Fusion Toxin Research Articles

Targeting Latent Human Cytomegalovirus (CMV) with a Novel Fusion Toxin Protein during Ex Vivo Lung Perfusion

Donor to recipient CMV mismatch leads to high incidence of CMV infection post lung transplant leading to inferior long-term patient outcomes. CMV latently infected cells ubiquitously express a surface marker, US28. We aimed to evaluate the therapeutic effects of a novel fusion toxin protein (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV reactivation post transplantation. Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected to evaluate viral reactivation using an in vitro assay that induces latent CMV to reactivate to an infectious phase. Since US28 has 38% homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied using flow cytometry for cell death assessment of CD34+ and CD14+ cells. Lung function on EVLP was evaluated as a safety endpoint. F49A-FTP was delivered through the PA on EVLP with no acute toxic events based on physiological parameters (Figure 1A). We performed an in vitro assay using tissue samples obtained after EVLP to stimulate CMV to reactivate in order to assess efficacy of the drug. After primary culture from lung tissue samples, relative reactivation events (assessed with IFF staining for IE protein of lytic CMV, Figure 1B) occurred in 100% of controls vs. only 0.04±0.02% of treated lungs. For off-target analysis, we performed flow cytometry and calculated the ratio of post:pre perfusion CD34+ and CD14+ cells (Figure 1C). There were no changes in number of cells for both groups: CD34+ ratio 0.8±0.5 vs. 0.6±0.5 (control and F49A-FTP, respectively, p=0.4974); CD14+ ratio 1.54±0.74 vs. 1.66±1.13 (control and F49A-FTP, respectively, p=0.8660). Our study demonstrates that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation. Ongoing ex vivo and in vivo reactivation experiments will confirm these findings.

Bispecific human IL2-CCR4 immunotoxin targets human cutaneous T-cell lymphoma.

The majority of clinically diagnosed cutaneous T‐cell lymphomas (CTCL) highly express the cell‐surface markers CC chemokine receptor 4 (CCR4) and/or CD25. Recently, we have developed diphtheria toxin‐based recombinant Ontak®‐like human IL2 fusion toxin (IL2 fusion toxin) and anti‐human CCR4 immunotoxin (CCR4 IT). In this study, we first compared the efficacy of the CCR4 IT vs IL2 fusion toxin for targeting human CD25+CCR4+ CTCL. We demonstrated that CCR4 IT was more effective than IL2 fusion toxin. We further constructed an IL2‐CCR4 bispecific IT. The bispecific IT was significantly more effective than either IL2 fusion toxin or CCR4 IT alone. The bispecific IT is a promising novel targeted therapeutic drug candidate for the treatment of refractory and recurrent human CD25+ and/or CCR4+ CTCL.

Application of therapeutic protein-based fusion toxins

Protein-based therapeutics have been applied for decades to remove most malignant tumors. Many anti-tumor drugs using antibodies have been developed and put to practical use. However, due to limitations of antibodies such as tolerance, high molecular weight, and poor tissue penetration, new types of fusion proteins have been developed for therapeutic purposes. In this study, we review the recent therapeutic trials and improvements of fusion protein toxins. As a targeting moiety, non-Ig scaffolds have not only the advantages of immunoglobulin such as high affinity and selectivity, but also small size, high stability, high yield expression. As a toxic moiety, non-immunologic and highly toxic endogenous proteins of human origin like proapoptotic protein or RNase are challenged. To lessen the adverse reactions of fusion toxins, several therapeutic strategies such as removal of epitopes, increase of serum half-life were developed.

Advances in the treatment of cytomegalovirus.

BackgroundHuman cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances.Sources of dataSources of data are recently published research papers and reviews about HCMV treatments.Areas of agreementCurrent antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA.Areas of controversyShould we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection?Growing pointsGrowing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells.Areas timely for developing researchWe need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.

