Abstract:
The present invention generally relates to a new approach for thetherapy of allergic responses, based on targeted elimination of cells expressing the FcεRI receptor by a chimeric cytotoxin FC 2′-3 -PE 40 . A sequence encoding amino acids 301-437 of the Fc region of the mouse IgE molecule was genetically fused to PE 40 ′—a truncated form of PE lacking the cell binding domain. The chimeric protein, produced in  E. coli,  specifically and efficiently kills mouse mast cell lines expressing the FcεRI receptor, as well as primary mast cells derived from bone marrow. The present invention provides a chimeric protein for targeted elimination of FcεRI expressing cells especially useful for the therapy of allergic responses. The said chimeric protein is comprised of a cell targeting moiety for FcεRI expressing cells and a cell killing moiety. The preferred killing moiety is the bacterial toxin Pseudomonas exotoxin (PE). This Pseudomonas exotoxin is a product of  Pseudomonas aeruginosa.  The present invention also relates to a method for the preparation of said protein. This chimeric protein is prepared by genetically fusing the Fc region of the mouse IgE molecule to PE 40 , a truncated form of PE lacking the cell binding domain. The present invention also provides pharmaceutical compositions, for the treatment of allergic diseases and for the treatment of hyperplasias and malignancies, comprising as an active ingredient the above mentioned chimeric protein and a conventional adjuvant product.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention generally relates to a novel approach for the therapy of allergic responses. More specifically the present invention relates to Fcε-PE chimeric protein for targeted elimination of FcεRI expressing cells, a method for its production, and pharmaceutical compositions containing the same. This chimeric protein is composed of cell targeting which is a part of IgE molecule linked to cell killing moieties for recognizing and distroying cells overexpressing the specific receptor. The killing moiety used in the chimeric protein of the present invention is the bacterial toxin Pseudomonas exotoxin (PE) (a product of  Pseudomonas aeruginosa ).  
         BACKGROUND OF THE INVENTION  
         [0002]    About twenty percent of the world population suffers from various allergic diseases such as asthma, allergic rhinitis, food allergies, atopic dermatitis and anaphylaxis. The alarming increase in the prevalence of allergic diseases over the past decade has led to a clear need for more effective treatment.  
           [0003]    The interaction between IgE and mast cells or basophils is the primary effector pathway in allergic responses. IgE binds to high-affinity receptor (FcεRI) for its constant region, found almost exclusively on the surface of these cells. The binding itself, in spite of the low dissociation rate, does not result in stimulation of the cell. However, cross-linkage of cell surface-bound IgE by multivalent antigen causes receptor aggregation, triggering explosive cellular degranulation whereby mediators of allergy such as cellular degranulation whereby mediators of allergy such as histamine and seretonin are released.  
           [0004]    The fact that distribution of the FcεRI receptor is restricted to cells participating in an allergic response makes it an attractive candidate for targeted immunotherapy by chimeric cytotoxins. Chimeric cytotoxins are a novel class of targeted molecules constructed by gene fusion techniques. These molecules are composed of cell targeting and cell killing moieties, enabling them to recognize and distroy cells overexpressing specific receptors.  
           [0005]    The bacterial toxin Pseudomonas exotoxin (PE) used in chimeric protein constructs, is a product of  Pseudomonas aeruginosa.  Having accessed the cytoplasm, PE inhibits protein synthesis by its ADP-ribosylation activity, thus causing cell death (Middlebrook, J. I., and Dorland, R. B. 1984. Bacterial toxins: cellular mechanisms of action. Microbiol. Rev. 48, 199.). Effective chimeric cytotoxins have been constructed by fusion of cDNAs encoding various growth factors or single chain antibodies with PE derivatives lacking intrinsic cell binding capacity. One of these chimeric proteins designated IL 2 -PE 40 , constructed to target and selectively eliminate activated T cells overexpressing IL 2  receptors, was shown to provide effective and selective immunosuppression in various models of autoimmune disorders, graft rejection and cancer (Lorberboum-Galski, H. 1994. Interleukin 2-Pseudomonas exotoxin A (IL2-PE40) chimeric protein for targeted immunotherapy and the study of immune responses. J. Toxicol.-Toxin Rewiewes, 13 (1), 105.).  
           [0006]    The entire recombinant constant region of IgE (Fcε) expressed in bacteria, have an affinity for FcεRI receptor comparable to that of the native IgE, as well as the capacity to sensitize basophils for anti-IgE indused histamine release. When recombinant fragments of human Fcεexpressed in bacteria, were tested for receptor binding, a peptide corresponding to residues 301-376 at the junctions of domains 2 and 3 of the constant region was found to be sufficient for high-affinity binding to the receptor. It was also reported that ε-chain dimerization was not required for receptor binding (Helm, B., Marsc, P., Vercelli, D., Padlan, E., Gould, H., and Geha, R. 1988. The mast cell binding site on human immunoglobulin E. Nature 331, 180.).  
           [0007]    The present invention generally relates to a novel approach for the therapy of allergic responses. At present the major known groups of drugs used in the treatment of asthma and allergic disorders are:  
           [0008]    1. β2 agonists—produce airway dilatation through simulation of β2 adrenergic receptors.  
           [0009]    2. Methylxantines—smooth muscle relaxants, produce bronchodilatation.  
           [0010]    3. Glucocorticoids—reduce inflammation.  
           [0011]    4. Cromolyn sodium—prevents mast cell degranulation.  
           [0012]    5. Antihistamines—prevents histamine action on it&#39;s target cells.  
           [0013]    Although widely used, all of these drugs have notable disadvantages in regard to:  
           [0014]    1. Specificity: The action of all of these drugs (except cromolyn sodium) is not mast cell specific. Therefore, they can not prevent the release of allergy mediators but rather reverse or block the effects caused by their action. The treatment by these drugs is symptomatic, it can be started only after the onset of the allergic reaction and thus can&#39;t be used in a prophylactic manner.  
           [0015]    2. Toxicity: Being non-specific, these drugs exert their action on various tissues and organs causing serious side effects. The major side effect of B2 agonists is tremor, but they also cause cardiac arrhythmias; Methylxantines stimulate the central nervous system, causing nervousness, nausea, vomiting, anorexia, headache and cardiac muscle-causing tachycardia. At high plasma levels there is a danger of seizures and arrhythmias. Antihistamines affect the central nervous system, causing sedation. Steroids are most harmful, causing suppression of the pituitary-adrenal function, fluid and electrolyte disturbances, hypertension, hyperglycemia, increased susceptibility to infections, osteoporosis and arrest of growth in children.  
