MXPA97009270A - Homologo de enzima conversion de interleucina-1 hum - Google Patents

Abstract

The present invention provides nucleotide and amino acid sequences that identify and encode a novel homolog of human interleukin-1 conversion enzyme (ICEY). The invention also provides antisense molecules to the nucleotide sequences, which encode ICEY, expression vectors for the production of purified ICEY, antibodies capable of specifically binding to ICEY, hybridization probes or oligonucleotides for the detection of nucleotide sequences encoding to ICEY, genetically engineered host cells for the expression of ICEY, diagnostic tests for the activation of monocyte / macromographs based on nucleic acid molecules that encode ICEY, and the use of protein to produce antibodies capable of specifically binding to the protein and the use of the protein to classify the inhibitor

Description

HOMOLOGO DE ENZIMA CONVERSIÓN DE INTERLEUCINA-1 HUMANA TECHNICAL FIELD The present invention is in the field of molecular biology; more particularly, the present invention describes the nucleic acid and amino acid sequences of a novel interleukin-1 conversion enzyme derived from activated THP-1 cells.

RELATED REQUESTS AND COPENDS This application is a continuation in part of Series No. 08 / 443,865 of E. U.A. , filed on May 31, 1995, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION To understand the conversion enzyme of interleukin-1 (ICE), it is helpful to first examine the role of interleukin-1 (I L-1), its enzyme substrate. I L- 1 facilitates the natural host immunity, predominantly those aspects related to the initiation of inflammatory reactions that protect the body against bacterial infection. (Ayala et al., (1994), J. Immunol 53: 2592-2599). At low concentrations in the bloodstream, IL-1 mediates local inflammation by inducing the synthesis of other cytokines, such as IL-6 and IL-8, and the synthesis of proteins that mediate leukocyte adhesion, and prostaglandin production. (Abbas et al. (1994) Cellular and Molecular Immunology, W.B. Saunders Company). At intermediate concentrations, IL-1 enters the bloodstream and can induce fever, the synthesis of acute plasma proteins by the liver, and metabolic waste (cachexia) (Abbas, supra). At even higher concentrations, IL-1 has been implicated in the destruction of tissue observed in numerous inflammatory-related diseases, including rheumatoid arthritis, septic shock, inflammatory bowel disease, and insulin-dependent diabetes mellitus. (Li et al. (1995) Cell 80: 401-411). The activity of IL-1 results from the expression and release of two gene products, IL-1a and IL-1β, predominantly from activated monocytes. (Howard et al. (1991) J. Immunology 147: 2964-2969). Both gene products are initially synthesized as inactive precursors of approximately 31 kDa in monocytes. Pre-IL-1ß is divided to an active 17 kDa form through the IL-1β conversion enzyme (ICE) before being released from the activated monocytes. Pre-IL-1a is probably divided to an active form of 17kDa through a calpain-like IL-1a conversion enzyme prior to release. (Carruth et al. (1991) J. Biol. Chem. 266: 12162-12167). In addition, ICE has been implicated in the release of IL-1a from activated monocytes but the mechanism is not understood (Li, supra). The gene product IL-1β is the predominant form of IL-1 which is present at high concentrations in the bloodstream during inflammatory diseases, such as rheumatoid arthritis, septic shock, inflammatory bowel disease, and insulin-dependent diabetes mellitus ( Li, supra). Since pre-I L-1 ß cleavage through ICE is coupled to the release of IL-1β and increased IL-1 activity in the bloodstream, the activity of ICE may be higher in these pathological conditions. The importance of regulating the activity of ICE to modulate the concentration of I L-1β to effect the host immune response has recently been confirmed. The product of the crmk gene of the pox virus prevents the proteolytic activation of I L-1β and inhibits the host inflammatory response. The smallpox virus containing the crmk gene is unable to suppress the inflammatory response, resulting in a reduction of cells infected by the virus and less damage to the host. (Miura et al., (1993) Cell 75: 653.-660). ICE is a novel cysteine protease that is known to specifically separate the inactive IL-1β precursor to its active form. (Ayala, supra). This protease recognizes the presence of Asp-X, where x is preferably a small hydrophobic amino acid residue, and separates the binding between Asp and X. However, many of the Asp-X links are not recognized by ICE suggesting that flanking frequencies or other criteria, such as accessibility, are also required for recognition and cleavage. In the case of IL-1β, the ICE separates the precursor to form I L-1 active β in two bonds of specific sequence, the link between the residues Asp-27 and Gly-28 and the link between the residues Asp-1 16 and Ala-1 17. The same ICE is synthesized and maintained in the cells as an inactive 45 kDa precursor, which is processed to the active ICE consisting of 20- and 10-kDa subunits, p20 and p10. (Ayala, supra). Both units are derived from the 45 kDa precursor, which is separated into four different fragments, a 13 kDa precursor domain, the p20 subunit, a 2 kDa separator, and the p 10 subunit. Since all these polypeptide fragments are flanked by the Asp-X residues in the intact 45 kDa precursor, it is possible that the ICE precursor is autocatalytically activated. (Ayala, supra). The three-dimensional structure of ICE has been determined from crystallographic studies. (Walker et al., (1994) Cell 78: 343-352). First, it is evident that the active form of ICE is a homodimer of catalytic domains, each of which consists of subunits p20 and p10. Second, although the active site cysteine residue is located at p20, both p20 and p10 are essential for the activity. The structures of subunits p20 and p10 are intertwined in order to create a single 6-strand beta-sheet core flanked on both sides by alpha helices. The first 4 beta chains are contributed by p20, while the remaining 2 beta chains are contributed by p10. The ICE genes of various forms have been sequenced and possess a total amino acid homology of 29% to the ced-3 product of the Caenorhabditis elegans gene, a gene with a possible role in apoptosis (Yuan et al. (1993) Cell 75: 641-652). In addition, the ICE gene contains a sequence region, which encodes residues 166 to 287, which shares a 43% homology with ced-3. It is not known if ced-3 acts as a cysteine protease, but it does contain the putative catalytic residues that are located in the active site of ICE (Cys285 and His237). The amino acid pentapeptide, Glu-Ala-Cys-Arg-Gly (QACRG), which contains the active site cysteine, is the longest peptide conserved between ICE, from mice and humans, and CDE-3, from three different nematodes . In addition, ced-3 contains the same four residues whose side chains are involved in the binding of the aspartate-carboxylate group of the substrate at the catalytic site (Arg-179, Gln-283, Ser 347 and Arg-341) (Yuang, supra) . Inhibitors with specific ICE inhibitor crnA blocks apoptosis induced by TNF and FAS. Therefore, ICE or a homolog is thought to be involved in apoptosis induced by TNF and FAS. In addition, ICE possesses a degree of homology to the mammalian gene Nedd-2 / lch-1, a gene product with a possible role in embryogenesis, which is expressed during the development of embryonic brain and is regulated in adult brain . (Yuan, supra). Nedd-2, ced-3 and the ICE gene products are approximately 27% homologous. The carboxy terminus of CED- and p10 possesses the highest degree of homology for Nedd-2. The Need-2 gene product does not contain the highly conserved cysteine or the highly conserved pentapeptide QACRG found in the ICE and is probably not a cysteine protease. To confirm the role of ICE in inflammation-related diseases by controlling levels of active IL-1β, Li, supra, created ICE-deficient shock mice. These genetically engineered mice were physiologically normal but lacked the ability to process the IL-1ß percussor to its active form when the monocytes were activated with microbial products, such as lipopolysaccharides (LPS). In addition, the production of IL-1 a was reduced, and the level of other cytokines, tumor necrosis factor (TN F) and I L-6, involved in inflammatory responses to microbial products was somewhat reduced. These mice were resistant to the lethal effects of septic shock, when exposed to LPS (Li, supra). Therefore, the inhibition of ICE activity to reduce the concentration of I L-1β in the bloodstream can be a method to treat diseases related to inflammation. The ICE can be used to help identify patients who are susceptible to these diseases. Since ICE shares sequence homology to ced-3 and overexpression of ICE appears to induce apoptosis, studies of ICE-deficient mice were important, since the mice appeared normal in terms of their development. If ICE played a strong role in apoptosis during development, ICE deficient mice might have had higher abnormalities in the brain, intestines, lymphoid and brain tissues (Li, supra). However, ICE can perform functions other than cleavage of the IL-1β precursor. ICE mRNA has been detected in a greater variety of tissues than mRNA of IL-1β (Miura, supra). ICE has attracted interest as a target for novel anti-inflammatory drugs, since the cytokine, which activates, IL-1β, is pro-inflammatory and has been implicated in the pathophysiology of several diseases, including rheumatoid arthritis, septic shock , inflammatory bowel disease and insulin-dependent diabetes mellitus (Dinarello and Wolf (1993) N Engl J. Med. 328: 106-13). The provision of a new ICE gene and polypeptide will also be a drug search to classify and design more effective and more specific inhibitors to this pro-inflammatory substance. Not surprisingly, this ICE homologue was found in a collection of activated monocytic cells, mainly, THP-1 cells treated with phorbol and endotoxin.

