PT1471144E - 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

BIOLOGICAL FACTOR VARIABILITY ASSOCIATED TO ASMA AAF2 (IL-9 RECEPTOR) USEFUL IN TREATMENT AND DIAGNOSIS OF ATOPICAL ALLERGIES INCLUDING ASTHMA AND RELATED DISTURBANCES "

FIELD OF THE INVENTION

This invention describes biological variability at the IL-9 receptor (Asthma Associated Factor 2) (SEQ ID NO: 1) and relates these sequence variants to susceptibility to asthma, atopic allergy and related disorders. Additionally, methods are described which utilize variants of IL-9 receptors in the development of asthma medications that depend on the regulation of IL-9 activity.

BACKGROUND OF THE INVENTION Inflammation is a complex process in which the body's defense system fights strange entities. Although the battle against strange entities may be necessary for the survival of the body, some defense systems respond improperly to strange entities, even harmless ones, as dangerous and thereby damage the surrounding tissue in the ensuing battle. Atopic allergy is a disorder where the genetic background dictates the response to environmental stimuli. The disorder is generally characterized by an increased ability of the lymphocytes to produce IgE antibodies in response to ubiquitous antigens. Activation of the immune system by these antigens also leads to allergic inflammation that may occur after ingestion, penetration through the skin or after inhalation. When this immune activation occurs and pulmonary inflammation follows, this disorder is generally characterized as asthma. Certain cells are important in this inflammatory reaction in the respiratory tracts and they include T cells and antigen presenting cells, IgE producing B cells, mast cells / basophils that store inflammatory mediators and bind to IgE, and eosinophils which release additional mediators. These inflammatory cells accumulate at the site of allergic inflammation and the toxic products they release contribute to tissue destruction related to the disorder.

Although asthma is generally defined as an inflammatory airway disorder, clinical symptoms arise from intermittent airflow obstruction. It is a chronic, disabling disorder that appears to be increasing in prevalence 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 Thus, a huge burden is placed on the present resources of health care in the treatment of these disorders. The diagnosis and treatment of asthma and related disorders are problematic.1 In particular, assessment of inflamed lung tissue is often difficult and often the cause of inflammation can not be determined. Although atopic asthma is an echogenic disorder, knowledge about particular only 2 variant genes has recently been identified. Methods for detecting these genetic variations and their role in inflammation, diagnosis and prognosis remain to be determined. What is required in the art is the development of technology to accelerate the diagnosis of atopic asthma which relates specifically to the variation in genes responsible for susceptibility / resistance to this atopic disease.

Current treatments suffer from their own set of disadvantages. The major therapeutic agents, β-agonists, reduce symptoms, i. e., transiently improve lung functions, but do not affect the underlying inflammation, so that lung tissue remains in danger. In addition, the constant use of β-agonists results in desensitization, which reduces their efficacy and safety.2 Agents that may decrease the underlying inflammation, anti-inflammatory steroids, have their own known list of side effects ranging from immunosuppression until bone loss.2

Because of the problems associated with conventional therapies, alternative treatment strategies were evaluated. 38-39 Glycophorin A, 37 cyclosporin 38 and a non-epitope fragment of IL-2.36 all inhibit interleukin-2-dependent T lymphocyte proliferation. It is known, however, that they have many other effects. For example, cyclosporin is used as an immunosuppressant after organ transplantation. Although these agents may represent alternatives to steroids in the treatment of asthmatics, 36-39 inhibit interleukin-2 dependent T lymphocyte proliferation and potentially critical immune functions associated with homeostasis. What is necessary in the art is technology to accelerate the development of therapy that is specifically designed to treat the cause and not the symptoms of atopic asthma. These therapies represent the most likely way to avoid toxicity associated with non-specific treatment. The therapies would selectively target a pathway that is downstream of immune functions, such as IL-2 mediated T lymphocyte activation that is necessary for the development of asthma and would explain the episodic nature of the disorder and its close association with allergy . Nature demonstrates that a pathway is the appropriate target for asthma therapy when there is normally biological variability in the pathway and individuals demonstrating the variability are not immunocompromised or diseased, with the exception of 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 under intense study. Although these disorders are heterogeneous and may be difficult to define, because they can take many forms, certain characteristics are common among asthmatics. Examples of such characteristics include abnormal skin test response to allergen challenge, lung eosinophilia, bronchial hyperresponsiveness (BHR), bronchodilator reversibility, and airflow obstruction.3-10 These expressions of asthma-related traits can be studied by quantitative or qualitative measures.

In many cases, elevated levels of IgE are correlated with BHR, an enhanced bronchoconstricting response to a variety of stimuli.4'6'8.9 BHR is thought to reflect the presence of airway inflammation, 6,8 and is considered a 4 risk factor for asthma.11 12 BHR is accompanied by bronchial inflammation, including infiltration of eosinophils in the lung and an allergic diathesis in asthmatic individuals.6'8'13-18

A number of studies document an inherited component for atopic asthma.4'10 Family studies, however, have been difficult to interpret since these disorders are significantly influenced by age and sex, as well as many environmental factors , such as allergens, viral infections and pollutants. Furthermore, because there is no known biochemical defect associated with susceptibility to these disorders, mutant genes and their abnormal gene 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 far beyond those originally recognized. Although the IL-9 pathway serves as a T cell growth stimulator, this cytokine is also known to mediate the growth of erythroid progenitors, B cells, mast cells, and fetal thymocytes. 22,23 The IL-9 pathway acts synergistically with IL-3, causing the activation and proliferation of mast cells.24 The IL-9 pathway also potentiates the IL-4 induced production of IgE, IgG and IgM by B lymphocytes normal human IgM and IL-4 induced release of IgE and IgG by murine B lymphocytes.26 A role for the IL-9 pathway in inflammatory mucosal response to parasitic infection has also been demonstrated.27,28 However, it is not known how the IL-9 receptor sequence correlates specifically with atopic asthma and bronchial hyperresponsiveness. IL-9 is known to bind to a specific receptor, expressed on the surface of target cells. 23,29'30 In fact, the receptor consists of two protein chains: a protein chain, known as the IL-9 receptor, binds specifically to IL-9; the other protein chain is shared in common with the IL-2 receptor. In addition, a cDNA encoding the human IL-9 receptor has been cloned and sequenced. This cDNA encodes a 522 amino acid protein that exhibits significant homology to the murine IL-9 receptor. The extracellular region of the receptor is highly conserved, with 67% homology existing between the murine and human proteins. The cytoplasmic region of the receptor is less highly conserved. The human cytoplasmic domain is much larger than the corresponding region of the murine receptor.23 The IL-9 receptor gene was also characterized.30 It is believed to exist as a single copy in the mouse genome and is composed of nine exons and eight introes. The human genome contains at least four pseudogenes of IL-9 receptor. The human IL-9 receptor gene was assigned to the 320 kb subtelomeric region of sex chromosomes X and Y.23

Despite these studies, no variants of the IL-9 receptor gene were identified. There is, therefore, a specific need for genetic information on atopic allergy, asthma, bronchial hyperresponsiveness and for elucidating the role of the IL-9 receptor in the etiology of these disorders. This information can be used to diagnose atopic allergy and related disorders using methods that identify genetic variants of this gene that are associated with these disorders. In addition, there is a need for methods using the IL-9 receptor variants in order to develop therapy to treat these disorders. 6

SUMMARY OF THE INVENTION We have identified natural variants of the human IL-9 receptor (also known as Factor Associated with Asthma 2 or AAF2) and have linked these variants to the pathogenesis of asthma and related disorders. These findings have led to the development of diagnostic methods and methods for identifying medicaments for the treatment of atopic asthma therapy. Additionally, we have determined that the IL-9 receptor is critical for a number of antigen-induced responses in mice, including bronchial hyperresponsiveness, eosinophilia, and high cell counts in bronchial lavage and elevated total serum IgE. These findings typify atopic asthma and associated allergic inflammation.

In addition, we have determined that a G-to-A nucleic acid variant occurs at position 1273 of the cDNA (SEQ ID NO: 2), which produces the predicted amino acid substitution of a histidine by an arginine at codon 344 of the precursor protein of the human IL-9 receptor. When the arginine residue occurs in both alleles in an individual, it is associated with less evidence of atopic asthma. Thus, we have identified the existence of a non-asthmatic phenotype, characterized by arginine at codon 344, when it occurs in both IL-9 receptor gene products in a subject. As a further significant corollary, the Applicant has identified the existence of susceptibility to an atopic asthmatic phenotype characterized by a histidine at codon 344. The Applicant has also determined that there is an excision variant of IL-9R, wherein the glutamine residue at position 173 of IL-9R precursor protein was deleted (SEQ ID NO: 3) (Figure 5). The candidate has further demonstrated that this variant is not capable of transcribing a signal via the Jak-Stat pathway (Figure 15) and is not able to induce cell proliferation after IL-9 stimulation (Figure 16); therefore, individuals with this allele would be less susceptible to atopic asthma and related disorders. We further determined that there is a variant of IL-9R genomic DNA, where nt 213, a thymine residue in intron 5 (213 nt upstream of exon 6), has been converted to a cytosine nucleotide. It is likely that such a variation may cause an increase in the frequency of the excision variant that removes the glutamine residue at the start of exon 6.

In addition, we have identified an IL-9R variant, in which exon 8 has been deleted (SEQ ID NO: 4), which results in a change in the reading phase and a premature termination codon in exon 9. Such a variant would be , most likely prevented from transmitting a signal via the Jak-Stat pathway, and therefore individuals with this allele would also be less susceptible to atopic asthma and related disorders. The biological activity of IL-9 results from its binding to the IL-9 receptor and the consequent propagation of a regulatory signal in specific cells; therefore, IL-9 functions can be disrupted by the interaction of IL-9 antagonists with IL-9 or its receptor. The negative regulation, i. i.e., the reduction of the functions controlled by IL-9, is achieved in several ways. Administration of antagonists that may disrupt IL-9 binding to its receptor is a key mechanism and such antagonists are within the claimed invention. Examples include administration of polypeptide products encoded by DNA sequences in a naturally occurring soluble form of the IL-9 receptor, wherein the DNA sequences encode a polypeptide comprising exons 2 and 3 (SEQ ID NO: 5). Two other variations may produce soluble IL-9R receptor forms comprising exons 2, 3 and 4 and, in one case, four amino acids from a different reading phase in exon 5 (SEQ ID NO: 6) (Figure 6) and In another case, there are 27 amino acids from a different reading phase in exon 5 (SEQ ID NO: 7) (Figure 7).

Methods for identifying agonists and antagonists of the IL-9 receptor pathway can be identified by evaluating receptor-ligand interactions, which are well described in the literature. These methods can be adapted to automated high-throughput assays that facilitate chemical screening and potential therapeutic identification. Agonists are recognized by identifying a specific interaction with the IL-9 receptor. Loss of binding to a putative ligand that is labeled when a 100 to 1000 fold excess of unlabeled ligand is used is generally accepted as evidence of receptor-specific binding. During these experiments many brands and detection schemes can be used. A similar loss of binding, when increasing concentrations of test compound are added to a known ligand and receptor, is also evidence for an antagonist. Knowledge of the variant receptors provides the means for constructing expression vectors that can be used to prepare the soluble receptor for receptor binding assays. Mutagenesis of these soluble receptors can be used to determine which amino acid residues are critical for ligand binding and aid in the design based on antagonist structure.

Cells depleted of human IL-9 receptor can be transiently or stably transfected with expression vectors containing a variant receptor and used to assay for IL-9 pathway activity. These activities may be cell proliferation or prevention of apoptosis, both of which were attributed to the IL-9 pathway. These cells can be used to identify agonists and receptor antagonists, as previously described.

The methods discussed above represent a number of effective methods, using the variant forms of IL-9 receptor, for the development of therapy for atopic asthma and other related disorders.

A number of techniques have been described that can be used to diagnose atopic asthma recognizing single nucleotide variants at the IL-9 receptor, including DNA sequencing, restriction fragment length polymorphisms (RFLPs), allele-specific oligonucleotide analyzes ( ASO), chain linkage reaction (CSF), chemical cleavage and single chain conformational polymorphism (SSCP) analyzes. One of skill in the art will recognize that the use of one or more of these techniques, as well as others in the literature, may be used to detect one or more variations in the IL-9 receptor mRNA gene or transcript. 10

Still other techniques for detecting amino acid variants at the IL-9 receptor, including ELISA, immunoprecipitations, Westerns and immunoblotting, may be used.

The methods discussed above represent several effective methods for the diagnosis of atopic asthma and other related disorders.

Thus, the inventors have provided methods that utilize the IL-9 receptor to identify antagonists that are capable of regulating the interaction between IL-9 and its receptor. More specifically the Applicant provides a method for assaying the functions of the IL-9 receptor in order to identify compounds or agents that may be administered in an amount sufficient for down-regulation of the expression or functions of the IL-9 pathway.

Having identified the role of the IL-9 pathway in atopic allergy, bronchial hyperresponsiveness and asthma, the applicant also provides a method for the diagnosis of susceptibility and resistance to atopic allergy, asthma, and related disorders.

The accompanying drawings which are incorporated and form a part of this disclosure, illustrate various embodiments of the invention and, together with the description, serve to explain the principle of the invention. 11

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic representation of the human IL receptor cDNA. The boxes represent exon 2 to 9 spanning the coding region (relative scaled size, except the 3 'untranslated portion of exon 9, underlined by dashed line). The transmembrane region is encoded by exon 7, the intracellular domain by exon 8 and 9, and the extracellular domain by exon 2 to 6. The arrows indicate aberrant polymorphisms or excisions affecting the partial exon sequence; 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 polymorphism 410 + n consisting of any of serines 8 or 9; *) complete deletion of exon 3, 4 or 8.

Figure 2: Translational cDNA sequence of the wild-type IL-9R precursor protein, with the Arg allele at codon 344 (nucleotides 1272-1274) and 8 Ser / 4 Asn repeats starting at codon 410 (nucleotides 1470-1472).

Figure 3: Sequence of cDNA translated from the precursor protein of IL-9R, with the His allele at codon 344 (nucleotides 1272-1274) and 9 Ser / 4 Asn repeats starting at codon 410 (nucleotides 1470-1472).

Figure 4: Translational cDNA sequence of the IL-9R precursor protein, with the deletion of exon 8 causing a phase shift in exon 9, the production of 11 non-wild type amino acids and a premature termination codon. 12

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

Figure 7. Translational cDNA sequence of the IL-9R precursor protein, with an alternative excision in exon 5 resulting in a premature termination codon and the production of 27 non-wild type amino acids.

