Recombinant Fusion Toxin Research Articles

Evaluation and Comparison of Clostridium Epsilon-Alpha Fusion Gene Expression Using Different Commercial Expression Vector.

Clostridium perfringens and Clostridium septicum are gram-positive, anaerobic, spore-forming rods and pathogens for humans and livestock, which are widespread in nature as well as human and animal digestive systems. C. perfringens produces numerous different exoproteins, which are various systems of action. The major C. perfringens toxins include alpha, beta, epsilon, and iota. C. perfringens are classified into five groups (A-E) on the basis of the production of these lethal toxins. Furthermore, toxins secreted from C. septicum include alpha, beta, delta, and gamma. Epsilon and alpha toxins of C. perfringens and C. septicum are the major causes of enterotoxemia and braxy in sheep and goats, respectively. The production of recombinant immunogenic proteins of these bacteria using suitable expression vectors and expression prokaryotic hosts can be a convenient method for the reduction of the costs and production time of clostridial anaerobic vaccines. In the present study, recombinant Escherichia coli strain TOP10 containing pJETεα was used for the evaluation of C. perfringens type D and C. septicum epsilon-alpha fusion protein using different commercial vectors. After the extraction of pJETεα from the recombinant cell, it was digested by NdeI and XhoI restriction enzymes and subcloned into pET22b (+), pET26b (+), and pGEM-B1 expression vectors in E. coli/Rosetta and E. coli/BL21 (DE3). The expression of recombinant fusion toxin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in three different temperatures, various isopropyl β-D-1-thiogalactopyranoside (IPTG) gradients, and different times using pGEMεα, pET22εα, and pET26εα vectors in E. coli/Rosetta and E. coli/BL21 (DE3). According to the obtained results, recombinant E. coli/Rosetta/pET22εα showed better expression at a temperature of 37°C after 6 h of induction by IPTG.

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.

DAB389IL-2 recombinant fusion toxin effect on lymphocyte- and macrophage-producing cytokine subpopulation cells in experimentally induced demyelinating disease in mice

Context: We have reported previously that DAB389IL-2 recombinant fusion toxin targets IL-2R bearing CD4+ cells, and suppresses demyelinating disease in acute (A) - and chronic (C) - experimental autoimmune encephalomyelitis (EAE) animal models of multiple sclerosis.Objectives: The present study was undertaken to investigate the effect of DAB389IL-2 treatment on various cytokine-secreting cell populations in A-EAE and C-EAE mice.Materials and methods: The effects of DAB389IL-2 at doses of 200-, 800-, or 1600 kU administered i.v. on days 11–13 and 15 post disease induction on the clinical score and cytokine-secreting cell populations were examined using flow cytometry.Results: C-EAE mice treated with 1600 kU DAB389IL-2, but not A-EAE mice treated with 800 kU had significantly reduced disease. The CD3+CD25+ sub-population in spleens and spinal cords of A-EAE mice treated with 800 kU DAB389IL-2 was increased, whereas in C-EAE mice treated with 1600 kU this population was decreased. DAB389IL-2 treatment reduced CD3+CD4+, CD3+CD8+, CD4+CD8+, CD3+IL-2+, CD3+IFN-γ+ and CD3+TNF-α+ T cell subpopulations in the spinal cord in A-EAE, and C-EAE mice on day 16. CD11b+ macrophages that were IL-2-, IFN-γ-, and TNF-α- positive were reduced in A-EAE mice. DAB389IL-2 treatment reduced CD19+ B-cells positive for IL-2 or CD11b+ in the spinal cord in acute and chronic disease. DAB389IL-2 treatment also reduced lymph node CD3+CD8+, CD4+CD8+, CD3+CD25+ populations on day 16, and lymph node CD3+IL-10+ and peripheral blood CD3+CD25+ populations on day 24.Discussion and conclusion: Our study demonstrates that DAB389IL-2 fusion toxin suppresses EAE in a dose-dependent manner, and alters inflammatory cell sub-populations during disease development.

Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.

Ontak-like human IL-2 fusion toxin

Ontak® is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak®.

Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin ZHER2:2891-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain.

Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (KD=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (KD=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.

Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer.

HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer.

Diphtheria toxin-based recombinant murine IL-2 fusion toxin for depleting murine regulatory T cells in vivo.

Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells which suppress immune responses of effector cells and are known to play a very important role in protection against autoimmune disease development, induction of transplantation tolerance and suppression of effective immune response against tumor cells. An effective in vivo Treg depletion agent would facilitate Treg-associated studies across many research areas. In this study, we have developed diphtheria toxin-based monovalent and bivalent murine IL-2 fusion toxins for depleting murine IL-2 receptor positive cells including CD25(+) Treg in vivo. Their potencies were assessed by in vitro protein synthesis inhibition and cell proliferation inhibition assays using a murine CD25(+) CTLL-2 cell line. Surprisingly, in contrast to our previously developed recombinant fusion toxins, the monovalent isoform (DT390-mIL-2) was approximately 4-fold more potent than its bivalent counterpart (DT390-bi-mIL-2). Binding analysis by flow cytometry demonstrated that the monovalent isoform bound stronger than the bivalent version. In vivo Treg depletion with the monovalent murine IL-2 fusion toxin was performed using C57BL/6J (B6) mice. Spleen Treg were significantly depleted with a maximum reduction of ∼70% and detectable as early as 12 h after the last injection. The spleen Treg numbers were reduced until Day 3 and returned to control levels by Day 7. We believe that this monovalent murine IL-2 fusion toxin will be an effective in vivo murine Treg depleter.

Recombinant TAT-gelonin fusion toxin: synthesis and characterization of heparin/protamine-regulated cell transduction.

Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.

Photochemical activation of the recombinant HER2-targeted fusion toxin MH3-B1/rGel; Impact of HER2 expression on treatment outcome

HER2 is overexpressed in 20–30% of breast tumors and is associated with aggressiveness and increased risk of recurrence and death. The HER2 protein is internalized as a part of its activity, and may therefore be utilized as a target for the specific intracellular delivery of drugs. Photochemical internalization (PCI) is a novel technology now undergoing clinical evaluation for its ability to improve the release into the cytosol of drugs entrapped in the endo/lysosomal compartment. PCI employs an amphiphilic photosensitizer which localizes in the membranes of endo/lysosomes. Subsequent light exposure (visible light) causes destabilization of the endo/lysosomal membranes. PCI has been proven highly effective for improving the cytosolic delivery of targeted toxins based on type I ribosome inactivating protein toxins such as gelonin. We examined the impact of the level of target antigen expression on PCI efficacy. Four human breast cancer cell lines (MDA-MB-231, BT-20, Zr-75-1 and SK-BR-3) covering a wide range of HER2 expression were included in the present study. PCI of the HER2-targeted fusion toxin MH3-B1/rGel was found to be highly effective in all four cell lines. The increase in PCI-mediated efficacy was not directly correlated with the cellular levels of HER2 as assessed by western blots, the overall uptake of MH3-B1/rGel as measured by flow cytometry, the amount of MH3-B1/rGel localized to endo/lysosomes assessed by confocal microscopy or the cell sensitivity to the photochemical treatment itself (photosensitizer and light without MH3-B1/rGel). However, correcting the PCI efficacy for the baseline cellular sensitivity to rGel revealed a linear correlation (R2=0.80) with HER2 expression. The present report therefore concludes the cellular sensitivity to the toxin as an important parameter for PCI efficacy and also indicates PCI of a HER2-targeted fusion toxin as an attractive treatment alternative for breast cancer patients with both HER2-low and -high expression.

Photochemical internalization augments tumor vascular cytotoxicity and specificity of VEGF121/rGel fusion toxin

Vascular targeting for cancer is increasingly recognized as a therapeutic strategy although the lack of objective responses and the development of resistance are major limitations for clinically-available drugs. Endothelial targeted toxins exert increased toxicity compared to antiangiogenic drugs and may therefore overcome these limitations. The specificity and toxicity of targeted toxins may be increased by utilization of a drug delivery system which provides selective release of the targeted toxins in the target cells. Photochemical internalization (PCI) is a non-invasive modality which causes translocation into the cytosol of agents that are trapped in endosomes. This study describes the first use of PCI in combination with a recombinant fusion toxin targeting tumor vasculature. Endothelial cells bearing VEGFR2 treated with VEGF121/rGel showed dramatic enhancement of toxicity after PCI utilizing the photosensitizer TPCS2a (Amphinex®). We compared the PCI of VEGF121/rGel to that of bleomycin which is currently under clinical evaluation. The VEGFR2 specificity of VEGF121/rGel was shown to be preserved by the PCI treatment. PCI of VEGF121/rGel was further shown to induce vascular collapse and edema in the invasive areas of CT26.CL25 colon carcinoma tumors as shown by CD31 IHC. Antitumor effects, as assessed by tumor growth delay were found for PCI of VEGF121/rGel and PCI of bleomycin with cure rates of 40% and 33% respectively. PCI of VEGF121/rGel was, however, better tolerated compared to PCI of bleomycin. Thus, PCI of vascular targeted toxins provides higher specificity and increased tolerability compared to PCI of bleomycin and may represent an interesting clinical future for the PCI technology.

