MXPA97007880A - Novelty chemiocine expressed in eosinofi - Google Patents

Abstract

The present invention relates to novel nucleotide and amino acid sequences for a novel C-C chemokine found initially in a blood cell cDNA library of an individual having Hypereosinophilic Syndrome. The present invention also provides anti-sense molecules for the nucleotide sequences encoding EEC, expression vectors for the production of purified EEC, antibodies capable of binding specifically to EEC, hybridization probes or oligonucleotides for the detection of nucleotide sequences that encode to EEC, diagnostic tests for chemokine activation based on molecules and antibodies of nucleic acid encoding EEC, capable of specifically binding to E

Description

NOVEDOSA OUIMIOCINA EXPRESSED IN EOSINOFILOS TECHNICAL BACKGROUND The present invention relates to novel nucleotide and amino acid sequences of a chemokine found in eosinophils, and to the use of these sequences in the diagnosis and treatment of diseases.

PREVIOUS TECHNIQUE Ouimiocins Chemokines are a family of cytokines that are produced when the immune system responds to antigens that are not its own, such as microorganisms or invading antigens of an incompatible tissue type, and are associated with the trafficking of leukocytes under abnormal conditions. inflammatory or disease. Chemokines mediate the expression of particular adhesion molecules in endothelial cells, and generate gradients of chemoattractant factors that activate specific cell types. In addition, chemokines stimulate the proliferation of specific cell types, and regulate the activation of cells that carry specific receptors. These activities demonstrate a high degree of target cell specificity. Chemokines are small polypeptides, generally about 70-100 amino acids long, 8-11 kD molecular weight and active over a concentration range of 1-100 ng / ml. Initially, these were isolated and purified from inflamed tissues and characterized in relation to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques, and have been characterized by structural as well as functional analysis. Chemokines are related through a four-cysteine motif, which is mainly based on the separation of the first two cysteine residues in the mature molecule. Currently, chemokines are assigned to one of two families, the C-C (a) chemokines and the C-X-C (ß) chemokines. Although there are exceptions, C-X-C chemokines generally activate neutrophils and fibroblasts, whereas C-C chemokines act on a more diverse group of target cells that include monocytes / macrophages, basophils, eosinophils, T lymphocytes, and others. The known chemokines of both families are synthesized by many different cell types, as reviewed in Thompson A. (1994) The Cytokine Handbook, 2nd Ed. Academic Press, NY. The two groups of chemokines in turn will be described. It appears that C-C chemokines have less N-terminal processing than C-X-C chemokines. The known C-C chemokines of human and / or murine include MlP-la and β; 1-309; RANTES and MCP-1. The alpha and beta inflammatory proteins (IP-lc and β) of the macrophage were first purified from the stimulated mouse macrophage cell line, and produced an inflammatory response when injected into normal tissue. At least three distinct and non-allelic genes encode human MIP-IQI and seven different genes encode MIP-ljS. The MlP-la and the MIP-ljS consist of 68-69 amino acids that are approximately 70 percent identical in their secreted acidic, mature forms. Both are expressed in T cells, B cells and stimulated monocytes, in response to mitogens, anti-CD3 and endotoxin, and both polypeptides bind to heparin. Although both molecules stimulate the monocytes, the MIP-IQI chemoattrates the CD-8 subset of T lymphocytes and eosinophils, while the MIP-1J chemoattrates the CD4 subset of T lymphocytes. In the mouse, these proteins are known to stimulate Myelopoiesis 1-309 was cloned from a human d-cell line and showed 42 percent identity amino acids with cloned T cell activation gene 3 (TCA3) from a mouse. There is considerable nucleotide homology between the 5 'flanking regions of these two proteins, and they share an extra pair of cysteine residues that are not found in other chemokines. These similarities suggest that 1-309 and TCA3 are homologous species that have differed over time in both sequence and function.

RANTES is another C-C chemokine that is expressed in T cells (but not in B cells), in platelets, in some tumor cell lines, and in stimulated rheumatoid sinuvial fibroblasts. In the latter, these are regulated by interleukins-1 and -4, the transformation nerve factor and interferon y. The straining cDNA of T cells encodes an 8 kD basic protein that lacks N-linked glycosylation and is capable of affecting lymphocytes, monocytes, basophils and eosinophils. The expression of RANTES mRNA is substantially reduced after stimulation of T cells. The monocyte chemotactic protein (MCP-1) is a protein of 76 amino acids that seems to be expressed in almost all cells and tissues, on stimulation by a variety of agents. The objectives of MCP-1, however, are limited to monocytes and basophils in which it induces a flux of MCP-1 receptor: calcium bound by protein G (Charo I, personal communication). Two other related proteins (MCP-2 and MCP-3) were purified from a human osteosarcoma cell line. MCP-2 and MCP-3 have 62 percent and 73 percent amino acid identity, respectively, with MCP-1, and share their chemoattractant specificity with monocytes. International Publication Number WO 95/17092, published June 29, 1995, and its priority document, Application of the United States of America Serial Number 08 / 208,339, filed on March 8, 1994, discloses the sequence of nucleotides and amino acids of MIP3, a chemokine found in a cDNA library of aortic endothelium that has 66 percent similarity to MIP-la. The chemokine molecules have been reviewed in Schall TJ (1994) Chemotactic Cytokines: Targets for Therapeutic Development, International Business Communications, Southborough, MA, pages 180-270; and in Paul WE (1993) Fundamental Immunology, Raven Press, New York City (NYC), pages 822-826.

Eosinophils Eosinophils are bi- or multinucleated white blood cells containing basophilic or eosinophilic granules formed during their development by highly active golgi and ribosomal machinery. The plasma membrane is not structurally distinct from that of other leukocytes, but is characterized by immunoglobulin (Ig) receptors, particularly IgG and IgE. These cells are formed throughout life from pluripotent stem cells, and play a crucial role in protecting the body's systemic defense of foreign microorganisms and proteins. In comparison to a total of 7,000 white blood cells per microliter of blood, the number of eosinophils is normally about 160 cells per microliter. Eosinophils, which usually last 6 days, are formed and stored in the bone marrow until they are recruited to the site of inflammation or invasion. Eosinophils have a special role in parasitic infections. These attack the parasitic larvae, presumably through their Ig receptors, and undergo degranulation in response to interleukin-5 (IL-5), IL-3, granulocyte / monocyte cell stimulation factor (GM-CSF) produced by activated T cells and host mast cells (Abu-Ghazaleh Rl, Kita H, Gleich GJ (1992) Immunol Ser 57: 137-67) or other factors produced by the parasite. Degranulation releases many active species including the following: 1) hydrolytic enzymes such as peroxidase, acid phosphatase, phospholipase, B glucuronidase, ribonuclease, arylsulfatase and cathepsin; 2) highly reactive superoxides; and 3) major basic protein (MBP), a potent arginine-rich larvicidal polypeptide and cationic eosinophilic protein (Capron M (1992) Mem Inst Oswaldo Cruz 87 (S5): 83-9). Eosinophils are produced in large quantities in people with helminthic infections such as hookworm, schistosomiasis, toxocariasis, trichuriasis, filariasis, strongyloidosis, echinococcosis, cysticercosis, and trichinosis, for example.

Large numbers of eosinophils are also collected in tissues such as the heart, lungs, central nervous system, breasts and skin, where allergic reactions commonly occur. These are chemoattracted at the site of inflammation or invasion by the eosinophilic chemotactic factor, platelet activation factor, complement 5a, or IL-5, which are released by mast cells and basophils during the allergic reaction. Eosinophils neutralize the slow-reacting substance of anaphylaxis (a mixture of leukotrienes) and histamine released by mast cells and basophils; produce the eosinophil-derived inhibitor that prevents mast cell degranulation; and phagocytose the antigen-antibody complexes, all of which down-regulate the hypersensitivity response. Eosinophilia, an excess of eosinophils, ie more than 500 per microliter of blood, is commonly observed in patients with allergies, hay fever, asthma and reactions to drugs as common as aspirin, sulfonamides and penicillins. Eosinophilia is also associated with rheumatoid arthritis and with cancers such as Hodgkins lymphoma, chronic myelogenous leukemia, and carcinomas of the lung, stomach, pancreas, ovaries, uterus, and liver. Eosinophilia can cause tissue damage from excessive degranulation, and is usually treated with glucocorticoid chemotherapy. Eosinophils, their morphology, function and relationship to disease are reviewed, inter alia, in Guyton AC (1991) Textbook of Medical Physiology, WB Saunders Co., Philadelphia PA; Isselbacher KJ et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York City, pages 1437-1504; and Zucker-Franklin D et al. (1988) Atlas of Blood Cells, Function and Pathology, Lea and Febiger, Philadelphia PA. Current techniques for the diagnosis of abnormalities in inflamed or diseased tissues depend mainly on the observation of clinical symptoms or serological analysis of body tissues or fluids for hormones, polypeptides or different metabolites. Patients often do not manifest any clinical symptoms early in the development of the disease. In addition, serological analyzes do not always differentiate between invasive diseases and genetic syndromes that have superimposed or very similar ranges. Current methods for the treatment of inflammatory conditions involve the administration of steroids and other drugs with multiple side effects. The discovery of novel chemokines involved in inflammatory conditions provides the basis for the development of safer and more accurate diagnostic and therapeutic compositions and methods.