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

Figure 8: Translational cDNA sequence of IL-9R precursor protein, with deletion of exon 4 producing 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. Arg / Arg individuals are homozygous for the Arg allele with the 8 Ser / 4 Asn repeats. The Arg / His individuals are heterozygous for the Arg allele, with the 8 Ser / 4 Asn repeats and the His allele with the 9 Ser / 4 Asn repeats, 9 Ser / 3 Asn repeats and 10 Ser / 2 Asn repeats in exon 9. His / His individuals are homozygous for the His allele, with 9 Ser / 4 Asn repeats, 9 Ser / 3 Asn repeats and 10 Ser / 2 repeats

Asn in exon 9. Arg / Arg individuals are protected from atopic allergy. Arg / His and His / His individuals are susceptible to atopic allergy (P = 0.002). 13

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

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

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

Figure 13: XY specific amplimers for specific amplification of the IL-9 receptor gene. 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 antibody neutralizing the IL-9 receptor with wild-type and Delta-Q receptors. Panel A): COS7 cells were transiently transfected with LXSN vector (A and B), wild-type IL-9R (C and D), wild-type 9-residue IL-9R. Be starting with codon 410 (E and F), Δ-Q variant 173 (G and H) and Δ-Q 173 with 9 residues Ser from codon 410 (I and J) and sequentially incubated with Î "MAB290 and anti-mouse IgG antibody conjugated to Texas red (B, D, F, H and J) as described 14 (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 Texas Red-conjugated anti-rabbit IgG antibody. Bar = 10 microns.

Figure 15: Activation of members of the Jak, Stat and Ir families by different variants of the human IL-9 receptor. TS1 cells expressing either GH9, AQGR8 or AQGH9 were starved for 6 hours and then treated for 5 minutes without cytokine (-), murine IL-9 (m) or human IL-9 (h ). Cell extracts were immunoprecipitated with various antibodies specific for different members of Jak, Stat and Irs families. Immunoblots were first reacted with an anti-phosphotyrosine antibody so as to detect only phosphorylated tyrosine proteins and then washed and re-probed with the same antibody used to immunoprecipitate each protein. GH9, AQGR8, AQGH9 are as indicated in Figure 16.

Figure 16: Proliferation of TS1 cells expressing different forms of human IL-9 receptor. Cells were seeded in quadruplicate in 96-well (1000 / well) plates and treated without either cytokine or murine or human IL-9 (5 ng / ml). A colorimetric assay was performed 7 days later in order to determine the number of cells and the ratio between treated / untreated cells (% control) was calculated to evaluate the growth rate. LxSN = cells transfected with vector 15 empty; GR8 is wild-type IL-9R: GH9 is the His 344 variant with 9 residues Ser is starting at codon 410; AQGR8 and AQGH9 are AQ173 variants on the wild-type background and His 344 + 9-Ser, respectively.

Figure 17: IL-9R intron 5 genomic DNA sequence with a nucleic acid variation, 213 nt upstream from exon 6, where a residue T is changed to a residue C as indicated by the arrow.

DETAILED DESCRIPTION OF THE INVENTION The Applicant has resolved the need in the art by elucidating an IL-9 pathway and compositions affecting that pathway which may be used in the treatment, diagnosis and development of methods for identifying agents for preventing or treating atopic 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 hyperresponsiveness (BHR), rhinitis, urticaria, allergic inflammatory bowel disorders and various forms of eczema. Atopy is a hypersensitivity to environmental allergens expressed as elevated serum total IgE or abnormal responses from skin tests to allergens, compared to controls. BHR refers to bronchial hyperresponsiveness, an intensified bronchoconstricting response to a variety of stimuli. 16

Through DNA analysis of individuals exhibiting atopic allergy and asthma-related disorders, we have identified polymorphisms in the IL-9 receptor gene (IL-9R) that can correlate with the expression of asthma. 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 a variety of functions involving the regulation of the myeloid and lymphoid systems humans. The human IL-9 receptor gene of the present invention is found in the subtelomeric region of the XY chromosomes.

By polymorphism, the applicant defines a change in a specific DNA sequence, designated " locus ", of the prevailing sequence. In general, a locus is defined as polymorphic when specialists have identified two or more alleles covering this locus and the less common allele exists at a frequency of 1% or more. Specific amplification of authentic IL-9R (gene encoding the biologically functional protein located in the pseudo-autosomic region XYq), using standard primer design, was not possible because IL-9R had four highly homologous unprocessed pseudogenes (> 90% nucleotide identity) at other loci in the human genome (chromosome 9, 10, 16, 18). Because of the high identity of these other genes, PCR genomic amplification using standard primer design resulted in the co-amplification of all genes thus rendering the authentic gene sequence analysis equivocal. To study the structure of authentic IL-9R, since it may relate to predisposition to disease, such as asthma, discussed 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), the inventors designed specific amplimers. Specific primers were found to be authentic for IL-9R amplification, without any amplification of the 4 pseudogenes. Primer sequences are shown in Example 2 and their specificity is demonstrated in Figure 13.

We also amplified, by RT-PCR, the entire coding region of the IL-9 receptor cDNA using RNA extracted from PBMC (peripheral blood mononuclear cells) purified from 50 donors. Figure 1 shows the most frequent variations observed in 50 individuals analyzed. Exons 3, 4, 5, 6 and 8 were affected by aberrant excision events in samples where full length cDNAs could also be cloned. Some transcripts showed complete deletion of exon 3 causing a phase shift, creating a stop codon after a segment of 79 unrelated residues. In the case of deletion of exon 4, a phase shift is also generated and the first codon in exon 5 is converted into a stop codon. In some other cDNAs, exon 5 showed partial deletion of the first 5 or 29 nucleotides, leading both deletions to phase shifts resulting in early termination codons within exon 5. For this reason, in all cases, the putative truncated protein would be devoid of most of the extracellular domain, as well as all transmembrane and cytoplasmic domains. If secreted, these forms could function as soluble receptors. Finally, the first three nucleotides of exon 6, corresponding to codon 173, were often excised, resulting in the deletion of glutamine at this codon, without any further changes in the remaining protein sequence. This excision variant is possibly related to a variant verified in intron 5 of genomic DNA (SEQ ID NO: 24), which would increase the frequency of the excision variant (Figure 17 and Example 12). We have also found limited allelic variations to the coding sequence of exon 9. Polymorphisms involving codon 310 and 410 have previously been disclosed. 29.30 (Kermouni, A., et al., Genomics, 371-382 (1995)). Codon 310 codes for arginine or glycine, depending on whether the first nucleotide at that codon is an adenine or a guanidine, respectively. In codon 410 (hereinafter referred to as " 410 + n ") begins a segment of 8 or 9 trinucleotide repeats of AGC, which would be translated into 8 or 9 serines, respectively. The Applicant has verified a new polymorphism at codon 344. Here, the second nucleotide is either adenine or guanidine, the two possible residues being encoded by this codon histidine or arginine, respectively. Moreover, a match between codon 344 and 410 + n was observed, where the arginine at codon 344 is consistently checked with 8 serines at codon 410 + n and the histidine at codon 344 is checked with 9 serines. Human IL-9 receptor cDNA, originally cloned from a human megakaryoblastic leukemic cell line, Mo7e, showed 9 serines at codon 410 + n and, unlike the clones of the applicants, an arginine at codon 344.29. Another megakaryoblastic leukemic cell line, UT-7, was reported to carry the same arginine / 9 serine allele. The inventors cloned 16 cDNAs from the Mo7e cell line and found that 6 had 8 serines at codon 410 + n and arginine at codon 344. The remaining ten clones showed the published sequence. We also genotyped the human acute myelogenous leukemia cell line, KG-1, and verified that it was homozygous for histidine / 9 serines. These DNA molecules and corresponding RNA are isolated using techniques which are standard in the art, such as Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1985). By isolated, the inventors have defined that the DNA is free of at least some of the contaminants associated with the nucleic acid or polypeptides occurring in a natural environment. The invention also includes the proteins encoded by the nucleic acid sequences as described in the claims. The invention further includes fragments of the molecules. For fragments, the inventor defined portions of the nucleic acid sequence that maintains the function of the complete sequence. As would be known in the art, the fragments result from deletions, additions, substitutions and / or modifications. The source of the IL-9 receptor variants of the invention is human. Alternatively, the DNA or fragment thereof may be synthesized using methods known in the art. It is also possible to produce the compound by genetic engineering techniques, by constructing DNA by any accepted technique, cloning the DNA in an expression vehicle, and transfecting the carrier into a cell that will express the compound. See, for example, the methods set forth in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1985). Demonstration of variant IL-9 receptor sequences which may be associated with atopic allergy and an asthma-like phenotype, and others which may be associated with the absence of an asthma-like phenotype, provides methods of diagnosing atopic asthma susceptibility and related disorders. Certain variants can produce soluble receptors that can be used for the treatment of these disorders.

A receptor is a component, soluble or membrane bound, which recognizes and binds to molecules and the IL-9 receptor of the invention is the component that recognizes and binds IL-9. The functions of the IL-9 receptor consist of binding to IL-9 or an IL-9-like molecule and propagation of its regulatory signal in specific cells. 29'30'34'35 human IL-9 has been shown to cause the phosphorylation of the IL-9 receptor itself and the activation of Jak-Stat, Jakl, Statl, Stat3, Stat5 and Irs2 pathways following IL-9 receptor binding (Demoulin, JB, et al., Molecular and Cellular Biology, pp. 4710-4716, Set of 1996). We examined whether IL-9R or its variants showed any variation in the activation of these proteins and extended the analysis to Jak3 and Irs1. The entire pathway proteins including Jak3 and Irs1 were determined to be phosphorylated by activation of IL-9R. It was also determined that IL-9R variants with alterations at codons 310, 344 and 410 + n provided the same up-regulation as wild-type IL-9R. Accordingly, one aspect of the invention is therapy for the treatment of atopic asthma which inhibits interactions in the Jak-Stat pathway.

Unlike the wild type receptor and the other variants tested, variant AQ173 could not activate any proteins in the Jak-Stat pathway (Figure 15). In addition, the AQ173 variant could not maintain cell proliferation after IL-9 stimulation (Figure 16). Therefore, individuals expressing the AQ173 variant are less likely to be susceptible to atopic asthma and related disorders. A 21 aspect of the invention therefore is a therapy that enhances expression of the AQ173 excision variant for the treatment of atopic asthma and related disorders.

One diagnostic embodiment involves the recognition of variations in the DNA sequence of the IL-9 receptor gene or transcript. One method involves the use of a nucleic acid molecule (also known as a probe) having a sequence complementary to the IL-9 receptors of the invention under sufficient hybridization conditions, as would be understood by those skilled in the art. In one embodiment, the nucleic acid molecule will specifically bind to the Arg344 codon of the mature IL-9 receptor protein, or His344 and, in another embodiment, will bind Arg344 and His344. In yet another embodiment, it will bind to the codon for Gin 173. These methods may also be used to recognize other variants of the IL-9 receptor. Another method of recognizing DNA sequence variation associated with these disorders is the DNA sequence analysis by multiple methods well known in the art.44 Another embodiment involves the detection of DNA sequence variation in the IL receptor gene -9 associated with these disorders. These include the polymerization chain reaction, restriction fragment length polymorphism (RFLP) analysis and single chain conformational analysis. In a preferred embodiment, the applicants specifically provide a method for recognizing, at a genetic level, the polymorphism at the IL-9 receptor associated with the His344 and Arg344 alleles using an ASO PCR. In other embodiments, the chain-linking reaction can be used to distinguish these IL-9 receptor gene alleles. 22

Also disclosed are methods for the identification of IL-9 antagonists and their receptor. Antagonists are compounds which are themselves devoid of pharmacological activity but cause effects through presentation of the action of an agonist. In order to identify an antagonist of the invention, it may be tested for competitive binding with a known agonist or for down-regulation of IL-9-like functions, as described herein and in the cited literature. 2,22-35

Specific assays may be used to screen for medicaments useful in the treatment of atopic allergy, based on the known role of the IL-9 receptor in T lymphocyte proliferation, IgE synthesis and release from mast cells. "Another assay involves the ability of the human IL-9 receptor to specifically induce rapid and transient tyrosine phosphorylation of multiple proteins in Mo7e cells. Because this response is dependent on 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 Stat3 transcriptional factor appears to be specifically related to IL-935 receptor functions and this response represents a simple method or assay for the characterization of compounds as described

Yet another method for characterizing IL-9 receptor function involves the use of the well-known murine clone TSla, transfected with a human receptor that can be used to evaluate the function of human IL-9 with a cell proliferation assay. These methods can be used to identify IL-9 receptor antagonists. 23

In a further embodiment, the invention includes the down-regulation of IL-9 expression or function through administration of soluble IL-9 receptor molecules as claimed to 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 binding of IL-9 to a cell-bound receptor and acts as an antagonist of IL-9. Soluble receptors were used to bind cytokines or other ligands in order to regulate their function. A soluble receptor is a form of a membrane bound receptor that occurs in solution or outside the membrane. Soluble receptors may occur, because of the segment of the molecule that generally associates with the membrane, being absent. This segment is generally 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. We have identified three variants of excision of the human IL-9 receptor that result in the production of proteins that could act as soluble receptors.

An excision variant resulted in the deletion of exon 4 which introduced a phase shift, resulting in a termination codon as the first codon of exon 5. This variant would yield a peptide of about 45 residues containing an epitope reactive with antibodies that block the IL-9 / IL-9R interaction. The other two variants contain deletions in exon 5 which will produce early terminus codons at the beginning of the exon but, in these cases, without the deletion of exon 4. These variants 24 would produce a protein of about 100 residues also containing the epitope recognized by blocking antibody.

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

For example, screening for peptides and single chain antibodies using phage display could be facilitated using a soluble receptor. The phage that binds to the soluble receptor can be isolated and the molecule identified by capture of affinity of the receptor and bound phage. In addition, composite screens for agents useful in the treatment of atopic asthma and related disorders may incorporate a soluble receptor and ligand that binds in the absence of an antagonist. Detection of ligand and receptor interaction occurs because of the proximity of these molecules. Antagonists are recognized by inhibition of these interactions.

Additionally, the invention 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 petroleum, of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Martin, E.W., 25

Remington's Pharmaceutical Sciences, specifically incorporated herein by reference.

The compounds used in the method of treatment may be administered systemically or topically, depending on considerations, such as the condition being treated, need for site specific treatment, amount of drug to be administered and similar considerations. Topical administration may be used. Any common topical formation, such as a solution, suspension, gel, ointment or ointment and the like may be employed. The preparation of such topical formulations is well described in the art of pharmaceutical formulations as exemplified by, for example, Remington's Pharmaceutical Science, Issue 17, Mack Publishing Company, Easton, Pa. For topical application these compounds could also be administered as a powder or particularly in aerosol form. In a preferred embodiment, the compounds of this invention may be administered by inhalation. For inhalation therapy, the compound may be in a solution useful for administration by metered dose inhalers, or in a form suitable for a dry powder inhaler. The active ingredient may be administered in pharmaceutical compositions adapted for systemic administration. As is known, if a drug is to be administered systemically, it may be made as a powder, pill, tablet or the like, or as a syrup or elixir for oral administration. For intravenous, intraperitoneal or intralesional administration, the compound will be prepared as a solution or suspension, capable of being administered by injection. In certain instances, it may be useful to formulate these compounds in the suppository form or as a sustained release formulation for depot under the skin or intermuscular injection.

