Search

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 μg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 μg/teat). MCF7 tumor-bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

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.

Background: In a multicenter phase 1 study of children with relapsed/refractory acute lymphoblastic leukemia (ALL), moxetumomab pasudotox, an anti-CD22 immunotoxin, demonstrated a manageable safety profile and preliminary evidence of clinical activity. A phase 2 study further evaluated efficacy., Procedure: This international, multicenter, phase 2 study enrolled children with relapsed/refractory B-cell precursor ALL who received moxetumomab pasudotox 40 µg/kg intravenously every other day, for six doses per 21-day cycle. The primary objective was to evaluate the complete response (CR) rate. Secondary objectives included safety, pharmacokinetics, and immunogenicity evaluations., Results: Thirty-two patients (median age, 10 years) were enrolled at 16 sites; 30 received study drug and were evaluable for safety; 28 were evaluable for response. The objective response rate was 28.6%, with three patients (10.7%) achieving morphologic CR, and five patients (17.9%) achieving partial response. Disease progression occurred in 11 patients (39.3%). Ten patients (33.3%) experienced at least one treatment-related serious adverse event, including capillary leak syndrome (CLS; n = 6), hemolytic uremic syndrome (HUS; n = 4), and treatment-related death (n = 1) from pulmonary edema. No differences were observed in inflammatory markers in patients who did or did not develop CLS or HUS., Conclusions: Despite a signal for clinical activity, this phase 2 study was terminated at interim analysis for a CR rate that did not reach the stage 1 target. Preclinical data suggest enhanced efficacy of moxetumomab pasudotox via continuous infusion or in combination regimens; thus, further studies designed to optimize the efficacy and safety of moxetumomab pasudotox in pediatric ALL may be warranted., (© 2020 Wiley Periodicals, Inc.)

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples., (Copyright © 2019. Published by Elsevier B.V.)

Staphylococcus aureus is a common pathogen causing infections in humans with various degrees of severity, with pneumonia being one of the most severe infections. In as much as staphylococcal pneumonia is a disease driven in large part by α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL), we evaluated whether active immunization with attenuated forms of Hla (HlaH35L/H48L) alone, PVL components (LukS-PVT28F/K97A/S209A and LukF-PVK102A) alone, or combination of all 3 toxoids could prevent lethal challenge in a rabbit model of necrotizing pneumonia caused by the USA300 community-associated methicillin-resistant S. aureus (MRSA). Rabbits vaccinated with Hla toxoid alone or PVL components alone were only partially protected against lethal pneumonia, whereas those vaccinated with all 3 toxoids had 100% protection against lethality. Vaccine-mediated protection correlated with induction of polyclonal antibody response that neutralized not only α-hemolysin and PVL, but also other related toxins, produced by USA300 and other epidemic MRSA clones., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Immunotoxins (ITs) are attractive anticancer modalities aimed at cancer-specific delivery of highly potent cytotoxic protein toxins. An IT consists of a targeting domain (an antibody, cytokine, or another cell-binding protein) chemically conjugated or recombinantly fused to a highly cytotoxic payload (a bacterial and plant toxin or human cytotoxic protein). The mode of action of ITs is killing designated cancer cells through the effector function of toxins in the cytosol after cellular internalization via the targeted cell-specific receptor-mediated endocytosis. Although numerous ITs of diverse structures have been tested in the past decades, only 3 ITs-denileukin diftitox, tagraxofusp, and moxetumomab pasudotox-have been clinically approved for treating hematological cancers. No ITs against solid tumors have been approved for clinical use. In this review, we discuss critical research and development issues associated with ITs that limit their clinical success as well as strategies to overcome these obstacles. The issues include off-target and on-target toxicities, immunogenicity, human cytotoxic proteins, antigen target selection, cytosolic delivery efficacy, solid-tumor targeting, and developability. To realize the therapeutic promise of ITs, novel strategies for safe and effective cytosolic delivery into designated tumors, including solid tumors, are urgently needed., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Mesothelin (MSLN) is a cell surface glycoprotein expressed at a high level on many malignancies, including pancreatic adenocarcinoma, serous ovarian cancer, and epithelioid mesothelioma. MSLN-targeted recombinant immunotoxins (RITs) consist of an anti-MSLN Fv fused to the catalytic domain of Pseudomonas exotoxin A. Recent data has also shown that MSLN is expressed at clinically relevant levels on the surface of colorectal cancer (CRC). In this study, CRC cell lines were tested for MSLN expression and susceptibility to MSLN-targeted RITs., Materials and Methods: CRC cell lines were tested for membranous MSLN expression via flow cytometry. Cell lines expressing MSLN were tested by WST-8 cell viability assay for sensitivity to various RITs and chemotherapeutic agents. CRC cell line SW-48 was tested in a mouse model for response to RIT as a single agent or in combination with actinomycin D and oxaliplatin., Results: CRC cell lines were susceptible to anti-MSLN RITs at half maximal inhibitory concentration levels comparable with those previously described in pancreatic cancer cell lines. In a nude mouse model, MSLN-targeted RIT treatment of SW48 CRC tumors resulted in a significant decrease in tumor volume. Although combination therapy with standard of care chemotherapeutic oxaliplatin did not improve tumor regressions, combination therapy with actinomycin D resulted in > 90% tumor volume reduction with 50% complete regressions., Conclusions: These data support the development of anti-MSLN RITs as well as other MSLN-targeted therapies for CRC., (Published by Elsevier Inc.)

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain‑derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA‑derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half‑life extension, can be used for specific killing of HER2‑expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50‑values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA‑derivatives are promising agents for targeted cancer therapy.