THP-1 cells THP-1 is a human leukemic cell line with distinct monocytic characteristics, derived from the blood of a one-year-old child with acute monocytic leukemia (Tsuchiya S. et al. (1980) Int. J. Cancer 26 : 171 -176). The monocytic nature of TH P-1 was established using the following cytological and cytochemical criteria: 1) a-naphthyl butyrate esterase activity, which could be inhibited by NaF (sodium fluoride), 2) lysozyme production, 3) phagocytosis (the absorption of extracellular materials) of latex particles and cells of pure blood of sensitized sheep, and 4) ability of THP-1 cells treated with mitomycin C to activate the T lymphocytes after a treatment with concanavalin A. Morphologically, the cytoplasm contained small azurophilic granules, the nucleus was indented with an irregular shape with deep folds, and the cell membrane had Fc and C3b receptors, which probably function in phagocytosis. Monocytes develop from monoblasts through promotions in the spinal cord and in their mature form they have a half-life of approximately 3 days. Approximately 75% of the circulating monocyte deposit was found along the walls of the blood vessels, although these cells were randomly migrated to present antigenic or phagocytic tissues. The antigen monocytes present include interdigitalization and follicular dendritic reticular cells of the lymph and skin nodes. Phagocytic monocytes are prominent as the Kupffer cells of the liver and in the alveoli and spinal cord. Since precursor monocytes are rich in cytoplasmic granules containing peroxidase, azurophilic, macrophages have numerous cell surface receptors through which they verify their environment. These include receptors for immunoglobulin, complement, growth factors, lipoproteins, peptides and polysaccharides. The binding of ligands to these receptors activates macrophage proliferation, chemotaxis, secretion and phagocytosis. Many human myeloid and myelomonocytic cell lines retain some ability to differentiate to more mature phenotypes in response to various internal stimuli, including growth factors, lymphokines, cytokines, vitamin D derivatives, and tumor promoters and external agents such as trauma, smoking, UV irradiation, exposure to asbestos, and steroids. TH P-1 cells treated with the 12-O-tetradecanoyl-phorbol-13 (TPA) acetate promoter are induced to arrest proliferation and differentiate into macrophage-like cells, which resemble monocyte-derived macrophages. native, both morphologically and physiologically. These monocyte / macrophage type cells exhibit changes in gene expression, such as the co -duction of C-fos and c-jun and the reduced regulation of c-myb (Auwerx (1991) Experientia 47: 22-31), increase the C3b receptor complement density, and reduce both the FcR and the adhesion molecule, CD4. In addition, TH P-1 cells produce lipoprotein lipase and polyprotein E associated with atherosclerotic lesions, secrete several pro-inflammatory cytokines, including IL-1β and TNF (Cochran FR and Finch-Arietta MB (1989) Agents and Actions 27 : 271-273), and can make powerful tissue destruction oxidants and proteases, such as the I L-1 conversion enzyme.

DESCRIPTION OF THE INVENTION The subject invention provides a unique nucleotide sequence, which encodes a novel human ICE homolog. The new gene, also known as ICEY, which was encoded within the Incyte 14775 clone, encodes the ICEY polypeptide and represents a human cysteine protease. The invention also comprises diagnostic tests for the physiological or pathological activity of activated monocytes or macrophages, which include the steps of testing a sample or an extract thereof with cDNA encoding an ICE homologue, fragments or oligomers thereof. Other aspects of the invention include the antisense of icey; cloning or expression vectors containing icey; cells or host organisms transformed with expression vectors containing icey; a method for the production and recovery of purified ICEY polypeptides from host cells; purified ICEY polypeptide; antibodies and inhibitors for ICEY, and pharmacological compounds that use ICEY antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B depict the nucleotide sequence for the ice homologue and the predicted amino acid sequence of the ICE homologue (SEQ ID NO: 5 and SEQ ID NO: 6, respectively). Figures 2A and 2B show the amino acid alignment of the novel ICE homolog with human ICE (Gen Bank locus HSU13697); Number Gl 717040). The alignments shown were produced using the multiple sequence alignment program of the DNASTAR software (DNASTAR I nc., Madicson Wl). Figure 3 presents an analysis of alpha regions of the homolog ICE (A), beta (B) regions, return regions (T), coil regions (C), hydrophilic character plot (H), alpha amphipathic regions (AA), amphipathic beta regions (BA), antigenic index ( Al) and surface probability point (S) based on the predicted amino acid sequence and composition. Figure 4 depicts the alignment of the partial cDNA sequences with the complete icey gene.

MODES FOR CARRYING OUT THE INVENTION Definitions As used herein, the term "ICE homologue" or "ICEY" refers to the polypeptide described in SEQ ID NO: 6, or to an active fragment thereof, which is encoded by a mRNA transcribed from the cDNA. of the ice counterpart of SEQ ID NO: 5. ICEY can be naturally occurring or chemically synthesized. As used herein, the "icey" in lowercase letters refers to a nucleic acid sequence, while a "ICEY" in uppercase refers to a protein, peptide or amino acid sequence. As used herein, the term "active" refers to those forms of ICEY, which retain the biological and / or immunological activities of ICEY of natural existence. As used herein, the term "ICEY of natural existence" refers to ICEY produced by human cells that have not been genetically engineered and specifically contemplate various forms of ICEY that arise from post-translational modifications of the polypeptide, including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. As used herein, the term "derivative" refers to polypeptides derived from naturally occurring ICEY through chemical modifications such as ubiquitination, labeling (eg, with radionuclides, various enzymes, etc.), pegylation ( derivatization with polyethylene glycol) or by insertion or substitution through chemical synthesis of the amino acid such as ornithine, which normally does not occur in human proteins. As used herein, the term "variant" or "recombinant variant" or "mutant" refers to any polypeptide other than an ICEY naturally occurring through amino acid insertions, deletions, and substitutions, created using DNA techniques. recombinant. The guide to determine which amino acid residues can be replaced, added or deleted without abolishing the activities of interest, such as enzymatic activity, can be found by comparing the sequence of the particular ICEY with that of homologous molecules and minimizing the number of changes of amino acid sequence made in regions of high homology or regions of known activity. Preferably, the amino acid "substitutions" are the result of replacing 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, in aspartate with glutamate, or a threonine with a serine, that is, conservative amino acid replacements. The "insertions" or "deletions" are typically on the scale of about 1 to 5 amino acids. The allowed variation can be experimentally determined by systematically making insertions, deletions or amino acid substitutions in ICEY using recombinant DNA techniques and analyzing the resulting recombinant variants for activity. When you want, the nucleic acid encoding ICEY or a variant of ICEY can be genetically engineered to contain a "leader or signal sequence" that can direct the polypeptide through the membrane of a cell. As will be understood by those skilled in the art, said sequence may be naturally occurring in the polypeptides of the present invention or provided with sources of heterologous protein by recombinant DNA techniques. As used herein, a "fragment" of ICEY, "portion", or "segment" refers to a stretch of amino acid residues, which is of sufficient length to exhibit biological and / or immunogenic activity and in preferred embodiments will contain at least about 5 amino acids, at least 7 amino acids , at least about 8 to 13 amino acids, and, in additional embodiments, about 17 or more amino acids. As used herein, an "oligonucleotide" or "polynucleotide fragment," "portion," or "segment" refers to any stretch of nucleic acid encoding ICEY, which is of sufficient length to be used as an initiator in the polymerase chain reaction (PCR) or various hybridization methods known to those skilled in the art, for the purpose of identifying or amplifying identical or related nucleic acids.

