MXPA97008030A - New captecin homologo - Google Patents

Abstract

The present invention provides nucleotide and amino acid sequences that identify and encode a novel cathepsin C (RCP) homologue expressed in THP-1 cells. The present invention also provides antisense molecules for the nucleotide sequences encoding PCR, expression vectors for the production of purified PCR, antibodies capable of specifically binding to PCR, hybridization probes or oligonucleotides for the detection of nucleotide sequences encoding PCR, genetically overlapping host cells for the expression of PCR, diagnostic tests for activation of monocyte / macrophages based on nucleic acid molecules encoding PCR, and use of the protein to produce antibodies capable of specifically binding to the protein and use of the protein for select inhibitor

Description

NEW HOMOLOGO DE CAPTECINA C 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 new homolog of cathepsin C derived from activated THP-l cells.

BACKGROUND OF THE ART Cathepsin C Cathepsin C, or dipeptidyl-aminopeptidase I, or dipeptidyl-transferase is a lysosomal cysteine proteinase that is capable of sequentially removing dipeptides from the amino terminus of peptide and protein substrates and is involved in the degradation of the cellular protein. Cathepsin C is involved in the functions of the alimentary tract, cell growth and activation of neuraminidase (Kuribayashi, 1993, J.Biochem., 113: 441-449). The activity of cathepsin C is present in levels - higher in the cytotoxic lymphocytes and in the myeloid cells, which indicates their intervention in the induction, development or differentiation of the cytolytic effector cells (Thiele et al., 1990, Proc. Nati, Acad. Sci. USA 87: 83-87). Kominami et al. (1992, Biol. Chem. 373: 367-37) reported the presence of rat cathepsin C mRNA in almost all tissues of the rat. Large amounts of transcript prevailed in liver, spleen, small and large intestine, lung and kidney; moderate amounts in the esophagus, stomach, and heart, and small amounts in the brain, pancreas, adrenal gland, and testicles. The prevalence of transcript seems to correlate with the expected presence and monocyte / macrophage activity in normal tissue function. Cultured macrophages have been used to study the processing of cathepsin C from its synthesis as a propeptide for the mature oligomeric enzyme. Both precursor and mature cathepsin C are phosphorylated and glycosylated, and oligomerization appears to occur prior to entry into the lysosome (Muño D et al. (1993) Arch. Biochem. Biophy. 306: 103-10). In studies using synthetic substrates, cathepsin C showed that it functions as an endopeptidase in the degradation of the intracellular protein and as an exopeptidase (dipeptidylamino peptidase) in cell growth and activation of neuraminidase (Kuribayashi M et al., Supra) . Cathepsin C plays a role in the degradation of collagen, laminin, the ina and other structural proteins that comprise the extracellular matrix of bones. As soon as the bone weakens, it is even more susceptible to bone resorption, tumor invasion and metastasis.

Cathepsin C forms oligomeric structures of approximately 200 kDa, and consists of eight subunits. The dipeptidyl aminopeptidase activity of cathepsin C requires halide ion as well as sulfhydryl groups for activity (Kuribayashi, supra). The nucleotide sequences for the human human spleen dipeptidyl peptidase-I are described in International Publication Number WO 93/24634-A, published on December 9, 1993. WO 93/24634-A describes a dipeptidyl peptidase sequence -I human lacking a signal sequence and a proprotein sequence and describes methods and compositions for inhibiting inflammatory states. Kominami, supra, argues that cysteine proteases can exist as two chain forms produced by dissociation of the mature enzyme and reports that the N-terminal analysis of rat purified cathepsin C showed two different sequences of N-terms which probably represent the N-terminal sequence of each of the chains. THP-1 cells THP-1 is a line of human leukemic cells with different monocytic characteristics derived from the blood of a 1-year-old male child with acute monocytic leukemia (Tsuchiya S et al. (1980) Int. J. Cancer 26 : 171-176). The monocytic nature of THP-1 was identified through stearase activity of a-naphthyl butyrate which could be inhibited by NaF (sodium fluoride); the production of lysozyme; phagocytosis (the encapsulation of extracellular materials) of latex particles and sensitized sheep red blood cells, - and the ability of THP-1 cells treated with mitomycin C to activate T lymphocytes after treatment with concanavalin A. Morphologically, the cytoplasm it contained small azurophilic granules, the nucleus was indented and irregularly shaped with deep folds, and the cell membrane had Fe and C3b receptors that probably function in phagocytosis. Typical monocytes develop from monoblasts through the promonocytes in the bone marrow and in mature form they have a half-life of approximately three days. Approximately 75 percent of the circulating monocyte combination was found along the blood vessel walls, although these cells randomly migrate into tissues and become antigen-presenting cells, or phagocytic cells. Monocytes presenting antigen include the interdigitation of the reticular and follicular dendritic cells of the lymph nodes and the skin. Phagocytic monocytes are prominent as the Kupffer cells of the liver and in the pulmonary alveoli and bone marrow. Many human myeloid and myelomonocytic cell lines retain some ability to differentiate into 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 radiation , exposure to asbestos, and steroids. THP-1 cells treated with the 12-0-tetradecanoyl-phorbol-13 (TPA) acetate tumor promoter are induced to arrest proliferation and differentiate into macrophage-like cells which mimic macrophages derived from native monocytes both morphologically as physiologically. These monocyte / macrophage-like cells exhibit changes in the expression of genes such as the co -duction of C-fos, c-jun and the down-regulation of c-myb (Auwerx J (1991) Experientia 47: 22-31), increase in C3b complement receptor density, and decrease in both the FcR and the adhesion molecule, CD4. In addition, THP-1 cells produce lipase lipoprotein and apolipoprotein E, associated with atherosclerotic lesions, secrete several proinflammatory cytokines, including IL-13 and TNF (Cochran FR and Finch-Arietta MB (1989) Agents and Actions 27: 271- 273), and can make powerful oxidants and proteases that destroy tissues, such as cathepsins. Rheumatoid arthritis is just one example of a monocyte / macrophage disorder; in others, macrophages participate in other ways. For example, in arterysclerosis, macrophages accumulate cholesterol from blood lipoproteins and become foam cells of human atherosclerotic lesions. Renegade activated monocytes have also been implicated in defective defense against infection, damage to the intestine, osteoporosis, toxic shock syndrome, and systemic lupus erythematosus. DESCRIPTION OF THE INVENTION The present invention relates to novel nucleotide and amino acid sequences for a novel homolog of cathepsin C initially found in a cDNA library made of RNA isolated from THP-1 cells cultured for 48 hours with 100 ng / milliliter of ester forbólico (PMA), followed by a culture of 4 hours in medium containing 1 ug / milliliter of LPS. