MXPA99007424A - Member of the tnf family useful for treatment and diagnosis of disease - Google Patents

Abstract

An isolated clone comprising sequences transcribed from the TREPA gene. Also provided are human polypeptides translated from said TREPA sequences and a procedure for producing such polypeptide by recombinant techniques. Also provided are a procedure for producing soluble biologically active TREPA, which may be used to treat deficiencies of TREPA and diseases conditions ameliorated by TREPA. Antibodies, antagonists and inhibitors of such polypeptide which may be used to prevent the action of such polypeptide and therefore may be used therapeutically to treat TREPA-associated diseases, tumors or metastasies are disclosed. Also disclosed is the use of said antibodies, agonists and inhibitors as well as the nucleic acid sequences to screen for, diagnose, prognosticate, stage and monitor conditions and diseases attributable to TREPA, especially inflammation. The use of said partial sequence to provide antibodies, agonists and inhibitors as well as partial nucleic acid sequences to screen for, diagnose, stage and monitor diseases associated with TREPA, including butnot limited to inflammation. Illustrative sequences and clone designations for TREPA are provided.

Description

MI EM BRO OF THE FAMI LIA OF TNF UTI L FOR TREATMENT AND DIAGNOSIS OF DISEASE The present application is a continuation in part of the pending US patent application serial no. 08 / 798,692, filed February 12, 1997, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION This invention relates generally to extracellular signal molecules, and more particularly, refers to a member of the tumor necrosis factor (TNF) family of molecules designated as TRACK, reagents and methods for its detection, as well as its use in therapy. The TNF family (tumor necrosis factor) is an expanding set of extracellular signaling molecules (ligands) with biological activities that are intimately associated with a variety of disease conditions. The prototypical member of this family, TNF, is well known as a mediator of septic shock, inflammation, and graft versus host disease. See, for example, A. Cerami, I mmunol Todav. 9: 28-31 (1988); M. Revel, Ciba Found Svmp. 129: 223-33 (1987); J. Cohen, J. Bone Marrow Transplant 3 (3): 1 93-197 (1988). In addition, due to its beneficial effects on the vasculature of solid tumors, the isolated perfusion of TNF as a therapeutic agent for patients with cancer is evaluated. M.W. Boehme, Eur. J. Clin. Invest. 26: 404-41 0 (1 996). The important role of the TNF family in immune regulation has been demonstrated by mutations in both mice and humans. For example, mice with a loss of mutation function in the member of the TNF family known as a Fas ligand, exhibit a variety of disorders including lymphadenopathy and autoimmune disease. See, for example, R. Watanabe-Fukunaga, Nature 356: 314-317 (1992); Takahishi et al. , CeN 76: 969-966, (1994), Adiachi et al, PNAS, 90: 1756 (1 993); Fisher et al. , CeM 81: 953-946 (1,995); F. Rieux-Laucat, Science 268: 1347 (1995). Another example of the role of immunoregulation is the mutation of the TNF family member, binding CD40 in humans. Spontaneous mutations of the CD40 ligand in human patients results in the hyper Ig-M syndrome, demonstrating the requirement of CD40 ligand for B cell maturation and isotype change. In addition, targeted disruption of the TNF Lta family member in mice results in failure to develop peripheral lymph nodes. P. De Togni, Science 264: 703-707 (1 994). Another property of this family of ligands, which is potentially clinically useful, is the ability to selectively induce apoptosis (programmed cell death) in a variety of cancer cells, but not in most normal cells. The two known members of the TNF family, which induce apoptosis in the widest variety of cell lines, are TRAIL (ligand that induces TNF-related apoptosis) and Fas ligand. See, for example, T. Suda et. to the. , Ce 75: 1 1 69-1 1 78 (1 993); Wiley et. to the. , Immunity 3: 673-682 (1995). This property is unique to the TNF family of ligands. Members of the TNF family of ligands can be identified by an amino acid conservation region, which is restricted to approximately 150 N-terminal amino acids. This region forms a pleated sheet structure in b, which trimerizes and interacts with related receptors. Within this N-terminal domain, there are isolated regions of homology, which correspond to the filaments of the pleated leaf in b. Accordingly, the identity of the total amino acid sequence between members of the TNF family is not high, but can be recognized by those skilled in the art. Given the crucial roles of the members of this molecule family in immunoregulation, research into the existence and identity of other members of the TNF family is desirable. The identification and characterization of these molecules provide means to identify and treat a variety of immune disorders.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a novel member of the TNF family of ligands, as well as the cDNA encoding this novel member, and expression vectors encoding a soluble form of this novel member. Since this novel member of the TNF family is able to induce proliferation in human umbilical endothelial cells, it is called TREPA, which means Proliferation Agent in Endothelium. Related to TN F.

A method for producing TREPA polypeptides involves expression in host cells transformed with a recombinant expression vector containing a soluble designed version of TREPA, as well as a form of TREPA expressed on the surface of the cell. The present invention also provides a method for detecting TREPA target polynucleotides in a test sample, which comprises contacting a TREPA-specific target polynucleotide with at least one TREPA-specific polynucleotide and fragment or complement thereof provided herein, and detect the presence of the target in the test sample. The polynucleotide comprises SECU ENCIA I D NO 1 and fragments or complements thereof. In addition, the TREPA polynucleotide can bind to a solid phase before performing the assay. The present invention also provides a method for amplifying the 5 'end cDNA of TREPA gene in a test sample, which comprises performing reverse transcription with random primers, amplifying the obtained cDNA by using another or other TREPA oligonucleotide primers as sense and antisense initiator (s) in a first-stage PCR to obtain amplified cDNA and detect the presence of TREPA amplicon in the test sample. The amplification can be performed by the polymerase chain reaction. In addition, the test sample can be attached to a solid phase before performing the method. In addition, the detection step may comprise using a detectable label capable of generating a measurable signal. The detectable label can bind to a solid phase.

The present invention further provides a method for detecting TREPA in a test sample suspected to contain TREPA, which comprises contacting said test sample with at least one polynucleotide as a sense primer and with at least one polynucleotide as a anti-sense primer and amplifying same to obtain a first-stage reaction product; contacting said first-step reaction product with at least one of said polynucleotides of the contacting step and a second polynucleotide, with the proviso that the second oligonucleotide is located 3 'for the first oligonucleotide used and is of opposite direction to said first oligonucleotide, and detect said TREPA as an indication of the presence of the disease. The amplification can be performed by polymerase chain reaction. The test sample can be attached to a solid phase before performing the method. The detection step also comprises using a detectable label capable of generating a measurable signal, and the detectable label can be attached to a solid phase. In addition, useful test sets for detecting TARGET objective in a test sample are provided, which comprise a container containing at least one polynucleotide selected from the group consisting of SEQUENCE I D NO 1, and fragments and complements thereof. These test sets further comprise containers containing useful tools for collecting test samples, such as blood, urine, saliva and bowel movements. Such tools include lancets and absorbent paper or cloth to collect and stabilize blood; swabs to collect and stabilize saliva; cups to collect and stabilize urine samples and stools. Optionally, collection materials, papers, fabrics, swabs, cups and the like, can be treated to avoid denaturation or irreversible adsorption of the sample. They can also be treated with or contain preservatives, stabilizers or antimicrobial agents to help maintain the integrity of the specimens. The present invention provides a purified polynucleotide or fragment thereof derived from the TREPA gene capable of selectively hybridizing the genome TREPA population or the complement thereof. The polynucleotide is selected from the group consisting of SEQUENCE ID NO 1, and fragments and complements thereof. In addition, the polynucleotide can be produced by recombinant techniques. This recombinant molecule comprises a sequence that encodes at least one TREPA epitope and is contained within a recombinant vector. The recombinant polynucleotide further comprises a host cell transformed with said vector. The present invention further provides a recombinant expression system comprising an open reading frame of DNA or RNA derived from the TREPA gene wherein said open reading frame comprises the test sample with at least one TREPA-specific polynucleotide or complement thereof, and detecting the presence of the TREPA target polynucleotide in the test sample. The TREPA specific polynucleotide has at least 50% identity for the S ECU ENCIA I D NO 1 polynucleotide and fragments, analogues or complements thereof. The TREPA target polynucleotide can be attached to a solid phase before carrying out the method. The present invention also includes a method for detecting TREPA mRNA in a test sample, which comprises (a) performing reverse transcription with at least one primer in order to produce cDNA; (b) amplifying the cDNA obtained from step (a) by using another TREPA oligonucleotide (s) primer (s) as sense and antisense initiator (s) in a first-stage amplification to obtain amplicon from TREPA; and (c) detecting the presence of TREPA amplicon in the test sample, wherein the TREPA oligonucleotide primers have at least 50% identity for SECU ENCIA I D NO 1 and fragments, analogs or complements thereof. The method further comprises reacting the test sample with a solid phase before performing step (a) or step (b) or step (c). In addition, the detection step may further comprise using a detectable label capable of generating a measurable signal. An additional method is provided for detecting the target TRREP polynucleotide in a test sample that is suspected to contain the target. This method comprises (a) contacting the target TREPA polynucleotide with at least one TREPA oligonucleotide as a sense primer and with at least one TREPA oligonucleotide as an anti-sense primer, and amplifying the same to obtain a product. of first stage reaction; (b) contacting said first step reaction product with at least one other TREPA oligonucleotide, with the proviso that the other TREPA oligonucleotide is located 3 'for the TREPA oligonucleotides used in step (a) and is complementary to the first stage reaction product; and (c) detecting the target TREPA polynucleotide, wherein the TREPA oligonucleotides used in step (a) and step (b) have at least 50% > of identity for the SEQUENCE ID NO 1 and fragments, analogs or complements thereof. In addition, the test sample may be reacted with a solid phase before performing step (a), or step (b) or step (c). In addition, the detection step may comprise using a detectable label capable of generating a measurable signal. Additionally, the detectable label can be bound to a solid phase. The present invention further provides a test set useful for detecting the TREPA polynucleotide in a test sample, comprising a container containing at least one TREPA polynucleotide having at least 50% identity for SEQUENCE ID No. 1 and fragments, analogs or complements thereof. In addition, the test set comprises a container containing useful tools for the collection of said sample selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups. A purified polynucleotide or fragment thereof derived from the TREPA gene is also provided. The purified polynucleotide is capable of selectively hybridizing to the nucleic acid of said TREPA gene, and the purified polynucleotide has at least 50% identity for SEC UENCE I D NO 1 and fragments, analogs or complements thereof. In addition, the purified polynucleotide can be produced by recombinant techniques. When produced by recombinant techniques, the purified polynucleotide may further comprise a sequence of at least one epitope encoded by TREPA. The present invention provides a recombinant expression system comprising a nucleic acid sequence encoding an open reading frame derived from TREPA, which is operably linked to a control sequence compatible with a desired host. The nucleic acid sequence has at least 50% identity for SEQUENCE ID NO 1 and fragments, analogs or complements thereof. This recombinant expression system further comprises a cell transformed with the recombinant expression system. The present invention further provides a polypeptide encoded by TREPA. The polypeptide has at least 35% identity for the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQUENCE I D NO 3 and fragments thereof. The polypeptide can be produced by recombinant technology or by synthetic techniques. A compound is provided, he . which inhibits the activation of the TREPA polypeptide. The TREPA polypeptide has at least 35% identity for an amino acid sequence selected from the group consisting of SECTION 1, NO 2, SECTION 1 and NO 3 and fragments thereof. The invention further provides a polypeptide as a soluble fragment of the TREPA protein, which is capable of binding a receptor for TREPA.

A method is provided for treating a patient who has a need to induce activation of the TREPA polypeptide, which comprises administering to the patient a therapeutically effective amount of a compound, which induces the activation of the TREPA polypeptide. The TREPA polypeptide has at least 35% identity for an amino acid sequence selected from the group consisting of SEQUENCE I D NO 2, SEQUENCE ID NO 3 and fragments thereof. The present invention provides a method for determining whether a compound is an agonist or antagonist of the TREPA protein. This method comprises (a) contacting a cell having TREPA protein expressed on its surface with said compound and a receptor ligand; (b) determining whether a biological effect is produced from the interaction of the ligand and the receptor; and (c) determining whether the compound is an agonist or antagonist. The protein has at least 35% identity for an amino acid sequence selected from the group consisting of SEQUENCE I D NO 2, SEQUENCE I D NO 3 and fragments thereof. The present invention further provides a method for determining whether a receptor binds to a TREPA ligand. This method comprises (a) contacting a mammalian cell which expresses the TREPA ligand with a receptor; (b) detecting the presence of the receptor; Y (c) determine if the receptor binds to the TREPA ligand. An antibody is provided herein, which binds specifically to at least one epitope encoded by TREPA. The antibody is polyclonal or monoclonal. The epitope comprises an amino acid sequence having at least 35% identity for an amino acid sequence selected from the group consisting of SEQUENCE I D NO 2, SEQUENCE ID NO 3 and fragments thereof. Also provided is a test set for determining the presence of TREPA antigen or antibody in a test sample, which comprises a container containing a TREPA polypeptide having at least 35% identity for an amino acid sequence selected from the group consisting of of SECU ENCIA ID NO 2, SEQUENCE ID NO 3 and fragments thereof. In addition, the polypeptide can bind to a solid phase. In addition, the test set further comprises a container containing useful tools for collection of said sample selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups. Another test set for determining the presence of TREPA antigen or antibody in a test sample, which is provided by the present invention, comprises a container containing an antibody which binds specifically to TREPA antigen, wherein the antigen it comprises at least one TREPA epitope having at least about 60% similarity for a sequence selected from the group consisting of SEQ ID NO. 2, SEQUENCE ID NO. 3 and fragments thereof. Additionally, the test set comprises a container containing useful tools for collection of said sample selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups. In addition, the antibody from the pool can bind to a solid phase.

The present invention provides a method for producing a polypeptide comprising at least one TREPA epitope. The method comprises incubating transformed host cells with an expression vector, wherein the vector comprises a polynucleotide sequence encoding a polypeptide, said polypeptide comprising an amino acid sequence having at least 35% identity for an amino acid sequence selected from the group which consists of SEQUENCE ID NO 2, SEQUENCE ID NO 3 and fragments thereof. The present invention further provides a method for detecting TREPA antigen in a test sample that is suspected to contain the TREPA antigen, comprising (a) contacting the test sample with an antibody or fragment thereof, which binds specifically to at least one TREPA antigen epitope selected from the group consisting of SEQUENCE ID NO 2, SEQUENCE ID NO 3 and fragments thereof, for a time and under conditions sufficient for the formation of antibody / antigen complexes; (b) detecting said complexes. The method further comprises the antibody bound to a solid phase. The invention provides a method for detecting antibodies, which bind to the TREPA antigen in a test sample suspected of containing the antibodies. The method comprises (a) contacting said test sample with a TREPA polypeptide containing at least one TREPA epitope comprising an amino acid sequence or fragment thereof having at least 35% identity for an amino acid sequence selected from the group which consists of SEQ ID NO 2, SEQUENCE ID NO 3 and fragments thereof, for a time and under conditions sufficient to allow the antigen / antibody complexes to be formed; and (b) detecting said complexes. The TREPA polypeptide can be further bound to a solid phase. Also provided herein is a cell grown in tissue culture comprising a nucleic acid sequence encoding at least one TREPA antigen epitope or a fragment thereof, wherein the nucleic acid sequence is transfected into the cell, and wherein the nucleic acid sequence is SEQ ID NO. 1 and fragments, analogs or complements thereof. In addition, the present invention provides a method for producing antibodies, which specifically bind to TREPA antigen. The method comprises administering to an individual an isolated immunogenic polypeptide or fragments thereof, wherein the isolated immunogenic polypeptide comprises at least one TREPA epitope and has at least 35% identity for a sequence selected from the group consisting of SEQ ID NO. 2, SEQUENCE ID NO 3 and fragments thereof, in sufficient quantity to produce an immune response. An additional method for producing antibodies is provided, which binds specifically to the TREPA antigen, which comprises administering to a mammal a plasmid comprising a sequence which encodes at least one TREPA epitope, wherein the TREPA sequence is selected from the group consisting of SEQUENCE ID NO 1 and fragments or complements thereof.

The present invention provides a composition of matter comprising a TREPA polynucleotide or fragment thereof, wherein the polynucleotide has at least 50% identity for SECU ENCIA ID NO 1 and fragments, analogies or complements thereof. Further provided is a composition of matter comprising a polypeptide containing at least one epitope encoded by TREPA, wherein the polypeptide has at least 35% identity for a sequence selected from the group consisting of SEQUENCE ID NO 2, SEQUENCE ID NO 3 and fragments of them. This subject polypeptide composition further comprises a soluble fragment of the TREPA protein and is capable of binding to a receptor for TREPA. The present invention also provides a gene or fragment thereof, which encodes TREPA protein, which comprises a sequence having at least 35% identity for SECU ENCIA ID NO 5. In addition, the present invention provides a gene or fragment of the same comprising DNA that has at least 35% identity for SECU ENCIA ID NO 4.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 presents an amino acid alignment of TREPA with several previously described members of the TNF family of ligands. hTREPA, or human THREAP is SEQUENCE ID NO 6, hTRAI L is SEQUENCE ID NO 7, mTRAI L is S ECU ENCE ID NO 8, hTNFa is SECU E NCIA ID NO 9, hTNFb is SEQUENCE ID NO 1 0 and h4-1 BBL is SEQUENCE ID NO 1 1.