The present invention includes ICEY polypeptides purified from natural or recombinant sources, vectors and host cells transformed with recombinant nucleic acid molecules encoding ICEY. Various methods for the isolation of ICEY polypeptides can be achieved by methods well known in the art. For example, said polypeptides can be purified by immunoaffinity chromatography using the antibodies provided by the present invention. Various other protein purification methods well known in the art include those described in Deutscher M (1990) Methods in Enzymology, Vol. 182, Academic Press, San Diego; and Scopes R (1982) Protein Purification: Principies and Practice. Spring-Verlag, New York, both incorporated here by reference. As used herein, the term "recombinant" refers to a polynucleotide, which encodes ICEY and is prepared using recombinant DNA techniques. The polynucleotide encoding ICEY may also include allelic or recombinant variants and mutants thereof. As used herein, the term "probe" or "nucleic acid probe" or "oligonucleotide probe" refers to a portion, fragment, or segment of icey that is capable of being hybridized to a desired target nucleotide sequence. . A probe can be used to detect, amplify or quantify cDNAs or endogenous nucleic acid encoding ICEY using conventional techniques in molecular biology. A probe may be of variable length, preferably from about 10 nucleotides to several hundred nucleotides. As will be understood by those skilled in the art, the conditions of hybridization and probe design will vary depending on the intended use. For example, a probe intended for use in PCR will generally be about 15 to 30 nucleotides in length, and may include up to 60 nucleotides, and may be part of a deposit of degeneration probes, ie, oligonucleotides that tolerate nucleotide mismatch but it adapts the union to an unknown sequence; while a probe for use in Southern or Northern hybridizations may be a single, specific nucleotide sequence, which is several hundred nucleotides in length. Accordingly, a preferred probe for the specific detection of icey will comprise a polynucleotide or oligonucleotide fragment from a non-conserved nucleotide region of SEQ ID NO: 5. As used herein, the term "non-conserved nucleotide region" refers to a nucleotide region that is unique to SEC I D NO: 5, and does not comprise a region that is conserved in the family of ICE genes. The probes can be single chain or double chain and can have a specific character in hybridizations based on solution, cell, tissue or membrane, including on-site ELISA type technologies. The present invention encompasses oligonucleotides, fragments or portions of the polynucleotides described herein, or their complementary strands used as probes. The nucleic acid probes may comprise portions of the sequence having fewer nucleotides than about 6 kd 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 ICEY, is present in a cell or tissue or to isolate identical or similar nucleic acid sequences of chromosomal A DN, as described by Walsh PS et al. (1992 PCR Methods Appl 1: 241-250) The nucleic acid probes of the present invention invention can be derived from naturally occurring nucleic acids or recombinant single or double chain or be chemically synthesized. They can be labeled through groove translation, Klenow fill 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. and others (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 here by reference. Alternatively, the recombinant variants encoding the polypeptides of the present invention or related polypeptides can be synthesized or identified through hybridization techniques known to those skilled in the art making use of "redundancy" in the genetic code. Several codon substitutions, such as silent changes that produce several restriction sites, can be introduced to optimize cloning to a plasmid or viral vector 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, polypeptide degradation or turnover rate. One example involves inserting a stop codon into the nucleotide sequence to limit the size of ICEY in order to provide a binding, a non-activated ligand of smaller molecular mass, which could serve to block the activity of the natural ICEY. "Activated monocytes", as used herein, refers to activated, mature monocytes or macrophages found in immunologically active tissues. "Monocyte / macrophage disorders" include, but are not limited to, inflammatory bowel disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, and sepsis. "Animal", as used herein, can be defined to include humans, domestic or agricultural animals (cats, dogs, cows, sheep, etc.) or test species (mouse, rat, rabbit, etc.). The present invention includes ICEY polypeptides purified from natural or recombinant sources, cells transformed with recombinant nucleic acid molecules encoding ICEY. Various methods for isolating the ICEY polypeptides can be achieved through methods well known in the art.

For example, said polypeptides can be purified through immunoaffinity chromatography using the antibodies provided by the present invention. Several other protein purification methods, well known in the art, include those described in Deutscher M (1990) Methods in Enzymology, Vol. 182, Academic Press, San Diego, CA; and Scopes R (1982) Protein Purification: Principies and Practice, Springer-Verlag, New York, both incorporated herein by reference. "ICE inhibitors," or ICE-like molecules that bind I L-1, but do not activate it, can be recombinantly synthesized by substituting the other cysteine for other natural (such as alanine) or synthetic amino acids at the active pentapeptide site. Alternatively, the cysteine can be chemically modified by routine methods in the art, in order to inhibit activation. ICE homologs, with a modified specific character, can be easily synthesized by replacing the cysteine-free residues of the conserved pentapeptide, GLU ALA CYS ARG GLY. The preparation of said recombinant mutants is well known in the art. Substitution of the four amino acids without cysteine of the pentapeptide with other natural or synthetic amino acids can be readily accomplished by those skilled in the art. The activity of the ICE mutants can be tested by methods known to those skilled in the art. The present invention provides a nucleotide sequence found within clone I ncyte 14775 only by identifying a new, human ICE homologue (ICEY) of the cysteine protease family, which was initially found expressed in TH P-1 cells treated with ester. of forbol. Since ICEY is expressed in monocytes, nucleic acids (icey), polypeptides (ICEY) and antibodies to ICEY, are useful in diagnostic assays based on the identification of ICEY associated with inflammatory processes or inflammatory disease. It was also found that ICEY was expressed in cDNA collections made of macrophages; ovarian tumor; colon; fetal placenta; chest; prostate tumor; synovium of a rheumatoid knee, an osteoarthritic knee and a rheumatoid doll; lymphocytes; lymphocytes from a mixed 24-hour lymphocyte reaction; spleen; spine; cervix; lung; peripheral blood granulocytes treated with fM LP; ganglioneuroma; Fetal liver / spleen; neonatal placenta; and eosinophil is from an individual who has hyper eosinophilia. Excessive ICEY expression can lead to tissue damage or destruction. Therefore, a diagnostic test for ICEY can accelerate the diagnosis and appropriate treatment of numerous inflammatory disorders including activated monocyte disorders, such as inflammatory bowel disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, septic shock and similar pathological problems. . The nucleotide sequences encoding ICEY (or its complement) have numerous applications in techniques known to those skilled in the field of molecular biology. These techniques include the use as hybridization probes, the use in the construction of oligomers for PCR, the use for chromosome and gene mapping, the use in recombinant production of ICEY, and the use in the generation of anti-sense DNA or RNA. , its chemical analogues and the like. The uses of nucleotides encoding I CEY described herein are illustrative of known techniques and are not intended to limit their use to any technique known to one of ordinary skill in the art. In addition, the nucleotide sequences described here can be used in molecular biology techniques that have not yet been developed, provided that the new techniques are based on properties of nucleotide sequences that are currently known, v. gr. , the triplet genetic code and the interactions of specific base pairs. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding ICEY, some carrying minimal homology to the nucleotide sequence of any known and naturally occurring gene, can be produced. The invention has specifically contemplated each of the possible variations of the nucleotide sequence that can be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the normal triplet genetic code as applied to the natural existence icey nucleotide sequence, and all these variations are considered to be specifically described. Although the nucleotide sequences, which encode ICEY and / or its variants, are preferably capable of hybridization to the naturally occurring icey nucleotide sequence under severe conditions, it may be advantageous to produce nucleotide sequences encoding ICEY or its derivatives possessing a substantially different use of codon. The codons can be selected to increase the rate at which the expression of the peptide occurs in a particular prokaryotic or eukaryotic host expression according to the frequency with which the codons are used by the host. Other reasons for substantially altering the nucleotide sequence encoding ICEY and / or its derivatives without altering the encoded amino acid sequence include the production of RNA transcribers having more desirable properties, such as a longer half-life, than the transcriptionists produced from the sequence of natural existence. The nucleotide sequences encoding ICEY can be linked to other nucleotide sequences through well-established recombinant DNA techniques (see Sambrook J et al., Supra). The nucleotide sequences for icey binding include a cloning vector classification, v. gr. , plasmids, cosmids, lambda 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 origin of replication in at least one organism, restriction endonuclease sensitive sites, and selectable markers for the host cell. Another aspect of the present invention is to provide icey-specific nucleic acid hybridization probes capable of hybridization to naturally occurring nucleotide sequences encoding ICEY. Said probes can also be used for the detection of similar ICEY-encoding sequences and preferably must contain at least 50% of the nucleotides of the conserved region or active site. The hybridization probes of the present invention can be derived from the nucleotide sequences of Figures 1 A and 1 B or from genomic sequences including promoters, enhancer elements and / or possible introns of the respective wild-type ice axes. Hybridization probes can be labeled through a variety of reporter groups, including radionuclides such as 3 P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin / biotin coupling systems, and the like. PCR as described in the patents of E.U.A. Us 4,683, 195; 4,800, 195; and 4,965, 188 provides additional uses for the oligonucleotides based on the nucleotide sequence encoding ICEY. Said probes used in the PCR can be of recombinant origin, can be chemically synthesized, or a mixture of both, and comprise a discrete nucleotide sequence for diagnostic use or a deposit of degeneration of possible sequences for the identification of closely related genomic sequences . Other means for producing hybridization probes specific for icey DNAs include the cloning of nucleic acid sequences encoding ICEY or ICEY derivatives to vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro through the addition of the appropriate RNA polymerase such as T7 or SP6 RNA polymerase and radioactively appropriate labeled nucleotides. It is now possible to produce a DNA sequence, or portions thereof, that encodes ICEY and its derivatives completely by synthetic chemistry, after which the gene can be inserted into any of many available DNA vectors using reagents, vectors and cells which are known in the art at the time of filing this application. In addition, synthetic chemistry can be used to introduce mutations in the icey sequences or any portion thereof. The nucleotide sequence of the nucleic acid encoding ICEY can be confirmed by DNA sequencing techniques. Methods for sequencing DNA are well known in the art. Conventional enzymatic methods employed a Klenow fragment of DNA polymerase, SEQUENASE® (US Biochemical Corp., Cleveland, OH) or Taq polymerase to extend the DNA strands of the oligonucleotide primer fortified to the DNA template of interest. Methods have been developed for the use of both single and double chain templates. The chain termination reaction products were electrophoresed on urea-acrylamide gels and detected either by autoradiography (for radionuclide-labeled precursors) or by fluorescence (for fluorescent-labeled precursors). Recent improvements in mechanized reaction preparation, sequencing and analysis using the fluorescent detection method have allowed the expansion in the number of sequences that can be determined per day (using machines such as the Catalyst 800 and the Applied DNA sequencer Biosystems 377 or 373). The nucleotide sequence can be used in an assay to detect inflammation or disease associated with abnormal levels of ICEY expression. The nucleotide sequence can be labeled by methods known in the art and added to a fluid or tissue sample of a patient under hybridization conditions. After an incubation period, the sample is washed with a compatible fluid, which optionally contains a dye (or other brand that requires 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 normal one. 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 icey can be used to construct hybridization probes for the mapping of that gene. the nucleotide sequence provided herein may be mapped to a particular chromosome or to specific regions of that chromosome using well-known genetic and / or chromosomal mapping technique. These techniques include in situ hybridization, ligation analysis against known chromosomal markers, hybridization classification with collections, chromosomal preparations selected by flow, or constructions of artificial chromosome YAC, P1 or BAC. The fluorescent in situ hybridization technique of chromosome extensions has been described, inter alia, in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York. The fluorescent in situ hybridization technique of chromosomal preparations and other physical chromosome mapping techniques can be correlated with genetic map data. Examples of genetic map data can be found in Genome Issue of Science 1994 (265: 1981 f). The correlation between the location of icey on a physical chromosomal map and a specific disease (or predisposition to a specific disease) can help to narrow down the region of DNA associated with that genetic disease. The nucleotide sequence of the present invention can be used to detect the difference in gene sequence between normal and carrier or affected individuals.

