ES2347962T3 - Biological variability of asthma associated factor, AAF2 (IL-9 receptor), useful in treating and diagnosing atopic allergies including asthma and related disorders. - Google Patents

Abstract

This invention relates to the diagnosis, treatment and methods for discovery of new therapeutics for atopic asthma and related disorders based on variants of Asthma Associated Factor (2). One embodiment of the invention is a variant of AAF2, wherein codon 173 is deleted resulting in the loss of glutamine 173 from the mature protein precursor. This single amino acid deletion results in a non-functional AAF2 protein and therefore the presence of this phenotype should be associated with less evidence of atopic asthma. Correspondingly, the lack of susceptibility to an asthmatic, atopic phenotype is characterized by the loss of glutamine at codon 173. The invention includes isolated DNA molecules which are variants of the wild type sequence as well as the proteins encoded by such DNA and the use of such DNA molecules and expressed protein in the diagnosis and treatment of atopic asthma.

Description

Field of the Invention

The present invention describes the biological variability in the IL-9 receptor (Asthma Associated Factor 2) (SEQ ID NO: 1) and relates these sequence variants to asthma sensitivity, atopic allergy and associated disorders. In addition, methods using variable IL-9 receptors in the development of pharmaceutical products for asthma that depend on the regulation of IL-9 activity are described.

Background of the Invention

Inflammation is a complex process in which the body's defense system fights foreign entities. Although the fight against foreign entities may be necessary for the survival of the body, some defense systems respond to them inappropriately, even to those that are harmless, and dangerous and thus damage the surrounding tissue in the subsequent fight.

Atopic allergy is a disorder in which the genetic background dictates the response to environmental stimuli. In general, the disorder is characterized by an increase in the ability of lymphocytes to produce IgE antibodies in response to ubiquitous antigens. The activation of the immune system by these antigens also leads to allergic inflammation, which may occur after ingestion, by penetration into the skin or after inhalation. When this immune activation takes place and leads to pulmonary inflammation, in general this disorder is classified as asthma. In this inflammatory reaction of the airways certain cells are important, including T cells as well as those that have antigens, B cells that produce IgE and mast cells / basophils that store inflammatory mediators and bind to IgE, as well as eosinophils that release other mediators. These inflammatory cells accumulate at the site of allergic inflammation and the toxic products they release contribute to the destruction of tissue related to the disorder.

Although asthma is generally defined as an inflammatory disorder of the airways, clinical symptoms arise from intermittent airflow obstruction. It is a chronic, disabling disorder that seems to increase in its frequency and severity. 1. It is estimated that 30-40% of the population suffers from atopic allergy and 15% of children and 5% of adults in the population suffer from asthma.1 Therefore, the treatment of these disorders represents a huge burden for our healthcare resources.

Both the diagnosis and treatment of asthma and its related disorders are problematic1. In particular, evaluation of inflamed lung tissue is often difficult and often the cause of inflammation cannot be determined. Although atopic asthma is an echogenic disorder, only recently has knowledge of particular gene variants been known. The methods to detect these genetic variations and their role in inflammation, diagnosis and prognosis remain to be determined. The technique requires the development of a technology to accelerate the diagnosis of atopic asthma that is specifically related to variations in the genes responsible for the sensitivity / resistance to this atopic disease.

Current treatments have their own set of drawbacks. The main therapeutic agents, β-agonists, reduce symptoms, that is, temporarily improve lung functions, but do not affect underlying inflammation, so that lung tissues remain at risk. In addition, the constant use of β-agonists results in desensitization, which reduces their efficacy and safety2. Agents that can reduce underlying inflammation, anti-inflammatory steroids, have their own list of known side effects, ranging from immunosuppression to bone loss2.

Due to the problems associated with conventional therapies, A37 have been

evaluated alternative treatment strategies38-39. Glycoforin, cyclosporine38 and a nonapeptide fragment of IL-226 inhibit all proliferation of interleukin-2-dependent T lymphocytes. It is known, however, that they have numerous other effects2. For example, cyclosporine is used as an immunosuppressant after organ transplantation. Although these agents may represent alternatives to steroids in the treatment of asthmatics 36-39, they inhibit the proliferation of interleukin-2-dependent T lymphocytes, as well as the potentially critical immune functions associated with homeostasis. A technique is needed in the art to accelerate the development of therapeutic products that are specifically designed to treat the cause and not the symptoms of atopic asthma. These therapies represent the most appropriate way to avoid toxicity associated with non-specific treatment. The therapies would selectively focus a path downstream of immune functions, such as the activation of IL-2-mediated T lymphocytes, which is necessary for the development of asthma and that would explain the episodic nature of the disorder and its close association with allergy. Nature demonstrates that a route is the appropriate target for asthma therapy when there is normally a biological variability in the pathway and individuals who show such variability are not immunocompromised or ill, except for their atopic asthma symptoms.

Due to the difficulties related to the diagnosis and treatment of atopic allergies, including asthma, the complex pathophysiology of these disorders is undergoing thorough study. Although these disorders are heterogeneous and can be difficult to define, since they can take many forms, certain common characteristics are found among asthmatics. Examples of such characteristics include the abnormal response to the skin test to the challenge with allergens, pulmonary eosinophilia, bronchial hyperreactivity (BHR), bronchodilator reversibility and airflow obstruction 3-10. These expressions of asthma-related characteristics can be studied through quantitative or qualitative measurements.

In many cases, high levels of IgE are correlated with BHR, a strong bronchoconstrictor response to a variety of stimuli4,6,8,9. It is believed that BHR reflects the presence of inflammation in the airways 6,8 and is considered a risk factor for asthma 11-12. BHR is accompanied by bronchial inflammation, including eosinophilic infiltration in the lung and an allergic diathesis in asthmatic individuals6,8,13-18.

Numerous studies document a hereditary component of atopic asthma4,10. However, it has been difficult to interpret the studies in families, since age and gender significantly influence these disorders, as well as many environmental factors such as allergens, viral infections and contaminants19-21. In addition, because there is no known biochemical defect associated with sensitivity to these disorders, the mutant genes and their abnormal genetic products can only be recognized by the anomalous phenotypes they produce.

The functions of IL-9 and the IL-9 receptor (the IL-9 pathway) now extend beyond what was originally recognized. Although the IL-9 pathway serves as a stimulant of T-cell growth, this cytokine is also known to mediate the growth of erythroid progenitors, B cells, mast cells and fetal thymocytes22,23. The IL-9 pathway acts synergistically with IL-3, causing mast cell activation and proliferation24. The IL-9 pathway also enhances the IL-4 induced production of IgE, IgG and IgM by normal human B lymphocytes25 and the IL-4 induced release of IgE and IgG by murine B lymphocytes26. The role of the IL-9 pathway in the inflammatory response to parasitic mucosal infection has also been demonstrated27,28.

However, it is not known how the IL-9 receptor sequence has specific correlation with atopic asthma, as well as bronchial hyperreactivity. It is known that IL-9 binds to a specific receptor expressed on the surface of the target cells23,29,30. The receptor is specifically composed of two protein chains: a protein chain, known as the IL9 receptor, specifically binds to IL-9; the other protein chain is shared in common with the IL-223 receptor. In addition, a cDNA encoding the human IL-9 receptor has been cloned and sequenced23,29,30. This cDNA encodes a 522 amino acid protein that shows significant homology with the murine IL-9 receptor. The extracellular region of the receptor is very conserved, with a homology of 67% between murine and human proteins. The cytoplasmic region of the receptor is less conserved. The human cytoplasmic domain is much larger than the corresponding region of the murine receptor23.

The IL-930 receptor gene has also been characterized. It is believed to exist as the only copy in the mouse genome and is composed of nine exons and eight introns30. The human genome contains at least four IL-9 receptor pseudogenes. The human IL-9 receptor gene has been mapped in the 320 kb subtelomeric region of the X and Y23 sex chromosomes.

Despite these studies, no variants of the IL-9 receptor gene have been discovered. Therefore, genetic information on atopic allergy, asthma, bronchial hyperreactivation, as well as clarifying the role of the IL-9 receptor in the etiology of these disorders is specifically needed. This information can be used to diagnose atopic allergy and its related disorders by methods that identify the genetic variants of this gene, which are associated with these disorders. In addition, methods that use IL-9 receptor variants are needed to develop therapeutic products in order to treat these disorders.

Summary of the Invention

Applicants have discovered natural variants of the human IL-9 receptor (also known as Asthma Associated Factor 2 or AAF2) and have linked these variants to the pathogenesis of asthma and its related disorders. These findings led to the development of diagnostic methods, as well as methods to discover pharmaceutical products for the therapeutic treatment of atopic asthma. In addition, applicants have determined that the IL-9 receptor is critical for a number of antigen-induced responses in mice, including bronchial hyperreactivity, eosinophilia and elevated cell counts in bronchial lavage, as well as elevated levels of total IgE in serum. These findings typify atopic asthma and associated allergic inflammation.

In addition, applicants have determined that a variant of the nucleic acid from G to A is produced at position 1,273 of the cDNA (SEQ ID NO: 2) that results in the predicted amino acid substitution of a histidine for an arginine in codon 344 of the human IL-9 receptor precursor protein. When arginine residue is found in both alleles in an individual, it is associated with less evidence to atopic asthma. Therefore, applicants have identified the existence of a non-asthmatic phenotype characterized by arginine in codon 344 when it is produced in both products of the IL-9 receptor gene in an individual. As a significant additional consequence, applicants have identified the occurrence of sensitivity to an atopic asthma phenotype characterized by a histidine in codon 344.

Applicants have also determined that there is an IL-9R splice variant in which the glutamine residue at position 173 of the IL-9R precursor protein has been removed (SEQ ID NO: 3) (Figure 5). Applicants have also shown that this variant cannot transcribe a signal via the Jak-Stat route (Figure 15) and that it is unable to induce cell proliferation when stimulated with IL-9 (Figure 16); therefore, individuals with this allele would be less susceptible to atopic asthma and its related disorders.