DETAILED DESCRIPTION OF THE INVENTION A novel protein designated TREPA is provided herein, together with the DNA encoding the TREPA receptor binding domain, and recombinant vectors to produce TREPA DNA. The present invention also provides antibodies that specifically bind to TREPA proteins. In one embodiment, these antibodies are monoclonal antibodies. The identification of a cDNA clone encoding human TREPA is described in Example 1 below. The nucleotide sequence of the human TREPA gene is presented in SEQUENCE ID No. 1, and the amino acid sequence of the protein is presented in the SEQUENCE ID NO 2 and SEQUENCE I D NO 3. The carboxyl-terminal amino acids of the members of the TNF family demonstrate the conservation of the sequence. Therefore, new members of the family can be recognized by sequence homology for known members of the family in that region. The amino acid sequences described herein reveal that TREPA is a member of the TNF family of ligands. Smith et. to the. , Cell 73: 1349 (1992); Suda et. al , CeM 75: 1 169 (1993); Smith et. to the. , CeJI 76: 959 (1994). Of all the known members of the TNF family, TREPA is very closely related to TNF-alpha. The conserved sequences located in the carboxyl terminal portion of the TNF family of ligands are identified in Smith et. to the. , Cel l. supra. FI GU RA 1 presents an amino acid alignment of TREPA (SEQUENCE I D NO 6) with several previously described members of the TNF family of ligands. These are, from top to bottom, human TRAIL (SEQUENCE ID No. 7), murine TRAI L (SEQUENCE ID NO 8), human TNF-a (SEQUENCE ID NO 9), murine TNF-b (SEQUENCE ID NO 1 0) , and ligand 4-1 human BB (SEQUENCE ID NO 1 1), respectively. Several highly conserved residues that are common for a majority of members of the TNF family are present in TREPA. Among these residues are amino acids 190 (A), 1 10 (H), 133 (W), 159 (G), 161 (Y), 166 (Q), 169 (F), 205 (P), 213 ( S), 234 (L), 243 (F), 244 (G), and 246 (F).

Therapeutic applications Endothelial cells are cells that delineate the walls of blood vessels. The proliferation of endothelial cells can result in the growth of blood vessels in a tissue, a process known as vascularization. There are several clinical conditions where it is beneficial to promote the vascularization of a tissue. This invention provides the use of TREPA as a therapeutic agent to promote vascularization in those situations. For example, wounded tissues require vascularization as part of the healing process. Since it has been shown that TREPA increases the vascularization of HUVEC cells (human umbilical vein endothelial cells) (see Example XX), this invention provides the use of TREPA as a therapeutic agent to promote wound healing. Another situation where tissue vascularization is desirable is in tissue grafts, where the grafted tissue must be vascularized by the host in order for the graft to be successful. Accordingly, the present invention provides the use of TREPA as a therapeutic agent to promote tissue grafts. Similarly, there are situations where it is desirable to inhibit vascularization. For example, the formation of solid tumors requires that the tumor be vascularized by the host or the tumor may not grow beyond a certain limited size. Therefore, inhibiting the vascularization of solid tumors can prevent their growth. The present invention provides TREPA blocking, by means of such as anti-sense RNA, blocking antibody, or compounds developed by focusing TREPA in order to prevent the vascularization of solid tumors. Programmed cell death (apoptosis) is a fundamental biological process. Inflammation is a biological process by which a region of the body is infiltrated by a variety of activated lymphocytes, in response to conditions such as bacterial infection or autoimmunity. Although this process is beneficial, there are disease conditions caused by excessive and inappropriate inflammation, which results in tissue damage. TNF has been implicated as a major cause of some of these conditions, such as sepsis, rheumatoid arthritis and inflammatory bowel disease. Since TREPA is closer to the TN member F-alpha of the TNF family, the present invention provides the use of TREPA as an indicator of inflammation, and for blocking of TREPA, by means such as anti-sense RNA, blockade, or compounds developed by focusing TREPA as a means to inhibit inflammation.

Programmed cell death (apoptosis) is a fundamental biological process by which, given appropriate and external signals, cells are induced to self-destruct. It has been described that this process occurs during embryogenesis, endocrine-dependent tissue atrophy, normal tissue disorder, immune and nervous system development. In the case of the immune system, T cells that recognize auto-epitopes are destroyed by apoptosis during the maturation of T cells in the thymus. It has been proposed that the failure of self-reactive T cells that undergo apoptosis is responsible for autoimmune disorders. Gammon et. to the. , I mmunology Today 12: 193 (1991). A feature of the TNF family is the ability of many family members to induce programmed cell death (apoptosis) in a variety of cells, both normal and tumor origin (Wiley et al., Immunity 3: 673-682 , (1995), and references therein). Accordingly, the present invention provides the use of TREPA as an anti-cancer agent to induce apoptosis in cancer and tumor-associated cells. The present invention also provides the use of TREPA as an agent for removing unprocessed unprocessed cells, such as self-reactive T cells. TREPA can be administered internally alone or in conjunction with other agents, or it can be used for ex vivo treatment and replacement of diseased tissues. For example, circulating lymphoma cells can be exposed to immobilized TREPA ex vivo, and the treated blood can be reintroduced into the patient.

Another feature of the TNF family of ligands is to increase the exposure of solid tumors to cells of the immune system by over-regulating vascular adhesion molecules, thereby attracting antitumor leukocytes to the region. M.W. Boehme, Eur. J. Clin. Invest. , supra. TNF also decreases the intestitial pressure of tumors, allowing concentrations of anti-cancer chemical agents or mixtures of anti-cancer chemical agents to enter the tumor at higher concentrations. The present invention provides the use of TREPA protein as an accessory to be used with anti-cancer chemotherapy agents for the treatment of tumors. In addition, normal viral infection cells can make them susceptible to apoptosis induced by members of the TNF family, such as TNF-alpha and Fas Ligand. See, for example, C.V. Paya et. to the. , I mmunol. 141: 1 989-1995 (1988); P. D. Katsiskas, J. Exp. Med. 1 81: 2029-2036 (1995). The present invention provides the use of TREPA protein as an anti-viral therapeutic agent, to be used alone or in conjunction with other agents, such as anti-viral compounds, or therapeutic protein agents. Since it has been shown that pretreatment of cells virally infected with g-interferon increases apoptosis induced by members of the TNF family, one modality of this application is to administer the TREPA protein together with other agents, such as, g- interferon. This invention also provides the use of TREPA protein to develop any disorder mediated (directly or indirectly) by insufficient amounts or production of defective TREPA protein. The purified human TREPA protein can be administered to a patient with such a condition. Alternatively, the gene therapy techniques for producing TREPA peptide in vivo are also provided. Pharmaceutical preparations of TREPA comprise purified TREPA protein and a carrier, diluent or "physiologically acceptable excipient." Such compositions include buffers, anti-oxidants, low molecular weight peptides, proteins, amino acids, carbohydrates, chelating agents, gluationone, and other excipients and stabilizers commonly found in pharmaceutical compositions The present invention provides methods for evaluating a test sample for products of a TREPA gene, which comprises making cDNA from mRNA in the test sample, and detecting the cDNA as an indication of the presence of the TREPA gene. The method may include an amplification step, wherein portions of the cDNA corresponding to the gene or fragment thereof are amplified. Methods for testing the translation products of mRNAs are also provided. Test samples, which can be assayed by methods provided herein include tissues, cells, body fluids and secretions. The present invention also provides reagents, such as oligonucleotide primers and polypeptides, which are useful for performing these methods. The portions of the nucleic acid sequences described herein are useful as primers for transcription-reverse RNA or for the amplification of cDNA.; or as probes to determine the presence of certain cDNA sequences in test samples. Also described are nucleic acid sequences which allow the production of encoded polypeptide sequences, which are useful as standards or reagents in diagnostic immunoassays, targets for pharmaceutical sorting assays and / or as target components or sites for various therapies. Monoclonal and polyclonal antibodies directed against at least one epitope contained within these polypeptide sequences are useful for diagnostic tests, as well as delivery agents for therapeutic agents and for classifying diseases or conditions associated with the TREPA gene provided herein, especially inflammation. The isolation of sequences from other portions of the gene of interest can be achieved by using probes or PCR primers derived from these nucleic acid sequences, thus allowing additional probes and polypeptides of the genome of interest to be established, which will also be useful in diagnosis , prognosis and / or treatment of diseases and conditions characterized by the TREPA gene described herein. Techniques for determining "similarity" of the amino acid sequence are well known in the art. In general, "similarity" means the exact comparison of amino acid to amino acid of two or more polypeptides at the appropriate place, where the amino acids are identical or possess similar chemical and / or physical properties, such as charge or hydrophobicity. Then a so-called "similarity percentage" can be determined between the polypeptide sequences compared. Techniques for determining the identity of amino acid and nucleic acid sequences are also well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amio acid sequence encoded in the same, and compare this with a second amino acid sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared in determining their "percent identity". Similarly, two amino acid sequences can be compared by determining their "percent identity". The programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl), for example, the GAP program, are capable of calculating both the identity between two polynucleotides and the identity and similarity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known in the art. The compositions and methods described herein will allow the identification of certain markers as indicative of TREPA disease; the information obtained from there will assist in the diagnosis, stage, monitoring, prognosis and / or therapy of diseases or conditions associated with the TREPA gene, especially inflammation, cancer, and graft-vs-host disease. Test methods include, for example, probe assays which utilize the sequence (s) provided herein, and which may also utilize nucleic acid amplification methods, such as, the reaction in polymerase chain (PCR), the ligase chain reaction (LCR), and hybridization. In addition, the nucleotide sequences provided herein contain open reading frames from which an immunogenic epitope can be found. It is believed that this epitope is unique to the disease state or condition associated with the TREPA gene. It is thought that the TREPA gene described herein is useful as a marker, either elevated in diseases such as inflammation., altered in diseases such as inflammation, or present as a normal protein but appearing in an inappropriate body compartment. The uniqueness of the epitope can be determined by its immunological reactivity with the specific TREPA gene, especially inflammatory diseases, such as sepsis. Methods for determining immunological reactivity are well known and include, but are not limited to, for example, radioimmunoassays (RIA), enzyme-linked immunosorbent assay (ELI SA), haemagglutination (HA), fluorescence polarization immunoassay (FPIA) , chemiluminescent immunoassay (CLIA), and others; several examples of suitable methods are described herein. Unless stated otherwise, the following terms should have the following meanings: A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence, which is comprised of a sequence of at least about 6 nucleotides , is preferably at least about 8 nucleotides, is more preferably at least about 10-1 2 nucleotides, and even more preferably is at least about 1 5-20 corresponding nucleotides, ie, identical to or complementary to, a region of the designated nucleotide sequence. The sequence may be complementary to or identical to a sequence, which is unique to a particular polynucleotide sequence, as determined by techniques known in the art. Comparisons for the sequences in data banks can be used, for example, as a method for determining the uniqueness of a designated sequence. Regions from which sequences can be derived include, but are not limited to, regions that encode specific epitopes, as well as non-translated and / or non-transcribed regions. The derived polynucleotide will not necessarily be physically derived from the nucleotide sequence of interest under study, but may be generated in any way, including but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region (s) from which the polynucleotide is derived; as tai, it can represent either a sense or antisense orientation of the original polynucleotide. In addition, the combinations of regions corresponding to that of the designated sequence can be modified in manners known in the art to be consistent with the intended use. The term "initiator" denotes a sequence of specific oligonucleotides complementary to a target nucleotide sequence and which is used to hybridize to the target nucleotide sequence and to serve as a starting point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase. The term "probe" denotes a defined nucleic acid segment (or analog segment of nucleotides, ie, PNA), which can be used to identify specific DNA present in samples that support the complementary sequence. A "polypeptide" or "amino acid" sequence derived from a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence or a portion thereof, wherein the portion consists of of at least 3 to 5 amino acids, and more preferably at least 8 to 10 amino acids, and even more preferably 15 to 20 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence. A "recombinant polypeptide" as used herein means at least one polypeptide which, by virtue of its origin or manipulation, is not associated with all or a portion of the polypeptide with which it is naturally associated and / or is linked to a polypeptide different from that to which it is naturally bound. A recombinant polypeptide or derivative is not necessarily translated from a designated nucleic acid sequence. It can also be generated in any way, including chemical synthesis or expression by a recombinant expression system.

The term "synthetic peptide" as used herein means a polymeric form of amino acids of any length, which can be synthesized chemically by methods well known to the practitioner. These synthetic peptides are useful in several applications. The term "polynucleotide" as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes single or double filament DNA, as well as single or double filament RNA. It also includes modifications, such as methylation or coronation, and unmodified forms of the polynucleotide. "A sequence corresponding to a cDNA" means that the sequence contains a polynucleotide sequence that is identical to or complementary to a sequence in the designated DNA. The degree (or "percentage") of identity or complementarity for the cDNA will be about 50% or greater, preferably it will be at least about 70% or greater, and more preferably will be at least about 90%. The corresponding sequence will be at least about 50 nucleotides in length, preferably it will be about 60 nucleotides in length, and more preferably, it will be at least about 70 nucleotides in length. The correspondence between the gene gene or fragment of interest and the cDNA can be determined by methods known in the art, which include, for example, a direct comparison of the sequenced material with the described cDNAs, or hybridization and digestion with filament nucleases. simple, followed by determination of the size of the digested fragments. "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof, which is essentially free, ie, contains less than about 50%, preferably less than about 70% », and more preferably, less than about 90% of the protein, with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well known in the art and include, for example, the disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide (s) and proteins by ion exchange chromatography, affinity chromatography , and sedimentation according to density In this manner, "purified polypeptide" means a polypeptide of interest or fragment thereof, which is essentially free, ie, contains less than about 50%, preferably less than about 70%, and more preferably, less than about 90% or of cellular components with which the polypeptide of interest is naturally associated The methods for purifying are known in the art The term "isolated" means that the material is removed. of its original environment (for example, the natural environment if it happens naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, which is separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide could be part of a vector and / or such a polynucleotide or polypeptide could be part of a composition, and still be isolated since the vector or composition is not part of its natural environment. "Polypeptide" as used herein, indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, they are included within the definition of polypeptide, peptides, oligopeptides and proteins. However, this term is not intended to refer to post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. "Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures", and other such terms denoting microorganism or higher eukaryotic cell lines cultured as unicellular entities, refer to cells which can be, or have been, used as receptors for recombinant vectors or other transferred DNA, and include the original progeny of the original cell that has been transfected. As used herein, "replicon" means any genetic element, such as a plasmid, a chromosome or a virus, which behaves as an autonomous unit of polynucleotide replication within a cell. A "vector" is a replicon in which another polynucleotide segment joins, so as to cause replication and / or expression of the attached segment.

The term "control sequence" refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism. In prokaryotes, such control sequences include, generally, promoter, ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some cases, enhancers. The term "control sequence" is intended to include, in this manner, at a minimum all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences. "Operably linked" refers to a situation where the described components are in a relationship that allows them to function in their intended manner. In this way, for example, a control sequence "operably linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences. The term "open reading frame" or "ORF" refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence. A "coding sequence" is a sequence of polynucleotides which is transcribed into m RNA and translocated into a polypeptide when placed under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation initiation codon at the 5 'end and a translation stop codon at the 3' end. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequence. The term "immunologically identifiable with / as" refers to the presence of epitope (s) and polypeptide (s), which are also present in and unique to the designated polypeptide (s). The immunological identity can be determined by antibody ligation and / or ligation competition. These techniques are known to the practitioner and are also described herein. The uniqueness of an epitope can also be determined by computer searches of known data banks, such as GenBank, for the polynucleotide sequences which encode the epitope, and by comparisons of amino acid sequences with other known proteins. As used herein, "epitope" means an antigenic determinant of a polypeptide. Conceivably, an epitope can comprise three amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually, consists of at least eight to ten amino acids. Methods for examining spatial conformation are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. A "conformational epitope" is an epitope that is comprised of a specific juxtaposition of amino acids in an immunogeographically recognizable structure, such amino acids being present in the same polypeptide in a contiguous or non-contiguous order or present in different polypeptides. A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. The immunological reactivity can be determined by antibody ligation, more particularly by the ligation kinetics of antibody, and / or by ligation competition using as competitor or competitors known polypeptides containing an epitope against which the antibody is directed. Methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

As used herein, the term "immunogenic polypeptide containing an epitope of interest" means naturally occurring polypeptides of interest or fragments thereof, as well as polypeptides prepared by other means, for example, by chemical synthesis or expression of the polypeptide in a recombinant organism. The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, without regard to the method used for insertion. For example, direct feedback, transduction or f-coupling are included. The exogenous polynucleotide can be maintained as a non-integrated vector, eg, a plasmid, or alternatively, it can be integrated into the host genome. "Treatment m" refers to profi l axis and / or tera pia.