The nucleotide sequences encoding ICEY can be used to produce purified ICEY using well known methods of recombinant DNA technology. Among the many purifications that teach methods for the expression of genes after they have been isolated is Goeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol. 185, Academic Press, San Diego CA. ICEY can be expressed in a variety of host cells, either prokaryotic or eukaryotic. The host cells can be from the same species where the icey nucleotide sequences are endogenous or from a different species. The advantages of producing ICEY by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures. Cells transformed with DNA encoding ICEY can be cultured under conditions suitable for the expression of ICEY and recovery of the protein from the cell culture. The ICEY produced through a recombinant cell can be secreted or it can be contained intracellularly, depending on the sequence of ICEY and the genetic construct 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. In addition to recombinant production, fragments of ICEY can be produced through direct peptide synthesis using solid phase techniques (see Stewart et al. (1969) Solid-Phase Synthesis, WH Freeman Co, San Francisco CA); Merrifield J (1963) J. Am. Chem. Soc. 85: 2149-2154). In vitro protein synthesis can be performed using manual techniques or by automation. Automatic synthesis can be achieved, for example, using the Applied Biosystems 431 A Peptide Synthetizer (Foster City, CA) in accordance with the instructions provided by the manufacturer. Several fragments of ICEY can be chemically synthesized separately and combined using chemical methods to produce the full-length molecule. ICEY for antibody induction does not require biological activity; however, the protein must be antigenic. The peptide used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids and preferably at least 10 amino acids. These must resemble a portion of the amino acid sequence of the protein and may contain the entire amino acid sequence of a small natural existence molecule such as ICEY. Short amino acid stretches of ICEY can be fused with those of another protein such as a key limpet hemocyanin and the chimeric molecule used for antibody production. Several methods are known to those skilled in the art to prepare monoclonal and polyclonal antibodies for ICEY. In one aspect, a denatured ICEY of the HPLC separation of reverse fas is obtained and can be used to immunize mice or rabbits using techniques known to those skilled in the art. Approximately 100 micrograms are suitable for the immunization of a mouse, while up to 1 mg is used for the immunization of a rabbit. To identify mouse hybridomas, the denatured protein can be radioiodinated and used to classify potential murine B-cell hybridomas for those that produce the antibody. This procedure requires only small amounts of protein, such as 20 mg could be sufficient to mark and classify the several thousand clones. In another aspect, the amino acid sequence of ICEY, as deduced from the translation of the DNA sequence, is analyzed to determine regions of high immunogenicity. For example, oligopeptides comprising hydrophilic regions, as shown in Figure 3, can be synthesized and used in suitable immunization protocols to produce antibodies. The analysis to select appropriate epitopes is described in Ausubel FM et al. (1989, Current Protocols in Molecular Biology, John Wiley & amp;; Sons, NYC). The optimal amino acid sequences for immunization are usually in the C-terminus, the N-terminus and those hydrophilic intervening regions of the polypeptide, which will likely 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 an Applied Biosystems Peptide Synthesizer Model 431A using fmoc chemistry and coupled to key limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N ester -hydroxy-succinimide (MBS; see Ausubel FM et al., supra). If necessary, a cysteine can be introduced at the N-terminus of the peptide to allow coupling to KLH and animals can be immunized with the peptide-KLH complex in a complete Freund's helper. The resulting antisera can be treated for antipeptide activity by binding the peptide to the plastic, blocking with 1% BSA, reacting with antisera, washing and reacting with specific goat anti-rabbit IgG, affinity purified, labeled (radioactive or fluorescent). ). Hybridomas can also be prepared and classified using normal techniques. Hybridomas of interest can be detected by classifying with labeled ICEY to identify those fusions that produce the monoclonal antibody with the desired specific character. In a typical protocol, the plate cavities (FAST, Becton-Dickinson, Palo Alto, CA) are coated with specific rabbit-anti-mouse antibodies (or suitable anti-species Ig) at 10 mg / ml. The coated cavities are blocked with 1% BSA, washed and exposed to supernatants of hybridomas. After incubation, the cavities are exposed to ICEY labeled at 1 mg / ml. The clone production antibodies will bind a marked amount of ICEY, which is detectable above the background. These clones can be expanded and subjected to 2 cloning cycles at a limiting dilution (1 cell / 3 cavities). Cloned hybridomas are injected into mice treated with pristane to produce ascites, and the monoclonal antibody can be purified from mouse ascites fluid by affinity chromatography using Protein A. Monoclonal antibodies with affinities of at least 108 M * 1, preferably from 109 to 1010 or stronger, typically will be done through normal procedures as described in Harlow and Lane (1988) Antibodies; A Laboratory Manual. Cold Spring Harbor Laboratory, New York; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, New York, both incorporated herein by reference. Antibodies specific for ICEY can be produced by inoculating an appropriate animal with the polypeptide or an antigenic fragment. An antibody is specific for ICEY if it is produced against all or a portion of ICEY and binds to all or a portion of ICEY. The induction of antibodies includes not only the stimulation of an immune response by injection to animals, but also analogous steps in the production of synthetic antibodies or other specific binding molecules such as the classification of recombinant immunoglobulin collections (Orlandi T 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 (1991) Nature 349: 293-299) provides a number of highly specific binding reagents based on the principles of antibody formation. These techniques can be adapted to produce molecules that specifically bind to ICEY. A further embodiment of the present invention is the use of ICEY-specific antibodies, inhibitors, receptors or their analogs as bioactive agents to treat activated monocyte disorders, such as inflammatory bowel disease., insulin dependent diabetes mellitus, reu matoid arthritis, septic shock or similar pathological problems. Bioactive compositions comprising agonists, antagonists, receptors or inhibitors of ICEY can be administered at a suitable therapeutic dose determined by any of several methodologies including clinical studies in mammalian species to determine the maximum tolerable dose and in normal human subjects for determine the safe dose. In addition, the bioactive agent can be complexed with a variety of well-established compounds or compositions, which improve stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered to the blood stream by intravenous infusion or any other effective means that may be used to treat conditions associated with the production and function of IC EY. Antibodies, inhibitors, or antagonists of ICEY (or other treatments for excessive production of ICEY, hereinafter referred to as "treatments"), may provide different effects when administered therapeutically. The treatments will be formulated in a non-toxic, inert, aqueous, pharmaceutically acceptable vehicle medium, preferably at a pH of about 5 to 8, most preferably 6 to 8, although the pH may vary according to the characteristics of the antibody, inhibitor, or antagonist that is being formulated and the condition that will be treated. The characteristics of the treatments for the excessive production of ICEY include the solubility of the molecule, half-life and immunogenicity; These and other characteristics can help define an effective vehicle. Native human proteins are preferred as treatments for the excessive production of ICEY, but organic or synthetic molecules resulting from drug classifications can be equally effective in particular situations.

