ADP-ribosylation Of Elongation Factor Research Articles

Identification of Novel Nontoxic Mutants of Diphtheria Toxin Unable to ADP-ribosylate EF2 Using Molecular Dynamics Simulations and Free Energy Calculations

Introduction: The Diphtheria vaccine is currently manufactured by chemical inactivation of Diphtheria toxin (DT) following large-scale cultivation of the pathogenic Corynebacterium diphtheriae. The development of non-toxic variants of DT in non-pathogenic recombinant hosts improves the process safety aspects considerably and is therefore desirable. The cytotoxicity of DT was exerted through ADPribosylation of elongation factor 2 (EF2) by the catalytic (C) domain of DT using NAD as the ADP donor. Aim: We, therefore, aim to design DT variants unable to perform ADP-ribosylation of EF2. Methods: We used molecular dynamics simulations (MDS) for docking of the C-domain of DT onto EF2 to identify interacting amino acid fluctuations using the root mean square fluctuation (RMSF). Amino acids of the C domain possessing low fluctuations and hence low flexibility were then specified and used for the mutant design. The C domain and the mutants were docked with NAD and ADENYLYL-3'-5'- PHOSPHO-URIDINE3'-MONOPHOSPHATE (APU) as a C domain inhibitor. G52E showing negligible NAD binding was also included in our study. Free binding energies of the complexes were calculated and used to select the desired mutants. Moreover, contact maps of the C domain and the selected mutants were compared to elucidate their structural differences. The SCRATCH tool was used to estimate their solubility upon recombinant expression in E. coli. ElliPro was further used to determine their B-cell epitopes. Results: Our results indicated that amino acids Y20, V80, V81, V83, and Y149 within the C domain showed low flexibility. The constructed mutants, including Y20E and Y80E were able to bind APU, whereas Y149E was not able to bind it. Both Y20E and Y149E were unable to bind NAD. Thus, these mutants did not have ADP-ribosylation activity and were nontoxic. These were structurally different from the C domain. However, their solubility in E. coli and their conformational B-cell epitopes were similar to those of the C domain. Conclusion: Therefore, Y20E and Y149E are applicable for the vaccine design with DT using a much safer process compared to the commercial process.

Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy.

Immunotoxins are a class of targeted cancer therapeutics in which a toxin such as Pseudomonas exotoxin A (PE) is linked to an antibody or cytokine to direct the toxin to a target on cancer cells. While a variety of PE-based immunotoxins have been developed and a few have demonstrated promising clinical and preclinical results, cancer cells frequently have or develop resistance to these immunotoxins. This review presents our current understanding of the mechanism of action of PE-based immunotoxins and discusses cellular mechanisms of resistance that interfere with various steps of the pathway. These steps include binding of the immunotoxin to the target antigen, internalization, intracellular processing and trafficking to reach the cytosol, inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (EF2), and induction of apoptosis. Combination therapies that increase immunotoxin action and overcome specific mechanisms of resistance are also reviewed.

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis

ABSTRACTCholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide in vitro and in vivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7+ vesicles and in basal exocytic Rab11+ vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular re-distribution that coincides with the re-organization of COPI+ and COPII+ vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals.One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate in acute myeloid leukemia