Background: Moxetumomab pasudotox is a promising new therapy for the treatment of patients with relapsed/refractory hairy cell leukemia (R/R HCL), but practical guidance relating to its administration is limited., Objectives: This article describes clinical guidelines for the administration of moxetumomab pasudotox to patients with R/R HCL and presents related case studies., Methods: A limited review of the literature on HCL was undertaken., Findings: Nursing care of patients prescribed moxetumomab pasudotox includes monitoring clinical and laboratory parameters, managing side effects, being aware of signs of serious side effects, and maintaining patient hydration during administration.

OBJECTIVE In this clinical trial report, the authors analyze safety and infusion distribution of IL13-Pseudomonas exotoxin, an antitumor chimeric molecule, administered via intratumoral convection enhanced delivery (CED) in pediatric patients with diffuse intrinsic pontine glioma (DIPG). METHODS This was a Phase I single-institution, open-label, dose-escalation, safety and tolerability study of IL13-PE38QQR infused via single-catheter CED into 5 pediatric DIPG patients. IL13-PE38QQR was administered to regions of tumor selected by radiographic findings. Two escalating dose levels were evaluated: 0.125 µg/mL in cohort 1 and 0.25 µg/mL in cohort 2. Real-time MRI was performed during intratumoral infusions, and MRI and MR spectroscopy were performed before and after the infusions. Clinical evaluations, including parent-reported quality of life (QOL), were assessed at baseline and 4 weeks post-infusion. RESULTS Direct infusion of brainstem tumor with IL13-PE using the CED technique in patients with DIPG produced temporary arrest of disease progression in 2 of 5 patients, both of whom subsequently received a second infusion. All 5 patients showed signs of disease progression by 12 weeks after initial infusion. Two patients experienced transient cranial nerve deficits and lethargy after infusion, and these deficits resolved with corticosteroid treatment in both cases. No patient had radiographic evidence of acute or long-term treatment toxicity. Parent-reported QOL was consistent with medical outcomes. CONCLUSIONS Even though IL13-PE delivered by CED did not reach the entire MRI-defined tumor volume in any patient, short-term radiographic antitumor effects were observed in 2 of the 5 patients treated. The patients’ performance status did not improve. Drug delivery using multiple catheters may produce improved outcomes. Clinical trial registration no.: NCT00088061 (clinicaltrials.gov) ABBREVIATIONS CED = convection-enhanced delivery; DIPG = diffuse intrinsic pontine glioma; IL-13 = interleukin 13; IL13R = IL-13 receptor; IPI = Impact of Pediatric Illness; KPS = Karnofsky Performance Status; LPS = Lansky Performance Status; MRS = MR spectroscopy; NAA = n-acetyl aspartate; QOL = quality of life; Vd = volume of distribution; Vi = volume of infusion.

Moxetumomab pasudotox-tdfk (LUMOXITI™), an anti CD22 recombinant immunotoxin, has been developed by MedImmune and its parent company AstraZeneca for the treatment of hairy cell leukaemia. The product, discovered at the National Cancer Institute, is an optimised version of immunotoxin CAT-3888. Moxetumomab pasudotox is composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A, PE38. The Fv portion of moxetumomab pasudotox binds to CD22, a cell surface receptor expressed on a variety of malignant B-cells, thereby delivering the toxin moiety PE38 directly to tumour cells. Once internalised, PE38 catalyses the ADP ribosylation of the diphthamide residue in elongation factor-2 (EF-2), resulting in the rapid fall in levels of the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1), leading to apoptotic cell death. This article summarizes the milestones in the development of moxetumomab pasudotox leading to this first approval for the treatment of adults with relapsed or refractory hairy cell leukaemia who received at least two prior systemic therapies, including treatment with a purine nucleoside analogue. Development of moxetumomab pasudotox for non-Hodgkin's lymphoma, chronic lymphocytic leukaemia and precursor cell lymphoblastic leukaemia/lymphoma was discontinued.

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tac (CD25) is expressed on multiple hematologic malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac and a 38-kDa fragment of Pseudomonas exotoxin A (PE38). Although LMB-2 has shown high cytotoxicity toward Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and nonspecific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T-cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site, which is required for cytotoxic activity. Although this strategy has been successfully implemented for mesothelin and CD22-targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of anti-Tac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II-deleted toxin variants. The domain II-deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored 5-fold of the cytotoxic activity and dramatically improved the MTD. Both of these improvements led to significantly increased antitumor efficacy in vivo We conclude that the next-generation anti-Tac immunotoxin is an improved candidate for targeting Tac-expressing malignancies. Mol Cancer Ther; 17(7); 1486-93. ©2018 AACR ., (©2018 American Association for Cancer Research.)

In many tumors, including prostate cancer, anti-apoptotic members of the Bcl-2 family are overexpressed and cause cell death resistance, which is a typical hallmark of cancer. Different therapeutic approaches, therefore, aim to restore the death mechanisms for enhanced apoptosis. Our recombinant immunotoxin D7(VL-VH)-PE40 is composed of the scFv D7(VL-VH) against the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer cells and of the cytotoxic domain of the bacterial toxin Pseudomonas Exotoxin A (PE40). Since Pseudomonas Exotoxin A-based immunotoxins are known to preferentially inhibit the expression of the anti-apoptotic protein Mcl-1, the rationale was to test our immunotoxin in combination with the BH3 mimetic ABT-737, which specifically inhibits Bcl-2, Bcl-xl, and Bcl-w for enhanced induction of apoptosis in prostate cancer cells. The immunotoxin showed high and specific binding and cytotoxicity against PSMA expressing prostate cancer cells marked by a direct inhibition of Mcl-1. The combination of the immunotoxin with a subtoxic concentration of ABT-737 caused additive or even synergistic effects, which were based on an enhanced apoptosis induction as detected by poly(ADP-ribose) polymerase (PARP) and Caspase-3 cleavage in Western blot. Our study shows that the combination therapy of immunotoxin plus ABT-737 is a promising approach for the future treatment of advanced prostate cancer to improve therapeutic efficacy and to reduce adverse side effects.