Treatments for overproduction of ICEY can be delivered through known routes of administration including, but not limited to, topical creams and gels, patches and transdermal dressings; injectable, intravenous and washing formulations; and orally adm ined liquids and pills, particularly formulated to resist stomach acid and enzymes. The particular formulation, the exact dose, and route of administration will be determined by the attending physician and will vary according to each specific situation. These determinations are made considering multiple variables such as the condition that will be treated, the treatment that will be administered, and the pharmacokinetic profile of the particular treatment. Additional factors that may be taken into account include the patient's disease status (eg, severity), age, weight, gender, diet, time of administration, drug combination, reaction sensitivities, and tolerance / response to the therapy. Long-acting formulations can be administered only once a day, or even less frequently: every 3 to 4 days, every week, or every two weeks, depending on the half-life and clear regimen of particular treatments.

The normal dose amounts may vary from 0.1 to 100, 000 micrograms, up to a total dose of approximately 1 g, depending on the route of administration. The guide for particular doses and methods of supply is provided in the literature; see patents of E. U.A. Nos. 4,657,760; 5,206, 344; 0 5,225,212. It is anticipated that different formulations will be effective for different treatments and that routine administration may require delivery in a form different from that of local administration to a specific organ or tissue. It is contemplated that conditions or diseases that activate monocytes, macrophages and possibly leukocytes can precipitate damage that is treatable with antibodies, inhibitors or antagonists of ICEY. Activated monocyte disorders can be specifically diagnosed by the tests discussed above, and such tests can be performed in cases of suspected inflammatory bowel disease, insulin dependent diabetes mellitus, rheumatoid arthritis, sepsis and similar physiological / pathological problems. The Examples presented below are provided to illustrate the present invention. These examples are provided by way of illustration only and are not included for the purpose of limiting the invention.

INDUSTRIAL APPLICABILITY I. Isolation of mRNA and Constructions of cDNA Collections The icey sequence was identified between the unique sequences 14775 (SEQ ID NO: 1) and 157811 (SEQ ID NO: 2) obtained from collections of human activated THP-1. THP-1 is a human leukemic cell line derived from the blood of a one-year-old child with acute monocytic leukemia. The cells used for the activated collection (or PMA + LPS) were cultured for 48 hours with 100 nm of PMA in DMSO and for 4 hours with 1 μg / ml of LPS. The activated THP-1 collection was constructed as usual by Stragene (Stragene, 11099 M. Torrey Pines Rd., La Jolla, CA 92037) essentially as described below. Stragene prepared the cDNA library using oligo d (T) initiation. The synthetic adapter oligonucleotides were ligated onto the cDNA molecules allowing them to be inserted into the Uni-ZAP ™ vector system (Stragene). This allowed a lambda collection construction of unidirectional efficiency (sense orientation) and the convenience of a plasmid system with blue / white color selection to detect clones with cDNA inserts. The quality of the cDNA library was classified using DNA probes, and then the phagemid pBluescript® (Stragene) was separated. This phagemid allows the use of a plasmid system to facilitate insert characterization, sequencing, site-directed mutagenesis, the creation of unidirectional deletions and expression of fusion polypeptides. Subsequently, the collection phage particles constructed as usual were infected with XL1-Blue® (Stragene) of the E. coli host strain. The high transformation efficiency of this bacterial strain increases the probability that the cDNA library will contain rare, under-represented clones. Alternative unidirectional vectors may include, but are not limited to, pcDNAl (Invitrogen, San Diego CA) and pSHIox-1 (Novagen, Madison Wl).

II. Isolation of cDNA Clones The phagemid forms of individual cDNA were obtained by the in vivo excision procedure, where it was coinfected XL1 -BLU E with an auxiliary phage f1. Proteins derived from both lambda phage and helper phage f1 initiated new DNA synthesis from defined sequences on the target DNA of lambda and created a DNA molecule phagemid circular single strand, smaller including all sequences Plasmid DNA of pBluescript and the insert of cDNA. The phagemid DNA was released from the cells and purified, then used to re-infect fresh bacterial host cells (SOLR, Stragene Inc.), where the double-stranded phagemid DNA was produced. Since the phagemid carries the gene for β-lactamase, the newly transformed bacteria were selected from the medium containing penicillin. The phagemid DNA was purified using QIAWELL-8 Plasmid Purification System from QIAGEN® DNA Purification System. This technique provides a fast and reliable high production method to use bacterial cells and isolate highly purified phagemid DNA. The DNA eluted from the purification resin was suitable for DNA sequencing and other analytical manipulations. lll. Sequence of cDNA clones The cDNA inserts of the randomized isolates from the THP-1 library were partially sequenced. CDNAs were sequenced by the Sanger method F. and Coulson AR (1975; J. Mol Biol. 94:. 441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno NV) in combination with four Peltier Thermal Cyclers ciclizadores (PCT200 from MJ Research, Waterown MA) and Applied Biosystems 377 or 373 DNA Sequencing Systems (Perkin Elmer) and the determined reading frame.

IV. Homology Search of cDNA Clones and Deducted Proteins Each sequence thus obtained was compared with sequences in GenBank using a search algorithm developed by Applied Biosystems I nc. , and incorporated into the I N H ER IT ™ 670 Sequence Analysis System. In this algorithm, a Pattern Specification Language (developed by TRW Inc.) was used to determine regions of homology. The three parameters that determine how the sequence comparisons performed window size, window deviation, and error tolerance. Using a combination of these three parameters, the DNA database was searched for sequences containing regions of homology to the target sequence, and the appropriate sequences were sorted with an initial value. Subsequently, these homologous regions were examined using dot matrix homology graphs to distinguish the regions of homology from those of coincidence of opportunity. Smith-Waterman alignments of the protein sequence were used to display the results of the homology search. This method did not immediately identify an ICEY counterpart. Subsequently, the human ICEY sequence was obtained from GenBank Gl number 717040 and compared to the sequences in the LI FESEQ ™ database (Incyte Pharmaceuticals, I nc.). Incyte clones 14775 and 15781 1 were determined to have homology to the sequence of GenBank, Gl 717040. The presence of icey collection of TH 1 activated P-, which represents activated macrophages is consistent with its expression in tissues with active immunological defenses, particularly inflamed and diseased tissues, including those previously defined with relation of disorders by activated monocyte. Peptide and protein sequence homologies were investigated using the INHERIT 670 Sequence Analysis System in a manner similar to that used in A DN sequence homologies. The Pattern Specification Language and the parameter windows were used to search the protein databases for sequences containing regions of homology, which are classified with an initial value. The dot matrix homology graphs were examined to distinguish the regions of significant homology from coincidence of opportunity. The nucleotide and amino acid sequences for the entire coding region of the human ICE homolog claimed in this invention are shown in Figures 1 A and 1 B.