DESCRIPTION OF THE INVENTION The present invention relates to novel sequences of nucleotides and amino acids for a chemokine initially found in a cDNA library made from the blood cells of a patient diagnosed with Hypereosinophilic Syndrome at the Mayo Clinic. The new gene, which is known as expressed eosinophilic chemokine, or eec (Incyte Clone 288236), encodes the polypeptide designated EEC, a new member of the C-C chemokine family. The present invention relates to the use of EEC nucleotide and amino acid sequences in the study, diagnosis and treatment of disease states related to the trafficking of leukocytes under abnormal, inflammatory or disease conditions, such as in Eosinophilia. Eosinophilia is defined as a dramatic increase in the number of eosinophils per microliter of blood. This has been observed in the following conditions: collagen vascular diseases such as rheumatoid arthritis, eosinophilic fasciitis, allergic angitis, periarteritis nodosa, and granulomatosis; malignancies such as Hodgkin's lymphoma, mycosis fungoides, chronic myelogenous leukemia and cancer of the lung, stomach, pancreas, ovaries or uterus, helminthic infections such as hookworm, schistosomiasis, toxocariasis, trichuriasis, filariasis, strongyloidosis, echinococcosis, cysticercosis, and trichinosis; hypereosinophilic syndromes such as Loeffler's syndrome, Loeffler's endocardiatis, eosinophilic leukemia, eosinophilic myalgia, and hypereosinophilic idiopathic syndrome; and allergies and asthma. The present invention is based in part on the amino acid homology that shares the EEC with other members of the C-C family of the chemokines, and in part in the presence of nucleotide sequences for the EEC in an eosinophilic cDNA library. The nucleotide and amino acid sequences for EEC have similarity to the nucleotide and amino acid sequences for MIP3 disclosed in International Publication Number WO 95/17092, published June 29, 1995, and its priority document, the Application of the United States. from North America with Serial No. 08 / 208,339, filed March 8, 1994. Therefore, the present invention is based on the discovery of a novel CC chemokine, EEC, which is associated with the trafficking of leukocytes under abnormal conditions, inflammatory or disease. The EEC and the nucleotide sequences that encode it, and the oligonucleotides, nucleic acid peptide (PNA), fragments portions or anti-sense molecules thereof, provide the basis for diagnostic methods for early and accurate detection and / or EEC quantification associated with inflammatory or disease conditions. For example, the nucleotide sequences eec described herein, encoding EEC, or fragments thereof, can be used in hybridization assays of cells or tissues that have undergone a biopsy, or of body fluids, for diagnose abnormalities in individuals who have or are at risk of having inflammation. An abnormal level of nucleotide sequences encoding EEC in a biological sample may reflect a chromosomal aberration, such as a deletion or mutation of nucleic acid. In accordance with the above, the nucleotide sequences encoding EEC provide the basis for probes that can be used diagnostically to detect chromosomal aberrations such as deletions, mutations or chromosomal translocations in the gene encoding EEC. Eec gene expression can be altered in those disease states, or there may be a chromosomal aberration present in the region of the gene encoding EEC. The present invention also provides anti-sense eec molecules or EEC antagonists that can be used to block the activity of EEC, ie, leukocyte trafficking, under conditions where it is desirable to block the activity of chemokine, such as inflammation. . Alternatively, the eec or EEC antagonist sense molecules can be used to improve leukocyte trafficking, such as in acute or chronic infection, where they can desirably increase leukocyte trafficking. the present invention also relates to expression vectors and genetically engineered host cells comprising eec nucleotide sequences for in vitro or in vivo production of the nucleotide and amino acid sequences. Additionally, the present invention relates to the use of an EEC polypeptide, or fragment or variant thereof, to produce anti-EEC antibodies and to screen for antagonists or inhibitors of the EEC polypeptide that can be used diagnostically to detect and quantify the levels of EEC protein in disease states. The present invention also relates to pharmaceutical compositions comprising effective amounts of inhibitors or antagonists of the EEC protein or the anti-sense nucleic acid of EEC, in conditions where it is desirable to reduce the activity of chemokine, for example, in the treatment of inflammation. The present invention also relates to compositions comprising effective amounts of EEC antagonists, or other molecules capable of improving the activity of EEC, for use in the treatment of conditions where it is desirable to improve leukocyte trafficking., for example, in infections. The invention further provides diagnostic assays and kits for the detection of CSE in cells and tissues comprising a purified EEC, which can be used as a positive control, and anti-EEC antibodies. These antibodies can be used in solution-based, membrane-based, or tissue-based technologies to detect any condition or condition of disease related to the expression of the protein or the expression of deletions or variants thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 visually displays the nucleotide sequence for the expressed eosinophilic chemokine, eec, and the predicted amino acid sequence of EEC. Figure 2 shows the amino acid alignment of EEC with other human chemokines of the C-C family, including MlP-la, SEQ ID NO: 3; MIP-lb, SEQ ID NO: 4; MCP-1, SEQ ID NO: 5, MCP-2, SEQ ID NO: 6, MCP-3, SEQ ID NO: 7; RANTES, SEQ ID NO: 8; and Majority, SEQ ID NO: 9. The alignments shown in Figures 2 and 5 were produced using the DNASTAR software multiple sequence alignment program (DNASTAR Inc., Madison Wl). Figure 3 visually displays an analysis of the hydrophobicity of EEC based on the predicted amino acid sequence and composition. Figure 4 shows a ratio tree of human C-C chemokines. The phylogenetic tree was generated by the phylogenetic tree program of the DNASTAR software, using the Clustal method with the residual weight table PAM250. Figure 5 shows the amino acid alignment of MIP-3 and EEC.

MODES FOR CARRYING OUT THE INVENTION The present invention relates to a novel CC chemokine receptor, designated herein as EEC, whose nucleotide sequence was initially found between the sequences of a cDNA library made from the blood cells of a patient diagnosed with Hypereosinophilic Syndrome at the Mayo Clinic. The present invention relates to the use of the nucleotide and amino acid sequences described herein in the study, diagnosis and treatment of disease states associated with the trafficking of leukocytes under abnormal, inflammatory or disease conditions, such as vascular diseases. collagen such as rheumatoid arthritis, eosinophilic fasciitis, allergic angitis, periarteritis nodosa, and granulomatosis, - malignancies such as Hodgkin's lymphoma, mycosis fungoides, chronic myelogenous leukemia and lung, stomach, pancreas, ovarian or uterine cancer, - helminthic infections such as hookworm, schistosomiasis, toxocariasis, trichuriasis, filariasis, strongyloidosis, echinococcosis, cysticercosis, and trichinosis; hypereosinophilic syndromes such as Loeffler's syndrome, Loeffler's endocardiatis, eosinophilic leukemia, eosinophilic myalgia, and hypereosinophilic idiopathic syndrome; and allergies and asthma. The present invention also relates to the use of EEC and genetically engineered host cells that express the EEC, to evaluate and track substances and compounds that modulate EEC activity. The present invention is based in part on the presence of nucleotide sequences encoding EEC in a random sample of 9576 sequences usable in a cDNA library made from peripheral blood cells obtained from a 48-year-old male patient diagnosed with Hypereosinophilic Syndrome at the Mayo Clinic. It was determined that the cell population was greater than 77 percent of eosinophils, by Wright's stain. The present invention is also based on the identification of a novel C-C chemokine, EEC, which is associated with the trafficking of leukocytes in a wide variety of diseases, including infection and inflammation. "Nucleic acid sequence" as used herein, refers to a sequence of oligonucleotides, nucleotides, or polynucleotides, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be double stranded or of a single chain, either representing the sense or anti-sense chain. As used herein, "amino acid sequence" refers to sequences of peptides or proteins or portions thereof. As used herein, "lower case" eec "refers to a nucleic acid sequences, while" upper case "ECC refers to a protein sequence. As used herein, peptide nucleic acid (PNA) refers to a class of informational molecules that have a neutral "peptide-like" base structure, with core bases that allow the molecules to hybridize to complementary DNA or RNA with a higher affinity and specificity than the corresponding oligonucleotides (PerSeptive Biosystems 1-800-899-5858). As used herein, EEC encompasses EECs of any mammalian species, including bovine, ovine, murine, porcine, equine and preferably human sources, in naturally occurring or variant form, or from any source, whether natural, synthetic, semi-synthetic or recombinant. As used herein, "naturally occurring" refers to an EEC with an amino acid sequence found in nature, and "biologically active" refers to an EEC that has structural, regulatory or biochemical functions of naturally occuring EEC, including immunological activity. The naturally occurring EEC also encompasses those CEEs that arise from post-translational modifications of the polypeptide, including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. As used herein, "immunological activity is defined as the ability of the natural, recombinant or synthetic EEC or any oligopeptide thereof to induce a specific immune response in appropriate animals or cells, and to bind with specific antibodies." "derivative", as used herein, refers to the chemical modification of an EEC Illustrative of such modifications is the replacement of hydrogen by an alkyl, acyl, or amino group. A polypeptide derivative of EEC retains essential biological characteristics of an Naturally occuring EEC Derived from EEC also refers to those EEC polypeptides derived from naturally occurring EEC, by chemical modifications such as ubiquitination, labeling (eg, with radionuclides, different enzymes, etc.), pegylation (derivatization with glycol of polyethylene), or by insertion or substitution by chemical synthesis of am inocides such as ornithine, which normally does not occur in human proteins. As used herein, the term "purified" refers to molecules, sequences of either nucleic acids or amino acids, that are removed from their natural environment and are isolated or separated from at least one other component with which they are naturally associated "Recombinant variant EEC" refers to any EEC polypeptide that differs from the naturally occurring EEC, by insertions, deletions, and amino acid substitutions, created using recombinant DNA techniques. A guide can be found to determine which amino acid residues should be replaced, added or deleted, without nullifying the activities of interest, such as cell adhesion and chemotaxis, by comparing the sequence of the particular EEC with that of the homologous cytokines, and minimizing the number of amino acid sequence changes made in regions of high homology. Preferably, amino acid "substitutions" are the result of the replacement of an amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a seriba, that is, conservative replacements of amino acids. The "insertions" or "deletions" are typically in the range of about 1 to 5 aa. The allowed variation can be determined experimentally by insertions, deletions, or systematically made substitutions of amino acids in an EEC molecule, using recombinant DNA techniques, and testing the resulting recombinant variants to see their activity. Where desired, a "leader sequence" can direct the polypeptide through the membrane of a cell. That sequence may be present naturally in the polypeptides of the present invention, or be provided from heterologous protein sources by recombinant DNA techniques. As used herein, a "fragment", "portion", or "segment" of EEC refers to a stretch of amino acid residues that had sufficient length to display biological and / or immunogenic activity, and in the preferred embodiments will contain at least about 5 amino acids, at least about 7 amino acids, at least about 8 to 13 amino acids, and, in additional embodiments, about 17 or more amino acids. As used herein, a "fragment", "portion", or "segment" of "oligonucleotide" or polynucleotide refers to any stretch of nucleic acids encoding EEC, that is of sufficient length to be used as a primer in the polymerase chain reaction (PCR) or in different Hybridization methods known to those of skill in the art, for the purpose of identifying or amplifying identical or related nucleic acids.