An effective amount is that amount which will down-regulate the functions controlled by the IL-9 receptor. A particular effective amount will vary from state to state and, in certain instances, may vary with the severity of the condition being treated and the patient's susceptibility to treatment. Consequently, a certain effective amount will be better determined at the time and place, through routine experimentation. It is envisaged, 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 usually 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 about 0.1 to 10 mg / kg, will affect a therapeutic result in most cases.

The inventors also provided a method for screening for compounds that negatively regulate the IL-9 receptor-controlled functions. It may be determined whether the functions expressed by the IL-9 receptor are down-regulated using standard techniques in the art. 29,30,34,35 In a specific embodiment, the inventors have provided a method of identifying compounds having 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 specifically induces rapid and transient tyrosine phosphorylation of multiple proteins in IL-9 responsive cells. The tyrosine phosphorylation of the transcriptional factor Stat3 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-like molecules depends on " stable expression " of IL-9 receptors in murine TS1 clones or TF1 clones which do not normally express the human receptor. These transfectants can be used to assess human IL-9 receptor function with a cell proliferation assay.29 The present disclosure also includes a simple screening assay for binding of specific and saturable ligand based on cell lines expressing the variants of the IL-9 receptor. 23,29 the IL-9 receptor is expressed on a wide variety of cell types, including K562, C8166-45, 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), the human megakaryoblastic leukemia cell line Mo7e34, TF1,29 macrophages , eosinophils, fetal thymocytes, the 29330 human kidney cell line, and the murine and embryonic 32D hippocampal progenitor cell lines.23'29'30 The practice of the present invention will employ the conventional terms and techniques of molecular biology, pharmacology, immunology, and biochemistry which are within the general knowledge of those 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. 28

However, the following basic background information is provided. The genetic material of the body, or DNA, is arranged on 46 chromosomes each comprising two arms joined by a centromere. Each chromosome is divided into segments, designated p or q. The p symbol 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 provided by the chromosome number (i.e., 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 DNA sequence information in areas known as the pseudo-autosomal regions. DNA, deoxyribonucleic acid, consists of two complementary nucleotide chains including the four different base compounds, adenine (A), thymine (T), cytosine (C) and guanine (G). 0A of one chain binds to the T of another chain, whereas the C of one chain binds to G of another, so as to form " base pairs " each pair having a base in each chain.

A sequential clustering of three nucleotides (a " codon ") encodes an amino acid. Thus, for example, the three CAG nucleotides encode the amino acid Glutamine. The 20 naturally occurring amino acids and their one-letter codes are as follows:

Ala A Arg R Asn N Asp D Asx B Cys C Gin Q Glu E Glx Z Gly G His H lie I Leu L Lys K Met M Phe F Pro P Be S Thr T Trp W Tyr Y Vai V

Alanine

Arginine

Asparagine Aspartic Acid

Asparagine or Aspartic Acid

Cysteine

Glutamine Glutamic Acid

Glutamine or glutamic acid

Glycine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Proline

Serina

Threonine

Tryptophan

Tyrosine

Valine

Amino acids comprise proteins. The amino acids may be hydrophilic, i. i.e., exhibiting an affinity for water, or hydrophobic, i. e., having an aversion to water. Thus, amino acids designated as G, A, V, L, I, P, F, Y, W, C and M are hydrophobic and the amino acids designated as 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 winding of a peptide chain and, consequently, the three-dimensional structure of a protein. DNA is protein related as follows: Genomic DNA - > MRNA - > protein! 1

CDNA Genomic DNA comprises all DNA sequences found in the cell of an organism. It is " transcribed " in messenger RNA (" mRNA "). Complementary DNA (" cDNA ") is a complementary copy of mRNA performed by reverse transcription of mRNA. Unlike genomic DNA, mRNA and cDNA contain only regions of the DNA encoding protein or encoding polypeptide, the so-called " exons ". Genomic DNA may also include " introns " which do not encode proteins.

In fact, eukaryotic genes are discontinuous with the proteins encoded by them, consisting of exons disrupted by introns. After transcription into RNA, the introns are removed by excision so as to produce mature messenger RNA (mRNA). The excision points between exons are typically determined by consensus sequences that act as signals for the excision process. Excision consists of a deletion of the intron from the primary RNA transcript and a ligation or fusion of the ends of the remaining RNA on both sides of the excised intron. The presence or absence of introns, intron composition and number of introns per gene may vary between strains of the same species and between species having the same basic functional gene. Although, in most cases, it is assumed that introns are non-essential and benign, their categorization is not absolute. For example, an intron of one gene may represent an exon of another. In some cases, alternative or different patterns of excision may produce different proteins from the same single segment of DNA. In fact, the structural characteristics of the introns and the underlying excision mechanisms form the basis for the classification of different types of introns.

As for the exons, they may correspond to discrete domains or motifs, such as functional domains, winding regions or structural elements of a protein; or to short polypeptide sequences, such as reverse folds, hooks, glycosylation signals and other signal sequences, or unstructured polypeptide adapter regions. The exon modules of the present combinatorial method may comprise nucleic acid sequences, corresponding to naturally occurring exon sequences or sequences of naturally occurring exons that have been mutated (e.g., point mutations, truncations, fusions).

Turning now to the manipulation of DNA, the DNA can be cut, excised and otherwise manipulated using " restriction enzymes " which cut off DNA at certain known sites and DNA ligases that bind DNA. Such techniques are well known to those skilled in the art, as set forth in such texts 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 may 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 within which a segment of DNA is inserted, so as to allow for expression of the DNA; i. i.e., production of the protein encoded by DNA. Expression vectors may be constructed in the laboratory, obtained from other laboratories, or purchased from commercial sources. The recombinant vector (known by various terms in the art) may be introduced into a host by a process generally known as " transformation ". Transformation means the transfer of an exogenous DNA segment by any of a number of methods, including infection, direct absorption, transduction, F-crossing, microinjection or electroporation in a host cell.

Unicellular host cells, variously known as recombinant host cells, cells and cell culture, include bacteria, yeast, insect cells, plant cells, mammalian cells and human cells. In particularly preferred embodiments, the host cells include E. coli, Pseudomonas, Bacillus, Streptomyces, Yeast, CHO, RL-1, BW, LH, COS-J, COS-7, BSC1, BSC40, BMT10 and S69. Yeast cells include, in particular, Saccharomyces, Pichia, Candida, Hansenula and Torulopsis.

As recognized in the art, expression of the DNA segment by the host cell requires appropriate regulatory sequences or elements. Regulatory sequences vary according to the host cell employed, but include, for example, in prokaryotes, a promoter, ribosome binding site and / or a transcription termination site. In eukaryotes, such regulatory sequences include a promoter and / or a transcription termination site. As well recognized by those in the art, the expression of the polypeptide can be improved, i.e. e., raised above standard levels, by careful selection and placement of these regulatory sequences.

In other embodiments, promoters that may be used include the human cytomegalovirus (CMV) promoter, tetracycline-inducible promoter, simian virus promoter (SV40), Moloney murine leukemia virus long-terminal (LTR) promoter, glucocorticoid-inducible murine mammary tumor virus (MMTV) promoter, herpes thymidine kinase promoter, murine and human actin promoters, HTLV1 and HIV 5 'flanking region of IL-9, 5' flanking region of the IL- Mouse promoter, bacterial tac promoter, and Drosophila thermal shock protein (SAR) support conjugation promoter elements. DNA may 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 biological molecular technique of interest for the present invention is " binding analysis ". Binding analysis is an analytical method used to identify the chromosome or chromosome region that correlates with a characteristic or disorder.47 Chromosomes are the basic units of genetic heritage on which genes are organized. In addition to the genes, the specialists have identified " DNA markers " on chromosomes. DNA markers are known DNA sequences whose identity and sequence can be readily determined. Binding analysis methodology was applied to the mapping of disease genes, for example, genes related to susceptibility to asthma, to 47 48 spectrromous chromosomes. '

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the disclosure and practice of the disclosed invention. The description and examples are intended to be exemplary, with a true scope of the invention being only indicated by the claims.

Having provided this background information, the applicant now describes the preferred aspects of the invention. EXAMPLE 1

Identification of IL-9 receptor transcript polymorphisms

A population of 52 individuals was randomly screened for asthma and atopy from the Philadelphia area of Pennsylvania. Serum total IgE was assayed by enzyme-linked immunosorbent assay (ELISA, Genzyme, Cambridge, Massachusetts).

In order to evaluate the structural forms of the human IL-9 receptor cDNA, PBMCs from these unrelated donors were isolated and cultured in the presence of PHA and PMA (described in Example 4). Earlier data from the applicant's laboratory demonstrated that expression kinetics for the IL-9 receptor message in primary cultures peaked at day 6 after mitogenic stimulation. The applicant cultured the cells, therefore, for 6 days, at which time the cells were harvested and their RNA and DNA isolated as described in Example 5.

RNAs were reverse transcribed and amplified by PCR using specific primers specific for full-length IL-9 receptor cDNA as described in Example 5. The amplification products of each individual were cloned into the TA PCR cloning vector and ten clones containing the expected inserts (as determined by digestion and gel electrophoresis) were sequenced in their entirety and analyzed for structural or sequence variation. 36 From the previous screening, seven major variants were identified. These cDNAs represent a codon deletion 173, a deletion of exon 4, two separate deletions in exon 5, one deletion of exon 8, and one full length cDNA containing a change of ARG in HIS at codon 344 of the mature protein. There are additional variants in each of these genetic backgrounds. The Arg allele is associated with 8 Ser / 4 Asn repeats and 7 Ser / 4 Asn repeats; the His allele is associated with 9 Ser / 4 Asn repeats, 9 Ser / 3 Asn repeats and 10 Ser / 2 Asn repeats. All these variants are represented in Figure 1.

The variants were cloned into the eukaryotic expression vector pCEP4 (Clontech) which contains a CMV promoter which 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 selection of eukaryotic cells containing the vector and, presumably, expression of the cloned cDNA under the control of the CMV promoter. Recombinant plasmids were analyzed sequentially and those plasmids containing the correct cDNA inserts were transfected into eukaryotic recipient cells, such as TK-tsl3 from Syrian hamster fibroblasts, T98G human glioblastoma, human TF-1 myeloid leukemia cell line, and 32D murine myelogen precursor cell line as described in Example 3. The function was biologically evaluated as a response to the growing IL-9 ligand and / or apoptosis (Examples 7 and 10).

Experiments in which the full-length IL-9 receptor cDNAs containing the ARG or HIS variants were performed are TK-tsl3 hamster fibroblasts or T98G human glioblastoma cells. Cells were transfected and analyzed 48 hours later by Western blotting and in situ staining using human-specific carboxyl end-points (Santa Cruz) antibodies (Example 8). In situ analysis demonstrated that both receptor forms appeared to be expressed in the hamster and human line (Figures 11 and 12). Interestingly, although Western blots of both forms appeared to be expressed at equal levels in the human and hamster line, a pattern of differential migration emerged between the ARG and HIS receptor forms in the TS1 cell line (Figure 12), suggesting a differential post-translational modification, such as glycosylation, phosphorylation, etc. Biochemical difference may be the mechanism by which altered function results in altered phenotype. The frequency of the various substitutions was used as a non-skewed estimate of the prevalence of each variant in the general population. The genotype was compared to phenotype evaluated by questionnaire. A diagnosis of allergy and asthma was determined by a physician reviewing the questionnaires. Homozygous individuals for Arg344 alleles demonstrated significantly less likely evidence for allergy and asthma as compared to heterozygous or homozygous 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 38 X / Y pseudo-autosomic regions and to exclude pseudogenes of the highly conserved IL-9 receptor located on chromosomes 9, 10, 16 and 18. First, sequence alignments between the two published pseudogenes and the genomic sequence of the IL-9 receptor genes were performed. The primers were then initially designed in regions diverging between authentic genes and pseudogenes and then analyzed by PCR using unique chromosome specific hybrids derived from the Coreill DNA Repository (Camden, NJ). If the primers produced only correct sized products from hybrids X and Y, they were then optimized for robust amplification. In several cases, the primers directed to the divergent regions were not XY-specific; therefore, the applicants introduced additional base changes in the particular primer, so as to further increase the number of mismatches against the pseudogenes, as compared to the IL-9 receptor gene sequence. Table 1 contains the primer sequence and optimum annealing temperatures for XY-specific amplification. The specificity of these primers for XY amplification is demonstrated in Figure 13.

