Link:  Pharm/Biotech Resources

Title:  FcεPE chimeric protein for targeted treatment of allergy responses, a method for its production and pharmaceutical compositions containing the same

United States Patent:  6,919,079

Issued:  July 19, 2005

Inventors:  Fishman; Ala (Haita, IL); Yarkoni; Shai (Kfar-Saba, IL); Lorberboumgalski; Haya (Jerusalem, IL)

Assignee:  Yissum Research Company of the Hebrew University of Jerusalem (IL)

Appl. No.:  091645

Filed:  December 18, 1996

PCT Filed:  December 18, 1996

PCT NO:  PCT/IL96/00181

371 Date:   June 18, 1998

102(e) Date:  June 18, 1998

PCT PUB.NO.:  WO97/22364

PCT PUB. Date:  June 26, 1997

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-PE₄₀. A sequence encoding amino acids 301-437 of the Fc region of the mouse IgE molecule was genetically fused to PE₄₀-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 Fc_εRI expressing cells especially useful for the therapy of allergic responses. The said chimeric protein is comprised of a cell targeting moiety for Fc_εRI 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 PE₄₀, 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.

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 PE₄₀-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 Fc_εRI expressing cells especially usefull for the therapy of allergic responses. The said chimeric protein is comprisesd of a cell targeting moiety for the Fc_εRI 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 PE₄₀, 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 PE₄₀ 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

The Fc-PE chimeric protein according to the present invention has a number of advantages over the existing known drugs.:

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-_PE40chimeric 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 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.

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-PE₄₀ 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′-C₃). We used also a sequence corresponding to a.a. 225-552, containing the whole C₂-C₄domains. 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 2×10⁶ cells/ml in the presence of Lipopolysaccharide (LPS, 10 μg/ml) and IL₄ (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.

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′, SEQ ID NO. 5, (sense, starting from nucleotide 406, according to gene bank sequence J00476) and 5′-GCG CCC ATA TGT GGG GTC TTG GTG ATG GAA C-3′, SEQ. ID NO. 6, (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′, SEQ. ID. NO. 7, (sense, starting from nucleotide 175) and 5′-GCG GAT CCC ATA TGG GAG GGA CGG AGG GAG G-3′, SEQ. ID. NO. 8, (antisense, starting from nucleotide 1167) for the Fcε_2-4 sequence.

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 MgSO₄ 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 IL₂-PE₄₀, 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 C₁′-C₃ and C₂-C₄ fragments respectivly, each fused 5′ to PE₄₀, 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-PE₄₀ 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.d.₆₀₀0.5 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 30s 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 suspention 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. 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 and against IgE (Serotec, England). 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'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 paticularly rich with chimeric protein. 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-PE₄₀ 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 Fc_εRI receptor. The cytotoxic activity of the chimeric protein was evaluated by inhibition of protein synthesis, as measured by [³H] Leucine incorporation. Various concentrations of the chimeric protein, diluted with 0.25% bovine serum albumim in phosphate-buffered saline, were added to 2×10⁴ 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 IL₃ for growth, C57, an IL₃ independent mast cell line originating in mouse bone marrow; and the Abelson-virus transformed mast cell line originating in mouse midgestation embryonic placenta. Fcε-PE₄₀ was found to be cytotoxic in a dose-dependent manner to all the cell lines tested. The MC-9 and C57 lines were extremely sensitive to the chimeric toxin, with an ID₅₀ of 50-75 ng/ml and 100-125 ng/ml, respectively. The Alelson cell line was much less sensitive (ID₅₀ of 1200-1500 ng/ml).

4. Specificity of Fcε-PE₄₀ Response.

To verify the specificity of Fc_2′-3-PE₄₀ activity, two control proteins, PE₄₀ 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.

PE₄₀, which has no intrinsic targeting capacity had, as expected, no effect on the target cell lines. Fc_2′-3-PE_40M which possesses a Fc_2′-3 moiety linked to a mutated, enzymatically inactive form PE₄₀, was also not cytotoxic to the target cells.

In addition, it was possible to block the cytotoxic effect of Fc_2′-3-PE₄₀ against target cells by whole mouse IgE (40 jμ/ml) or by a αPE polyclonal antibody (10 μg/ml).

The effect of Fc_2′-3-PE₄₀ was also tested on various mouse non-target cell lines. 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 ID₅₀ values were twenty-fold higher than those of the MC-9 cells.