The present invention includes CEE polypeptides purified from natural or recombinant sources, vectors and host cells transformed with nucleic acid molecules encoding EEC. Different methods for the isolation of EEC polypeptides can be performed by methods well known in the art. For example, these polypeptides can be purified by immunoaffinity chromatography, using the antibodies provided by the present invention. Other different methods of protein purification, well known in the art, include those described in Deutscher M (1990) Methods in Enzymology, Volume 182, Academic Press, San Diego; and Scopes R (1982) Protein Purification: Principles and Practice, Springer-Verlag, NYC, both incorporated herein by reference. As used herein, the term "recombinant" refers to a polynucleotide that encodes EEC, and is prepared using recombinant DNA techniques. The polynucleotide encoding a? EC may also include allelic or recombinant variants and mutants thereof. As used herein, the term "probe" or "nucleic acid probe" or "probe and oligonucleotide" refers to a portion, fragment, or segment of eec that is capable of being hybridized to a desired target nucleotide sequence. . A probe can be used to detect, amplify, or quantify the endogenous cDNAs or nucleic acids encoding EEC, by employing conventional techniques in molecular biology. A probe may be of variable length, preferably from about 10 nucleotides to many hundreds of nucleotides. As will be understood by those skilled in the art, the hybridization conditions and the design of the probe will vary depending on the intended use. For example, a probe that is intended to be used in PCR will be approximately 15 to 60 nucleotides in length, and may be part of a group of degenerate probes, ie, oligonucleotides that tolerate mismatching of nucleotides, but are accommodated by binding to an unknown sequence, whereas a probe for use in Southern or Northern hybridizations can be a single, specific nucleotide sequence that is many hundreds of nucleotides in length. Nucleic acid probes may comprise portions of the sequence having fewer nucleotides of about 6 kb, and usually less than about 1 kb. The oligonucleotide and nucleic acid probes of the present invention can be used to determine whether the nucleic acid encoding EEC is present in a cell or tissue, or to isolate identical or similar nucleic acid sequences from chromosomal DNA, as described Walsh PS et al. (1992 PCR Methods Appl 1: 241-250).

In accordance with the foregoing, a preferred probe for the specific detection of eec will comprise a polynucleotide or oligonucleotide fragment of a non-conserved nucleotide region of SEQ ID NO: 1. As used herein, the term "non-conserved nucleotide region" refers to a nucleotide region that is unique to SEQ ID NO: 1, and does not comprise a region that is conserved in the C-C chemokine family. The probes may be single-stranded or double-stranded, and may have specificity in hybridizations based on solution, cell, tissue, or membrane, including in situ technologies and similar to the enzyme-linked immunosorbent assay. In a modality described herein, a nucleotide probe for the detection of EEC encoding nucleotide sequences, is derived from nucleotide sequences encoding amino acid residues at the position of amino acid residues 22 to 63, inclusive, of SEQ ID NO: 2. The nucleic acid probes of the present invention can be derived from acids naturally occurring or recombinant nucleic acids, single-stranded or double-stranded, or chemically synthesized. These can be labeled by nick translation, Klenow filling reaction, PCR or other methods well known in the art. The probes of the present invention, their preparation and / or labeling are elaborated in Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, NYC, both incorporated herein by reference. Alternatively, the recombinant variant nucleotide sequences encoding the polypeptides of the present invention can be synthesized or identified through hybridization techniques known to those of skill in the art, through the use of "redundancy" in the genetic code. Different codon substitutions, such as silent changes that produce different restriction sites, can be introduced to optimize cloning within a chiral vector plasmid or expression in a particular prokaryotic or eukaryotic system. Mutations can also be introduced to modify the properties of the polypeptide, to change ligand-binding affinities, interchain affinities, or degradation of the polypeptide, or rate of change.

Description of EEC Coding Sequences Figure 1 shows the nucleotide sequence of human eec (SEQ ID NO: 1). The coding region for EEC was initially identified within a DNA library (Incyte EOSIHET02 cDNA library) made from the blood cells of a patient diagnosed with Hypereosinophilic Syndrome at the Mayo Clinic, where it was found seven times in approximately 9576 usable sequences. A BLAST search (Acronym in English for Basic Local Alignment Search Tool; Altschul SF (1993) J. Mol. Evol. 36: 290-300; Altschul SF et al. (1992) J. Mol. Biol. 215: 403- 410), comparing the cDNAs of the EOSIHE02 library against the GenBank primate database, identified Clone 288236 of Incyte as a non-exact coupling for a human 464.2 mRNA for a cytokine effector (Gl G34750). Nucleotide sequences encoding EEC were also found, once at approximately 2553 in a cDNA library (UTRSNOT01 from INCYTE) made from tissue from the uterus. Because EEC is expressed in eosinophils, nucleic acids (eec), polypeptides (EEC) and antibodies to EEC are useful in diagnostic assays based on the production of chemokine in cases of inflammation or disease that affects the number and function of eosinophils. Excessive expression of EEC can also activate monocytes, macrophages, basophils, T lymphocytes and / or other cells that respond to chemokines and the result is the production of abundant proteases and other molecules that can lead to tissue damage or destruction. Therefore, a diagnostic test for the excess of expression of EEC can accelerate the diagnosis and appropriate treatment of eosinophilia, an abnormal condition caused by viral, bacterial, fungal or parasitic infections; the mechanical injury associated with the trauma; hereditary diseases such as allergies and asthma; infiltrative diseases such as leukemias and lympholas, - or other physiological and pathological problems associated with changes in the numbers of eosinophils. Methods for DNA sequencing are well known in the art, and employ enzymes such as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland OH), Taq polymerase (Perkin Elmer, Norwalk CN), the thermostable T7 polymerase (Amersham, Chicago IL), or combinations of recombinant polymerases and proofreading exonucleases, such as the Amplification ELONGASE marketed by Gibco BRL (Gaithersburg MD). Methods have been developed to extend the DNA of a quenched oligonucleotide primer with the DNA template of interest, for both single-stranded and double-stranded templates. The chain termination reaction products were separated using electrophoresis, and detected by their incorporated labeled precursors. Recent improvements in reaction preparation, sequencing and mechanized analysis have allowed the expansion in the number of sequences that can be determined per day. Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno NV), Peltier Thermal Cycler (PTC200, MJ Research Watertown MA) and the ABI Catalyst 800 and 377, and DNA sequencers 373 (Perkin Elmer, Norwalk CN). The quality of any particular cDNA library containing the polynucleotides encoding EEC can be determined by conducting a pilot scale analysis of the cDNAs and verifying the percentages of clones containing the vector, DNA lamda or E. coli, mitochondrial or repetitive DNA, and clones with exact or homologous links to public databases. The nucleotide sequences encoding EEC (or its complement) have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include their use as hybridization probes, its use in the construction of oligomers for PCR, its use for mapping of chromosomes and genes, its use in the recombinant production of EEC, and its use in the generation of anti-sense DNA or RNA, its chemical analogs and the like. The uses of the nucleotides encoding EEC described herein are examples of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art. In addition, the nucleotide sequences described herein can be used in molecular biology techniques that have not yet been developed, with the proviso that the new techniques depend on the properties of the nucleotide sequences that are currently known, for example, the genetic code of triplet, specific interactions of base pairs, and so on. Those skilled in the art will note that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding EEC can be produced, some of which carry minimal homology to the nucleotide sequence of any known and naturally occurring gene. The invention has specifically contemplated each and every possible variation of the nucleotide sequence that could be made by selecting combinations based on possible selections of codons. These combinations are made in accordance with the standard triplet genetic code as applied to the naturally occurring eec nucleotide sequence, and all these variations will be considered as being specifically described. Although the nucleotide sequences encoding EEC and / or their variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring eec under conditions of stringency, it may be desirable to produce nucleotide sequences encoding EEC or its derivatives that possess a substantially different codon usage. The codons can be selected to increase the rate at which the expression of the peptide occurs in a prokaryotic or eukaryotic expression host, in accordance with the frequency with which the host uses the particular codons. Other reasons for substantially altering the sequence of nucleotides encoding EEC and / or its derivatives, without altering the encoded amino acid sequence, include the production of RNA transcripts that have more desirable properties, such as a longer half-life, than the transcripts produced from the naturally occurring sequence. The nucleotide sequences encoding EEC can be linked to a variety of other nucleotide sequences by well-established recombinant DNA techniques (cf Sambrook J et al., Supra). Nucleotide sequences useful for binding to eec include a classification of cloning vectors, for example, plasmids, cosmids, lamda phage derivatives, phagemids, and the like, which are well known in the art. Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and the like. In general, the vectors of interest may contain a functional replication origin in at least one organism, suitable sensitive sites, sensitive to the restriction endonuclease, and selectable markers for the host cell. One aspect of the present invention is to provide nucleic acid hybridization probes specific for eec, capable of hybridizing with naturally occurring nucleotide sequences encoding EEC. These probes can also be used for the detection of similar chemokine encoding sequences, and should preferably contain at least 50 percent of the nucleotides of a C-C encoding a sequence. Hybridization probes of the present invention can be derived from nucleotide sequences of SEQ ID NO: 1, or from genomic sequences including promoters, improving elements and / or possible introns of the respective naturally occurring eecs. Hybridization probes can be labeled by a variety of reporter groups, including radionuclides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe by avidin / biotin coupling systems, and the like. PCR, as described in the Patents of the United States of America Numbers 4,638,195; 4,800,195; and 4,965,188 provides additional uses for the oligonucleotides based on the nucleotide sequence encoding EEC. These probes used in PCR may be of recombinant origin, they may be chemically synthesized, or a mixture of both, and comprise a discrete nucleotide sequence for diagnostic use, or a degenerate group of possible sequences for the identification of closely related genomic sequences. . Other means for producing hybridization probes specific for eec DNAs include cloning of nucleic acid sequences encoding EEC or EEC derivatives into vectors for the production of mRNA probes. These vectors are known in the art and are commercially available, and can be used to synthesize RNA probes in vitro, by means of the addition of the appropriate RNA polymerase, such as T7 or SP6 RNA polymerase, and the appropriate radioactively labeled nucleotides. . It is now possible to produce a DNA sequence, or portions thereof, by encoding EEC and its derivatives entirely by synthetic chemistry, after which the gene can be inserted into any of the many available DNA vectors, using reagents, vectors and cells that are known in the art at the time of filing this application. On the other hand, synthetic chemistry can be used to introduce mutations within the eec sequences or any portion thereof. The nucleotide sequence can be used in an assay to detect inflammation or disease associated with abnormal levels of EEC expression. The nucleotide sequence can be labeled by methods known in the art, and added to a sample of fluid or tissue from a patient, under hybridization conditions. After an incubation period, the sample is washed with a compatible fluid optionally containing a dye (or other label requiring a developer), if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed, the dye is quantified and compared to a standard. If the amount of dye is significantly elevated, the nucleotide sequence has been hybridized with the sample, and the assay indicates the presence of inflammation and / or disease. The nucleotide sequence for eec can be used to construct hybridization probes to map that gene. The nucleotide sequence provided herein can be mapped to a particular chromosome, or to specific regions of that chromosome, using well-known genetic and / or chromosomal mapping techniques. These techniques include in situ hybridization, binding analysis against known chromosomal markers, hybridization screening with libraries, chromosomal preparations sorted by flow, or constructions of YAC artificial chromosomes or Pl constructs. The fluorescent in situ hybridization technique of chromosome dispersions has been described, among other places, in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City. Fluorescent in situ hybridization of chromosomal preparations, and other physical chromosome mapping techniques, can be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981f). The correlation between the location of eec on a physical chromosomal map and a specific disease (or predisposition to a specific disease) can help to narrow the region of DNA associated with that genetic disease. The nucleotide sequence of the present invention can be used to detect differences in gene sequence between normal and carrier or affected individuals.