Moxetumomab pasudotox (Moxe) is a chimeric protein composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A and kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia, but many children with relapsed/refractory acute lymphoblastic leukemia (ALL) either respond transiently or are initially resistant. Resistance to Moxe in cultured cells is due to low expression of diphthamide genes (DPH), but only two of six ALL blast samples from resistant patients had low DPH expression. To develop a more clinically relevant model of resistance, we treated NSG mice bearing KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells were killed by Moxe, but relapse occurred from discrete bone marrow sites. The resistant cells would no longer grow in cell culture and showed major chromosomal changes and changes in phenotype with greatly decreased CD22. RNA deep sequencing of resistant KOPN-8 blasts revealed global changes in gene expression, indicating dedifferentiation toward less-mature B cell precursors, and showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival increased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells and is prevented by treatment with 5-azacytidine., Competing Interests: Conflict of interest statement: A.S.W. and I.P. are coinventors on patents assigned to the NIH for the investigational products. A.S.W. has received research support, honorarium, and travel support from Medimmune; and honorarium and travel support from Pfizer, Kite Pharma, and Spectrum Pharmaceuticals.

Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the first time, we report the synthesis and evaluation of polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Four different conjugates were synthesized using recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA) as the carrier proteins, using either direct reductive amination or an intermediate linker molecule, adipic acid dihydrazide (ADH). Upon injection into mice, a significantly higher antibody titer was observed in mice administrated with conjugate-1 (OSP-HSA) (P=0.0001) and conjugate 2 (OSP-rEPA) (P≤0.0001) as compared to OSP alone. In contrast, the antibody titer elicited by conjugate 3 (OSP ADH -HSA) and conjugate 4 (OSP ADH -rEPA) were insignificant (P=0.1684 and P=0.3794, respectively). We conclude that reductive amination is the superior method to prepare the S. Typhi OSP glycoconjugate. Moreover, rEPA was a better carrier protein than HSA. Thus OSP-rEPA conjugate seems to be efficacious typhoid vaccines candidate, it may be evaluated further and recommended for the clinical trials., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Purpose: Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA') was generated., Methods: We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa., Results: Alexa Fluor ® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells., Conclusions: Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Although the studies on the pathogenesis and prognosis of leukemia have made revolutionary progress, the long-term survival remains unsatisfactory. Alternative techniques are being developed to target leukemia. Several decades after researchers' work, a variety of bacteria toxins are being explored as potential anti-leukemia agents, either to provide direct effects or to deliver therapeutic proteins to leukemia. LukS-PV, a component of Panton-Valentine Leukocidin secreted by S. aureus, has been tested in acute myeloid leukemia as a novel experimental strategy. Further researches about the targeting mechanisms of LukS-PV are required to make it a complete therapeutic approach for leukemia treatment. The function of this article is to provide clinicians and experimentalists with a chronological and comprehensive appraisal of use of LukS-PV as an experimental strategy for leukemia therapy., (Copyright © 2016. Published by Elsevier B.V.)

Tumor-targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)-targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC-10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co-expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile. Biotechnol. Bioeng. 2016;113: 2698-2711. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc., (© 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

DARPins fused with other proteins are promising non-immunoglobulin scaffolds for specific binding to target cells. In this study HER2-specific DARPin (DARPin&#95;9-29) was used as a tumor-targeting moiety for the delivery of a cytotoxic agent - the fragment of Pseudomonas aeruginosa exotoxin A. It was determined that DARPin-PE40 possesses a considerable cytotoxic activity and induces apoptosis in HER2-positive cells. Cytotoxic effect of DARPin-PE40 strongly correlates with the HER2 expression level. The effect of intravenous administration of DARPin-PE40 was tested in the xenograft model of breast cancer. It was shown that treatment of animals with DARPin-PE40 caused strong and prolonged suppression of xenograft tumor growth., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: In previous years, immunotoxins have been shown to be a greatly promising therapeutic tool for brain malignancies, such as gliomas. Human mesenchymal stem cells (hMSCs) exhibit tropism to tumor tissue. However, the effect of bispecific immunotoxins in malignant gliomas is still unknown. The aim of this study was to investigate the function of bispecific immunotoxins in human malignant gliomas., Materials and Methods: In the present study, the bispecific immunotoxin VEGF165-ephrin A1-PE38KDEL was established using deoxyribonucleic acid shuffling and cloning techniques. The VEGF165-ephrin A1-PE38KDEL was delivered by hMSCs to mouse malignant gliomas. The effects of the bispecific immunotoxins on glioma-derived blood vessels and vasculogenic mimicry to elucidate the molecular mechanisms underlying the antitumorigenic effects of immunotoxins were examined in vivo., Results: In vitro, transfected hMSCs significantly inhibited the cell viability of gliomas cell lines U87 and U251 in a dose-dependent manner compared with untransfected hMSCs (P<0.01). In vivo, the intratumoral injection of engineered hMSCs was effective at inhibiting tumor growth in a malignant glioma tumor model., Conclusion: The bispecific immunotoxin secreted from hMSCs acts as a novel strategy for improving treatment options for malignant gliomas in the clinic.