Purification and Characterization of DT389GCSF Fusion Protein: A Unique Immunotoxin Against the Human Granulocyte-Colony Stimulating Factor Receptor

Immunotoxin is a recombinant fusion toxin which has been developed to kill cancer cells selectively. DT389GCSF as a new immunotoxin consists of a truncated diphtheria toxin linked to granulocyte colony stimulating factor (GCSF) via the SerGly4SerMet flexible linker. In this study, DT389GCSF was expressed in inclusion body form in Escherichia coli BL21 (DE3) then it was purified. After refolding, the structure of refolded protein was assessed by Native-PAGE and spectroscopic techniques. In the following, the refolded protein’s nuclease activity and its cytotoxicity toward HL-60 were evaluated. The results showed that the yield of DT389GCSF expression and purification was about 30% and 95% respectively, using CLIQS software. Also structural and functional studies confirmed that DT389GCSF obtained its intact structure and also function. MTT assay revealed that DT389GCSF can induce the death of HL-60 cell line in vitro. IC50 value upon 48 h of exposure of DT389GCSF toward HL-60 was 5.2 × 10−7±0.00011 M. The specific toxicity of DT389GCSF was determined by its disability to kill the K562 cell line. Findings from the present study indicate that DT389GCSF is toxic to human leukemia (HL-60) cells, supporting its use as an effective agent towards the study and treatment of patients with AML.

Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxinA.

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxinA(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 receptor2 (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 Escherichiacoli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26nM) 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) 1to 3nM and 2to 10nM, respectively]. The invitro 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.

MDNA55: A locally administered IL4 guided toxin as a targeted treatment for recurrent glioblastoma.

2039 Background: IL4 receptor (IL4R) is frequently and intensely expressed on a variety of human cancers and is associated with poor survival outcomes. Determining the role of the IL4R biomarker in glioblastoma (GBM) will be important for treatment with targeted therapies such as the IL4 fusion toxin MDNA55. Methods: A classification for IL4Rα expression in GBM tissues by H-Score was developed using a validated immunohistochemistry-based approach. MDNA55-05 is an open-label study of MDNA55 administered intratumorally via convection enhanced delivery in recurrent GBM. Levels of IL4Rα expression were assessed retrospectively in 24 subjects in the clinical trial and were correlated with GBM history, imaging responses and survival outcomes following treatment with MDNA55 to explore clinical validation. Results: Range, linearity, specificity and sensitivity testing using a rabbit polyclonal antibody to IL4Rα were performed using normal cortex (negative control) and a panel of normal human tissues and GBM cases from tissue banks. A total of 41 GBM samples were screened and grouped by reactivity thresholds: H-Scores ≥50 were observed in 95% of cases (39/41), H-Scores ≥200 were observed in 51% of cases (21/41), and H-Scores ≥250 were observed in 24% of cases (10/41). GBM tissues obtained at initial diagnosis from subjects enrolled in the trial show that moderate/high IL4R expression (H-Score > 75) was associated with shorter time to first relapse when compared to subjects with low IL4R expression (H-Score ≤ 75) (10.3 mos vs. 16.7 mos, respectively) after upfront standard-of-care treatment, consistent with published findings that IL4R expression is associated with more aggressive disease. Remarkable decreases in tumor size seen in some subjects following MDNA55 treatment were associated only with moderate/high IL4R expression and survival rate at 12 months in this group was also improved (OS12 = 55%) compared to subjects with low IL4R expression (OS12 = 30%). Conclusions: Treatment options for patients with recurrent GBM are very limited and positive outcomes remain rare. Targeting therapies such as MDNA55 by IL4R status may improve patient outcomes and help guide patient selection strategies for future clinical studies. Clinical trial information: NCT02858895.

An IL-2 proaerolysin fusion toxin that selectively eliminates regulatory t cells to enhance antitumor immune response.

Recentsuccess with immune-checkpoint inhibitors in some tumor types has highlighted the power of the immune system to control and eradicate human cancer cells. However, these therapies have demonstrated a limited activity in prostate cancer, which has a more immunosuppressive microenvironment that can be because of the presence of a variety of inhibitory cell types, such as myeloid-derived suppressor cells, mesenchymal stem cells, and regulatory T cells (Tregs). One strategy to improve the efficacy of immune-based therapies for prostate cancer is to selectively eliminate these immunosuppressive cells within the tumor microenvironment. We developed and characterized a chimeric protein consisting of the cytokine IL-2 fused to binding mutant of the highly toxic bacterial toxin proaerolysin (ie IL2-R336A). The IL2-R336A fusion protein selectively kills immunosuppressive Tregs that express the IL-2 receptor while having little to no effect on cells negative for this target. IL2-R336A depleted Tregs in both tumor bearing and nontumor bearing mice. Tumor bearing mice vaccinated with a GMCSF-expressing CT-26 GVAX vaccine had reduced tumor growth when given IL2-R336A before vaccination. IL2-R336A also enhanced immune response to a model hemagglutinin antigen (HA) in HA-tolerized mice. These results suggest that this IL2-R336A toxin may be a useful in improving the therapeutic efficacy of antitumor vaccines by enhancing the immune response against target tumor antigens.