           [0016]    3. Duration of the effect: β-adrenergic agonists, aminoxantines and antihistamines are mostly short-acting drugs, and as such must be administered frequently. Steriods which are long-acting drugs, have also long induction time and are of little value in emergencies.  
           [0017]    The only existing mast cell specific drug is Cromolyn sodium. This drug can be used prophilactically, essentially without side effects. However, it has a very short half life, very long induction time, it can be applied only locally and only part of the patients respond to it. All these make use of Cromolyn sodium very limited.  
           [0018]    A number of attempts to interfere with interaction between IgE and it&#39;s high-affinity receptor, as a basis for anti-allergy therapy, have been reported in recent years. Recombinant peptides comprising structural elements from IgE (Helm, B., Kebo, D., Vercelli, D., Glovsky, M. M., Gould, H., Ishizaka, K., Geha, R., and Ishizaka, T. 1989. Blocking the passive sensatization of human mast cells and basophil granolocytes with IgE antibodies by a recombinant human ε-chain fragment of 76 amino acids. Proc. Natl. Acad. Sci. USA 86, 9465.) or FCεRI (Ra, C., Kuromitsu, S., Hirose, T., Yasuda, S., Furuichi, K., and Okumura, K. 1993. Soluble human high affinity receptor for IgE abrogates the IgE-mediated allergic reaction. Int. Immunol. 5, 47.;Haak-Frendscho, M., Ridgway, J., Shields, R., Robbins, K., Gorman, C., and Jardieu, P. 1993. Human IgE receptor a-chain IgG chimera blocks passive cutaneous anaphylaxis reaction in vivo. J. Immunol. 151, 351.) have been investigated as competitive inhibitors of the IgE-FcεRI interaction. Monoclonal antibodies generated against IgE (Baniyash, M., and Eshhar, Z. 1984. Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE. Eur. J. Immunol. 14, 799) or FcεRI (Kitani, S., Kraft, D., Fischler, C., Mergenhagen, S. E., and Siraganian, R. P. 1988. Inhibition of allergic reactions with monoclonal antibody to the high affinity IgE receptor. J. Immunol. 140, 2585.), capable of blocking IgE binding to the receptor, without causing mast cell degranulation have also been tested. However, the affinity of IgE for FcεRI is very high (K M =10 −20 M), so that once it is bound to it&#39;s receptor, the IgE molecule remains attached to the cell membrane for several weeks. Moreover, mast cell can be activated at low receptor occupancy: the cross-linkage of as few as 5% of receptors is sufficient to cause mast cell degranulation. These two properties of the system impede inhibition by competitive agents, thus limiting their clinical value. Our anti-allergy molecule depends to a much lesser extent on the ability to compete with IgE. Once having entered the target cell through a non-occupied IgE receptor, the chimeric protein affects the target cell. Moreover, early expression of the receptor in the maturation course of mast calls should allow the elimination of immature target cells before they are capable of mediator release. As the receptor is not expressed on stem cells, no damage to bone marrow is expected on the whole.  
           [0019]    The IgE system is quite complex and diverse. Interactions between IgE and its binding structures have many functions apart from the allergic response, some of which are only beginning to emerge. Monoclonal antibodies against IL-4, the IL-4 receptor or the low-affinity IgE receptor eliminate expression of IgE in mice but have more general immunosupressive effects. The advantage of the present invention in which the high-affinity IgE receptor is targeted and not the overall IgE system, is therefore evident.  
         SUMMARY OF THE INVENTION  
         [0020]    The present invention generally relates to a new approach for therapy of allergic responses, based on targeted elimination of cells expressing the FcεRI receptor by a chimeric cytotoxin Fc 2′-3 -PE 40 . A sequence encoding amino acids 301-437 of the Fc region of the mouse IgE molecule was genetically fused to PE 40 —a truncated form of PE lacking the cell binding domain. The chimeric protein, produced in  E. coli,  specifically and efficiently kills mouse mast cell lines expressing the FcεRI receptor, as well as primary mast cells derived from bone marrow.  
           [0021]    The present invention provides a chimeric protein for targeted elimination of Fc&lt;RI expressing cells especially usefull for the therapy of allergic responses. The said chimeric protein is comprisesd of a cell targeting moiety for the FcεRI expressing cells and a cell killing moiety. The preferred killing moiety is the bacterial toxin Pseudomonas exotoxin (PE). This Pseudomonas exotoxin is a product of  Pseudomonas aeruginosa.    
           [0022]    The present invention also relates to a method for the preparation of said protein. This chimeric protein is prepared by genetically fusing the Fc region of the mouse IgE molecule to PE 40 , a truncated form of PE lacking the cell binding domain.  
           [0023]    The present invention also provides a pharmaceutical compositions, for the treatment of allergic diseases and for the treatment of hyperplasias and malignancies, comprising as an active ingredient the above mentioned chimeric protein and a conventional adjuvant product.  
           [0024]    The present invention further relates to the method for the preparation of these pharmaceutical compositions comprising genetically fused Fc region of the mouse IgE molecule to PE 40  and adding, if needed, a conventional adjuvant product. The pharmaceutical compositions according to the present invention may be in any suitable form for injection, for toppical application, or for oral administration.  
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0025]    The Fc-PE chimeric protein according to the present invention has a number of advantages over the existing known drugs.:  
         [0026]    1. Specificity: Fc-PE is highly specific, affecting the cells (mast cells and basophils) responsible for the release of allergic mediators. As it prevents the allergic attack, it can be of great value as a prophylactic treatment.  
         [0027]    2. Toxicity: As it acts on affector cells and not on it&#39;s target organs, Fc-PE is expected to have little, if any, side effects. Moreover, as the receptor is not expressed on stem cells, no damage to bone marrow and immunosupression are anticipated. Re-institution of a normal physiological state is expected to occur within several weeks after the end of the treatment.  
         [0028]    3. Duration of the effect: Because maturation of mast cells takes several weeks, the effect of Fc-PE is predicated to be long-standing, eliminating the need for frequent administration. Moreover, as in vitro studies indicate that reduction of 80% in cellular protein systhesis is observed in less than 4 hours, induction time of Fc-PE is expected to be relatively short, enabling it&#39;s usage in acute phase allergic reactions.  