Applicants have also determined that there is a variant of the genomic DNA of IL-9R in which nt-213, a thymine residue in intron 5 (213 nt upstream of exon 6), has become a cytosine nucleotide. It is likely that such variation may cause an increase in the frequency of the splice variant that removes the glutamine residue at the beginning of exon 6.

In addition, applicants have discovered a variant of IL-9R in which exon 8 (SEQ ID NO: 4) has been suppressed, resulting in a change in the reading frame and a premature stop codon in exon 9 Said variant would probably be prevented in the transmission of a signal by the Jak-Stat route and, therefore, individuals with this allele would also be less sensitive to atopic asthma and its related disorders.

The biological activity of IL-9 results from its binding to the IL-9 receptor and the subsequent propagation of a regulatory signal in specific cells; therefore, the functions of IL-9 can be interrupted by the interaction of IL-9 antagonists with IL-9 or its receptor. Sub-regulation, that is, the reduction of functions controlled by IL-9, is achieved in different ways. The administration of antagonists that can interrupt the binding of IL-9 to its receptor is a key mechanism, said antagonists being within the scope of the claims of the invention. Examples include the administration of polypeptide products encoded by DNA sequences in a naturally occurring soluble form of the IL-9 receptor, where DNA sequences encode a polypeptide comprising exons 2 and 3 (SEQ ID NO: 5 ). Two other variations can produce soluble forms of the IL-9R receptor comprising exons 2, 3 and 4 and in one case four amino acids from a different reading frame in exon 5 (SEQ ID NO: 6) (Figure 6) and in the other case there are 27 amino acids from a different reading frame in exon 5 (SEQ ID NO: 7) (Figure 7).

Methods for identifying agonists and antagonists of the IL-9 receptor pathway can be carried out by evaluating the receptor-ligand interactions described in the literature. These methods can be adapted to high-performance automated assays that facilitate chemical tracing and potential therapeutic identification. Agonists are recognized by the identification of a specific interaction with the IL-9 receptor. Generally, the loss of binding of a putative ligand that is labeled when 100 to 1,000 times excess of unlabeled ligand is used as evidence of specific binding to the receptor is accepted. During these experiments, very different labels and detection schemes can be used. A similar loss of binding is also evidence of an antagonist when the concentrations of the test compound added to a known ligand and receptor are increased.

Knowledge of the variable receptors provides the means to construct expression vectors that can be used to make the soluble receptor for receptor binding assays. The mutagenesis of these soluble receptors can be used to determine which amino acid residues are critical to bind the ligand and assist in the design of the antagonists based on their structure.

Cells that lack a human IL-9 receptor can be transiently or stably transfected with expression vectors containing a variable receptor and used to test the activity of the IL-9 pathway. These activities can be cell proliferation

or of prevention of apoptosis, both attributed to the IL-9 pathway. These cells can be used to identify agonists and receptor antagonists as described above.

The methods mentioned above represent various effective methods that use the variable forms of the IL-9 receptor to develop therapeutic products against atopic asthma and other related disorders.

Multiple techniques have been described that can be used to diagnose atopic asthma and recognize single nucleotide variants in the IL-9 receptor, including DNA sequencing, restriction fragment length polymorphisms (RFLPs), analysis of Allele-specific oligonucleotides (ASO), ligase chain reaction (CSF), chemical cleavage and conformational analysis of single-stranded polymorphisms (SSCP). A specialist will recognize that one or more of these techniques, as well as others mentioned in the literature, can be used to detect one or more variations in the IL-9 receptor gene or in the mRNA transcript.

Even other techniques can be used to detect amino acid variants in the IL-9 receptor, which include ELISA, immunoprecipitation, Western and Immunoblotting.

The methods mentioned above constitute effective methods to diagnose atopic asthma and other related disorders.

Thus, applicants have provided methods that use the IL-9 receptor to identify antagonists that are capable of regulating the interaction between IL-9 and its receptor. More specifically, applicants provide a method for testing the functions of the IL9 receptor in order to identify those compounds or agents that can be administered in an amount sufficient to sub-regulate the expression or functions of the IL-pathway. 9.

With the identification of the role of the IL-9 pathway in atopic allergy, bronchial hyperreactivity and asthma, applicants also provide a method for the diagnosis of sensitivity and resistance to atopic allergy, asthma and related disorders.

The attached figures that are incorporated and form part of this specification illustrate several embodiments of the invention and, together with the description, serve to explain the principle thereof.

Brief Description of the Figures

Figure 1: Schematic representation of the human IL receptor cDNA. The boxes describe exon 2 to 9 covering the coding region (size relative to scale, except for the untranslated part 3 ’of exon 9, outlined with a dashed line). The transmembrane region is encoded by exon 7, the intracellular domain by exons 8 and 9 and the extracellular by exons 2 a

6. Arrows indicate aberrant polymorphisms or splices that affect the partial sequence of the exon; a) deletion of the first 5 or 29 nucleotides in exon 5; b) deletion of the first 3 nucleotides in exon 6 (codon 173); c) arg / gly polymorphism at codon 310; d) arg / his polymorphism at codon 344; e) codon 410 + n polymorphism consisting of 8 or 9 serines; *) complete deletion of exon 3, 4 or 8.

Figure 2: Translated cDNA sequence of the wild-type IL-9R precursor protein with the Arg allele at codon 344 (nucleotides 12721274) and 8 Ser / 4 Asn repeats beginning at codon 410 (nucleotides 1470-1472 ).

Figure 3: Translated cDNA sequence of the wild-type IL-9R precursor protein with the His allele at codon 344 (nucleotides 12721274) and 9 Ser / 4 Asn repeats starting at codon 410 (nucleotides 1470-1472 ).

Figure 4: Translated cDNA sequence of the IL-9R precursor protein with the exon 8 deletion that causes a change of framework in exon 9, the production of 11 non-wild type amino acids and a premature stop codon.

Figure 5: Translated cDNA sequence of the IL-9R precursor protein with Glutamine deletion at codon 173.

Figure 6: Translated cDNA sequence of the IL-9R precursor protein with an alternative splice in exon 5 resulting in a premature stop codon and the production of 4 non-wild type amino acids.

Figure 7: Translated cDNA sequence of the IL-9R precursor protein with an alternative splice in exon 5 resulting in a premature stop codon and the production of 27 wild-type amino acids.

Figure 8: Translated cDNA sequence of the IL-9R precursor protein with the deletion of exon 4 that produces a stop codon as the first codon of exon 5.

Figure 9: Table showing the association between the IL-9 receptor genotype and atopic allergy. Individuals with Arg / Arg are homozygous for the Arg allele with the repetitions of 8 Ser / 4 Asn. Individuals with Arg / His are heterozygous for the Arg allele with the repetitions of 8 Ser / 4 Asn and the His allele with the repetitions of 9 Ser / 4 Asn, repetitions of 9 Ser / 3 Asn and repetitions 10 Ser / 2 Asn in exon 9. Individuals with His / His are homozygous for the His allele with repetitions of 9 Ser / 4 Asn, repetitions of 9 Ser / 3 Asn, and repetitions of 10 Ser / 2 Asn in exon 9. Individuals with Arg / Arg are protected against atopic allergy.

Individuals with Arg / His and His / His are susceptible to allergy

atopic (P = 0.002).

Figure 10: Map of the IL-9 receptor expression construct.

Figure 11: Western blot of recombinant IL-9 receptor proteins (left: Arg allele with the 8 Ser / 4 Asn repeats; right: His allele with the 9 Ser / 4 Asn repeats) using the C- antibody probe terminal in the transfected TK cell line.

Figure 12: Expression of human IL-9 receptor variants in TS1 cells, showing the differential mobility between the Arg 344 variant with the 8 Ser / 4 Asn (GR8) repeats and the His 344 variant with the 9 repetitions Ser / 4 Asn (GH9). A change in mobility is observed that demonstrates the differential post-translational modification of these two forms of IL-9 receptor variants.

Figure 13: XY specific amplimers for specific amplification of the IL-9 receptor gene. The pseudogenes on chromosomes 9, 10, 16 or 18 are not amplified by PCR (M is mouse DNA, H is human DNA, and C is hamster DNA).

Figure 14: Immunoreactivity of an anti-human neutralizing antibody against the IL-9 receptor with wild-type and Delta-Q receptors. Panel A): COS7 cells were transiently transfected with the LXSN vector alone (A and B), wild-type IL-9R (C and D), wild-type IL-9R with 9 Ser residues starting at codon 410 (E and F), variant of Δ-Q 173 (G and H) and Δ-Q 173 with 9 Ser residues starting at codon 410 (I and J) and sequentially incubated with MAB290 and anti-mouse IgG Texas Red conjugate antibody ( B, D, F, H and J) as described (Example 8). DAPI staining (A, C, E, G and I) was included to visualize each cell in the photographed field. Panel B): as in A) except that the cells were first fixed / permeabilized and then incubated with a specific C-terminal antibody (sc698) followed by incubation with the anti-rabbit IgG Texas Red conjugate antibody. Bar = 10 microns.

Figure 15: Activation of members of the Jak, Stat and Irs families through different variants of the human IL-9 receptor. TS1 cells expressing GH9, ΔQGR8, or ΔQGH9 were deprived of nutrients for 6 hours and then treated for 5 minutes without cytokine (-), with murine IL-9 (m) or with human IL-9 (h). Cell extracts were immunoprecipitated with several specific antibodies from different members of the Jak, Stat and Irs families. The immunoblots were first reacted with an antiphosphotyrosine antibody to detect only phosphorylated proteins in tyrosine and then stripped and re-hybridized with the same antibody used to immunoprecipitate each protein. GH9, ΔQGR8, or ΔQGH9 are as indicated in Figure 16.

Figure 16: Proliferation of TS1 cells expressing different forms of the human IL-9 receptor. Cells were seeded in quadruplicate in 96-well plates (1,000 / well) and treated without cytokine or with murine or human IL-9 (5 ng / ml). A colorimetric test was performed 7 days later to determine the number of cells and the relationship between treated and untreated cells (% control) was calculated to assess the growth rate. LxSN = cells transfected with the empty vector; GR8 is wild-type IL-9R: GH9 is the His 344 variant with 9 Ser residues starting at codon 410; ΔQGR8 and ΔQGH9 are the variants of ΔQ173 in the wild type and the background of His 344 + 9 - Ser, respectively.