Outcomes of acute myeloid leukemia (AML) remain poor and warrant the development of novel therapeutic agents. CD123 (interleukin-3 [IL-3] receptor alpha subunit), is overexpressed on AML blasts and leukemia progenitor/stem cells (LSCs) compared to normal hematopoietic cells (Jordan et al. Leukemia 2000). SL-101 is a novel anti-CD123 antibody-conjugate comprised of anti-CD123 scFv fused to a truncated and optimized pseudomonas exotoxin (PE) lacking its native targeting domain. We have previously demonstrated SL-1019s cell killing efficacy in AML cell lines (Han et al. ASH 2013). Here we report the anti-tumor efficacy of SL-101 against primary AML cells and the underlying mechanisms of its cytotoxicity. Fourteen genetically diverse primary AML samples were treated with various doses of SL-101 for 48 h. Most samples express high levels of CD123 (median 89.9%, range 20.4-99.3%) and intermediate levels of CD131 (median 54.0%, range 9.0-91.1%), the IL-3 beta subunit. SL-101 was highly active against AML samples, with an IC50 of 0.19 µg/ml (range 0.003 - 0.98 µg/ml). No significant correlation was found between SL-101 activity and levels of CD123 or CD131 (n=12). SL-101 also selectively and significantly suppressed AML colony formation (69.5% ± 15.0% inhibition of total colonies, n=7) while sparing normal bone marrow (5.6% ± 3.3% inhibition, n=4; p=0.0001) (Fig. 1A). We next investigated the mechanisms of the cytotoxic activity of SL-101. Using annexinV/DAPI flow cytometry, we first evaluated the induction of apoptosis in AML blasts and phenotypically defined CD45dimCD34+CD38-CD123+ LSCs. After 48 h treatment, SL-101 at 1.0 µg/ml induced higher specific apoptosis in the AML LSCs (51.2% ± 25.4%) than in blasts (39.4% ± 19.0%, p=0.006, n=10). Quantification of the annexinV–/DAPI– viable cells using counting beads demonstrated further reduction of cell numbers by SL-101 (LSCs 72%, blasts 64.6%), indicating additional mechanisms of cell growth inhibition. It was recently demonstrated that upon internalization, PE traffics through the endoplasmic reticulum to the cytosol, where it inactivates protein synthesis by catalyzing ADP ribosylation of elongation factor 2 and causes non-apoptotic cell death (Wayne et al. Blood 2014). To examine the contribution of the direct inhibition of protein synthesis by PE, we first studied SL-101 internalization utilizing DyLight 680-labeled SL-101 by flow cytometry and fluorescence imaging. In CD123-expressing AML cell lines MV4-11 and MOLM13, the intracellular median intensity of DyLight 680 signal increased 5.4- and 3.5-fold, respectively, within 4 h of treatment and 22.3- and 16.3-fold after 24 h (Fig 1B). Fluorescence imaging confirmed cytosolic localization of SL-101 in both cell lines, demonstrating efficient cellular uptake of SL-101. We also examined the efficacy of SL-101 in inhibiting nascent protein synthesis in MV4-11 cells using an AHA Alexa Fluor 488 protein synthesis assay. SL-101 significantly reduced protein synthesis (40.3%, p=0.0005) within 4 h (Fig 1C), even at low concentrations (0.01 µg/ml), which was comparable to the positive control cycloheximide (44.8% ± 7.9%, p=0.0001). These findings confirmed the potential of SL-101 to efficiently internalize and promote cell death through protein synthesis blockade. We further investigated the ability of SL-101 to inhibit intracellular signaling in response to IL-3. To this end, cytokine-dependent Mo7e leukemia cells were serum starved and pre-treated with SL-101 at 1.0 µg/ml overnight, followed by stimulation with IL-3. SL-101 significantly suppressed IL-3-induced activation of p-STAT5 (57.1% ± 2.6% inhibition, p=0.003) and modestly inhibited p-AKT (17.4% ± 5.4% inhibition, p=0.04), but not p-ERK signaling (Fig. 1D). In summary, our data demonstrate that the novel anti-CD123 antibody-conjugate, SL-101, is highly active in AML and induces growth arrest and apoptosis in AML blasts and LSCs by inhibiting protein synthesis and interfering with IL-3 signal transduction pathways. Ongoing studies that will be reported at this meeting investigate the in vivo anti-leukemia efficacy of SL-101 in NSG mice engrafted with primary AML cells. In conclusion, SL-101 is a novel, potent antibody-conjugate directed against AML blasts and LSCs, and our studies warrant further development of this agent. Disclosures Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership.

Anti-Leukemia Efficacy and Mechanisms of Action of SL-101, a Novel Anti-CD123 Antibody-Conjugate in Acute Myeloid Leukemia

