AA), we developed a CAFs and breast cancer cells dual-targeting nano drug delivery system to transport the LightOn gene express system, a spatiotemporal controlled gene expression consisting of a light-sensitive transcription factor and a specific minimal promoter. We adopted RGD (Arg-Gly-Asp) to selectively bind to the αvß3 integrin on activated vascular endothelial cells and tumor cells. After the LightOn system has reached the tumor site, LightOn gene express system can spatiotemporal controllably express toxic Pseudomonas exotoxin An under blue light irradiation. The LightOn gene express system, combined with multifunctional nanoparticles, achieved high targeting delivery efficiency both in vitro and in vivo. It also displayed strong tumor and CAFs inhibition, anti-angiogenesis ability and anti-metastasis ability, with good safety. Moreover, it improved survival rate, survival time, and lung metastasis rate in a mouse breast cancer model. This study proves the efficacy of combining the LightOn system with targeted multifunctional nanoparticles in tumor and anti-metastatic therapy and provides new insights into tumor microenvironment regulation.

New insights on Pseudomonas aeruginosa exotoxin A-based immunotoxins in targeted cancer therapeutic delivery.

Pseudomonas aeruginosa exotoxin A-based immunotoxins (PE-ITs) are fusion proteins that harness targeting and toxin moieties. Structural optimizations in PE and targeting moieties were implemented to lower their immunogenicity and alleviate undesirable side effects. PE moiety was engineered to lack its cell-binding domain and T cell epitope regions, whereas single chain (scFv) and disulfide Fv portions (dsFv), nanobodies, and monobodies were utilized as targeting moieties. This review discusses applications of PE-ITs on different types of cancer, structural optimizations to reduce PE-ITs drawbacks, and recent modifications applied for efficient therapeutic delivery. Finally, we draw attention to the possibility of combining radiotherapy, radionuclides, and RGDs with PE-IT to improve overall response rates of IT-based treatments and reduce cancer cell resistance.

Exotoxin A-immunotoxins are proteins that have been used in cancer treatments. The building components of these proteins are very poisonous to both cancer and normal cells. Also, unfavorable body reactions and side effects were seen with their usage. To allow the safe use of these proteins, changes were made in their building components. These changes made them damaging only to cancer cells while being safe to normal non-cancerous cells. This review will talk about the use of exotoxin A-Immunotoxins in different cancer treatments, and how they are created to limit the poisonous effect of their building components to only cancer cells.

Development of anti DLL4 Nanobody fused to truncated form of Pseudomonas exotoxin: As a novel immunotoxin to inhibit of cell proliferation and neovascularization.

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.

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.

Recent development and optimization of pseudomonas aeruginosa exotoxin immunotoxins in cancer therapeutic applications.

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.

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.

[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.

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.

The pharmacological management of hairy cell leukemia.

INTRODUCTION: Hairy cell leukemia (HCL) is a B-cell lymphoid malignancy that accounts for approximately 2% of all leukemias. Treatment with purine nucleoside analogs (PNA) results in a high response rate and remains the standard of care. Long term follow-up shows that most patients relapse and require retreatment. Newer combination strategies and agents have emerged to try to reduce the relapse rate and to address cases of PNA refractoriness. AREAS COVERED: The authors reviewed the literature on the pharmacological management of HCL, including recent studies that led to new agents being incorporated into practice. EXPERT OPINION: Combination of cladribine plus rituximab produces a high rate of measurable residual disease-negative complete remission. In our center, newly diagnosed patients are offered cladribine followed by 8 weekly doses of rituximab in an ongoing phase II trial. Patients in first relapse are also offered this combination if they were initially treated with a single-agent PNA, or if the remission duration was ≥5 years after first-line cladribine plus rituximab. Patients who relapse within 5 years are offered therapy with a novel agent that may include the BRAF inhibitor vemurafenib, alone or in combination with rituximab, dabrafenib in combination with trametinib, the BTK inhibitor ibrutinib, or moxetumomab pasudotox.

Engineered Anti-GPC3 Immunotoxin, HN3-ABD-T20, Produces Regression in Mouse Liver Cancer Xenografts Through Prolonged Serum Retention.

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 KD 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.

Contextualizing the Use of Moxetumomab Pasudotox in the Treatment of Relapsed or Refractory Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is an indolent B-cell malignancy characterized by high initial sensitivity to purine analog chemotherapy, minimal residual disease (MRD) frequently accompanying complete remission (CR), and relapses requiring additional treatment. Repeat chemotherapy shows decreasing efficacy and increasing toxicity with each course. Newer therapies targeting BRAF/MEK or Bruton's tyrosine kinase are effective but generally leave MRD. Rituximab has modest activity as a single agent and can achieve MRD-negative CR in combination with purine analogs, but there is significant toxicity from the chemotherapy. Moxetumomab pasudotox-tdfk (Moxe) is a biologic containing an antibody fragment (Fv) binding to CD22, attached to a portion of Pseudomonas exotoxin A. Binding to CD22 enables the toxin to enter and kill cells. Moxe is administered by 30-minute infusions on days 1, 3, and 5 of up to six cycles spaced 4 weeks apart. In phase I testing, 64% of 33 patients at the highest dose level achieved CR, most without MRD. Lack of MRD correlated with prolonged CR duration; of 11 MRD-negative CRs, 10 were still in CR after a median of 42 months of observation. In pivotal testing, 75% of 80 patients had a hematologic response, 41% with CR; 82% (27/33) of CRs were MRD-negative, and only 4 of the 27 MRD-negative patients relapsed during the follow-up period. Hemolytic uremic syndrome and capillary leak syndrome were each observed in 9% of patients, all reversible. In September 2018, the U.S. Food and Drug Administration approved Moxe for the treatment of relapsed/refractory HCL after ≥2 prior therapies. Moxe is undergoing further development in combination with rituximab. IMPLICATIONS FOR PRACTICE: Hairy cell leukemia (HCL) has effective treatments including purine analogs with and without rituximab, and oral inhibitors of BRAF, MEK and Bruton's tyrosine kinase (BTK). Despite these therapies, relapse occurs, and moxetumomab pasudotox has an important role in relapsed and refractory HCL because of its ability to achieve high rates of complete remissions (CRs) without chemotherapy; most of these CRs are without minimal residual disease (MRD). CR duration is enhanced in patients who achieve eradication of MRD. To improve the efficacy of this recombinant immunotoxin, a phase I trial is underway in combination with rituximab to reduce tumor burden and decrease immunogenicity.

Moxetumomab pasudotox for hairy cell leukemia: preclinical development to FDA approval.

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.

Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA; Leukothera®): Mechanisms of Action and Therapeutic Applications.

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 ß2 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.

Moxetumomab pasudotox for the treatment of relapsed and/or refractory hairy cell leukemia.

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).

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy.

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