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old male child with acute monocytic leukemia (ref: Int. J. Cancer 26 (1980): 17l). The new gene, which is designated herein as rep (Incyte Clone 14284), encodes the polypeptide designated RCP, a novel cathepsin C. The present invention relates to the use of nucleotide and amino acid sequences of PCR in the study, diagnosis and treatment of disease states related to inflammatory disease.; autoimmune disease; and malignancy of myeloid cell origin in disease states such as arteriosclerosis, leukemia, systemic lupus erythematosus, osteoporosis, rheumatoid arthritis, and toxic shock syndrome as well as rejection of grafts and graft-versus-host disease. The present invention is based in part on the nucleotide and amino acid homology that shares PCR with rat cathepsin C isolated from rat kidney cDNA (Kominami E et al. (1912) Biol Chem 373: 367-73); the presence of the conserved residues of the catalytic triad, Cys in 258, His in 405, and Asn in 427, and the NNS glycosylation site in the hydrophobic region that are common to captesin C; and in part in the presence of nucleotide sequences for PCR in the THP-1 cDNA library made from a source of monocytic leukemia. The present invention is, therefore, based on the discovery of a novel cathepsin C homologue that is associated with monocytes. PCR and the nucleotide sequences encoding it and oligonucleotides, peptide nucleic acid (PNA), fragments, portions or antisense molecules thereof, provide the basis for diagnostic methods for the early and accurate detection and / or quantification of CPR associated with inflammatory disease; autoimmune disease; and malignancy of myeloid cell origin in disease states such as arteriosclerosis, leukemia, systemic lupus erythematosus, osteoporosis, rheumatoid arthritis, and toxic shock syndrome as well as graft rejection and graft-versus-host disease. For example, the RCP nucleotide sequences described herein, which encode the PCR, or fragments thereof, can be used in hybridization assays of biopsied cells or tissues or body fluids to diagnose abnormalities in individuals who have or are in risk of inflammation. An abnormal level of nucleotide sequences encoding the PCR in a biological sample may reflect a chromosomal aberration, such as a nucleic acid deletion or mutation. In accordance with the above, the nucleotide sequences encoding PCR provide the basis for probes that can be used diagnostically to detect chromosomal aberrations such as deletions, mutations or chromosomal transpositions in the gene encoding the PCR. The expression of the RCP gene may be altered in those disease states or there may be a chromosomal aberration present in the region of the gene encoding the PCR. The present invention also provides RCP antisense molecules or RCP antagonists that can be used to block the activity of PCR, ie, protease activity, under conditions where it is desirable to block protein degradation, such as inflammation. The present invention also relates to the expression vectors and the engineered host cells comprising RCP nucleotide sequences for the in vitro or in vivo production of the nucleotide and amino acid sequences. Additionally, the present invention relates to the use of a PCR polypeptide, or a fragment or variant thereof, to produce anti-PCR antibodies and to look for antagonists or inhibitors of the PCR polypeptide that can be used in a diagnostic manner to detect and quantify CPR protein levels in disease states. The present invention also relates to pharmaceutical compositions comprising effective amounts of inhibitors or antagonists of the RCP protein or antisense nucleic acid for use in conditions where it is desirable to reduce the activity of human cathepsin C described herein, for example, in the treatment of inflammation and other disease states. The invention further provides assays and diagnostic equipment for the detection of PCR in cells and tissues comprising a purified PCR which can be used as a positive control, and anti-RCP antibodies.4 Those antibodies can be used in solution-based technologies , membrane-based, or tissue-based 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 Figures la-Ib represent the nucleotide sequence for rcp (SEQ ID NO: l) and the predicted amino acid sequence (SEQ ID NO: 2) of the RCP polypeptide. Figures 2a-2b show the amino acid alignment of RCP with rat captain C. The alignments shown in Figures 2 and 4 were produced using the software multi-sequence alignment program of DNASTAR (DNASTAR Inc., Madison, Wl). Figure 3 represents an analysis of the hydrophobicity of PCR based on the predicted sequence and composition of amino acids. Figure 4 depicts the alignment of PCR with the human dipeptidyl peptidase-I (DPI) described in WO 93/24634.

MODES FOR CARRYING OUT THE INVENTION The present invention relates to a novel homolog of captesin C, designated herein as RCP, the nucleotide sequence of which was initially found between the sequences of a cDNA library made of RNA isolated from THP-1 cells cultured for 48 hours with 100 ng / milliliter of forbolic ester (PMA), followed by a 4-hour culture in medium containing 1 ug / milliliter of LPS. The THP-1 (ATCC TIB 202) is a human pro-oocyte line derived from the peripheral blood of a 1-year-old male child with acute monocytic leukemia (ref: Int. J. Cancer 26 (1980): 171). 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 inflammatory disease, autoimmune disease; and malignancy of myeloid cell origin in disease states such as arteriosclerosis, leukemia, systemic lupus erythematosus, osteoporosis, rheumatoid arthritis, and toxic shock syndrome as well as rejection of grafts and graft-versus-host disease. The present invention also relates to the use of PCR and genetically engineered host cells that express CPR to evaluate and select substances and compounds that modulate CPR activity. The present invention is based in part on the presence of nucleotide sequences encoding PCR in a random sample of 2214 sequences usable in a cDNA library made of THP-1-cells treated with 100 ng / milliliter of forbolic ester, followed by a 4 hours culture in a medium containing 1 ug / milliliter of LPS. The present invention is further based in part on the amino acid homology that RCP shares with rat cathepsin C, as illustrated in Figure 2. Based on the sequence of rat cathepsin C described in Kominami, supra, in addition to the combination of hydrophobicity depicted in Figure 3, the signal sequence for the homologous cathepsin depicted in Figures 1A-1B appears to end in the region between amino acid residues 21 (Ala) and 22 (Val) and the region between the residues of amino acids 28 (Ala) and 29 (Asn). Therefore, the proprotein region of the human Cathepsin C homolog represented in Figures 1A-1B appears to start between amino acid residues 22 and 28, inclusive, and terminate at amino acid residue 230 (His). Based on the sequence of captesin C of reta, the sequence of the mature protein for human captesin C, shown in Figures 1A and IB, starts at amino acid residue 231 (Leu) and ends at amino acid residue 463 (Leu) . Kominami, supra, argues that cysteine proteases can exist as two chain forms produced by dissociation of the mature enzyme and reports that the N-terminal analysis of rat purified cathepsin C showed two different sequences of N-terms which probably represent the N-terminal sequence of each of the chains. In addition, cathepsin C from hepatoma cells 7777 is reported to be synthesized and processed into mature forms of 18 kDa and 6 kDa. By homology with rat Captesin C, the homologue of mature human Captesin C depicted in Figures 1A-1B, appears to contain two different sequences of N-terms that can represent the N-terminal sequence of each of the two chains. The present invention, therefore, is based on the identification of a new homologue of Captesin C, RCP, which is associated with inflammation and disease. In a specific embodiment herein, the proprotein region of the Cathepsin C homologue starting between amino acid residues 22 and 29, inclusive, and terminating at amino acid residue 230, is administered as an antagonist of the cathepsin homologue. C in conditions where it is desirable to block its activity such as in inflammatory disease; autoimmune disease; and malignancy of myeloid cell origin in disease states such as arteriosclerosis, leukemia, systemic lupus erythematosus, osteoporosis, rheumatoid arthritis, and toxic shock syndrome as well as rejection of grafts and graft-versus-host disease. "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 or chain simple, either by representing the chain with sense or antisense. As used herein, "amino acid sequence" refers to sequences of peptides or proteins or portions thereof.