         [0029]    Fcε-PE can also be valuable in the treatment of hyperplasias and malignancies of mast cells and basophils, like systemic mastocytosis (in both benign and malignant forms) and basophilic leukemia. Chemotherapy is not appropriate for patients with benign mastocytosis due to severe side effects. On the other hand, there is no good clinical protocol for the treatment of the malignant diseases. Fcε-PE chimeric protein, being highly potent and selective can be used for both benign and malignant conditions involving cells expressing the FcεRI receptors.  
         [0030]    The following experimental results indicate that the Fc 2′-3-PE 40chimeric protein according to the present invention is a promising candidate for effective and selective allergy therapy.  
         [0031]    The present invention provides a FCε-PE chimeric cytotoxin protein for the targered elimination of FcεRI expressing cells, useful especially for the therapy of allergic responses such as asthma, allergic rhinitis, food allergies, atopic dermatitis, and anaphylaxis.  
         [0032]    The said invention will be further described in detail by the following experiments. These experiments do not intend to limit the scope of the invention but to demonstrate and clarify it only.  
         [0033]    1. Construction of Fcε-PE 40  chimeric proteins.  
         [0034]    For the targeting moiety of the chimeric proteins fragments of the mouse IgE constant region (Fcε) are used as it binds both to human and to mouse high affinity IgE receptors (Conrad, D. H., Wingard, J. R., and Ishizaka, T. 1983 The interaction of human and rodent IgE with the human basophil IgE receptor. J. Immunol. 130, 327.).  
         [0035]    We used a sequence corresponding to a.a. 301-437, containing the COOH terminus of domain 2 and the entire domain 3(C 2 ′-C 3 ). We used also a sequence corresponding to a.a. 225-552, containing the whole C 2 -C 4 domains. The cDNA for these fragments was obtained by RT-PCR, using RNA isolated from mouse B cells which were isotopically switched to secrete IgE and a specific set of primers. B cells obtained from the spleen of a 6-week-old BALB/C mouse were separated by negative selection using anti-Thy1.2 and rabbit complement. Cells were incubated at 2×10 6  cells/ml in the presence of Lipopolysaccharide (LPS, 10 μg/ml) and IL 4  (500 u/ml) for 5 days to induce isotypic switching for IgE production. After 5 days, total cellular RNA was isolated (RNAzol TM B isolation kit produced by BIOTECK Laboratories, Houston, USA.). Total RNA (2.5 μg) was then reverse transcribed into first strand cDNA, using the reverse transcription System (Promega, USA) under conditions, recomended by the manfacturer. The cDNA was diluted to a total volume of 1 ml with TE buffer (10 mM Tris-HCL, pH 7.6, 1 mM EDTA) and stored at 4° C. until used.  
         [0036]    Fcε fragmants were generated by PCR, using cDNA and a pair of synthetic oligonucleotide primers 5′-GCG GAT CCC ATA TGG AGC AAT GGA TGT CGT-3′ (sense, starting from nucleotide 406, according to gene bank sequence J00476) and 5′-GCG GAT CCC ATA TGT GGG GTC TTG GTG ATG GAA C-3′ (antisense, starting from nucleotide 813) for the Fcε 2′-3  sequence and 5′-GCG GAT CCC ATA TGC GAC CTG TCA ACA TCA CTG-3′ (sense, starting from nucleotide 175) and 5′-GCG GAT CCC ATA TGG GAG GGA CGG AGG GAG G-3′(antisense, starting from nucleotide 1167) for the Fcε 2 - 4  sequence.  
         [0037]    Synthetic oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer and purified on oligonucleotide purification cartridges. The vent polymerase enzyme (Biolabs) was used for amplification. The reaction mixture was incubated in a DNA thermal cycler (MJ Research, Inc, USA.) for 33 cycles. Each cycle consisted of 1 min. at 95° C., 1 min. at the annealing temperature and 2 min. at 72° C. The MgSO 4  concentration and the annealing temperature used for each primer pair were: 2.5 mM and 61° C. for Fc 2′-3′ , 2 mM and 57° C. for Fc 2-4 .  
         [0038]    The pHL 906 plasmid, which encodes IL 2 -PE 40 , was described previously (Fishman, A., Bar-Kana, Y., Steinberger, I., and Lorberboum-Galski, H. 1994. Increased cytotoxicity of IL2-PE chimeric proteins containing targeting signal for lysosomal membranes. Biochem. 33, 6235.). The pHL906 plasmid was cut with Ndel, obtaining the larger fragment of 3596 bp. The above Fcε fragment was inserted into the Ndel site of pHL906. The resulting plasmids, pAF2302 and pAF2415, coding for the C 2 ′-C 3  and C 2 -C 4  fragments respectivly, each fused 5′ to PE 40 , were characterized by restriction and sequence analysis (results not shown).  Escherichia coli  strain HB101 was used for transformation and preparation of the plasmids.  
         [0039]    2. Expression and partial purification of the chimeric proteins.  
         [0040]    The newly designed chimeric protein, Fcε-PE 40  encoded by plasmid pAF2302 was expressed in  E. coli  strain BL21(lambda-DE3) which carries a T7 RNA polymerase gene in a lysogenic and inducible form. Induction was performed at O.D. 600 0.5 or 180 min. in the presence of isopropyl β-D-thiogalactoside (IPTG, 1 mM final concentration). A pellet expressing cells was suspended in TE buffer (50 mM Tris pH 8.0, 1 mM EDTA) containing 0.2 mg/ml lysosyme, sonicated (three 30s bursts) and centrifuged at 30,000Xg for 30 min. The supernatant (soluble fraction) was removed and kept for analysis. The pellet was denatured in extraction buffer (6 M guanidine-hydrochloride, 0.1 M Tris pH 8.6, 1 mM EDTA, 0.05 M NaCl and 10 mM DTT) and stirred for 30 min. at 4° C. The suspention was cleared by centrifugation at 30,000Xg for 15 min. and the pellet discarded. The supernatant was; then dialysed against 0.1 M Tris (pH 8.0), 1 mM EDTA, 0.25 mM NaCl and 0.25 mM L-Arginine for 16 h. The dialysate was centrifuged at 15,0000Xg for 15 min. and the resultant supernatant (insoluble fraction, guanidine-hydrochiloride treated) was used as a source of the chimeric proteins. Proteins were characterized by gel electrophoresis (FIG. 2). The protein profile of whole cell extracts revealed the high expression level of the chimeric protein.  
         [0041]    The protein was further characterized by Western blot analysis using antibodies against PE (FIG. 3A) and against IgE (Serotec, England) (FIG. 3B). The electrophoresed samples were transfered onto nitrocellulose and immunoblotted as described (Lorberboum-Galski, H., Fitzgerald, D. J., Chaudhary, V., Ashya, S., and Pastan, I. 1988. Cytotoxic activity of an interleukin 2 —Pseudomonas exotoxin chimeric protein produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 1992.). A Vectastain ABC Kit (Vector Laboratories, USA) was used according to the manufacturer&#39;s instructions. The chimera reacted with both antibodies, thus confirming the cloning and production of in-frame full-length chimeric protein.  
         [0042]    Subcellular fractionation of expressing cells revealed that the insoluble fraction (inclusion bodies) was paticularly rich with chimeric protein (FIG. 2). This fraction was therefore used as the source of the chimeric protein.  