Figure 17: Genomic DNA sequence of intron 5 of IL-9R with a variation in nucleic acid 213 nt upstream of exon 6 where a residue T is converted to a residue C as indicated by an arrow.

Detailed description of the invention

Applicants have resolved these needs of the technique by determining an IL-9 pathway as well as the compositions that affect this pathway, which can be used in the treatment, diagnosis and development of methods to identify agents to prevent or treat atopic asthma. and related disorders. The invention is described in the appended claims.

Asthma encompasses inflammatory airway disorders with reversible airflow obstruction. Atopic allergy refers to atopy and related disorders including asthma, bronchial hyperreactivity (BHR), rhinitis, hives, allergic inflammatory bowel disorders and various forms of eczema. Atopy is a hyperreactivity to environmental allergens expressed as elevation of total serum IgE or abnormal skin test responses to allergens compared to a control. BHR refers to bronchial hyperreactivity, increased bronchoconstrictor response to a variety of stimuli.

By analyzing the DNA of individuals who show atopic allergy and asthma-related disorders, applicants have identified polymorphisms in the IL-9 receptor gene (IL-9R) that may correlate with asthma expression. The IL-9 receptor gene (also known as Asthma Associated Factor 2 or AAF2) refers to the genetic locus of the interleukin-9 receptor, a cytokine receptor associated with various functions that involve the regulation of lymphoid and myeloid systems humans. The human IL-9 receptor gene of the present invention is in the subtelomeric region of the XY chromosomes.

By polymorphism, applicants refer to a change in a specific DNA sequence, called "locus," of a predominant sequence. In general, a locus is defined as polymorphic when those skilled in the art have identified two or more alleles that encompass that locus and the less common allele appears with a frequency of 1% or higher.

Specific amplification of authentic IL-9R (gene coding for the biologically functional protein located in the pseudoautosomal region XYq) using the standard primer design was not possible because IL9R has four highly homologous unprocessed pseudogenes (> 90% of nucleotide identity) in other loci in the human genome (chromosome 9, 10, 16, 18). Due to the high identity of these other genes, genomic PCR amplification using the standard primer design resulted in a co-amplification of all genes, thus causing the analysis of the authentic gene sequence to be equivocal. To study the authentic structure of IL-9R, as it may be related to the predisposition to a disease, such as asthma, which is described in this application, or other diseases such as cancer (Renauld et al., Oncogene, 9: 1327-1332, 1994; Gruss et al., Cancer Res., 52: 1026-1031, 1992), applicants have designed specific amplimers. It was found that the specific primers were authentic for amplification of IL-9R without amplification of the 4 pseudogenes. The primer sequences and their specificity are shown in Example 13 in Figure 13.

Applicants have also amplified, by RT-PCR, the entire coding region of the IL-9 receptor cDNA using RNA extracted from purified PBMCs (peripheral blood mononuclear cells) from 50 donors. Fig. 1 illustrates the most frequent variations found in 50 individuals analyzed. Exons 3, 4, 5, 6 and 8 were affected by aberrant splicing events in samples in which full-length cDNAs could also be cloned. Some transcripts showed the complete deletion of exon 3, which causes a reading frame change that creates a stop codon after an expansion of 79 unrelated residues. In case of deletion of exon 4, a change of the reading frame is also generated and the first codon in exon 5 becomes a stop codon. In some other cDNAs, exon 5 exhibited partial deletion of the first 5 or 29 nucleotides, both deletions leading to changes in reading frames that resulted in early stop codons within exon 5. Therefore, in all cases, The putative truncated protein would lack most of the extracellular domain as well as all transmembrane and cytoplasmic domains. If secreted, these forms can function as soluble receptors. Finally, the first three nucleotides of exon 6, which corresponds to codon 173, were often unpacked, resulting in the deletion of glutamine in this codon without any other change in the remaining protein sequence. This splice variant possibly relates to a variant found in intron 5 of the genomic DNA (SEQ ID NO: 24) that would increase the frequency of the splice variant (Figure 17 and Example 12).

Applicants have also found allelic variations limited to the coding sequence of exon 9. Polymorphisms involving codon 310 and 41029.30 (Kermouni, A et al., Genomics, 371-382 (1995)) have been previously described. Codon 310 encodes arginine or glycine, depending on whether the first nucleotide in that codon is an adenine or a guanidine, respectively. In codon 410 (hereinafter referred to as "410 + n") an expansion of 8 or 9 repeats of AGC trinucleotides begins which would result in 8 or 9 serines, respectively. Applicants have discovered a new polymorphism at codon 344. Here, the second nucleotide is adenine.

or guanidine, the two possible residues encoded by this histidine or arginine codon being respectively. In addition, a correspondence between codon 344 and 410 + n was observed where arginine in codon 344 is consequently found with 8 serines in codon 410 + n and histidine in codon 344 meets 9 serines. The cDNA of the human IL-9 receptor originally cloned from a human megakaryoblastic leukemic cell line, Mo7e, had 9 serines at codon 410 + n and, unlike the applicant's clones, an arginine at codon 34429. It has reported that another UT-7 megakaryoblastic leukemic cell line carried the same 9 serine / arginine allele30. Applicants cloned 16 cDNAs from the Mo7e cell line and found that 6 had 8 serines in codon 410 + n and arginine in codon

344. The remaining 10 clones presented the published sequence. Applicants also genotyped the KG-1 acute human myelocytic leukemia cell line and discovered that it was homozygous histidine / 9 serines. These corresponding DNA and RNA molecules are isolated by techniques that are standard in the art, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1985). In isolation, applicants refer to the fact that DNA is free of at least some of the contaminants associated with nucleic acid and polypeptides that appear in a natural environment.

The invention also includes proteins encoded by nucleic acid sequences as described in the claims. The invention also includes fragments of the molecules. By fragments, applicants refer to portions of the nucleic acid sequence that maintain the function of the total sequence. As is known in the art, the fragments result from deletions, additions, substitutions and / or modifications. The origin of the IL-9 receptor variants of the invention is human. Alternatively, the DNA or a fragment thereof can be synthesized by methods known in the art. It is also possible to produce the compound by genetic engineering techniques, by constructing DNA by any accepted technique, by cloning DNA into an expression vehicle and transfecting the vehicle into a cell that will express the compound. See for example the methods mentioned in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1985).

The demonstration of the variant sequences of the IL-9 receptor, which may be associated with atopic allergy and an asthmatic phenotype, and others that may be associated with the lack of an asthmatic phenotype, provides diagnostic methods for atopic asthma susceptibility and related disorders. Certain variants can produce soluble receptors that can be used to treat these disorders.

A receptor is a soluble or membrane bound component that recognizes and binds to molecules, and the IL-9 receptor of the invention is the component that recognizes and binds to IL-9. The functions of the IL-9 receptor consist of binding to IL-9 or a type 9 molecule and propagating its regulatory signal in specific cells29,30,34,35. It has been shown that human IL-9 causes phosphorylation of the IL-9 receptor itself and the activation of Jak-Stat, Jak1, Stat1, Stat3, Stat5 and Irs2 pathway proteins, to the binding of the human IL receptor -9 (Demoulin, JB et al., Molecular and Cellular Biology,

p. 4710-4716, Sept. nineteen ninety six). Applicants have examined whether IL-9R or its variants showed any tendency to activate these proteins and extended the analysis to Jak3 and Irs1. It was determined that all proteins in the pathway, including the Jak3 and Irs1 pathways, were phosphorylated by the activation of IL-9R. It was also determined that IL-9R variants with changes in codons 310, 344 and 410 + n provided the same over-regulation as wild-type IL-9R. Therefore, one aspect of the invention consists of therapeutic products for the treatment of atopic asthma that inhibit interactions in the Jak-Stat pathway.

Unlike the wild-type receptor and the other variants tested, the ΔQ173 variant could not activate proteins in the Jak-Stat pathway (Figure 15). In addition, the ΔQ173 variant could not support cell proliferation upon stimulation of IL-9 (Figure 16). Therefore, individuals expressing the ΔQ173 variant are probably less sensitive to atopic asthma and related disorders. Thus, one aspect of the invention relates to therapeutic products for increasing the expression of the ΔQ173 splice variant for the treatment of atopic asthma and related disorders.

A diagnostic embodiment involves the recognition of variations in the DNA sequence of the IL-9 receptor gene or its transcript. One method involves the use of a nucleic acid molecule (also known as a probe) that has a complementary sequence of the IL-9 receptors of the invention under sufficient hybridization conditions, as will be understood by those skilled in the art. In one embodiment, the nucleic acid molecule will specifically bind the codon for Arg344 of the mature IL-9 receptor protein, or His 344, and in another embodiment it will bind both Arg344 and His344. In still another embodiment, the codon will be linked to Gln 173. These methods can also be used to recognize other variants of the IL-9 receptor. Another method to recognize the variation in the DNA sequence associated with these disorders is the direct analysis of the DNA sequence by various methods well known in the art44. Another embodiment involves the detection of the variation in the DNA sequence in the IL-9 receptor gene associated with these disorders40-44. These methods include polymerase chain reaction, restriction fragment length polymorphism (RFLP) analysis and single strand conformational analysis. In a preferred embodiment, the applicants specifically provide a method for recognizing, at the genetic level, polymorphism in the IL-9 receptor associated with the His344 and Arg344 alleles using a PCR ASO. In other embodiments, the ligation chain reaction can be used to distinguish these alleles from the IL-9 receptor genes.

Methods for the identification of IL-9 antagonists and their receptor are also described. Antagonists are compounds that are devoid of pharmacological activity themselves, but cause effects when presenting the action of an agonist. In order to identify an antagonist of the invention, competitive binding with a known agonist or sub-regulation of IL-9 type functions can be tested as described herein and in the literature mentioned 2,22-35.