Outcomes of acute myeloid leukemia (AML) remain poor and warrant the development of novel therapeutic agents. CD123 (interleukin-3 [IL-3] receptor alpha subunit), is overexpressed on AML blasts and leukemia progenitor/stem cells (LSCs) compared to normal hematopoietic cells (Jordan et al. Leukemia 2000). SL-101 is a novel anti-CD123 antibody-conjugate comprised of anti-CD123 scFv fused to a truncated and optimized pseudomonas exotoxin (PE) lacking its native targeting domain. We have previously demonstrated SL-101’s cell killing efficacy in AML cell lines (Han et al. ASH 2013). Here we report the anti-tumor efficacy of SL-101 against primary AML cells and the underlying mechanisms of its cytotoxicity.Fourteen genetically diverse primary AML samples were treated with various doses of SL-101 for 48 h. Most samples express high levels of CD123 (median 89.9%, range 20.4-99.3%) and intermediate levels of CD131 (median 54.0%, range 9.0-91.1%), the IL-3 beta subunit. SL-101 was highly active against AML samples, with an IC50 of 0.19 µg/ml (range 0.003 - 0.98 µg/ml). No significant correlation was found between SL-101 activity and levels of CD123 or CD131 (n=12). SL-101 also selectively and significantly suppressed AML colony formation (69.5% ± 15.0% inhibition of total colonies, n=7) while sparing normal bone marrow (5.6% ± 3.3% inhibition, n=4; p=0.0001) (Fig. 1A).We next investigated the mechanisms of the cytotoxic activity of SL-101. Using annexinV/DAPI flow cytometry, we first evaluated the induction of apoptosis in AML blasts and phenotypically defined CD45dimCD34+CD38-CD123+ LSCs. After 48 h treatment, SL-101 at 1.0 µg/ml induced higher specific apoptosis in the AML LSCs (51.2% ± 25.4%) than in blasts (39.4% ± 19.0%, p=0.006, n=10). Quantification of the annexinV–/DAPI– viable cells using counting beads demonstrated further reduction of cell numbers by SL-101 (LSCs 72%, blasts 64.6%), indicating additional mechanisms of cell growth inhibition.It was recently demonstrated that upon internalization, PE traffics through the endoplasmic reticulum to the cytosol, where it inactivates protein synthesis by catalyzing ADP ribosylation of elongation factor 2 and causes non-apoptotic cell death (Wayne et al. Blood 2014). To examine the contribution of the direct inhibition of protein synthesis by PE, we first studied SL-101 internalization utilizing DyLight 680-labeled SL-101 by flow cytometry and fluorescence imaging. In CD123-expressing AML cell lines MV4-11 and MOLM13, the intracellular median intensity of DyLight 680 signal increased 5.4- and 3.5-fold, respectively, within 4 h of treatment and 22.3- and 16.3-fold after 24 h (Fig 1B). Fluorescence imaging confirmed cytosolic localization of SL-101 in both cell lines, demonstrating efficient cellular uptake of SL-101.We also examined the efficacy of SL-101 in inhibiting nascent protein synthesis in MV4-11 cells using an AHA Alexa Fluor 488 protein synthesis assay. SL-101 significantly reduced protein synthesis (40.3%, p=0.0005) within 4 h (Fig 1C), even at low concentrations (0.01 µg/ml), which was comparable to the positive control cycloheximide (44.8% ± 7.9%, p=0.0001). These findings confirmed the potential of SL-101 to efficiently internalize and promote cell death through protein synthesis blockade.We further investigated the ability of SL-101 to inhibit intracellular signaling in response to IL-3. To this end, cytokine-dependent Mo7e leukemia cells were serum starved and pre-treated with SL-101 at 1.0 µg/ml overnight, followed by stimulation with IL-3. SL-101 significantly suppressed IL-3-induced activation of p-STAT5 (57.1% ± 2.6% inhibition, p=0.003) and modestly inhibited p-AKT (17.4% ± 5.4% inhibition, p=0.04), but not p-ERK signaling (Fig. 1D).In summary, our data demonstrate that the novel anti-CD123 antibody-conjugate, SL-101, is highly active in AML and induces growth arrest and apoptosis in AML blasts and LSCs by inhibiting protein synthesis and interfering with IL-3 signal transduction pathways. Ongoing studies that will be reported at this meeting investigate the in vivo anti-leukemia efficacy of SL-101 in NSG mice engrafted with primary AML cells. In conclusion, SL-101 is a novel, potent antibody-conjugate directed against AML blasts and LSCs, and our studies warrant further development of this agent. [Display omitted] DisclosuresRowinsky:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership.