The term "individual" as used herein refers to vertebrates, particularly members of the mammalian species and It includes, but is not limited to, pets, sport animals, primates and humans; More specifically, the term refers to humans. The term "sense filament" or "positive filament" (or "+") as used herein, denotes a nucleic acid that contains the sequence encoding the polypeptide. The term "antisense filament" or "negative strand" (or "-") denotes a nucleic acid that contains a sequence that is complementary to that of the "positive" strand. The term "test sample" refers to a component of an individual's body which is the source of the analyte (such as antibodies of interest or antigens of interest). These components are well known in the art. These test samples include biological samples which can be tested by the methods of the present invention described herein and include animal and human body fluids, such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymphatic fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids, such as cell culture supernatants; specimens of fixed tissues; and specimens of fixed cells. "Purified product" refers to a preparation of the product, which has been isolated from the cellular constituents with which the product is normally associated, and from other cell types, which may be present in the sample of interest. "PNA" denotes a "peptide nucleic acid analog", which can be used in a method such as an assay described herein to determine the presence of a target. "MA" denotes a "morpholino analog", which can be used in a method, such as, an assay described herein to determine the presence of a target. See, for example, US patent no. 5, 378, 841, which is incorporated herein by reference. The PNAs are neutrally charged portions, which can be directed against DNA or RNA targets. PNA probes used in assays instead of, for example, the DNA probes of the present invention offer advantages not achievable when DNA probes are used. These advantages include manufacturability, large scale labeling, reproducibility, stability, insensitivity to changes in ionic strength and resistance to enzymatic degradation, which is present in methods using DNA or RNA. These PNAs can be labeled with signal generating compounds such as fluorescein, radionucleotides, chemo-lysine compounds, and the like. In this manner, PNAs or other nucleic acid analogues, such as Mas, can be used in assay methods instead of DNA or RNA. Although assays are described herein using DNA probes, it is within the scope of the practitioner that PNAs or Mas can be replaced by RNA or DNA with appropriate changes if and as necessary in assay reagents.

"Analyte", as used herein, is the substance to be detected, which may be present in the test sample. The analyte can be any substance by which there is a specific binding member that occurs naturally (such as, an antibody), or by which a specific binding member can be prepared. Thus, an anaiite is a substance that can bind to one or more specific ligation members in an assay. "Analyte" also includes any antigenic substance, haptens, antibodies and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means of specific binding partners (pairs) that occur naturally, such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of vitamin B 12, the use of folate binding protein to determine folic acid, or the use of a lectin as a member of a specific ligation pair for the determination of a carbohydrate. The analyte may include a protein, a peptide, an amino acid, a target nucleotide, and the like. "Inflammation" or "inflammatory disease", as used herein, refers to the infiltration of activated lymphocytes, such as neutrophils, eosinophils, macrophages, T cells and B cells, into a host tissue that results in damage to the organism. Guest. Examples of inflammatory disease include, but are not limited to, conditions such as inflammatory bowel disease, sepsis, and rheumatoid arthritis.

An "Expressed Sequence Tag" or "EST" refers to the partial sequence of a cDNA insert, which has been made by reverse transcription of m RNA extracted from a tissue, followed by insertion into a vector. A "transcript image" refers to a table or list giving the quantitative distribution of ESTs in a library and represents the active genes in the tissue from which the library was made. The present invention provides assays, which utilize speci? C ligation members. A "specific ligation member", as used herein, is a member of a specific ligation pair. That is, two different molecules where one of the molecules through chemical or physical means binds specifically to the second molecule. Consequently, in addition to specific ligation pairs of antibody and antigen from common immunoassays, other specific ligation pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Additionally, specific ligation pairs can include members that are analogous to the original specific ligation members, e.g., an analyte-analog. Immunoreactive specific ligation members include antigens, fragments of antigens, antibodies and antibody fragments, both monoclonal and pliclonal, and complexes thereof, including those formed by recombinant DNA molecules. The term "hapten", as used herein, refers to a partial antigen or non-protein binding member, which is capable of binding to an antibody, but which is not capable of producing antibody formation unless is coupled to a carrier protein. A "capture reagent", as used herein, refers to a specific unlabelled binding member, which is specific either to the analyte as in a "sandwich" assay, to the indicator reagent or analyte as in a competitive assay, or for a specific auxiliary binding member, which is itself specific to the analyte, as in an indirect assay. The capture reagent can be linked directly or indirectly to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample. The "indicator reagent" comprises a "signal generating compound" ("label"), which is capable of generating and generating a measurable signal detectable by external means, conjugated ("joined") to a specific binding member. A "specific ligation member", as used herein, means a member of a specific ligation pair. That is, two different molecules where one of the molecules through chemical or physical means binds specifically to the second molecule. In addition to being an antibody member of a specific ligation pair, the indicator reagent can also be a member of any specific ligation pair, including either hapten-anti-hapten systems, such as biotin or anti-biotin, avidin or biotin , a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. A specific immuno-reactive ligation member can be an antibody, an antigen, or an antibody / antigen complex, which is capable of binding to either a polypeptide of interest as in a sandwich assay, to the capture reagent as in a test competitive, or the auxiliary specific ligation member as in an indirect trial. When probes and probe assays are described, the term "reporter molecule" may be used. A reporter molecule comprises a signal generating compound as described hereinbefore, conjugated to a specific ligation member of a specific ligation pair, such as carbazole or adamantane. The various "signal generating compounds" (labels) contemplated include chromogens, catalysts, such as enzymes, luminescent compounds, such as fluorescein and rhodamine, chemiluminescent compounds, such as dioxetanes, acridiniums, phenanthridines and luminol, radioactive elements, and direct visual labels. . Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but will be able to produce a signal either on its own or in conjunction with one or more additional substances. "Solid phases" ("solid supports") are known to those in the art and include the cavity walls of a reaction tray, test tubes, polystyrene beads, magnetic beads, nytocellulose strips, membranes, microparticles, as, latex particles, g red lobules of sheep (or other animal), and Duracytes® (red blood cells "fixed" by formaldehyde and pyruvic aldheido, available from Abbott Laboratories, Abbott Park, IL) and others. The "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtitre cavities, glass or silicon flakes, red blood cells of sheep (or other suitable animals) and Duracytes® are all suitable examples . Suitable methods for immobilizing peptides in solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase", as used herein, refers to any material which is insoluble, or can be rendered insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor, which has the ability to attract and immobilize the capture reagent. The additional receptor may include a charged substance that is charged in an opposite manner with respect to the capture reagent by itself or a charged substance conjugated to the capture reagent. Still as another alternative, the receptor molecule can be any specific ligation member, which is immobilized on (bound to) the solid phase, and which has the ability to immobilize the capture reagent through a specific ligation reaction. The receptor molecule enables the indirect ligation of the capture reagent to a solid phase material before performance of the assay or during the performance of the assay. A) Yes, the solid phase can be a plastic, derived plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtitre cavity, leaf, bead, microparticle, leaflet, red blood cells of sheep (or other animal) suitable), Duracytes® and other configurations known to those of ordinary skill in the art. It is contemplated and within the scope of the present invention that the solid phase also comprises any suitable porous material with sufficient porosity to allow access by detection of antibodies and a suitable surface affinity for binding antigens. Generally, the microporous structure is preferred but materials with gel structure can also be used in the hydrated state. Such useful solid supports include, but are not limited to, nitrocellulose and nylon. It is contemplated that such porous solid supports described herein, are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 1 5 microns. The surface of such supports can be activated by chemical processes, which cause covalent binding of the antigen or antibody to the support. Irreversible ligation of the antigen or antibody is obtained, however, in general, by adsorption in the porous material by poorly understood hydrophobic forces. Other suitable solid supports are known in the art.

Reagents The present invention provides reagents, such as polynucleotide sequences derived from a TREPA of interest, polypeptides encoded thereby, and antibodies developed from these polypeptides. The present invention also provides reagents such as oligonucleotide fragments derived from the described polynucleotides and complementary nucleic acid sequences for these polynucleotides. The polynucleotides or polypeptides or antibodies of the present invention can be used in the diagnosis, prognosis and / or treatment of individuals with conditions associated with the TREPA gene, such as inflammation, or to identify a predisposition to this condition. The sequences described herein represent unique polynucleotides, which can be used in assays or to produce a specific profile of gene transcription activity. The selected TREPA-derived polynucleotides can be used in the methods described herein for the detection of normal or altered gene expression. Such methods may employ the TREPA-derived polynucleotides described herein or oligonucleotides, fragments or derivatives thereof, or complementary nucleic acid sequences for these polynucleotides. The polynucleotides described herein, their complementary sequences or fragments of either can be used in assays to detect, amplify or quantify genes, cDNAs or mRNAs in relation to TREPA disease or conditions associated therewith. They can also be used to identify a whole or partial coding region, which encodes a TREPA polypeptide. In addition they can be provided in individual containers in the form of a set for tests, or provided as individual compositions. If provided in a set for testing, other suitable reagents, such as buffers, conjugates and the like, may be included. The polynucleotide (s) may be in the form of mRNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA and synthetic DNA are within the scope of the present invention. The DNA can be double filament or single filament, and if it is single filament it can be the coding filament (sense) or non-coding filament (anti-sense). The coding sequence, which encodes the polypeptide, may be identical to the coding sequence provided herein or may be a different coding sequence, said coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the DNA provided herein. This polynucleotide can include only the coding sequence for the polypeptide, or the coding sequence for the polypeptide and additional coding sequence, such as a leader or secretory sequence or a proprotein sequence, or the coding sequence for the polypeptide (and optionally the additional coding sequence) and the non-coding sequence, such as a 5 'and / or 3' non-coding sequence of the coding sequence for the polypeptide. In addition, the invention includes variant polynucleotides containing modifications such as deletions, substitutions or additions of polynucleotides; and any polypeptide modification resulting from the variant polynucleotide sequence. A polynucleotide of the present invention may also have a coding sequence, which is an allelic variant that occurs naturally from the coding sequence provided herein. In addition, the coding sequence for the polypeptide can be fused in the same reading frame for a polynucleotide sequence, which assists in the expression and secretion of a polypeptide from a host cell, for example, a leader sequence, which it functions as a secretory sequence to control the transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cut by the host cell to form the polypeptide. Polynucleotides can also encode a proprotein, which is the protein plus additional 5 'amino acid residues. A protein that has a prosequence is a proprotein and may, in some cases, be an inactive form of the protein. Once the prosequence is cut off, an active protein remains. Thus, the polynucleotide of the present invention can encode a protein, or a protein having a prosequence or a protein having both a pre-sequence (leader sequence) and a prosequence. The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence, which allows purification of the polypeptide of the present invention. The marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for the purification of the polypeptide fused to the tag in the case of a bacterial host, or, for example, the tag sequence can be a tag of hemagglutinin ( HA) when a mammalian host is used, for example, COS-7 cells. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein. See, for example, I. Wilson, et. to the. , CeN 37: 767 (1984). It is contemplated that the polynucleotides will be considered to hybridize to the sequences provided herein if there is at least 50%, and preferably at least 70%, identity between the polynucleotide and the sequence. The present invention also provides an antibody produced by using a purified TREPA gene polypeptide of which at least a portion of the polypeptide is encoded by the TREPA gene polynucleotide selected from the polynucleotides provided herein. These antibodies can be used in the methods provided herein for the detection of TREPA polypeptides in test samples. The antibody can also be used for therapeutic purposes, for example, to neutralize the activity of a TREPA polypeptide under conditions associated with altered or abnormal expression. The present invention also relates to a TREPA polypeptide, which has the amino acid sequence deduced as provided herein, as well as fragments, analogs and derivatives of such a polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural purified polypeptide or a synthetic polypeptide.

The fragment, derivative or analog of the TRE PA polypeptide may be one, in which one or more of the amino acid residues is substituted with a conserved or non-conserved amino acid residue (preferably, a conserved amino acid residue), and such a residue of substituted amino acid may or may not be one encoded by the genetic code; or it may be one in which one or more of the amino acid residues includes a substituent group; or it may be one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol); or it can be one in which additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed by purification of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are within the scope of the present invention. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated, and preferably purified, form. Thus, a polypeptide of the present invention may have an amino acid sequence that is identical to that of the polypeptide that occurs naturally or that is different by minor variations due to one or more amino acid substitutions. The variation may be a "conservative change" usually in the range of about 1 to 5 amino acids, wherein the substituted amino acid has similar chemical or structural properties, for example, replacement of leucine with isoleucine or threonine with serine. In contrast, variations may include non-conservative changes, for example, replacement of a glycine with a tryptophan. Similar minor variations may also include deletions or insertions of amino acids or both. The guide for determining which and how many amino acid residues can be substituted, inserted or deleted without changing the biological or immunological activity can be found using computer programs well known in the art, for example, computer program DNASTAR (DNASTAR Inc., Madison Wl ). Probes constructed in accordance with the polynucleotide sequences of the present invention can be used in various assay methods to provide various types of assays. For example, such sndas can be used in Fluorescent In Situ Hybridization (FISH) technology, to perform chromosomal analysis, and can be used to identify cancer-specific structural alterations in chromosomes, such as deletions or translocations that are visible from chromosome disseminations or detectable using allele-specific oligonucleotide probes and / or generated by PCR, allele-specific amplification or by direct sequencing. The probes can also be labeled with radioisotopes, haptens directly or indirectly detectable, or fluorescent molecules, and used in in situ hybridization studies to evaluate mRNA expression of the gene comprising the polynucleotide in fixed tissue or cell specimens. This invention also provides teachings regarding the production of the polynucleotides and polypeptides provided herein.

Probe assays The sequences provided herein can be used to produce probes, which can be used in assays for the detection of nucleic acids in test samples. The probes may be designated from conserved nucleotide regions of the polynucleotides of interest or from non-conserved nucleotide regions of the polynucleotides of interest. The design of such probes for optimization in trials is within the skill of the practitioner. In general, nucleic acid probes are developed from single or non-conserved regions when maximum specificity is desired, and nucleic acid probes are developed from conserved regions when testing nucleotide regions that are closely related to, for example, different members of a multi-gene family or in related species such as mouse and man. The polymerase chain reaction (PCR) is a technique for amplifying a desired nucleic acid sequence (target) contained in a nucleic acid or mixture thereof. In PCR, a pair of excess primers is used to hybridize at the outer ends of the complementary films of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become objective sequences by themselves, following the dissociation of the original target filament. Then new primers are hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. PCR is described in U.S. Patents 4,683, 1 95 and 4,683,202, which are incorporated herein by reference. The Ligase Chain Reaction (LCR) is an alternative method for the amplification of nucleic acids. In LCR, pairs of probes are used which include two primary probes (first and second) and two secondary probes (third and fourth), all being used in molar excess to the target. The first probe hybridizes to a first segment of the target filament and the second probe hybridizes to a second segment of the target filament, the first and second segments being contiguous so that the primary probes border one another in relation to 5 'phosphate-3' hydroxyl , and so that a ligase can covalently fuse or join two probes in a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting manner. Of course, if the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first case. Once the attached filament of primary probes is separated from the target filament, it will hybridize with the third and fourth probes, which can be joined to form a secondary, complementary ligated product. It is important to realize that the attached products are functionally equivalent to either the objective or its complement. Through repeated cycles of hybridization and binding, the target sequence is achieved. This technique is described more fully in EP-A-320 308 for K. Backman, published on June 16, 1989 and EP-A-439 1 82 for K. Backman et. to the. , published July 31, 1 991, both incorporated herein by reference. For amplification of mRNAs, it is within the scope of the present invention to reverse-transcribe mRNA in cDNA followed by polymerase chain reaction (RT-PCR); or, using a simple enzyme for both steps as described in US Pat. No. 5,332,770, which is incorporated herein by reference; or reverse transcribing mRNA in cDNA followed by asymmetric opening ligase chain reaction (RT-AGLCR) as described by R. L. Marshall, et. to the. , PCR Methods and Applications 4: 80-84 (1994), which are also incorporated herein by reference. Other known amplification methods, which may be used herein may include, but are not limited to, the so-called "NASBA" or "3SR" technique described in PNAS USA 87: 1874-1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European patent application (EPA) No. 454461 0; filament displacement amplification (as described in GT Walker et al., Clin. Chem. 42: 9-1 3 (1 996) and European patent application No. 684315; and target-mediated amplification, as described by PCT publication WO 9322461. In one embodiment, the present invention generally comprises the steps of contacting a test sample suspected of containing a target polynucleotide sequence with amplification reaction reagents, comprising an amplification initiator and a detection probe that can hybridize with an internal region of the amplicon sequences The probes and primers employed according to the method provided herein are marked with capture and detection tags, wherein the probes are labeled with a type of label and the primers are marked with the other type of tag.In addition, the primers and probes are selected so that the probe sequence There is a lower melting temperature than the starter sequences. The amplification reagents, detection reagents and test sample are placed under amplification conditions whereby, in the presence of target sequence, copies of the target sequence (an amplicon) are produced. In the usual case, the amplicon is double filament because the primers are provided to amplify an objective sequence and its complementary filament. Then, the double-filament amplicon is thermally denatured to produce simple filament amplicon members. Upon formation of the single filament amplicon members, the mixture is cooled to allow complex formation between the probes and simple filament amplicon members. After the single filament amplicon member / probe hybrids are formed, they are detected. Standard heterogeneous assay formats are suitable for detecting hybrids using the detection labels and capture tags present in the primers and probes. The hybrids can be attached to a solid phase reagent by virtue of the capture tag and detected by virtue of the detection tag. In cases where the detection label is directly detectable, the presence of the hybrids in the solid phase can be detected by causing the label to produce a detectable signal, if necessary, and to detect the signal. In cases where the tag is not directly detectable, the captured hybrids may be contacted with a conjugate, which generally comprises a binding member attached to a directly detectable tag. The conjugate binds to the complexes and the presence of the conjugate in the complexes can be detected with the directly detectable label. Thus, the presence of the hybrids in the solid phase reagent can be determined. Those skilled in the art will recognize that wash steps may be employed to wash the unhybridized probe or amplicon, as well as the unbound conjugate. A test sample is usually anything that is suspected to contain an objective sequence. Test samples can be prepared using methodologies well known in the art, such as, by obtaining a specimen from an individual and, if necessary, breaking any cell contained therein to release target nucleic acids. Although the target sequence is described as single filament, it is also contemplated to include the case where the target sequence is indeed double stranded, but is simply separated from its complement prior to hybridization with the primer amplification sequences. In the case where PCR is employed in this method, the ends of the target sequences are usually known. In cases where CSF is used or a modification thereof in the preferred method, the complete target sequence is usually known.