As used herein, "rcp" with lower case refers to nucleic acid sequence, while "RCP" with upper case refers to protein sequence. As used herein, peptide nucleic acid (PNA) refers to a class of information molecules that have a neutral "peptide-like" structure with nucleobases that allow molecules to hybridize with DNA or RNA. complementary with greater affinity and specificity than the corresponding oligonucleotides (PerSeptive Biosystems 1-800-899-5958). As used herein, RCP encompasses CPR from any human source, in natural occurrence or in variant form, or from natural, synthetic, semi-synthetic or recombinant sources. As used herein, the term "activated monocytes" refers to mature monocytes or activated macrophages found in immunologically active tissues. As used herein, the term "monocyte / macrophage disorders" includes, but is not limited to arteriosclerosis, leukemia, systemic lupus erythematosus, osteoporosis, rheumatoid arthritis, and toxic shock syndrome. As used herein, "naturally occurring" refers to PCR with an amino acid sequence that is found in nature, and "biologically active" refers to PCR that has the structural, regulatory or biochemical functions of the PCR that occurs naturally, including immunological activity. Naturally occurring PCR also encompasses the PCRs 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 natural, recombinant or synthetic RCP or any oligopeptide thereof, to induce a specific immune response in the appropriate animals or cells and to bind with specific antibodies. The term "derivative" as used herein refers to the chemical modification of a PCR. Illustrative of these modifications is the replacement of hydrogen by an alkyl, acyl, or amino group. A RCP polypeptide derivative retains essential biological characteristics of a naturally occurring PCR. A derivative of RCP also refers to RCP polypeptides derived from PCR that occur naturally through chemical modifications such as ubiquitination, labeling (eg with radionuclides, various enzymes, etc.) pegylation (derivatization with polyethylene glycol), or by the insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins. As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences, 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 PCR" refers to any PCR polypeptide that differs from the naturally occurring PCR by insertions, deletions, and amino acid substitutions created using recombinant DNA techniques. The guide to determining which amino acid residues can be replaced, added or deleted without abolishing activities of interest, such as cell adhesion and chemotaxis, can be found by comparing the sequence of the particular PCR with that of the homologous cytokines and minimizing the number of changes in the sequence of amino acids made in regions of high homology. Preferably, amino acid "substitutions" are the result of replacing an amino acid with another amino acid having structural and / or chemical properties, such as the replacement of leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine, that is, conservative amino acid replacements. "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The allowed variation can be determined experimentally by systematically inserting, deletions or substitutions of amino acids in a PCR molecule using recombinant DNA techniques and testing the activity of the resulting recombinant variants. When desired, a "signal or leader sequence" can direct the polypeptide through the membrane of a cell. This sequence may be naturally present in the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques. As used herein, a reference to "fragment," "portion," or "segment" of PCR is an extension of amino acid residues that is of sufficient length to display biological and / or immunogenic activity and in the preferred embodiments will contain less 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 "fragment," "portion," or "segment" of polynucleotide refers to any extension of amino acid residues that is of sufficient length to display biological and / or immunogenic activity and in Preferred embodiments will contain at least 5 amino acids, at least about 7 amino acids, at least about 8 amino acids, and, in additional embodiments, about 17 or more amino acids.

As used herein, an "oligonucleotide" or "fragment" "portion" or "segment" of polynucleotide, refers to any extension of nucleic acids encoding PCR that is of sufficient length to be used as a primer in the reaction Polymerase chain (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 PCR polypeptides purified from natural or recombinant sources, vectors and host cells transformed with nucleic acid molecules encoding PCR. Various methods for the isolation of PCR polypeptides can be obtained by methods well known in the art. For example, those 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: Principles and Practice , Springer-Verlag, NYC, both incorporated herein by reference. As used herein, the term "recombinant" refers to a polynucleotide that encodes PCR and is prepared using recombinant DNA techniques. The polynucleotide encoding PCR 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 PCR that is capable of being hybridized to a nucleotide sequence. desired goal. A probe can be used to detect, amplify or quantify cDNAs or endogenous nucleic acid encoding PCR, using conventional techniques in molecular biology. A probe may be of variable length, preferably from 10 nucleotides to several hundred nucleotides. As will be understood by those skilled in the art, the conditions of hybridization and the design of the probe will vary depending on the intended use. For example, a probe intended for use in PCR will be approximately 15 to 60 nucleotides in length and may be part of a combination of degenerate probes, ie, oligonucleotides that tolerate mating errors but adapt to bind an unknown sequence, - wherein a probe for use in Southern or northern hybridizations can be a single, specific nucleotide sequence, which is several hundred nucleotides in length. Nucleic acid probes may comprise portions of the sequence having fewer nucleotides of about 6 kb and usually less than 1 kb. The oligonucleotide and nucleic acid probes of the present invention can be used to determine whether the nucleic acid encoding PCR is present in a cell or tissue or to isolate identical or similar nucleic acid sequences from chromosomal DNA as described by Walsh PS and collaborators (1992 PCR Methods Appl 1: 241-250). Accordingly, a preferred probe for specific PCR detection will comprise a polynucleotide or oligonucleotide fragment from a non-conserved nucleotide region of SEQ ID NO: 1. As used herein, the term "non-conserved nucleotide region" refers to a region of nucleotides that is unique to SEQ ID NO: 1 and does not comprise a region that is retained in cathepsin C. Probes can be chain single or double-stranded and may have specificity in hybridizations based on solution, cell, tissue or membrane, including technologies in itself and similar to ELISA. The nucleic acid probes of the present invention can be derived from naturally occurring nucleic acids or recombinant nucleic acids of single chain or double chain, or be chemically synthesized. These may be labeled by nick translation, Klenow fill reaction, PRC 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 nucleotide sequences encoding the polypeptides of the present invention can be synthesized or identified through hybridization techniques known to those skilled in the art making use of "redundancy" in the genetic code. Various codon substitutions, such as silent changes that produce several restriction sites, can be introduced to optimize cloning within a plasmid or viral vector or expression in a prokaryotic or eukaryotic system. Mutations may also be introduced to modify the properties of the polypeptide, for example, to change the degradation of the polypeptide and the rate of turnover. Description RCP coding sequences The nucleotide sequence of human PCR (SEQ ID NO: l) is shown in Figure l. Part of the coding region for PCR was initially identified within the cDNA library made from THP-1 cells treated with PMA and LPS (library INCYTE THP1PLB01), where once in approximately 2214 usable sequences were found. As used herein, the term "usable sequences" refers to the total number of clones in a cDNA library after vector removal, nucleotide repeats, contamination, and mitochondrial DNA. A BLAST search (Basical Local Alignment Search Tool; Altschul SF (1993) J. Mol. Evol. 36: 290-300; Altschul SF et al. (1990) J. Mol. Biol. 215: 403-410) comparing cDNAs of the THP1PLB01 library against the GenBank primate database identified the Clona Incyte 14284 identified as a cathepsin C. The nucleotide sequences encoding PCR have been found in cDNA libraries made of bladder tissue (library INCYTE BLADNOT01), - mononuclear cells (2 hours of culture) adhering to plastic, isolated from regulator coating units obtained from the Stanford Blood Bank (INCYTE MPHGNOT03 library), - rheumatoid hip synovial tissue (INCYTE SYNORAB01 library); normal placenta (INCYTE PLACNOB01 library); plastic adherent mononuclear cells harvested on the second day of a mixed lymphocyte culture (MLR) (INCYTE library MMLR2DT01); THP-1 cells cultured for 48 hours with 100 ng / milliliters of forbolic ester (PMA) (library INCYTE THP1PEB01), • synovial tissue of rheumatoid elbow (library INCYTE SYNORAT01); hNT2 cell line derived from a human tetracarcinoma, which exhibited characteristic properties of a committed neuronal precursor at an early stage of development, treated with 10 μm of retinoic acid for 24 hours before RNA isolation (INCYTE library HNT2RAT01); 1 T / B lymphoblast cells from a leukemic source (INCYTE TBLYNOT01 library), - neonatal keratinocytes derived from leg skin (INCYTE KERANOT01 library), non-adherent and adherent peripheral blood mononuclear cells, 96-hour mixed lymphocyte reaction (MLR) (library) INCYTE TMLR3DT01), - hNT2 cell line derived from a human tetracarcinoma (INCYTE library HNT2NOT01), - lung tissue (INCYTE LUNGNOT01 library), - apheresis peripheral blood cells from a 48-year-old male patient diagnosed with hypereosinophilic syndrome (INCYTE library) EOSIHET01); mononuclear cells derived from a population of combined donors after Ficoll Hypaque centrifugation and 72 hours in vivo culture INCYTE MMLR3DT01), and sigmoid colon tissue from an individual having Crohn's disease (INCYTE COLNNOT05 library). Because PCR is expressed in THP cells, ie monocytic monocytic leukemia, the blood of an individual with Hypereosinophilic Syndrome, colon tissue from an individual having Crohn's disease, cells derived from a human tetratocarcinoma, T / B cells of leukemia and rheumatoid synovium, nucleic acids (rcp), polypeptides (PCR) and antibodies against PCR are useful for diagnostic assays for the detection of nucleotide or amino acid sequences of CPR associated with inflammation and disease and can accelerate diagnosis and treatment appropriate. 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), T7 polymerase. thermostable (Amersham, Chicago IL), or combinations of recombinant polymerases and reading test exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg MD). Methods for extending the DNA of an annealed oligonucleotide primer corresponding to the DNA tempering of interest have been developed for both single and double stranded annealing. The products of the chain termination reaction were separated using electrophoresis and detected via their labeled precursors, incorporated. Recent improvements in the preparation of the mechanized reaction, sequencing and 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 Catalyst and DNA Sequencers 377 and 373 (Perkin Elmer, Norwalk CN).