EEC Expression The nucleotide sequences encoding EEC can be used to produce purified EEC, using well-known methods of recombinant DNA technology. Among the many publications that teach methods for gene expression after these have been isolated, is Goeddel (1990) Gene Expression Technology, Methods and Enzymology, Volume 185, Academic Press, San Diego CA. The EEC can be expressed in a variety of host cells, whether prokaryotic or eukaryotic. The host cells may be of the same species in which the nucleotide sequences of eec are endogenous, or of a different species. The advantages of producing EEC by recombinant DNA technology include obtaining adequate amounts of the protein for purification, and the availability of simplified purification procedures. Expression of eec can be performed by subcloning the cDNAs within the appropriate expression vectors, and transfecting the vectors within appropriate expression hosts. As described in Example VII, a preferred expression vector is one that allows the expression of a fusion protein comprising EEC and containing nucleic acid encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site . Histidine residues facilitate purification in IMIAC (affinity chromatography of immobilized metal ion, as described in Porath et al. (1992) Protein Expression and Purification 3: 263-281) while the enterokinase cleavage site provides a means to purify chemokine from the fusion protein. The cloning vector previously used for the generation of the tissue library also allows the expression of the eec sequence in E. coli. Since the inserts of the cDNA clone are generated by an essentially random process, there is a possibility in three that the included cDNA will be located in the correct frame for the proper translation. If the cDNA is not in the proper reading frame, it can be obtained by deletion or insertion of the appropriate number of bases, by well-known methods, including in vitro mutagenesis, exonuclease III digestion or mongo bean nuclease, or inclusion of linker of oligonucleotide. The eec cDNA can be enclosed within other vectors known to be useful for the expression of protein in specific hosts. The amplimers containing cloning sites, as well as a DNA segment sufficient to hybridize to stretches at both ends of the target cDNA (25 bases) can be synthesized chemically by standard methods. These primers can then be used to amplify the desired gene segments by PCR. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions, and can be isolated by gel electrophoresis. Alternatively, similar gene segments can be produced by digesting the cDNA with appropriate restriction enzymes, and filling the lost gene segments with chemically synthesized oligonucleotides. Segments of the coding sequence of more than one gene can be ligated together and cloned into appropriate vectors to optimize the expression of the recombinant sequence. Suitable expression hosts for those chimeric molecules include, but are not limited to mammalian cells such as Chinese Hamster Ovary (CHO) and 293 human cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae , and bacteria such as E. coli. For each of these cellular systems, a useful expression vector may also include a replication origin, to allow propagation in the bacterium, and a selectable marker such as the antibiotic resistance gene 3-lactamase, to allow selection in the bacteria. In addition, the vectors may include a second selectable marker such as the neomycin phosphotransferase gene, to allow selection in transfected eukaryotic host cells. Vectors for use in eukaryotic expression hosts may require RNA processing elements such as 3 'polyadenylation sequences, if these are not part of the cDNA of interest. Additionally, the vector may contain promoters or enhancers that increase gene expression. These promoters are host specific and include MMTV, SV40, or metallothionine promoters for CHO cells; promoters of trp, lac, tac or T7 for bacterial hosts, or alpha factor, alcohol oxidase or promoters of PGH for yeast. Transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used in mammalian host cells. Once the homogeneous cultures of the recombinant cells are obtained by standard culture methods, large quantities of EEC, produced recombinantly, from the conditioned medium can be recovered and analyzed using chromatographic methods known in the art. Cells transformed with DNA encoding EEC can be cultured under conditions suitable for chemokine expression and recovery of the cell culture protein. The EEC produced by a recombinant cell can be secreted or it can be contained intracellularly, depending on the particular genetic construction used. In general, it is more convenient to prepare recombinant proteins in secreted form. The purification steps vary with the production process and the particular protein produced. The EEC can be expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate the purification of protein. These purification facilitation domains include, but are not limited to, metal chelation peptides, such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (Immunex Corp., Seattle WA). The inclusion of a dissociable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and the eec sequence may be useful to facilitate the expression of EEC. In addition to recombinant production, fragments of EEC can be produced by direct synthesis of the peptide using solid phase techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco CA; Merrifield J (1963) J Am Chem Soc 85: 2149-2154. The synthesis of the protein in vitro can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, by using the Peptide Synthesizer of Applied Biosystems 431A (Foster City, CA), in accordance with the instructions provided by the manufacturer. Different fragments of EEC can be chemically synthesized separately, and combined using chemical methods to produce the full-length molecule.

EEC antibodies The EEC for antibody induction does not require biological activity, - however, the protein must be immunogenic. The peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. These must be a portion of the amino acid sequence of the protein and may contain the entire amino acid sequence of a naturally occurring, small molecule such as EEC. Short stretches of EEC amino acids can be fused with those of another protein such as orifice limpet hemocyanin and the chimeric molecule used for the production of antibodies. Antibodies specific for EEC can be produced by inoculating an appropriate animal with the polypeptide or an antigenic fragment. An antibody is specific for EEC if it is produced against an epitope of the polypeptide and binds at least part of the natural or recombinant protein. The production of antibodies includes not only the stimulation of an immune response by injection into animals, but also analogous steps in the production of synthetic antibodies or other fixative-specific molecules, such as the screening of recombinant immunoglobulin libraries (cf Orlandi R et al (1989) PNAS 86: 3833-3837, or Huse WD et al (1989) Science 256: 1275-1281), or in vitro stimulation of lymphocyte populations. Current technology (Winter G and Milstein C (1991) Nature 349: 293-299) provides many highly specific binding reagents, based on the principles of antibody formation. These techniques can be adapted to produce molecules that specifically bind to EECs. Those skilled in the art are aware of different methods for preparing monoclonal and polyclonal antibodies to EEC. In one approach, denatured EEC is obtained from the reverse phase HPLC separation, and is used to immunize mice or rabbits, using techniques known to those skilled in the art. Approximately 100 micrograms are suitable for immunization of a mouse, while up to 1 milligram can be used for immunization of a rabbit. To identify mouse hybridomas, the denatured protein can be radioiodinated, and used to screen potential murine B-cell hybridomas for those that produce antibodies. This procedure requires only small amounts of protein, so that 20 milligrams would be enough to label and track many thousands of clones. In another approach, the amino acid sequence of EEC is analyzed, as deduced by the translation of the cDNA sequence, to determine regions of high immunogenicity. Oligopeptides comprising hydrophilic regions, as shown in Figure 3, are synthesized and used in suitable immunization protocols for culturing antibodies. The analysis for selecting the appropriate epitopes is described in Ausubel et al. (1989, Current Protocols in Molecular Biology, John Wiley &Sons, NYC). The optimal amino acid sequences for immunization are usually in the C-terminus, the N-terminus and those intermediate hydrophilic regions of the polypeptide that are likely to be exposed to the external environment when the protein is in its natural conformation. Typically, the selected peptides, approximately 15 residues in length, are synthesized using a Model 431A Peptide Synthesizer from Applied Biosystems, using fmoc chemistry, and coupled to the orifice 1-mimetic hemocyanin (KLH, Sigma) by reaction with M ester. -maleimidobenzoyl-N-hydroxysuccinimide (MBS; see Ausubel FM et al., supra). If necessary, cysteine can be introduced at the N-terminus of the peptide, to allow coupling with the orifice limpet hemocyanin, and the animals can be immunized with the peptide-hemocyanin complex orifice limpet in complete Freund's adjuvant. You can test the resulting antiserum to see the antipeptide activity, by fixing the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antiserum, washing and reacting with specific goat anti-rabbit IgG, purified by affinity, labeled (radioactive or fluorescent). Hybridomas can also be prepared and screened using standard techniques. Hybridomas of interest can be detected by tracing with labeled EEC, to identify those fusions that produce the monoclonal antibody with the desired specificity. In a typical protocol, plate wells are covered (FAST; Becton-Dickinson, Palo Alto, CA) with specific rabbit-anti-mouse antibodies (anti-adequate species Ig) at 10 milligrams / milliliter. The covered wells are blocked with 1% bovine serum albumin, washed and exposed to supernatants of the hybridomas. After incubation, the wells are exposed to labeled EEC at 1 milligram / milliliter. The clones that produce antibodies will set an amount of labeled EEC, which can be detected on the background. These clones can be expanded and subjected to 2 cloning cycles at limiting dilution (1 cell / 3 wells). Cloned hybridomas are injected into previously treated mice, to produce ascites, and monoclonal antibodies can be purified from ascites fluid by affinity chromatography, using Protein A. Monoclonal antibodies with affinities of at least 108 M "1, preference 109 to 1010 or stronger, will typically be made by standard procedures, as described in Harlow and Lane (1988) ASntibodies: A Laboratory Manual. Cold Spring Harbor Laboratory New York; and in Goding (1986) Monoclonal Antobodies: Principies and Practice, Academic Press, New York City, both incorporated herein by reference.