         [0043]    The ADP-ribosylation activity of tested samples was measured using wheat germ extracts enriched in elongation factor 2 as substrate, as described previously, and revealed that the novel chimeric protein was enzymatically active (results not shown).  
         [0044]    3. Effect of Fc 2′-3 -PE 40  chimeric protein on mouse mast cell lines.  
         [0045]    The cytotoxic effect of the chimeric protein was tested on various mouse mast cell lines known to express the FcεRI receptor. The cytotoxic activity of the chimeric protein was evaluated by inhibition of protein synthesis, as measured by [ 3 H] Leucine incorporation. Various concentrations of the chimeric protein, diluted with 0.25% bovine serum albumin in phosphate-buffered saline, were added to 2×10 4  cells/0.2 ml seeded in 96-well plates for 20 h., followed by an 8 h pulse with 2 μCi of [ 3 H]-Leucine. The results are expressed as a percentage of the control experiments in which the cells were not exposed to the chimeric protein. All assays were carried out in triplicate in three separate experiments.  
         [0046]    Three target cell lines expressing the FcεRI receptor were used: MC-9, a mast cell line originating in mouse fetal liver and dependent on IL 3  for growth, C57, an IL 3  independent mast cell line originating in mouse bone marrow; and the Abelson-virus transformed mast cell line originating in mouse midgestation embryonic placenta. Fcε-PE 40  was found to be cytotoxic in a dose-dependent manner to all the cell lines tested (FIG. 4). The MC-9 and C57 lines were extremely sensitive to the chimeric toxin, with an ID 50  of 50-75 ng/ml and 100-125 ng/ml, respectively. The Alelson cell line was much less sensitive (ID 50  of 1200-1500 ng/ml).  
         [0047]    4. Specificity of FCε-PE 40  response.  
         [0048]    To verify the specificity of Fc 2′-3 PE 40  activity, two control proteins, PE 40  and Fc 2′-3 -PE 40M , were generated and evaluated for their effect on target and non target cells. To construct Fc 2′-3 -PE 40M , the region coding for the 122 amino acids at the C-terminal of PE was exised with EcoRI and BamHI and replaced by a corresponding fragment carrying a deletion at amino acid 553.  
         [0049]    PE 40 , which has no intrinsic targeting capacity had, as expected, no effect on the target cell lines (FIG. 4). Fc 2′-3 -PE 40M  which possesses a Fc 2′-3  moiety linked to a mutated, enzymatically inactive form PE 40 , was also not cytotoxic to the target cells (FIG. 4).  
         [0050]    In addition, it was possible to block the cytotoxic effect of Fc 2′-3 -PE 40  against target cells by whole mouse IgE (40 μg/ml, FIG. 5A) or by a αPE polyclonal antibody (10 μg/ml, FIG. 5B).  
         [0051]    The effect of Fc 2′-3 -PE 40  was also tested on various mouse non-target cell lines (Table 1). All cell lines of hemopoetic origin were unaffected by the chimeric protein. Suprisingly, fibroblast and hematoma cell lines exhibited some sensitivity to chimeric toxin, although the ID 50  values were twenty-fold higher than those of the MC-9 cells (Table 1).  
         [0052]    The above data demonstrates that the toxic effect of Fc 2′-3-PE 40 on mast cell lines is due to a specific response mediated by the Fc 2′-3  moiety which targets the cytotoxic part of the chimera (PE 40 ) into the cell.  
         [0053]    5. Effect of chimeric proteins on primary mast cells.  
         [0054]    As it is likely that fresh murine mast cells react differently from established cell lines, we also tested primary mast cells obtained from normal mice for their sensitivity to Fc 2′-3 -PE 40 . When cultured in the presence of IL 3  for two weeks, mouse bone marrow differentiates into an almost pure population of cells with the morphology of immature mast cells, containing granules and expressing the FcεRI receptor.  
         [0055]    BALB/C mice aged 4-6 weeks were sacrified and their bone marrow was aseptically flushed from femurs into 0.9% cold NaCl. The cell suspension was washed twice with 0.9% NaCl, centrifuged for 10 min. at 300Xg and finally resuspended in RPMI 1640 medium containing 10% heat inactivated fetal calf serum, 4 mM L-glutamine, 1 mM sodium piruvate, 0.1 mM nonessential amino acids, 5×10 −5  M β-mercaptoethanol, 100 u/ml penicillin, 100 μg/ml streptomycin and 20 u/ml recombinant mouse IL 3 . Cells were grown in tissue culture flasks at a density of 10 6  cells/ml, at 37° C. in a 5% CO 2  humidified atmosphere for 2-3 weeks. The media were changed every 7 days. Recombinant IL 4  (10u/ml) was added starting from day 7 in culture.  
         [0056]    To follow the degree of maturation, cells were mounted on slides, stained with acidic Toluidine Blue (pH 1.0) and examined microscopically under oil.  
         [0057]    The effect of chimeric proteins was tested on bone marrow derived mast cells (BMMC) on the 16th day of cultures. As shown in FIG. 6, Fc 2′-3 -PE 40  was cytotoxic to BMMC in a dose dependent manner, with an ID 50  of 125 ng/ml. At a high chimeric protein dose, there was nearly 100% inhibition of protein synthesis. None of the control proteins Fc 2′-3 -PE 40M  or PE 40  displayed cytotoxicity against BMMC (FIG. 6). Thus, primary mast cells respond towards the chimeric protein similarly to the established mast cell lines (FIGS. 4 and 6).  
         [0058]    6. Receptor specificity of Fc 2′-3 -PE 40 .  
         [0059]    Aside from the high affinity FcεRI receptor, three other membrane surface structures were reported to bind IgE with low affinity′the low affinity FcεRII receptor, the εBP galactoside-binding protein (also termed MAC-2 or CBP35) and the FcγRII/III receptor. These structures appear on various cell types, mainly of hemopoethic origin, but also on fibroblasts (εBP). FcγRII/III and εBP appear on mast cell membranes in addition to FcεRI. As our aim was to target only mast cells, it was essential to prove that the chimeric protein does not recognize these structures and thus can not be internalized through them. Theoretically our chimeric protein does not fulfill the binding requirements of the low-affinity IgE binding structure FcεRII, εBP and FcγRII/III. FcεRII binds only disulfide linked ε-chain dimmers, while our protein lacks domain 4 which is essential for dimerization. εBP binds only glycosylated IgE; Fc 2′-3 -PE 40  being produced in bacteria, is not glycosylated. FcγRII/III binds IgE-immunocomplexes but not free IgE. Nevertheless, the issue of receptor binding was challenged experimentally.  
         [0060]    Experiments involving εBP and FcγRII/III were performed on C57 mast cells, known to express these receptors in addition to FcεRI. To test whether the chimeric protein can enter the cell via the FcγRII/III receptors, cells were preincubated with the 2.4G2 antibody (Pharmigen) (50 μg/m) prior to addition of the chimeric protein. This monoclonal antibody, which binds to the extracellular domains of both FcγRII and the FcγRIII receptors was shown to be a competitive inhibitor of IgE binding. As can be seen in FIG. 7A, there was no difference in the cellular response to Fc 2′-3 -PE 40  between control cells and cells preincubated with the antibody.  