The quality of any particular cDNA library in which the polynucleotides encoding PCR is found can be determined by conducting a pilot scale analysis of the cDNAs and verifying percentages of clones containing vector, lambda or E. coli DNA, mitochondrial DNA or repetitive, and clones with exact matches or homologous with respect to public databases. The nucleotide sequences encoding PCR (or its complement) have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include the use as hybridization probes, the use in the construction of oligomers for PCR, the use of chromosome and gene mapping, use in the recombinant production of PCR, and use in the generation of antisense DNA or RNA, their chemical analogs. and similar. The uses of nucleotides encoding PCR described herein are exemplary of known techniques and are not intended to limit their use in any known technique to one 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, provided that the new techniques are based on properties of nucleotide sequences that are currently known, for example, the code Genetic triplet and the specific interactions of base pairs.

Those skilled in the art will appreciate that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding RCP may be produced, some which bear minimal homology to the nucleotide sequence of any known and naturally occurring gene. The invention has specifically contemplated each and every possible variation of nucleotide sequence that can encode PCR by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring CPR, and all these variations will be considered as being specifically described. Although the nucleotide sequences that encode PCR and / or its variants are preferably capable of hybridizing to the nucleotide sequence of the PCR that occurs naturally under stringent conditions, it may be advantageous to produce nucleotide sequences encoding PCR or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host according to the frequency with which the host uses particular codons. Other reasons for substantially altering the nucleotide sequence encoding PCR 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 sequence that occurs naturally. The nucleotide sequences encoding PCR can be linked to a variety of other nucleotide sequences by well established recombinant DNA techniques (cf Sambrook J. et al. Supra). Useful nucleotide sequences for binding to the PCR include a classification of cloning vectors, for example, plasmids, cosmids, lambda phage derivatives, fagomides, 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, suitable restriction endonuclease sensitive sites, and selectable markers for the host cell. The knowledge of the complete sequence, correct, cDNA of the new gene of captesin C will allow its use in antisense technology in the investigation of the function of the gene. Oligonucleotides, genomic or cDNA fragments comprising the antisense strand of rcp can be used either in vitro or in vivo to inhibit the expression of the protein. This technology is well known in the art and probes can be designed at various locations along the nucleotide sequence. By treating whole test cells or animals with these antisense sequences, the gene of interest can effectively be stopped. Frequently the function of the gene can be checked by observing the behavior at the cellular, tissue or organism level (for example lethally, loss of differentiated function, changes in morphology, etc.). In addition to using sequences constructed to interrupt transcription of the open reading frame, modifications of gene expression can be obtained by designating antisense sequences in intron regions, promoter / enhancer elements, or even transactivating regulatory genes. Similarly, inhibition can be achieved using Hogeboom base pair methodology, also known as "triple helix" base pairing. One aspect of the present invention is to provide PCR-specific nucleic acid hybridization probes capable of hybridization to naturally occurring nucleotide sequences encoding PCR. These probes can also be used for the detection of similar sequences that encode cathepsin C and should preferably contain at least 50 percent of the nucleotides of SEQ ID NO: 1. The hybridization probes of the present invention can be derived from the nucleotide sequences of SEQ ID NO: 1 or from genomic sequences including promoters, enhancer elements and / or possible introns of the respective naturally occurring PCRs. Hybridization probes can be labeled by a variety of reporter groups, including radionuclides such as J P or JS. or enzymatic labels such as alkaline phosphatase coupled with the probe via avidin / biotin coupling systems, and the like. The polymerase chain reaction as described in the Patents of the United States of North America Numbers: 4,683,195; 4,800,195; and 4, 965, 188 provides additional uses for oligonucleotides based on the nucleotide sequence encoding PCR. These probes used in polymerase chain reaction 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 degenerate combination of possible sequences for the identification of closely related genomic sequences . Another means for producing specific hybridization probes for PCR DNA includes the cloning of nucleic acid sequences encoding PCR or PCR 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 the addition of the appropriate RNA polymerase such as T7 or SP6 RNA polymerase and radioactively labeled nucleotides. It is now possible to produce a DNA sequence, or portions thereof, that encodes PCR 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 which are known in the art at the time of filing this application. Moreover, synthetic chemistry can be used to introduce mutations into the PCR sequences of any portion thereof. The nucleotide sequence can be used in any assay to detect inflammation or disease associated with abnormal levels of CPR expression. The nucleotide sequence can be labeled by methods known in the art and added to a fluid or tissue sample of a patient, such as, for example, rheumatoid synovium, under hybridization conditions. After an incubation period, the sample is washed with a compatible fluid that optionally contains a dye (or other label 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 standard. If the amount of dye is significantly elevated, the nucleotide sequence has been hybridized with the sample, and the test indicates the presence of inflammation and / or disease. The nucleotide sequence for PCR can be used to construct hybridization probes to map 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 techniques. These techniques include site hybridization, binding analysis against known chromosomal markers, hybridization analysis with libraries, chromosomal preparations sorted by flow, or constructions of artificial chromosomes, YAC or Pl constructs. The fluorescent in situ hybridization technique of chromosome propagation and other physical chromosome mapping techniques have been described can be correlated with additional genetic mapping data. Examples of genetic mapping data can be found in the 1994 Genome Issue of Science (265, -l981f). The correlation between the location of CPR 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 affected or affected individuals. Expression of PCR The nucleotide sequences encoding PCR can be used to produce purified PCR using well-known methods of recombinant DNA technology. In one embodiment, the nucleic acid encoding the mature PCR is expressed, in another embodiment, the nucleic acid encoding the proprotein sequence and the mature PCR is expressed, and in yet another embodiment, the nucleic acid encoding the proprotein sequence is expressed. Among the many publications that show 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. RCP can be expressed in a variety of host cells, either prokaryotic or eukaryotic. The host cells can be of the same species from which the nucleotide sequences of PCR or a different species are endogenous. The advantages of producing PCR by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures. The expression of PCR can be raised by subcloning the cDNAs into suitable expression vectors and transfecting the vectors into suitable expression hosts. In one embodiment, an expression vector is one that provides expression of a fusion protein comprising PCR and contains 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 ions as described in Porath et al. (1992) Protein Expression and Purification 3: 263-281) while the enterokinase cleavage site provides a means for purify the chemokine from the fusion protein. The cloning vector previously used for the generation of the tissue library also provides for the expression of the PCR sequence in E. coli. Since insertions of cDNA clones are generated by an essentially random process, there is an opportunity in three that the included cDNA falls into the correct box 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, digestion with exonuclease III or mung bean nuclease, or a link inclusion of oligonucleotides. The PCR cDNA can be transported in other vectors known to be useful for the expression of protein in specific hosts. Oligonucleotide amplimers containing cloning sites as well as a DNA segment sufficient to hybridize in 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 polymerase chain reaction. The resulting new gene segments can be digested with suitable restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, similar gene segments can be produced by digesting the cDNA with suitable restriction enzymes and filling the missing gene segments with chemically synthesized oligonucleotides. Segments of the coding sequence of more than one gene can be ligated together and cloned into suitable vectors to optimize the expression of the recombinant sequence. Suitable expression hosts for these chimeric molecules include but are not limited to mammalian cells such as Chinese Hamster Ovary (CHO) 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, an expression vector useful as an origin of replication to allow propagation in bacteria and a selectable marker such as the β-lactamase antibiotic resistance gene can be included 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 they are not part of the cDNA of interest. Additionally, the vector may contain promoters or enhancers that increase the expression of the gene. These promoters are host specific and include MMTV, SV40, or metallothionin promoters for Chinese hamster ovary cells, - trp, lac, or T7 promoters from bacterial hosts, or alpha factor, alcohol oxidase, or PGH promoters for yeast. Transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used in mammalian host cells. Once homogenous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced PCR can be recovered from the conditioned medium and analyzed using chromatographic methods known in the art. Cells transformed with DNA encoding PCR can be cultured under conditions suitable for the expression of cathepsin C and recovery of the protein from the cell culture. PCR produced by a recombinant cell can be secreted or it can be contained intracellularly, depending on the particular 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. The PCR can be expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate purification of the protein. These purification facilitation domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobile metals, protein A domains that allow purification on immobile 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 PCR sequence can be useful to facilitate the expression of PCR. In addition to the production of recombinants, PCR fragments can be produced by direct peptide synthesis, using solid phase techniques (cf Steward et al. (1989) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA). Merrifield J (1963) J. Am. Chem. Soc. 85: 2149-2154 In vi tro protein synthesis can be performed using manual techniques or by automation Automated synthesis can be achieved, for example, by using a synthesizer. 