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins' ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation.

Abstract 5611: Photochemical internalization of VEGF121/rGel: a strategy for optimizing tumor-vasculature targeting.

Abstract Photochemical internalization (PCI) is a modality for intracellular delivery of drugs which lack an effective transport mechanism into the cell. Such drugs are taken up by means of endocytosis and are transported to the lysosomes where they are degraded before they have exerted their biological effect. PCI utilizes photosensitizers which accumulate in the membranes of these endo/lysosomal compartments. Light exposure causes rupture of these membranes and subsequent cytosolic release of the drug which freely can interact with its target. PCI of the conventional cytostatic drug Bleomycin has been shown to be highly effective in vivo and is currently evaluated in clinical trials with promising results. Bleomycin is, however, probably not optimal for PCI mediated delivery, and therefore cannot fully take use of the technology. Recombinant targeted toxins may be designed to possess all characteristics for an optimal drug to be delivered by PCI: (1) Exclusive uptake in tumor cells, (2) Intracellular target, (3) Not able to penetrate the plasma membrane, (4) Suboptimal escape from endocytic compartments without PCI, (5) Able to diffuse into the tumor tissue. We here present PCI of a recombinant fusion toxin composted of VEGF121, and gelonin, a type I ribosome inactivating protein toxin. VEGF121/rGel administration has previously been shown to be effective in suppressing tumor xenografts and metastasis. Severe adverse effects may, however, limit the possibilities to obtain complete responses with VEGF121/rGel monotherapy. PCI was shown to increase the selectivity of VEGF121/rGel in VEGFR1 and VEGFR2 transfected porcine endothelial cells (PAE) in vitro. The PCI treatment also resulted in a dramatic reduction in the dosage of VEGF121/rGel to 1/100 as measured by a LD90 of 10 nM for the fusion toxin alone compared to 100 pM with the PCI treatment in VEGFR2 expressing PAE cells. For in vivo experiments the murine colon cell line CT26.CL25 was injected subcutaneously in the left hip of BALB c mice and PCI of VEGF121/rGel was administered one week after implantation. By day 24 after PCI all animals in the non-treatment control group were sacrificed due to tumor size (>1000m3),while a 50% complete response (CR) was found with PCI of VEGF121/rGel. The animals were followed up to day 60 when the experiment was terminated. No tumor regression was observed in CR animals in this time-frame. About 50%CR was also detected in PCI of bleomycin-treated animals. Animals receiving PCI of bleomycin showed an average weight loss of 15% compared to no significant weight loss in the PCI of the VEGF121/rGel treated group. This is the first report on PCI of a tumor vascular targeting drug. In conclusion, the present results indicate PCI of VEGF121/rGel as a highly selective method for destroying tumor vasculature. The project is supported by the Norwegian Cancer Society. Research was conducted, in part, by the Clayton Foundation for Research Citation Format: Anette Weyergang, Lawrence H. Cheung, Michael Rosenblum, Khalid Mohamedali, Johannes Waltenberger, Kristian Berg. Photochemical internalization of VEGF121/rGel: a strategy for optimizing tumor-vasculature targeting. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5611. doi:10.1158/1538-7445.AM2013-5611