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Strain-alleviation model of ADP-ribosylation

ADP-ribosylation was one of the first molecular mechanisms described to be used by bacterial protein toxins to target eukaryotic cells. Most potent and devastating toxins belong to this group, including diphtheria toxin and Pseudomonas exotoxins A, which block protein synthesis by ADP-ribosylation of elongation factor 2. Cholera toxin from Vibrio cholerae, which causes several thousand cases of death worldwide every year, is another ADP-ribosylating toxin, of which ADP-ribosylate Gs proteins is involved in receptor signaling (1). All of these toxins modify host proteins by transferring ADP ribose from NAD+ onto specific eukaryotic target proteins, thereby altering their physiological functions. Although ADP-ribosylating toxins share only limited sequence similarity, crystal structures available from various toxins are amazingly similar (2). However, so far the precise molecular reaction by which these toxins catalyze the ADP-ribosylation of their target proteins has remained largely enigmatic. In an exciting study published in PNAS (3), Tsurumura et al. describe several crystal structures of Clostridium perfringens iota toxin, in complex with its protein substrate actin, which are obtained during different phases of the ADP-ribosylation process. The authors’ findings are groundbreaking not only for the understanding of toxin-induced ADP-ribosylation but also for comprehension of the molecular reaction induced by an increasing number of endogenous ADP ribosyltransferases.

Endogenous ADP-ribosylation of elongation factor-2 by interleukin-1β

Eukaryotic elongation factor-2 (eEF-2) catalyses the motion of the growing peptide chain relative to the mRNA at the ribosomes during protein synthesis. This highly conserved G-protein is the specific target of two lethal bacterial toxins, Pseudomonas aeruginosa exotoxin A and diphtheria toxin. These toxins exert their detrimental action by ADP-ribosylating a biologically unique posttranslationally modified histidine residue (diphthamide(715)) within eEF-2, thus inactivating the enzyme. Diphthamide(715) is also the target of endogenous (mono) ADP-ribosyl transferase activity. In this article, we report the first known activator of endogenous ADP-ribosylation of eEF-2, interleukin-1β (IL-1β). Thereby, systemic inflammatory processes may link to protein synthesis regulation.

Phase I trial of recombinant immunotoxin CAT-8015 (HA22) in multiply relapsed hairy cell leukemia.

6523 Background: CAT-8015 (HA22) contains the Fv domains of the anti-CD22 Mab RFB4 and truncated pseudomonas exotoxin, inducing cell death after binding, internalization, and ADP-ribosylation of elongation factor 2. CAT-8015 is a high-affinity mutant of BL22 (CAT-3888), with increased cytotoxicity toward chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL) cells ex vivo. Methods: To determine the clinical activity of CAT-8015, separate phase I trials were initiated in CLL, HCL, and non-Hodgkin lymphoma, and the HCL trial completed accrual. Eligible patients had several prior therapies including purine analogs and needed treatment for cytopenias or symptoms. CAT-8015 was administered over 30 min iv days 1, 3 and 5 (QODx3) at 28-day intervals for up to 10 cycles. Results: 28 HCL patients received CAT-8015: 3 each at 5, 10, 20, and 30 ug/kg QODx3, 4 at 40 and 12 at 50 ug/kg QODx3. Patients had 2-7 (median 3) prior therapies. Patients received 1-9 (median 4) cycles of CAT-8015 each. Dose-limiting toxicity (DLT) was not observed. Two patients, with cycle 3 of 30 and cycle 5 of 50 ug/Kg QOD x3, had a grade 2 reversible hemolytic uremic syndrome (HUS) with peak creatinine of 1.53-1.66 mg/dL and platelet nadir of 111,000-120,000/mm3. Drug-related toxicities in 25-60% of the 28 patients included (in decreasing frequency) grade 1-2 hypoalbuminemia, edema, fever, ALT and AST elevations, headaches, and nausea. Of 25 evaluable patients, 44% made antibodies neutralizing > 75% of the cytotoxicity of 1,000 ng/mL of CAT-8015. Responses were achieved at all dose levels. The overall response rate was 79% including 12 (43%) of 28 HCL patients achieving complete remission (CR). Of the 12 CRs, 11 (92%) are ongoing 7-29 (median 21) months after enrollment. Conclusions: CAT-8015 is active in relapsed/refractory HCL and is without DLT after completion of expansion at the highest dose level (50 ug/Kg x3) to 12 patients. These results justify further treatment of patients at this dose level to further explore efficacy and safety. Dose-escalation will continue in a separate trial to determine if the increased cytotoxic activity of CAT-8015 compared to CAT-3888 will be associated with significant efficacy in CLL and non-Hodgkin lymphoma. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration MedImmune

Abstract 4356: Cytotoxicity of the anti-CD22 immunotoxin HA22 against pediatric acute lymphoblastic leukemia (ALL)