Typically, the target sequence is a sequence of nucleic acids, such as, for example, RNA or DNA. The method provided herein can be used in well-known amplification reactions including thermal cycle reaction mixtures, particularly in PCR and GLCR. Amplification reactions usually employ primers to repeatedly generate copies of a target nucleic acid sequence, said target sequence usually being a small region of a much larger nucleic acid sequence. The primers are themselves nucleic acid sequences which are complementary to regions of an objective sequence. Under amplification conditions, these primers hybridize or bind to complementary regions of the target sequence. Typically, copies of the target sequence are generated by the primer extension and / or binding process, which uses enzymes with polymerase or ligase activity, separately or in combination, to add nucleotides to the hybridized primers and / or ligate pairs of adjacent probes. Nucleotides that are added to the primers or probes, such as preformed monomers or oligomers, are also complementary to the target sequence. Once the primers or probes have been extended and / or bound sufficiently, they are separated from the target sequence, for example, by heating the reaction mixture to a "melting temperature", which is one in which they dissociate. the filaments of complementary nucleic acids. Thus, a sequence complementary to the target sequence is formed.

A new amplification cycle can then take place to further amplify the number of target sequences by separating any double strand sequence, allowing primers or probes to hybridize to their respective targets, extending and / or joining the hybridized primers or probes and re-separating . The complementary sequences that are generated by amplification cycles can serve as templates for primer extension or filling of the opening of two probes to further amplify the number of target sequences. Normally, a reaction mixture is subjected to cycles between 20 and 1000 times, more usually, a reaction mixture is subjected to cycles between 25 and 50 times. The numbers of cycles can be determined by the practitioner. In this way, multiple copies of the target sequence and its complementary sequence are produced. Thus, the primers begin amplification of the target sequence when it is present under amplification conditions. In general, two primers are used in PCR, which are complementary to a portion of a target filament and its complement. For CSF, four probes are usually used, two of which are complementary to a target sequence and two of which are similarly complementary to the target complement. In addition to the previously mentioned sets of initiators and enzymes, a nucleic acid amplification reaction mixture may also comprise other reagents, which are well known and include, but are not limited to: enzyme cofactors, such as, manganese; magnesium; you go out; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs), such as, for example, deoxyadenin triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate. Although the amplification primers begin amplification of the target sequence, the detection probe (or hybridization) is not involved in the amplification. Detection probes are generally nucleic acid sequences or analogs of uncharged nucleic acids, such as, for example, peptide nucleic acids, which are described in the international patent application WO 92/20702; morpholino analogs, which are described in US Pat. Nos. 5, 185,444, 5, 034, 506 and 5, 142,047; and similar. Depending on the type of label carried by the probe, the probe is used to capture or detect the amplicon generated by the amplification reaction. The probe is not involved in the amplification of the target sequence and therefore may have to be made "non-stretchable" since additional dNTPs can not be added to the probe. In and of analogues of themselves, they are usually non-stretchable and the nucleic acid probes can be rendered non-stretchable by modifying the 3 'end of the probe, so that the hydroxyl group is no longer able to participate in the elongation. For example, the 3 'end of the probe can be functionalized with the capture or detection label or consumed by the eel or otherwise block the hydroxyl group. Alternatively, the 3 'hydroxyl group can simply be cut, replaced or modified. The serial patent application no. 07 / 049,061 filed April 1, 1993 and incorporated herein by reference, describes modifications which may be used to make a non-stretchable probe. According to this, the ratio of primers to probes is not important. Thus, any of the probes or initiators can be added to the reaction mixture in excess, whereby the concentration of one would be greater than the concentration of the other. Alternatively, the primers and probes can be used in equivalent concentrations. However, preferably, the initiators are added to the reaction mixture in excess of the probes. Thus, starter to probe ratios of, for example, 5: 1 and 20: 1 are preferred. Although the length of the primers and probes may vary, the sequences of the probes are selected so that they have a lower melting temperature than the initiator sequences. Hence, the initiator sequences are generally longer than the probe sequences. Normally, the initiator sequences are in the range of between 20 and 50 nucleotides long, more usually in the range of between 20 and 30 nucleotides long. The normal probe is in the range of 10 to 25 nucleotides long. Various methods for synthesizing primers and probes are well known in the art. Similarly, methods for attaching tags to primers or probes are also well known in the art. For example, it is a routine matter to synthesize nucleic acid primers or probes using conventional nucleotide-phosphoramidite chemistry and instruments available from Appl ied Biosystems, Inc., (Foster City, CA),, Dupont (Wilmington, DE), or Milligen (Bedford MA). Many methods have been described for labeling oligonucleotides such as the primers or probes of the present invention. Enzo Biochemical (New York, NY) and Clontech (Palo Alto, CA) have described and marketed both probe labeling techniques. For example, a primary amine can be attached to a 3 'end oligo using 3'-Amine-ON CPGMR (Clontech, Palo Alto, CA). Similarly, a primary amine can be linked to an end 5 'oligo using Aminomodifier I I (Clontech). The amines can be reacted to several haptens using conventional binding and activation chemistries. In addition, the US codependent serial applications nos. 625,566, filed December 1, 1990 and 630,908, filed December 20, 1990, which are incorporated herein by reference, teach methods for labeling probes at their 5 'and 3' ends, respectively. Publications WO92 / 10505, published June 25, 1992 and WO 92/1 1388, published July 9, 1992, teach methods for labeling probes at their 5 'and 3' ends, respectively. According to a known method for labeling an oligonucleotide, a label reagent-phosphoramidite is prepared and used to add the label to the oligonucleotide during its synthesis. See, for example, N .T. Thuong et. to the. , Tet. Letters 29 (46): 590.5-5908 (1988); or J.S. Cohen et. to the. , U.S. patent application published 07 / 246,688 (NTIS ORDER No. PAT-APPL-7-246,688) (1989). Preferably, the probes are labeled at their 3 'and 5' ends. The capture tags are carried by the primers or probes, and can be a specific ligation member that forms a ligation pair with the specific ligation member of the solid phase reagent. It will be understood, of course, that the initiator or probe itself can serve as the capture tag. For example, in the case where a solid phase reagent ligation member is a nucleic acid sequence, it can be selected such that a complementary portion of the primer or probe is attached to thereby immobilize the initiator or probe to the solid phase. In cases where the probe itself serves as the ligation member, those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to single filament amplicon members. In the case where the primer itself serves as the capture tag, at least a portion of the primer is free to hybridize with a nucleic acid in a solid phase, because the probe is selected so as not to be complementary to the nucleic acid. initiating sequence. Generally, simple filament amplicon probe / member complexes can be detected using techniques commonly employed to perform heterogeneous immunoassays. Preferably, in this embodiment, detection is performed in accordance with the protocols used by the commercially available Abbott LCx® instrumentation (Abbott Laboratories, Abbott Park, IL). The primers and probes described herein are useful in normal PCR assays, where the test sample is contacted with a pair of primers, amplification is performed, the hybridization probe is added, and detection is performed.

Another method provided by the present invention comprises contacting a test sample with a plurality of polynucleotides, wherein at least one polynucleotide is provided herein, annealing the test sample with the plurality of polynucleotides and detecting the hybridization complexes. Hybridization complexes are identified and quantified to compile a profile, which is indicative of TREPA disease. The expressed RNA sequences can be further detected by reverse transcription and amplification of the DNA product by methods well known in the art, including the polymerase chain reaction (PCR).