         [0061]    We next examined whether εBP is involved in the cytotoxicity of Fc 2′-3 -PE 40 . As εBP is attached to membrane carbohydrate determinants, addition of lactose to the culture medium causes its dissociation from the cell surface. We found no difference in the cellular response to Fc 2′-3 -PE 40  in the presence or absence of lactose (25mM, FIG. 7B).  
         [0062]    Additional experiments in the presence of 2.4G2 antibody and lactose were performed on fibroblast cell lines that were found partially responsive to the chimeric protein (Table 1). Again, there was no difference in FC 2′-3 -PE 40  cytotoxicity against treated and control cells (results not shown).  
         [0063]    To test whether Fc 2′-3 -PE 40  affects FcεRII-bearing cells, we used the 0.12A3 cell line, a mouse B cell hybridoma expressing the FcεRII receptor. The 0.12A3 cells were totally non responsive to Fc 2′-3 -PE 40 , even at high doses (&gt;5000 ng/ml, FIG. 8A). As this line loses the receptor upon long term culture, the assay was followed by FACS analysis with the B3B4 antibody against the receptor (Pharmigin). The results showed that the receptor was expressed on 54% of the cells (results not shown).  
         [0064]    An additional experiment was performed on fresh mouse B splenocytes preincubated for 16 h. with LPS (50 μg/ml) to stimulate expression of FcεRII. Fc 2′-3 -PE 40  has no effect on these B splenocytes (FIG. 8B), although 69% of the cells expressed the receptor, as determined by FACS analysis.  
         [0065]    Collectively, these results suggest that Fc 2′-3 -PE 40  does not bind to the low affinity IgE-binding structures, namely FcεRII, FcγRII/III and εBP.  
         [0066]    7. Effect of Fc 2′-3 -PE 40  on cellular degranulation.  
         [0067]    Because of the possible clinical applicability of Fc 2′-3 -PE 40 , it was important to test whether treatment of mast cells with Fc 2′-3 -PE 40  results in the release of allergic mediators triggered upon FcεRI binding by the chimetric protein.  
         [0068]    C57 cells prelabelled overnight with [ 3 H]-hydroxytryptamine (10 μci/ml) were washed, plated at 2×10 5  cells/well in DMEM containing 10% FCS, in 96-well tissue culture plates and incubated with Fc 2′-3 -PE 40  (10 μg/ml) at 37° C. At various time points, supernatants were separated and release of seretonin into the supernatant was measured. Unlabled cells were also incubated with Fc 2′-3 -PE 40  and at the same time intervals were pulsed 1 hr with [ 3 H] leucine to measure protein systhesis inhibition by chimeric toxin. There was no difference in supernatant [3H] seretonin content between Fc 2′-3 -PE 40  treated and untreated cells at ½, 4 or 8 hr following chimeric protein addition (FIG. 9A). Inhibition of protein synthesis reached 80% at 4 h. and a value of 90% by 8 h. (FIG. 9B). These results suggest that Fc 2′-3 -PE 40  does not cause release of allergic mediators during receptor binding or upon inhibition of protein synthesis.  
         [0069]    8.Electrophoretic characterization of Fcε-PE40  
         [0070]    Western blot analysis of electrophoresed samples run under non-reducing conditions (omitting 2-mercaptoethanol from the sample buffer) revealed that the Fc2′-3-PE40 chimeric protein is predominantly present as a monomer (FIG. 10 b ). For native PAGE, 2-mercaptoethanol was omitted from the sample buffer and the samples were not heated. In addition, SDS was replaced with equivalent volumes of water in the gel, sample buffer and electrode running buffer. Under non-denaturing conditions the chimeric protein runs as a broad band (FIG. 10 c ). A single native system can not distinguish the effects of molecular weight, charge and conformation on protein electrophoretic mobilities. However, the proximity of the molecules in the band indicates that they can not differ much in these parameters.  
         [0071]    9. Internalization assay  
         [0072]    In vitro activity of the chimeric protein is achieved only upon it&#39;s internalization. To rest whether the chimeric protein is internalysed, 5×10 5  cells/3 ml were incubated for 1 hour with 20 μg of the chimeric protein at 37° C. After 3 washes with cold PBS the pellet was treated with 0.5 ml of acid solution (0.15M NaCl, 0.15M acetic acid (pH 3)) for 3 min on ice to remove membrane-bounded chimeric protein. The pH was then neutrilised by addition of 50% FCS following by three washed with RPMI/10% FCS. The cell pellet was lysed with 0.3 ml of RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM tris-HCl pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 15% SDS, 1% deoxycholyc acid, 1% Nonidet P-40). Various samples were electrophoresed and immunoblotted using α-PE and the ECL detection system (Amersham). Western blot analysis revealed undoubtfully that Fc2′-3-PE40 chimeric protein is internalized into the target cells (FIG. 11).  
         [0073]    10. Effect of Fc 2′-3 -PE 40  on cellular degranulation  
         [0074]    C57 cells were incubated overnight with [ 3 H]-Hydroxytryptamine (10 μci/ml) at 37° C. Cells were washed 3 times to remove free [3H]-Hydroxytryptamine, plated in Tyrod&#39;s buffer (10mM Hepes pH 7.4, 130 mM NaCl, 5 mM KCl, 5.6 mM Glucose, 0.5% BSA) at 2.5×10 5  cells/0.5 ml in 24 well tissue culture plates and incubated with IgE (10 μg/ml) for 1 hour at 4° C. MgCl 2  and CaCl 2  were then added to the final concentration of 1 mM and 1.6 mM respectively, following by incubation with Dinitrophenyl-human serum albumin (DNP-HSA, 50 ng/ml) for 30 minutes or with the different concentrations of chimeric protein for various times at 37° C. Cell-free supernatants were collected by centrifugation and amount of [ 3 H]-Hydroxytryptamine released was measured. No degranulation was observed with any concentration of chimeric protein tested (FIG. 12 a ). As a control, cells preincubated with IgE were exposed to DNP under the same conditions. The effect of triggering degranulation by DNP is clearly visible (FIG. 12 a ). Fc 2′-3 -PE 40  did not cause any degranulation also at later stages of it&#39;s interaction with the target cell (FIG. 12 b ), while it inhibits protein synthesis by over 80% (FIG. 12 c ). Our results demonstrate that Fc 2′-3 -PE 40 _does not_trigger degr anulation at any stage during it&#39;s interaction with the cell.  
                                                       TABLE 1                           Cytotoxicity of Fc 2′-3 -PE 40  chimeric protein against various mouse cells                Cell line   Cell Origin   ID 50  (ng/ml)                        TARGET       MC-9   Mast cells    50-100       CELLS       C57   Mast cells   100-125               BMMC   Primary bone                   marrow-derived                   mast cells               Abelson   Transformed mast   1,200-1,500                   cells       NON-   HEMO-   L 10 A   B cell,   &gt;10,000       TARGET   POETIC       non-secreting       CELLS       X 16 B   B cell,   &gt;10,000                   non-secreting               UT   B cell,   &gt;10,000                   non-secreting               PD1.1   T cell, immature   &gt;10,000               EL-4   T cell, mature   &gt;10,000               Erythro-       &gt;10,000               leukemia           CONNECTIVE   LTK   Fibroblast   1900           TISSUE   Hepatoma       1500                  
 