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti–PD-1 in melanoma

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

Targeting MCF-7 Cell Line by Listeriolysin O Pore Forming Toxin Fusion with AHNP Targeted Peptide.

Background:Tumor-targeting peptides are attracting subjects in cancer therapy. These peptides, which are widely studied, deliver therapeutic agents to the specific sites of tumors. In this study, we produced a new form of recombinant listeriolysin O (LLO) with genetically fused Anti-HER2/neu peptide (AHNP) sequence adding to its C-terminal end. The aim of the study was to engineer this pore-forming toxin to make it much more specific to tumor cells.Materials and Method and Results:Two forms of the toxin (with and without peptide) were subcloned into a bacterial expression plasmid. Subcloning was performed using a polymerase chain reaction (PCR) product as a megaprimer in a quick-change PCR to introduce the whole insert gene into the expression plasmid. After expression of two recombinant forms of LLO in BL21 DE3 cells, purification was performed using Ni-NTA affinity column. MDA-MB-231 and MCF-7 cell lines (as negative and positive controls, respectively) were treated with both LLO toxins to evaluate their cytotoxicity and specificity. The IC50 of LLO on MDA-MB-231 and MCF-7 cells was 21 and 5 ng/ml, respectively. In addition, IC50 for the fusion AHNP-LLO toxin was 140 and 60 ng/ml, respectively. It was found that the cytotoxicity of the new engineered AHNP-LLO toxin has decreased by about 9x compared to the wild-type toxin and the specificity of the AHNP-LLO toxin has been also reduced.Conclusions:Results show that the C-terminal of the LLO should not be modified and it seems that N-terminal of the toxin should be preferred for engineering and adding peptide modules.

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Primary cutaneous lymphomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Primary cutaneous lymphomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Light-enhanced VEGF121/rGel: A tumor targeted modality with vascular and immune-mediated efficacy

Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4 T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel –PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.

Abstract LB-293: IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model

Abstract In tumor immunotherapy, regulatory T lymphocytes (Tregs) play an important role in the suppression of beneficial anti-tumor responses. In the non-immunogenic B16-F10 mouse melanoma model, tumors are found to be highly infiltrated by Tregs. DAB389IL-2 (Ontak, denileukin diftitox) is an IL-2 diphtheria toxin-related fusion protein that has been showed to deplete Tregs in both humans and mice. We treated mice with established B16F10 tumors with s-DAB389IL-2 (produced as a secreted protein from C. diphtheria) and observed a significant decrease in tumor growth rate in C57BL/6 mice. Also, s-DAB389IL-2 treatment increased the frequency of CD8+IFNγ+ T cells (TILs) in tumors and spleens. S-DAB389IL-2 treatment followed by PD-1 blockade resulted in greater tumor growth inhibition than either monotherapy alone. These data suggest that s-DAB389IL-2 plus anti-PD1 treatment can improve anti-tumor responses in non-immunogenic B16F10 melanoma and inhibit tumor growth. Additionally, we mutated a vascular leak inducing motif to construct s-DAB389IL-2 (V6A), which cause less vascular leak in vitro. S-DAB389IL-2 (V6A) inhibited tumor growth with similar efficacy to the original fusion toxin. As vascular leak is the major adverse effect caused by Ontak, DAB389IL-2 (V6A) shows promise as a better tolerated drug for further studies as a cancer immunotherapy. Citation Format: Juan Fu, Drew Pardoll, Laurene Cheung. IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-293.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Evaluating the Effects of Cholinergic Depletion in the PPTg, LDTg, and VTA on Acoustic Startle Response and Morphine‐Related Reward