Background: Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors., Purpose: Construct a new IT-targeting CCKR-overexpressing colon cancers., Methods: To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice., Results: The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18-26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8-3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice., Conclusions and Discussion: All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.

Typhoid fever caused by Salmonella Typhi is still a major public health problem in developing countries. In this study, we constructed a genetically modified Salmonella Typhi strain expressing O-specific polysaccharides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conjugates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccharides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.

Pseudomonas aeruginosa is an opportunistic bacterium that causes serious nosocomial infection in immunocompromised patients. The aim of this study was to prepare a fusion protein consisting of exotoxin A (ExoA) and flagellin (Fla) from P. aeruginosa and to evaluate its potential as a vaccine candidate against P. aeruginosa infection. The genes encoding for ExoA and Fla proteins were cloned in-frame and expressed in Escherichia coli. The recombinant ExoA-Fla fusion protein was purified by Ni-NTA affinity chromatography. Mice were immunized subcutaneously with ExoA, Fla, and ExoA-Fla fusion proteins, and the humoral immune response was evaluated by ELISA method. The immunized and control group mice were challenged with a 2× LD50 (7.5 × 10(7) CFU) of P. aeruginosa for the protection assay. The results indicated that vaccination with Fla, ExoA, and ExoA-Fla fusion proteins produced a significant amount of specific immunoglobulin G antibodies. Immunization of mice with ExoA-Fla fusion protein showed significant protection against intraperitoneal challenge with 7.5 × 10(7) CFU (2× LD50) P. aeruginosa. Results of this study suggest that recombinant ExoA-Fla fusion protein is a highly immunogenic protective protein showing promise as a vaccine candidate against P. aeruginosa.

This study establishes an experimental model for subclinical mastitis induced by Gram-positive (G+) exosecretions of Staphylococcus aureus origin or Gram-negative (G-) endotoxin of Escherichia coli origin to examine its effects on follicular growth and steroid concentrations in Holstein dairy cows. Cows were synchronized with the Ovsynch protocol followed by a series of follicular cycles that included GnRH and PGF2α doses administered every 8 days. Cows received small intramammary doses of either G+ (10 μg, n = 10) or G- (0.5 μg, n = 6) toxin, or saline (n = 6; uninfected control) every 48 hours for 20 days. Follicular fluids were aspirated from preovulatory follicles before (aspiration one: control), at the end of (aspiration two: immediate effect), and 16 days after the end of (aspiration three: carryover effect) toxin exposure. During the 3 weeks of subclinical mastitis induced by G+ or G-, no local inflammatory signs were detected in the mammary gland and no systemic symptoms were noted: body temperatures of the treated cows did not differ from controls; plasma cortisol and haptoglobin concentrations were not elevated and did not differ among groups. Somatic cell count was higher in the treated groups than in controls, and higher in the G- versus G+ group. For analysis of reproductive responses, cows were further classified as nonaffected or affected based on an more than 20% decline in follicular androstenedione concentration in aspiration two or three relative to the first, control aspiration. Most G- (5/6) and 40% of G+ (4/10) cows were defined as affected by induced mastitis. An immediate decrease in the number of medium-size follicles was recorded on Day 4 of the induced cycle, toward the end of the 20-day mastitis induction, in the affected G+ compared with uninfected control group (1.0 ± 0.5 vs. 3.0 ± 0.4 follicles; P < 0.05); the affected G- and nonaffected G+ subgroups exhibited a similar numerical decline in the number of follicles. A carryover (but not immediate) decrease to 51% and 62% in follicular estradiol concentrations in G- affected group and G+ affected group was detected relative to controls (P < 0.05). The nonaffected G+ subgroup did not differ from its control counterparts. Based on the current experimental model, subclinical IMI induced by G+ or G- toxin disrupts follicular functions, and it seems that the ovarian pool of early antral follicles is susceptible to subclinical mastitis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Impaired apoptosis is often a key element in tumor development. Therefore, drugs mimicking prosurvival antagonists offer promise as cancer therapeutics. When ABT-737, a BH3-only mimetic, was added to KB3-1 human cervical adenocarcinoma cells, we noted an induction of an endoplasmic reticulum (ER) stress response and the dislocation of ER luminal proteins, including chaperones, to the cell cytosol. Furthermore, when immunotoxin (antibody-toxin chimeric molecule) and ABT-737 combinations were added to cells, there was enhanced toxin-mediated inhibition of protein synthesis, consistent with enhanced translocation of toxin to the cytosol. A similar enhancement was not seen with thapsigargin, suggesting that ER stress alone was not responsible for enhanced translocation. Cytosol preparations from ABT-737-treated but not from thapsigargin-treated cells revealed the presence of greater amounts of processed 37-kDa toxin fragment compared with the addition of immunotoxin alone. As early as 4 hours after the addition of ABT-737 and immunotoxin, there was release of mitochondrial cytochrome c and activation of caspase-3/7 indicating that the combination caused apoptotic cell death. These results were reflected in decreased cellular ATP levels that were noted with combinations of ABT-737 and immunotoxin but not with either agent alone or with combinations of thapsigargin and immunotoxin. We conclude that ABT-737 increases ER permeability, promoting the dislocation of toxin from the ER to the cytosol resulting in early apoptotic cell death. These mechanistic insights suggest why this class of BH3-only mimetic synergizes in a particular way with Pseudomonas exotoxin-based immunotoxins., (©2014 American Association for Cancer Research.)