         [0075]    [0075] 
     
       
       
         1 
         
           
             8  
           
           
             1  
             1512  
             DNA  
             Mouse  
             
               CDS  
               (1)..(1512)  
               1. The mouse IgE constant region (=F(Ce)) 
      2. Pseudomonas aeruginosa Endotoxin (PE40)  
             
           
            1 

atg gag cag caa tgg atg tct gaa agc acc ttc acc tgc aag gtc acc       48 
Met Glu Gln Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Lys Val Thr 
1               5                   10                  15 

tcc caa ggc gta gac tat ttg gcc cac act cgg aga tgc cca gat cat       96 
Ser Gln Gly Val Asp Tyr Leu Ala His Thr Arg Arg Cys Pro Asp His 
            20                  25                  30 

gag cca gcc ggt gtg att acc tac ctg atc cca ccc agc ccc ctg gac      144 
Glu Pro Ala Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp 
        35                  40                  45 

ctg tat caa aac ggt gct ccc aag ctt acc tgt ctg gtg gtg gac ctg      192 
Leu Tyr Gln Asn Gly Ala Pro Lys Leu Thr Cys Leu Val Val Asp Leu 
    50                  55                  60 

gaa agc gag aag aat gtc aat gtg acg tgg aac caa gag aag aag act      240 
Glu Ser Glu Lys Asn Val Asn Val Thr Trp Asn Gln Glu Lys Lys Thr 
65                  70                  75                  80 

tca gtc tca gca tcc cag tgg tac act aag cac cac aat aac gcc aca      288 
Ser Val Ser Ala Ser Gln Trp Tyr Thr Lys His His Asn Asn Ala Thr 
                85                  90                  95 

act agt atc acc tcc atc ctg cct gta gtt gcc aag gac tgg att gaa      336 
Thr Ser Ile Thr Ser Ile Leu Pro Val Val Ala Lys Asp Trp Ile Glu 
            100                 105                 110 

ggc tac ggc tat cag tgc ata gtg gac cac cct gat ttt ccc aag ccc      384 
Gly Tyr Gly Tyr Gln Cys Ile Val Asp His Pro Asp Phe Pro Lys Pro 
        115                 120                 125 

att gtg cgt tcc atc acc aag acc cca cat atg gcc gaa gag ggc ggc      432 
Ile Val Arg Ser Ile Thr Lys Thr Pro His Met Ala Glu Glu Gly Gly 
    130                 135                 140 

agc ctg gcc gcg ctg acc gcg cac cag gct tgc cac ctg ccg ctg gag      480 
Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu 
145                 150                 155                 160 

act ttc acc cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg gag cag      528 
Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln 
                165                 170                 175 

tgc ggc tat ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg gcg cgg      576 
Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg 
            180                 185                 190 

ctg tcg tgg aac cag gtc gac cag gtg atc cgc aac gcc ctg gcc agc      624 
Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser 
        195                 200                 205 

ccc ggc agc ggc ggc gac ctg ggc gaa gcg atc cgc gag cag ccg gag      672 
Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu 
    210                 215                 220 

cag gcc cgt ctg gcc ctg acc ctg gcc gcc gcc gag agc gag cgc ttc      720 
Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe 
225                 230                 235                 240 

gtc cgg cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc gac      768 
Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp 
                245                 250                 255 

gtg gtg agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg ggc ccg      816 
Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro 
            260                 265                 270 

gcg gac agc ggc gac gcc ctg ctg gag cgc aac tat ccc act ggc gcg      864 
Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala 
        275                 280                 285 

gag ttc ctc ggc gac ggc ggc gac gtc agc ttc agc acc cgc ggc acg      912 
Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr 
    290                 295                 300 

cag aac tgg acg gtg gag cgg ctg ctc cag gcg cac cgc caa ctg gag      960 
Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu 
305                 310                 315                 320 

gag cgc ggc tat gtg ttc gtc ggc tac cac ggc acc ttc ctc gaa gcg     1008 
Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala 
                325                 330                 335 

gcg caa agc atc gtc ttc ggc ggg gtg cgc gcg cgc agc cag gac ctc     1056 
Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu 
            340                 345                 350 

gac gcg atc tgg cgc ggt ttc tat atc gcc ggc gat ccg gcg ctg gcc     1104 
Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala 
        355                 360                 365 

tac ggc tac gcc cag gac cag gaa ccc gac gca cgc ggc cgg atc cgc     1152 
Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg 
    370                 375                 380 

aac ggt gcc ctg ctg cgg gtc tat gtc ccg cgc tcg agc ctg ccg ggc     1200 
Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly 
385                 390                 395                 400 

ttc tac cgc acc agc ctg acc ctg gcc gcg ccg gag gcg gcg ggc gag     1248 
Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu 
                405                 410                 415 

gtc gaa cgg ctg atc ggc cat ccg ctg ccg ctg cgc ctg gac gcc atc     1296 
Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile 
            420                 425                 430 

acc ggc ccc gag gag gaa ggc ggg cgc ctg gag acc att ctc ggc tgg     1344 
Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp 
        435                 440                 445 

ccg ctg gcc gag cgc acc gtg gtg att ccc tcg gcg atc ccc acc gac     1392 
Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp 
    450                 455                 460 

ccg cgc aac gtc ggc ggc gac ctc gac ccg tcc agc atc ccc gac aag     1440 
Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys 
465                 470                 475                 480 

gaa cag gcg atc agc gcc ctg ccg gac tac gcc agc cag ccc ggc aaa     1488 
Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys 
                485                 490                 495 

ccg ccg cgc gag gac ctg aag taa                                     1512 
Pro Pro Arg Glu Asp Leu Lys 
            500 

 
           
             2  
             503  
             PRT  
             Mouse  
           
            2 

Met Glu Gln Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Lys Val Thr 
1               5                   10                  15 

Ser Gln Gly Val Asp Tyr Leu Ala His Thr Arg Arg Cys Pro Asp His 
            20                  25                  30 

Glu Pro Ala Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp 
        35                  40                  45 

Leu Tyr Gln Asn Gly Ala Pro Lys Leu Thr Cys Leu Val Val Asp Leu 
    50                  55                  60 

Glu Ser Glu Lys Asn Val Asn Val Thr Trp Asn Gln Glu Lys Lys Thr 
65                  70                  75                  80 

Ser Val Ser Ala Ser Gln Trp Tyr Thr Lys His His Asn Asn Ala Thr 
                85                  90                  95 

Thr Ser Ile Thr Ser Ile Leu Pro Val Val Ala Lys Asp Trp Ile Glu 
            100                 105                 110 