Table 1: Specific X / Y Amplifiers of the IL-9 Receptor EXAO INITIATOR INITIATOR DIRECTION ΑΝΤΙ-SENSE TEMP. SIZE 2 5'-GCA GGT GGG GAC CCA TG -3 '(SEQ ID NO: 8) 5'-AGG CTT GAC ATC GGA CAA C-3' (SEQ ID NO: 9) 68 * 300 bp 3 5'-CTG GCC TGA AGT ACT TAC C -3 '(SEQ ID NO: 10) 5'-CTG CTT CAA TCC TGG GGA A-3' (SEQ ID NO: 11) 62 * 222 bp 4 5'-CTG AGT TCC CCA GGA TTG A-3 '(SEQ ID NO: 12) 5'-CAA GGC CCT GCT CCA AA -3' (SEQ ID NO: 13) 64 * 335 pb 5 5'-TGG GGC TTC AGC CTC ACA TG -3 '(SEQ ID NO: ID No. 14) 5'-TAT GTA GAG TGG GGA GTC TA 3 '(SEQ ID NO: 15) 62 * 259 bp 6 5'-TGT ATT CTC GAG GGC TGA C-3' (SEQ ID NO: 16) 5'-TGA GGT GAA CAG GGG AGA A 3 '(SEQ ID NO: 17) 62 * 337 bp 7 5'-CCC TGG GCC CTT CAT GT -3' (SEQ ID NO: 18) 5'-ACA AGG GCG GCC TTT GAT -3 '(SEQ ID NO: 19) OO to 262 bp 8 5'-AGG GAC GAG GTG GGC GGA C-3' (SEQ ID NO: 20) (SEQ ID NO: 21) 58 * 376 bp 9 5'-ATG CTA CCT GAG CCC TTC C -3 '(SEQ ID NO: 22) 5'-GGA CAT GAT GCA TCT GGC G -3' (SEQ ID NO: 23) 62 * 664 bp

These primers represent an innovative method for the analysis of DNA sequence variation in these genes that can be used for the diagnosis of susceptibility or resistance to atopic asthma and related disorders. 40

Using this technology, each exon was examined by DNA sequence analyzes for individuals in the applicant populations in order to detect sequence variation in the IL-9 receptor gene. The sequence polymorphisms were distinguished from artifacts by repeated analyzes. An association of receptor variants with allergic phenotypes is discussed in Figure 9. The sequence of the receptor alleles is shown in Figures 1-8. EXAMPLE 3

Expression of IL-9 receptor and purified recombinant IL-9 ligand binding assay and potentially IL-9-like compounds in structure or function are fluorescently labeled for high specific activity through the use of commercially available techniques according to the invention. supplier's recommendations (Pierce). Human and murine 32D Mo7e cells are cultured and resuspended at 37øC in 0.8 ml of Dulbecco's modified Eagle's medium supplemented with 10% (vol / vol) fetal bovine serum, 50 mM 2-mercaptoethanol, L 0.55 mM -arginine, 0.24 mM L-asparagine and 1.25 mM L-glutamine or RPMI supplemented with 10% (vol / vol) fetal bovine serum, 50 mM 2-mercaptoethanol, L- 0.55 mM arginine, 0.24 mM L-asparagine and 1.25 mM L-glutamine, respectively. TF1.1 cells (TF1 cells lacking human IL-9 receptors), T98G, TK and murine 32D (Examples 7 and 10) were used as they were or after transfection with the human IL-9 receptor constructs as shown in Fig. described below. Plasmid DNA containing one of the full length or truncated forms of IL-9 receptors was cloned into plasmid pCEP4 (Clontech) and purified by centrifugation through Qiagen columns (Qiagen). Plasmid DNA (50 micrograms) was added to cells in 0.4 cm cuvettes, just prior to electroporation. After a dual electrical pulse (750V / 74 ohms / 40 microfarad and 100 V / 74 ohms / 2100 microfarad), the cells are immediately diluted in new medium, supplemented with IL-9. Stably transfected cells were generated after 14 days of selection with hygromycin B (400 pg / ml at 1.6 mg / ml). Hygromycin-resistant clones were analyzed for IL-9 receptor expression by Western blots and in situ staining as described in Example 8. The cell receptor binding is visualized directly in real time with fluorescence microscopy. Binding and internalization are followed over time in control (non-transfected) cells and with cells transfected with each of the known IL-9 receptor variants. An excess of unlabeled ligand or blocking antibody is used in parallel experiments, in order to demonstrate specific binding. The soluble IL-9 receptor, including amino acids 44 to 270, with or without a HA ditag, is also incubated with different forms of recombinant human labeled IL-9. Variable amounts of FLAG tail ligand (IBI) are incubated in PBS at room temperature for 30 minutes with 0.5 pg soluble receptor. EBC buffer (50 mM Tris, pH 7.5, 0.1æ NaCl, 0.5% NP40) (300æl) is added together

with 1 pg of anti-HA antibody or anti-FLAG monoclonal antibody 42 (ΙΒΙ) and incubated for 1 hour on ice. Forty microliters of protein A sepharose solution were added to each sample and mixed for 1 hour at 4 ° C. Samples were centrifuged for 1 minute at 11000 X G and the pellets were washed 4 times with 500 μl EBC. The pellets were dissolved in 26 æl of 2X SDS buffer, boiled for 4 minutes and electrophoresed through an 18% SDS polyacrylamide gel. Western blots were probed with an anti-IL-9 receptor antibody (Santa Cruz Inc.) or anti-FLAG monoclonal antibody (IBI) against FLAG-tail rhIL-9. Therapeutic candidates are evaluated by measuring the antagonism of ligand binding. The receptor expression is shown in Figures 11 and 14. EXAMPLE 4

Isolation and cell culture

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by density gradient centrifugation using endotoxin-tested Ficoll-Paque PLUS according to the manufacturer (Pharmacia Biotech, AB Uppsala, Sweden). PBMC (5x106), mouse spleen cells (5x106) or 5x106 Mo7e cells were cultured in 7ml RPMI-1640 (Bethesda Research Labs (BRL), Bethesda, MD) supplemented to a final concentration of 10%, with isogenic human serum or heat inactivated FBS. Cells were cultured for 24 h at 37 ° C, unstimulated or stimulated with 5 pg / mL / 5 pg / mL PMA, or 5 pg / mL PHA and 50 ng / mL rhIL2 (R & D Systems, Minneapolis, MN). EXAMPLE 5

DNA Isolations & RNA, rtPCR, cloning and sequencing of PCR products, cytoplasmic RNA and genomic DNA were extracted after 6 days of mitogenic stimulation of cultured PBMC as described by Nicolaides and Stoeckert.46 One g of RNA from each source was denatured for> , 10 minutes at 70 ° C, and then reverse transcribed (V +) into cDNA using 2.5 units of Superscript II reverse transcriptase (GIBCO, BRL), 1 U / I RNAse Inhibitor, oligo d 1) 16 to 2.5 mM, 1 mM of each dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCl, pH 7, 0.25 mM MgCl 2, at 37øC, for one hour. A false reverse transcription reaction was used as a negative control.

One twentieth of the rt reaction was used in PCR (50æL) containing 6.7 mM MgCl2, 16.6 mM (NH4) 2S04, 67 mM Tris-HCl, pH 8.8, 10 mM 2-mercaptoethanol , 6% DMSO, 1.25 mM of each dNTP, 2.5 U of Amplitaq DNA polymerase and 300 ng of each of the oligonucleotides representing exon 2 IL-9 human cDNA (5 '-GCT GGA CCT TGG AGA GTG- 3 ') (SEQ ID NO: 1) and exon 9 (5'-GTC TCA GAC AAG GGC TCC AG-3' reverse) (SEQ ID NO: 2). The reaction mixture was subjected to the following PCR conditions: 120 sec at 95øC, then 35 cycles at: 30 sec at 94øC; 90 seconds at 58 ° C; 90 seconds at 72 ° C. Finally, the reaction mixture was cyclized once, for 15 minutes, at 72 ° C for extension.

PCR products representing hIL-9 receptor cDNA were subjected to gel electrophoresis through 1.5% agarose gels and visualized using ethidium bromide staining. The products of a false reverse transcriptase reaction, in which H20 replaced RNA, were used as a negative control amplification in all experiments.

The PCR products were subcloned into the TA Cloning vector (Invitrogen, San Diego, CA). Amplification of the human cDNA provided a 1614 bp product. Plasmids containing hIL-9 receptor cDNA inserts were isolated by standard techniques (Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York). After amplification, the DNA sequence including and surrounding each insert was analyzed by sequencing (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463) using fluorescent dideoxy terminators and an automated sequencer (ABI 377, Perkin Elmer), for determination of PCR-induced or cloning induced errors. The hIL-9 receptor cDNA inserts with no cloning and / or polymerase induced sequence errors were subcloned into expression vectors. EXAMPLE 6

Cloning and expression of IL-9 receptor constructs in vitro

The hIL-9 receptors were subcloned into the eukaryotic epitope expression vector pCEP4 (Clontech). The inserts were cloned as BamHI-XhoI fragments into the polypeptide pCEP4, in the sense orientation to the CMV promoter, using standard techniques (Figure 10). The constructs were expressed in cellular hosts as described. EXAMPLE 7

Cell assays using (Mo7e, 32D, TF1.1, TK ts! 3 and T98G)

Cell lines were used to evaluate the function of IL-9 receptor variants and for the screening of compounds potentially useful in the treatment of atopic asthma. The compounds were tested for their ability to antagonize IL-9-deduced baseline anti-apoptotic or proliferative response. After the establishment of an anti-apoptotic or proliferative baseline response in a given cell line, a statistically significant loss of response in repeated triplicate triplicates was considered evidence for antagonism. A true antagonistic response was differentiated from cell toxicity by direct observation, trypan blue staining (a technique well known to one of ordinary skill in the art) or loss of acid phosphatase activity. The antagonistic specificity is checked for each compound by assessing whether the activity is demonstrated against other proliferative agents, such as interleukin 3 or interleukin 4. Recombinant IL-9 and potentially IL-9-like compounds in structure or function have been purified and prepared for appropriate means. The putative agonists and antagonists were prepared in water, saline or 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 h of growth factor deprivation, cells were incubated without (control) or with varying amounts of IL -9 and compounds potentially similar to IL-9 in structure or function. Cell proliferation was assayed using the Abacus Cell Proliferation kit (Clontech, Paio Alto, CA) which determines the amount of intracellular acid phosphatase present as an indication of cell numbers. The p-nitrophenyl phosphate substrate (pNPP) was converted by acid phosphatase into p-nitrophenol, which was measured as an enzyme concentration indicator. The pNPP was added to each well and incubated at 37 ° C for one hour. 1 N sodium hydroxide was then added to quench the enzymatic reaction and the amount of p-nitrophenol was quantitated using a Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, VA) at 410 nm wavelength. Standard curves comparing the number of cells with the optical absorbance were used to determine the linear range of the assay. Test results were only used when absorbance measurements were within the linear range of the assay. Briefly, assays were run 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 degrees C. The acid phosphatase was used as a measure of the number of cells present. All experiences are repeated at least twice. Apoptosis was assayed using the Annexin V kit as described by the supplier (Clontech), which determines dexamethasone-induced apoptosis through the recognition of extracellular phosphatidylserine, an early marker for apoptosis. The number of apoptotic cells was obtained by fluorescence microscopy as a percentage of cells stained with Annexin V. The Mo7e line is a human megakaryoblastic cell line, grown in RPMI 1640 (GIBCO / BRL, Gaithersburg, MD), Serum

Fetal bovine (Hyclone) at 20% and IL-3 (R & D Systems, Minneapolis, MN) at 10 ng / ml. The T98G line is a human glioblastoma cell line cultured in RPMI 1640 (GIBCO / BRL). The TK-tsl3 line of hamster fibroblasts, as well as the murine 32D cell line, a murine myelogen precursor line, were also cultured in RPMI 1640 (GIBCO / BRL) containing 10% fetal bovine serum (Hyclone) , further 1 ng / ml IL-3 was used with the 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 Western blotting and rtPCR) but, in comparison with its predecessor (TF1), no longer carries the receptor of IL-9, by rtPCR analysis, immunostaining and Western blotting. TF1.1 is cultured in RPMI 1640 (GIBCO / BRL) and 10% fetal bovine serum (Hyclone). All cell lines respond to multiple cytokines, including IL-9. The 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) cofactor (GIBCO / BRL , Gaithersburg, MD). Cells were counted using a hemocytometer and diluted to a concentration of 1 X 105 cells / mL and plated on a 96-well microtiter plate. Each well contained 0.15 or 0.2 mL, giving a final concentration of 10 to 50,000 cells per well, depending on the cell. Mo7e cells were stimulated with only 50 ng / mL Sera Cell Factor (SCF) (R & D Systems, Minneapolis, MN), 50 ng / mL SCF plus IL-3 (R & D Systems, Minneapolis, MN ) at 50 ng / ml or 50 ng / ml SCF plus 50 ng / ml IL-9. A control containing only cells and basal media was included. Serial dilutions of test compounds (i.e., 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 an independent control for response and non-specific cytotoxicity. Cultures were incubated for 72-96 hours at 37øC in 5% CO2. EXAMPLE 8

In situ analyzes & Western blot of exogenous IL-9 receptor in transfected cell lines In situ staining of the IL-9 receptor was performed as follows. Cells were plated for 24 hours and then lamellae containing the adherent cells were washed twice in phosphate buffered saline containing calcium and magnesium (PBS) (Gibco / BRL). For intracellular staining of the IL-9 receptor, cells were fixed in 4% paraformaldehyde / PBS plus 0.1% triton-X for 15 minutes at room temperature prior to treatment with IL-9 receptor antibody anti-human; for extracellular staining, cells were treated with antibody prior to fixation. The cells were then washed twice in PBS and blocked with 7.5% BSA in dH20 for 30 minutes at room temperature. PBS-washed cells were then incubated with a 10æg / ml solution of anti-human IL-9 receptor (polyclonal antibody directed against the carboxyl end of the IL-9 receptor) in 1% BSA / PBS for 1 hour at room temperature. The cells were washed three times in PBS and then incubated in 10æg / ml solution of a rhodamine-conjugated anti-rabbit antibody in 1% BSA / PBS for 30 minutes at room temperature. Cells were then washed three times in PBS and counted using 1æg / ml DAPI for 1 minute at room temperature. Cells were washed three times in dH20 and fixed to a slide microscope and analyzed by fluorescence microscopy. The results for the transfected COS7 cells are shown in Figure 14.

Western blots were performed on protein lysates obtained from direct lysis of cell extracts in 0.5% lysis buffer (50 mM Tris, 150 mM NaCl, NP40), 1 mM DTT and protease inhibitors) and boiled for 5 minutes. Samples were electrophoresed on tris-glycine SDS gels (Novex) at 4-20% in tris-glycine run buffer. The proteins were then transferred to nitrocellulose by electrotransfer using the Trans Blot II apparatus (Bio Rad). 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 room temperature. The blots were then probed using a polyclonal antibody directed to the carboxyl end of the IL-9 receptor (1æg / ml) in TBS-T for 1 hour. The blots were then washed three times in TBS-T for 10 minutes and probed using a horseradish peroxidase conjugated anti-rabbit secondary antibody (1: 10000) in TBS-T for 30 minutes. The blots were washed as above 50 and then incubated with Luminol / stimulator solution (Pierce), a chemiluminescent substrate, for 5 minutes at room temperature and then exposed to film for 1-60 seconds. See Figures 11 and 12. EXAMPLE 9 Methods for Genomic Amplification of Authentic IL-9R The specific amplification of authentic IL-9R (gene encoding the biologically functional protein located in the XYq pseudo-autosomic region) using standard primer design, was not possible because IL-9R had four highly homologous (> 90% nucleotide identity) unprocessed pseudogenes at other loci in the human genome (chromosome 9, 10, 16, 18). Because of the high identity of these other genes, PCR genomic amplification using standard primer design resulted in the co-amplification of all genes thus rendering the authentic gene sequence analysis equivocal. To study the authentic structure of IL-9R, since it may relate to disease predisposition, such as asthma, discussed in this application, or other diseases, such as cancer (Renauld, et al., Oncogene, 9: 1327 Gruss, et al., Cancer Res., 52: 1026-1031, 1992), specific amplimers were designed as follows:

The sequences of the pseudogene IL-9R and authentic genes were aligned using the Mac Vector software. The intronic sequences surrounding each exon were then inspected for regions of diversity between the authentic gene and pseudogenes. The primers were then designed against these regions and used to PCR amplify human / rodent hybrid DNAs containing individual human chromosomes. The products were run on 3% agarose gels and analyzed for authentic IL-9R amplification, without any amplification of the 4 pseudogenes. Specific PCR amplification conditions were also optimized by varying the annealing temperature and buffer conditions (DMSO content of 5-10%). In cases where amplification of pseudogenes still occurred, nucleotide changes were introduced into primers in order to cause greater divergence of pseudogenes compared to the authentic gene. Primer sequences are shown in Example 2 and their specificity is demonstrated in Figure 13. EXAMPLE 10