Specific assays can be used to screen pharmaceutical products useful for the treatment of atopic allergy based on the known role of the IL-9 receptor in the proliferation of T lymphocytes, IgE synthesis and mast cell release29,30,33-35 . Another assay involves the ability of the human IL-9 receptor to specifically induce rapid and transient phosphorylation of multiple protein tyrosine in Mo7e34 cells. Because this response depends on the expression and activation of the IL-9 receptor, it represents a simple method or assay for the characterization of potentially valuable compounds. Tyrosine phosphorylation of the Stat3 transcription factor appears to be specifically related to the functions of the IL-935 receptor and this response represents a simple method or assay for the characterization of the compounds as described.

Yet another method for characterizing the function of the IL-9 receptor involves the use of the well-known murine TS1 clone transfected with a human receptor, which can be used to evaluate the function of human IL-9 in a cell proliferation assay29. These methods can be used to identify IL-9 receptor antagonists.

In another embodiment, the invention includes the subregulation of the expression

or IL-9 function by administration of molecules of the soluble IL-9 receptor that, as claimed, bind to IL-9. Applicants and Renauld et al.29 have shown the existence of a soluble form of the IL-9 receptor. This molecule can be used to prevent IL-9 binding to the cell-bound receptor and act as an IL-9 antagonist. Soluble receptors have been used to bind cytokines or other ligands to regulate their function45. A soluble receptor is a form of a membrane bound receptor that appears in solution

or outside the membrane. Soluble receptors may occur because the segment of the molecule that is commonly associated with the membrane is absent. This segment is commonly referred to in the art as the transmembrane domain of the gene or membrane binding segment of the protein. Thus, in one embodiment of the invention, a soluble receptor may represent a fragment or an analog of a membrane bound receptor.

Applicants have identified three variants of human IL-9 receptor splicing that result in the production of proteins that could act as soluble receptors.

A variant of the splicing resulted in the deletion of exon 4 which introduced a change of the reading frame, resulting in a stop codon as the first codon of exon 5. This variant would produce a peptide of approximately 45 residues containing an epitope reactive with the antibodies that block the IL-9 / IL-9R interaction. The other two variants contain deletions in exon 5, which will produce premature stop codons early in the exon, but, in these cases, without deletion of exon 4. These variants would produce a protein of approximately 100 residues that also contains the epitope recognized by the blocking antibody.

Soluble IL-9 receptors can be used to track potential therapeutic products, including the antagonist, useful in the treatment of atopic asthma and related disorders.

For example, the screening of single chain peptides and antibodies by means of phage display could be facilitated with the use of a soluble receptor. The phage that binds to the soluble receptor can be isolated and the molecule can be identified by affinity capture of the receptor and the bound phage. In addition, screening of compounds for agents useful in the treatment of atopic asthma and related disorders may incorporate a soluble receptor and a ligand that bind in the absence of an antagonist. The detection of the ligand and receptor interaction is due to the proximity of these molecules. Antagonists are recognized for inhibiting these interactions.

The invention further includes pharmaceutical compositions comprising the compounds of the invention together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be sterile liquids, such as water and oils, including those from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred vehicle when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid vehicles, in particular for injectable solutions. Pharmaceutically suitable vehicles are described in Martin, E.W., Remington’s Pharmaceutical Sciences, specifically incorporated herein by reference.

The compounds used in the treatment method can be administered systemically or topically, depending on considerations such as the condition to be treated, the need for a site-specific treatment, the amount of medication to be administered and similar considerations.

Topical administration can be used. Any common topical form, such as solution, suspension, gel, ointment or balm and the like can be used. The preparation of such topical formulations is well described in the art of pharmaceutical formulations, as illustrated, for example, in Remington's Pharmaceutical Science, Edition 17, Mack Publishing Company, Easton, Pa. For topical application, they can also be administered these compounds as powder or spray, particularly in aerosol form. In a preferred embodiment, the compounds of the present invention can be administered by inhalation. For inhalation therapy, the compound can be found in a solution useful for administration by means of metered dose inhalers, or in a form suitable for a dry powder inhaler. The active ingredient can be administered in pharmaceutical compositions adapted for systemic administration. As is known, if a medication should be administered systemically, it can be prepared in the form of powder, pill, tablet or the like, or as syrup or elixir for oral administration. For intravenous, intraperitoneal or intralesional administration, the compound can be prepared as a solution or suspension to be administered by injection. In certain cases, it may be useful to formulate these compounds as a suppository or as a prolonged release formulation for deposition under the skin or by intermuscular injection.

An effective amount is that amount that will sub-regulate the functions controlled by the IL-9 receiver. A certain effective amount will vary from condition to condition and, in certain cases, may vary depending on the severity of the condition being treated and the patient's sensitivity to treatment. Consequently, a certain effective amount will be determined at the time and place by routine experimentation. It is anticipated, however, that in the treatment of asthma and related disorders according to the present invention, a formulation containing between 0.001 and 5 percent by weight, preferably about 0.01 to 1%, will normally constitute a therapeutically effective amount. When administered systemically, an amount between 0.01 and 100 mg per kg of body weight per day, but preferably around 0.1 to 10 mg / kg, will have a therapeutic result in most cases.

Applicants also provide a method to select the compounds that sub-regulate the functions controlled by the IL-9 receptor. It can be determined whether the functions expressed by the IL-9 receptor are sub-regulated by means of techniques standard in the art29,30,34,35. In a specific embodiment, applicants provide a method of identifying compounds of functions comparable to IL-9. In one embodiment, the functions of the IL-9 receptor can be evaluated in vitro. As is known to those skilled in the art, activation of the human IL-9 receptor induces rapid and transient phosphorylation of multiple protein tyrosine in IL-9 sensitive cells. The tyrosine phosphorylation of the Stat3 transcription factor appears to be specifically related to the actions of the IL-9 pathway. Another method for characterizing the function of IL-9 and IL-9 type molecules depends on the "stable expression" of IL-9 receptors in murine TSI clones or TF1 clones, which do not normally express the human receptor. These transfectants can be used to evaluate the function of the human IL-9 receptor with a cell proliferation assay29.

The present specification also includes a simple screening assay for saturable and specific binding of ligands based on cell lines expressing variants of the IL-923.29 receptor. The IL-9 receptor is expressed in a wide variety of cell types, including K562, C816645, KG-1 transfected with human IL-9 receptors, B cells, T cells, mast cells, HL60, clone 5 of HL60, TS1 transfected with human IL-9 receptors, 32D transfected with human IL-9 receptors, neutrophils, megakaryocytes (UT-7 cells) 30, the human megakaryoblastic leukemia cell line Mo7e34, TF129, macrophages, eosinophils, fetal thymocytes, the line 29330 and 32D murine human renal cell, as well as embryonic hippocampal progenitor cell lines23,29,30.

The practice of the present invention employs the conventional terms and techniques of molecular biology, pharmacology, immunology and biochemistry that are part of the normal specialization of those skilled in the art. See for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, or Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.

However, we offer the following basic preliminary information. The body's genetic material, or DNA, is arranged in 46 chromosomes, each comprising two arms joined by a centromere. Each chromosome is divided into segments designated p or q. The symbol p is used to identify the short arm of a chromosome, as measured from the centromere to the nearest telomere. The long arm of a chromosome is designated by the symbol q. The location on a chromosome is given by the chromosome number (that is, chromosome 5) as well as the coordinates of the p or q region (i.e., q31-q33). In addition, the body carries the sex chromosomes, X and Y. During meiosis, the X and Y chromosomes exchange information on DNA sequences in areas known as pseudoautosomal regions.

The DNA, deoxyribonucleic acid, is composed of two complementary strands of nucleotides, which include the four different base compounds, adenine (A), thymine (T), cytosine (C) and guanine (G). A of one strand joins T of the other strand, while C of one strand joins G of the other to form complementary "base pairs," each pair having a base in each strand.

A sequential grouping of three nucleotides (a "codon") codes for an amino acid. Thus, for example, the three CAG nucleotides encode the amino acid Glutamine. The 20 natural amino acids, and their codes of one

lyrics, are the following:

To the girl

To TO

Arginine

Arg R

Asparagine

Asn N

Aspartic acid

Asp D

Asparagine or Aspartic Acid

Asx B

Cysteine

Cys C

Glutamine

Gln Q

Glutamine Acid

Glu AND

Glutamine or Glutamic Acid

Glx Z

Glycine

Gly G

Histidine

His H

Isoleucine

Ile I

Leucine

Leu L

Lysine

Lys K

Methionine

Met M

Phenylalanine

Phe F

Proline

Pro P

Serina

Be S

Threonine

Thr T

Tryptophan

Trp W

Tyrosine

Tyr Y

Valine

Val V

Amino acids comprise proteins. The amino acids can be hydrophilic, that is, have an affinity for water, or hydrophobic, that is, dislike water. Thus, the amino acids designated by G, A, V, L, I, P, F, Y, W, C and M are hydrophobic and the amino acids designated by S, Q, K, R, H, D, E, N and T are hydrophilic. In general, the hydrophilic or hydrophobic nature of the amino acids affects the folding of a peptide chain and, consequently, the three-dimensional structure of a protein.

DNA is related to protein as follows:

Genomic DNA mRNA protein

image 1

CDNA

Genomic DNA comprises all the DNA sequences found in a cell in the body. It is "transcribed" into messenger RNA ("mRNA"). Complementary DNA ("cDNA") is a complementary copy of mRNA made by reverse transcription of mRNA. Unlike genomic DNA, both mRNA and cDNA contain only the protein coding or DNA polypeptide coding regions, the so-called "exons." Genomic DNA can also include "introns" that do not encode proteins.

In fact, eukaryotic genes are discontinuous with the proteins encoded by them, consisting of exons interrupted by introns. After transcription in RNA, introns are removed by splices to generate mature messenger RNA (mRNA). Splicing points between exons are typically determined by consensus sequences that act as signals for the splicing process. The splicing process consists of a deletion of the intron from the primary RNA transcript and a junction or fusion of the ends of the remaining RNA on each side of the suppressed intron. The presence or absence of introns, the composition of introns and the number of introns per gene can vary between strains of the same species and between species with the same basic functional gene. Although, in most cases introns are assumed to be non-essential and benign, their categorization is not absolute. For example, an intron from one gene can represent an exon from another. In some cases, alternative or different models of splices can generate different proteins of the same single DNA extension. In fact, the structural characteristics of introns and the mechanisms of underlying joints form the basis of the classification of different types of introns.