431A peptides from Applied Biosystems (Foster City, CA) according to the instructions provided by the manufacturer Several PCR fragments can be chemically synthesized separately and combined using chemical methods to produce the full-length molecule. for the induction of antibodies does not require biological activity, - however, the protein must be antigen.The peptides used to induce specific antibodies can n have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. They must mimic a portion of the amino acid sequence of the protein and can contain the entire amino acid sequence of a naturally occurring small molecule such as PCR. The short stretches of RCP amino acids can be fused with another protein such as keyhole limpet hemocyanin and the chimeric molecule used for the production of antibodies. Antibodies specific for PCR can be produced by inoculating a suitable animal with the polypeptide or an antigen fragment. In the present invention, the polypeptide may be the mature PCR sequence, the proprotein sequence or the proprotein and mature PCR sequence or a polypeptide comprising a signal sequence, the proprotein sequence and the mature sequence. An antibody is specific for PCR if it is produced against an epitope of a 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 in animals, but also analogous steps in the production of synthetic antibodies or other specific binding molecules such as the selection 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 the stimulation in ly of lymphocyte populations. Current technology (Winter G and Milstein C (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 CPR. Various methods for preparing monoclonal and polyclonal antibodies for PCR are known to those skilled in the art. In an approximation form, denatured PCR of the reverse phase high performance liquid chromatography separation is obtained and 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, whereas up to 1 milligram could be used for immunization of a rabbit. To identify mouse hybridomas, the denatured protein can be radioiodinated and used to screen for potential murine B-cell hybridomas potential for those predicting antibody. This procedure requires only small amounts of protein, so 20 milligrams would be enough to label and select several thousand clones. In another form of approximation, the amino acid sequence of PCR, as deduced from the translation of the cDNA sequence, is analyzed 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. Analyzes for selecting suitable epitopes are described by Ausubel FM et al. (1989, Current Protocols in Molecular Biology, John Wiley &Sons, NYC). The optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and the hydrophilic, intervening regions of the polypeptide that are most likely to be exposed to the external environment when the protein is in its natural form. 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 keyhole limpet hemocyanin (KLH, Sigma) by reaction with M ester. -maleimidobenzoyl-N-hydroxysuccinimide (MBS; see Ausubel FM et al., supra). If necessary, a cysteine can be introduced into the N-terminus of the peptide to allow coupling with KLH and animals can be immunized with the peptide-KLH complex in complete Freund's adjuvant. The resulting antiserum can be tested for anti-peptide activity by binding the peptide to plastic, blocking with 1 percent bovine serum albumin, reacting with antiserum, washing and reacting with goat-specific anti-rabbit immunoglobulin, purified affinity, labeled radioactive or fluorescent). Hybridomas can also be prepared and analyzed using standard techniques. Hybridomas of interest can be detected by selecting labeled PCR to identify the fusions that produce the monoclonal antibody with the desired specificity. In a typical protocol, tray wells (FAST, Becton-Dickinson, Palo Alto, CA) are coated with rabbit anti-mouse specific antibodies with purified affinity (or convenient antispecies Ig) at 10 milligrams / milliliter. The coated wells were blocked with 1 percent bovine serum albumin, washed and exposed to supernatants of hybridomas. After incubation, wells were exposed to labeled PCR at 1 milligram / milliliter. The clones that produce antibodies bound a labeled amount of PCR which is detectable above the background. These clones can be expanded and subjected to 2 cloning cycles at the limiting dilution (1 cell / 3 wells). Cloned hybridomas are injected into mice treated with prisans to produce ascites, and monoclonal antibody can be purified from mouse ascites fluid by affinity chromatography using protein A. Monoclonal antibodies with affinities of at least 10 ° M " , preferably 109 to 1010 or stronger, will typically be made by standard 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 City, both incorporated herein by reference. Uses of nucleotide and amino acid sequences for PCR Antibodies, inhibitors, or antagonists of PCR (or other treatments for excessive production of PCR) can provide different effects when administered therapeutically in the treatment of inflammatory diseases; autoimmune diseases, and malignancies of myeloid cell origin in disease states such as arteriosclerosis, leukemia, systemic lupus erythematosus, osteoporosis, rheumatoid arthritis, and toxic shock syndrome as well as graft rejection and graft-versus-host disease. Treatments for excessive production of PCR, such as antibodies, inhibitors, or PCR antagonists, will be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous vehicle medium, preferably at a pH of about 5 to 8, more 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 to be treated. In a specific embodiment herein, the antagonist is the proprotein sequence that begins between amino acid residues 22 and 29 and terminates at amino acid residue 230 of SEQ ID NO: 2. Characteristics of those treatments include solubility of the molecule , half-life and anigenicity / immuno-genicity; These and other characteristics can help to define an effective vehicle. Native human proteins are preferred as overproduction treatments for CPR, but synthetic or organic molecules that result from drug discrimination can be equally effective in particular situations. Treatments for excessive production of CPR can be administered by known routes of administration including but not limited to topical creams and gels; spray and transmucosal spray, patch and transdermal bandage; injectable, intravenous and lavage formulations; and orally administered fluids and pills, formulated particularly to resist acid and stomach enzymes. The particular formulation, 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 to be treated, the treatment to be administered, and the pharmacokinetic profile of the particular treatment. Additional factors that may be taken into account include disease status (eg, severity) of the patient, age, weight, gender, diet, time of administration, combination of drugs, reaction sensitivities, and tolerance / response to therapy. Long-acting formulations could be administered every 3 to 4 days, every week or once every two weeks depending on the half-life and I read the particular treatment relief regimen. 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. The guidance regarding particular dosage methods of administration is provided in the literature; see Patents of the United States of North America Numbers: 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatments and that systemic administration may need application in a manner different from that of localized administration. The examples below are given 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 rcp nucleotide sequence was identified among the sequences comprising the human THP-1 library. THP-1 is a leukemic human cell line derived from the blood of a 1-year-old male child with acute monocytic leukemia. The cells used for the PMA-LPS library were cultured for 48 hours with 100 nm PMA in DMSO and for 4 hours with 1 μg / milliliter of LPS. The THP-1 library was built especially by Stratagene (Stratagene, 11099 M. Torrey Pines Rd., La Jolla, CA 92037) essentially as described below. Stratagene prepared the cDNA library using oligo d (T) primer. The synthetic adapter oligonucleotides were ligated with the cDNA molecules allowing them to be inserted into the Uni-ZAP vector system (Stratagene). This allowed the construction of unidirectional high efficiency lambda library (orientation in sense) 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 explored using DNA probes, then, the phagemid pBluescript® (Stratagene) was separated. This phagemid allows the use of a plasmid system for easy characterization of insertion, sequencing, site-directed mutagenesis, creation of unidirectional deletions and expression of fusion polypeptides. Subsequently, phage particles from custom-built libraries were infected in the E. coli XLl-Blue® host strain (Stratagene). The high transformation efficiency of this bacterial strain increases the likelihood that the cDNA library will contain rare, underrepresented clones. Alternative unidirectional vectors could include, but are not limited to, pcDNAI (Invitrogen, San Diego CA) and pSHlox-1 (Novagen, Madison Wl). II Isolation of cDNA clones The phagemid forms of individual cDNA clones were obtained by the separation process in vivo, in which the XL1-BLUE was coinfected with a helper phage fl. Proteins derived from both lambda phage and helper phage fl initiated new DNA synthesis from defined sequences in the lambda target DNA and create a smaller, single chain circular phagemid DNA molecule that includes all sequences of plasmid pBluescript DNA and the cDNA insert. The phagemid DNA was released from the cells and purified, then used to reinfect fresh bacterial host cells (SOLR, Stratagene Inc), where the double-stranded phagemid DNA was produced. Because the phagemid carries the gene for the / 3-lactamase, the newly transformed bacteria were selected in the 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 method of high production to use bacterial cells and isolate highly purified phagemid DNA. The DNA eluted from the purification resin was convenient for DNA sequencing and other analytical manipulations. The cDNA inserts from randomized isolates from the THP-1 library were partially sequenced. 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 273 from Applied Biosystems (Perkin Elmer) and was determined reading chart.