Uses of Nucleotide Sequences and Amino Acids for EEC A further embodiment of the present invention is the use of antibodies, inhibitors, EEC-specific receptors or their analogs as bioactive agents to treat eosinophilia, inflammation or disease involving an altered number of eosinophils, including, but not limited to, viral, bacterial, fungal or parasitic infections; mechanical injury associated with trauma, - hereditary diseases such as allergies and asthma, - infiltrative diseases such as leukemias and lympholas, - or other physiological and pathological problems associated with changes in the numbers of eosinophils. Knowledge of the correct, complete cDNA sequence of the novel expressed chemokine gene will enable its use in anti-sense technology in the investigation of gene function. Oligonucleotides, genomic fragments or cDNAs comprising the anti-sense chain of eec, can be used either in vitro or in vivo to inhibit protein expression. This technology is now well known in the art, and the probes can be designated in different locations along the nucleotide sequence. By treating the whole test cells or animals, with these anti-sense sequences, the gene of interest can be effectively disabled. Frequently, the function of the gene can be ascertained by observing the behavior at the cellular, tissue or organism level (eg, lethality, loss of differentiated function, changes in morphology, etc.). In addition to the use of sequences constructed to interrupt the transcription of the open reading frame, modifications of gene expression can be obtained by designating anti-sense sequences to regions of introns, promoter / enhancer elements, or even regulatory genes. trans-acting. Similarly, inhibition can be achieved using the Hogeboom base pair methodology, also known as "triple helix" pareo. Antibodies, inhibitors, receptors or antagonists of EEC (or other treatments for excessive production of chemokine, hereinafter abbreviated as TEC), can provide different effects when administered therapeutically. The TECs will be formulated in an aqueous, non-toxic, inert, pharmaceutically acceptable carrier medium, preferably at a pH of about 5 to 8, more preferably 6 to 8, although the pH may vary in accordance with the characteristics of the antibody, inhibitor, receptor, or antagonist being formulated, and the condition to be treated. TEC characteristics include molecule solubility, half-life, and antigenicity / immunogenicity; These and other characteristics can help in the definition of an effective carrier. Native human proteins are preferred as TECs, but in particular situations organic or synthetic molecules that are the result of drug traces can be equally effective. The TECs can be applied by known routes of administration including, but not limited to, topical creams and gels, transmucosal spray and spray, patch and transdermal dressing; injectable, intravenous and washing formulations; and orally administered liquids and pills, particularly formulated to resist gastric acids and enzymes. The particular formulation, the exact dose, and the route of administration will be determined by the attending physician, and will vary according to each specific situation. These determinations are made by considering multiple variables such as the condition to be treated, the TEC to be administered, and the particular pharmacokinetic profile of the TEC. Additional factors that can be taken into consideration include the disease status (for example, severity) of the patient, age, weight, sex, diet, time of administration, combination of drugs, reaction sensitivities, and tolerance / response to therapy. Long-acting ECT formulations can be administered every 3 or 4 days, every week, or once every two weeks, depending on the average life and the rate of evacuation of the particular ECT. The amounts of normal doses may vary from 0.1 to 100,000 micrograms, up to a total dose of approximately 1 gram, depending on the route of administration. Guidance is given in the literature regarding the particular dosage and methods of application, - see United States of America Patents Numbers 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different TECs, and that the administration directed to the eosinophil may need an application in a different way than that of another organ or tissue. It is contemplated that the conditions or diseases of eosinophils that activate monocytes, macrophages, basophils, eosinophils or other leukocytes can damage that can be treated with ECTs. Eosinophilia can be diagnosed specifically by the tests discussed above, and these tests should be performed in cases of suspected viral, bacterial, fungal or parasitic infections, as cited here, - mechanical injuries associated with trauma, - hereditary diseases such as allergies, asthma, and rheumatoid arthritis; cancers such as the aforementioned carcinomas, leukemias, and lympholas, - or other physiological or pathological problems associated with changes in the numbers of eosinophils. All publications and patents mentioned in the specification above are incorporated herein by reference. It is considered that the above written specification is sufficient to enable an expert in the art to practice the invention. In fact, it is intended that the various modifications of the modes described above for carrying out the invention, which are readily apparent to those skilled in the art of molecular biology or related fields, are within the scope of the following claims. The examples below are provided to illustrate the present invention. These examples are provided by way of illustration, and are not included for the purpose of limiting the invention.

INDUSTRIAL APPLICABILITY I Isolation of mRNA and Construction of cDNA libraries The sequence of eec was identified between the sequences of a human eosinophil library. The eosinophils used for this library were obtained by the aphorresis of a 56-year-old male Caucasian patient at the Mayo Clinic (Rochester MN) who had been diagnosed with Hypereosinophilic Syndrome. The cells were washed twice in phosphate-buffered saline, and immediately destroyed by the action of the lysines in a pH regulator containing guanidinium isothiocyanate. The lysate was centrifuged on a CsCl mattress, precipitated with ethanol, resuspended in water, and treated with DNase for 15 minutes at 37 ° C. The RNA was extracted with phenol chloroform and precipitated with ethanol. The polyadenylated messages were isolated, using the Qiagen Oligotex (QIAGEN Inc., Chatsworth CA), and the cDNA library was constructed by Stratagene (11011 North Torrey Pines Road, La Jolla CA 92037). The first cDNA chain synthesis was achieved using an oligo d (T) primer / linker that also contained an XhoI restriction site. The second chain synthesis was performed using a combination of DNA polymerase I, E. coli ligase and RNase H, followed by the addition of an EcoRI adapter to the blunt-ended cDNA. After digested the double-stranded cDNA, adapted by EcoRI with the restriction enzyme Xhol, it was extracted with phenol chloroform, and fractionated in size in Sephacryl S400. The DNA of the appropriate size was then ligated to dephosphorylated Zap Lamda® arms (Stratagene) and packed using Gigapack extracts (Stratagene). The pBluescript phagemid DNAs (Stratagene) were excised in bulk from the eosinophil library, and the individual plasmid DNAs were made using Miniprep kits supplied by Advanced Genetic Technologies Corporation (Gaithersburg MD). These kits provide a 96-well format and sufficient reagents for 960 purifications. The recommended protocol supplied with each kit has been used, except for the following changes. First, the 96 wells are each filled with only 1 milliliter of excellent sterile broth with carbenicillin at 25 milligrams / liter and 0.4 percent glycerol. After inoculating the wells, the bacteria were cultured for 24 hours and destroyed by the lysines with 60 microliters of lysis pH buffer. A centrifugation step was performed (2900 revolutions per minute for 5 minutes) before adding the contents of the block to the main filter plate. The optional step of adding isopropanol to the pH regulator TRIS is not done routinely. After the last step in the protocol, the samples were transferred to a block of 96 Beckman wells for storage. The quality of the cDNA library was determined by conducting a pilot scale analysis of 192 cDNAs, and verifying the percentages of clones containing the vector alone, mitochondrial or repetitive DNA sequences and clones originating from DNA lamda or from E. coli. The exact / homologous link numbers for the public databases were also recorded, as well as the number of unique sequences, that is, those that do not have any known coupling in any available database.

II Isolation of cDNA Clones The phagemid forms of the individual cDNA clones were obtained by the in vivo excision process, in which XL1-BLUE was coinfected with a helper phage fl. Proteins derived from both lamda phage and helper phage fl, initiated new DNA synthesis from defined sequences in the lamda target DNA, and created a smaller single-chain circular phagemid DNA molecule that includes all the plasmid DNA sequences pBluescript and the cDNA insert. The Phagemid DNA from the cells, and purified, then used to re-infect fresh bacterial host cells (SOLR, Stratagene Inc), where the double-stranded phagemid DNA was produced. Because the phagemid carries the gene for β-lactamase, the newly transformed bacteria was selected in medium containing ampicillin. The phagemid DNA was purified using the QIAWELL-8 Plasmid Purification System of the QIAGEN® DNA Purification System. This technique provides a fast and reliable high performance method for the destruction by the action of the lysins of the bacterial cells and for the isolation of highly purified phagemid DNA. The leached DNA from the purification resin was suitable for DNA sequencing and other analytical manipulations. The cDNA inserts were sequenced in part from random isolates of the human eosinophilic library. The cDNAs were sequenced by the method of Sanger F. and AR Coulson (1975; J. Mol. Biol. 94: 441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno NV), in combination with four Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown MA) and DNA Sequencing Systems 377 or 373 from Applied Biosystems (Perkin Elmer) and by reading the given framework.