Classification of medicament and gene therapy The present invention also encompasses the use of gene therapy methods for the introduction of anti-sense TREPA gene-derived molecules, such as polynucleotides or oligonucleotides of the present invention in patients with conditions associated with abnormal expression of polynucleotides related to TREPA disease, including cancer. These molecules, including antisense RNA and DNA fragments and ribozymes, are designed to inhibit the translation of a TREPA-derived polynucleotide mRNA, and can be used therapeutically in the treatment of conditions associated with altered or abnormal expression of a TREPA-derived polynucleotide. Alternatively, the oligonucleotides described above can be delivered to cells by methods in the art, so that the antisense DNA or RNA can be expressed in vivo to inhibit the production of TREPA-derived polypeptide in the manner described above. Accordingly, antisense constructs for TREPA-derived polynucleotides reverse the action of TREPA-derived transcripts and can be used to treat TREPA disease conditions, such as inflammation. These antisense constructs can also be used to treat tumor metastasis. Effects on the tumor vasculature, such as those associated with this family of molecules (MW Boehme, Eur. J. Clin, Invest. 26: 404-410 1996), are useful in the treatment of both primary and metastatic solid tumors, including carcinomas of the breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gall bladder and ducts, small intestine, urinary tract (including kidney, bladder, and urothelium), female genital tract (including cervix) , uterus and ovaries, as well as, choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including thyroid, adrenal and pituitary glands), and skin, as well such as hemangiomas, melanomas, sarcomas (including those arising from soft and bony tissues, as well as kaposi's sarcoma) and tumors of the brain, nerves, eyes and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas). Such proteins may also be useful for treating solid tumors arising from hematopoietic malignancies, such as leukemias (ie, chloromas, plasmacytomas and plaques and mycosis fungoides tumors and cutaneous T-cell lymphoma / leukemia), as well as in the treatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas). In addition, these proteins or genes which encode their expression, may be useful in the prevention of metastasis of the tumors described above, either when used alone or in combination with radiotherapy and / or other chemotherapeutic agents. In addition, uses include the treatment and prophylaxis of autoimmune diseases, such as, rheumatoid, immune and degenerative arthritis; various eye diseases, such as diabetic retinopathy, premature retinopathy, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, hypoxia, and other abnormal neovascularization conditions of the eye; skin diseases, such as psoriasis; blood vessel diseases, such as, hemagiomas, and capillary proliferation within atherosclerotic plaques; Osler-Webber syndrome; myocardial angiogenesis; plate neovascularization; telangiectasia; heophiliac joints; angiofibroma; and granulation of wounds. Other uses include the treatment of diseases characterized by excessive or abnormal stimulation of endothelial cells, including, but not limited to, intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e. Another use is as a birth control agent, by inhibiting ovulation and the establishment of the placenta. TRE PA is also useful in the treatment of diseases that have angiogenesis as a pathological consequence, such as cat scratch disease (Róchele minalia quintosa) and ulcers (Helicobacter pylorO.) TREPA can be used in combination with other compositions and procedures for Treatment of diseases For example, a tumor can be treated conventionally with surgery, radiation or chemotherapy combined with TREPA, and then TREPA can be subsequently administered to the patient to extend the inactivity of micrometastases and to stabilize and inhibit the growth of any primary tumor. Additionally, TREPA, TREPA fragments, TREPA antiserum, TREPA receptor agonists, TREPA receptor antagonists, or combinations thereof, may be combined with pharmaceutically acceptable excipients, and optionally sustained release matrices, such as biodegradable polymers, to form compositions tera A sustained release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by acid-base or enzymatic hydrolysis or by dissolution. Once inserted into the body, the matrix is activated by enzymes and body fluids. Conveniently, a sustained release matrix is chosen from biocompatible materials, such as, liposomes, polylactides (polylactic acid), polyglycolide (glycolic acid polymer), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyan Iridides, poly (ortho) esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids, such as, phenylalanine, tyrosine, isoieucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of any one of either polylactide, polyglycolide, or polylactide co-glycolide (copolymers of lactic acid and glycolic acid). Cytotoxic agents, such as, ricin, can be linked to TREPA, and high affinity TREPA peptide fragments, thereby providing a tool for the destruction of cells that bind to TREPA. Peptides linked to cytotoxic agents can be instilled in a manner designed to maximize delivery to the desired location. For example, high affinity TREPA fragments linked to ricin can be delivered through a cannula into vessels supplying the target site or directly into the target. Such agents can also be delivered in a controlled manner through osmotic pumps coupled to the infusion cannulas. A combination of TREPA antagonists can be co-applied with angitogenesis stimulators to increase tissue vascularization. Therapeutic regimens of this type could provide an effective means of destroying metastatic cancer. The present invention also encompasses gene therapy, whereby the gene encoding TREPA is regulated in a patient. Various methods to transfer or deliver DNA to cells for expression of the gene product protein, otherwise referred to as gene therapy, are described in "Gene Transfer into Mammalian Somatic Cells in. vivo", N. Yang, Crit. Rev. Biotechn. 12 (4): 335-356 (1992), which is incorporated herein by reference. Gene therapy encompasses the incorporation of DNA sequences into somatic cells or germline cells for use either in ex vivo or in vivo therapy. The functions of gene therapy to replace genes, increases the function of normal or abnormal gene, and combats infectious diseases and other pathologies. Strategies to treat these medical problems with gene therapy include therapeutic strategies, such as, identifying the defective gene and then adding a functional gene either to replace the defective gene function or to increase a slightly functional fene; or prophylactic strategies, such as adding a gene which encodes a protein product that will treat the condition or that will make the tissue or organ more susceptible to a treatment regimen. As an example of a prophylactic strategy, a gene encoding TREPA can be placed in a patient and thus prevent the occurrence of angiogenesis; or a gene that makes the tumor cells more susceptible to radiation could be inserted, so that tumor radiation would cause an increased death of the tumor cells. Many protocols for TREPA DNA transfer or TREPA regulatory sequences are provided in this invention. Transfection of promoter sequences, other than those specifically associated with TREPA, or other sequences which would increase the production of TREPA protein are also envisioned as gene therapy methods. An example of this technology is found in Transkaryotic Therapies, Inc., of Cambridge, Massachusetts, using homologous recombination to insert a "genetic change" that turns on an erythropoietin gene in cells. See Genetic Engineering News, April 15, 1994. Such "genetic changes" could be used to activate TREPA (or a TREPA receptor) in cells that do not normally express these proteins. Gene transfer methods for gene therapy fall into three broad categories: (1) physical (eg, electroporation, direct gene transfer and particle bombardment), (2) chemical (eg, lipid-based carriers and others). non-viral vectors), and (3) biological (for example, vectors derived from viruses). For example, non-viral vectors, such as liposomes coated with DNA can be injected directly intravenously into the patient. It is believed that liposome / DNA complexes are concentrated in the liver, where they deliver DNA to macrophages and Kupffer cells. Additionally, vectors or the "naked" DNA of the gene can be injected directly into the desired organ, tissue or tumor for focused delivery of the therapeutic DNA. Gene therapy methodologies can also be described by delivery site. The fundamental ways to deliver genes include ex vivo gene transfer, gene transfer in vivo, and in vitro gene transfer. In the ex vivo gene transfer, the cells are taken from the patient and grown in a cell culture. The DNA is transfected into the cells, the transfected cells are expanded in number and then reimplanted in the patient. In in vitro gene transfer, the transformed cells are cells that grow in culture, such tissue culture cells and not particular cells of a particular patient. These "laboratory cells" are transfected, and the transfected cells are selected and expanded either for implantation in a patient or for other uses. Gene transfer in vivo involves introducing the DNA into the patient's cells when the cells are inside the patient. The three broad categories described above can be used to achieve gene transfer in vivo, ex vivo, and in vitro. Mechanical (ie, physical) methods of DNA delivery can be achieved by direct injection of DNA, such as, microinjection of DNA into somatic or germ cells, pneumatically delivered DNA coated particles, such as gold particles used in a " gene gun ", and inorganic chemical approaches, such as, calcium phosphate transfection. It has been found that the physical injection of plasmid DNA into muscle cells produces a high percentage of cells, which are transfected and have a sustained expression of marker genes. The plasmid DNA may or may not be integrated into the genome of the cells. The non-integration of transfected DNA would allow the transfection and expression of gene product proteins in non-proliferative tissues, terminally differentiated over a prolonged period without fear of insertions, deletions or mutational alterations in the cellular or mitochondrial genome. The long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells can provide treatments for genetic diseases or for prophylactic use. The DNA could be periodically reinjected to maintain the level of gene product without mutations occurring in the genomes of the recipient cells. The non-integration of the exogenous DNAs may allow the presence of several different exogenous DNA constructs within a cell, all the constructs expressing several gene products. Gene transfer mediated by particles can also be used to inject DNA into cells, tissues and organs. With a particle bombing device, or "gene gun", a driving force is generated to accelerate high density particles coated with DNA (such as gold or tungsten) at a high velocity that allows the penetration of organs, tissues or target cells. Gene transfer electroporation uses an electrical current to make cells or tissues susceptible to gene transfer mediated by electroporation. A brief electrical pulse with a given field strength is used to increase the permeability of a membrane in such a way that the DNA molecules can penetrate the cells. The techniques of particle-mediated gene transfer and electroporation are well known to those of ordinary skill in the art. Chemical methods of gene therapy involve carrier-mediated gene transfer through the use of fusogenic lipid vesicles, such as liposomes or other vesicles for membrane fusion. A carrier harboring a DNA of interest can be conveniently introduced into body fluids or the blood stream and then specifically targeted to a site for the target organ or tissue in the body. For example, liposomes can be developed, which are specific cell or specific organ. The foreign DNA carried by the liposome will be thus taken up by those specific cells. The injection of immunoliposomes that are targeted to a specific receptor in certain cells can be used as a convenient method to insert the DNA into the cells that carry the receptor. Another carrier system that has been used is the asialoglyprotin / polylysine conjugate system to carry DNA to hepatocytes for gene transfer in vivo. The transfected DNA can also complex with other classes of carriers, so that the DNA is taken to the recipient cell and then resides in the cytoplasm or nucleoplasm of the recipient cell. DNA can be coupled to nuclear carrier proteins in specifically designed vesicle complexes and can be carried directly into the nucleus. Carrier-mediated gene transfer can also involve the use of lipid-based proteins, which are not liposomes. For example, lipofectins and cytofectins are positive ions based on lipids that bind negatively charged DNA, forming a complex that can transport DNA through a cell membrane. Another method of carrier-mediated gene transfer involves receptor-based endocytosis. In this method, a ligand is made (specific for a cell surface receptor) to form a complex with a gene of interest and then injected into the bloodstream; the target cells that have the cell surface recepotr will bind specifically to the ligand and transport the ligand-DNA complex in the cell. Biological gene therapy methodologies usually employ viral vectors to insert genes into cells. The term "vector" as used herein in the context of biological gene therapy means a carrier that can contain or associate with, specific polynucleotide sequences, and which functions to carry the specific polynucleotide sequences in a cell. The transfected cells can be cells derived from the normal tissue of the patient, the diseased tissue of the patient, or they can be non-patient cells. Examples of vectors include plasmids and infectious microorganisms such as viruses, or non-viral vectors, such as the ligand-DNA conjugates, liposomes and lipid-DNA complexes discussed above. It may be convenient that a recombinant DNA molecule comprising a TREPA DNA sequence is operably linked to an expression control sequence to form an expression vector capable of expressing TREPA. Alternatively, TREPA gene regulation can be achieved by administering proteins that bind to the 'kringle' gene, or control regions associated with the TREPA gene, or their corresponding RNA transcript to modify the rate of transcription or translation. Viral vectors that have been used for gene therapy protocols include, but are not limited to, retroviruses, other RNA viruses, such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes virus, SV 40 , vaccinia and other DNA viruses. Retroviral replication-defective murine vectors are the most widely used gene transfer vectors. The murine leukemia retroviruses are composed of a single filament RNA complexed with a nuclear core protein and polymerase (pol) enzymes, enclosed by a protein core (gag) and surrounded by a glycoprotein (env) envelope that determines the range of the guest. The genomic structure of retroviruses includes the gag, pol, and env genes enclosed by 5 'and 3' long terminal repeats (LTR). The retroviral vector systems exploit the fact that a minimal vector containing the 5 'and 3' LTRs and the packaging signal are sufficient to allow vector packaging, infection and integration into target cells, provided that the viral structural proteins are supplied in trans on the packaging cell line. The fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, integration of precise single copy vector into target cell chromosomal DNA, and ease of manipulation of the retroviral genome. For example, altered retrovirus vectors have been used in ex vivo methods to introduce genes into lymphocytes that infiltrate tumors and peripherals, heptocytes, epidermal cells, myocytes, and other somatic cells (which can then be introduced into the patient to provide the gene product of D NA inserted). The adenovirus is composed of linear, double-stranded DNA complexed with nucleus proteins and surrounded by capsid proteins. Advances in molecular virology have led to the ability to exploit the biology of these organisms to create vectors capable of transducing novel genetic sequences in target cells in vivo. Adenovirus-based vectors will express gene product peptides at high levels. Adenoviral vectors have high infectivity efficiencies, even with low virus titers. Additionally, the virus is completely infectious as a cell-free virion, so that injection of producer cell lines is not necessary. Another potential advantage for adenoviral vectors is the ability to achieve long-term expression of heterologous genes in vivo. Viral vectors have also been used to insert genes into cells using in vivo protocols. To direct specific tissue expression of foreign genes, regulatory elements that act on cis or promoters that are known to be of specific tissue can be used. Alternatively, this can be achieved by using in situ delivery of DNA or viral vectors to specific anatomical sites in vivo. A viral vector can be delivered directly to the site in vivo, by means of a catheter, for example, thus allowing only certain areas to be infected by the virus, and providing long-term specific site gene expression. In vivo gene transfer has also been demonstrated using retrovirus vectors in breast tissue and liver tissue by injecting the altered virus into blood vessels leading to the organs. When used in the above treatment or other treatments, a therapeutically effective amount of one of the proteins of the present invention may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form. By a "therapeutically effective amount" of the protein of the invention is meant a sufficient amount of the protein to treat an angiogenic disease, (for example, to limit tumor growth or to decrease or block tumor metastasis) to a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily use of the proteins and compositions of the present invention will be decided by the attending physician within the scope of the sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific protein used; the specific composition used, the age, body weight, general health, sex and diet of the patient; the administration time, route of administration and rate of excretion of the specific protein used; the duration of treatment; medications used in combination or coincident with the specific protein used; and similar factors well known in the medical arts. For example, it is within the skill of the art to start with doses of the protein at levels lower than those required to achieve the therapeutic effect and gradually increase the dosage until the desired effect is achieved. The proteins of the present invention can be used in the form of salts derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, caforsulfonato, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate , bicarbonate, p-toluenesulfonate and undecanoate. In this way, dispersible or soluble products in oil or water are obtained. Examples of acids, which can be employed to form pharmaceutically acceptable acid addition salts, include such inorganic acids as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids such as maleic acid, succinic acid and citric acid. Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium or with organic base. Preferred salts of the proteins of the invention include phosphate, tris and acetate. The total daily dose of the proteins of this invention administered to a human or lower animal may vary from about 0.0001 to about 1 mg / kg of a patient's body mass / day. If desired, the effective daily dose can be divided into multiple doses for administration purposes; consequently, single dose compositions may contain such or submultiple amounts thereof to make the daily dose.

Alternatively, a protein of the present invention can be administered as a pharmaceutical composition containing the protein of interest in combination with one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, i-solid or liquid filler, diluent, encapsulating material or formulation aid of any type. The compositions can be administered parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), rectally or buccally. The term "parenteral" as used herein, refers to modes of administration which include infusion and intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection. Pharmaceutical compositions for parenteral injection comprise sterile, pharmaceutically acceptable, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution in sterile injectable solutions or dispersions just before use. Examples of suitable carriers, diluents, solvents or aqueous and non-aqueous vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil). , and injectable organic esters, such as ethyl oleate. The proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain auxiliaries, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microencapsulated drug matrices in biodegradable polymers, such as polylactide-polyglycolide, poly (orthoesters) and poly (anhydrides). Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by trapping the drug in liposomes or microemulsions, which are compatible with body tissues. Topical administration includes administration to the skin or mucosa, including lung and eye surface. Compositions for topical administration, including those for inhalation, can be prepared as a dry powder, which can be pressurized or non-pressurized. In the non-pressurized powder compositions, the active ingredient in finely divided form can be used in admixture with a larger pharmaceutically acceptable inert carrier, comprising particles having a size, for example, up to 100 micrometers in diameter. Suitable inert carriers include sugars, such as lactose. Conveniently, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 microns. Alternatively, the composition can be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied propellant medium and indeed the total composition, preferably is such that the active ingredient does not dissolve therein to any substantial degree. The pressurized composition may also contain an active surface agent, such as, a solid or liquid nonionic active surface agent, or it can be a solid anionic active surface agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt. An additional form of topical administration is to the eye. A protein of the invention is delivered in a pharmaceutically acceptable ophthalmic carrier, such that the protein is maintained in contact with the ocular surface for a sufficient period, to allow the protein to penetrate the cornea and inner regions of the eye, such as, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / cilium, crystalline, choroid / retina and sclera. The pharmaceutically acceptable ophthalmic vehicle can, for example, be an ointment, vegetable oil or an encapsulating material. Alternatively, the protein of the invention can be injected directly into the vitreous and aqueous humus.

Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the proteins of this invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or a suppository wax, which are solids at room temperature but liquid at body temperature, and therefore melt in the rectum or vaginal cavity and release the active protein. The proteins of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in the form of liposomes may contain, in addition to a protein of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology. Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq. Although the proteins of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more agents, which are conventionally administered to patients to treat angiogenic diseases. For example, when used in the treatment of solid tumors, the proteins of the invention can be administered with anti-neoplastic agents, such as, alpha interferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, m BACOD (methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and desametasone), PRO-MACE / MOPP (prednisone, methotrexate (w / rescue leucovine), doxorubicin, cyclophosphamide, taxol, etoposide / mechlorethamine, vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibin , TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like. The total daily dose of TREPA (administered in combination with a protein of this invention) to be administered to a human or other mammalian host in single or divided doses may be in amounts, for example, from 0.0001 to 300 mg / kg of weight daily body and more usually from 1 to 300 mg / kg of body weight. It will be understood that agents, which can be combined with the protein of the present invention for the inhibition, treatment or prophylaxis of angiogenic diseases are not limited to those listed above, but include in principle any agent useful for the treatment or prophylaxis of diseases angiogenic The fragments of synthetic TREPA peptides can also be produced and used in a variety of applications. As examples, different fragments of TREPA peptides can be used (1) as active agonists and antagonists at TREPA binding sites, (2) as a means to isolate a TREPA receptor, (3) as antigens for the development of antiserum specific, (4) as peptides to be used in diagnostic sets, and (5) as peptides linked to, or used in combination with, cytotoxic agents (for targeted death of cells that bind TREPA). The amino acid sequences comprising these peptides can be selected based on their position in the outer regions of the molecule, which are accessible to bind to antiserum. Additionally, these peptide sequences can be compared to known sequences using protein sequence databases, such as GenBank, Brookhaven Protein, SWISS-PROT, and PI R to determine potential sequence homologies. This information facilitates the elimination of sequences that exhibit a high degree of sequence homology for other molecules, thereby intensifying the potential for high specificity in the development of antisera, agonists and antagonists for TREPA. The systematic susbstitution of amino acids within these synthesized peptides can produce high affinity peptide agonists and antagonists for the TREPA receptor, which enhance or decrease the binding of TREPA to its receptor. Such agonists can be used to suppress the growth of micrometastases, thereby limiting the spread of cancer. In cases of inadequate vascularization, TRPA antagonists can be applied to block the inhibitory effects of TREPA and promote angiogenesis. For example, this type of treatment can have therapeutic effects by promoting the healing of wounds in diabetics.

TREPA peptides can also be employed to develop affinity columns for isolation of a TREPA receptor in, for example, cultured endothelial cells. As is known in the art, the isolation and purification of a TREPA receptor can be followed by amino acid sequencing to identify and isolate polynucleotides, which encode the TREPA receptor. Such polynucleotides can then be cloned into a suitable expression vector and transfected into tumor cells. Expression of the receptor by transfected tumor cells would intensify the sensitivity of these cells to endogenous or exogenous TREPA, thereby decreasing the rate of metastatic growth. Additionally, recombinant expression of this receptor would allow larger amounts of receptor to be produced, for example, to produce an amount sufficient to be used in high throughput screening assays to identify smaller antagonists, which mimic the action of TREPA. The TREPA peptides of the present invention can also be used as antigens to generate polyclonal or monoclonal antibodies that are specific for the TREPA inhibitor. One way in which such antibodies could be used is in methods and diagnostic sets to detect or quantify TREPA in a body fluid or tissue. The results of these tests could be used to diagnose or determine the importance of prognosis of TREPA. TREPA peptides can be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent, chemiluminescent, bioluminescent, and other proteins for a variety of applications. For example, a TREPA polypeptide can be labeled to facilitate testing for its ability to bind TREPA antisera or to detect cell types, which possess a TREPA receptor. The coupling technique is generally chosen based on functional groups available in the amino acids of the TREPA sequence including, but not limited to, amino, sulfhydryl, carboxyl, amide, phenol and imidazole. Various reagents used to effect such couplings include, among others, glutaraldehyde, diazodized benzidine, carbodiimide and p-benzoquinone. The efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabelling of a TREPA peptide with I 125 can be achieved using chloramine T and Nal125 of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted in disposable columns. The labeled peptide is levigated from the column, and the fractions are collected. Aliquots of each fraction are removed and the radioactivity is measured in a gamma counter. In this manner, a labeled TREPA peptide can be obtained, which is free of unreacted Nal125. Another application of peptide conjugation is for the production of polyclonal antisera. For example, TREPA peptides containing lysine residues can be bound to purified bovine serum albumin using glutaraldehyde. The efficiency of this reaction can be determined by measuring the incorporation of radiolabelled peptide. The unreacted peptide and glutaraldehyde can be separated by dialysis and the conjugate can be stored for subsequent use.

The production of antiserum against TREPA, TREPA analogs, TREPA peptide fragments and the TREPA receptor can be performed using established techniques known to those skilled in the art. For example, polyclonal antisera can be raised in rabbits, sheep, goats or other animals. TREPA peptides conjugated to a carrier molecule, such as bovine serum albumin, or TREPA by itself, can be combined with an auxiliary mixture, emulsified and injected subcutaneously at multiple sites in the back, neck, flanks and sometimes in the bearings of the legs of a suitable host. Generally, reinforcing injections are then given at regular intervals, such as, every 2 to 4 weeks. Approximately 7 to 10 days after each injection, blood samples are obtained by venipuncture, using, for example, the marginal veins of the ear after dilation. The blood samples are allowed to coagulate overnight at 4CC and are centrifuged at approximately 2400 X g at 4 ° C for approximately 30 minutes. The serum is removed, taken to the tablets, and stored at 4 ° C for immediate use or at -20 to -90 ° C for subsequent analysis. Samples of polyclonal antisera generation serum or samples of monoclonal antisera production media can be analyzed for antibody titre determination and, in particular, for the determination of high titre antisera. Subsequently, TREPA antisera of higher titres can be tested to establish the following: a) optimal dilution of antiserum for higher specific binding of the antigen and lower nonspecific binding, b) ability to bind increasing amounts of TREPA peptide in a standard displacement curve, c) potential cross-reactivity with related peptides and proteins, including plasminogen and also TREPA of related species, and d) ability to detect TREPA peptides in extracts from plasma, urine, tissues and cell culture media. The titer can be established through various means known in the art, such as by "dot blot" and density analysis, and also in precipitation of radiolabeled antibody-peptide complexes using protein A, secondary antisera, cold ethanol or carbon-dextran. followed by measurement of activity with a gamma counter. If desired, antisera of higher titre can be purified on affinity columns. For example, TREPA peptides can be coupled to a commercially available resin and used to form an affinity column. The antiserum samples can then be passed through the column, so that the TREPA antibodies bind (via TREPA) to the column. These bound antibodies are subsequently levigated, collected and evaluated for titer determination and specificity. The present invention also provides a method for classifying a plurality of compounds for specific ligation to a TREPA-derived polypeptide, or any fragment thereof, to identify at least one compound which specifically binds to the TREPA-derived polypeptide. Such a method comprises the steps of providing at least one compound; combining the TREPA-derived polypeptide with each compound under suitable conditions for a sufficient time to allow ligation; and detecting TREPA polypeptide binding to each compound. Antisense technology can be used for the expression of control genes through the formation of the triple helix or RNA or antisense DNA, both of which methods are based on the ligation of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which encodes the polypeptide of the present invention, is used to design an antisense RNA oligonucleotide from 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription, thereby preventing transcription and production of the TREPA-derived polypeptide. For the triple helix, see, for example, Lee et. to the. , Nucí. Acids Res. 6: 3073 (1979); Cooney et. to the. , Science 241: 456 (1988); and Dervan et. to the. , Science 251: 360 (1 991). The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation of a mRNA molecule to the polypeptide derived from TREPA. For antisense, see, for example, Okano, J. Neurochem. 56: 560 (1991); and "Oligodeoxynucleotides as Antisense I nhibitors of Gene Expression" (Oligodeoxynucleotides as antisense inhibitors of gene expression), CRC Press, Boca Raton, FA. (1988). Antisense oligonucleotides act most effectively when modified to contain artificial internucleotide linkages, which render the molecule resistant to nucleolytic cleavage. Such artificial internucleotide linkages include, but are not limited to, internucleotide linkages of phosphoroamidate, phosphorothiolate and methylphosphonate.