With respect to exons, these may correspond to discrete domains or motifs such as, for example, functional domains, folding regions or structural elements of a protein, or short polypeptide sequences, such as reverse turns, loops, glycosylation signals and others. signal sequences, or binding regions of unstructured polypeptides. The exon modules of the present combinatorial method may comprise nucleic acid sequences that correspond to natural exon sequences or to natural exon sequences that have been mutated (eg, point mutations, truncations, fusions).

Returning now to DNA manipulation, the DNA can be cut, spliced and otherwise manipulated using "restriction enzymes", which cut the DNA at certain known sites and the DNA ligases that bind to the DNA. Those skilled in the art are well aware of these techniques, as set forth in texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1985) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

DNA of a specific size and sequence can then be inserted into a "replicon", which is any genetic element, such as a plasmid, cosmid or virus that is capable of replication under its own control. A "recombinant vector" or "expression vector" is a replicon into which a segment of DNA is inserted to allow expression of the DNA; that is, the production of the protein encoded by the DNA. Expression vectors can be constructed in the laboratory, obtained from other laboratories or purchased from commercial sources.

The recombinant vector (known by several terms in the art) can be introduced into a host by a process generically known as "transformation." Transformation means the transfer of an exogenous DNA segment by any one of the methods that include infection, direct uptake, transduction, F-pairing, microinjection or electroporation in a host cell.

Unicellular host cells, known in different ways as recombinant host cells, cells and cell culture, include bacteria, yeasts, insect cells, plant cells, mammalian cells and human cells. In particularly preferred embodiments, the host cells include E. coli, Pseudomonas, Bacillus, Streptomyces, Yeast, CHO cells, RI-1, B-W, LH, COS-J, COS-7, BSC1, BSC40, BMT10 and S69. Yeast cells especially include Saccharomyces, Pichia, Candida, Hansenula and Torulopsis.

As is known to those skilled in the art, expression of the DNA segment by the host cell requires suitable regulatory sequences or elements. Regulatory sequences vary according to the host cell employed, but include, for example, in prokaryotes, a promoter, a ribosomal binding site and / or a transcription termination site. In eukaryotes, such regulatory sequences include a promoter and / or a transcription termination site. As is known to those skilled in the art, the expression of the polypeptide can be improved, that is, increased at standard levels, by careful selection and placement of these regulatory sequences.

DNA can be expressed as a polypeptide of any length such as peptides, oligopeptides and proteins. Polypeptides also include translational modifications such as glycosylations, acetylations, phosphorylations and the like.

Another molecular biological technique of interest for the present invention is "genetic linkage analysis". Link analysis is an analytical method used to identify the chromosome or chromosomal region that correlates with a characteristic or disorder47. Chromosomes are the basic units of inheritance on which genes are organized. In addition to genes, specialists have identified "DNA markers" on chromosomes. DNA markers are known sequences of DNA whose identity and sequence can be easily determined. The methodology of analysis of genetic links has been applied to the mapping of disease genes, for example, genes related to asthma sensitivity, to specific chromosomes47,48. Other embodiments of the invention will be apparent to those skilled in the art when considering the specification and practice of the described invention. It is intended that the specification and examples be considered as illustrative only, the claims indicating the true scope of the invention.

Having provided this preliminary information, applicants now describe the preferred aspects of the invention.

Example 1: Identification of transcription polymorphisms of the IL-9 receptor

A population of 52 individuals was randomly determined with respect to asthma and atopy in the region of Philadelphia, Pennsylvania. Total serum IgE was tested by enzyme-linked immunosorbent assay (ELISA, Genzyme, Cambridge, Massachusetts).

To assess the structural forms of the human IL-9 receptor cDNA, PBMCs from these 52 unrelated donors were isolated and cultured in the presence of PHA and PMA (described in Example 4). Previous data from the applicants' laboratory demonstrated the expression kinetics for the IL-9 receptor message in the peak of primary cultures on day 6 after mitogen stimulation. Thus, the applicants cultured the cells for 6 days, at which time the cells were harvested and their RNA and DNA were isolated as described in Example 5.

RNAs were subjected to reverse transcription and PCR amplification using specific full-length cDNA primers of the IL-9 receptor as described in Example 5. The amplification products from each individual were cloned into the TA cloning vector. by PCR, ten clones containing the expected insertions (as determined by digestion and gel electrophoresis) were sequenced in their entirety and analyzed for structural or sequence variation.

From the previous screening, seven main variants were identified. These cDNAs represent a deletion in codon 173, a deletion in exon 4, two separate deletions in exon 5, a deletion in exon 8, and a full-length cDNA containing a change of ARG in HIS in codon 344 of the mature protein. There are additional variants in each of these genetic backgrounds. The Arg allele is associated with the 8 Ser / 4 Asn repetitions and the 7 Ser / 4 Asn repetitions; the His allele is associated with the 9 Ser / 4 Asn repetitions, the 9 Ser / 3 Asn repetitions, and 10 Ser / 2 Asn. All these variants are described in Figure 1.

The variants were cloned into the eukaryotic expression vector pCEP4 (Clontech) containing a CMV promoter that directs the expression of the cloned cDNA followed by an SV40 polyadenylation signal. The vector also contains a hygromycin B resistance gene, which is used for the selection of eukaryotic cells that contain the vector and probably express the cDNA cloned under the control of the CMV promoter. Recombinant plasmids were analyzed sequentially and those plasmids containing correct cDNA insertions were transfected into eukaryotic receptor cells, such as Syrian Hamster TK-ts13 fibroblast, human T98G glioblastoma, TF-1 human myeloid leukemia line, and line 32D myeloid precursor murine cell, as described in Example 3. The function was evaluated biologically in response to the IL9 ligand in growth and / or apoptosis (Example 7 and 10).

Experiments were carried out with the full length cDNAs of the IL-9 receptor containing the ARG or HIS variants are TKts13 hamster fibroblasts or human T98G glioblastoma cells. The cells were transfected and analyzed 48 hours later by Westernblot and staining in situ using specific carboxy-terminal human antibodies (Santa Cruz) (Example 8). In situ analysis showed that both forms of the receptor appeared to be expressed in both hamster and human lines (Figures 11 and 12). It is interesting to note that, while Westernblots of both forms seemed to express themselves at equal levels in both human and hamster lines, a differential migration model appeared between the ARG and HIS receptor forms in the TS1 cell line (Figure 12), which suggests a differential post-translational modification such as glycosylation, phosphorylation, etc. The biochemical difference may be the mechanism by which the altered function results in the altered phenotype.

The frequency of the various substitutions was used as a point estimate of the prevalence of each variant in the general population. The genotype was compared with the phenotype evaluated by questionnaire. A doctor determined a diagnosis of allergy and asthma by reviewing the questionnaires. Homozygous individuals for Arg344 alleles were less likely to show remarkable evidence of allergy and asthma compared to heterozygotes or homozygotes with His344 (Figure 9).

Example 2: Genomic analysis of IL-9 receptor genes

In order to perform genomic analyzes of allergic and / or asthmatic individuals, the following strategy was designed to create specific PCR primers for authentic IL-9 receptor genes located in the X / Y pseudoautosomal regions and to exclude highly conserved pseudogenes from the IL-9 receptor located on chromosomes 9, 10, 16 and 18. First, sequence alignments were preformed between the two published pseudogenes and the genomic sequence of the IL-9 receptor genes. Primers were then initially designed in divergent regions between the authentic genes and the pseudogenes, and then analyzed by PCR using unique chromosome-specific hybrids from the Coriell DNA Deposit

(Camden, NJ). If the primers produce only correctly sized products from the X and Y hybrids, then they are optimized for robust amplification. In several cases, primers targeting divergent regions were not specific to XY; therefore, applicants 5 introduced additional base changes in the particular primer to increase a higher number of mismatches against pseudogenes compared to the sequence of the IL-9 receptor gene. Table 1 contains the sequence of the primers and the optimal alignment temperatures for the specific amplification of XY. The specificity of these primers for the

10 XY amplification is shown in Figure 13.

Table 1 IL-9 Receiver Specific Amplifiers

Exon

Sense primer Antisense primer T Size

2

5’- AGG CTT GAC ATC GGA CAA C -3 ’(SEQ ID NO 9) 68 * 300 bp

3

62 * 222 bp

4

5’- CAA GGC CCT GCT CCA AA 3 ’(SEQ ID NO 13) 64 * 335 bp

5

62 * 259 bp

6

5’-TGAGGTGAACAGGGGAGAA3 ’(SEQIDNO17) 62 * 337 bp

7

5’- ACA AGG GCG GCC TTT GAT -3 ’(SEQ ID NO 19) 60 * 262 bp

8

5 * - CCT GCC CCC CAT GTT CTT -3 ’(SEQ ID NO 21) 58 * 376 bp

9

5’-GGACATGATGCATCTGGCG3 ’(SEO IDNO23) 62 * 664 bp

These primers represent a new method to analyze the variation in the DNA sequences of these genes, and can be used to diagnose sensitivity or resistance to atopic asthma and related disorders.

Using this technology, each exon was examined by analysis of DNA sequences for individuals in populations of applicants to detect variation in the sequences of the IL-944 receptor gene. Sequence polymorphisms were distinguished from the artifact by repeated analyzes. An association of receptor variants with allergic phenotypes is established in Figure 9. The sequence of receptor alleles is established in Figures 1-8.