Object: Coinfused surrogate imaging tracers can provide direct insight into the properties of convection-enhanced delivery (CED) in the nervous system. To better understand the distributive properties of CED in a clinical circumstance, the authors analyzed the imaging findings in pediatric diffuse intrinsic pontine glioma (DIPG) patients undergoing coinfusion of Gd-DTPA and interleukin-13-Pseudomonas exotoxin (IL13-PE)., Methods: Consecutive patients undergoing CED (maximal rates of 5 or 10 μl/minute) of Gd-DTPA (1 or 5 mM) and IL13-PE (0.125 μg/ml or 0.25 μg/ml) for DIPG were included. Real-time MRI was performed during infusions, and imaging results were analyzed., Results: Four patients (2 males, 2 females; mean age at initial infusion 13.0 ± 5.3 years; range 5-17 years) underwent 5 infusions into DIPGs. Brainstem infusions were clearly identified on T1-weighted MR images at 1-mM (1 infusion) and 5-mM (4 infusions) coinfused Gd-DTPA concentrations. While the volume of distribution (Vd) increased progressively with volume of infusion (Vi) (mean volume 2.5 ± 0.9 ml; range 1.1-3.7 ml), final Vd:Vi ratios were significantly reduced with lower Gd-DTPA concentration (Vd:Vi for 1 mM of 1.6 compared with a mean Vd:Vi ratio for 5 mM of 3.3 ± 1.0) (p = 0.04). Similarly, anatomical distribution patterns were affected by preferential flow along parallel axial fiber tracts, into prior infusion cannula tracts and intraparenchymal air pockets, and leak back around the infusion cannula at the highest rate of infusion., Conclusions: Magnetic resonance imaging of a coinfused Gd-DTPA surrogate tracer provided direct insight into the properties of CED in a clinical application. While clinically relevant Vds can be achieved by convective delivery, specific tissue properties can affect distribution volume and pattern, including Gd-DTPA concentration, preferential flow patterns, and infusion rate. Understanding of these properties of CED can enhance its clinical application. Part of clinical trial no. NCT00880061 ( ClinicalTrials.gov ).

Recombinant immunotoxins (RIT) are agents being developed for cancer treatment. They are composed of an Fv that binds to a cancer cell, fused to a 38-kDa fragment of Pseudomonas exotoxin A. SS1P is a RIT that targets mesothelin, a protein expressed on mesothelioma as well as pancreatic, ovarian, lung, and other cancers. Because the protein tyrosine kinase family regulates a variety of cellular processes and pathways, we hypothesized that tyrosine kinases might regulate susceptibility to immunotoxin killing. To investigate their role, we used siRNAs to lower the level of expression of the 88 known tyrosine kinases. We identified five tyrosine kinases, INSR, HCK, SRC, PDGFRβ, and BMX that enhance the activity of SS1P when their level of expression is lowered by siRNAs. We further investigated the Src family member HCK in this study. Knocking down of SRC slightly increased SS1P killing in A431/H9 cells, but knocking down HCK substantially enhanced killing by SS1P. We investigated the mechanism of enhancement and found that HCK knockdown enhanced SS1P cleavage by furin and lowered levels of Mcl-1 and raised Bax. We then found that Src inhibitors mimic the stimulatory effect of HCK knockdown; both SU6656 and SKI-606 (bosutinib) enhanced immunotoxin killing of mesothelin-expressing cells by SS1P and CD22-expressing cells by HA22 (moxetumomab pasudotox). SU6656 also enhanced the antitumor effects of SS1P and HA22 in mouse xenograft tumor models. Our data suggest that the combination of immunotoxin with tyrosine kinase inhibitors may be an effective way to treat some cancers.

Instillation of silica into the lungs of rodents results in pathological changes that strongly mimic human silicosis, an occupational lung disease marked by restrictive airway obstruction, inflammation, and fibrosis. Because IL-13 is a pivotal proinflammatory and fibrogenic cytokine, we examined whether a recombinant immunotoxin comprised of human IL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE) might affect pathological features of experimental silicosis. Mice received a single intranasal instillation of silica particles and were treated with intranasal IL-13-PE every other day from days 21 to 27 postsilica. The sensitivity of putative cell targets to IL-13-PE was also assessed in in vitro settings. Upregulation of IL-13, its receptor subunits IL-13Rα1 and IL-13Rα2, and shared receptor IL-4Rα were associated with development of granulomatous lung inflammation triggered by silica. IL-13-PE inhibited silica-induced granuloma and fibrotic responses noted at 24 h and 15 d after the last treatment. Upregulation of TNF-α, TGF-β, and chemokines, as well as increased collagen deposition and airway hyperreactivity to methacholine were all clearly sensitive to IL-13-PE. In addition, IL-13-PE inhibited both IL-13-induced proliferation of cultured lung fibroblasts from silicotic mice and silica-induced IL-8 generation from A549 cells. In conclusion, our findings show that therapeutic treatment with IL-13-PE can reverse important pathological features caused by inhalation of silica particles, suggesting that this recombinant immunotoxin is a promising molecular template in drug discovery for the treatment of silicosis.