The polypeptide or peptide fragment employed in such a test can be either free in solution, fixed to a solid support, carried on a cell surface or located intracellularly. A method of classifying medicament utilizes eukaryotic or prokaryotic host cells, which are stably transformed with recombinant nucleic acids, which can express the polypeptide or peptide fragment. The drugs can be classified against such transformed cells in competitive ligation assays. For example, the formation of complexes between a polypeptide and the agent being tested can be measured in either viable or fixed cells. Thus, the present invention provides methods for classifying drugs or any other agent, which can be used to treat diseases associated with the TREPA gene by measuring the effect of the drug on the amount of TREPA protein or nucleotide produced or biological effects of TREPA. TRIP Examples of these types of measurements include, but are not limited to, measuring Ca ++ effluvium, cAMP production, aptipsis, etc. These measurements are known to those of ordinary skill in the art. The present invention is also provided to measure the effect of the drug in a recombinant reporter gene designed to respond to TREPA. These methods comprise measuring the effect of applying the drug to a genetically engineered or natural experimental organism, such as cultured cells, bacteria, or laboratory animals and measuring the amount of TREPA protein or nucleotide produced, or biological effects of TNF gamma, or Recombinant reporter gene amount, such as, luciferase. The present invention thus provides methods for classifying drugs or any other agent, which can be used to treat diseases associated with the TREPA gene. These methods comprise contacting the medicament with a polypeptide or fragment thereof, and assaying either for the presence of a complex between the agent and the polypeptide, or for the presence of a complex between the polypeptide and the cell. In competitive ligation assays, the polypeptide is typically labeled. After a suitable incubation, the free (or non-complexed) polypeptide or fragment thereof is separated from that present in bound form, and the amount of free or non-complexed tag is a measure of the ability of the particular drug to bind to polypeptide or to interfere with the polypeptide / cell complex. The present invention also encompasses the use of competitive drug classification assays, in which neutralizing antibodies capable of binding polypeptide specifically compete with a test drug to bind to the polypeptide or fragment thereof. In this form, antibodies can be used to detect the presence of any polypeptide in the test sample, which shares one or more antigenic determinants with a polypeptide provided herein. Another technique for classifying drugs provides high performance classification for compounds having adequate binding affinity to at least one polypeptide described herein. Briefly, large numbers of different small peptide test compounds are synthesized in a solid phase, such as plastic fasteners or some other surface. The peptide test compounds are reacted with polypeptide and washed. The polypeptide thus bound to the solid phase is detected by methods well known in the art. The purified polypeptide can also be coated directly on plates for use in the drug classification techniques described herein. In addition, non-neutralizing antibodies can be used to capture the polypeptide and immobilize it on the solid support. See, for example, EP 84/03564, published on September 1, 1 984, which is incorporated herein by reference. The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules including agonists, antagonists, or inhibitors with which they interact. Such structural analogs can be used to form drugs, which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo. J. Hodgson, Bio / Technology 9: 19-21 (1991), incorporated herein by reference. For example, in one approach, the three-dimensional structure of a polypeptide, or of a polypeptide-inhibitor complex, is determined by X-ray crystallogging, by computer modeling or, most commonly, by a combination of the two approaches. Both the form and the charges of the polypeptide can be ascertained to elucidate the structure and determine the active site (s) of the molecule. Less frequently, useful information regarding the structure of a polypeptide can be gained by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design molecules similar to analogous polypeptides or to identify efficient inhibitors. Useful examples of rational drug design may include molecules, which have improved activity or stability, as shown by S. Braxton et. to the. , Biochemistry 31: 7796-7801 (1992), or which act as inhibitors, agonists or antagonists of natural peptides as shown by S. B. P. Athauda et. to the. , J. Biochem. (Tokyo) 1 13 (6): 742-746 (1993), incorporated herein by reference. It is also possible to isolate a specific target antibody, selected by an assay as described hereinabove, and then determine its crystal structure. In principle, this approach produces a pharmacophore on which the subsequent drug design can be based. In addition it is possible to deviate the protein crystallography completely by generating anti-idiotypic antibodies ("anti-ids") to a pharmacologically active, functional antibody. Like a mirror image of a mirror image, the anti-id binding site is an analogue of the original receiver. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides can then act as the pharmacophore (i.e., a prototype pharmaceutical medicament). A sufficient amount of a recombinant polypeptide of the present invention can be made available for performing analytical studies, such as X-ray crystallography. In addition, knowledge of the polypeptide amino acid sequences, which are derivable from the nucleic acid sequence provided herein, they will provide guidance for those who employ computer modeling techniques in place of, or in addition to, X-ray crystallography. Antibodies specific to the TREPA-derived polypeptide may also be used to inhibit the biological action of the polypeptide at join the polypeptide. In this manner, antibodies can be used in therapy, for example, to treat TREPA diseases including inflammation. In addition, such antibodies can detect the presence or absence of TREPA-derived polypeptide and, therefore, are useful as diagnostic markers for the diagnosis of TREPA disease, especially inflammation. Such antibodies can also function as diagnostic markers for TREPA disease conditions, such as inflammation. The present invention is also directed to antagonists and inhibitors of the polypeptides of the present invention. Antagonists and inhibitors are those that inhibit or eliminate the function of the polypeptide. In this way, for example, an antagonist can bind to a polypeptide of the present invention and inhibit or eliminate its function. The antagonist, for example, could be an antibody against the polypeptide which eliminates the activity of the TREPA-derived polypeptide by binding to the TREPA-derived polypeptide, or in some cases, the antagonist can be an oligonucleotide. Examples of small molecule inhibitors include, but are not limited to, small peptides or peptide-like molecules. Antagonists and inhibitors can be employed as a composition with a pharmaceutically acceptable carrier, including but not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The administration of TREPA-derived polypeptide inhibitors are preferably systemic. The present invention also provides an antibody, which inhibits the action of such polypeptides.

Recombinant Technology The present invention provides host cells and expression vectors comprising polynucleotides of the present invention and methods for the production of polypeptides that they encode. Such methods comprise coating the host cells under conditions suitable for expression of the TREPA-derived polynucleotide and recovering the TREPA-derived polypeptide from the cell culture. The present invention also provides vectors, which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the present invention and production of polypeptides of the present invention by recombinant techniques. The host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention, which can be cloning vectors or expression vectors. The vector can be in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in modified conventional nutrient media as appropriate to activate promoters, select transformants or amplify genes derived from TREPA. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the practitioner of ordinary skill. The polynucleotide of the present invention can be used to produce a polypeptide by recombinant techniques. Thus, the polynucleotide sequence can be included in any of a variety of expression vehicles, in particular, vectors or plasmids, to express a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV40 derivatives; bacterial plasmids; Phage DNA; yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, domestic pustular rash virus, and pseudo-rabies. However, any other plasmid or vector can be used, as long as it is replicable and viable in the host.

The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into appropriate restriction endonuclease sites by methods known in the art. Such procedures and others are considered to be within the reach of those skilled in the art. The DNA sequence in the expression vector is operably linked to one or more appropriate expression control sequences (promoter) to direct mRNA synthesis. Representative examples of such promoters include, but are not limited to, SV40 or LTR promoter, trp or lac promoters from E. coli. the phage lambda promoter P sub L and other known promoters for gene control expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome ligation site for translation initiation and a transcription terminator. The vector may also include appropriate sequences to amplify the expression. In addition, the expression vectors preferably contain a gene to provide a phenotypic treatment for the selection of transformed host cells, such as, resistance to neomycin or dihydrofolate reductase for culture of eukaryotic cells, or such as, resistance to tetracycline or ampicillin in E coli. The vector containing the appropriate DNA sequence as described hereinabove, as well as an appropriate promoter or control sequence, can be employed to transform an appropriate host to allow the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Salmonella typhimurium and Streptomyces sp.; fungal cells, such as, yeast; insect cells, such as Drosophila and Sf9; animal cells, such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is considered to be within the reach of those skilled in the art from the teachings provided herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, in which a sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences including, for example, a promoter operably linked to the sequence. Those skilled in the art know large amounts of suitable vectors and promoters and are commercially available. The following vectors are provided by way of example. Bacteria: pSPORTI (Life Technologies, Gaithersburg, MD), pQE70, pQE60, pQE-9 (Quiagen) pBs, phaescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH 16a, pNH 18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

The promoter regions can be selected from any desired gene using CAT vectors (chloramphenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacl, lacZ, T3, SP6, T7, gpt, lambda P sub R, P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV), immediately initial herpes simplex virus (HSV), thymidine kinase, early and late SV40, retrovirus LTRs, and mouse metallothionein-1. The selection of the appropriate vector and promoter is within the level of ordinary skill in the art. In a further embodiment, the present invention provides host cells containing the above described construct. The host cell can be a larger eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the construct into the host cell can be effected by transfection of calcium phosphate, transfection mediated by DEAE-Dextran, or electroporation (L. Davis et al., "Basic Methods in Molecular Biology" (Basic Methods in Molecular Biology), 2nd edition, Appleton and Lang, Paramount Publishing, East Norwaik, CT (1994).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be produced synthetically by conventional peptide synthesizers.

The proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et. to the. , Molecular Cloning: A Laboratorv Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), which is incorporated herein by reference. The transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Intensifiers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the last side of the replication origin (pb 1 00 to 270), an early promoter enhancer of cytomegalovirus, a polyoma enhancer on the latter side of the replication origin, and adenovirus enhancers. In general, the recombinant expression vectors will include origins of replication and selectable markers allowing the transformation of the host cell, for example, the ampicillin resistance gene of the TRP1 gene of E. coli and S. cerevisiae, and a promoter derived from a highly expressed gene to direct the transcription of a downstream structure sequence. Such promoters may be derivatives of operons encoding licolytic enzymes, such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translational initiation and termination sequences, and preferably, a leader sequence capable of directing the secretion of translated protein in the periplasmic space or extracellular environment. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, for example, stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure the maintenance of the vector and, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis, Salmonella typhitnurium and several species within the genera Pseudomonas. Streptomyces. and Staphylococcus, although others may also be used as a routine matter of choice. Useful expression vectors for bacterial use comprise a selectable marker and bacterial origin of replication derived from plasmids, comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Other vectors include, but are not limited to. PKK223-3 (Pharmacia fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wl). These "skeleton" sections of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed by appropriate means (e.g., temperature change or chemical induction), and the cells are cultured for a period of time. additional. The cells are normally harvested by centrifugation, broken by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used in the expression of proteins can be broken by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell-using agents; Such methods are well known to the ordinary practitioner. Various mammalian cell culture systems may also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, such as the lines of C 1 27, 3T3, CHO, HeLa and BHK cells. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding site, polyadenylation site, donor and splice receptor sites, transcription termination sequences, and non-interfering sequences. transcribed flanking 5 '. DNA sequences derived from the SV40 viral genome, eg, SV40 origin, early promoter, enhancer, splice and polyadenylation sites can be used to provide the required non-transcribed genetic elements. Useful, representative vectors include pRc / CMV and pcDNA3 (available from Invitrogen, San Diego, CA). The TREPA polypeptide is recovered and purified from recombinant cell cultures by known methods, including precipitation with ethanol or ammonium sulfate, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography or lectin chromatography. It is preferred to have low concentrations (approximately 0.1-5 mM) of calcium ion present during purification (Price, et al., J. Biol. Chem. 244: 917 (1969)). Protein re-folding steps may be used, as necessary, to complete the configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be used for final purification steps. The polypeptides of the present invention may be naturally purified products expressed from a high expression cell line, or a product of synthetic chemical procedures, or may be produced by recombinant techniques from a prokaryotic or eukaryotic host (e.g. by bacteria cells, yeasts, higher plants, insects and mammals in culture). Depending on the host employed in a recombinant production process, the polypeptides of the present invention can be glycosylated with mammalian carbohydrates or other eukaryotic carbohydrates or can be non-glycosylated. The polypeptides of the invention may also include an initial methionine amino acid residue. The present invention further includes modified versions of the TREPA polypeptide to prevent glycosylation while allowing the expression of a reduced carbohydrate form of the protein in yeast, insect or mammalian expression systems. Known methods for inactivating glycosylation sites include, but are not limited to, those presented in US Pat. No. 5,071,972 and EP 276,846, which are incorporated herein by reference. Other variants included in the present invention include the removal of sequences encoding cysteine residues, thereby preventing the formation of incorrect intramolecular disulfide bridges, which decrease the biological activity of the protein product. The present invention also includes the removal of proteolytic processing sites, allowing expression in a system, which contains the problematic protease, for example, the KEX2 protease in yeast. Known methods for removing such protease sites include, but are not limited to, a method for removing KEX2 sites presented in EP21 2,914. The present invention includes TREPA peptides in the form of higher order oligomers, dimers, trimers and oligomers. Oligomers can be formed by various means including, but not limited to, disulfide bonds between peptides, non-covalent interactions between peptides, and polyethylene glycol linkages between peptides. Also encompassed by this invention is the fusion of TREPA peptides to peptide linkers or peptides that are capable of promoting oligomers. Such peptides include, but are not limited to, leucine closures and antibodies derived from peptides, such as described in Landschulz et. to the. , Science 240: 1 759 (1988); Hollenbaugh and Aruffo, "Construction of Immunoglobin Fusion Proteins", in Current Protocols in Immunology, Supplement 4, pages 1 0.19.1 -10.19.1 1 (1992) John Wiley and Sons, New York, NY. The start plasmids can be constructed from available plasmids according to known, published procedures. In addition, equivalent plasmids for those described are known in the art and will be apparent to the skilled practitioner. The following is the general procedure for the isolation and analysis of cDNA clones. In a particular embodiment described herein, mRNA was isolated from TREPA and used to generate the cDNA library. Synovio TREPA was obtained from patients with rheumatoid arthritis by surgical removal. A cDNA insert of a TREPA isolate was sequenced in its entirety, analyzed in detail as set forth in the Examples and described in the Sequence Listing as S ECUENCE I D NO 1. These polynucleotides encode a sufficient portion of the gene of interest to encode a biologically active molecule. The lack of full-length clones is attributed to the fact that many genes are several hundred, and sometimes several thousand, of bases in length and, with current technology, can not be cloned in their entirety due to vector limitations, transcription Incomplete reverse of the first filament, or incomplete replication of the second filament. Secondary clones, contiguous, containing additional nucleotide sequence can be obtained using a variety of methods known to those skilled in the art. Methods for sequencing DNA are well known in the art. Conventional enzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (US Biochemical Corp, Cleveland, OH) or Taq polymerase to extend DNA strands of a quenched oligonucleotide primer to the DNA template of interest. Methods have been developed for the use of both single filament and double filament templates. The chain termination reaction products can be subjected to electrophoresis in urea / polyacrylamide gels and detected either by autoradiography (by labeled precursors of radionucleotides) or by fluorescence (by fluorescently labeled precursors). Recent improvements in the preparation of mechanized reaction, 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 Applied Biosystems 377 DNA Sequencers (Applied biosystems, Foster City, CA). The reading frame of the nucleotide sequence can be investigated by several types of analysis. First, the reading frames contained within the coding sequence can be analyzed by the presence of ATG start codon and stop codons TGA, TAA or TAG. Normally, one reading frame will continue through the largest portion of a cDNA sequence while the other two reading frames tend to contain numerous stop codons. In such cases, the determination of reading frame is direct. In other more difficult cases, additional analysis is required. The last confirmation of a correct open reading frame is achieved by using the nucleotide sequence to produce a biologically active molecule, by such methods that are familiar to those skilled in the art.