Abstract Cure rates for children with acute lymphoblastic leukemia (ALL) have steadily improved, but a proportion of patients still relapse despite intensive and prolonged chemotherapy. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. We have developed recombinant anti-CD22 immunotoxins composed of the Fv portion of the anti-CD22 mAb RFB4 linked to a 38kDa truncated protein derived from Pseudomonas exotoxin A (PE38). These produce cell death through a unique and highly specific mechanism of action, entailing binding to cell surface CD22, rapid internalization, intracellular processing, and inhibition of protein synthesis through ADP-ribosylation of elongation factor-2. In phase I and II trials of patients with hairy cell leukemia the first generation agent, BL22, produced complete responses (CR) of 61% and 47% respectively. However, activity was modest in children with ALL (Blood 2007;110:262a). A modified agent, HA22, was derived by complementarity-determining region mutagenesis resulting in a significantly higher binding affinity for the CD22 antigen. We cultured pre-B ALL cells from newly diagnosed (n = 13) and relapsed children (n= 22), as well as human ALL cells derived from xenografts in immunodeficient mice (n = 4) and the cell line KOPN-8 on telomerase-immortalized human bone marrow mesenchymal cells. On day 2 of culture, HA22 was added at concentrations ranging from 0 to 500ng/ml and the cells were incubated for an additional 72 hours. Dexamethasone 10umol/L was used as a positive control. The number of viable CD19+ cells at the end of the cultures was determined by flow cytometry. Viability was assessed using gates set for light-scatter properties and Annexin-PE/7-AAD staining on Days 2 and 5. The percentage of CD19+ viable cells relative to the control was determined and the 50% inhibitory concentration (IC50) calculated. Mesenchymal-supported cell cultures maintained ALL cell viability (mean 77%, median 52%, range 6% - 280% viable cells at 96 hours). ALL samples varied in their sensitivity to HA22. IC50s ranged from 1-80 ng/mL in 29 of 40 samples. In 11 samples 50% killing was not achieved. Resistance to cytotoxicity from dexamethasone was also observed, which correlated with resistance to HA22 for samples from newly diagnosed patients (r = 0.55, p = 0.053). Peripheral blood blasts appeared to be more sensitive to HA22 in comparison to bone marrow derived blasts (p = 0.008). Blasts with higher CD22 site densities showed increased cytotoxicity to HA22 (r = −0.34, p = 0.16). These results indicate that most cases of childhood B-lineage ALL are sensitive to HA22 at concentrations well below clinically achievable blood levels. Clinical testing of HA22 in children with drug-resistant ALL has been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4356.

Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell Intoxication

Exotoxin A is a major virulence factor of Pseudomonas aeruginosa. This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of the toxin proteins with different unfolding abilities so that they inhibited or did not inhibit endosomal translocation of the chimera. We then assayed the amount of toxin fragments delivered to the cytosol during cell intoxication. Finally we used furin inhibitors and examined the fate and intracellular localization of the toxin and its receptor. The results showed that exotoxin cytotoxicity results largely from endosomal translocation of the entire toxin. We found that the C-terminal fragment was unstable in the cytosol.

Optimizing Denileukin Diftitox (Ontak ® ) Therapy

Denileukin diftitox (Ontak) is a novel recombinant fusion protein consisting of peptide sequences for the enzymatically active and membrane translocation domain of diphtheria toxin linked to human IL-2. Denileukin diftitox specifically binds to IL-2 receptors on the cell membrane, is internalized via receptor-mediated endocytosis and inhibits protein synthesis by ADP ribosylation of elongation factor 2, resulting in cell death. This article focuses on the clinical trial that led to the US FDA approval of the drug for cutaneous T-cell lymphoma in 1999, and other investigational studies for hematologic malignancies, recurrent and refractory chronic lymphocytic leukemia, non-Hodgkin B-cell lymphoma, graft-versus-host disease and autoimmune disease, demonstrating the activity and adverse effects of the drug.

