Abstract:
The present invention generally relates to a new approach for the 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. 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:
This application is a continuation application of U.S. application Ser. No. 10/096,840 filed on Mar. 14, 2002, which is a divisional of U.S. application Ser. No. 09/091,645 filed on Jun. 18, 1998, which issued as U.S. Pat. No. 6,919,079, all of which are incorporated by reference herein as if fully set forth. 
    
    
     FIELD OF THE INVENTION 
     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 
     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. 
     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. 
     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. 
     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.). 
     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 Fcε expressed 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.). 
     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: 
     1. β2 agonists—produce airway dilatation through simulation of β2 adrenergic receptors. 
     2. Methylxantines—smooth muscle relaxants, produce bronchodilatation. 
     3. Glucocorticoids—reduce inflammation. 
     4. Cromolyn sodium—prevents mast cell degranulation. 
     5. Antihistamines—prevents histamine action on it&#39;s target cells. 
     Although widely used, all of these drugs have notable disadvantages in regard to: 
     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. 
     2. Toxicity: Being non-specific, these drugs exert their action on various tissues and organs causing serious side effects. The major side effect of β2 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. 
     3. Duration of the effect: β-adrenergic agonists, aminoxantines and antihistamines are mostly short-acting drugs, and as such must be administered frequently. Steroids which are long-acting drugs, have also long induction time and are of little value in emergencies. 
     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. 
     A number of attempts to interfere with interaction between IgE and it&#39;s high-affinity receptor, as a basis for antiallergy 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.; HaakFrendscho, 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. 
     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 immunosuppressive 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 
     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′ -PE40. 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 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.    
     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 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. 
     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 topical application, or for oral administration. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1 : Schematic representation of plasmids coding for the Fc 2′-3′ -PE 40  and Fc 2-4 -PE40 chimeric proteins. 
         FIG. 2 : SDS-polyacrylamide gel electrophoresis analysis of cell fractions containing Fcε-PE 40  chimeric proteins. Samples containing 5 μg. total protein were loaded onto 10% gels. (A) Cells expressing Fc 2′-3′ -PE 40  Lane 1, markers; Lane 2 whole cell extract; Lane 3, soluble fraction; Lane 4, insoluble fraction. (B) Cells expressing Fc 2-4 -PE40. Fractions are as described in A. 
         FIG. 3 : Immunoblotting of insoluble fractions containing Fcε-PE 40  chimeric proteins with antibodies against PE (A) and IgE (B). Samples containing 1 μg of total protein were loaded onto 10% SDS-polyacrylamide gels. Electrophoresed samples were transferred onto nitrocellulose and processed as described in Materials and Methods. (A) αPE: Lane 1, Fc 2′-3′ -PE 40 ; Lane 2: Fc 2-4 -PE 40  (B) αlgE: Lanes are as described in A. 
         FIG. 4 : Cytotoxic activity of various chimeric proteins against MC-9 cells (A), C57 cells (B), and Abelson cells (C). Cells were incubated for 20 h with insoluble fractions containing the chimeric proteins -▪ Fc 2′-3′ -PE 40 ; -•-Fc 2-4 -PE40; -▴-Fc 2′-3′ -PE 40M ; or -□-PE 40  at various concentrations (according to total protein concentration). Experiments with MC-9 cells were performed in the presence of IL 3  (20 u/ml) and IL 4  (10 u/ml). [ 3 H] Leucine incorporation into cell proteins was measured as described in Materials and Methods. The results are expressed as the percentage of protein synthesis of control cells not exposed to chimeric proteins. 
         FIG. 5 : Inhibition of Fc 2′-3′ -PE 40  cytotoxicity by (A) IgE and (B) αPE. Cells were incubated with whole IgE (40 mg/ml) for 1 h at 4° C. before the addition of Fc 2′-3′ -PE 40  αPE (10 mg/ml) was added a few minutes prior to the addition of Fc 2′-3′ -PE 40 . All other experimental conditions were as described in  FIG. 4 . ▪ 
         FIG. 6 : Cytotoxic activity of various chimeric proteins against bone marrow derived primary mast cells (BMMC). Bone marrow was cultured as described in Materials and Methods. Experiments were performed on the 16 th  day of culture, as described in  FIG. 4 , in the presence of IL 3  (20 u/ml) and IL 4  (10 u/ml). 