Algorithms have been created to analyze the occurrence of individual nucleotide bases in each of three putative codons. See, for example, J.W. Fickett, Nuc Acids Res 1 0: 5303 (1 982). Coding DNA for particular organisms (bacteria, plants and animals) tends to contain certain nucleotides within certain periodicities of triples, such as a significant preference for pyrimidines at the third codon position. These preferences have been incorporated into widely available computer programs, which can be used to determine the coding potential (and frame) of a given stretch of DNA. The information derived from the algorithm combined with the start / stop codon information can be used to determine the appropriate frame with a high degree of certainty. This, in turn, easily allows cloning the sequence in the correct reading frame into appropriate expression vectors.

The nucleic acid sequences described herein can be joined in a variety of other polynucleotide sequences and vectors of interest by well-established recombinant DNA techniques. See J. Sambrook et al. , supra. Vectors of interest include cloning vectors, such as plasmids, cosmids, phage derivatives, fagomids, as well as sequencing, replication and expression vectors and the like. In general, such vectors contain a functional origin of replication in at least one organism, convenient restriction endonuclease digestion sites, and selectable markers appropriate for particular host cells. The vectors can be transferred by a variety of means known to those of skill in the art in suitable host cells, which then produce the desired DNA, RNA or polypeptides. Occasionally, random reverse transcription or sequencing errors will mask the presence of the open reading frame or appropriate regulatory element. In such cases, it is possible to determine the correct reading frame when attempting to express the polypeptide and determine the amino acid sequence by standard peptide sequencing and tracing techniques. See, F. M. Ausubel, et al. , Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989). Additionally, the current reading frame of a given nucleotide sequence can be determined by transfection of host cells with vectors containing all three potential reading frames. Only those cells with the nucleotide sequence in the correct reading frame will produce a peptide of the predicted length.

The nucleotide sequences provided herein have been prepared by current, automated methods of the art, and as such, may contain unidentified nucleotides. These will not present a problem for those skilled in the art, who wish to practice the invention. Various methods employing standard recombinant techniques, described in J, may be used. Sambrook (supra) or periodic updates of them, can be used to complete missing sequence information. The same techniques used to obtain a full-length sequence, as described herein, can be used to obtain the nucleotide sequence. The expression of a particular cDNA can be achieved by subcloning the cDNA into an appropriate expression vector and transfecting this vector into an appropriate expression host. The cloning vector used for the generation of the TREPA cDNA library can be used to transcribe mRNA from a particular cDNA. Immediately following these eight residues is a bacteriophage promoter designed for artificial initiation and transcription and a number of unique restriction sites, including EcoR I, for cloning. The vector can be transfected into an appropriate E. coli host strain. Induction of the isolated bacterial strain with isopropylthiogalactoside (I PTG) using standard methods will produce a fusion protein, which contains the first seven residues of beta-galactosidase, about 15 linker residues, and the peptide encoded within the cDNA. Since the insertions of cDNA clones are generated by an essentially random process, there is a three chance that the included cDNA will fall into the correct frame for proper translation. If the cDNA is not in the proper reading frame, the correct frame can be obtained by deletion or insertion of an appropriate number of bases by well-known methods, including in vitro mutagenesis, digestion with exonuclease I II or 'mung bean' nuclease, or inclusion of oligonucleotide linker. The cDNA can be launched into other vectors known to be useful for protein expression in specific hosts. Oligonucleotide primers containing cloning sites and DNA segments sufficient to hybridize to stretches at both ends of the target cDNA can be chemically synthesized by standard methods. These primers can then be used to amplify the desired gene segments by PCR. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternatively, segments of similar genes can be produced by digestion of the cDNA with appropriate restriction enzymes and filled into missing gene segments with chemically synthesized oligonucleotides. Segments of the coding sequence of more than one gene can be joined and cloned into appropriate vectors to optimize recombinant sequence expression. Suitable expression of hosts for such 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 cellular systems, a useful expression vector may also include an origin of replication to allow propagation in bacteria and a selectable marker, such as, the beta-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 the addition of 3 'poly A tail if the sequence of interest lacks poly A, and / or the addition of an introns sequence, which promotes splicing and mRNA processing, but it does not alter the amino acid sequence of the gene product. In addition, this invention encompasses expression vectors by which the secretion of the host cell outer protein is achieved by fusing into frame DNA encoding signal peptide sequences to the open reading frame encoding TREPA. These sequences may be of prokaryotic, eukaryotic or viral origin. Additionally, the vector may contain promoters or enhancers which increase the expression of genes. Such promoters are host specific and include, but are not limited to, MMTV, SV40 or metallothionin promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase or promoters of PG H for yeast. Adenoviral vectors with or without transcription enhancers, such as rous sarcoma virus (RSV), can be used to drive protein expression in mammalian cell lines. Once homogenous cultures of recombinant cells are obtained, large quantities of recombinantly produced protein can be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant milk protein produced by cows, goats, transgenic sheep, etc. The poiypeptides and closely related molecules can be expressed recombinantly in such a way as to facilitate the purification of proteins. An approach involves the expression of a chimeric protein, which includes one or more domes of additional polypeptides present unnaturally in human polypeptides. Such domains that facilitate purification include, but are not limited to, metal chelating peptides, such as, histidine-tryptophan domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the FLAGS extension / affinity purification system (I mmunex Corp, Seattle, WA). The inclusion of a collatable linker sequence, such as, Factor XA or enterokinase from I nvitrogen (San Diego, CA) between the polypeptide sequence and the purification domain may be useful for recovering the polypeptide.

Immunoassays Polypeptides, including their fragments or derivatives or analogs thereof of the present invention, or cells expressing them, can be used in a variety of assays, many of which are described herein, for the detection of TREPA antibodies. They can also be used as an immunogen to produce antibodies. These antibodies can be, for example, polyclonal or monoclonal antibodies, humanized and single chain, chimeric antibodies, as well as Fab fragments, or the product of a Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments. For example, antibodies raised against a polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an aminal or by administering the polypeptide to an animal, such as a mouse, rabbit, goat or human. A mouse, rabbit or goat is preferred. The antibody thus obtained will then bind to the polypeptide by itself. In this way, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies that bind to the natural polypeptide. Such antibodies can then be used to isolate the polypeptide from the test samples, such as, tissue that is suspected to contain the polypeptide. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples that include the hybridoma technique as described by Kohler and Milstein, Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique as described by Kozbor et. al , Immun. Today 4:72 (1983), and the EBV hybridoma technique for producing human monoclonal antibodies as described by Cole, et. to the. , in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, I nc, New York, NY, pp. 77-96 (1985). The techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. See, for example, US Patent no. 4, 946,778, which is incorporated herein by reference. Various assay formats can utilize the antibodies of the present invention, including sandwich immunoassays and probe assays. For example, the monoclonal antibodies or fragments thereof of the present invention can be employed in various assay systems to determine the presence, if any, of TREPA-derived polypeptide in a test sample. For example, in a first assay format, a polyclonal or monoclonal antibody or fragment thereof, or a combination of these antibodies, which has been coated on a solid phase, is contacted with a test sample, to form a first mix This first mixture is incubated for a time and under conditions sufficient to form antigen / antibody complexes. Then, an indicator reagent comprising a monoclonal antibody or a polyclonal or a fragment thereof, or a combination of these antibodies, to which a signal generating compound has been bound, is contacted with the antigen / antibody complexes to form a second mix. This second mixture was then incubated for a time and under conditions sufficient to form antibody / antigen / antibody complexes. The presence of a TREPA-derived polypeptide antigen present in the test sample and captured in the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound. The amount of TREPA derived from the polypeptide antigen present in the test sample is proportional to the signal generated. Or, a polypeptide antibody derived from TREPA, polyclonal or monoclonal or fragment thereof, or a combination of these antibodies which is bound to a solid support, the test sample and an indicator reagent comprising a monoclonal or polyclonal antibody or fragments thereof, which specifically binds to TREPA-derived polypeptide antigen, or a combination of these antibodies to which a signal generating compound binds, are contacted to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antibody / antigen / antibody complexes. The presence, if any, of TREPA-derived polypeptide present in the test sample and captured in the solid phase, is determined by detecting the measurable signal generated by the signal generating compound. The amount of TREPA-derived polypeptide proteins present in the test sample is proportional to the signal generated. In another assay format, an antibody or a combination of at least two monoclonal antibodies of the invention can be used as a competitive probe for the detection of antibodies to TREPA-derived polypeptide protein. For example, TREPA-derived polypeptide proteins, such as the recombinant antigens described herein, either alone or in combination, are coated on a solid phase. A test sample suspected to contain antibody to the TREPA-derived polypeptide antigen is then incubated with an indicator reagent comprising a signal generating compound and at least one monoclonal antibody of the invention for a time and under conditions sufficient to form Antigen / antibody complexes from either the test sample and indicator reagent bound to the solid phase or the indicator reagent bound to the solid phase. The reduction in ligation of the monoclonal antibody to the solid phase can be measured quantitatively. In yet another detection method, each of the monoclonal or polyclonal antibodies of the present invention can be used in the detection of TREPA-derived polypeptide antigens in sections of fixed tissues, as well as fixed cells by immunohistochemical analysis. Also within the scope of the present invention are cytochemical assays where these antibodies are directly labeled (with, for example, fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.), or are labeled by using anti-body antibodies. tagged secondary species (with several tags as exemplified herein) to track the histopathology of the disease. In addition, these monoclonal antibodies can bind to matrices similar to CN Br-activated Sepharose and be used for the affinity purification of TREPA-derived polypeptide proteins specific to cell cultures or biological tissues, in order to purify proteins and polypeptide antigens from NATURAL TREPA. The monoclonal antibodies of the invention can also be used for the generation of chimeric antibodies for therapeutic use or other similar applications. The antibodies or monoclonal fragments thereof can be provided individually to detect TREPA-derived polypeptide antigens. Combinations of the monoclonal antibodies (and fragments thereof) provided herein may also be used together as components in a mixture or "cocktail" of at least one TREPA-derived polypeptide antibody of the invention with antibodies to other regions of the invention. polypeptides derived from TREPA, each having different binding specificities. Thus, this cocktail may include the monoclonal antibodies of the invention, which are directed to TREPA-derived polypeptide proteins of TREPAs and other monoclonal antibodies to other antigenic determinants of TREPA-derived polypeptide genome. The polyclonal antibody or fragment thereof, which can be used in the assay formats, must specifically bind to a polypeptide region derived from TREPA or other polypeptide proteins derived from TREPA used in the assay. The polyclonal antibody used is, preferably of mammalian origin; polyclonal anti-TREPA derived peptide antibodies can be used from human, goat, rabbit or sheep. Most preferably, the polyclonal antibody is polyclonal antibody derived from rabbit polyclonal anti-TREPA. The polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different TREPA-derived polypeptide specificity, they would be useful for diagnosis, evaluation and prognosis of TREPA-derived polypeptide conditions, as well as for studying specificity and differentiation of polypeptide protein derived from TREPA. It is contemplated and within the scope of the present invention that the TREPA-derived polypeptide may be detectable in assays by the use of a recombinant antigen, as well as by the use of a synthetic peptide or purified peptide, which contains an amino acid sequence of TREPA-derived polypeptide. It is also within the scope of the present invention that different synthetic, recombinant or purified peptides that identify different epitopes of the TREPA-derived polypeptide can be used in combination in an assay to diagnose, evaluate or predict the TREPA disease condition. In this case, these peptides can be coated on a solid phase, or each peptide separately can be coated on separate solid phases, such as microparticles, and then combined to form a mixture of peptides, which can then be used in assays. . Additionally, it is contemplated that multiple peptides that define epitopes of different polypeptides can be used with combination to make a diagnosis, evaluation or prognosis of TREPA disease. It is then allowed that peptides coated in solid phases or labeled with detectable labels compete with peptides from a patient sample for a limited amount of antibody. A ligation reduction of synthetic, recombinant or purified peptides for the antibody (or antibodies) is an indication of the presence of polypeptides secreted by TREPA in a patient sample, which in turn indicates the presence of TREPA gene in the patient . Such variations of assay formats are known to those of ordinary skill in the art and are discussed below in the present. In another assay format, the presence of antibody and / or antigen for TREPA-derived polypeptide can be detected in a simultaneous assay, as follows. A test sample is contacted simultaneously with a capture reagent of a first analyte, wherein said capture reagent comprises a first binding member specific for a first analyte bound to a solid phase and a capture reagent for a second analyte. , wherein said capture reagent comprises a first binding member for a second analyte attached to a second solid phase, thereby forming a mixture. This mixture is incubated for a time and under conditions sufficient to form capture reagent / first analyte and capture reagent / second analyte complexes. These complexes thus formed are then contacted with an indicator reagent comprising a member of a ligation pair specific for the first analyte labeled with a signal generating compound and an indicator reagent comprising a specific ligation pair member for the labeled second analyte. with a signal generating compound to form a second mixture. This second mixture is incubated for a time and under conditions sufficient to form capture reagent / first analyte / indicator reagent and capture reagent / second analyte / indicator reagent complexes. The presence of one or more analytes is determined by detecting a signal generated in connection with the complexes formed in either or both solid phases, as an indication of the presence of one or more analytes in the test sample. In this assay format, recombinant antigens derived from human expression systems can be used, as well as monoclonal antibodies produced from proteins derived from mammalian expression systems, as described herein. Such assay systems are described in greater detail in EP No. 0473065. Still in other assay formats, the polypeptides described herein can be used to detect the presence of anti-TREPA-derived polypeptide in the test samples. For example, a test sample is incubated with a solid phase to which at least one recombinant protein has been bound. These are reacted for a time and under conditions sufficient to form antigen / antibody complexes. Following the incubation, the antigen / antibody complex is detected. The indicator reagents can be used to facilitate detection, depending on the chosen test system. In another assay format, a test sample is contacted with a solid phase to which a recombinant protein produced as described herein is bound and also contacted with a monoclonal or polyclonal antibody specific for the protein. , which has been marked preferably with an indicator reagent. After incubation, for a time and under conditions sufficient to form antibody / antigen complexes, the solid phase is separated from the free phase, and the label is detected either in the solid or free phase as an indication of the presence of the polypeptide antibody derived from TREPA. Other assay formats are contemplated using the recombinant antigens described herein. These include contacting a test sample with a solid phase to which at least one antigen has been bound from a first source, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen complexes. / antibody, and then contacting the solid phase with a labeled antigen, said antigen is derived from a second source different from the first source. For example, a recombinant protein derived from a first source, such as, E. coli is used as a capture antigen on a solid phase, a test sample is added to the solid phase thus prepared, and a recombinant protein derived from a Different source (ie, not E. coli) is used as a part of an indicator reagent. Likewise, combinations of a recombinant antigen in a solid phase and synthetic peptide in the indicator phase are also possible. A test format, which uses a TREPA-derived polypeptide-specific antigen from a first source such as the capture antigen and a TREPA-derived polypeptide-specific antigen from a different second source are contemplated. Thus, various combinations of recombinant antigens, as well as the use of synthetic peptides, purified proteins, and the like, are within the scope of this invention. Assays such as this and others are described in U.S. Pat. 5,254,458, which enjoys common property and is incorporated herein by reference. Other embodiments are also contemplated, which utilize various other solid phases, and are within the scope of the invention. For example, ion capture methods can be employed to immobilize an immobilizable reaction complex with a negatively charged polymer (described in EP 0326100 and EP Publication No. 0406473) according to the present invention to effect an immunochemical reaction of fast solution phase. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the immune complex / poly-anions and the positively charged porous matrix., previously treated, and is detected by using several previously described signal generating systems, including those described in chemiluminescent signal measurements as described in the EPO publication no. 0 273, 1 1 5. In addition, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi-automated systems, wherein the solid phase comprises a microparticle (magnetic or non-magnetic) ). Such systems include those described in published EPO applications nos. EP 0 425 633 and EP 0 424 634, respectively.