Cell proliferation and cytokine stimulation assay

In order to determine the growth response of TS1 cells expressing various forms of human IL-9 receptor, the cells were washed with PBS and resuspended in D-MEM, 10% fetal bovine serum. 103 cells per well were seeded in triplicate in 96-well microtiter plates and, if appropriate, recombinant human IL-9 or murine IL-9 (R & D

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

Immunoprecipitations, immunoblots and antibodies

Typically, 20-50x10 6 cells were lysed in 1 ml 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 1x complete protease inhibitor mixture " 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 recovered and transferred to a new tube. For immunoprecipitations, 1-5 Âμg of the antibody was added to the lysate and incubated overnight at 4Â ° C. 20 mL of Protein A + agarose G conjugated beads were added for 2 h, followed by four washes using RIPA buffer. The beads were resuspended in Laemmli buffer and boiled for 3 minutes prior to electrophoresis. Proteins were transferred onto membrane 53

Iwmobilion-P (Millipore) and detected using a secondary antibody conjugated to horseradish peroxidase, followed by a chemiluminescence detection assay (Pierce). Antibodies specific for murine and human IL-9 receptor (sc698), murine Jak1, Irs1, Irs2, Stat1, Stat2, Stat3, Stat4, Stat5 and phosphotyrosine (PY) were purchased from Santa Cruz (Santa Cruz, CA). Anti-Jak3 MAB290 and anti-human monoclonal IL-9 receptor were purchased from Upstate Biotechnology and R & D Systems, respectively. Figure 15 demonstrates the activation of members of the Jak family by means of different variants of the human IL-9 receptor. EXAMPLE 12

Identification of genomic polymorphisms of IL-9 receptor

Genomic DNAs were isolated from PBMC from volunteer donors as described (Nicolaides and Stoeckert, Biotechniques 8: 154-156, 1990). Intron sequence analysis of the human IL-9R gene was performed by PCR using sequence primers ID No. 14 and sequence ID No. 17 which resulted in a product having a molecular size of approximately 1243 base pairs. Amplifications were performed at 94 ° C for 30 seconds, 62 ° C for 1.5 minutes, 72 ° C for 1.5 minutes for 35 cycles in buffers described previously (Nicholaides et al., Genomics 30: 195- 206, 1995). The products were then purified and sequenced using a standard sequence protocol. Inspection of the intron 5 sequences in multiple individuals found a nucleotide change at nt -213, upstream of the exon 6, 54 sequences which resulted in a nucleotide change of thymidine (published sequence) into cytosine. An example of this change is shown in Figure 17. Although the invention has been described and illustrated herein by references to various specific materials, processes and examples, it is understood that the invention is not limited to the particular combinations of materials and processes selected for that purpose . As will be understood by those skilled in the art, numerous variations of such details may be implied.

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(C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent Number Launch 1.0,