         FIG. 7 : Cytotoxic activity of various chimeric proteins against the C57 cells in the presence of: αFcγRII/III (2.4G2). Cells were incubated with 2.4G2 (50 μg/ml) or galactose (25 mm) for 30 min. at 37° C. prior to the addition of Fc 2′-3′ -PE 40 . All other experimental conditions were as described in  FIG. 4 . (A) Fc 2′-3′ -PE 40  in the absence (-•-) or presence (—O—) of 2.4G2. (B): Fc 2′-3′ -PE 40  in the absence (-Δ-) or presence (-▴-) of galactose. 
         FIG. 8 : Cytotoxic activity of various chimeric proteins against FcεRII bearing cells. (—O—) B splenocytes.-▪-0.12A3 B cell hybridoma. B splenocytes were preincubated for 16 h. with LPS (50 μg/ml) and IL 4  (50 u/ml). All other experimental conditions were as described in  FIG. 4 . 
         FIG. 9(A) : The effect of Fc 2′-3′ -PE 40  on seretonin release from C57 cells. Cells were labeled overnight with [ 3 H] Hydroxytryptamine creatinine sulfate. The cells were then washed and incubated with Fc 2′-3′ -PE 40  (10 μg/ml). Control cells were not exposed to any protein. At different time points [ 3 H] Hydroxytryptamine creatinine sulfate release into the medium was measured.-▪-control, -□-Fc 2′-3′ -PE 40    
         FIG. 9  (B): Time-dependant cytotoxycity of Fc 2′-3′ -PE 40  against C57 cells. Unlabeled cells were incubated as in (A). At the same time points, cells were pulsed for 1 h with [ 3 H] Leucine and its incorporation into cellular proteins was measured. The results are expressed as the percentage of protein synthesis of control cells not exposed to chemeric proteins. 
         FIG. 10 : Immunoblotting of Fc2′-3′-PE40 chimeric protein electrophoresed under the following conditions with anti-PE: A) in SDS under reducing conditions, B) in SDS under nonreducing conditions and C) a nondenaturing gel (i.e. no reduction, no SDS). 
         FIG. 11 : Internalization of Fc 2′-3′ -PE 40  chimeric protein by MC-9 cells. Samples containing 20 μl of each of the following fractions were loaded onto SDS-10% polyacrylamide gels: lane 1, 40 ng Fc 2′-3′ -PE 40 ; lane 2, supernatant of the cells; lane 3, last wash before the acid treatment; lane 4, acid wash supernatant; lane 5, last wash after acid treatment; and lane 6, lysed cells. 
         FIG. 12(A) : The effect of Fc 2′-3′ -PE 40  on serotonin release from C57 cells. A) Cells were labeled overnight with [ 3 H] hydroxytryptamine creatinine sulfate. The cells were then washed and exposed to various concentrations of Fc 2′-3′ -PE 40  for 30 minutes. Control cells were pre-incubated with IgE and exposed to DNP and [ 3 H] hydroxytryptamine creatinine sulfate released into the medium was measured: 
       ▪ Control, □ IgE-DNP, ▪100 g, □ 250, 
       □ 1000 ng, or □ 5000 ng Fc 2′-3′ -PE 40    
         FIG. 12  (B): Cells were incubated with Fc 2′-3′ -PE 40  at different time points [ 3 H] hydroxytryptamine sulfate release into the medium was measured; legends as in  FIG. 12(A) . 
         FIG. 12  (C): Time dependent cytotoxicity of Fc 2′-3′ -PE 40  against C57 cells. Unlabeled cells were incubated as in  FIG. 12(B) . At the same time points cells were pulsed for 1 h with [ 3 H] leucine and its incorporation into cellular proteins was measured. The results are expressed as the percentage of protein synthesis of control cells not exposed to chimeric proteins. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The Fc-PE chimeric protein according to the present invention has a number of advantages over the existing known drugs: 
     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. 
     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 immunosuppression are anticipated. Re-institution of a normal physiological state is expected to occur within several weeks after the end of the treatment. 
     3. Duration of the effect: Because maturation of mast cells takes several weeks, the effect of Fc-PE is predicted to be long-standing, eliminating the need for frequent administration. Moreover, as in vitro studies indicate that reduction of 80% in cellular protein synthesis 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. 
     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. 
     The following experimental results indicate that the Fc 2′-3′ -PE40 chimeric protein according to the present invention is a promising candidate for effective and selective allergy therapy. 
     The present invention provides a Fcε-PE chimeric cytotoxin protein for the targeted elimination of FcεRI expressing cells, useful especially for the therapy of allergic responses such as asthma, allergic rhinitis, food allergies, atopic dermatitis, and anaphylaxis. 
     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. 
     1. Construction of Fcε-PE 40  Chimeric Proteins. 
     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.). 
     We used a sequence corresponding to a.a. 301-437, containing the COOH terminus of domain 2 and the entire domain 3(C2′-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-Thyl.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 β 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, recommended by the manufacturer. 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. 
     Fcε fragments 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), SEQ ID NO: 5, 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, SEQ ID NO: 6, and 5′-GCG GAT CCC ATA TGC GAC CTG TCA ACA TCA CTG-3′ (sense, starting from nucleotide 175), SEQ ID NO: 7, 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, SEQ ID NO: 8. 
     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 . 
     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 respectively, 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. 