IV Search for homology of cDNA clones and deduced proteins Each sequence thus obtained was compared to sequences in the Gen Bank using a search algorithm developed by Applied Biosystems Inc. and incorporated into the INHERIT ™ 670 Sequence Analysis System. algorithm, the pattern specification language (developed by TRW Inc.) was used to determine regions of homology. The three parameters that determine how sequence comparisons operate are window size, window offset, and error tolerance. Using a combination of these three parameters, the DNA database, and the appropriate sequences are scored with an initial value. Subsequently, these homologous regions are examined using dot matrix homology graphs to distinguish regions of opportunity coincidence homology. The Smith-Waterman alignments of the protein sequence were used to display the results of the homology search. Peptide and protein sequence homologies were tested 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 for sequences containing regions of homology that were graded with an initial value. The dot matrix homology plots were examined to distinguish regions of significant homology from timely matches. BLAST, which is a search tool for basic local alignment (Altschul SF (1993) J. Mol.Evol 36: 290-300; Altschul, SF et al. (1990) J. Mol. Biol 215: 403- 10), was used to look for local sequence alignments. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of the alignments, BLAST is especially useful for determining exact matches or for identifying homologs. BLAST is useful for matches that do not contain gaps. The fundamental unit of the output of the BLAST algorithm is the High Qualification Segment Pair (HSP). A highly qualified segment pair consists of two fragments of sequences of arbitrary but equal lengths whose alignment is locally maximum and for which the alignment qualification reaches or exceeds a cut-off threshold set by the user. The BLAST approach is to search for pairs of highly rated segments between a query sequence and a database sequence, to evaluate the statistical significance of any match found, and to report only matches that satisfy the threshold selected by the user of significance. Parameter E establishes the statistically significant threshold for reporting the sequence matches of the database. E is interpreted as the upper limit of the expected frequency of the occurrence of opportunity of a highly rated segment pair (or fixed high-scoring segment pair) within the context of the entire database search any base sequence of data whose match satisfies E is reported in the output of the program. The nucleotide and amino acid sequences for the entire coding region of the human cathepsin C homolog, RCP, are shown in Figure 1. V Identification and sequencing of total length of the genes Of all the clones chosen at random and sequenced from the THP-1 library, the rcp sequence was homologous to but clearly different from any known cathepsin C molecule. The complete nucleotide sequence for rcp was translated, and the translation within the table is shown in Figure 1. When the three possible predicted translations of the sequence are searched against protein databases like SwissProt and PIR, no exact matches were found to the possible translations of rcp. Figure 2 shows the comparison of the amino acid sequence of RCP with rat cathepsin C. The substantial regions of homology between these molecules include the catalytic triad residues C25g, H405, and N42? and site276_27g and NNS glycosylation common among cysteine proteases. The hydrophobicity plots for PCR are shown as Figure 3. VI Antisense Analysis The PCR sequence, or any part thereof, is used to inhibit the expression of endogenous CPR in vivo or in vi tro. Although the use of antisense oligonucleotides, which consists of about 20 base pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments. An oligonucleotide based on the PCR coding sequence is used to inhibit the expression of endogenous PCR. Using Oligo 4.0, the complementary oligonucleotide is designed from the conserved 5 'sequence and used to inhibit any transcription, by sending the binding of the promoter to the upstream untranslated sequence, or the translation of an RCP transcript preventing the ribosome from binding to the mRNA. VII Diagnostic test using specific antibodies for CPR Particular PCR antibodies are useful for diagnosing conditions, and chronic or acute diseases that are characterized by differences in the amount or distribution of CPR. PCR was initially found in a THP-1 cell line and can be used to detect abnormalities or pathologies that activate monocytes. Diagnostic tests for PCR include methods that use the antibody and a label to detect PCR in human body fluids, tissues or extracts from these tissues. The polypeptides and antibodies of the present invention can be used with or without modification. Frequently, polypeptides and antibodies will be labeled by joining 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 reported in both the scientific and patent literature. Suitable labels include the Patents of the United States of North America Numbers: 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Recombinant immunoglobulins can also be produced as shown in U.S. Patent Number: 4,816,567, incorporated herein by reference. A variety of protocols for measuring soluble or membrane-bound PCR are known in the art, using either polyclonal or monoclonal antibodies specific for the respective protein. Examples include enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA) and fluorescent activated cell sorting (FACS). A two-site monoclonal-based immunological test using monoclonal antibodies reactive to two epitopes that do not interfere with PCR is preferred, but a competitive binding test may be employed. These tests are described, inter alia, in Maddox, DE et al. (1983, J. Exp. Med. 158: 1211). VIII Purification of native PCR using specific RCP RCP native or recombinant antibodies is purified by immunoaffinity chromatography using antibodies specific for PCR. In general, an immunoaffinity column is constructed by covalently coupling the anti-PCR antibody to an activated chromatographic resin. Polyclonal immunoglobulins are prepared from immune serum either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). In the same way, monoclonal antibodies are prepared from mouse ascites fluid by precipitation of 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 according to the manufacturer's instructions. These immunoaffinity columns are used in PCR purification by preparing a fraction of the cells containing PCR in a soluble form. This preparation is derived by the solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, the soluble PCR containing a signal sequence is secreted in a useful amount in the medium in which the cells are cultured. A soluble preparation containing PCR is passed over the immunoaffinity column, and the column is washed under conditions that allow preferential absorbance of cathepsin C (for example, highly ionic strong regulators in the presence of detergent). The column is then eluted under conditions that disrupt the antibody / PCR binding (eg, a pH 2-3 buffer or a high concentration of a chaotrope such as urea or thiocyanate ion), and PCR is collected. IX CPR Activity Purified or expressed CPR activity can be tested by mixing a known amount of enzyme with a matrix material such as collagen in a biologically acceptable medium and allowing the PCR to digest the collagen over a period of time. A zymogram, consisting of a non-denaturing polyacrylamide gel soaked in collagen on which different concentrations are deposited, preferably between 10 and 100 ng / μl, of PCR, can be used to demonstrate the RCP activity. The stain of the gel per protein after digestion will demonstrate these deposits in which the concentration of collagen (lighter stain) or completely rinsed has been reduced (Paech et al. (1993) Anal. Biochem. 