A major challenge associated with allogeneic hematopoietic stem cell transplantation is effective prevention and/or attenuation of symptoms associated with acute graft-versus-host disease (aGVHD) that can result from a failure of either host and/or donor CD4(+)CD25(+) regulatory T (Tr) and CD8(+)CD28 suppressor T (Ts) cells to dampen immunopathogenic responses mediated by alloreactive donor CD4(+)CD28(+) Th1 (Th1) and CD8(+)CD28(-) Tc1 (Tc1) cell-mediated inflammatory processes. Considering the crucial role of CD28/B7-1 costimulatory signal pathway in development, activation, differentiation, and function of these T subsets, we developed a targeted DNA vaccine encoding the Pseudomonas exotoxin-A and the B7-1 molecule that could act as both an antagonist to Th1-mediated and Tc1-mediated responses while concomitantly acting as an agonist stimulating Tr-mediated and Ts-mediated responses as a strategy for reducing aGVHD-associated lethality. A single intramuscular injection with this vaccine significantly increased Tr and Ts levels in a murine aGVHD model. In addition, immunized mice presented with significantly diminished Th1-cytokines interferon-γ and interleukin-2 response and a moderately upregulated Th2-cytokine interleukin-10 and Th3-cytokine transforming growth factor-β response. More importantly, vaccination significantly reduced aGVHD, measured by significantly extended mean survival times, decreased mean weight loss, recovery of peripheral leukocyte numbers, disease presentation associated with mild-moderate histopathologic changes, a balanced Th1-Th2-Th3-cytokine responses and functional Tr-mediated and Ts-mediated regulatory/suppressor mechanisms at levels more potent than observed in animals treated with cyclosporine A+methotrexate. Our data first provide the proof-of-principal that B7-1-PE40KDEL targeted DNA vaccine represents a prophylactic approach for reducing alloreactive Th1-mediated and Tc-mediated aGVHD lethality by CD28/B7-1 axis.

Moxetumomab pasudotox is an immunotoxin currently being investigated in patients for the treatment of CD22-expressing B-cell malignancies. A single-cycle pharmacokinetic (PK)-pharmacodynamic (PD) study was conducted in cynomolgus monkeys for PK comparability assessment and population PK-PD modeling after major manufacturing process and site changes. Primates were randomized by body weight and baseline CD22 lymphocyte counts to receive intravenous administrations of 1 mg/kg moxetumomab pasudotox (n = 12/group) on Days 1, 3, and 5. PK and B-lymphocyte count data were modeled using a population approach. The 90% confidence intervals of the geometric mean ratios of PK exposure were within the 80%-125% range. The B lymphocytes were depleted to a similar extent, and the immunogenicity incidences were similar across the two groups. The B-cell depletion was described by a novel lifespan model in which moxetumomab pasudotox induced random destruction of B cells in each aging compartment. The endogenous de novo influx from bone marrow was subject to a negative feedback mechanism. The estimated B cell apparent lifespan was 51 days. Covariate analysis confirmed that the manufacturing change had no impact on PK or PD of moxetumomab pasudotox. Results from this study supported continued clinical investigation of moxetumomab pasudotox using the new material., (Copyright © 2012 Wiley Periodicals, Inc.)

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo., Methods: We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models., Results: Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain., Conclusions: Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation.

Purpose: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2., Experimental Design: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays., Results: In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose., Conclusions: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies., (©2011 AACR.)

Overexpressed receptors, characteristic of many cancers, have been targeted by various researchers to achieve a more specific treatment for cancer. A common approach is to use the natural ligand for the overexpressed receptor as a cancer-targeting agent which can deliver a chemically or genetically conjugated toxic molecule. However, it has been found that the therapeutic efficacy of such ligand-drug molecular conjugates can be limited, since they naturally follow the intracellular trafficking pathways of the endogenous ligands. Therefore, a thorough understanding of the intracellular trafficking properties of these ligands can lead to novel design criteria for engineering ligands to be more effective drug carriers. This review presents a few commonly used ligand/receptor systems where intracellular trafficking considerations can potentially improve the therapeutic efficacy of the ligand-drug molecular conjugates.

Purpose: Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)-specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A., Experimental Design: DARPin Ec4 was genetically fused to a truncated form of Pseudomonas aeruginosa exotoxin A (ETA″) and expressed in Escherichia coli. The cytotoxicity of Ec4-ETA″ was measured against tumor cell lines of various histotypes in vitro. Tumor localization and antitumor activity were determined in mice bearing 2 different EpCAM-positive tumor xenografts., Results: Ec4-ETA″ expressed very well in soluble form in the cytoplasm of E. coli and yielded up to 40 mg after purification per liter of culture. The protein was monomeric and the disulfides of ETA″ formed spontaneously. Ec4-ETA″ bound to EpCAM with low nanomolar affinity, similar to free Ec4. Furthermore, it was highly cytotoxic against various EpCAM-positive tumor cell lines in vitro with IC(50) values less than 0.005 pmol/L. This effect was competed by free Ec4, but not by unspecific DARPins. Upon systemic administration in athymic mice, Ec4-ETA″ efficiently localized to EpCAM-positive tumors to achieve maximum accumulation 48 to 72 hours after injection, whereas an irrelevant control fusion toxin did not accumulate. Tumor targeting with Ec4-ETA″ resulted in a strong antitumor response including complete regressions in some animals., Conclusions: Our data show for the first time the potential of DARPins for the generation of protein therapeutics for tumor targeting, and that Ec4-ETA″ deserves attention for clinical development., (©2010 AACR.)

Rheumatoid arthritis is a proinflammatory autoimmune disease attributed to failure of both CD4(+)CD25(+) regulatory T (Tr) and CD8(+)CD28(-) suppressor T (Ts) cells to control autoreactive CD4(+)CD28(+) Th1 (Th1) and autoantibody-producing B cells. Here we show a single intramuscular injection of our novel targeted DNA vaccine encoding Pseudomonas exotoxin A and costimulatory molecule B7-2 without autoantigens in a collagen-induced arthritis model simultaneously increased Tr and Ts cells and selectively decreased autoreactive Th1 cells. The vaccine induced a shift from Th1 to Th2 and Th3 cellular and cytokine profiles and a decrease in CD4(+)/CD8(+) cell ratios. Importantly, the vaccine showed potent antirheumatic activity by clinical and other examinations such as X-ray, histopathology, and anti-type II collagen IgG levels and was comparable to methotrexate, the current "gold standard" treatment. As an effective stimulator of both Tr and Ts cells and a specific suppressor of autoreactive Th1 cells, this vaccine is a promising therapeutic approach for rheumatoid arthritis.