III Homology Search of cDNA Clones and Deduced Proteins Each sequence thus obtained was compared with sequences in the GenBank using a search algorithm developed by Applied Biosystems Inc., and incorporated into the Sequence Analysis System INHERITMR 670. In this algorithm , the Pattern Specification Language (developed by TRW Inc.) was used to determine regions of homology. The three parameters that determine how sequence comparisons run were window size, window offset, and error tolerance. Using a combination of these three parameters, the DNA database was searched for the sequences containing regions of homology to the screening sequence, and the appropriate sequences were classified with an initial value. Subsequently, these homologous regions were examined using dot matrix homology planes to distinguish regions of homology from probable couplings. Smith-Waterman alignments were used to visually display the results of the homology search. Peptide and protein sequence homologies were determined using the INHERIT 670 Sequence Analysis System in a manner similar to that used in DNA sequence homologies. The Pattern Specification Language and the parameter windows were used to search for protein databases to search for the sequences containing regions of homology that were classified with an initial value. The point matrix homology planes were examined to distinguish significant regions of homology from probable couplings. BLAST was used, which is the acronym in English for Basic Local Alignment Search Tool (Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, SF and collaborators (1990) J Mol Biol 215: 403-10), to look for local sequence alignments. BLAST produces sequence alignments of both nucleotides and amino acids to determine sequence similarity. Due to the local nature of the alignments, BLAST is especially useful in the determination of exact couplings, or in the identification of homologs. BLAST is useful for couplings that do not contain separations. The fundamental unit of the output of the BLAST algorithm is the High Class Segment Pair (HSP). A Pair of High Class Segments consists of two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximum, and for which the alignment classification meets or exceeds a threshold or limitation classification established by the user. The BLAST approach is to look for High Class Segment Pairs between a query sequence and a database sequence, to evaluate the statistical significance of any links found, and to report only those links that satisfy the threshold of meaning selected by the user. Parameter E establishes the statistically significant threshold for reporting the database sequence couplings. E is interpreted as the upper link of the expected frequency of probable occurrence of a High Class Segment Pair (or set of High Class Segment Pairs) within the context of the entire database search. Any sequence of the database whose coupling satisfies E is reported in the output of the program. In Figure 1 the nucleotide and amino acid sequences are shown for the entire coding region of the chemokine expressed in eosinophils, EEC.

IV Identification and Full-Length Sequencing of the Genes Of all the clones chosen and randomly sequenced from the human eosinophil library, the eec sequence was homologous to, but clearly different from, any known C-C chemokine molecule. The complete sequence of nucleotides for eec was moved, and in Figure 1 the translation in frame is shown. When looking for the three possible predicted translations of the sequence against protein databases such as SwissProt and PIR, no exact coupling was found for possible translations of eec. Figure 2 shows the comparison of the amino acid sequence of EEC with those of other C-C chemokine molecules. The substantial regions of homology between these molecules that include the definitive C-C motif are shaded. The hydrophobicity planes for EEC are shown as Figure 3. The phylogenetic analysis (Figure 4) shows how closely the eec is related to other well characterized human C-C chemokines. The most related of these molecules are grouped together on the right hand side of the figure.

V Anti-Sense Analysis The sequence of EEC, or any part thereof, is used to inhibit in vivo or in vitro expression of endogenous CSE. Although the use of anti-sense oligonucleotides, which consist of approximately 20 base pairs, is specifically described, essentially the same procedure is used with longer cDNA fragments. An oligonucleotide based on the EEC coding sequence is used to inhibit the expression of endogenous EEC. Using Oligo 4.0, the complementary oligonucleotide is designated from the conserved 5 'sequence, and is used to inhibit any transcription, by preventing the promoter from binding to the sequence not translated upstream, or translating an EEC transcript, by preventing the ribosome from binding to the mRNA.

VI EEC Expression The nucleotide sequences encoding EEC were cloned into an expression vector comprising a T7 promoter followed by an initiation methionine (ATG) codon, followed by six histidine codons, followed by the E TrxA gene. coli (which codes for thioredoxin), followed by a sequence encoding an enterokinase dissociation site and nucleotide sequences encoding EEC. Empirical studies associated with signal sequence dissociation indicate that dissociation occurs at or near the C-terminus of a predicted hydrophobic region located at the N-terminus of the full-length protein. In Figure 3 the profile of hydrophobicity of EEC is shown, and based on this profile, it appears that residue 21 of SEQ ID NO: 2 (Alanine), is the amino acid residue with N-terminal for mature EEC expression. The presence of a N-terminal residue, 50 amino acid residues of the C-C residues characteristic of a C-C chemokine may reflect a N-terminal extension for EEC that imparts novel activity. The expression vectors described above containing the 6 histidine codons were used to transform a host cell, the host cell culture was induced with IPTG and the expressed protein was subjected to electrophoresis with denaturing SDS polyacrylamide gel. The nucleic acid was partially purified from the expression vector, using the miniprep procedure of Sambrook supra, which produced super-coiled DNA. 100 ng of DNA was used to transform the bacterial host cell, W3110 / DE3. W3110 / DE3 was constructed using ATCC W3110 and the DE3 lamda lysogenization kit, commercially available with Novagen. Frequently lysogens are less competent than their mother, W3110, and adapt to use super-coiled DNA for efficient transformation. A single transformant of each chemokine transformation was selected and used to inoculate a 5 milliliter culture of L broth containing ampicillin. They were grown overnight (12-15 hours) every 5 milliliters of culture at 37 degrees Cwith agitation. The next day, 1 milliliter of the overnight culture was used to inoculate a 100 milliliter culture of L broth with ampicillin in a 500 milliliter flask, and allowed to be cultured at 37 degrees C with shaking, until the OD600 crop reached 0.4-0.6. If the inoculated cells were allowed to grow by passing an OD600 of 0.6, they would begin to reach the stationary phase, and induction levels would be reduced. At the time of inoculation, a 5 milliliter sample was removed, placed on ice and used as a previous induction sample (or 0 hours). When the cell culture reached an OD600 of 0.6, 400 microliters of a 100 mg IPTG stock solution was added for a final concentration of 0.4mM. The cultures were allowed to cultivate for 3 hours at 37 degrees C with shaking. The induction analysis was determined by means of sampling aliquots of 5 milliliters of the culture at intervals of 1 hour to 6 hours, and analyzing on an electrophoresis with denaturing SDS polyacrylamide gel. It appears that the fusion protein accumulated in the insoluble fraction of the cells. The maximum induction of EEC occurred during 2 hours. Growth beyond 4 hours resulted in the destruction by action of lysines in the culture and reduced overall yields of the desired protein, due to proteolysis. Five milliliter aliquots were obtained from the cell cultures at 0, 1 and 2 hours, and centrifuged for 5 minutes at 3000 revolutions per minute at 4 degrees C. The supernatant was aspirated, and the pills were subjected to a freezing step- defrosting to help the lysis of the cells. The pill was re-suspended in TE [Tris-HC1 of lOmM with pH of 8.0, EDTA of 1 mM with pH of 8.0] at 4 degrees C, at a volume calculated as: vol of TE (ul) = (OD600) ( 250), and an equivalent volume of 2x SDS of Sample Load pH Regulator (Novex) was added to each sample. The samples were boiled for 5 minutes and 10 microliters of each sample per line was loaded. The expected molecular weight of the fusion protein comprising EEC is 19,233 Daltones. The analysis of the EEC expressed on a 14 percent SDS-polyacrylamide gel shows an apparent molecular weight of approximately 20kDa. A second fusion protein lacking six histidine residues was constructed and expressed, and had an expected molecular weight of 18,410 Daltones. The EEC protein appears predominantly in the insoluble fraction of cell lysates.

VII Recombinant EEC Isolation The EEC is expressed as a chimeric protein having six histidines followed by thioredoxin (TrxA from E. coli) with an enterokinase cleavage site between the TrxA protein and EEC. The histidines are added to facilitate the purification of the protein. The presence of histidine allows purification in IMIAC chromatography (Porath supra).

VIII Diagnostic Testing Using EEC Specific Antibodies The particular EEC antibodies are useful for the diagnosis of prepatological conditions, and chronic or acute diseases that are characterized by differences in the amount or distribution of EEC. The EEC was initially found in the human eosinophil library and is diagnostic for abnormalities or pathologies associated with the trafficking of leukocytes and eosinophils. Diagnostic tests for EEC use the antibody and a label to detect the EEC in body fluids, tissues or extracts from those human tissues. The polypeptides and antibodies of the present invention are used with or without modification. Polypeptides and antibodies are labeled by binding them, either covalently or non-covalently, with a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known, and have been widely reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of these labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, recombinant immunoglobulins can be produced as shown in U.S. Patent No. 4,816,567, incorporated herein by reference. A variety of protocols for measuring soluble or membrane bound EEC are known in the art, using either polyclonal or monoclonal antibodies specific for the respective protein. Examples include the enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell (FACS) selection. A two-site monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes in EEC is preferred, but a competitive binding assay can be employed. These assays are described, inter alia, in Maddox, DE et al. (1983, j Exp Med 158: 1211).

IX Purification of Native EEC Using Specific Antibodies Native or recombinant EEC is purified by immunoaffinity chromatography, using antibodies specific for EEC. An immunoaffinity column is constructed by covalently coupling the anti-EEC antibody with an activated chromatographic resin. Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate, or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Monoclonal antibodies are prepared from mouse ascites fluid, by precipitation with ammonium sulfate, or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently bound to a chromatographic resin such as Sepharose activated by CnBr (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derived resin is washed in accordance with the manufacturer's instructions. These immunoaffinity columns are used in the purification of EEC by preparing a fraction from cells containing EEC in a soluble form. This preparation is derived by the solubilization of the whole cell or of a subcellular fraction obtained by differential centrifugation, by the addition of detergent, or by other methods well known in the art. Alternatively, the soluble EEC containing a signal sequence can be secreted in a useful amount into the medium in which the cells are cultured. A soluble preparation containing EEC is passed over the immunoaffinity column, and the column is washed under conditions that allow preferential absorbency of the chemokines (eg, high ionic strength pH regulators in the presence of detergent). The column is leached under conditions that break the antibody / chemokine binding (eg, a pH regulator with a pH of 2-3 or an elevated concentration of a chaotrope such as urea or thiocyanate ion), and the EEC is harvested.

X Chemotaxis Induced by EEC or Cellular Activation The chemotactic activity of the EEC is measured in 48-well microchemotaxis chambers (Falk WR et al. (1980) J Immunol Methods 33: 239). In each well, two compartments are separated by a filter that allows the passage of cells in response to a chemical gradient. The cell culture medium such as RPMI 1640 (Sigma, St.