208: 249-54). XIII Drug Discrimination This invention is particularly useful for discriminating compounds using PCR polypeptide or binding fragments thereof in any variety of drug discrimination techniques. The PCR polypeptide or the fragment used in this test can be free in solution, fixed to a solid support, carried on a cell surface or located intracellularly. A method of drug discrimination utilizes eukaryotic or prokaryotic host cells that are stably transformed with recombinant nucleic acids that express the polypeptide or fragment. The drugs are discriminated against these transformed cells in competitive binding tests. These cells, either in a viable or fixed form, can be used for standard binding tests. One can measure, for example, the formation of complexes between CPR and the agent being tested. Alternatively, one can examine the decrease in complex formation between the PCR and its cell, for example a monocyte, caused by the agent being tested. Thus, the present invention provides methods of discriminating drugs or any other agent that can affect inflammation and disease. These methods comprise contacting the agent with a PCR polypeptide or fragment thereof and testing (i) to see the presence of a complex between the agent and the PCR fragment or polypeptide, or (ii) to see the presence of a complex between the PCR or fragment polypeptide and the cell, by methods well known in the art. In these competitive binding assays, the PCR polypeptide or fragment is typically labeled. After convenient incubation, the free RCP polypeptide or fragment is separated from that which is present as a link, and the amount of free or uncomplexed tag is a measure of the ability of the particular agent to bind to CPR or to interfere with the CPR and the complex agent. Another technique for discriminating drugs provides high product discrimination for compounds that have convenient binding affinity to the PCR polypeptide and is described in detail in European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. reference. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic nails or some other surface. The peptide test compounds are reacted with PCR polypeptide and washed. The bound RCP polypeptide is then detected by methods well known in the art. The purified PCR can also be coated directly on plates for use in the aforementioned drug discrimination techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug discrimination assays in which the neutralizing antibodies capable of binding the PCR specifically compete with a test compound to bind the RCP 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 PCR. In accordance with the above, the present invention provides a method for discriminating a plurality of compounds to view their specific binding affinity with the polypeptide of Claim 8 or any portion thereof, comprising the steps of providing a plurality of compounds, - combining PCR with each of a plurality of compounds for a sufficient time to allow bonding under convenient conditionsM and detecting the PCR link with each of the plurality of compounds, thereby identifying the compounds that specifically link PCR. XIV Rational drug design The goal of rational drug design is to produce structural analogs 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 that are more active or more stable forms of the polypeptide or that increase or interfere with the function of a polypeptide in vivo (see Hodgson J (1991) Bio / Technology 9: 19-21, incorporated herein by reference). In an approximation form, the three-dimensional structure of a protein of interest, or of a protein inhibitor complex, is determined by X-ray crystallography, by computer model, or, more typically, by a combination of these two approaches . Both the form and the charges of the polypeptide are checked to elucidate the structure and to determine the active sites of 1 molecule. Useful information is obtained with respect to the structure of a polypeptide modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous cathepsin C molecules or to identify efficient inhibitors. Useful examples of rational drug design include molecules having enhanced activity or stability as shown in Braxton S. and Wells JA (1992 Biochemistry 31: 7796-7801) or acting 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 specific target antibody, selected by functional test, as described above, and then solve its crystal structure. This form of approximation, in principle, gives a farmanucleus on which the subsequent drug design can be based. It is possible to deviate protein crystallography together with generating anti-idiotypic antibodies (anti-ids) for a pharmacologically active, functional antibody. Like an image in the mirror of an image in the mirror, the binding site of the anti-ids would be expected to be analog of the original receiver. The anti-id could then be used to identify and isolate peptides from chemically or biologically produced peptide libraries. The isolated peptides would then act as the farmanucleus. By virtue of the present invention, sufficient amount of polypeptide can be made available for carrying out analytical studies such as X-ray crystallography. In addition, knowledge of the PCR amino acid sequence provided herein will provide guidance to those employing modeling techniques. computer instead of or in addition to X-ray crystallography. All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to the preferred specific embodiments, it should be understood that the invention as claimed should in no way be limited to these specific embodiments. Undoubtedly, various modifications of the modes described for carrying out the invention that are obvious to those skilled in 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. (Ü) TITLE OF THE INVENTION: "NEW CATEPSIN C HOMOLOGIST" (iii) SEQUENCE NUMBER: 2 (iv) CORRESPONDENCE DIRECTION: (A) RECIPIENT: Incyte Pharmaceuticals, Inc. (B) STREET: 3174 Porter Drive (C) ) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: UNITED STATES OF NORTH AMERICA (F) POSTAL CODE: 94304 (v) LEGIBLE FORM BY COMPUTER: (A) MEDIUM TYPE: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: To be assigned (B) DATE SUBMITTED: 19-April-1996 (C) CLASSIFICATION: (viii) INFORMATION OF ATTORNEY / REPRESENTATIVE: (A) NAME: Luther, Barbara J. (B) REGISTRATION NUMBER: 33954 (C) REFERENCE / FILE NUMBER: PF -0032 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-852-0195 (2) INFORMATION FOR THE SEQUENCE ID. NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1389 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) LIBRARY: THP-l (B) CLONA: 14284. { xi) DESCRIPTION OF THE SEQUENCE: SEQ. ID. NO: 1: ATGGGTGCTG GGCCCTCCTT GCTGCTCGCC GCCCTCCTGC TGCTTCTCTC CGGCGACGGC 60 GCCGTGCGCT GCGACACACC TGCCAACTGC ACCTATCTTG ACCTGCTGGG CACCTGGGTC 120 TTCCAGGTGG GCTCCAGCGG TTCCCAGCGC GATGTCAACT GCTCGGTTAT GGGACCACAA 180 GAAAAAAAAG TAGTGGTGTA CCTTCAGAAO CTGGATACAG CATATGATGA CCTTGGCAAT 240 TCTGGCCATT TCACCATCAT TTACAACCAA