A guide to taming a toxin--recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer

Pseudomonas exotoxin A (PE) is a highly toxic protein secreted by the opportunistic pathogen Pseudomonas aeruginosa. The modular structure and corresponding mechanism of action of PE make it amenable to extensive modifications that can redirect its potent cytotoxicity from disease to a therapeutic function. In combination with a variety of artificial targeting elements, such as receptor ligands and antibody fragments, PE becomes a selective agent for the elimination of specific cell populations. This review summarizes our current understanding of PE, its intoxication pathway, and the ongoing efforts to convert this toxin into a treatment for cancer.     

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.     

Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation

Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.     

A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

Pseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains. Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death. To investigate the role of the six alpha-helices (A-F) that form the translocation domain, we deleted them successively one at a time. All mutants showed native cell-binding and catalytic activities, indicating that deletions specifically affected translocation activity. This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectly by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication. Deletion of B, C and D helices resulted in non-toxic and non-translocating molecules, whereas mutants lacking the A or E helix displayed significant cytotoxicity albeit 500-fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three- to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtained until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule.     

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.     

Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.     

Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.     

Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.     

Crystallization of exotoxin A from Pseudomonas aeruginosa

Exotoxin A from Pseudomonas aeruginosa has been crystallized in a form suitable for high resolution diffraction analysis. The crystals, grown in the presence of high concentrations of polyethylene glycol (20%, w/v) and of NaCl (1.5 m), are monoclinic and contain one monomeric toxin molecule per asymmetric unit. The space group is P2₁, with $\text{a = 60.6}\text{A}\text{?}\text{, b = 100.2}\text{A}\text{?}\text{, c = 59.8}\text{A}\text{?}\text{, β = 98.6 °}$.     

Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.     

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.     

Influence of deletions within domain II of exotoxin A on its extracellular secretion from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for P. aeruginosa exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six alpha-helices (A to F). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within the periplasm. The conformational rearrangement induced in this variant is predicted from the three-dimensional PE structure, and the molecular modification is confirmed by gel filtration experiments. Helix E is in the core of the molecule and creates close contact with other domains (I and III). Deletion of the surface-exposed helix F has no effect on secretion, indicating that no secretion information is contained in this helix. Finally, we concluded that disruption of a structured domain II yields an extended form of the molecule and prevents formation of the conformational secretion motif.     

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

 We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Δ34–220), which had most of the Ia domain deleted but retained amino acid residues 1–33 and 221–252 of this domain, was constructed. EGF-PE(Δ34–220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Δ34–220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(Δ1–252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(Δ34–220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(Δ34–220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Δ34–220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells. Received : 20 May 1994 / Received last revision : 9 September 1994 / Accepted : 28 September 1994     

重组铜绿假单胞菌外毒素结构域Ia片段的佐剂功效研究

采用分子克隆技术,将铜绿假单胞菌PA103株编码的外毒素结构域Ia(Domain Ia)的基因重组于原核表达载体pET-42b( )上,构建了pET-EPA103蛋白表达载体。转化感受态大肠杆菌DE3。经IPTG诱导表达,初步纯化表达蛋白,用以免疫BALB／c纯系小鼠。制备小鼠脾淋巴细胞悬液,经刀豆素A(ConA)刺激后,用MTT比色法检测特异性淋巴细胞增殖反应。通过rEPA皮下注射BALB／c小鼠耳廓,诱导小鼠迟发型过敏反应(DTH)。采用特异性淋巴细胞增殖反应和DTH试验来检测pET-EPA103表达蛋白所引起的小鼠细胞免疫应答水平,淋巴细胞增殖情况与DTH均可间接反映细胞免疫应答水平,进而评价重组铜绿假单胞菌外毒素(rEPA)Domain Ia蛋白片段的佐剂功效。     

The acid-triggered entry pathway of Pseudomonas exotoxin A

In this study we examined the pH requirements and reversibility of early events in the Pseudomonas toxin entry pathway, namely, membrane binding, insertion, and translocation. At pH 7.4, toxin binding to vesicles and insertion into the bilayer are very inefficient. Decreasing the pH greatly increases the efficiencies of these processes. Acid-treated toxin exhibits pH 7.4 binding and insertion levels. This indicates that hydrophobic regions that become exposed upon toxin acidficiation become buried again when the pH is raised to 7.4. In contrast, the change in toxin conformation that occurs upon membrane binding is irreversible. Returning samples to pH 7.4, incubation with excess toxin, or dilution with buffer up to 1000-fold leads to very little loss of bound toxin. Bound toxin exhibits an extremely high susceptibility to trypsin compared to free toxin (at both pH 4 and pH 7.4). At pH 4, membrane-associated toxin slowly proceeds to a trypsin-protected state; neutralization halts this process. At low pH, toxin was found to bind and insert into DMPC vesicles very efficiently at temperatures both above and below 23 degrees C, the lipid melting point. With fluid targets, the proportion of bound toxin that was photolabeled from within the bilayer peaked rapidly and then decreased with time. With frozen targets, the efficiency of photolabeling peaked but then remained fairly constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the structure-function relationship of Pseudomonas aeruginosa exotoxin A

Biochemical and genetic techniques have provided considerable insight into the structure-function relationship of one of the ADP-ribosyl transferases produced by Pseudomonas aeruginosa, exotoxin A. Exotoxin A contains a typical prokaryotic signal sequence which, in combination with the first 30 amino-terminal amino acids of the mature protein, is sufficient for exotoxin A secretion from P. aeruginosa. Determination of the nucleotide sequence and crystalline structure of this prokaryotic toxin allowed a molecular model to be constructed. The model reveals three structural domains of exotoxin A. Analysis of the identified domains shows that the amino-terminal domain (domain I) is involved in recognition of eukaryotic target cells. Furthermore, the central domain (domain II) is involved in secretion of exotoxin A into the periplasm of Escherichia coli. Evidence also implicates the role of domain II in translocation of exotoxin A from the eukaryotic vesicle which contains the toxin after it becomes internalized into susceptible eukaryotic cells via receptor-mediated endocytosis. The carboxy-terminal portion of exotoxin A (domain III) encodes the enzymatic activity of the molecule. The structure of this domain includes a cleft which is hypothesized to be the catalytic site of the enzyme. Several residues within domain III have been identified as having a direct role in catalysis, while others are hypothesized to play an important structural role.     

Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP-ribosyl, which catalyze the covalent transfer of an ADP-ribose moiety of NAD⁺ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD⁺ binding site of exotoxin A: the binding of NAD⁺ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.