Louis MO) containing the expressed chemokine, on one side of a filter, usually polycarbonate, and the cells suspended in the same medium are placed on the opposite side of the filter. Sufficient incubation time is allowed for the cells to pass through the filter in response to the concentration gradient across the filter. The filters are recovered from each well, and the cells that adhere to the side of the filter against chemokine are typed and quantified. The specificity of the chemoattraction is determined by performing the chemotaxis assay on specific cell populations. First, the blood cells obtained from the venipuncture are fractionated by density gradient centrifugation, and the chemotactic activity of EEC is tested in the populations of neutrophils, peripheral blood mononuclear cells, monocytes and lymphocytes. Optionally, these enriched cell populations are further fractionated using the CD8 + and CD4 + specific antibodies for negative selection of CD4 + and CD8 + enriched T cell populations, respectively. Another assay clarifies the chemotactic effect of? EC on activated T cells. Unfractionated T cells or subsets of fractionated T cells are cultured for 6 to 8 hours in tissue culture blood vessels covered with CD-3 antibody. After this activation of CD-3, the chemotactic activity of EEC is tested as described above. Many other methods for obtaining enriched cell populations are known in the art. Some chemokines also produce a non-chemotactic cell activation of neutrophils and monocytes. This is tested by standard measurements of neutrophil activation such as actin polymerization, increase in respiratory burst activity, de-granulation of the azurophilic granule, and mobilization of Ca ** as part of the signal transduction path. The test to see the mobilization of Ca ** involves previously loading the neutrophils with a fluorescent probe whose emission characteristics have been altered by the Ca fixation. When the cells are exposed to an activation stimulus, the flow of Ca ** is determined by observing the cells in a fluorometer. Measurement of Ca2 mobilization has been described in Grynkievicz G et al. (1985) J Biol Chem 260: 3440, and McColl S et al. (1993) J Immunol 150: 4550-4555, incorporated herein by reference. The degranulation and respiratory burst responses are also measured in monocytes (Zachariae COC et al. (1990) J Exp Med 171: 2177-82). Other measures of monocyte activation are the regulation of the expression of the adhesion molecule and the production of cytokine (Jiang Y et al. (1992) J Immunol 148: 2423-8). The expression of the adhesion molecules also varies with the activation of lymphocytes (Taub D et al. (1993) Science 260: 355-358).

XI Drug Screening E? C, or biologically active fragments thereof, are used to screen compounds in any of a variety of drug screening techniques. The chemokine polypeptide or fragment employed in this test can be either free in solution, fixed to a solid support, originated on a cell surface, or localized intracellularly. A method of drug screening utilizes eukaryotic or prokaryotic host cells that are stably transformed with recombinant nucleic acids that express the polypeptide or fragment. The drugs are screened against these transformed cells in competitive binding assays. These cells, in a viable or fixed form, can be used for standard fixation tests. One can measure, for example, the formation of complexes between E? C and the agent being tested. Alternatively, one could examine the decrease in complex formation between ΔC and its target cell, monocyte, etc., caused by the agent being tested. In this manner, the present invention provides screening methods for drugs or any other agents that can affect inflammation and disease. These methods comprise contacting the agent with a C-polypeptide or fragment thereof and performing tests (i) to see the presence of a complex between the agent and the C-polypeptide or fragment, or (ii) to see the presence of a complex between the EEC polypeptide or fragment and the cell, by methods well known in the art. Typically, in these competitive binding assays, the chemokine polypeptide or fragment is labeled. After adequate incubation, the EEC polypeptide or free fragment is separated from that present in fixed form, and the amount of free or un-complexed tag is a measure of the ability of the particular agent to bind to EEC, or to interfere with the EEC and the agent complex. Another embodiment of the present invention relates to a method for screening a plurality of compounds to view the affinity of specific binding to the polypeptide of Claim 8 or any portion thereof, comprising the steps of: a) providing a plurality of compounds; b) combining Splenic Chemokine Expressed in Eosinophils (EEC) with each of a plurality of compounds for a sufficient time to allow fixation under suitable conditions, - and c) detect the binding of EEC to each of the plurality of compounds, identifying by the same the compounds that are specifically set to EEC.

Another technique for drug screening provides high throughput screening for compounds that have adequate binding affinity to the C-polypeptide, and is described in detail in the European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with EEC polypeptide and washed. The fixed E? C polypeptide is then detected by methods well known in the art. The purified EC can also be directly coated onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding to E? C compete specifically with a test compound for binding to chemokine polypeptides or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with ΔC.

XII Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or small molecules with which they interact, for example, agonists, antagonists, or inhibitors. Any of these examples can be used to design drugs that are more active or stable forms of the polypeptide, or that improve or interfere with the function of a polypeptide in vivo (cf Hodgson J (1991) Bio / Technology 9: 19-21, incorporated to the present as reference). In one approach, the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex, is determined by X-ray crystallography, by computer modeling or, more typically, by a combination of the two approaches. Both the manner and charges of the polypeptide must be ascertained to clarify the structure and to determine the active site (s) of the molecule. Less frequently, useful information can be obtained with respect to the structure of a polypeptide, by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design similar molecules similar to chemokine, or to identify efficient inhibitors. Useful examples of rational drug design include molecules that have improved their activity or stability, as shown in Braxton S and Wells JA (1992 Biochemistry 31: 7796-7801) or that act as inhibitors, agonists, or antagonists of native peptides, as shown in Athauda SB et al. (1993 J Biochem 113: 742-746), incorporated herein by reference. It is also possible to isolate a target specific antibody, selected by functional assay, as described above, and then dissolve its crystal structure. This approach, in principle, produces a farmanucleus on which a subsequent drug design can be based. It is possible to avoid protein crystallography completely, by generating anti-idiotypic antibodies (anti-ids) for a functional, pharmacologically active antibody. As a mirimage of a mirimage, one would expect the anti-ids binding site to be an analogue of the original receptor. In the present invention, an ani-id antibody from EEC is used to identify and isolate peptides from chemically or biologically produced peptide libraries. Then the isolated peptides would act like the farmanucleus. By virtue of the present invention, a sufficient amount of polypeptides can be made available for performing analytical studies such as X-ray crystallography. In addition, knowledge of the amino acid sequence of EEC will provide guidance for those who employ computer modeling techniques. instead of, or in addition to, X-ray crystallography.