Example 3: IL-9 receptor expression and ligand binding assay

Purified recombinant IL-9 and compounds potentially similar to IL-9 in structure or function were labeled by fluorescence with high specific activity by commercial techniques in accordance with the supplier's recommendations (Pierce). Human Mo7e and murine 32D cells are cultured and resuspended at 37 ° C in 0.8 ml of Dulbecco's modified Eagle medium completed by 10% (vol / vol) of fetal bovine serum, 50 mM of 2-mercaptoethanol, 0.55 mM of L-arginine, 0.24 mM of L-asparagine and 1.25 mM of L-glutamine or RPMI completed by 10% (vol / vol) of fetal bovine serum, 50 mM of 2-mercaptoethanol, 0.55 mM of L-arginine, 0.24 mM of L-asparagine, and 1.25 mM of L-glutamine, respectively. Cells such as TF1.1 (TF1 cells lacking human IL-9 receptors), T98G, TK-murine 32D, (Example 7 and 10) were used or after transfection with human IL receptor constructs -9 as described below. Plasmid DNA containing one of the full-length or truncated forms of the IL-9 receptors was cloned into plasmid pCEP4 (Clontech) and purified by centrifugation on Qiagen columns (Qiagen). Plasmid DNA (50 micrograms) was added to the cells in 0.4 cm cuvettes just before electroporation. After a double electrical impulse (750 V / 74 ohm / 40 microfarads and 100 V / 74 ohm / 2100 microfarads), the cells are immediately diluted in fresh medium completed with IL-9.

Stable transfected cells were generated after 14 days of selection with hygromycin-B (400 μg / ml to 1.6 mg / ml). Hygromycin-resistant clones were analyzed by Westernblot and staining for IL-9 receptor expression as described in Example 8.

Cellular receptor binding is visualized directly in real time with fluorescence microscopy. Binding and internalization are monitored over time in control cells (not transfected) and with cells transfected with each of the known variants of the IL-9 receptor. An excess of unlabeled ligand or blocking antibody is used in parallel experiments to demonstrate specific binding.

The soluble IL-9 receptor that includes amino acids 44 to 270 with or without a double (ditag) HA tag is also incubated with different forms of labeled recombinant human IL-9. Variable amounts of FLAG labeled ligand (IBI) are incubated in PBS at room temperature for 30 minutes with 0.5 μg of soluble receptor. EBC buffer (300 μl) (50 mM Tris with pH 7.5; 0.1 M NaCl; 0.5% NP40) is added together with 1 μg of anti-HA antibody or anti-FLAG monoclonal antibody (IBI ) and incubate for 1 hour on ice. Forty microliters of a solution of sepharose-protein A is added to each sample and mixed for 1 hour at 4 ° C. The samples are centrifuged for 1 minute 1,000xg and the granules are washed 4 times with 500 µl of EBC. The granules are dissolved in 26 µl of 2 x SDS buffer, boiled for 4 minutes and electrophoresed in a polyacrylamide gel with 18% SDS. Westernblotts are tested with an anti-IL-9 receptor antibody (Santa Cruz Inc.) or an anti-FLAG monoclonal antibody (IBI) against FLAG-labeled rhIL-9. Therapeutic candidates are evaluated by measuring ligand binding antagonism. The expression of the receptor is shown in Figures 11 and 14.

Example 4: Cell isolation and culture

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by density gradient centrifugation using Ficoll-Paque PLUS tested for endotoxin according to the manufacturer (Pharmacia Biotech, AB Uppsala Sweden). PBMC (5x106), mouse spleen cells (5x106), or Mo7e cells (5x106) were cultured in 7 ml RPMI-1640 (Bethesda Research Labs (BRL), Bethesda, MD) completed to one 10% final concentration with isogenic human serum or heat-inactivated FBS. Cells were cultured for 24 hours at 37 ° C without stimulating, or stimulated with 5 μg / ml PMA / 5 μg / ml PHA, or 5 μg / ml PHA and 50 ng / ml rhlL2 (R&D Systems, Minneapolis, MN).

Example 5: Isolation of DNA & RNA, rtPCR, cloning and sequencing of PCR products

Cytoplasmic RNA and genomic DNA were extracted after 6 days of stimulation with mitogens from cultured PBMCs, as described in Nicolaides and Stoeckert46. One μg of RNA from each source was denatured for more than 10 minutes at 70 ° C and then subjected to reverse transcription (V +) in cDNA using 2.5 units of Superscript II reverse transcriptase (GIBCO, BRL), 1 U / l of RNase inhibitor, 2.5 mM oligo d (T) 16 primer, 1mM each of dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCl, pH 7.0, 25 mM of MgCl2 at 37 ° C for one hour. A simulation reverse transcription reaction was used as a negative control.

The twentieth part of the rt reaction was used in the PCR (50 μl) containing 6.7 mM MgCl2, 16.6 mM (NH4) 2SO4, 67 mM Tris-HCl, pH 8.8, 10 mM 2-mercaptoethanol, 6% DMSO, 1.25 mM each of dNTP, 2.5 U of Amplitaq DNA polymerase, and 300 ng of each of the oligonucleotides representing exon 2 of human IL-9 cDNA ( forward 5'-GCT GGA CCT TGG AGA GTG-3 ') (SEQ ID NO: 1) and exon 9 (reverse 5'-GTC TCA GAC AAG GGC TCC AG-3') (SEQ ID NO: 2). The reaction mixture was subjected to the following PCR conditions: 120 seconds at 95 ° C, then 35 cycles at: 30 seconds at 94 ° C, 90 seconds at 58 ° C; 90 seconds at 72 ° C. Finally, the reaction mixture was cycled once for 15 minutes at 72 ° C for extension.

PCR products representing hIL-9 receptor cDNA were subjected to gel electrophoresis with 1.5% agarose gels and visualized using ethidium bromide staining. The products of a simile rhinase transcriptase reaction, in which H2O was replaced by RNA, were used as amplification of the negative control in all experiments.

The PCR products were subcloned into the TA cloning vector (Invitrogen, San Diego, CA). Amplification of the human cDNA gave a product of 1614 bp. Plasmids containing hIL-9 receptor cDNA inserts were isolated by conventional techniques (Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York). After amplification, the DNA sequence that included and surrounded each insert was analyzed by sequencing (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463) using dideoxy fluorescent terminators in an automatic sequencer (ABI 377, Perkin Elmer) for the determination of errors induced by PCR or induced by cloning. The hIL-9 receptor cDNA inserts without cloning and / or errors in polymerase-induced sequences were subcloned into expression vectors.

Example 6: Cloning and expression of the IL-9 receptor constructs

in vitro

The hIL-9 receptors were subcloned into the episomal eukaryotic expression vector pCEP4 (Clontech). The inserts were cloned as BamH1-Xhol fragments in the polylinker of pCEP4 in the sense of orientation to the CMV promoter using standard techniques (Figure 10). The constructs were expressed in host cells as described.

Example 7: Cellular assays using Mo7e, 32D, TF1.1, TK-ts13 and T98G

Cell lines were used to evaluate the function of variable IL-9 receptors and for the selection of potentially useful compounds in the treatment of atopic asthma. Compounds were tested for their ability to antagonize the antiapoptotic or proliferative response of the baseline caused by IL-9. Once a proliferative or antiapoptotic response of the baseline was established in a certain cell line, a statistically significant loss of response in repeated tests three times in triplicate was considered as evidence of antagonism. A real antagonistic response was differentiated from cellular toxicity by direct observation, trypan blue staining (technique well known to a person skilled in the art), or by loss of acid phosphatase activity. The specificity of the antagonism is evaluated for each compound by evaluating whether activity is demonstrated against other proliferative agents, such as interleukin-3 or interleukin-4.

The recombinant IL-9 and potential compounds similar to IL-9 in structure or function were purified and prepared for use in appropriate media. Putative agonists and antagonists were prepared in water, in saline solution or in DMSO and water. Cells were used as they were or after transfection with the IL-9 receptor constructs as described in Example 3. After 24 hours of deprivation of growth factors, cells were incubated without (control) or with varying amounts of purified IL-9 and potential compounds analogous to IL-9 in structure or function.

Cell proliferation was tested by the Abacus Cell Proliferation Kit (Clontech, Palo Alto, CA) which determines the amount of intracellular acid phosphatase present as an indication of cell number. The p-nitrophenyl phosphate substrate (pNPP) was converted, by acid phosphatase, into p-nitrophenol, which was measured as an indicator of enzymatic concentration. PNPP was added to each well and incubated at 37 ° C for one hour. Then 1N sodium hydroxide was added to interrupt the enzymatic reaction and the amount of p-nitrophenol was quantified using a Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, VA) at a wavelength of 410 nm. Standard curves comparing cell number with optical absorbance were used to determine the linear range of the assay. Test results were used only when absorbance measurements were in the linear range of the test. Briefly, the tests were performed with quadruplicate samples of cells in flat-bottom microtiter plates (150 or 200 microliter wells) with or without ligand for 72 to 96 hours at 37 ° C. Acid phosphatase was used as a measure of the number of cells present. All experiments were repeated at least twice.

Apoptosis was tested using the Annexin V kit as indicated by the supplier (Clontech), which determines dexamethasone-induced apoptosis by recognizing extracellular phosphatidylserine, an early marker of apoptosis. The apoptotic cell number was obtained by fluorescence microscopy as a percentage of cells stained with Annexin

V.

The Mo7e line is a megakaryoblastic human cell line, cultured in RPMI 1640 (GIBCO / BRL, Gaithersburg, MD), 20% Bovine Fetal Serum (Hyclone) and 10 ng / ml of IL-3 (R&D Systems, Minneapolis, MN) . The T98G line is a human glioblastoma cell line cultured in RPMI 1640 (GIBCO / BRL). The TK-ts13 hamster fibroblast line was also used as well as the 32D murine cell line, a myeloid murine precursor line, and both were cultured in RPMI 1640 / GIBCO / BRL) containing 10% fetal calf serum (Hyclone); In addition, 1 ng / ml of IL-3 was used with 32D cell lines. TF1.1 is a line of human myeloid leukemia known to express the gamma subunit of the IL-2 receptor (confirmed by Westernblot and rtPCR), but, in comparison to its predecessor (TF1), it no longer supports the IL-receptor 9 by rtPCR, immunostaining and Westernblot analysis. TF1.1 is grown in RPMI 1640 (GIBCO / BRL) and 10% fetal bovine serum (Hyclone). All cell lines respond to multiple cytokines, including IL-9. Cell lines were fed and re-seeded at 2 x 105 cells / ml every 72 hours.