V. Identification and Sequencing of Full Length of the Genes The Incyte 14775 and 15781 1 clones (SEQ ID NO: 1 and 2, respectively) were resected with M 13 and reverse primers M 13 to obtain an additional 3 'and 5' nucleotide sequence. The additional sequences did not overlap, indicating that the coding region was incomplete. Since only clone 14775 appeared to have the entire coding region, the clone was only initiated with the newly designated oligos (S EC I D NOS: 3 and 4) to obtain an internal sequence to terminate the nucleotide sequence of the gene. The additional sequence, which has been obtained from clone 15781 1 was used to confirm and edit the sequences of clone 14775. The confirmed icey sequence was homologous to, but clearly different from, any known I CE molecule. The complete nucleotide sequence for icey was translated, and the frame translation is shown in Figures 1A and 1B. When all three possible predicted translations of the sequence were searched against protein databases, such as SwissProt and PI R, no exact matches were found to any of the possible icey translations. Figures 2A and 2B show the comparison of the amino acid sequence of ICEY with the Gl 717040 number of GenBank of human ICE. The amino acids in agreement are highlighted in black. The expressed structure / alpha, beta, and flexible regions), as well as hydrophilic character and antigenicity plots for ICEY are shown in Figure 3. Figure 4 shows the alignments of the complete gene sequence (upper line) (SEC ID N0: 5) with the partial cDNA sequences of the clones listed 14775 and 15781 1 (SEQ ID NOS: 1 and 2, respectively). The sequence alignment of SEC I D NO: 1 is shown on the left and that of SEC I D NO: 2 on the right.

SAW. Antisense Analysis Knowledge of the correct, complete cDNA sequence of the new icey gene allows its use in antisense technology in the investigation of gene function. Oligonucleotides, genomic or cDNA fragments comprising the icey antisense strand are used either in vitro or m vivo to inhibit the expression of the protein. Such technology is now well known in the art, and probes are designed at various locations along the icey nucleotide sequence. By treating cells or test animals complete with said antisense sequences, the gene of interest can be effectively closed. Frequently, the function of the gene is ascertained by observing the behavior at the cellular, tissue or organism level (eg, lethality, loss of differentiated function or changes in morphology, for example). In addition to using sequences constructed to interrupt the transcription of the open reading frame, modifications of gene expression are obtained by designing antisense sequences to intron regions, promoter / enhancer elements, or even to trans-act regulation genes. Similarly, inhibition of activity is achieved using the Hogeboom base pair methodology, also known as "triple helix" base pairs.

Vile. Expression of ICEY The expression of icey is achieved by subcloning the cDNAs to appropriate expression vectors and transfecting the vectors to appropriate expression hosts. The cloning vector used for the generation of the tissue collection is used for the expression of the icey sequence in E ^ coli. Upstream of the cloning site, this vector contains a promoter for β-galactosidase, followed by the nucleotide sequence encoding the amino-terminal Met and the subsequent 7 residues of β-galactosidase. Immediately after these eight residues is a bacteriophage engineered promoter useful for artificial initiation and transcription and a number of unique restriction sites, including Eco Rl, for cloning. Induction of the strain of isolated, transfected bacteria with I PTG using normal methods produces a fusion protein corresponding to the first seven residues of β-galactosidase, approximately 15 residues of the "linker", and the protein encoded by the cDNA. Since the cDNA clone inserts are generated by an essentially random procedure, there is an opportunity in three that the included cDNA is in the correct frame for the appropriate translation. If the cDNA is not in the proper reading frame, the appropriate reading frame is obtained by eliminating or inserting the appropriate number of bases through well-known methods including in vitro mutagenesis, exonuclease digestion, and "mung bean" nuclease. , or inclusion of the oligonucleotide linker. The icey cDNA is released to other vectors that are known to be useful for the expression of protein in specific hosts. The oligonucleotide amplimers containing cloning sites as well as a DNA segment sufficient for the hybridization of stretches at both ends of the target cDNA (25 base pairs) are chemically synthesized by normal methods. These primers are used to amplify the desired gene segments by PCR. The resulting new gene segments are digested with appropriate restriction enzymes under normal conditions and isolated by gel electrophoresis. Alternatively, similar gene segments are produced by digesting the cDNA with appropriate restriction enzymes and filling in the missing gene segments with chemically synthesized oligonucleotides. The segments of more than one gene are ligated together and cloned into appropriate vectors to optimize the expression of the recombinant sequence. Suitable expression hosts for said chimeric molecules include but are not militated to mammalian cells such as Chinese Hamster Ovary (CHO) cells and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae. , and bacteria such as E. coli. For each of these cell systems, a useful expression vector also includes an origin of replication to allow propagation in bacteria and a selectable marker, such as the ß-lactamase antibiotic resistance gene, to allow selection in 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 such are not part of the cDNA of interest. Additionally, the vector may contain promoters or enhancers, which increase the expression of the gene. Such promoters are specific hosts and include MMTV, SV40, or metallothionin promoters for CHO cells; trp, lac, or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase or PGH promoters for yeast. Transcription enhancers, such as the sarcomo rous virus (RSV) enhancer, are used in mammalian host cells. Once homogenous cultures of recombinant cells are obtained through normal culture methods, large quantities of recombinantly produced ICEY are recovered from the conditioned medium and analyzed using chromatographic methods known in the art, since the expression of the ICEY can be lethal to certain cell types, care must be taken in the selection of an appropriate host species and growth conditions. Alternatively, the protein is expressed in the inactive form, such as in inclusion bodies. The inclusion bodies are separated from the cells, the protein is solubilized and duplicated to an active form.

VIII. Isolation of Recombinant ICEY The ICEY purification is achieved by creating a chimeric protein having one or more polypeptide domains added to the amino acids of ICEY to facilitate the purification of the protein. Said domains that facilitate purification include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules, which allow the purification of protein A domains, immobilized metals, which allow the purification of immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (Immunex Corp. Seattle WA). The inclusion of a cleavage linker sequence such as Facto XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and the icey sequence is used to purify ICEY from the fusion protein complex.

IX. Diagnostic Test Using Specific ICEY Antibodies Particular ICEY antibodies are useful for the diagnosis of prepatological conditions, and chronic or acute diseases, which are characterized by differences in the amount or distribution of ICEY. ICEY has been found in the activated THP-1 collection and is thus associated with abnormalities or pathologies, which activate monocytes. Diagnostic tests for ICEY include methods that use the antibody and a label to detect ICEY in fluids of the human body, tissues or extracts of said tissues. The polypeptides and antibodies of the present invention are used with or without modification. Frequently, polypeptides and antibodies will be labeled or denoted, either covalently or non-covalently, with a substance, which provides a detectable signal. A wide variety of brands and conjugation techniques are known and have been widely reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescent agents, magnetic particles and the like. The patents that teach the use of said marks include the E patents. U .A. Nos. 3,817,837; 3, 850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149; and 4,366,241. Also, recombinant immunoglobulins can be produced as shown in the patent of E. U.A. No. 4,816,567, incorporated herein by reference. A variety of protocols for measuring soluble ICEY or membrane binding, using polyclonal or monoclonal antibodies specific to the respective protein, is known in the art. Examples include the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site monoclonal-based immunoassay is preferred using monoclonal antibodies reactive to two epitopes without interference to ICEY, but a competitive binding assay may be employed. These assays are described, inter alia, in Maddox, DE et al. (1983, J. Exp. Med. 158: 121 1).

X. Purification of ICEY Native Using Specific Antibodies The native or recombinant ICEY was purified by immunoaffinity chromatography using antibodies specific for ICEY. In general, an immunoaffinity column is constructed by covalently coupling the anti-ICEY antibody to an activated chromatographic resin.

Polyclonal immunoglobulins will be prepared from immune serum 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 by precipitation of ammonium sulfate or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently linked to a chromatographic resin such as activated CnBr-Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions. Said immunoaffinity columns are used in the purification of ICEY by preparing a fraction of cells containing ICEY in a soluble form. This preparation is derived by solubilization of the whole cell or a sub-cellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, the ICEY containing a signal sequence is secreted in a useful amount to the medium in which the cells grow. A preparation containing soluble ICEY is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of ICEY (eg, pH regulators of high ionic strength in the presence of detergent). Then, the column is the uida under conditions that break the binding of the antibody / ICEY (eg, a pH regulator with a pH of 2-3 or a high concentration of a chaotrope to the urea or thiocyanate (ion), and ICEY is collected XI Activity ICEY The purified or expressed ICEY activity is tested by methods known to those skilled in the art. One method advises the stimulation of macrophages with LPS to induce the expression of pre-I L-1β and then treat with ATP and ICEY. The levels of I L-1β in the medium are measured by the immunosorbent enzyme-linked immunoassay (ELISA), as described in Li et al. (1995) Cell 80: 401-41 1.