It has been suggested that alveolar and interstitial macrophages play a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) by producing proinflammatory and/or fibrogenic cytokines. We showed that inflammatory macrophages expressed folate receptor beta (FRbeta) while resident macrophages in normal tissues expressed no or low levels of FRbeta. In the present study, we examined the distribution of FRbeta-expressing macrophages in the lungs of patients with usual idiopathic pulmonary fibrosis (UIP) and mice with bleomycin-induced pulmonary fibrosis (PF) and tested whether the depletion of FRbeta-expressing macrophages could suppress bleomycin-induced PF in mice. Immunostaining with anti-human or -mouse FRbeta monoclonal antibody (mAb) revealed that FRbeta-expressing macrophages were present predominantly in fibrotic areas of the lungs of patients with UIP and mice with bleomycin-induced PF. Intranasal administration of a recombinant immunotoxin, consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRbeta mAb and truncated Pseudomonas exotoxin A, increased survival significantly and reduced levels of total hydroxyproline and fibrosis in bleomycin-induced PF. In immunohistochemical analysis, decreased numbers of tumour necrosis factor-alpha-, chemokines CCL2- and CCL12-producing cells were observed in the immunotoxin-treated group. These findings suggest a pathogenic role of FRbeta-expressing macrophages in IPF. Thus, targeting FRbeta-expressing macrophages may be a promising treatment of IPF.

Convection-enhanced delivery (CED) of cintredekin besudotox (CB) was compared with Gliadel wafers (GW) in adult patients with glioblastoma multiforme (GBM) at first recurrence. Patients were randomized 2:1 to receive CB or GW. CB (0.5 microg/mL; total flow rate 0.75 mL/h) was administered over 96 hours via 2-4 intraparenchymal catheters placed after tumor resection. GW (3.85%/7.7 mg carmustine per wafer; maximum 8 wafers) were placed immediately after tumor resection. The primary endpoint was overall survival from the time of randomization. Prestated interim analyses were built into the study design. Secondary and tertiary endpoints were safety and health-related quality-of-life assessments. From March 2004 to December 2005, 296 patients were enrolled at 52 centers. Demographic and baseline characteristics were balanced between the 2 treatment arms. Median survival was 36.4 weeks (9.1 months) for CB and 35.3 weeks (8.8 months) for GW (P = .476). For the efficacy evaluable population, the median survival was 45.3 weeks (11.3 months) for CB and 39.8 weeks (10 months) for GW (P = .310). The adverse-events profile was similar in both arms, except that pulmonary embolism was higher in the CB arm (8% vs 1%, P = .014). This is the first randomized phase III evaluation of an agent administered via CED and the first with an active comparator in GBM patients. There was no survival difference between CB administered via CED and GW. Drug distribution was not assessed and may be crucial for evaluating future CED-based therapeutics.

Aim: To obtain recombinant atoxic form of Pseudomonas aeruginosa exotoxin A and assess its protective properties during simultaneous administration with recombinant protein F in experiment., Materials and Methods: Genetic methods were employed for construction of deletion variant of P. aeruginosa exotoxin A gene, whereas Escherichia coli cells were used for transformation. Purification of proteins was performed by common method. White outbred mice were used for immunization and experimental infection was produced by intraperitoneal administration of live virulent culture of P. aeruginosa strain PA103., Results: The gene coding defect form of P. aeruginosa exotoxin A with lacked 106 C-terminal aminoacid residues was cloned. Synthesized protein was nontoxic and immunogenic. Recombinant variants of anatoxin A and outer membrane protein F of P. aeruginosa protected animals from experimental infection caused by toxigenic strain PA103 of P. aeruginosa if were administered separately or concomitantly. Nonetheless, the highest protective effect was observed after immunization with both proteins., Conclusion: Studied recombinant toxoid and OprF could be the candidates for inclusion in vaccines for prevention of pseudomonas infection.

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

This study was done to determine if intranasal vaccination of weaned beef calves with a chimeric protein containing the immunodominant surface epitope of Mannheimia haemolytica PlpE (R2) and the neutralizing epitope of leukotoxin (NLKT) covalently linked to truncated cholera toxin (CT) subunit B (CTB) could stimulate secretory and systemic antibodies against M. haemolytica while enhancing resistance of cattle against M. haemolytica intrabronchial challenge. Sixteen weaned beef calves were intranasally vaccinated with CTB-R2-NLKT chimeric (SAC102) or with R2-NLKT-R2-NLKT chimeric (SAC89) protein with or without native CT on days 0 and 14 and were challenged intrabronchially on day 28. In vitro, SAC102 bound the CT receptor molecule, GM(1)-ganglioside. Mean IgA antibodies to M. haemolytica whole cells (WC) and to LKT were high on day 0. A small, yet significant increase (p<0.05) was found in mean nasal antibodies to M. haemolytica WC for the SAC89+CT and SAC102 vaccinates after the second vaccination. SAC102 stimulated significant (p<0.05) mean serum antibody responses to all three antigens by day 28. Following challenge, mean antibodies to WC and LKT significantly increased (p<0.05) for the SAC102, SAC89 and SAC89+CT groups with the mean antibody responses to rPlpE stimulated by SAC102 vaccination being significantly higher (p<0.05) than for the other vaccinated and control groups. On day 1 after challenge, mean clinical score for the control group was significantly higher (p<0.05) than for the SAC102 and SAC89+CT vaccinates, and by day 2 after challenge, clinical score for the control group was significantly higher (p<0.05) than for all three chimeric vaccinated groups. Therefore, intranasal vaccination with CTB-R2-NLKT (SAC102) and R2-NLKT-R2-NLKT (SAC89) chimeric proteins enhanced resistance against intrabronchial challenge with the bacterium as well as stimulating antibody responses to M. haemolytica antigens.