The cells were centrifuged for 10 minutes at 2000 rpm and resuspended in RPMI 1640 with 0.5% bovine serum albumin (GIBCO / BRL, Gaithersburg, MD) and insulin-transferrin-selenium (ITS) cofactors (GIBCO / BRL, Gaithersburg, MD). The cells were counted using a hemocytometer, diluted to a concentration of 1 x 105 cells / ml and placed in 96-well microtiter plates. Each well contained 0.15 or 0.2 ml, giving a final concentration of 10 to 50 thousand cells per well, depending on the cell. Mo7e cells were stimulated with 50 ng / ml of Mother Cell Factor (SCF) (R&D Systems, Minneapolis, MN) alone, 50 ng / ml of SCF plus 50 ng / ml of IL-3 (R&D Systems, Minneapolis, MN ), or 50 ng / ml of SCF plus 50 ng / ml of IL-9. A control containing cells and basal media only was included. Serial dilutions of the test compounds (ie, recombinant IL-9 proteins, peptides, small molecules) were added to each test condition in triplicate. TF1.1 cells that were not transfected with IL-9 receptors were used as independent control for non-specific response and cytotoxicity. Cultures were incubated for 72-96 hours at 37 ° C in 5% CO2.

Example 8: In situ and Western analysis of the exogenous IL-9 receptor in transfected cell lines

In situ staining of the IL-9 receptor was carried out as follows. Cells were grown on coverslips for 24 hours and then coverslips containing adherent cells were washed twice with a phosphate buffered saline solution containing calcium and magnesium (PBS) (GIBCO / BRL). For intracellular staining of the IL-9 receptor, cells were fixed in 4% PBS / paraformaldehyde plus 0.1% triton-X for 15 minutes at room temperature before treatment with the IL-receptor anti-human antibody 9; for extracellular staining, the cells were treated with the antibody before fixation. Then, the cells were washed twice in PBS and blocked with 7.5% BSA in dH2O for 30 minutes at room temperature. The cells washed in PBS were then incubated with a 10 μg / ml solution of the IL-9 anti-human receptor (polyclonal antibody directed against the carboxy-terminal of the IL-9 receptor) in 1% PBS / BSA for 1 hour at room temperature. The cells were washed three times in PBS and then incubated in a solution of 10 μg / ml of a rhodamine-conjugated anti-rabbit antibody in PBS / 1% BSA for 30 minutes at room temperature. The cells were then washed three times in PBS and counterstained using 1 μg / ml DAPI for 1 minute at room temperature. The cells were washed three times in H2Od, fixed on a microscope slide and analyzed by fluorescence microscopy. The results of COS7 transfected cells are shown in Figure 14.

Western blots were performed on protein lysates obtained from direct lysis of cell extracts in a 0.5% lysis buffer ((50 mM Tris, 150 mM NaCl, NP40), 1 mM DTT and protease inhibitors) and boiled for 5 minutes The samples were electrophoresed in SDS gels with 4-20% Tris-glycine (Novex) in a Tris-glycine pass buffer. The proteins were then transferred to nitrocellulose by electroblot using the Trans Blot II (Bio Rad) apparatus. After transfer, the membrane was blocked in TBS-T ((20 mM Tris, 137 mM NaCl, pH 7.6) plus 0.05% Tween 20) plus 5% blotto for 1 hour at temperature ambient. The blots were then probed using a polyclonal antibody directed against the carboxy-terminal of the IL-9 receptor (1 μg / ml) in TBS-T for 1 hour. The blots were then washed three times in TBS-T for 10 minutes and probed using a secondary antibody conjugated to anti-rabbit horseradish peroxidase (1: 10,000) in TBS-T for 30 minutes. The blots were washed as mentioned above and then incubated with a solution boosted by Luminol (Pierce), a chemiluminescent substrate, for 5 minutes at room temperature, and then exposed to a film for 1-60 seconds. See Figures 11 and 12.

Example 9: Methods for Genomic Amplification of Authentic IL-9R

Specific amplification of the authentic IL-9R (gene coding for the biologically functional protein located in the pseudoautosomal region XYq) by means of the design of standard primers was not possible because IL-9R has four highly homologous pseudogenes (> 90% nucleotide identity), not processed in other loci of the human genome (chromosome 9, 10, 16, 18). Due to the high identity of these other genes, the genomic amplification by PCR by means of the design of standard primers resulted in the co-amplification of all the genes, thus converting the analysis of the sequences of the authentic gene into an equivocation. In order to study the authentic structure of IL-9R, as it may be related to the predisposition to diseases such as asthma, as stated in this application, or other diseases such as cancer (Renauld et al., Oncogene, 9: 1327-1332, 1994; Gruss et al., Cancer Res., 52: 1026-1031, 1992), specific amplimers were designed as follows:

The pseudogen sequences of IL-9R and authentic genes were aligned using Mac Vector software. The intronic sequences surrounding each exon were then examined in the regions of diversity between the authentic gene and the pseudogenes. Primers against these regions were then designed and used to amplify by PCR the hybrid human / rodent DNAs containing individual human chromosomes. The products were made in 3% agarose gels and analyzed for authentic IL-9R amplification without amplification of the 4 pseudogenes. The specific PCR amplification conditions were also optimized by varying the alignment temperature as well as the buffer conditions (5-10% DMSO content). In cases where pseudogenes amplification continued to occur, nucleotide changes were introduced in the primer sequences to cause greater divergence of the pseudogenes compared to the authentic gene. The primer sequences are shown in Example 2 and their specificity is demonstrated in Figure 13.

Example 10: Cell Proliferation and Cytokine Stimulation Assay

To determine the growth response of TS1 cells expressing various forms of the human IL-9 receptor, the cells were washed with PBS and resuspended in D-MEM, 10% fetal bovine serum. 103 cells were seeded per well, in triplicate, in 96-well microplates and, where appropriate, recombinant human IL-9 or murine IL-9 (R&D Systems, Minneapolis, MN) was added at a final concentration of 5 ng / ml. Cell proliferation was evaluated at 7 days using an acid phosphatase assay. Briefly, 50 μl of a buffer containing 0.1M sodium acetate (pH 5.5), 0.1% Triton X-100 and 10 mM p-nitrophenyl phosphate (Sigma 104 phosphatase substrate) was added to each well . The plate was incubated for 1-1 / 2 hours at room temperature, the reaction was stopped with 10 µl / well of 1N sodium hydroxide and the absorbance was read on a Dynatech Model MR600 at 410 nm. To analyze tyrosine phosphorylation in signal transduction cascade proteins with cytokine stimulation, TS1 cells expressing various forms of the human IL-9 receptor were washed with PBS, resuspended in D-MEM, serum albumin 0.5% bovine and incubated for 6 hours at 37 ° C. Successively, 20 x 106 cells were treated for 5 minutes with human IL-9 or murine IL-9 (100 ng / ml) and washed immediately with cold PBS. The cells were lysed in RIPA buffer as described in Example 11.

Example 11: Immunoprecipitations, immunoblotting and antibodies

Typically, 20-50 x 106 cells were lysed in 1 ml of RIPA buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 50 mM sodium fluoride, 1 nM sodium orthovanadate and 1 x "complete" mixture of protease inhibitors, Cat. No. 1697498 Boehringer Mannheim) and incubated for 45 minutes on ice. The lysates were centrifuged for 20 minutes in an Eppendorf microcentrifuge and the supernatant was recovered, which was transferred to a fresh tube. For immunoprecipitation, 1-5 μg of antibody was added to the lysate and incubated overnight at 4 ° C. 20 ml of pearls conjugated with A + G protein agarose were added for 2 hours, followed by four washes using the RIPA buffer. The beads were resuspended in Laemmli buffer and boiled for 3 minutes before electrophoresis. The proteins were transferred to an Immobilion-P membrane (Millipore) and detected by means of a secondary antibody conjugated to horseradish peroxidase, followed by a chemiluminescence detection assay (Pierce). Antibodies specific for the murine and human IL-9 receptor (sc698), Jak1, Irs1, Irs2, Stat1, Stat2, Stat3, Stat4, Stat5 and phosphotyrosine (PY) were purchased from Santa Cruz (Santa Cruz, CA). The anti-Jak3 and the IL-9 monoclonal anti-human receptor, MAB290, were purchased from Upstate Biotechnology and R&D Systems, respectively. Figure 15 demonstrates the activation of members of the Jak family through different variants of the human IL-9 receptor.

Example 12: Identification of the genomic polymorphisms of the IL-9 receptor

Genomic DNAs from PBMCs from voluntary donors were isolated as described in Nicolaides and Stoeckert, Bio-techniques 8: 154-156, 1990. Sequence analysis of intron 5 of the human IL-9R gene was performed by PCR using primers of sequence ID NO: 14 and sequence ID NO: 17, which resulted in a product with an approximate molecular size of 1243 base pairs. Amplifications were carried out at 94 ° C for 30 seconds, 62 ° C for 1.5 minutes, 72 ° C for 1.5 minutes for 35 cycles in the buffers described above (Nicholaides et al., Genomics 30: 195-206, 1995). The products were then purified and sequenced by means of a standard protocol for sequencing. The inspection of

5 sequences from intron 5 in multiple individuals discovered a nucleotide change in -213nt upstream of exon 6 sequences, which resulted in a nucleotide change of a thymidine (published sequence) in a cytosine. An example of this change is shown in Figure 17.

Although the invention has been described and illustrated herein with reference to various specific materials, procedures and examples, it is understood that the invention is not limited to the particular combinations of materials and procedures selected for this purpose. Numerous variations of these details may be implicit, as specialists will appreciate.

15 in the art.

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SEQUENCE LIST

(one)

GENERAL INFORMATION

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APPLICANT: MAGAININ, PHARMACEUTICALS INC.