XII. Drug Classification ICEY, or immunologically active fragments thereof, are used to select compounds in any variety of drug classification techniques. The ICEY polypeptide or fragment employed in said test is either free in solution, fixed to a solid support, exits a cell surface, or is located intracellularly. A method for classifying drugs uses eukaryotic or prokaryotic host cells, which are stably transformed with recombinant nucleic acids expressing ICEY or a fragment thereof. The drugs are classified against said transformed cells in competitive binding assays. Said cells, either in a viable or fixed form, can be used for normal binding assays. One can measure, for example, the formation of complexes between ICEY and the agent being tested. Alternatively, one can examine the decrease in complex formation between ICEY and its target cell, the monocyte or macrophage, caused by the agent being treated. Thus, the present invention provides methods for classifying drugs, natural inhibitors or any other agents that can affect inflammation and disease. These methods comprise contacting said agent with an ICEY polypeptide or fragment thereof and analyzing, 1) the presence of a complex between the agent and the ICEY polypeptide or fragment, or 2) the presence of a complex between the polypeptide of ICEY or fragment and the cell, through methods well known in the art. In such competitive binding assays, the ICEY polypeptide or fragment is typically labeled. After a suitable incubation, the ICEY polypeptide or fragment was separated from that present in the binding form, and the amount of free label or complex is a measure of the ability of the particular agent to bind to ICEY or to interfere with the ICEY complex / cell and agent complex. Another technique for classifying the drug provides a high throughput classification for compounds having adequate affinity to the ICEY polypeptide and is described in detail in European Patent Application 84/03564, published September 1, 1984, incorporated here by reference. In summary, a plurality of different peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the ICEY polypeptide and washed. The bound ICEY polypeptide is then detected by methods well known in the art. The purified ICEY can also be coated directly onto plates for use in the aforementioned drug classification techniques. In addition, antibodies without neutralization can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug classification assays wherein the neutralizing antibodies capable of binding ICEY, specifically compete with a test compound for binding to IC EY polypeptides or a fragment thereof. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with ICEY.

XIV. Rational Drug Design The objective of rational drug design is to produce structural analogues of biologically active polypeptides of interest or of small molecules with which they interact, for example, agonists, antagonists, or inhibitors. Any of these examples can be used to design drugs, which are more active or stable forms of the polypeptide or which improve or interfere with the function of a polypeptide in vivo (see Hodgson (1991) Bio / Technology 9: 19-21, incorporated herein by reference). In one aspect, the three-dimensional structure of a protein of interest, or of a protein inhibitor complex, is determined through x-ray crystallography, through computer modeling or, more typically, through a combination of the two aspects. Both the form and charges of the polypeptide must be ascertained to see the structure and to determine the active sites of the molecule. Useful information regarding the structure of a polypeptide can be gained through modeling based on the structure of homologous proteins. The crystal structure of an ICE protein (Walker, 1994, supra) can be used as a starting point. Relevant structural information is used to design analog ICEY-like molecules or to identify efficient inhibitors. Useful examples of rational drug design include molecules having a different specific character or improved activity or stability as shown by Braxton S and Wells JA (1992 Biochemistry 31: 7796-7801) or which acts as inhibitors, agonists or antagonists of native peptides as shown by Athauda SB et al. (1993 J. Biochem.1 13: 742-746), incorporated herein by reference.