Gly Tyr Gly Tyr Gln Cys Ile Val Asp His Pro Asp Phe Pro Lys Pro 
        115                 120                 125 

Ile Val Arg Ser Ile Thr Lys Thr Pro His Met Ala Glu Glu Gly Gly 
    130                 135                 140 

Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu 
145                 150                 155                 160 

Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln 
                165                 170                 175 

Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg 
            180                 185                 190 

Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser 
        195                 200                 205 

Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu 
    210                 215                 220 

Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe 
225                 230                 235                 240 

Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp 
                245                 250                 255 

Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro 
            260                 265                 270 

Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala 
        275                 280                 285 

Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr 
    290                 295                 300 

Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu 
305                 310                 315                 320 

Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala 
                325                 330                 335 

Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu 
            340                 345                 350 

Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala 
        355                 360                 365 

Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg 
    370                 375                 380 

Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly 
385                 390                 395                 400 

Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu 
                405                 410                 415 

Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile 
            420                 425                 430 

Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp 
        435                 440                 445 

Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp 
    450                 455                 460 

Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys 
465                 470                 475                 480 

Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys 
                485                 490                 495 

Pro Pro Arg Glu Asp Leu Lys 
            500 

 
           
             3  
             2031  
             DNA  
             Pseudomonas aeruginosa  
             
               CDS  
               (1)..(2031)  
               1. The mouse IgE constant region (=F(Ce)) 
      2. Pseudomonas aeruginosa Endotoxin (PE40)  
             
           
            3 

atg cga cct gtc aac atc act gag ccc acc ttg gag cta ctc cat tca       48 
Met Arg Pro Val Asn Ile Thr Glu Pro Thr Leu Glu Leu Leu His Ser 
1               5                   10                  15 

tcc tgc gac ccc aat gca ttc cac tcc acc atc cag ctg tac tgc ttc       96 
Ser Cys Asp Pro Asn Ala Phe His Ser Thr Ile Gln Leu Tyr Cys Phe 
            20                  25                  30 

att tat ggc cac atc cta aat gat gtc tct gtc agc tgg cta atg gac      144 
Ile Tyr Gly His Ile Leu Asn Asp Val Ser Val Ser Trp Leu Met Asp 
        35                  40                  45 

gat cgg gag ata act gat aca ctt gca caa act gtt cta atc aag gag      192 
Asp Arg Glu Ile Thr Asp Thr Leu Ala Gln Thr Val Leu Ile Lys Glu 
    50                  55                  60 

gaa ggc aaa cta gcc tct acc tgc agt aaa ctc aac atc act gag cag      240 
Glu Gly Lys Leu Ala Ser Thr Cys Ser Lys Leu Asn Ile Thr Glu Gln 
65                  70                  75                  80 

caa tgg atg tct gaa agc acc ttc acc tgc aag gtc acc tcc caa ggc      288 
Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Lys Val Thr Ser Gln Gly 
                85                  90                  95 

gta gac tat ttg gcc cac act cgg aga tgc cca gat cat gag cca cgg      336 
Val Asp Tyr Leu Ala His Thr Arg Arg Cys Pro Asp His Glu Pro Arg 
            100                 105                 110 

ggt gtg att acc tac ctg atc cca ccc agc ccc ctg gac ctg tat caa      384 
Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Gln 
        115                 120                 125 

aac ggt gct ccc aag ctt acc tgt ctg gtg gtg gac ctg gaa agc gag      432 
Asn Gly Ala Pro Lys Leu Thr Cys Leu Val Val Asp Leu Glu Ser Glu 
    130                 135                 140 

aag aat gtc aat gtg acg tgg aac caa gag aag aag act tca gtc tca      480 
Lys Asn Val Asn Val Thr Trp Asn Gln Glu Lys Lys Thr Ser Val Ser 
145                 150                 155                 160 

gca tcc cag tgg tac act aag cac cac aat aac ggc aca act agt atc      528 
Ala Ser Gln Trp Tyr Thr Lys His His Asn Asn Gly Thr Thr Ser Ile 
                165                 170                 175 

acc tcc atc ctg cct gta gtt gcc aag gac tgg att gaa ggc tac ggc      576 
Thr Ser Ile Leu Pro Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly 
            180                 185                 190 

tat cag tgc ata gtg gac cac cct gat ttt ccc aag ccc att gtg cgt      624 
Tyr Gln Cys Ile Val Asp His Pro Asp Phe Pro Lys Pro Ile Val Arg 
        195                 200                 205 

tcc atc acc aag acc cca ggc cag cgc tca gcc ccc gag gta tat gtg      672 
Ser Ile Thr Lys Thr Pro Gly Gln Arg Ser Ala Pro Glu Val Tyr Val 
    210                 215                 220 

ttc cca cca cca gag gag gag agc gag gac aaa cgc aca ctc acc tgt      720 
Phe Pro Pro Pro Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu Thr Cys 
225                 230                 235                 240 

ttg atc cag aac ttc ttc cct gag gat atc tct gtg cag tgg ctg ggg      768 
Leu Ile Gln Asn Phe Phe Pro Glu Asp Ile Ser Val Gln Trp Leu Gly 
                245                 250                 255 

gat ggc aaa ctg atc tca aac agc cag cac agt acc aca aca ccc ctg      816 
Asp Gly Lys Leu Ile Ser Asn Ser Gln His Ser Thr Thr Thr Pro Leu 
            260                 265                 270 

aaa tcc aat ggc tcc aat caa ggc ttc ttc atc ttc agt cgc cta gag      864 
Lys Ser Asn Gly Ser Asn Gln Gly Phe Phe Ile Phe Ser Arg Leu Glu 
        275                 280                 285 

gtc gcc aag aca ctc tgg aca cag aga aaa cag ttc acc tgc caa gtg      912 
Val Ala Lys Thr Leu Trp Thr Gln Arg Lys Gln Phe Thr Cys Gln Val 
    290                 295                 300 

atc cat gag gca ctt cag cat atg gcc gaa gag ggc ggc agc ctg gcc      960 
Ile His Glu Ala Leu Gln His Met Ala Glu Glu Gly Gly Ser Leu Ala 
305                 310                 315                 320 

gcg ctg acc gcg cac cag gct tgc cac ctg ccg ctg gag act ttc acc     1008 
Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr 
                325                 330                 335 

cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg gag cag tgc ggc tat     1056 
Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr 
            340                 345                 350 

ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg gcg cgg ctg tcg tgg     1104 
Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp 
        355                 360                 365 

aac cag gtc gac cag gtg atc cgc aac gcc ctg gcc agc ccc ggc agc     1152 
Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser 
    370                 375                 380 

ggc ggc agc ctg ggc gaa gcg atc cgc gag cag ccg gag cag gcc cgt     1200 
Gly Gly Ser Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg 
385                 390                 395                 400 

ctg gcc ctg acc ctg gcc gcc gcc gag agc gag cgc ttc gtc cgg cag     1248 
Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln 
                405                 410                 415 

ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc gac gtg gtg agc     1296 
Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser 
            420                 425                 430 

ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg ggc ccg gcg gac agc     1344 
Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser 
        435                 440                 445 

ggc gac gcc ctg ctg gag cgc aac tat ccc act ggc gcg gag ttc ctc     1392 
Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu 
    450                 455                 460 

ggc gac ggc ggc gac gtc agc ttc agc acc cgc ggc acg cag aac tgg     1440 
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp 
465                 470                 475                 480 

acg gtg gag cgg ctg ctc cag gcg cac cgc caa ctg gag gag cgc ggc     1488 
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly 
                485                 490                 495 

tat gtg ttc gtc ggc tac cac ggc acc ttc ctc gaa gcg gcg caa agc     1536 
Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser 
            500                 505                 510 

atc gtc ttc ggc ggg gtg cgc gcg cgc agc cag gac ctc gac gcg atc     1584 
Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile 
        515                 520                 525 

tgg cgc ggt ttc tat atc gcc ggc gat ccg gcg ctg gcc tac ggc tac     1632 
Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr 
    530                 535                 540 

gcc cag gac cag gaa ccc gac gca cgc ggc cgg atc cgc aac ggt gcc     1680 
Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala 
545                 550                 555                 560 

ctg ctg cgg gtc tat gtg ccg cgc tcg agc ctg ccg ggc ttc tac cgc     1728 
Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg 
                565                 570                 575 

acc agc ctg acc ctg gcc gcg ccg gag gcg gcg ggc gag gtc gaa cgg     1776 
Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg 
            580                 585                 590 

ctg atc ggc cat ccg ctg ccg ctg cgc ctg gac gcc atc acc ggc ccc     1824 
Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro 
        595                 600                 605 

gag gag gaa ggc ggg cgc ctg gag acc att ctc ggc tgg ccg ctg gcc     1872 
Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala 
    610                 615                 620 

gag cgc acc gtg gtg att ccc tcg gcg atc ccc acc gac ccg cgc aac     1920 
Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn 
625                 630                 635                 640 

gtc ggc ggc gac ctc gac ccg tcc agc atc ccc gac aag gaa cag gcg     1968 
Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala 
                645                 650                 655 

atc agc gcc ctg ccg gac tac gcc agc cag ccc ggc aaa ccg ccg cgc     2016 
Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg 
            660                 665                 670 

gag gac ctg aag taa                                                 2031 
Glu Asp Leu Lys 
        675 

 
           
             4  
             676  
             PRT  
             Pseudomonas aeruginosa  
           
            4 

Met Arg Pro Val Asn Ile Thr Glu Pro Thr Leu Glu Leu Leu His Ser 
1               5                   10                  15 

Ser Cys Asp Pro Asn Ala Phe His Ser Thr Ile Gln Leu Tyr Cys Phe 
            20                  25                  30 

Ile Tyr Gly His Ile Leu Asn Asp Val Ser Val Ser Trp Leu Met Asp 
        35                  40                  45 

Asp Arg Glu Ile Thr Asp Thr Leu Ala Gln Thr Val Leu Ile Lys Glu 
    50                  55                  60 

Glu Gly Lys Leu Ala Ser Thr Cys Ser Lys Leu Asn Ile Thr Glu Gln 
65                  70                  75                  80 

Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Lys Val Thr Ser Gln Gly 
                85                  90                  95 

Val Asp Tyr Leu Ala His Thr Arg Arg Cys Pro Asp His Glu Pro Arg 
            100                 105                 110 

Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Gln 
        115                 120                 125 

Asn Gly Ala Pro Lys Leu Thr Cys Leu Val Val Asp Leu Glu Ser Glu 
    130                 135                 140 

Lys Asn Val Asn Val Thr Trp Asn Gln Glu Lys Lys Thr Ser Val Ser 
145                 150                 155                 160 

Ala Ser Gln Trp Tyr Thr Lys His His Asn Asn Gly Thr Thr Ser Ile 
                165                 170                 175 

Thr Ser Ile Leu Pro Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly 
            180                 185                 190 

Tyr Gln Cys Ile Val Asp His Pro Asp Phe Pro Lys Pro Ile Val Arg 
        195                 200                 205 

Ser Ile Thr Lys Thr Pro Gly Gln Arg Ser Ala Pro Glu Val Tyr Val 
    210                 215                 220 

Phe Pro Pro Pro Glu Glu Glu Ser Glu Asp Lys Arg Thr Leu Thr Cys 
225                 230                 235                 240 

Leu Ile Gln Asn Phe Phe Pro Glu Asp Ile Ser Val Gln Trp Leu Gly 
                245                 250                 255 

Asp Gly Lys Leu Ile Ser Asn Ser Gln His Ser Thr Thr Thr Pro Leu 
            260                 265                 270 

Lys Ser Asn Gly Ser Asn Gln Gly Phe Phe Ile Phe Ser Arg Leu Glu 
        275                 280                 285 

Val Ala Lys Thr Leu Trp Thr Gln Arg Lys Gln Phe Thr Cys Gln Val 
    290                 295                 300 

Ile His Glu Ala Leu Gln His Met Ala Glu Glu Gly Gly Ser Leu Ala 
305                 310                 315                 320 

Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr 
                325                 330                 335 

Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr 
            340                 345                 350 

Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp 
        355                 360                 365 

Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser 
    370                 375                 380 

Gly Gly Ser Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg 
385                 390                 395                 400 

Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln 
                405                 410                 415 

Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser 
            420                 425                 430 

Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser 
        435                 440                 445 

Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu 
    450                 455                 460 

Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp 
465                 470                 475                 480 

Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly 
                485                 490                 495 

Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser 
            500                 505                 510 

Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile 
        515                 520                 525 

Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr 
    530                 535                 540 

Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala 
545                 550                 555                 560 

Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg 
                565                 570                 575 

Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg 
            580                 585                 590 

Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro 
        595                 600                 605 

Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala 
    610                 615                 620 

Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn 
625                 630                 635                 640 

Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala 
                645                 650                 655 

Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg 
            660                 665                 670 

Glu Asp Leu Lys 
        675 

 
           
             5  
             30  
             DNA  
             Synthetic Oligonucleotide  
           
            5 

gcggatccca tatggagcaa tggatgtcgt                                      30 

 
           
             6  
             33  
             DNA  
             Synthetic Oligonucleotide  
           
            6 

gcggatccca tatgtggggt cttggtgatg gaa                                  33 

 
           
             7  
             33  
             DNA  
             Synthetic Oligonucleotide  
           
            7 

gcggatccca tatgcgacct gtcaacatca ctg                                  33 

 
           
             8  
             30  
             DNA  
             Synthetic Oligonucleotide  
           
            8 

gcggatccca tatgggaggg acggagggag                                      30