GGCTTTGAGA TTsTGTTGAA TGACTACAAG 300 TGGTTTGCCT TTTTTAAGTA TAAAGAAGAG GGCAGCAAGG TGACCACTTA CTGCAACGAG 360 ACAATGACTG GGTGGGTGCA TGATGTGTTG GGCCGGAACT GGGCTTGTTT CACCGGAAAG 420 AAGGTGGGAA CTGCCTCTGA GAATOTOTAT GTCAACACAG CACACCTTAA GAATTCTCAG 480 GAAAAGTATT CTAATAGGCT CTACAAGTAT GATCACAACT TTGTsAAAGC TATCAATGCC 540 ATTCAGAAGT CTTGGACTGC AACTACATAC ATGGAATATG AGACTCTTAC CCTGGGAGAT 600 ATGATTAGGA G? AGTGGTßO CCACAGTCGA AAAATCCCAA GGCCCAAACC TGCACCACTG 660 ACTGCTGAAA TACAGCAAAA GATTTTGCAT TTGCCAACAT CTTGGGACTß GAGAAATGTT 720 CATGGTATCA ATTTTGTCAß TCCTGTTCGA AACCAAGCAT CCTGTGGCAG CTGCTACTCA 780 TTGCTTCA TGGGTATGCT AGAAGCGAßA ATCCGTATAC TAACCAACAA TTCTCAGACC 840 CCAATCCTAA GCCCTCAGGA GGTTGTGTCT TGTAGCCAGT ATGCTCAAGG CTGTGA? GGC 900 GGCTTCCCAT ACCTTATTGC AGG? AAGTAC GCCCAAGATT TTGGGCTGGT GGAAGAAGCT 960 TGCTTCCCCT ACACAGGCAC TGATTCTCCA TGCAAAATGA AGGAAGACTG CTTTCGTTAT 1020 TACTCCTCTG AGTACCACTA TGTAGGAGGT TTCTATGGAO GCTGCAATGA AGCCCTGATG 1080 AAGCTTGAGT TGGTCCATCA TGGGCCC? Tß GCAßTTGCTT TTGAAGTAT? TGATG? CTTC 1140 CTCCACTACA AAAAGGGGAT CTACCACCAC ACPGGTCTAA GAGACCCTTT CAACCCCTTT 1 00 GAGCTGACTA ATCATGCTGT TCTGCTTOTß GGCTATGGCA CTG? CTCAGC CTCTGGGATG 1260 GATTACTGGA TTGTTAAAAA CAGCTGGOGC ACCGOCTGGO GTGAGAATGG CTACTTCCGG 1320 ATCCGCAGAG GAACTGATGA GTGTGCAATT GAGAOCATAß CAGTGOCAGC CACACCAATT 1380 CCTAAATTG 1389 (2) INFORMATION FOR THE SEQUENCE ID. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 463 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ. ID. NO: 2: Mee Gly Ala Gly Pro Ser Leu Leu Leu Ala Ala iu Leu Leu Leu Leu 1 5 10 15 Ser Gly Aap Gly Wing Val Arg Cyß Aap Thr Pro Wing Aßn Cya Thr Tyr 20 25 30 Leu Asp Lau Lau Gly Thr Trp Val Phß Gln Val Gly Ser Sar Gly Ser 35 40 45 Gln Arg Asp Val Asn Cyß Ser Val Mae ßly Pro Gln Glu Lys Lyß Val 50 55 60 Val Val Tyr Leu Gln Lya Leu Asp Thr Wing Tyr Asp Asp Leu Gly Asn 65 70 75 80 Ser Gly His Phe Thr lie lie Tyr Aßn Gln Gly Phß Glu lie Val Leu 85 90 95 Asn Asp Tyr Lyß Trp Phe Ala Phß Phß Lyß Tyr Lyß Glu Glu Gly S r 100 105 110 Lys Val Thr Thr Tyr Cys Asn Glu Thr Mßt Thr Gly Trp Val His? Sp 115 120 125 Val Leu Gly Arg Asn Trp Wing Cyß Phß Thr Gly Lys Lys Val Gly Thr 130 135 140 Wing Ser Glu Aßn Val Tyr Val Asn Thr Wing His Leu Lys Asn Ser Gln 145 150 155 160 Glu Lys Tyr Ser Aßn Arg Leu Tyr Lys Tyr Asp His Asn Phe Val Lys 165 170 175 Ala lie Asn Ala? L? Gln Lys Ser Trp Thr Ala Thr Thr Tyr Met Glu 180 185 190 Tyr Glu Thr Leu Thr Leu Gly Asp Met He Arg Arg Ser Gly Gly His 195 200 205 Ser Arg Lys He Pro Arg Pro Lyß Pro Ala Pro Leu Thr Wing Glu lie 210 215 220 Gln Gln Lya Ha Leu Hiß Leu Pro Thr Ser Trp Aßp Trp Arg? ßn Val 225 230 235 240 Hiß Gly? Lß Aßn Phß Val Ser Pro Val Arg Aßn Gln Ala Ser Cyß Gly 245 250 255 Ser Cyß Tyr Ser Phß Wing Being Met Gly Mß Leu ßlu Wing Arg He Arg 260 265 270 He Leu Thr Asn? ßn Ser Gln Thr Pro? L? Leu Ser Pro Gln ßlu Val 275 280 285 Val Ser Cyß Ser Gln Tyr Wing Gln Gly Cyß Glu Gly ßly Phe Pro Tyr 290 295 300 Leu He Ala ßly Lyß Tyr Ala Gln ßp Phe Gly Leu Val Glu Glu? La 305 310 315 320 Cyß Phe Pro Tyr Thr ßly Thr? ßp Ser Pro Cyß Lyß Met Lyß ßlu? ßp 325 330 335 Cyß Phe? Rg Tyr Tyr Ser Ser ßlu Tyr Hiß Tyr Val ßly ßly Phß Tyr 340 345 350 Gly Gly Cyß? ßn ßlu? The Leu.Met Lyß Leu ßlu Leu Val Hiß Hiß ßly 355 360, 365 Pro Met the Val? Phß ßlu Val Tyr? Sp? ßp Phß Leu Hiß Tyr Lyß 370 375 380 Lyß ßly He Tyr His Hiß Thr ßly Leu? Rg? ßp Pro Phß? ßn Pro Phß 385 390 395 400 Glu Leu Thr? ßn Hiß? The Val Leu Leu Val ßly Tyr ßly Thr? ßp Ser 405 410 415? The Ser ßly Mee? ßp Tyr Trp Zlß Val Lyß? ßn Ser Trp ßly Thr ßly 420 425 430 Trp Gly Glu? ßn Gly Tyr Phß? Rg He? Rg? Rg ßly Thr? ßp ßlu Cy? 435 440 445 Wing He Glu Ser He? Val?? Thr Pro He Pro Ly? Leu 450 455 460

Claims (3)

1. CLAIMS 1. A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence as depicted in SEQ ID NO: 2, or its complement.
2. 2. The polynucleotide of claim 1 wherein the nucleic acid sequence consists of SEQ ID NO: 1.
3. 3. A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence as depicted in SEQ ID NO: 2, starting at between residues 22 and 29, inclusive, and ending at residue 463. 5 A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence as depicted in SEQ ID NO: 2, starting at amino acid residue 231 and terminating at amino acid residue 463. 6. A vector of expression comprising the polynucleotide of claim 1. 7. An expression vector comprising the polynucleotide of claim 3. 8. An expression vector comprising the polynucleotide of claim 5. 9. A host cell comprising the expression vector of claim 6. 10. A host cell comprising the expression vector of claim 7. 11. A host cell comprising the expression vector of claim 8. 12. A probe of nucleic acid comprising a non-conserved fragment of the polynucleotide of claim 2. A method for producing a polypeptide comprising the sequence as depicted in SEQ ID NO: 2, the method comprising: a) culturing the host cells of claim 6 under conditions suitable for the expression of the polypeptide; and b) recover the polypeptide from the cell culture. 14. A purified polypeptide having the amino acid sequence as depicted in SEQ ID NO: 2. 15. A purified polypeptide having the amino acid sequence as depicted in SEQ ID NO: 2 from the amino acid residue that begins between residues 22 and 29, inclusive and terminates with amino acid residue 230. 16. A purified polypeptide having the amino acid sequence as depicted in SEQ ID NO: 2 from the amino acid residue beginning between residue 22 and 29, inclusive and ending with amino acid residue 463. 17. A purified polypeptide having the amino acid sequence as depicted in SEQ ID NO: 2 from the amino acid residue beginning at residue 231 and terminates at amino acid residue 463. 18. An antibody specific for the purified polypeptide of claim 14. 19. An antibody specific for the purified polypeptide of claim 17. 20. A diagnostic composition for the detection of amino acid sequences encoding Cathepsin C comprising the polynucleotide of claim 5. 21. A diagnostic test for the detection of nucleic acid sequences encoding PCR in a biological sample, comprising the steps of: a) combining the biological sample with a polynucleotide which comprises the amino 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 sequence of nucleic acids of SEQ ID NO: 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 wherein the presence of an abnormal level of the complex Hybridization correlates positively with a condition associated with inflammation. 22. A diagnostic test for the detection of nucleotide sequences encoding PCR 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 primers comprise fragments of non-conserved regions of the nucleotide sequence of SEQ ID NO: l: b) detect amplified nucleotide sequences; and c) comparing the amount of nucleotide sequences amplified in the biological sample with a standard thereby determining whether the number of nucleotide sequences varies from the standard, where the presence of an abnormal level of the nucleotide sequence is positively correlated with a condition associated with inflammation. 23. A method for discriminating a plurality of compounds to view specific binding affinity with the polypeptide of Claim 14 or any portion thereof, comprising the steps of: a) providing a plurality of compounds, - b) combining PCR with each one of a plurality of compounds for a time sufficient to allow bonding under convenient conditions; and c) detecting the PCR link with each of the plurality of compounds, thereby identifying the compounds that specifically bind to the PCR.