The above data demonstrates that the toxic effect of Fc₂′-3-_PE40 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 (PE₄₀) 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-PE₄₀. When cultured in the presence of IL₃ 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 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 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 IL₃. Cells were grown in tissue culture flasks at a density of 10⁶ cells/ml, at 37° C. in a 5% CO₂ humidified atmosphere for 2-3 weeks. The media were changed every 7 days. Recombinant IL₄ (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.  Fc_2′-3-PE₄₀ was cytotoxic to BMMC in a dose dependent manner, with an ID₅₀ 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 PE₄₀ displayed cytotoxicity against BMMC. Thus, primary mast cells respond towards the chimeric protein similarly to the established mast cell lines.

6. Receptor Specificity of Fc_2′-3-PE₄₀.

Aside from the high affinity Fc_εRI receptor, three other membrane surface structures were reported to bind IgE with low affinity-the low affinity Fc_εRII 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-PE₄₀ 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.   There was no difference in the cellular response to Fc_2′-3-PE₄₀ between control cells and cells preincubated with the antibody.

We next examined whether εBP is involved in the cytotoxicity of Fc_2′-3-PE₄₀. 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-PE₄₀ in the presence or absence of lactose (25 mM).

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. Again, there was no difference in Fc_2′-3-PE₄₀ cytotoxicity against treated and control cells (results not shown).

To test whether Fc_2′-3-PE₄₀ affects Fc_εRII-bearing cells, we used the 0.12A3 cell line, a mouse B cell hybridoma expressing the Fc_εRII receptor. The 0.12A3 cells were totally non responsive to Fc_2′-3-PE₄₀, even at high doses (>5000 ng/ml). 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-PE₄₀ has no effect on these B splenocytes, although 69% of the cells expressed the receptor, as determined by FACS analysis.

Collectively, these results suggest that Fc_2′-3-PE₄₀ does not bind to the low affinity IgE-binding structures, namely Fc_εRII, FcγRII/III and εBP.

7. Effect of Fc_2′-3-PE₄₀ on Cellular Degranulation.

Because of the possible clinical applicability of Fc_2′-3-PE₄₀, it was important to test whether treatment of mast cells with Fc_2′-3-PE₄₀ results in the release of allergic mediators triggered upon Fc_εRI binding by the chimetric protein.

C57 cells prelabelled overnight with [³H]-hydroxytryptamine 10 μci/ml) were washed, plated at 2×10⁵ cells/well in DMEM containing 10% FCS, in 96-well tissue culture plates and incubated with Fc_2′-3-PE₄₀ (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-PE₄₀ and at the same time intervals were pulsed 1 hr with [³H] leucine to measure protein systhesis inhibition by chimeric toxin. There was no difference in supernatant [³H] seretonin content between Fc_2′-3-PE₄₀ treated and untreated cells at ½4 or 8 hr following chimeric protein addition. Inhibition of protein synthesis reached 80% at 4 h. and a value of 90% by 8 h. These results suggest that Fc_2′-3-PE₄₀ 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 Fc2′-3-PE40 chimeric protein is predominantly present as a monomer. 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. 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's internalization. To test whether the chimeric protein is internalysed, 5×10⁵ 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.

10. Effect of Fc_2′-3-PE₄₀ on Cellular Degranulation

C57 cells were incubated overnight with [³H]-Hydroxytryptamine (10 μci/ml) at 37° C. Cells were washed 3 times to remove free [³H]-Hydroxytryptamine, plated in Tyrod's buffer (10 mM Hepes pH 7.4, 130 mM. NaCl, 5 mM KCl, 5.6 mM Glucose, 0.5% BSA) at 2.5×10⁵ cells/0.5 ml in 24 well tissue culture plates and incubated with IgE (10 μg/ml) for 1 hour at 4° C. MgCl₂ and CaCl₂ 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 [³H]-Hydroxytryptamine released was measured. No degranulation was observed with any concentration of chimeric protein tested. 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. Fc_2′-3-PE₄₀ did not cause any degranulation also at later stages of it's interaction with the target cell, while it inhibits protein synthesis by over 80%. Our results demonstrate that Fc_2′-3-PE_{40 -}does not_trigger degr anulation at any stage during it's interaction with the cell.
 

Claim 1 of 7 Claims

1. A chimeric protein for therapy of allergic responses by targeted elimination of Fc_εRI expressing cells; wherein said chimeric protein is comprised of a cell targeting moiety comprising an Fc region of a mouse IgE molecule; and a cell killing moiety.

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