(V) APPLICATION NUMBER: PCT / US97 / 21992 (B) DATE OF REGISTRATION: 02-DEC-1997 (C) CLASSIFICATION: (vi) DATA OF THE PREVIOUS ORDER: ( (Vii) DATA OF THE PREVIOUS ORDER: (A) APPLICATION NUMBER: US 08/980872 (B) DATE OF PRESENTATION: 01 DATE OF REGISTRATION: 02-DEC-1996 SEQUENCE CHARACTERISTICS: (A) LENGTH: 1947 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY : linear (ii) MOLECULE TYPE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: AGCAGCTCTG TAATGCGCTT GTGGTTTCAG ATGTGGGCGG CCTGTGTGAACCTGTCGTGC 60 AAAGCTCACG TCACCAACTG CTGCAGTTAT CTCCTGAATC AGGCTGAGGGTCTTTGCTGT 120 GCACCCAGAG ATAGTTGGGT GACAAATCAC CTCCAGGTTG GGGATGCCTCAGACTTGTGA 180 TGGGACTGGG CAGATGCATC TGGGAAGGCT GGACCTTGGA GAGTGAGGCCCTGAGGCGAG 240 ACATGGGCAC CTGGCTCCTG GCCTGCATCT GCATCTGCAC CTGTGTCTGCTTGGGAGTCT 300 CTGTCACAGG GGAAGGACAA GGGCCAAGGT CTAGAA CCTT CACCTGCCTCACCAACAACA 360 TTCTCAGGAT CGATTGCCAC TGGTCTGCCC CAGAGCTGGG ACAGGGCTCCAGCCCCTGGC 420 TCCTCTTCAC CAGCAACCAG GCTCCTGGCG GCACACATAA GTGCATCTT6CGGGGCAGTG 480 AGTGCACCGT CGTGCTGCCA CCTGAGGCAG TGCTCGTGCC ATCTGACAATTTCACCATCA 540 CTTTCCACCA CTGCATGTCT GGGAGGGAGC AGGTCAGCCT GGTGGACCCGGAGTACCTGC 600 CCCGGAGACA CGTTAAGCTG GACCCGCCCT CTGACTTGCA GAGCAACATCAGTTCTGGCC 660 ACTGCATCCT GACCTGGAGC ATCAGTCCTG CCTTGGAGCC AATGACCACACTTCTCAGCT 720 ATGAGCTGGC CTTCAAGAAG CAGGAAGAGG CCTGGGAGCA GGCCCAGCACAGGGATCACA 780 TTGTCGGGGT GACCTGGCTT ATACTTGAAG CCTTTGAGCT GGACCCTGGCTTTATCCATG 840 62 AGGCCAGGCT GCGTGTCCAG ATGGCCACAC TGGAGGATGA TGTGGTAGAGGAGGAGCGTT 900 ATACAGGCCA GTGGAGTGAG TGGAGCCAGC CTGTGTGCTT CCAGGCTCCCCAGAGACAAG 960 GCCCTCTGAT CCCACCCTGG GGGTGGCCAG GCAACACCCT TGTTGCTGTGTCCATCTTTC 1020 TCCTGCTGAC TGGCCCGACC TACCTCCTGT TCAAGCTGTC GCCCAGGGTGAAGAGAATCT 1080 TCTACCAGAA CGTGCCCTCT CCAGCGATGT TCTTCCAGCC CCTCTACAGTGTACACAATG 1140 GGAACTTCCA GACTTGGATG GGGGCCCACA GGGCCGGTGT 12 GCTGTTGAGCCAGGACTGTG 00 CTGGCACCCC ACAGGGAGCC TTGGAGCCCT GCGTCCAGGA GGCCACTGCACTGCTCACTT 1260 GTGGCCCAGC GCGTCCTTGG AAATCTGTGG CCCTGGAGGA GGAACAGGAGGGCCCTGGGA 1320 CCAGGCTCCC GGGGAACCTG AGCTCAGAGG ATGTGCTGCC AGCAGGGTGTACGGAGTGGA 1380 GGGTACAGAC GCTTGCCTAT CTGCCACAGG AGGACTGGGC CCCCACGTCCCTGACTAGGC 1440 CGGCTCCCCC AGACTCAGAG GGCAGCAGGA GCAGCAGCAG CAGCAGCAGCAGCAGCAACA 1500 ACAACAACTA CTGTGCCTTG GGCTGCTATG GGGGATGGCA CCTCTCAGCCCTCCCAGGAA 1560 ACACACAGAG CTCTGGGCCC ATCCCAGCCC TGGCCTGTGG CCTTTCTTGTGACCATCAGG 1620 GCCTGGAGAC CCAGCAAGGA GTTGCCTGGG TGCTGGCTGG TCACTGCCAGAGGCCTGGGC 1680 TGCATGAGGA CCTCCAGGGC ATGTTGCTCC CTTCTGTCCT CAGCAAGGCTCGGTCCTGGA 1740 CATTCTAGGT CCCTGACTCG CCAGATGCAT CATGTCCATT TTGGGAAAATGGACTGAAGT 1800 TTCTGGAGCC CTTGTCTGAG ACTGAACCTC CTGAGAAGGG GCCCCTAGCAGCGGTCAGAG 1860 GTCCTGTCTG GATGGAGGCT GGAGGCTCCC CCCTCAACCC CTCTGCTCAGTGCCTGTGGG 1920 GAGCAGCCTC TACCCTCAGC ATCCTGG 1947 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1947 base pairs (B) TYPE: ác nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear 63 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2: AGCAGCTCTG TAATGCGCTT GTGGTTTCAG ATGTGGGCGG CCTGTGTGAACCTGTCGTGC 60 AAAGCTCACG TCACCAACTG CTGCAGTTAT CTCCTGAATC AGGCTGAGGGTCTTTGCTGT 120 GCACCCAGAG ATAGTTGGGT GACAAATCAC CTCCAGGTTG GGGATGCCTCAGACTTGTGA 180 TGGGACTGGG CAGATGCATC TGGGAAGGCT GGACCTTGGA GAGTGAGGCCCTGAGGCGAG 240 ACATGGGCAC CTGGCTCCTG GCCTGCATCT GCATCTGCAC CTGTGTCTGCTTGGGAGTCT 300 CTGTCACAGG GGAAGGACAA GGGCCAAGGT CTAGAACCTT CACCTGCCTCACCAACAACA 360 TTCTCAGGAT CGATTGCCAC TGGTCTGCCC CAGAGCTGGG ACAGGGCTCCAGCCCCTGGC 420 TCCTCTTCAC CAGCAACCAG GCTCCTGGCG GCACACATAA GTGCATCTTGCGGGGCAGTG 480 AGTGCACCGT CGTGCTGCCA CCTGAGGCAG TGCTCGTGCC ATCTGACAATTTCACCATCA 540 CTTTCCACCA CTGCATGTCT GGGAGGGAGC AGGTCAGCCT GGTGGACCCGGAGTACCTGC 600 CCCGGAGACA CGTTAAGCTG GACCCGCCCT CTGACTTGCA GAGCAACATCAGTTCTGGCC 660 ACTGCATCCT GACCTGGAGC ATCAGTCCTG CCTTGGAGCC AATGACCACACTTCTCAGCT 720 ATGAGCTGGC CTTCAAGAAG CAGGAAGAGG CCTGGGAGCA GGCCCAGCACAGGGATCACA 780 TTGTCGGGGT GACCTGGCTT ATACTTGAAG CCTTTGAGCT GGACCCTGGCTTTATCCATG 840 AGGCCAGGCT GCGTGTCCAG ATGGCCACAC TGGAGGATGA TGTGGTAGAGGAGGAGCGTT 900 ATACAGGCCA GTGGAGTGAG TGGAGCCAGC CTGTGTGCTT CCAGGCTCCCCAGAGACAAG 960 GCCCTCTGAT CCCACCCTGG GGGTGGCCAG GCAACACCCT TGTTGCTGTGTCCATCTTTC 1020 TCCTGCTGAC TGGCCCGACC TACCTCCTGT TCAAGCTGTC GCCCAGGGTGAAGAGAATCT 1080 TCTACCAGAA CGTGCCCTCT CCAGCGATGT TCTTCCAGCC CCTCTACAGTGTACACAATG 1140 GGAACTTCCA GACTTGGATG GGGGCCCACA GGGCCGGTGT GCTGTTGAGCCAGGACTGTG 1200 CTGGCACCCC ACAGGGAGCC TTGGAGCCCT GCGTCCAGGA GGCCACTGCACTGCTCACTT 1260 GTGGCCCAGC GCATCCTTGG AAATCTGTGG CCCTGGAGGA GGAACAGGAGGGCCCTGGGA 1320 CCAGGCTCCC GGGGAACCTG AGCTCAGAGG ATGTGCTGCC AGCAGGGTGTACGGAGTGGA 1380 GGGTACAGAC GCTTGCCTAT CTGCCACAGG AGGACTGGGC CCCCACGTCCCTGACTAGGC 1440 CGGCTCCCCC AGACTCAGAG GGCAGCAGGA GCAGCAGCAG CAGCAGCAGCAGCAGCAACA 1500 ACAACAACTA CTGTGCCTTG GGCTGCTATG GGGGATGGCA CCTCTCAGCCCTCCCAGGAA 1560 ACACACAGAG CTCTGGGCCC ATCCCAGCCC TGGCCTGTGG CCTTTCTTGTGA CCATCAGG 1620 GCCTGGAGAC CCAGCAAGGA GTTGCCTGGG TGCTGGCTGG TCACTGCCAGAGGCCTGGGC 1680 TGCATGAGGA CCTCCAGGGC ATGTTGCTCC CTTCTGTCCT CAGCAAGGCTCGGTCCTGGA 1740 CATTCTAGGT CCCTGACTCG CCAGATGCAT CATGTCCATT TTGGGAAAATGGACTGAAGT 1800 TTCTGGAGCC CTTGTCTGAG ACTGAACCTC CTGAGAAGGG GCCCCTAGCAGCGGTCAGAG 1860 GTCCTGTCTG GATGGAGGCT GGAGGCTCCC CCCTCAACCC CTCTGCTCAGTGCCTGTGGG 1920 GAGCAGCCTC TACCCTCAGC ATCCTGG 1947 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1944 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID No. 3: AGCAGCTCTG TAATGCGCTT GTGGTTTCAG ATGTGGGCGG CCTGTGTGAACCTGTCGTGC 60 AAAGCTCACG TCACCAACTG CTGCAGTTAT CTCCTGAATC AGGCTGAGGGTCTTTGCTGT 120 GCACCCAGAG ATAGTTGGGT GACAAATCAC CTCCAGGTTG GGGATGCCTCAGACTTGTGA 180 TGGGACTGGG CAGATGCATC TGGGAAGGCT GGACCTTGGA GAGTGAGGCCCTGAGGCGAG 240 ACATGGGCAC CTGGCTCCTG GCCTGCATCT GCATCTGCAC CTGTGTCTGCTTGGGAGT CT 300 CTGTCACAGG GGAAGGACAA GGGCCAAGGT CTAGAACCTT CACCTGCCTCACCAACAACA 360 TTCTCAGGAT CGATTGCCAC TGGTCTGCCC CAGAGCTGGG ACAGGGCTCCAGCCCCTGGC 4 20 TCCTCTTCAC CAGCAACCAG GCTCCTGGCG GCACACATAA GTGCATCTTGCGGGGCAGTG 4 80 AGTGCACCGT CGTGCTGCCA CCTGAGGCAG TGCTCGTGCC ATCTGACAATTTCACCATCA 540 CTTTCCACCA CTGCATGTCT GGGAGGGAGC AGGTCAGCCT GGTGGACCCGGAGTACCTGC 600 CCCGGAGACA CGTTAAGCTG GACCCGCCCT CTGACTTGCA GAGCAACATCAGTTCTGGCC 660 ACTGCATCCT GACCTGGAGC ATCAGTCCTG CCTTGGAGCC AATGACCACACTTCTCAGCT 720 ATGAGCTGGC CTTCAAGAAG CAGGAAGAGG CCTGGGAGGC CCAGCACAGGGATCACATTG TCGGGGTGAC 780 65 840 CTGGCTTATA CTTGAAGCCT TTGAGCTGGA CCCTGGCTTTATCCATGAGG CCAGGCTGCG TGTCCAGATG GCCACACTGG AGGATGATGT GGTAGAGGAGGAGCGTTATA 900 CAGGCCAGTG GAGTGAGTGG AGCCAGCCTG TGTGCTTCCA GGCTCCCCAGAGACAAGGCC 960 CTCTGATCCC ACCCTGGGGG TGGCCAGGCA ACACCCTTGT TGCTGTGTCCATCTTTCTCC 1020 TGCTGACTGG CCCGACCTAC CTCCTGTTCA AGCTGTCGCC CAGGGTGAAGAGAATCTTCT 1080 ACCAGAACGT GCCCTCTCCA GCGATGTTCT TCCAGCCCCT CTACAGTGTACACAATGGGA 1140 ACTTCCAGAC TTGGATGGGG GCCCACAGGG CCGGTGTGCT GTTGAGCCAGGACTGTGCTG 1200 GCACCCCACA GGGAGCCTTG GAGCCCTGCG TCCAGGAGGC CACTGCACTGCTCACTTGTG 1260 GCCCAGCGCG TCCTTGGAAA TCTGTGGCCC TGGAGGAGGA ACAGGAGGGCCCTGGGACCA 1320 GGCTCCCGGG GAACCTGAGC TCAGAGGATG TGCTGCCAGC AGGGTGTACGGAGTGGAGGG 1380 TACAGACGCT TGCCTATCTG CCACAGGAGG ACTGGGCCCC CACGTCCCTGACTAGGCCGG 1440 CTCCCCCAGA CTCAGAGGGC AGCAGGAGCA GCAGCAGCAG CAGCAGCAGCAGCAACAACA 1500 ACAACTACTG TGCCTTGGGC TGCTATGGGG GATGGCACCT CTCAGCCCTCCCAGGAAACA 1560 CACAGAGCTC TGGGCCCATC CCAGCCCTGG CCTGTGGCCT TTCTTGTGACCATCAGGGCC 1620 TGGAGACCCA GCAAGGAGTT GCCTGGGTGC TGGCTGGTCA CTGCCAGAGGCCTGGGCTGC 1680 ATGAGGACCT CCAGGGCATG TTGCTCCCTT CTGTCCTCAG CAAGGCTCGGTCCTGGACAT 1740 TCTAGGTCCC TGACTCGCCA GATGCATCAT GTCCATTTTG GGAAAATGGACTGAAGTTTC 1800 TGGAGCCCTT GTCTGAGACT GAACCTCCTG AGAAGGGGCC CCTAGCAGCGGTCAGAGGTC 1860 CTGTCTGGAT GGAGGCTGGA GGCTCCCCCC TCAACCCCTC TGCTCAGTGCCTGTGGGGAG 1920 CAGCCTCTAC CCTCAGCATC CTGG 1944 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF SEQUÊNCI A: (A) LENGTH: 1862 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: straight (D) TOPOLOGY: linear 66 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: No. 4: AGCAGCTCTG TAATGCGCTT GTGGTTTCAG ATGTGGGCGG CCTGTGTGAACCTGTCGTGC 60 AAAGCTCACG TCACCAACTG CTGCAGTTAT CTCCTGAATC AGGCTGAGGGTCTTTGCTGT 120 GCACCCAGAG ATAGTTGGGT GACAAATCAC CTCCAGGTTG GGGATGCCTCAGACTTGTGA 180 TGGGACTGGG CAGATGCATC TGGGAAGGCT GGACCTTGGA GAGTGAGGCCCTGAGGCGAG 240 ACATGGGCAC CTGGCTCCTG GCCTGCATCT GCATCTGCAC CTGTGTCTGCTTGGGAGTCT 300 CTGTCACAGG GGAAGGACAA GGGCCAAGGT CTAGAACCTT CACCTGCCTCACCAACAACA 360 TTCTCAGGAT CGATTGCCAC TGGTCTGCCC CAGAGCTGGG ACAGGGCTCCAGCCCCTGGC 420 TCCTCTTCAC CAGCAACCAG GCTCCTGGCG GCACACATAA GTGCATCTTGCGGGGCAGTG 480 AGTGCACCGT CGTGCTGCCA CCTGAGGCAG TGCTCGTGCC ATCTGACAATTTCACCATCA 540 CTTTCCACCA CTGCATGTCT GGGAGGGAGC AGGTCAGCCT GGTGGACCCGGAGTACCTGC 600 CCCGGAGACA CGTTAAGCTG GACCCGCCCT CTGACTTGCA GAGCAACATCAGTTCTGGCC 660 ACTGCATCCT GACCTGGAGC ATCAGTCCTG CCTTGGAGCC AATG ACCACACTTCTCAGCT 720 ATGAGCTGGC CTTCAAGAAG CAGGAAGAGG CCTGGGAGCA GGCCCAGCACAGGGATCACA 780 TTGTCGGGGT GACCTGGCTT ATACTTGAAG CCTTTGAGCT GGACCCTGGCTTTATCCATG 840 AGGCCAGGCT GCGTGTCCAG ATGGCCACAC TGGAGGATGA TGTGGTAGAGGAGGAGCGTT 900 ATACAGGCCA GTGGAGTGAG TGGAGCCAGC CTGTGTGCTT CCAGGCTCCCCAGAGACAAG 960 GCCCTCTGAT CCCACCCTGG GGGTGGCCAG GCAACACCCT TGTTGCTGTGTCCATCTTTC 1020 TCCTGCTGAC TGGCCCGACC TACCTCCTGT TCAAGCTGTC GCCCAGACTTGGATGGGGGC 1080 CCACAGGGCC GGTGTGCTGT TGAGCCAGGA CTGTGCTGGC ACCCCACAGGGAGCCTTGGA 1140 GCCCTGCGTC CAGGAGGCCA CTGCACTGCT CACTTGTGGC CCAGCGCGTCCTTGGAAATC 1200 TGTGGCCCTG GAGGAGGAAC AGGAGGGCCC TGGGACCAGG CTCCCGGGGAACCTGAGCTC 1260 AGAGGATGTG CTGCCAGCAG GGTGTACGGA GTGGAGGGTA CAGACGCTTGCCTATCTGCC 1320 ACAGGAGGAC TGGGCCCCCA CGTCCCTGAC TAGGCCGGCT CCCCCAGACTCAGAGGGCAG 1380 CAGGAGCAGC AGCAGCAGCA GCAGCAGCAG CAACAACAAC AACTACTGTGCCTTGGGCTG 1440 CTATGGGGGA TGGCACCTCT CAGCCCTCCC AGGAAACACA CAGAGCTCTGGGCCCATCCC AGCCCTGGCC TGTGGCCTTT 1500 1560 CTG CTTGTGACCA TCAGGGCCTG GAGACCCAGCAAGGAGTTGC GGTGCTG GCTGGTCACT GCCAGAGGCC TGGGCTGCAT GAGGACCTCCAGGGCATGTT 1620 GCTCCCTTCT GTCCTCAGCA AGGCTCGGTC CTGGACATTC TAGGTCCCTGACTCGCCAGA 1680 67 TGCATCATGT CCATTTTGGG AAAATGGACT GAAGTTTCTG GAGCCCTTGTCTGAGACTGA 1740 ACCTCCTGAG AAGGGGCCCC TAGCAGCGGT CAGAGGTCCT GTCTGGATGGAGGCTGGAGG 1800 CTCCCCCCTC AACCCCTCTG CTCAGTGCCT GTGGGGAGCA GCCTCTACCCTCAGCATCCT 1860 GG 1862 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCTGGACCTT GGAGAGTGAG GCCCTGAGGC GAGACATGGG CACCTGGCTCCTGGCCTGCA 60 TCTGCATCTG CACCTGTGTC TGCTTGGGAG TCTCTGTCAC AGGGGAAGGACAAGGGCCAA 120 GGTCTAGAAC CTTCACCTGC CTCACCAACA ACATTCTCAG GATCGATTGCCACTGGTCTG 180 CCCCAGAGCT GGGACAGGGC TCCAGCCCCT GGCTCCTCTT CACCAG 226 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 513 base pairs (B) TYPE: acid (c) STRANDEDNESS: SEQ ID NO: 6: GCTGGACCTT GGAGAGTGAG GCCCTGAGGC GAGACATGGG CACCTGGCTCCTGGCCTGCA 60 TCTGCATCTG CACCTGTGTC TGCTTGGGAG TCTCTGTCAC AGGGGAAGGACAAGGGCCAA 120 GGTCTAGAAC CTTCACCTGC CTCACCAACA ACATTCTCAG GATCGATTGCCACTGGTCTG 180 CCCCAGAGCT GGGACAGGGC TCCAGCCCCT GGCTCCTCTT CACCAGCAACCAGGCTCCTG 240 GCGGCACACA TAAGTGCATC TTGCGGGGCA GTGAGTGCAC CGTCGTGCTGCCACCTGAGG 300 CAGTGCTCGT GCCATCTGAC AATTTCACCA TCACTTTCCA CCACTGCATGTCTGGGAGGG 360 AGCAGGTCAG CCTGGTGGAC CCGGAGTACC TGCCCCGGAG ACACGCTGGACCCGCCCTCT 420 GACTTGCAGA GCAACATCAG TTCTGGCCAC TGCATCCTGA CCTGGAGCATCAGTCCTGCC 480 TTGGAGCCAA TGACCACACT TCTCAGCTAT GAG 513 (2) INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: (A) LENGTH: 513 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GCTGGACCTT GGAGAGTGAG GCCCTGAGGC GAGACATGGG CACCTGGCTCCTGGCCTGCA 60 TCTGCATCTG CACCTGTGTC TGCTTGGGAG TCTCTGTCAC AGGGGAAGGACAAGGGCCAA 120 GGTCTAGAAC CTTCACCTGC CTCACCAACA ACATTCTCAG GATCGATTGCCACTGGTCTG 180 CCCCAGAGCT GGGACAGGGC TCCAGCCCCT GGCTCCTCTT CACCAGCAACCAGGCTCCTG 240 69 GCGGCACACA TAAGTGCATC TTGCGGGGCA GTGAGTGCAC CGTCGT6CTGCCACCTGAGG 300 CAGTGCTCGT GCCATCTGAC AATTTCACCA TCACTTTCCA CCACTGCATGTCTGGGAGGG 360 AGCAGGTCAG CCTGGTGGAC CCGGAGTACC TGCCCCGGAG ACACGAGCAACATCAGTTCT 420 GGCCACTGCA TCCTGACCTG GAGCATCAGT CCTGCCTTGG AGCCAATGACCACACTTCTC 480 AGCTATGAGC TGGCCTTCAA GAAGCAGGAA GAG 513 (2) INFORMATION FOR SEQ ID NO 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY : linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GCAGGTGGGG ACCCATG 17 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 19 base pairs ( B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 70 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9: AGGCTTGACA TCGGACAAC 19 SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 nucleotides (B) ) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 71 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 11: CTGCTTCAAT CCTGGGGAA 19 (2) INFORMATION SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLE (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) SEQUENCE CHARACTERISTICS: (a) LENGTH: another nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 12: GTGAGTTCCC CAGGATTGA 19 (2) INFORMATION FOR SEQ ID NO. ) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 72 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 13: CAAGGCCCTG CTCCAAA 17 (2) INFORMATION SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : (a) LENGTH: 20 base pairs (B) (SEQ ID NO: 14): SEQ ID NO: 14: TGGGGCTTCA GCCTCACATG 20 (2) INFORMATION FOR SEQ ID NO: TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 73 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 15: TATGTAGAGT GGGGA GTCTA 20 (2) INFORMATION FOR SEQ ID NO 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TGTATTCTCG AGGGCTGAG 19 (2) INFORMATION FOR SEQ ID NO. 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 74 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 17: TGAGGTGAAC AGGGGAGAA SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear ( ii) MOLECULE TYPE: another nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CCCTGGGCCC TTCATGT 17 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) COMPRIMENT (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 75 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 : (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: ACAAGGGCGG CCTTTGAT 18 (2) INFORMATION FOR SEQ ID NO. 20: (i) SEQUENCE CHARACTERISTICS: : linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: AGGGACGAGG TGGGCGGAC 19 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 76 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CCTGCCCCCC ATGTTCTT 18 (2) INFORMATION FOR SEQ ID NO 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: lin (i) SEQUENCE CHARACTERISTICS: (a) LENGTH: (i) SEQUENCE CHARACTERISTICS: (i) SEQUENCE CHARACTERISTICS: (i) SEQUENCE CHARACTERISTICS: (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid 77 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GGACATGATG CATCTGGCG 19 (2) INFORMATION FOR SEQ ID NO 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1944 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AGCAGCTCTG TAATGCGCTT GTGGTTTCAG ATGTGGGCGG CCTGTGTGAACCTGTCGTGC 60 AAAGCTCACG TCACCAACTG CTGCAGTTAT CTCCTGAATC AGGCTGAGGGTCTTTGCTGT 120 GCACCCAGAG ATAGTTGGGT GACAAATCAC CTCCAGGTTG GGGATGCCTCAGACTTGTGA 180 TGGGACTGGG CAGATGCATC TGGGAAGGCT GGACCTTGGA GAGTGAGGCCCTGAGGCGAG 240 ACATGGGCAC CTGGCTCCTG GCCTGCATCT L CATCTGCAC CTGTGTCTGCTTGGGAGTCT 300 CTGTCACAGG GGAAGGACAA GGGCCAAGGT CTAGAACCTT CACCTGCCTCACCAACAACA 360 TTCTCAGGAT CGATTGCCAC TGGTCTGCCC CAGAGCTGGG ACAGGGCTCCAGCCCCTGGC 420 TCCTCTTCAC CAGCAACCAG GCTCCTGGCG GCACACATAA GTGCATCTTGCGGGGCAGTG 480 AGTGCACCGT CGTGCTGCCA CCTGAGGCAG TGCTCGTGCC ATCTGACAATTTCACCATCA 540 CTTTCCACCA CTGCATGTCT GGGAGGGAGC AGGTCAGCCT GGTGGACCCGGAGTACCTGC 600 CCCGGAGACA CGTTAAGCTG GACCCGCCCT CTGACTTGCA GAGCAACATCAGTTCTGGCC 660 ACTGCATCCT GACCTGGAGC ATCAGTCCTG CCTTGGAGCC AATGACCACACTTCTCAGCT 720 ATGAGCTGGC CTTCAAGAAG CAGGAAGAGG CCTGGGAGGC CCAGCACAGGGATCACATTG 780 78 TCGGGGTGAC CTGGCTTATA CTTGAAGCCT TTGAGCTGGA CCCTGGCTTTATCCATGAGG 840 CCAGGCTGCG TGTCCAGATG GCCACACTGG AGGATGATGT GGTAGAGGAGGAGCGTTATA 900 CAGGCCAGTG GAGTGAGTGG AGCCAGCCTG TGTGCTTCCA GGCTCCCCAGAGACAAGGCC 950 CTCTGATCCC ACCCTGGGGG TGGCCAGGCA ACACCCTTGT TGCTGTGTCCATCTTTCTCC 1020 TGCTGACTGG CCCGACCTAC CTCCTGTTCA AGCTGTCGCC CAGGGTGAAGAGAATCTTCT 1030 ACCAGAACGT GCCCTCTCCA GCGATGTTCT TCCAGCCCCT CTACAGTGTAC ACAATGGGA 1140 ACTTCCAGAC TTGGATGGGG GCCCACAGGG CCGGTGTGCT GTTGAGCCAGGACTGTGCTG 1200 GCACCCCACA GGGAGCCTTG GAGCCCTGCG TCCAGGAGGC CACTGCACTGCTCACTTGTG 1250 GCCCAGCGCG TCCTTGGAAA TCTGTGGCCC TGGAGGAGGA ACAGGAGGGCCCTGGGACCA 1320 GGCTCCCGGG GAACCTGAGC TCAGAGGATG TGCTGCCAGC AGGGTGTACGGAGTGGAGGG 1330 TACAGACGCT TGCCTATCTG CCACAGGAGG ACTGGGCCCC CACGTCCCTGACTAGGCCGG 1440 CTCCCCCAGA CTCAGAGGGC AGCAGGAGCA GCAGCAGCAG CAGCAGCAGCAGCAACAACA 1500 ACAACTACTG TGCCTTGGGC TGCTATGGGG GATGGCACCT CTCAGCCCTCCCAGGAAACA 1550 CACAGAGCTC TGGGCCCATC CCAGCCCTGG CCTGTGGCCT TTCTTGTGACCATCAGGGCC 1620 TGGAGACCCA GCAAGGAGTT GCCTGGGTGC TGGCTGGTCA CTGCCAGAGGCCTGGGCTGC 1680 ATGAGGACCT CCAGGGCATG TTGCTCCCTT CTGTCCTCAG CAAGGCTCGGTCCTGGACAT 1740 TCTAGGTCCC TGACTCGCCA GATGCATCAT GTCCATTTTG GGAAAATGGACTGAAGTTTC 1800 TGGAGCCCTT GTCTGAGACT GAACCTCCTG AGAAGGGGCC CCTAGCAGCGGTCAGAGGTC 1860 CTGTCTGGAT GGAGGCTGGA GGCTCCCCCC TCAACCCCTC TGCTCAGTGCCTGTGGGGAG 1920 CAGCCTCTAC CCTCAGCATC CTGG 1944 (2) INFORMATION FOR SEQ ID NO: 25: (i) AC SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear 79 (ii) TYPE OF MOLECULE: other nucleic acid (xi) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF SEQ ID NO: 25: GCTGGACCTT GGAGAGTG 18 (2) INFORMATION FOR SEQ ID NO. CHEMISTRY: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GTCTCAGACA AGGGCTCCAG 20 80 (2) INFORMATION FOR SEQ ID NO. 27: SEQUENCE CHARACTERISTICS: (A) LENGTH: 501 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Met Gly Thr Trp Leu Leu Ala Cys Ile Cys Ile Cys Thr Cys Vai Cys 1 5 10 15 Leu Gly Will Be Go Gly Gly Gly Gly Gly Pro Arg Arg Ser Thr 20 25 30 Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile Asp Cys His Trp Ser 35 40 45 Ala Pro Glu Leu Gly Gin Gly Ser Ser Pro Trp Leu Leu Phe Thr Ser 50 55 60 Asn Gin Ala Pro Gly Gly Thr His Lys Cys Ile Leu Arg Gly Ser Glu 65 70 75 80 Cys Thr Go Leu Pro Pro Glu Ala Leu Pro Will Be Asp Asn 85 90 95 Phe Thr Ile Thr Phe His His Cys Met Ser Gly Arg Glu Gin Will Be 100 105 110 Leu Will Asp Pro Glu Tyr Leu Pro Arg Arg His Vai Lys Leu Asp Pro 115 120 125 Pro Ser Asp Leu Gin Ser Asn Ile Ser Ser Gly His Cys Ile Leu Thr 130 135 140 81