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Characterization of a Cloned Temperature-Sensitive Construct of the Diphtheria Toxin A Domain

Our goal was to determine how the DTM mutant construct of the A domain of diphtheria toxin (DTx) causes temperature-sensitive effects in Drosophila and yeast [Bellen, H. J., D'Evelyn, D., Harvey, M., Elledge, S. J. (1992) Development 114, 787-796]. Because DTM fortuitously bears the same point mutation as found in the A chain of CRM197, an ADP-ribosyltransferase (ADPrT)-deficient form of DTx, we hypothesized that the dramatic low-temperature-sensitive effects did not stem from ADP-ribosylation of elongation factor 2 (EF-2). To rule out acquisition of ADPrT activity at low temperatures, we assayed mutant forms of the A domain of DTx produced by in vitro transcription/translation and found that DTM has no ability to ADP-ribosylate EF-2 at 18 or 30 degrees C. Because the DTM gene results in a protein with a 23-amino acid missense carboxy-terminal extension, we also constructed a form without this extension. Assays for nuclease activity revealed that nuclease activity comigrated with the two distinguishable E. coli-cloned mutant proteins DTM and DTM-23, regardless of whether electrophoresis was conducted under denaturing or nondenaturing conditions in gels embedded with DNA. Studies with CRM197 showed that Ca(2+) and Mg(2+) promote single-strand DNA nicks, whereas Mn(2+) promotes double-strand DNA breaks. Evidence that the cation-dependent nuclease and NAD-dependent ADPrT enzymic sites are distinct is that NAD protected only the A domain of DTx from proteolytic cleavage, whereas DNA protected the A domains of both DTx and CRM197. We conclude that the nuclease activity of DTM is responsible for the temperature-sensitive effects associated with its expression in both yeast and Drosophila.

Enhancement of Diphtheria Toxin-induced Apoptosis in Vero Cells by Combination Treatment with Brefeldin A and Okadaic Acid.

In the present study, we compared the abilities of ricin and diphtheria toxin to induce apoptosis in Vero cells. The cytolysis and DNA fragmentation by ricin paralleled its protein synthesis inhibitory activity. However, unlike ricin, diphtheria toxin could induce neither cytolysis nor DNA fragmentation in Vero cells up to very high concentration, in spite of the fact that Vero cells were even more sensitive to protein synthesis inhibition by diphtheria toxin than ricin. Interestingly, coexistence of brefeldin A (BFA) and okadaic acid (OA) significantly enhanced diphtheria toxin-mediated cytolysis and DNA fragmentation without affecting the activity of protein synthesis inhibition. Ammonium chloride almost completely abolished the ability of diphtheria toxin to induce apoptosis in the presence of BFA and OA as well as the protein synthesis inhibitory activity. The mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2), failed to induce apoptosis in Vero cells even in the presence of BFA and OA. Thus, translocation of diphtheria toxin into the cytosol and subsequent enzymatic inactivation of EF-2 may be necessary steps to induce apoptosis. Taken together our results suggest that protein synthesis inhibition by toxins is not sufficient to induce apoptosis, and underlying mechanisms of apoptosis induction may be distinct between ricin and diphtheria toxin. Since a morphological change in the Golgi complex was observed in Vero cells treated with BFA and OA, modulation of the Golgi complex by these reagents may be partly responsible for enhanced apoptosis induction by diphtheria toxin.

Diphtheria toxin NAD affinity and ADP ribosyltransferase activity are reduced at tryptophan 153 substitutions for alanine or phenylalanine

Previous studies on chemical modifications of diphtheria toxin (DT) fragment A have suggested that the Trp153 amino acid residue is essential for the ADP ribosylation of elongation factor 2. We verified this experimentally after replacing Trp153 by Phe or Ala residues through in vitro mutagenesis of a cloned toxin gene fragment. Each of the mutant fragment A forms were found to reveal a reduced ADP ribosyl transferase (ADPRT) activity as well as lower affinity for NAD. Both ADPRT activity and NAD affinity of DT fragment A were only partially destroyed by nearly synonymous Trp153 => Phe153 substitution, but dramatically destroyed by Ala153 substitution. At the same time, each of the mutant fragment A forms appeared to be thermostable, suggesting that the mutations do not dramatically destroy the structure of the protein. These results clearly demonstrate that Trp153 is not highly specific for DT fragment A structure maintenance, but is highly specific for the key toxin functions such as ADP ribosylation of elongation factor 2 and NAD binding. We suggest that the Trp153 role in DT functioning may be that of binding the ribose moiety of NAD, which is crucial for DT catalytic activity and hence for toxicity.

Requirement for Prolonged Action in the Cytosol for Optimal Protein Synthesis Inhibition by Diphtheria Toxin

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.

Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A.

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T(H)1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA(mut) gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.

Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T H1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA mut gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.

On the Mode of Inhibition of Eukaryotic Protein Synthesis by ADP-Ribosylation of Elongation Factor 2

On the Mode of Inhibition of Eukaryotic Protein Synthesis by ADP-Ribosylation of Elongation Factor 2