Convection-enhanced drug delivery (CED) enables achieving a drug concentration within brain tissue and brain tumors that is orders of magnitude higher than by systemic administration. Previous phase I/II clinical trials using intratumoral convection of interleukin-4 Pseudomonas exotoxin (PRX321) have demonstrated an acceptable safety and toxicity profile with promising signs of therapeutic activity. The present study was designed to assess the distribution efficiency and toxicity of this PRX321 using magnetic resonance imaging (MRI) and to test whether reformulation with increased viscosity could enhance drug distribution. Convection of low- [0.02% human serum albumin (HSA)] and high-viscosity (3% HSA) infusates mixed with gadolinium-diethylenetriamine pentaacetic acid and PRX321 were compared with low- and high-viscosity infusates without the drug, in normal rat brains. MRI was used for assessment of drug distribution and detection of early and late toxicity. Representative brain samples were subjected to histological examination. Distribution volumes calculated from the magnetic resonance images showed that the average distribution of 0.02% HSA was larger than that of 0.02% HSA with PRX321 by a factor of 1.98 (p < 0.02). CED of 3.0% HSA, with or without PRX321, tripled the volume of distribution compared with 0.02% HSA with PRX321 (p < 0.015). No drug-related toxicity was detected. These results suggest that the impeded convection of the PRX321 infusate used in previous clinical trials can be reversed by increasing infusate viscosity and lead to tripling of the volume of distribution. This effect was not associated with any detectable toxicity. A similar capability to reverse impeded convection was also demonstrated in a CED model using acetic acid. These results will be implemented in an upcoming phase IIb PRX321 CED trial with a high-viscosity infusate.

VB4-845 is a recombinant immunotoxin that is comprised of a truncated form of Pseudomonas exotoxin A (ETA) genetically-linked to a humanized scFv fragment, (4D5MOCB), specific to epithelial cell adhesion molecule (EpCAM). EpCAM is overexpressed on a wide variety of human tumors and thus represents a suitable target antigen for immunotoxin therapy. Preclinical studies were used to evaluate the benefit of locoregional administration of an ETA-based immunotoxin versus systemic delivery. Repeated subcutaneous (s.c.) administration of VB4-845 (up to 77.8 microg/kg) in rats resulted in minimal adverse effects, except for injection-site reactions, while repeated systemic administration elicited symptoms consistent with vascular leak syndrome. S.c. weekly doses of the drug in cynomolgus monkeys resulted in minimal adverse effects limited to injection-site reactions and a transient elevation of liver enzymes in 1 animal. Toxicokinetics showed rapid clearance of the drug, with the development of an immune response by day 14 following repeated injections. These results argue that the local administration of VB4-845 has advantages with respect to safety over systemic administration and may be an effective alternative method for targeting those cancers that are amenable to local routes of administration.

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. Despite treatment with surgery, radiation therapy, and chemotherapy, the prognosis remains poor, particularly for glioblastoma, which has a median survival of 12 to 15 months. An important impediment to finding effective treatments for malignant gliomas is the presence of the blood brain barrier, which serves to prevent delivery of potentially active therapeutic compounds. Multiple efforts are focused on developing strategies to effectively deliver active drugs to brain tumor cells. Blood brain barrier disruption and convection-enhanced delivery have emerged as leading investigational delivery techniques for the treatment of malignant brain tumors. Clinical trials using these methods have been completed, with mixed results, and several more are being initiated. In this review, we describe the clinically available methods used to circumvent the blood brain barrier and summarize the results to date of ongoing and completed clinical trials.

Background: Pseudomonas aeruginosa is an important cause of nosocomial infection and may lead to septicemia and death. We evaluated the immunogenicity of semi-purified exotoxin A from the bacterium in a mouse burn model., Methods: The toxoid was prepared from exotoxin A taken from toxigenic strains of P. aeruginosa (PA 103). 50 mice were immunized with the toxoid, burned with hot metal and infected with 1 x 10(8) CFU of toxigenic strains of P. aeruginosa (experimental group); 25 non-immunized mice were also burned and infected (control group). The mortality rate and presence of any exotoxin and P. aeruginosa in the sera, liver and spleen were determined., Results: In the experimental group, 2 mice died before the burns were administered and were excluded from the study. The remainder (48 mice) were challenged with a lethal dose of P. aeruginosa and followed for 70 days. 3 of these mice died. Neither P. aeruginosa nor exotoxin A was not detected in the liver, spleen or sera of the surviving mice. The protective efficacy of toxoid vaccination was therefore 93.8%. In the control group, all mice died from bacteremia and septicemia, most (80%) within 6 days, and P. aeruginosa and exotoxin A were isolated from sera, spleen and liver., Conclusion: Active immunization of mice using a semi-purified exotoxin A derived from P. aeruginosa was 93.8% effective at protecting mice from subsequent P. aeruginosa infections in a mouse burn model.