Trp Ser Ile Ser Pro Ala Leu Glu Pro Met Thr Thr Leu Leu Ser Tyr 145 150 155 160 Glu Leu Ala Phe Lys Lys Gin Glu Glu Ala Trp Glu Gin Ala Gin His 165 170 175 Arg Asp His Ile val Gly Val Thr Trp Leu Ile Leu Glu Ala Phe Glu 180 185 190 Leu Asp Pro Gly Phe Ile His Glu Ala Arg Leu Arg Val Gin Met Ala 195 200 205 Thr Leu Glu Asp Asp Val Val Glu Glu Glu Arg Tyr Thr Gly Gin Trp 210 215 220 Ser Glu Trp Ser Gin Pro Val Cys Phe Gin Ala Pro Gin Arg Gin Gly 225 230 235 240 Pro Leu Ile Pro Pro Trp Gly Trp Pro Gly Asn Thr Leu Val Ala Val 245 250 255 Ser Ile Phe Leu Leu Leu Thr Gly Pro Thr Tyr Leu Leu Phe Lys Leu 260 265 270 Ser Pro Arg Val Lys Arg Ile Phe Tyr Gin Asn Val Pro Ser Pro Al 275 280 285 Met Phe Phe Gin Pro Leu Tyr Ser Val His Asn Gly Asn Phe Gin Thr 290 295 300 Trp Met Gly Ala His Arg Ala Gly Val Leu Leu Ser Gin Asp Cys Ala 305 310 315 320 Gly Thr Pro Gly Gly Ala Leu Glu Pro Cys Val Gin Glu Ala Thr Ala 325 330 335 Leu Leu Thr Cys Gly Pro Ala Arg Pro Trp Lys Ser Val Ala Leu Glu 340 345 350 Glu Glu Gin Glu Gly Pro Gly Thr Arg Leu Pro Gly Asn Leu Ser Ser 355 360 365 Glu Asp Go Leu Pro Ala Gly Cys Thr Glu Trp Arg Val Gin Thr Leu 370 375 380 Wing Tyr Leu Pro Gin Glu Asp Trp Ala Pro Thr Ser Leu Thr Arg Pro 385 390 395 400 Ala Pro Pro Asp Ser Glu Gly Ser Arg Ser Ser Ser Ser Ser Ser Ser Ser 405 410 415 Ser Ser Asn Asn Asn Asn Tyr Cys Ala Leu Gly Cys Tyr Gly Gly Trp 420 425 430 His Leu Ser Ala Leu Pro Gly Asn Thr Gin Ser Ser Gly Pro Ile Pro 435 440 445 Ala Leu Cys Gly Leu Ser Cys Asp His Gin Gly Leu Glu Thr Gin 4 50 455 4 60 82

Gin Gly 4 65 Vai Ala Trp Val 470 Leu Ala Gly His Cys 475 Gin Arg Pro Gly Leu 4 80 His Glu Asp Leu Gin 485 Gly Met Leu Leu Pro 490 Ser Val Leu Ser Lys 495 Ala Arg Ser Trp Thr 500 Phe (2) SEQUENCE CHARACTERISTICS: (A) LENGTH: 501 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Met Gly Thr Trp Leu Leu Ala Cys Ile Cys Ile Cys Thr Cys Val Cys 1 5 10 15 Leu Gly Val Ser Val Thr Gly Gly Gly Gin Gly Pro Arg Ser Arg Thr 20 25 30 Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile Asp Cys His Trp Ser 35 40 45 Ala Pro Glu Leu Gly Gin Gly Ser Ser Pro Trp Leu Leu Phe Thr Ser 50 55 60 Asn Gin Ala Pro Gly Gly Thr His Lys Cys Ile Leu Arg Gly Ser Glu 65 70 75 80 Cys Thr Val Val Leu Pro Pro Glu Ala Val Leu Val Pro Ser Asp Asn 85 90 95 Phe Thr Ile Thr Phe His His Cys Met Ser Gly Arg Glu Gin Val Ser 100 105 110 83

Leu Vai Asp Pro 'Glu i Tyr Leu i Pro Arg Arg His Vai Lys Leu Asp Pro 115 120 125 Pro Ser Asp Leu Gin i Ser Asn Ile Ser Ser Gly His Cys Ile Leu Thr 130 135 140 Trp Ser Ile Ser Pro > Ala Leu Glu Pro Met Thr Thr Leu Leu Ser Tyr 145 150 155 160 Glu Leu Ala Phe Lys Lys Gin Glu Glu Ala Trp Glu Gin Ala Gin His 165 170 175 Arg Asp His Ile Go Gly Go Thr Trp Leu Ile Leu Glu Ala Phe Glu 180 185 190 Leu Asp Pro Gly Phe Ile His Glu Ala Arg Leu Arg Vai Gin Met Ala 195 200 205 Thr Leu Glu Asp Asp Asp Glu Glu Glu Arg Tyr Thr Gly Gin Trp 210 215 220 Ser Glu Trp Ser Gin Pro Val Cys Phe Gin Wing Pro Gin Arg Gin Gly 225 230 235 240 Pro Leu Ile Pro Pro Trp Gly Trp Pro Gly Asn Thr Leu Vai Ala Go 245 250 255 Ser Ile Phe Leu Leu Leu Thr Gly Pro Thr Tyr Leu Leu Phe Lys Leu 260 265 270 Ser Pro Arg Go Lys Arg Ile Phe Tyr Gin Asn Go Pro Pro Ala 275 280 285 Met Phe Phe Gin Pro Leu Tyr Ser Vai His Asn Gly Asn Phe Gin Thr 290 295 300 Trp Met Gly Ala His Arg Ala Gly Vai Leu Leu Ser Gin Asp Cys Ala 305 310 315 320 Gly Thr Pro G in Gly Ala Leu Glu Pro Cys Go Glu Ala Thr Ala 325 330 335 Leu Leu Thr Cys Gly Pro Ala His Pro Trp Lys Ser Vai Ala Leu Glu 340 345 350 Glu Glu Gin Glu Gly Pro Gly Thr Arg Leu Pro Gly Asn Leu Ser Ser 355 360 365 Glu Asp Go Leu Pro Ala Gly Cys Thr Glu Trp Arg Vai Gin Thr Leu 370 375 380 Ala Tyr Leu Pro Gin Glu Asp Trp Ala Pro Thr Ser Leu Thr Arg Pro 385 390 395 400 84

Ala Pro Pro Asp Ser Glu Gly Ser Arg Ser Ser Ser Ser Ser Ser Ser Ser 405 410 415 Ser Ser Asn Asn Asn Asn Tyr Cys Ala Leu Gly Cys Tyr Gly Gly Trp 420 425 430 His Leu Ser Ala Leu Pro Gly Asn Thr Gin Ser Ser Gly Pro Ile Pro 435 440 445 Ala Leu Ala Cys Gly Leu Ser Cys Asp His Gin Gly Leu Glu Thr Gin 450 455 4 60 Gin Gly Go Ala Trp Go Leu Ala Gly His Cys Gin Arg Pro Gly Leu 465 470 475 480 His Glu Asp Leu Gin Gly Met Leu Leu Pro Ser Vai Leu Ser Lys Wing 485 490 4 95

(A) LENGTH: 500 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (D) TOPOLOGY: (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: Met 1 Gly Thr Trp Leu 5 Leu Ala Cys Ile Cys 10 Ile Cys Thr Cys Val 15 Cys Leu Gly Will Be 20 Go Thr Gly Glu Gly 25 Gin Gly Pro Arg Ser 30 Arg Thr Phe Thr Cys 35 Leu Thr Asn Asn Ile 40 Leu Arg Ile Asp Cys 45 His Trp Ser 85

Ala Pro 50 Glu Leu Gly Gin Gly 55 Ser As η 65 Gin Ala Pro Gly Gly 70 Thr His Cys Thr Vai Vai Leu 85 Pro Pro Glu Phe Thr Ile Thr 100 Phe His His Cys Leu Vai Asp 115 Pro Glu Tyr Leu Pro 120 Pro Ser 130 Asp Leu Gin Ser Asn 135 Ile Trp 145 Ser Ile Ser Pro Ala 150 Leu Glu Glu Leu Ala Phe Lys 165 Lys Gin Glu Asp His Ile Val 180 Gly Val Thr Trp Asp Pro Gly 195 Phe Ile His Glu Ala 200

Ser Pro Trp Leu Leu Phe Thr Ser 60 Lys Cys Ile Leu Arg Gly Ser Glu 75 80 Ala Val Leu Val Pro Ser Asp Asn 90 95 Met Ser Gly Arg Glu Gin Val Ser 105 110 Arg Arg His Val Lys Leu Asp Pro 125 Ser Ser Gly His Cys Ile Leu Thr 140 Pro Met Thr Thr Leu Leu Ser Tyr 155 160 Glu Ala Trp Glu Ala Gin His Arg 170 175 Leu Ile Leu Glu Ala Phe Glu Leu 185 190 Arg Leu Arg Val Gin Met Ala Thr 205

Leu Glu Asp Asp Val Val Glu Glu 210 215 Glu Trp Ser Gin Pro Val Cys Phe 225 230 Leu Ile Pro Pro Trp Gly Trp Pro 245 Ile Phe Leu Leu Leu Thr Gly Pro 260 Pro Arg Val Lys Arg Ile Phe Tyr 275 280 Phe Phe Gin Pro Leu Tyr Ser Val 290 295 Met Gly Ala His Arg Ala Gly Val 305 310

Glu Arg Tyr Thr 220 Gly Gin Trp Ser Gin Ala Pro 235 Gin Arg Gin Gly Pro 240 Gly Asn 250 Thr Leu Val Ala Val 255 Ser Thr 265 Tyr Leu Leu Phe Lys 270 Leu Ser Gin Asn Val Pro Ser 285 Pro Ala Met His Asn Gly Asn 300 Phe Gin Thr Trp Leu Leu Ser 315 Gin Asp Cys Ala Gly 320 86