A truncated diphtheria toxin based recombinant porcine CTLA-4 fusion toxin

Targeted cell therapies are possible through the generation of recombinant fusion proteins that combine a toxin, such as diphtheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the fusion protein to the cell of interest, the diphtheria toxin is internalized which results in protein synthesis inhibition and subsequent cell death. We have recently expressed and purified the recombinant soluble porcine CTLA-4 both with and without N-glycosylation in yeast Pichia pastoris for in vivo use in our preclinical swine model. The glycosylated and non-N-glycosylated versions of this recombinant protein each bind to a porcine CD80 expressing B-cell lymphoma line (LCL13271) with equal affinity (KD=13nM). In this study we have linked each of the glycosylated and non-N-glycosylated soluble porcine CTLA-4 proteins to the truncated diphtheria toxin DT390 through genetic engineering yielding three versions of the porcine CTLA-4 fusion toxins: 1) monovalent glycosylated soluble porcine CTLA-4 fusion toxin; 2) monovalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin and 3) bivalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin. Protein synthesis inhibition analysis demonstrated that while all three fusion toxins are capable of inhibiting protein synthesis in vitro, the non-N-glycosylated porcine CTLA-4 isoforms function most efficiently. Binding analysis using flow cytometry of the porcine CTLA-4 fusion toxins to LCL13271 cells also demonstrated that the non-N-glycosylated porcine CTLA-4 isoforms bind to these cells with higher affinity compared to the glycosylated fusion toxin. The monovalent non-N-glycosylated porcine CTLA-4 fusion toxin was tested in vivo. NSG (NOD/SCID IL-2 receptor γ−/−) mice were injected with porcine CD80+ LCL13271 tumor cells. All animals succumbed to tumors and those treated with the monovalent non-N-glycosylated porcine CTLA-4 fusion toxin survived longer based on a symptomatic scoring system compared to the untreated controls. This recombinant protein may therefore provide a novel approach for in vivo depletion of porcine antigen presenting cells (APCs) for studies investigating the induction of transplantation tolerance, autoimmune disease and cancer treatment.

A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages

BackgroundThe C3bot1 protein (∼23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes.Methodology/Principal FindingsHere, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages.Conclusions/SignificanceIn summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.

Abstract 3633: Photochemical internalization (PCI) of the vascular targeting immunotoxin VEGF121/rGel, a novel method for selective targeting of tumor vasculature

Abstract Vascular endothelial growth factor (VEGF-A) is a major mediator in tumor angiogenesis. VEGF-A binds to the receptors VEGFR-1 and VEGFR-2, both known to be overexpressed on tumor endothelial cells. Here we present a highly selective method for the targeting of VEGFRs by PCI of a VEGFR targeting fusion toxin. PCI is a novel drug delivery system for macromolecules that accumulate in the cells endosomes and lysosomes. PCI is based on light sensitive compounds, photosensitizers, which can absorb light at appropriate wavelengths and thereby generate reactive oxygen species (ROS). Photosensitizers utilized for PCI are amphiphilic and localize intracellularly to endocytic vesicles. Light exposure activates the photosensitizer and causes rupture of the vesicles so that drugs trapped within can be released into the cytosol. PCI was employed to deliver VEGF121/rGel, a recombinant VEGFR targeting fusion toxin composted of VEGF121, an isoform of VEGF-A, and gelonin, a type I ribosome inactivating protein toxin. VEGF121/rGel has been shown to be effective in suppressing tumor xenografts. Severe adverse effects may limit the possibilities to obtain complete responses with VEGF121/rGel monotherapy. We evaluated whether PCI could improve the efficacy and thereby reduce the dosage required for VEGF121/rGel to kill tumor vascular endothelial cells. Porcine aortic endothelial cells (PAE) transfected with either VEGFR-1 (PAE/FLT-1) or VEGFR-2 (PAE/KDR) served as a model system for endothelial cells. Clonal cell survival and MTT assay studies showed that PCI treatment resulted in a dramatic reduction in the dosage of VEGF121/rGel to 1/100 as measured by a LD90 of 10nM for the fusion toxin alone compared to 100pM with the PCI treatment in PAE/KDR cells. VEGF121/rGel alone exerted a higher cytotoxicity in PAE/KDR cells than in PAE/FLT-1 cells. Surprisingly, no difference in cytotoxicity was observed between the cell lines after PCI of the fusion toxin. Fluorescent microscopy of Alexa Fluor 488-labeled VEGF121/rGel as well as Western blots of VEGF121/rGel following PCI treatment showed, however, a ∼9 fold higher uptake of the IT in PAE/KDR cells compared to the PAE/FLT-1 cells. The results therefore indicate activation of different intracellular pharmacologic pathways in these cell lines as the cause of similar PCI mediated cytotoxicity. This is the first report on PCI of a tumor vascular targeting drug. In conclusion, the present results indicate PCI of VEGF121/rGel as a highly attractive method for destroying tumor vasculature as it shows high cytotoxicity to both VEGFR-1 and VEGFR-2 expressing cells. An animal study of PCI of VEGF121/rGel is already initiated in mice. The project is supported by the Norwegian Cancer Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3633. doi:10.1158/1538-7445.AM2011-3633

Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA' against CLDN-3,4-overexpressing ovarian cancer cells

The aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer. CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells. Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml. The C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

Targeted toxins and saponins – a powerful cooperation

Saponins from Gypsophila species are known to enhance the cytotoxicity of targeted toxins. In general targeted toxins consist of two components: a toxin moiety with enzymatic activity and a ligand moiety, which mediates specific binding to cancer cells. It was shown that saponins from Gypsophila paniculata L. enhanced the toxicity of a targeted toxin, composed of the N-glycosidase saporin from Saponaria officinalis L. and human epidermal growth factor up to 2,500 000-fold in primary breast cancer cells. Meanwhile the efficiency of the combined application of saponins and targeted toxins was shown in a mouse tumour model. Compared to the single application of the targeted toxin (SA2E), the combination with saponins lead to a tremendous reduction (at least 50-fold) of the SA2E dosage. By this dose reduction side effects were only moderate. This represents an enormous step forward in the targeted anti tumour therapy with recombinant fusion toxins. However, in all these studies a mixture of saponins was used. For the further successful development of a combinatorial anti cancer therapy with targeted toxins and saponins pure saponins are mandatory. This study was intended to isolate pure saponins from Gypsophila paniculata L. in order to scruntinize the toxicity enhancing properties of isolated Gypsophila saponins on targeted toxins. One of the four isolated saponins showed strong, one moderate and two no cytotoxicity enhancing properties. On the basis of these results we determined the structural prerequisites for the saponin-mediated toxicity enhancement of targeted toxins.

Correlates of capillary leak syndrome (CLS) in patients with cutaneous T-cell lymphoma (CTCL) during treatment with denileukin diftitox (DD) in a placebo (PBO)-controlled phase III trial.

e18509 Background: Interleukin-2 therapy is associated with capillary leakage which manifests as hypoalbuminemia, hypotension/intravascular signs of dehydration, with concurrent edema/fluid retention. DD is a recombinant fusion toxin composed of sequences of human IL-2 and diphtheria toxin and is approved for the treatment of CD25+ CTCL. In open-label studies of DD, CLS has been noted. To learn more about the possible association of DD with CLS, we analyzed data from a Phase III trial in CTCL pts with recurrent or persistent stage Ia-III disease. Methods: We examined the clinical reporting of adverse events in any single cycle of therapy for the components of CLS of any severity, identifying 1) hypoalbuminema, 2) dehydration or low blood pressure, and 3) fluid retention or edema. The trial included 144 pts, 44 receiving PBO (mean cycles per pt = 5.1), 45 receiving DD 9 μ g/kg qd × 5 every 21 days (LD) (mean cycles per pt = 5.7), and 55 receiving DD 18 μ g/kg daily × 5 every 21 days (HD) (mean cycles per p...

Suppression of murine experimental autoimmune encephalomyelitis by interleukin-2 receptor targeted fusion toxin, DAB 389IL-2

Previously we have shown that DAB 389IL-2, a recombinant fusion toxin targeting IL-2R bearing cells, suppressed disease in the rat experimental autoimmune encephalomyelitis (EAE) model of acute multiple sclerosis (MS). Our present study demonstrates that DAB 389IL-2 can also effectively suppress acute (A)-EAE, relapsing (R)-EAE and chronic (C)-EAE in mouse demyelinating models. DAB 389IL-2 significantly suppressed mitogenic proliferation of spleen cells while mutant fusion proteins DA glu53B 389IL-2 and DAB 389IL-2 8–10 did not. EAE was successfully suppressed when DAB 389IL-2 was administered in various regimens between days 1 and 15 post immunization in all three models. CD4 +IL-2R + cells were reduced in the spleen but not in the lymph nodes of DAB 389IL-2-treated mice during A-EAE while the number of CD8 + cells was unchanged. DAB 389IL-2 also significantly reduced the number of CD4 +, CD8 +, CD25 +, TCRγδ + phenotype and CD11b + macrophages/microglia within spinal cord lesions. These data strongly suggest that DAB 389IL-2 specifically targeted myelin protein-activated CD4 + T cells and strengthens the argument for the use of DAB 389IL-2 in treatment strategies for MS.