     2. Expression and Partial Purification of the Chimeric Proteins. 
     The newly designed chimeric protein, Fcε-PE 40  encoded by plasmid pAF2302 was expressed in  E. coli  strain BL21 (lambdaDE3) which carries a T7 RNA polymerase gene in a lysogenic and inducible form. Induction was performed at O.D. 600 0.5 for 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 30 s bursts) and centrifuged at 30,000×g 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 sustention was cleared by centrifugation at 30,000×g 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 15,000×g for 15 min. and the resultant supernatant (insoluble fraction, guanidine-hydrochloride 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. 
     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 transferred 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. 
     Subcellular fractionation of expressing cells revealed that the insoluble fraction (inclusion bodies) was particularly rich. with chimeric protein ( FIG. 2 ). This fraction was therefore used as the source of the chimeric protein. 
     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). 
     3. Effect of Fc 2′-3′ -PE 40  Chimeric Protein on Mouse Mast Cell Lines. 
     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 [3H]-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. 
     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). 
     4. Specificity of Fcε-PE 40  Response. 
     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 existed with EcoRI and BamHI and replaced by a corresponding fragment carrying a deletion at amino acid 553. 
     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 ). 
     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 ). 
     The effect of Fc 2′-3′ -PE 40  was also tested on various mouse non-target cell lines (Table 1). All cell lines of hemopoietic origin were unaffected by the chimeric protein. Surprisingly, 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). 
     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. 
     5. Effect of Chimeric Proteins on Primary Mast Cells. 
     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. 
     BALB/C mice aged 4-6 weeks were sacrificed 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 300×g 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  (10 u/ml) was added starting from day 7 in culture. 
     To follow the degree of maturation, cells were mounted on slides, stained with acidic Toluidine Blue (pH 1.0) and examined microscopically under oil. 
     The effect of chimeric proteins was tested on bone marrow derived mast cells (BMMC) on the 16th day of culture. 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 ). 
     6. Receptor Specificity of Fc 2′-3′ -PE 40 . 
     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 homeopathic 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. 
     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. 
     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 (25 mM,  FIG. 7B ). 
     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). 
     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). 
     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. 
     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. 
     7. Effect of Fc 2′-3′ -PE 40  on Cellular Degranulation. 
     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. 
     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. Unlabeled 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 synthesis inhibition by chimeric toxin. There was no difference in supernatant [ 3 H) 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. 
     8. Electrophoretic Characterization of Fcε-PE40 
     Western blot analysis of electrophoresed samples run under non-reducing conditions (omitting 2-mercaptoethanol from the sample buffer) revealed that the Fc 2′-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. 
     9. Internalization Assay 
     In vitro activity of the chimeric protein is achieved only upon it&#39;s internalization. To test 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 neutralised 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 Fc 2′-3′ -PE40 chimeric protein is internalized into the target cells ( FIG. 11 ). 
     10. Effect of Fc 2′-3′ -PE 40  on Cellular Degranulation 
     C57 cells were incubated overnight with [ 3 H]-Hydroxytryptamine (10 μci/ml) at 37° C. Cells were washed 3 times to remove free [ 3 H]-Hydroxytryptamine, plated in Tyrod&#39;s buffer (10 mM 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 degranulation 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 cells 
                 1,200-1,500 
               
               
                 NON- 
                 HEMOPOETIC 
                 L 10 A 
                 B cell, non-secreting 
                 &gt;10,000 
               
               
                 TARGET 
                   
                 X 16 B 
                 B cell, non-secreting 
                 &gt;10,000 
               
               
                 CELLS 
                   
                 UT 
                 B cell, non-secreting 
                 &gt;10,000 
               
               
                   
                   
                 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