Opiate abuse is a worldwide epidemic that costs nations billions of dollars to treat. Rewarding effects of opioids and other drugs of abuse are partially reliant on the activity of dopaminergic neurons located in reward centers of the brain such as the ventral tegmental area (VTA). The VTA receives cholinergic input from only two sources, the pedunculopontine tegmentum (PPTg) and the laterodorsal tegmentum (LDTg). Excitotoxic lesions to the PPTg in rats have previously been shown to reduce the rewarding effect of opiates (Bechara & van der Kooy, 1989; Olmstead & Franklin, 1997; Parker & van der Kooy, 1995). These studies were unable to identify the specific neuronal subtype responsible for the reduced rewarding effect, though they suggested the cholinergic neurons were responsible. By using a diphtheria‐UII fusion toxin to selectively ablate cholinergic neurons in the PPTg and LDTg we were able to test the role of cholinergic projections to the VTA. The toxin was injected bilaterally into the PPTg, LDTg, or VTA of male Sprague‐Dawley rats through stereotaxic surgery. Injection into the VTA creates a retrograde lesion that specifically ablates the cholinergic neurons of the PPTg and LDTg that innervate the VTA.The animals were subjected to an acoustic startle reflex/pre‐pulse inhibition paradigm in weeks 4, 8, and 10 post‐surgery. Animals with cholinergic depletion in the PPTg were unable to startle to the point where pre‐pulse inhibition was unable to be measured. While not significant, there was a trend toward an increase in startle in animals that received a VTA retrograde lesion. These findings suggest that the cholinergic PPTg is necessary for integrating sensory processing and motor output. Cholinergic input to the VTA from both the PPTg and LDTg produced a novel effect. Ten weeks post‐surgery the rats were subjected to a morphine conditioned place preference paradigm (CPP), an extinction period, and then a cocaine CPP paradigm. Animals with lesions to the LDTg were able to form cocaine‐mediated CPP but not morphine‐mediated CPP. PPTg lesioned animals formed preference for both morphine and cocaine. Sham animals and the VTA retrograde animals also formed preference for both drugs. These findings suggest that cholinergic neurons in the LDTg that project to an area that is not the VTA likely play a role in morphine‐related reward, and that the cholinergic output from the PPTg is not required in the formation of morphine conditioned place preference. Taking the results of both experiments together, it can be inferred that the cholinergic LDTg plays a role in reward while the cholinergic PPTg plays a role in sensory processing. It is likely that these two areas work in concert to modulate a variety of behaviors. While these findings don't provide a treatment for opiate abuse, they help to further our understanding of the neuronal mechanisms that drive drug addiction.Support or Funding InformationR00DA024754This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting oncogenic Ras by the Clostridium perfringens toxin TpeL.

Clostridium perfringens toxin TpeL belongs to the family of large clostridial glycosylating toxins. The toxin causes N-acetylglucosaminylation of Ras proteins at threonine35 thereby inactivating the small GTPases. Here, we show that all main types of oncogenic Ras proteins (H-Ras, K-Ras and N-Ras) are modified by the toxin in vitro and in vivo. Toxin-catalyzed modification of Ras was accompanied by inhibition of the MAP kinase pathway. Importantly, TpeL inhibited the paradoxical activation of the MAP kinase pathway induced by the BRAF inhibitor Vemurafenib in the human melanoma cell line SBCL2. The toxin also blocked Ras signaling in a zebrafish embryo model expressing oncogenic H-RasG12V, resulting in a reduction of melanocyte number. By using the binding and translocation component of anthrax toxin (protective antigen), the glucosyltransferase domain of TpeL was effectively introduced into target cells that were not sensitive to native TpeL toxin. To reach a higher specificity towards cancer cells, a chimeric TpeL toxin was engineered that possessed the knob region of adenovirus serotype 35 fiber, which interacts with CD46 of target cells frequently overexpressed in cancer cells. The chimeric TpeL fusion toxin efficiently inhibited Ras and MAP kinases in human pancreatic cancer Capan-2 cells, which were insensitive to the wild-type toxin. The data reveal that TpeL and TpeL-related immunotoxins provide a new toolset as Ras-inactivating agents.