XIII Identification of EEC Receptors Purified E? C are useful for the characterization and purification of specific cell surface receptors and other binding molecules. Cells that respond to the EEC by chemotaxis or other specific responses are likely to express a receptor for EEC. Radioactive labels are incorporated into the EEC by various methods known in the art. A preferred embodiment is the labeling of primary amino groups in EEC with the Bolton-Hunter 125I reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529), which has been used to label other chemokines, without concomitant loss of biological activity (Hebert CA et al. (1991) J Biol Chem 266: 18989; McColl S et al. (1993) J Immunol 150: 4550-4555). Cells carrying receptors are incubated with the labeled chemokine molecule. The cells are then washed to remove unbound chemokine, and the tagged molecule bound to the receptor is quantified. The data obtained using different concentrations of E? C are used to calculate the values for the number and affinity of receptors. The EEC labeling is also useful as a reagent for the purification of your specific receptor. In an affinity purification mode, the chemokine is covalently coupled to a chromatography column. Cells carrying receptors are removed, and the extract is passed over the column. The receptor is fixed to the column by virtue of its biological affinity for EEC. The receptor is recovered from the column and subjected to N-terminal protein sequencing. This amino acid sequence is then used to design degenerate oligonucleotide probes for the cloning of the receptor gene. In an alternative method, mRNA is obtained from cells that carry receptors and are introduced into a cDNA library. The library is transfected into a population of cells, and those cells expressing the receptor are selected, using fluorescently labeled E? C. The specific C receptor is identified by means of the recovery and sequencing of recombinant ADÑ from highly labeled cells. In another alternative method, the antibodies are cultured against the surface of cells bearing receptors, specifically monoclonal antibodies. Monoclonal antibodies are screened to identify those that inhibit the binding of labeled E? C. These monoclonal antibodies are then used in affinity purification or cloning of receptor expression. In a similar manner, soluble receptors or other soluble binding molecules are identified. The labeling is incubated with extracts or other appropriate materials derived from the eosinophil. After incubation, EEC complexes (which are larger than the size of the purified chemokine molecule) are identified by a size classification technique such as size exclusion chromatography or density gradient centrifugation, and purify by methods known in the art. The soluble receptors or binding protein (s) are subjected to N-terminal sequencing, to obtain sufficient information for the identification of the database, if the soluble protein is known, or for cloning, if the soluble protein is unknown. All publications and patents mentioned in the above specification are incorporated herein by reference. It is considered that the above written specification is sufficient to enable an expert in the art to practice the invention. Indeed, it is intended that the various modifications of the modes described above for carrying out the invention, which are obvious to those skilled in the art of molecular biology or related fields, are within the scope of the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: INCYTE PHARMACEUTICALS, INC. (ii) TITLE D? THE INVENTION: NOVELTY CHEMIOCINE EXPRESSED IN EOSINOPHILES (iii) SEQUENCE NUMBER: 9 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: INCYT? PHARMACEUTICALS, INC. (B) STREET: 3174 Porter Drive (C) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: UNITED STATES (F) ZIP: 94304 (v) computer readable FORM: (A) MEDIUM TYPE : 5 1/4 disk (B) cOMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Release # 1.0, version # 1.30 (vi) CURRENT APPLICATION DATA: (A) TCP REQUEST NUMBER: To be Assigned (B) FBCHA D? SUBMISSION: April 13, 1995 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Luther, Barbara J. (B) REGISTRATION NUMBER: 33954 (C) NUMBER D? REFERENCE / ATTORNEY: PF-0031 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-852-0195 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: ( A) GENOTECA: EOSINOFILOS (B) CLON: 288236 (xi) DESCRIPTION OF THE SEQUENCE: S? Q ID NO: 1: ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC C? CATGCTTG TTACTGCCCT TGGATCCCAG 60 GCCCGGGTCA CAAAAGATGC AGAGACAGAG TTCATGATGT CAAAGCTTCC AT GGAAAA? 123 CCAGTACTTC TGGACATGCT CTGGAGGAGA AAGATTGGTC CTCAGATGAC CCTTTCTCAT 133 GCTGCAGGAT TCCATGCTAC TAGTGCTGAC TGCTGCATCT CCTACACCCC ACGAAGCAT S4C CCGTGTTCAC TCCTGGAGAG TTACTTTGAA ACGAACAGCG AGTGCTCCAA GCCGGCTGTC 333 ATCTTCCTCA CCAAGAAGGG GCGACGTTTC TGTGCCAACC CCAGTGATAA GCAAG77CAG 353 GTTGGCATGA GAATGCTGAA (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 137 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOL? CULA: protein (vii) IMMEDIATE SOURCE: ÍA) GENOTECA: EOSINOFILOS (B) CLON: 288236 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Mee Lys Val Ser Val Ala Ala Leu Ser Cys Leu Mßt Leu Val Thr Ala 1 5 10 15 Leu Gly Ser Gln Wing Arg Val Thr Lys Aßp Wing Glu Thr Glu Phe Met 20 25 30 Mßt Ser Lys Leu Pro Leu Glu Asn Pro Val Leu Leu Asp Mßt Leu Trp 35 40 45 Arg Arg Lys llß Gly Pro Gln Mee Thr Leu Ser His Ala Ala Gly Phe 50 '55 60 His Ala Thx Ser Ala Asp Cyß Cys'llß Ser Tyr Thr Pro Arg Ser llß 65 70 75 30 Pro Cys Ser Leu Leu Glu Ser Tyr Phß Glu Thr Asn Ser Glu Cys Ser 85 90 95 Lys Pro Gly Val He Phe Leu Thr Lys Lys Gly Arg Arg Phß Cys Ala 100 105 110 Asn Pro Ser Asp Lys Gln Val Gln Val Cys Mßt Arg Mßt Leu Lys Leu 115 120 125 Asp Thr Arg He Lys Thr Arg Lys Asn 130 135 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Met G n Val Ser Thr Wing Wing Leu Wing Val Leu Leu Cys Thr Mee Wing 1 5 10 15 Leu Cys Asn Gln Phe Ser Wing Being Leu Wing Wing Asp Thr Pro Thr Wing 20 25 30 Cys Cys Phe Ser Tyr Thr Ser Arg Gln lie Pro Gln Asn Phß He Ala 35 40 45 Asp Tyr Phß Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val le Phe 50 55 60 Leu Thr Lys Arg Ser Arg Gln Val Cys Wing Asp Pro Ser Glu Glu Trp 65 70 75 30 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Wing 85 90 '2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE S? CU? NCIA: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: Mßt Lys Leu Cys Val Thr Val Leu Ser Leu Leu Mßt Leu Val Ala Ala 1 5 - 10 15 Phß Cys Ser Pro Ala Leu Ser Ala Pro Mßt Gly Ser Asp Pro Pro Thr 20 25 30 Ala Cys Cys Phß Ser Tyr Thr Ala Arg Lys Leu Pro Arg; Asn Phe Val 35 40 45 Val Asp Tyr Tyr Glu Thr Ser Ser Leu Cys Ser Gln Pro Wing Val Val 50 55 60 Phe Gln Thr Lys Arg Ser Lys Gln Val Cys Wing Asp Pro Sex Glu Ser 65 70 75 30 Trp Val Gln Glu Tyr Val Tyr Asp Leu Glu Leu Asn 85 90 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 99 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu He Ala Ala Thr 1 5 10 15 Phß He Pro Gln Gly Leu Wing Gln Pro Asp Wing He Asn Wing Pro Val 20 25 30 Thr Cys Cys Tyr Asn Phß Thr Asn Arg Lys He Ser Val Gln Arg Leu 35 40 45 Wing Ser Tyr Arg Arg He Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60 He Phß Lys Thr He Val Ala Lys Glu He Cys Ala Asp Pro Lys Gln 65 70 75 30 Lys Trp Val Gln Asp Ser Mßt Asp His Leu Asp Lys Gln Thr Gln Thr 85 90 95 Pro Lys Thr (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 77 amino acids (B) TYPE: amino acid (C) CHAIN TIFO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (Xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: Wing Gln Pro Asp Ser Val Ser He Pro He Thx Cys Cys Phß Asn Val 5 10 15 He Asn Arg Lys He Pro He Gln Arg Leu Glu Ser Tyr Thr Arg He 20 25 '30 Thr Asn He Gln Cys Pro Lys Glu Ala Val He Phß Lys Thr Lys Arg 35 40 45 Gly Lys Glu Val Cys Wing Asp Pro Lys Glu Arg Trp Val Arg Asp Ser 50 55 60 Met Lys His Leu Asp Gln He Phß Gln Asn Leu Lys Pro 65 70 75 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 109 amino acids (B) TYPE: amino acid (C) TYPE 2E CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE O? MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: Mßt Trp Lys Pro Mßt Pro Ser Pro Ser Asn Mee Lys Wing Ser Ala Ala 1 5 10 15 Leu Leu Cys Leu Leu Leu Thr Ala Ala Ala Phß Ser Pro Gln Gly Leu 20 25 30 Wing Gln Pro Val Gly He Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe 35 40 45 He Asn Lys Lys He Pro Lys Gln Arg Leu Glu Ser Tyr Arg Arg Thr 50 55 60 Thr Ser Ser His Cys Pro Arg Glu Wing Val He Phß Lys Thr Lys Leu 65 70 75 30 Asp Lys Glu He Cys Wing Asp Pro Thr Gln Lys Trp Val Gln Asp Phß 85 90 95 Mee Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys Leu 100 IOS (2) INFORMATION FOR THE S? Q ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 91 amino acids (B) TYPE: amino acid (OR CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 * C Lyl Val S # r A1 * A1 * A * S - «Ala Val He Leu He Ala Ala Thr Ala 5 10. fifteen Leu Cys Wing Pro Wing Being Wing Pro Pro Tyr S «r Ser Asp Thr Thr? -o 0 25 30 Cys Cys Ph? Wing Tyr lie Wing Arg Pro Leu Pro Arg Wing HIS He 'vs 35 40 45 Glu Tyr Ph? Tyr Thr Ser Gly Lys Cys Ser Asn Pro Wing Val Val Phß 50 55 60 Val Thr Arg Lys Asn Arg Gln Val Cys Wing Asn Pro Glu Lys Lys Trp 65 70 75 80 Val Arg Glu Tyr He Asn Ser Leu Glu Met Ser (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 86 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TI PO OF MOLECULE: protein (xi) DESCRI PTION OF THE SEQUENCE: S? Q ID NO: 9 Mßt Lys Val Ser Val Ala Wing Leu Ser Val Leu Leu Leu Val Wing Ala 1 5 10 13 Leu Cys Asp Ala Gln Pro Thr Thr Cys Cys Phß Ser Tyr Thr Asn Arg 20 25 30 Lys He Pro Arg Gln Arg Leu Glu Ser Tyr Phß Glu Thr Ser Ser Gln 35 40 45 Cys S «t Lys Pro Ala Val He Phß Lys Thr Lyß Arg Gly Lys Glu Val 50 55- 60 Cys Ala Asp Pro Ser Glu Lys Trp Val Gln Asp Tyr Met Lys Leu Glu 65 70 75 30 Leu Asp Lys Gln Thr Lys 85

Claims (9)

1. A purified polynucleotide comprising a sequence of nucleic acids encoding the polypeptide having the sequence as depicted in SEQ ID NO: 2, or its complement.

2. The polynucleotide of Claim 1, wherein the nucleic acid sequence consists of SEQ ID NO: 1.

3. An expression vector comprising the polynucleotide of Claim 2.

4. A host cell comprising the expression vector of Claim 3.

5. A nucleic acid probe comprising a non-conserved fragment of the polynucleotide of the invention. Claim 2.

6. The nucleic acid probe of Claim 5, comprising a nucleotide sequence encoding the amino acid residues of amino acid 22 to 63, inclusive.

7. An anti-sense molecule comprising a polynucleotide sequence complementary to at least a portion of the polynucleotide of Claim 2.

8. A method for producing a polypeptide comprising the sequence as depicted in SEQ ID NO: 2, comprising the method: a) culturing the host cells of Claim 4, under conditions suitable for expression of the polypeptide, and b) recovering the polypeptide from the cell culture.

9. A chemokine expressed in Eosinophils that has the amino acid sequence as represented in the SEQ ID NO: 2. 11. An antibody specific for the purified polypeptide of Claim 9. 12. A diagnostic composition for the detection of nucleic acid sequences encoding the Splenic Chemokine Expressed in Eosinophils, comprising the nucleic acid probe of Claim 6. 13. A diagnostic test for the detection of nucleic acid sequences encoding Aβ in a biological sample, comprising the steps of: a) combining the biological sample with a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1, or a fragment thereof, under conditions suitable for the formation of a nucleic acid hybridization complex between the nucleic acid sequence of S? Q ID N0: 1 and a complementary nucleic acid sequence in said sample, b) detecting the hybridization complex, and c) comparing the amount of the hybridization complex with a standard, where the presence of an abnormal level of the complex Hybridization correlates positively with a condition associated with inflammation. 14. A diagnostic test for the detection of nucleotide sequences that encode chemokine from Spleen Expressed in Eosinophils in a biological sample, comprising the steps of: a) combining the biological sample with polymerase chain reaction primers under conditions suitable for amplification of the nucleic acid, wherein the former comprise fragments of non-conserved regions of the nucleotide sequence of SEQ ID N0: 1; b) detect the amplified nucleotide sequences; and c) comparing the amount of nucleotide sequences amplified in the biological sample with a standard, determining by the same whether the amount of said nucleotide sequence varies from the standard, where the presence of an abnormal level of the nucleotide sequence is positively correlated with a condition associated with the aberrant expression of EEC. 15. A method for screening a plurality of compounds for specific binding affinity with the polypeptide of Claim 8 or any portion thereof, comprising the steps of: a) providing a plurality of compounds; b) combining Splenic Chemokine Expressed in Eosinophils (E? C) with each of a plurality of compounds for a sufficient time to allow fixation under suitable conditions; and c) detecting the binding of EEC to each of the plurality of compounds, thereby identifying the compounds that specifically bind to EEC.