Thr Pro Gin Gly Ala Leu Glu Pro Cys Go Gin Glu Ala Thr Ala Leu 325 330 335 Leu Thr Cys Gly Pro Ala Arg Pro Trp Lys Ser Vai Ala Leu Glu Glu 340 345 350 Glu Glu Glu Gly Pro Gly Thr Arg Leu Pro Gly Asn Leu Ser Ser Glu 355 360 365 Asp Go Leu Pro Ala Gly Cys Thr Glu Trp Arg Vai Gin Thr Leu Ala 370 375 380 Tyr Leu Pro Gin Glu Asp Trp Ala Pro Thr Ser Leu Thr Arg Pro Ala 385 390 395 400 Pro Pro Asp Ser Glu Gly Ser Arg Ser Ser Ser Ser Ser Ser Ser Ser Ser 405 410 415 Ser Asn Asn Asn Asn Tyr Cys Ala Leu Gly Cys Tyr Gly Gly Trp His 4 20 425 4 30 Leu Ser Ala Leu Pro Gly Asn Thr Gin Ser Ser Gly Pro Ile Pro Ala 435 440 445 Leu Ala Cys Gly Leu Ser Cys Asp His Gin Gly Leu Glu Thr Gin Gin 450 455 4 60 Gly Go Ala Trp Go Leu Ala Gly His Cys Gin Arg Pro Gly Leu His 465 470 475 4 80 Glu Asp Leu Gin Gly Het Leu Leu Pro Ser Vai Leu Ser Lys Ala Arg 485 490 495

Serum Trp Thr Phe 500 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 286 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (Ii) MOLECULE TYPE: peptide (xi) SEQ ID NO: 30: Met Gly Thr Trp Leu Leu Ala Cys Ile Cys Ile Cys Thr Cys Val Cys 1 5 10 15 Leu Gly Will Be Go To Thr Gly Glu Gly Gin Gly Pro Arg Ser Arg Thr 20 25 30 Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile Asp Cys His Trp Ser 35 40 45 Ala Pro Glu Leu Gly Gin Gly Ser Ser Pro Trp Leu Leu Phe Thr Ser 50 55 60 Asn Gin Ala Pro Gly Gly Thr His Lys Cys Ile Leu Arg Gly Ser Glu 65 70 75 80 Cys Thr Vai Vai Leu Pro Pro Glu Ala Vai Leu Vai Pro Ser Asp Asn 85 90 95 Phe Thr Ile Thr Phe His His Cys Met Ser Gly Arg Glu Gin Val Ser 100 105 110 Leu Asp Pro Glu Tyr Leu Pro Arg Arg His Val Lys Leu Asp Pro 115 120 125 Pro Ser Asp Leu Gin Ser Asn Ile Ser Ser Gly His Cys Ile Leu Thr 130 135 140 Trp Ser Ile Ser Pro Ala Leu Glu Pro Met Thr Thr Leu Leu Ser Tyr 145 150 155 160 Glu Leu Ala Phe Lys Lys Gin Glu Glu Ala Trp Glu Gin Ala Gin His 165 170 175 Arg Asp His Ile Go Gly Go Go Thr Trp Leu Ile Leu Glu Ala Phe Glu 180 185 190 Leu Asp Pro Gly Phe Ile His Glu Ala Arg Leu Arg Val Gin Met Ala 195 200 205 Thr Leu Glu Asp Asp Go Vai Glu Glu Glu Arg Tyr Thr Gly Gin Trp 210 215 220 Ser Glu Trp Ser Gin Pro Val Cys Phe Gin Ala Pro Gin Arg Gin Gly 225 230 235 240 Pro Leu Ile Pro Pro 'Trp Gly Trp Pro Gly Asn Thr Leu Val Ala Val 245 250 255 Ser Ile Phe Leu Leu Leu Thr Gly Pro Thr Tyr Leu Leu Phe Lys Leu 260 265 270 Ser Pro Arg Arg Gly 'Trp Gly Pro Thr Gly Pro Val Cys Cys 275 280 285 88 (2) INFORMATION FOR SEQ ID N ° 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 31: Met Gly Thr Trp Leu Leu Ala Cys Ile Cys Ile Cys Thr Cys Vai Cys 1 5 10 15 Leu Gly Will Be Go Thr Gly Gly Gly Gin Gly Pro Arg Ser Arg Thr 20 25 30 Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile Asp Cys His Trp Ser 35 40 (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 128 amino acids (B) TYPE: (a) LENGTH: (A) LENGTH: 128 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 89 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Met Gly Thr Trp Leu Leu Ala Cys Ile Cys Ile Cys Thr Cys Vai Cys 1 5 10 15 Leu Gly Will Be Go Gly Gly Gly Gly Gly Pro Arg Arg Ser Thr 20 25 30 Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile Asp Cys His Trp Ser 35 40 45 Ala Pro Glu Leu Gly Gin Gly Ser Ser Pro Trp Leu Leu Phe Thr Ser 50 55 60 Asn Gin Ala Pro Gly Gly Thr His Lys Cys Ile Leu Arg Gly Ser Glu 65 70 75 80 Cys Thr Go Go Leu Pro Pro Glu Ala Go Leu Go Pro Be Asp Asn 85 90 95 Phe Thr Ile Thr Phe His His Cys Met Ser Gly Arg Glu Gin Will Be 100 105 110 Leu Go Asp Pro Glu Tyr Leu Pro Arg Arg His Ala Gly Pro Ala Leu 115 120 125 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 150 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Met 1 Gly Thr Trp Leu 5 Leu Ala Cys Ile Cys 10 Ile Cys Thr Cys 15 Cys Leu Gly Will Be 20 Thr Gly Glu Gly 25 Gin Gly Pro Arg Ser 30 Arg Thr 90

Phe Thr Cys Leu Thr Asn Asn Ile Leu Arg Ile Asp Cys His Trp Ser 35 40 45 Ala Pro Glu Leu Gly Gin Gly Ser Ser Pro Trp Leu Leu Phe Thr Ser 50 55 60 Asn Gin Ala Pro Gly Gly Thr His Lys Cys Ile Leu Arg Gly Ser Glu 65 70 75 80 Cys Thr Val Val Leu Pro Pro Glu Ala Val Leu Val Pro Ser Asp Asn 85 90 95 Phe Thr Ile Thr Phe His His Cys Met Ser Gly Arg Glu Gin Val Ser 100 105 110 Leu Val Asp Pro Glu Tyr Leu Pro Arg Arg His Glu Gin His Gin Phe 115 120 125

Trp Pro Leu His Pro Asp Leu Glu His Gin Ser Cys Leu Gly Ala Asn 130 13S 140

Asp His Thr Ser Gin Leu 145 150

Lisbon, August 6, 2010 91

Claims (23)

An 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, wherein the nucleotides corresponding to positions 613 to 641 of the wild-type IL-9 receptor (SEQ ID NO: 1) were deleted, isolated nucleic acid encoding the human interleukin-9 (IL-9) receptor, wherein the nucleotides corresponding to positions 613 to 617 of the receptor wild-type IL-9 (SEQ ID NO: 1) were deleted, their fragments containing the deletion and their deletion containing complements. An isolated nucleic acid encoding the human interleukin-9 (IL-9) receptor of claim 1, wherein the nucleotides corresponding to positions 613 to 641 of the wild-type IL-9 receptor (SEQ ID NO: 1) were deleted . The isolated nucleic acid of claim 1, wherein the deletion-containing fragment encodes a protein or peptide that binds to IL-9. The isolated nucleic acid of claim 1, wherein the deletion-containing fragment encodes the amino acid sequence of SEQ ID No. 33. The isolated nucleic acid of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 7. The isolated nucleic acid of claim 1, wherein the nucleotide sequence consists of SEQ ID NO: 7. The isolated nucleic acid of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 6. 8. Isolated nucleic acid of claim 1, wherein the nucleotide sequence consists of SEQ ID NO: 6. The isolated nucleic acid of claim 1, wherein the nucleic acid is DNA or RNA. The isolated nucleic acid of claim 9, wherein the DNA is cDNA. The isolated nucleic acid of claim 9, wherein the RNA is mRNA. A vector comprising the nucleic acid of any one of claims 1 to 11. The vector of claim 12, comprising a plasmid, cosmid or virus. The vector of claim 12 which is a recombinant or expression vector. The vector of claim 12 further comprising a regulatory sequence. The vector of claim 15, wherein the calling sequence is a promoter, ribosome binding site, or transcription termination site. The vector of claim 16, wherein the promoter is selected from the group consisting of human cytomegalovirus promoter, tetracycline inducible promoter, simian virus promoter, Moloney murine leukemia virus long terminal repeat promoter, tumor virus promoter glucocorticoid-inducible murine mammary gland, herpes thymidine kinase promoter, murine and human actin promoters, HTLV 1 and HIV 5 'flanking region 5', human or mouse IL-9 receptor 5 'flanking region, promoter tac protein and Drosophila thermal shock protein (SAR) support conjugation promoter elements. A host cell transformed by the vector of any of claims 12 to 17. The host cell of claim 18 which is transformed by infection, direct uptake, transduction, F-crossing, microinjection or electroporation. The host cell of claim 18 which is selected from the group consisting of bacterial cells, yeast cells, insect cells, plant cells and mammalian cells. The host cell of claim 20 which is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, CHO, RI-1, BW, LH, COS-J, COS-7, BSC1, BSC40, BMT10 cells or S69. The host cell of Claim 20, wherein the yeast cells are selected from the group consisting of Saccharomyces, Pichia, Candida, Hansenula or Torulopsis. An isolated human IL-9 receptor protein encoded by the nucleic acid of Claim 1. The isolated human protein of claim 23, comprising the amino acid sequence SEQ ID No. 33. The isolated human protein of claim 23, consisting of amino acid sequence SEQ ID NO: 33. The isolated human protein of any one of claims 23 to 25, wherein the protein binds to IL-9. The protein of claim 26, wherein the protein is soluble. A method of preparing a protein comprising culturing a host cell transformed with the nucleic acid of any one of claims 1 to 11 under conditions in which the protein encoded by said nucleic acid is expressed. A pharmaceutical composition comprising the isolated nucleic acid of any of claims 1 to 11. A pharmaceutical composition comprising the protein of any of claims 23 to 27. The composition of claims 29 or 30, wherein the composition further comprises a pharmaceutically acceptable carrier. The composition of claims 29 or 30, wherein the composition is formulated for topical administration. The composition of claims 29 or 30, wherein the composition is formulated for inhalation. The composition of claims 29 or 30, wherein the composition is formulated for systemic administration. The composition of claims 29 or 30, wherein the composition is formulated for intravenous, intraperitoneal or intralesional administration. 5/24 HUMAN IL-9 RECEPTOR cDNA a b c d e FIG.1 SEQUENCE RANGE: 243 to 1944 21 1SI 111 1 1)! 1-yl) -3-methyl-1H-imidazol-3-yl] I i i i i i ti i i ti i i i i i i ti i i i i i i i ti i ti t i i ti (I). t 1 t! ! I 1! t t! 1 I, t t t t t t t t t t t t t t t t t t t t t t i i i i i i t i i t i t i t i t t i t! , | , t t t! T I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I- 3/24 TCTACCAGAA CGTGCCCTCT CCAGCGATGT TCTTCCAGCC CCTCTACAGT GTACACAATG GGAACTTCCA GACTTGGATG GGGGCCCACA GGGCCGGTGT GCTGTIGAGC CAGGACTGTG CTGGCACCCC ACAGGGAGCC C - <C. . The "= &gt; r · n u- * λ: o B cg THE CP HERE CP-6 '= &gt; ctZ rO C n Ln The c / 5 cr &gt; 13CJ B CP O ss CVJ CNJ &lt; jd 1 =: & Úõ &lt; ^ 3 CP 12 C_J co ω cj &gt; You are SEQUENCE RANGE: 243 to 1944 251 1 211 11 15! 1 I 121 111 1 151 1 111 III I | | IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII VIII 1 IIIIIIIIIII i TC IACCAGAA CGTGCCCTCT CCAGCGATGT TCTTCCAGCC CCTCTACAGT GTACACAATG GGAACTTCCA GACTTGGATG GGGGCCCACA GGGCCGGTGT GCTGTTGAGC CAGGACTGTG CTCGCACCCC ACAGGGAGCC 9tB [G SffBKUclQ λ 1103 H ι ι ι ι ι ι ι ι ι 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 110 (II) (II): (II): (II): (II): [(I)] | I I I I I FIG. 4: SEQUENCE CHARACTERISTICS: 243 λ 1944 aIII II: II: III: IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII m sg mm mm mm mm &gt; 1 1 I 7/24 I I I I I I I I I I I I I I I | | I I I I I I I RG.5-1 / 24 ACCAGAACGT GCCCTCTCCA GCGATGTTCT TCCAGCCCCT CTACAGTGTA CACAATGGGA ACTTCCAGAC TTGGATGGGG GCCCACAGGG CCGGTGTGCT GTTGAGCCAG GACTGTGCTG GCACCCCACA GGGAGCCTTG 9/24 m O O &lt; 3 Cg rs Coyote | 3 μ c5 co § CP Ca] 10/24 C-9 SEQUENCE RANGE: 243 A 437 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 17 s / τ τ FIG.8 12/24 INTERLEUCIN-9 'I RECEPTOR VARIANS GENERY PHENOTYPE ARG / ARG ARG / HIS HIS / HIS ALLERGIC 1/18 8/18 * 1/18 * NON-ALLERGIC 7/18 1 / 18 0/18 FISHER EXACT TEST P = 0.002 ** * INCLUDES ASTHMATICS ** PRACTICES THAT ALLERGY IS AN AUTHENTIC DOMINANT TRACE FIG.9 13/24 FIG. 10 14/24 14/24 FIG. II 15/24 &quot; s / hlL9 80kD 4H-NMR (DMSO-d6):? 12 exon 2 exon 3 exon 4 exon 5 exon 6 exon 7 exon 8 exon 9 FIG. I4A-1 FIG. I4C-1 FIG. I4G-1 FIG. 141-1 18/24 FIG. I4B-1 FIG. I4D-1 FIG. I4H-1 FIG. I4J-1 19/24 19/24 FI6. Ι4Α-2 FI6. I4C-2 FIG. I4G.-2 FIG. 141-2 20/24 Β FIG. FIG. I4D-2 FIG. I4H-2 FIG. I4J-2 FIG. I5A 21/24 AQGR8AQGH9 GH9 IL9 - m h - m h - m co eo -j PY FI6. I5D im fljl SC m p Stat3 22/24 AQGR8 AQGH9 GH9 no r .....:; ι: ι iL9 - mn-mh-rnn 23/24 CONTROL FIG. 16 dP CELL LINE! 5ng / ml J mlL-9 5 ng / ml hlL-9 HUMAN IL-9 PECEPTQR HRA0 5 POTATOTISM3 SEQUENCE OF IL-9 RECEPTCRM. 0 mi nro «M u ery mm SEQUENCE OF IL-9 Μ0 REC RECEFECT 17 S / 17 2