(ii)

TITLE OF THE INVENTION: IL-9 Receptor Variants, Useful in the Treatment and Diagnosis of Atopic Allergies that Include Asthma and Related Disorders

(iii) NUMBER OF SEQUENCES: 33

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LEGIBLE FORM BY COMPUTER:

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 TYPE OF MEDIA: Flexible disk

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 COMPUTER: IBM PC compatible

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 OPERATING SYSTEM: PC-DOS / MS-DOS

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 SOFTWARE: Patentln Program # 1.0, Version # 1.30

(v)

DATA OF THIS APPLICATION:

(TO)

 APPLICATION NUMBER: PCT / US97 / 21992

(B)

 DATE OF SUBMISSION: DECEMBER 02, 1997

(C)

 CLASSIFICATION:

(saw)

INFORMATION PRIOR TO THE APPLICATION:

(TO)

 APPLICATION NUMBER: US 60 / 032.224

(B)

 SUBMISSION DATE: DECEMBER 02, 1996

(vii) INFORMATION PRIOR TO THE APPLICATION:

(TO)

 APPLICATION NUMBER: US 08 / 980,872

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 DATE OF PRESENTATION: DECEMBER 01, 1997

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(A) LENGTH: 513 base pairs

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(A) LENGTH: 17 base pairs

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GCAGGTGGGG ACCCATG

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AGGCTTGACA TCGGACAAC 19

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CTGGCCTGAA GTACTTACC 19

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CTGCTTCAAT CCTGGGGAA 19

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GTGAGTTCCC CAGGATTGA 19

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 LENGTH: 17 base pairs

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CAAGGCCCTG CTCCAAA 17

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 LENGTH: 20 base pairs

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TGGGGCTTCA GCCTCACATG 20

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 LENGTH: 20 base pairs

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TATGTAGAGT GGGGAGTCTA 20

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 LENGTH: 19 base pairs

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TGTATTCTCG AGGGCTGAG 19

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 LENGTH: 19 base pairs

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SEQUENCE DESCRIPTION: SEQ ID NO: 17

TGAGGTGAAC AGGGGAGAA 19

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INFORMATION FOR SEQ ID NO: 18

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 LENGTH: 17 base pairs

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SEQUENCE DESCRIPTION: SEQ ID NO: 18

CCCTGGGCCC TTCATGT 17

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INFORMATION FOR SEQ ID NO: 19

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 LENGTH: 18 base pairs

(B)

 TYPE: nucleic acid

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(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 19

ACAAGGGCGG CCTTTGAT 18

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INFORMATION FOR SEQ ID NO: 20

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CHARACTERISTICS OF THE SEQUENCE:

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 LENGTH: 19 base pairs

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 TYPE: nucleic acid

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SEQUENCE DESCRIPTION: SEQ ID NO: 20

AGGGACGAGG TGGGCGGAC 19

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INFORMATION FOR SEQ ID NO: 21

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 LENGTH: 18 base pairs

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SEQUENCE DESCRIPTION: SEQ ID NO: 21

CCTGCCCCCC ATGTTCTT 18

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 LENGTH: 19 base pairs

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SEQUENCE DESCRIPTION: SEQ ID NO: 22

ATGCTACCTG AGCCCTTCC 19

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INFORMATION FOR SEQ ID NO: 23

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 LENGTH: 19 base pairs

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 TYPE: nucleic acid

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SEQUENCE DESCRIPTION: SEQ ID NO: 23

GGACATGATG CATCTGGCG 19

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 LENGTH: 1944 base pairs

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SEQUENCE DESCRIPTION: SEQ ID NO: 24

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INFORMATION FOR SEQ ID NO: 25

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CHARACTERISTICS OF THE SEQUENCE:

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 LENGTH: 18 base pairs

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SEQUENCE DESCRIPTION: SEQ ID NO: 25

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GCTGGACCTT GGAGAGTG 18

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INFORMATION FOR SEQ ID NO: 26

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CHARACTERISTICS OF THE SEQUENCE:

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 LENGTH: 20 base pairs

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 TYPE: nucleic acid

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SEQUENCE DESCRIPTION: SEQ ID NO: 26

GTCTCAGACA AGGGCTCCAG 20

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INFORMATION FOR SEQ ID NO: 27

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CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 501 amino acids

(B)

 TYPE: amino acid

(C)

 STRANDEDNESS: unique

(D)

 TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 27

(ii)

TYPE OF MOLECULE: Peptide

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 28

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 29

image 1

image 1

(2)

INFORMATION FOR SEQ ID NO: 28

5

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 501 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unique

(D) TOPOLOGY: linear

image2

image 1

5

(2) INFORMATION FOR SEQ ID NO: 29

(i)

CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 500 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unique

10

(D) TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

image 1

image 1

(2)

INFORMATION FOR SEQ ID NO: 30

(i)

CHARACTERISTICS OF THE SEQUENCE:

5

(A) LENGTH: 286 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unique

(D) TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

10

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 30

image 1

(2)

INFORMATION FOR SEQ ID NO: 31

5

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 64 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unique

(D) TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 31

image2

(2)

INFORMATION FOR SEQ ID NO: 32

(i)

CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 128 amino acids

10

(B) TYPE: amino acid

(C) STRANDEDNESS: unique

(D) TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

fifteen

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32

image 1

(2)

INFORMATION FOR SEQ ID NO: 33

(i)

CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 150 amino acids

(B) TYPE: amino acid

5

(C) STRANDEDNESS: unique

(D) TOPOLOGY: linear

(ii)

TYPE OF MOLECULE: Peptide

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO: 33

10

image 1

Claims (33)

one.

Isolated nucleic acid encoding the human interleukin-9 (IL-9) receptor selected from the group consisting of isolated nucleic acid encoding the human interleukin-9 (IL-9) receptor characterized in that the nucleotides corresponding to the positions 613 to 641 of the wild-type IL-9 receptor (SEQ ID NO: 1) have been suppressed, isolated nucleic acid encoding the human interleukin-9 (IL-9) receptor characterized in that the nucleotides corresponding to the positions 613 to 617 of the IL-9 wild type receptor (SEQ ID NO: 1) have been deleted, fragments thereof containing the deletion and complements thereof containing the deletion.

2.

Isolated nucleic acid encoding the human interleukin-9 (IL-9) receptor according to claim 1, characterized in that the nucleotides corresponding to positions 613 to 641 of the wild-type IL-9 receptor (SEQ ID NO: 1) They have been removed.

3.

Isolated nucleic acid according to claim 1, characterized in that the fragment containing the deletion encodes a protein or peptide that binds to IL-9.

Four.

Isolated nucleic acid according to claim 1, characterized in that the fragment containing the deletion encodes the amino acid sequence of SEQ ID NO: 33.

5.

Isolated nucleic acid according to claim 1, characterized in that the nucleotide sequence comprises SEQ ID NO: 7.

6.

Isolated nucleic acid according to claim 1, characterized in that the nucleotide sequence consists of SEQ ID NO: 7.

7.

Isolated nucleic acid according to claim 1, characterized in that the nucleotide sequence comprises SEQ ID NO: 6.

8.

Isolated nucleic acid according to claim 1, characterized in that the nucleotide sequence consists of SEQ ID NO: 6.

9.

Isolated nucleic acid according to claim 1, characterized in that the nucleic acid is DNA or RNA.

10.

Isolated nucleic acid according to claim 9, characterized in that the DNA is cDNA.

eleven.

Isolated nucleic acid according to claim 9, characterized in that the RNA is mRNA.

12. A vector comprising the nucleic acid of any one of claims 1 to 11. 13. Vector according to claim 12 comprising a plasmid, a cosmid or a virus. 14. Vector according to claim 12 which is a recombinant or expression vector.

fifteen.

Vector according to claim 12 further comprising a regulatory sequence.

16.

Vector according to claim 15, characterized in that the sequence

Regulatory is a promoter, a ribosomal binding site or a transcription termination site. 17. Vector according to claim 16, characterized in that the promoter is selected from the group consisting of the human cytomegalovirus promoter, tetracycline inducible promoter, simian virus promoter, long terminal repeats promoter of the virus 20 Moloney murine leukemia, glucocorticoid-inducible murine mammary tumor virus promoter, herpes thymidine kinase promoter, human and murine actin promoters, 5 'IL-9 flanking region of HTLV-1 and HIV, flanking region 5 'human or mouse IL-9 receptor, tac bacterial promoter and enhancer elements 25 of the junction region of scaffolding proteins associated with thermal shock in Drosofila (SAR).

18.

Host cell transformed by the vector of any one of claims 12 to 17.

19.

Host cell according to claim 18 which is transformed by

30 infection, direct uptake, transduction, F mating, microinjection or electroporation.

twenty.

Host cell according to claim 18 which is selected from the group consisting of bacterial cells, yeast cells, insect cells, plant cells and mammalian cells.

twenty-one.

Host cell according to claim 20 which is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, CHO cells, RI-1, BW, LH, COS-J, COS-7, BSC1, BSC40, BMT10 or S69

22

Host cell according to claim 20, characterized in that the yeast cells are selected from the group consisting of Saccharomyces, Pichia, Candida, Hansenula or Torulopsis.

2. 3.

Isolated human IL-9 receptor protein encoded by the nucleic acid of claim 1.

24.

Isolated human protein according to claim 23 comprising the amino acid sequence SEQ ID NO: 33.

25.

Isolated human protein according to claim 23 consisting of the amino acid sequence SEQ ID NO: 33.

26.

Isolated human protein according to any one of claims 23 to 25 characterized in that the protein binds to IL-9.

27.

Protein according to claim 26, characterized in that the protein is soluble.

28.

Method for making a protein comprising the culture of a host cell transformed with the nucleic acid according to any one of claims 1 to 11 under conditions in which the protein encoded by said nucleic acid is expressed.

29.

Pharmaceutical composition comprising the isolated nucleic acid according to any one of claims 1 to 11.

30

Pharmaceutical composition comprising the protein according to any of claims 23 to 27.

31.

Composition according to claims 29 or 30, characterized in that the composition further comprises a pharmaceutically acceptable carrier.

32

Composition according to claims 29 or 30, characterized in that the composition is formulated for topical administration.

33.

Composition according to claims 29 or 30, characterized in that the composition is formulated for inhalation.

Composition according to claims 29 or 30, characterized in that the composition is formulated for systemic administration. 35. Composition according to claims 29 or 30, characterized in that the composition is formulated for intravenous, intraperitoneal administration or intralesional.