It is also possible to isolate a specific antibody on the target, selected by functional assay, as described above, and to resolve its crystal structure. This aspect, in principle, produces a farmanucleus on which the subsequent drug design can be based. It is possible to derive protein crystallography by generating anti-iodotypic (anti-ids) antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids could be expected to be an analogue of the original receptor. The anti-id can then be used to identify and isolate peptides from chemically or biologically produced peptide libraries. The isolated peptides could then act as the farmanucleus. Using the methods described infra. a sufficient amount of ICEY can be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the ICEY amino acid sequence provided herein will provide guidance for those who employ computer modeling techniques instead of or in addition to X-ray crystallography. All publications and patents mentioned in the above specification are incorporated herein by reference. The above written specification is considered sufficient to allow one skilled in the art to practice the invention. In fact, several modifications of the modes described above, which are readily apparent to those skilled in the field of molecular biology or related fields, are intended to be within the scope of the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: INCYTE PHARMACEUTICALS, INC. (ii) TITLE OF THE INVENTION: HOMOLOGO DE ENZIMA DE CONVERSIÓN DE INTERLEUCINA-1 HUMANA (iii) NUMBER OF SEQUENCES: 6 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: INCYTE PHARMACEUTICALS, INC. (B) STREET: 3330 Hillview Avenue (C) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: USA (F) CODE: 94304 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Résease # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: will be assigned (B) DATE OF SUBMISSION: submitted with the same (vii) DATA FROM THE PREVIOUS APPLICATION: (A) SERIES NO. OF REQUEST: 08 / 443,865 (B) DATE OF SUBMISSION: May 31, 1995 (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: LUTHER, BARBARA J. (B) NUMBER OF REG. : 33954 (C) NO. REF. / PERMANENT: PF-0037 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-852-0195 (2) INFORMATION FOR SEC ID NO: 1: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 303 base pairs (B) TYPE: nucleic acid (C) STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) COLLECTION: THP -1 (B) CLON: 014775 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ACASTCTTTA CTTTTATTOA AATACCAAAT ATTGACTATG CAAQCTATAC TGßTAAA OT 60 CTCTTTOATG TTGACAG? ßß AGOGCTOGOC TGCCTGTGQT TTCATTTTCA ATTOCCAaQA 1 0 AAGAGQTAG? AATCTCTTGT CAAGsTTGCT CGTTCTATGG. TOßGCATCTQ QOCTTTAßCC 180 TßTGGAACTT CAAATSATTT CTQTACCTTC CQAAATATTT CCATTAGOTQ OCAGCAOCAA 240 G-AATATTTCT GOAAOCATOT OATGAOTTCC OTAATGAGAT GOAGCCCCTT TOCßOTCTCT 300 TTC 303 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 167 base pairs (B) TYPE: nucleic acid (C) STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vi) ORIGINAL SOURCE: (E) HAPLO TYPE: THP-1 (vii) IMMEDIATE SOURCE: (A) COLLECTION: 157811 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAAACACTTC TCAATATOOA CCA ??? OATC ACCAGTGTAA AACCTCTTCT GCAAATCGAß 60 OCTQOCCACC TOCAOCAOAT CTCAAATATA CTCAAACTTT OTCCTCßTOA AOA? TTCCTO 120 ASACTGTGTA A? AAAAATC? TGATOAGATC TATCCAAT ?? AAAAOAO 167 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: CCCCCAOGTT CTCAGATGAC TQT 23 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACAGTCATCT GAOAACCTGO 000 23 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1274 base pairs (B) TYPE: nucleic acid (C) STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) COLLECTION: THP-1 (B) CLONE: 014775 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TOCATTCOßA COAGOTTAOC TATOOCTOAA GACAACCACA AAAAAAAAAC AGTTAAß TQ 60? TTOGAAT? CC TGaßCAAAOA TOTTCTTCAT GOTOTTTTTA ATTATTTOßC AAAACACQAT 120 • GTTCTOACAT TaAAGQAAOA GGAAAAaAAA AAATATTATQ ATACCAAAAT TOAAGACAAO 180 OCCCTOATCT TOOTAflACTC TTTsQAAAOA ATCOCßTGßT CATCAAATQT TTACCCAAAC 240 ACTTCTCAAT ATsOACCAAA ASATCACCAO TQTAAAACCT CTTCTOCAA? TCAQOTOß? 300 CCACCTOAOT CAOCAOAATC TAC ??? TAT? CTCAAACTTT OTCCTCOTOA AßAATTCCTO 360 AGACTGTGTA AAAAAAATCA TQATQAQATC TATCCAATAA AAAAGAOAQA GGACCGCAGA 420 CGCCTOGCTC TCATCATATG CAATACAAAG TTTGATCACC TßCCTOCAAß OAATGßOOCT 480 CACTATß? C? TCOTOOQOAT OA ??? aaCTO CTTCAAOOCC TGGGCTAC? C TGTOGTTGCC 540 GAAAAGAATC TCACAGCCAO Oa? T? TQß? S TCAGTOCTOA GaGCATTTQC TOCCAß? CC? 600 GAQCAC ?? QT CCTCTOACAO C? COTTCTTO OTACTCATOT CTCATOGCAT CCTAGAGOGA 660 ATCTOCOaAC CTOCOCATAA AA? O ?????? CCGßATQTOC TGCTTTATQA C? CC? TCTTC 720 CAGATATTCA ACAACCOCAA CTGCCTCAGT CTA ?? GSS? CA AACCCAAOOT CATCATTOTC 780 CAGOCCTOCA GAGGTOAAAA? CATGQOAAC TCTGGTCAGA OACTCTCCAC ACCTTQCATC 840 ATCTCTTCAC AGTCATCTGA GAACCTOOAG QCAOATTCTO TTTTCAAGAC CCCQAQOAQA 900 AGQACTTCAT TGCTOTTCTQ TTCTTCAACA CCACATAACQ TßTCCTGG? O? OACCGCAC? 960 AOGOQCTCCA TCTTCATTGO GQAACTCATG CACATGCTTC CAGAAATATT CTTCTCTCCA 1020 CCTAATOOAA ATATTTCQO? GOTACAG? AA TCATTTGAAG TTCCAC? ßOC T ?? AGCCCAG 1080 ATGCCCACCA TAGAACGAGC AACCTTGACA AGAGATTTCT ACCTCTTTCC TGGCAATTGA 1140 A ?? TGAAACC ACAGGCAQCC CAGCCCTCCT CTGTCAACAT CAAAGAGCAC ATTTACCAGT 1200 ATASCTTOCA TAGTCAATAT TTGJTATTTC AATAAAAGTA AAOACTQTAT CTTTTTAAAA 1260 AAAA ?? A ??? AAAA 1274 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 372 amino acids (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) DESCRIPTION SEQUENCE: SEQ ID NO: 6: Met the Olu SSP sn Hiss Lyss Lyss Lyss Thr Val Lys Mat Leu Glu Tyr 1 5 October 15 Leu Gly Lys sp Val Leu HLSS ßly Val Phe SSN Tyr Leu la Lyss????? Hiss 20 25 30 ASSP Val Leu Thr Leu Lyss Glu SSLU Glu Lyss Lyss Lyss Tyr Tyr ASSP Thr 35 40 45 Lyss Xle Glu? SSPs Lyss? Leu Xle Leu Val? SSPs Ser Leu SSLU? rg Xle 50 55 60? Trp Ser Ser? ßn Val Tyr Pro? ßn Thr Ser Oln Tyr Gly Pro Lyß 65 70 75 80 Aßp Hiß ßln Cyß Lys Thr Ser Ser? La? ßn? Rg Gly Gly Pro Pro ßlu 85 90 95 Be? Glu Ser Thr? An Xle Leu Lyss Leu CYSS Pro Arg SSLU SSLU Phe 100 105 110 Leu Arg Leu CYSS Lyss Lyss? SSN Hiss? SSPs Glu Xlß Tyr Pro Xle Lyss Lyss 115 120 125 Arg Glu ASSP Arg? Rg? Rg Leu? La Leu lie Xle Cyß? ßn Thr Lyß Phe 130 135 140? ßp Hi? Leu Pro? La? Rg? ßn Gly? The Hi? Tyr? ßp Xle Val Gly Met 145 150 155 160 Lyß? Rg Leu Leu Gln ßly Leu ßly and Tyr Thr Val Val? ßlu Lyß Aßn 165 170 175 Leu Thr? La? Rg Aßp Met ßlu Ser Val Leu Arg? La Phe? La? La? Rg 180 185 190 Pro ßlu Hiß Lys Ser Ser Aßp Ser Thr Phe Leu Val Leu Met Ser Hiß 195 200 205 Gly lie Leu Glu Gly lie Cyß Gly Pro Ala Hiß Lyß Lys Lyß Lyß Pro 210 215 220? ßp Val Leu Leu Tyr Aßp Thr lie Phe Gln Xle Phe Aßn Aßn? Rg? ßn 225 230 235 240 Cyß Leu Sßr Leu Lyß? Sp Lyß Pro Lys Val lie lie Val Gln? The Cyß 245 250 255 Arg QLY Glu Lys Hiss Gly Assn Ser Gly Gln? Rg Leu Ser Thr Pro Cys 260 265 270 Xle Xle Ser Ser Gln Ser Ser Glu? SSN Leu Glu? The? SSPs Ser Val Phe 275 280 285 Lyss Thr Pro? Rg? Rg? rg Thr Ser Leu Leu Phe Cyß Ser Ser Thr Pro 290 295 300 Hiß? ßn Val Ser Trp? rg? ßp? rg Thr? rg Gly Ser lie Phe Xle Gly 305 310 315 320 Glu Leu Met His Met Leu Pro Glu Xle Phe Phe Pro Pro? ßn Gly 325 330 335? ßn Xle Ser Glu Val ßln Lyß Ser Phe Glu Val Pro sln? The Lya? The 340 345 350 Oln Met Pro Thr lie Olu? Rg ? the Thr Leu Thr? rg? ßp Phe Tyr Leu 355 360 365 Phe Pro Oly? ßn

Claims (3)

1. CLAIMS 1 . - A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence presented in SEQ ID NO: 6, or its complement. 2 - The polynucleotide according to claim 1, wherein the nucleic acid sequence consists of SEQ ID NO: 5. 3. An expression vector comprising the polynucleotide of claim 2. 4 - A host cell comprising the The expression vector of claim 3. 5. - A nucleic acid probe comprising a non-conserved fragment of the polynucleotide of claim 2. 6. - An antisense molecule comprising a polynucleotide sequence complementary to at least a portion of the polynucleotide of claim 2. A method for producing a polypeptide comprising the sequence as depicted in SEQ ID NO: 6, said method comprising: a) culturing the host cells of claim 4 under conditions suitable for the expression of the polypeptide, and b) recovering said polypeptide from the cell culture. 8 - A homologue of purified interleukin conversion enzyme (ICEY) having the amino acid sequence presented in SEQ ID NO: 6. 9 -. 9 - An antibody specific for the purified polypeptide of claim 8. 10.- A diagnostic composition for the detection of nucleic acid sequences encoding the interleukin conversion enzyme homologue (ICEY) comprising the nucleic acid probe of claim 5. 11.- A diagnostic test for the detection of nucleotide sequences encoding the interleukin conversion enzyme homologue (ICEY) in a biological sample, a) by combining the biological sample with a polynucleotide, which comprises the nucleotide sequence of SEQ ID NO: 5 or a fragment thereof, wherein said fragment is derived from a non-conserved region of said nucleotide, under conditions suitable for the formation of a nucleic acid hybridization complex between the nucleic acid sequence SEQ ID NO: 5 and a complementary nucleic acid sequence in said sample; b) detecting said hybridization complex; and c) comparing the amount of said hybridization complex with a normal one, wherein the presence of an abnormal level of said hybridization complex correlates positively with a condition associated with inflammation. 12 - The diagnostic test according to claim 12, wherein the polynucleotide is labeled with a reporter molecule and the hybridization complex is detected by measuring said reporter molecule. 13. - A diagnostic test for the detection of nucleotide sequences encoding the interleukin conversion enzyme homologue (ICEY) in a biological sample, comprising the steps of: a) combining the biological sample with polymerase chain reaction primers under conditions suitable for nucleic acid amplification, wherein said primers comprise fragments of non-conserved regions of the nucleotide sequence of SEQ ID NO: 5; b) detect the amplified nucleotide sequences; and c) comparing the amount of amplified nucleotide sequences in said biological sample with a normal one thus determining whether the amount of said nucleotide sequence varies from said normal, wherein the presence of an abnormal level of said nucleotide sequence is positively correlated with a condition associated with inflammation. 14. A method for classifying a plurality of compounds for specific binding affinity with the polypeptide of claim 8 or any portion thereof, comprising. a) providing a plurality of compounds; b) combining the interleukin conversion enzyme homolog (ICEY) with each of a plurality of compounds for a sufficient time to allow binding under suitable conditions; and c) detecting the binding of ICEY to each of the plurality of compounds, thereby identifying the compounds that specifically bind ICEY.