The use of scanning probe microscopy (SPM) for immunoassays is also a technology to which the monoclonal antibodies of the present invention are readily adaptable. In scanning probe microscopy, in particular, in atomic force microscopy, the capture phase, for example, at least one of the monoclonal antibodies of the invention, adheres to a solid phase and a tracking probe microscope is used. to detect antigen / antibody complexes, which may be present on the surface of the solid phase. The use of scanning tunneling microscopy eliminates the need for tags, which should normally be used in many immunoassay systems to detect antigen / antibody complexes. The use of SPM to monitor specific ligation reactions can occur in many forms. In one embodiment, a member of a specific binding partner (analyte-specific substance which is the monoclonal antibody of the invention) is attached to a suitable surface for tracking. The binding of the analyte-specific substance can be by adsorption to a test piece, which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art. Or, covalent attachment of a specific binding partner (analyte-specific substance) can be used to a test piece, said test piece comprising a solid phase of glass, silicon, metal or plastic derivative. Covalent attachment methods are known to those skilled in the art and include a variety of means for irreversibly binding specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be activated before joining the specific binding partner. In addition, poielectrolyte interactions can be used to immobilize a specific ligation partner on a surface of a test piece by using techniques and chemistries. The preferred method of binding is by covalent means. Following the binding of a specific ligating member, the surface can be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific ligation. The surface can also be traced to verify its adaptability for testing purposes. The tracking process does not anticipate altering the specific binding properties of the test piece. Although the present invention discloses the preference for the use of solid phases, it is contemplated that reagents, such as antibodies, proteins and peptides of the present invention can be used in non-solid phase assay systems. These test systems are known to those skilled in the art, and are considered to be within the scope of the present invention. It is contemplated that the reagent employed for the assay may be provided in the form of a test set with one or more containers such as bottles or flasks, each container containing a separate reagent, such as a probe, initiator, monoclonal antibody. or a cocktail of monoclonal antibodies, or a polypeptide (either recombinant or synthetic) used in the assay. Other components, such as dampers, controls and the like, known to those of ordinary skill in the art, can be included in such test sets. It is also contemplated to provide test assemblies, which have means for collecting test samples comprising accessible body fluids, eg, blood, urine, saliva and evacuations. Such useful tools for collection "collection materials" include lancets and absorbent paper or cloth to collect and stabilize blood; swabs to collect and stabilize saliva; cups to collect and stabilize urine samples or stools. The collection materials, papers, fabrics, swabs, cups and the like, can be treated optionally to avoid denaturation or irreversible adsorption of the sample. The collection materials can also be treated with, or contain, preservatives, stabilizers or antimicrobial agents, to help maintain the integrity of the specimens. Test sets designed for the collection, stabilization and preservation of test specimens obtained by surgery or needle biopsy are also useful. It is contemplated that all assemblies can be configured in two components, which can be provided separately; a component for collection and transport of the specimen, and the other component for the analysis of the specimen. For example, the collection component can be provided to the open market user although the components for the analysis can be provided to others, such as laboratory personnel for determination of the presence, absence or amount of analyte. In addition, sets for the collection, stabilization and preservation of test specimens can be configured for use by untrained personnel and may be available on the open market for use at home with subsequent transportation to a laboratory for analysis of the test sample. The bacterium E. coli (clone 1 235095) has been deposited at American Type Culture Coliection (ATCC), 12301 Parkiawn Drive, Rockville, Maryland 20852, dated September 26, 1996, under the terms of the Budapest Treaty and will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last application for the deposit, or for the required period of the US patent, whichever is longer. The deposit and any other deposited material described herein are provided for convenience only, and are not required to practice the present invention in view of the teachings provided herein. The cDNA sequence in all deposited material is incorporated herein by reference. Clone 1235095 was agreed as A.T.C. C. Deposit No. 981 84. The present invention will now be described in the form of examples, which are intended to illustrate, but not limit, the scope of the present invention.EXAMPLES Example 1: Identification of EST clones of TREPA library A. Comparison of library of expressed sequence tags (ESTs) or transcript images. The partial sequences of insertions of cDNA clones, so-called tags of expressed sequences (ESTs), were derived from cDNA libraries made of numerous tissues, both diseased and normal, and entered into a database (LIFESEQMR database, available from Incyte Pharmaceutícals, Palo Alto, CA) as images of gene transcripts. The sequences were then evaluated to identify EST sequences, whose predicted amino acid sequences were representative of the TNF family of genes. Once such EST was identified, the known sequence of the gene represented by the EST was expanded by searching all databases of available human DNA sequences, both publicly and privately, available for overlapping sequences with significant homology . This expanded sequence will be described here as "contig". Having done this, the most widespread 5 'end clone available at the time was obtained from the EST provider (Incyte Pharmaceuticals). None of these EST sequences that overlap either in publicly available or Incyte databases were scored by the sequence producers as being members of the TNF family of ligands. In addition, none of these sequences is sufficient to produce a soluble, active form of the protein.

Example 2: Sequencing of clones containing EST The DNA sequences for clones comprising the majority of ESTs upstream of the contig are determined using dideoxy termination sequencing with either initiators labeled with dyes, dye terminators, or radiolabelled n-nucleotides, following known methods. See, for example, F. Sanger et. al , Proc. Nati Acad. Sci. U.S.A. 74: 5463 (1977). Because the vector pSPORTI (Life Technologies, Gaithersburg, MD) and pI NCY, based on pSPORT-1, contain universal initiation sites just adjacent to the 3 'and 5' ligation junctions of the inserts, the inserts are sequenced in both directions using universal initiators. The sequencing reactions are run on a denaturing polyacrylamide gel, and the sequences are determined by an Applied Biosystems 377 Sequencer sequencer (available from Applied Biosystems, Foster City, CA) or other sequencing apparatus. Based on other members of the TNF family of ligands, the sequence of clone ID # 690050 (SEQUENCE ID NO 1) contains the extracellular homology domain of whole TNF, and therefore, it is sufficient to form a biologically active molecule, a example of which is SECU ENCIA ID NO 3.

Example 3: Preparation of nucleic acids A. Extraction of RNA from tissue. Total RNA is isolated from solid tissues or cells from patients with prostate cancer and non-tumor tissues using a lithium chloride / urea technique known in the art and described by N. Kato et. to the. , J. Viroiogy 61: 21 82-21 91 (1988). Non-tumor tissues are used as negative controls. The mRNA can be further purified from total RNA using commercially available pools, such as oligo dT cellulose spin columns (RediColMR from Pharmacia, Uppsala, Sweden) for the isolation of poly-adenylated RNA. The total RNA or mRNA is then dissolved in lysis buffer (5M guanidine thiocyanate, 0.1M EDTA, pH 7.0) for analysis in the ribonuclease protection assay. B. Extraction of RNA from blood. RNA is prepared from blood samples from patients with or without associated disease TREPA diagnosed using the standard QUIAmp RNA protocol (Quiagen, Chattsworth, CA). Briefly, 25 μl of blood is mixed with 280 μl of Qiagen AVL buffer and incubated at room temperature for 1 5 min. Then 280 μl of 100% ethanol is added to the mixture, and the whole mixture is transferred to a spin column QlAamp. The column is then rotated at 6000 x g for 2 min, washed twice with 500 μl of Qiagen AW / ethanol buffer and rotated at 6,000 x g for 2 min. The column is rotated an additional 3 min to >; 1 0.000 x g. The RNA is reacted by adding 100 μl of RNase-free water preheated at 80 ° C to the column and rotating at 6,000 x g for 2 min. C. Extraction of RNA from polysomes. The tissue is comminuted in saline at 4 ° C and mixed with 2.5 volumes of 0.8 M sucrose in a solution of TK150M (50 mM KCI, 5 mM MgCl2, 50 mM Tris-HCl, pH 7.4) containing 6 m 2-mercaptoethanol. M. The tissue is homogenized in a Potter Teflon-glass homogenizer with five strokes at 100-200 rpm followed by six strokes in a Dounce homogenizer, as described by B. Mechler, Methods in Enzvmoloqy 152: 241 -248 (1987) . The homogenate is then centrifuged at 1 2,000 x g for 15 min at 4 ° C to pellet the nuclei. The polysomes are isolated by mixing 2 ml of supernatant with 6 ml of 2.5 M sucrose in TK150M and layered this mixture on 4 ml of 2.5 sucrose in TK150M in a 38 ml polyalomer tube. Two additional solutions of TK150M sucrose are successively layered on the extract fraction; a first layer of 13 ml of 2.05 M sucrose followed by a second layer of 6 ml of 1.3 M sucrose. The polysomes are isolated by centrifuging the gradient at 90,000 x g for 5 h at 4 ° C. The fraction is then taken from the interfacial zone of 1.3 M sucrose / sucrose 2.05 M with a siliconized pasteur pipette and diluted in an equal volume of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). An equal volume of SDS buffer of 90 ° C (SDS 1%, 200 mM NaCl, 20 mM Tris-HCl, pH 7.4) is added, and the solution is incubated in a boiling water bath for 2 min. The proteins are then digested with a proteinase-K digestion (50 mg / ml) for 15 min at 37 ° C. The mRNA is purified with 3 equal volumes of phenol-chloroform extractions followed by precipitation with 0.1 volume of 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at -20 ° C overnight. The precipitated RNA is coated by centrifugation at 12,000 x g for 10 min at 4 ° C. The RNA is dried and resuspended in TE, pH 7.4 or distilled water. The resuspended RNA can then be used in a dot blot or dot blot hybridization assay to verify the presence of m RNA containing EST sequences (see Example 6). The quality of nucleic acid and proteins is dependent on the method of preparation used. Each sample may require a different preparation technique to maximize the isolation efficiency of the target molecule.

Example 4: Ribonuclease Protection Assay A. Marking of complementary RNA hybridization (cRNA) probes. Sense and antisense riboprobes labeled are transcribed from the EST sequence, which contains a 5 'RNA polymerase promoter, such as SP6 or T7. The sequence can be from a vector containing the appropriate EST insert or a product generated by PCR of the insert using PCR primers, which incorporate a 5 'RNA polymerase promoter sequence. The transcripts are prepared in a 20 μl reaction volume containing 1 μg of DNA template, 2 μl of 1 00 mM dithiothreitol, 0.8 μl of Rnasin (10-40U), 500 μM of each of ATP, CTP, GTP , 5 μl of (alpha 32P) UTP or 100-500 μM biotinylated UTP, and 1 μl of RNA polymerase in transcription buffer (40 mM Tris-HCl, pH 7.5, 6 mM MgCl 2, 2 mM spermidine HCL, NaCl 5 mM). Following the incubation at 37 ° C for one hour, the transcripts are treated with DNase I (15 U) for an additional 30 min to digest the template. The probes are then isolated by spin columns, salt precipitation or electrophoresis techniques, which are well known in the art. Finally, the probes are dissolved in lysis buffer (5M guanidine thiocyanate, 0. 1M EDTA, pH 7.0). B. Hybridization of labeled probe to target. Approximately 20 μg of total cellular RNA extracted, as obtained in example 3 above, is mixed in 10 μl of lysis buffer with either (i) 1 x1 05 cpm of radioactively labeled probe, or (ii) 250 pg of non-isotopically probe ml < arcade, each in 2 μl of lysis buffer. The mixture is then incubated at 60 ° C for 5 min and hybridized overnight at room temperature. See, T. Kaabache et. to the. , Anal. Biochem. 2332: 225-230 (1995). C. RNase digestion. Hybridizations are terminated by incubation with 380 μl of a solution containing 40 μg / ml of RNase A and 625 units / ml of T1 RNase in 1 mM EDTA, 300 mM NaCl, 30 mM Tris-HCl, pH 7.4 for 45-60 min at room temperature. The RNase digestion is then terminated by the addition of 60 μl of proteinase-K (1.7 mg / ml) containing SDS 3.3%, followed by incubation for 30 min at 37 ° C. The digested mixture is then extracted with phenochloroform, isoamyl alcohol to remove the protein. The hybrid mRNA: cRNA is precipitated from the aqueous phase by the addition of 4 μg of yeast tRNA and 800 μl of ethanol, and incubation at -80 ° C for 30 min. The precipitates are collected by centrifugation. D. Analysis of fragments. The precipitates are dissolved in 5 μl denaturing gel that charges dye (80% formamide, 10 mM EDTA, pH 8.0, 1 mg / ml xylene cyanol, 1 mg / ml bromophenol blue) and electrophoresed in TBE of polyacrylamide 6%, denaturing gels of 8 M urea. The gels are dried under vacuum and autoradiographed. The quantification can be done by comparing the accounts obtained from the test samples with a calibration curve that was generated by using calibrators that are the sense filament. In cases where non-isotopic labels are used, the hybrids are transferred from the gels to membranes (nylon or nitrocellulose) by "blotting" and then analyzed using detection systems that employ streptavidin-alkaline phosphatase conjugates and chemiluminescent reagents or chemofluorescent The high level of mRNA expression corresponding to a sequence selected from the group consisting of SEQUENCE I D NO 1, or fragments or complements thereof, is then an indication of the presence of the TREPA gene.

Example 5: Northern blottinq The northern blot technique is used to identify an RNA fragment of a specific size from a complex population of RNA using gei electrophoresis and nucleic acid hybridization. Northern blotting is a technique well known in the art. Briefly, up to 20 μg of extracted RNA (see Example 3) are incubated in 20 μl of a solution containing 40 mM morpholine-propanol sulfonic acid (MOPS), pH 7.0, 1 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, 50% formamide v / v, for 1 5 min at 55 ° C. The denatured RNA is mixed with 2 μl of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue (glycerol 50%, EDTA 1 mM, bromophenol blue 0.4%, xylene cyanol 0.4%) and loaded in a denaturing 1.5% agarose gel containing 40 mM morpholine-propanol sulfonic acid (MOPS), pH 7.0, 1 mM sodium acetate, 1 mM EDTA; and formaldehyde 2.2 M. The gel is subjected to electrophoresis for an appropriate time, transferred to a washing and washing tray with five changes of RNase-free water for 5 min, followed by a 45-minute soak at room temperature in NaOH 50 mM and 10 mM NaCl. The gel is neutralized by soaking for 45 min in 0. 1 M Tris-HCl, pH 7.5. After soaking for 1 h in 20X SSC buffer (3 M NaCl, 300 mM trisodium citrate), the gel is transferred onto a matrix based on nitrocellulose or nylon. After the transfer is completed, the filter was washed in 3X SSC, dried with air for 2 h and measured in the oven at 80 ° C for 4 h under vacuum. The mRNAs are detected as in Example 4, supra. Again, the high level of mRNA expression corresponding to a sequence selected from the group consisting of SECU ENCIA ID NO 1 or fragments or complements thereof, is an indication of the presence of the TREPA gene.

Example 6: Dot blot / slot blot Dot and slot blot assays are quick methods for evaluating the presence of a specific nucleic acid sequence in a complex mixture of nucleic acids. To do this, up to 20 μg of RNA were mixed in 50 μl of formamide 50%, formaldehyde 7%, 1 X SSC, incubated 1 5 min at 68 ° C and chilled on ice. Then 100 μl of 20X SSC was added to the RNA mixture and loaded under vacuum onto a multiple apparatus having a prepared nylon or nitrocellulose membrane. The membrane is soaked in water, 20X SSC for 1 hour, placed in two sheets of Whatman # 3 pre-wetted 20X SSC filter paper, and loaded in a slot blot or dot blot vacuum manifold. The slot blot is analyzed with probes prepared and marked as in Example 4 above. The detection of mRNA corresponding to a sequence selected from the group consisting of SEQUENCE I D NO 1, or fragments or complements thereof, as an indication of the presence of the TREPA gene, suggesting diagnosis of the inflammatory disease. Other methods and buffers not specifically detailed for Examples 5 and 6 are described in J. Sambrook et. to the. , supra.

Example 7: In Situ Hybridization This method is useful for directly detecting specific target nucleic acid sequences in cells using detectable nucleic acid hybridization probes. The tissues are prepared with crosslinking agents, such as paraformaldehyde or glutaraldehyde for maximum retention of cellular RNA. See, L. Angerer et. al., Methods in Cell Biol. 35: 37-71 (1991). Briefly, the tissue is placed in more than 5 volumes of 1% glutaraldehyde in 50 mM sodium phosphate, pH 7.5 at 4 ° C for 30 min. The solution is changed with fresh solution for about 30 min of additional fixation. The fixation solution should have an osmolality of approximately 0.375% NaCl. The tissue is washed once in isotonic NaCl to remove the phosphate. The fixed tissues are then embedded in paraffin, as follows. The tissue is dehydrated through a series of ethanol concentrations for 15 min each: 50% twice, 70% twice, 85% 90% and 100% twice. The tissue is then soaked in two xylene changes for 20 min each at room temperature; then it is soaked in two changes of 1 xylene: 1 paraffin for 20 min each at 60 ° C; and then soaked in three final changes in paraffin for 1 5 min each. The tissue is then cut into 5 μm sections using a standard microtome and placed on a slide previously treated with the tissue adhesive 3-aminopropythrietoxysilane. The paraffin is removed from the tissue by means of two xylene soaks of 10 min, and rehydrated in a series of ethanol concentrations: 99% twice, 95%, 85%, 70%, 50%, 30% and distilled water twice . Sections are pre-treated with 0.2 M HCl for 10 min and are permeated with 2 μg / ml Proteinase-K at 37 ° C for 15 min. The labeled riboprobes transcribed from the EST plasmid pSPORTI (see Example 4) are hybridized to the prepared tissue sections and hybridized overnight at 56 ° C in 3X standard saline extract and 50% formamide. Excess probe is removed by washing in 2X standard saline citrate and 50% formamide followed by digestion with 100 μg / ml RNase A at 37 ° C for 30 min. The fluorescence probe is visualized by illumination with UV light under a microscope. Fluorescence in the cytoplasm is indicative of mRNA production. Fluorescence in the nucleus detects the presence of genomic material. Alternatively, the sections can be visualized by autoradiography.

Example 8: Reverse transcription PCR A. One-step RT-PCR assay. Target-specific primers are designed to detect the above target sequence by reverse transcription PCR by methods known in the art. The one-step RT-PCR is a sequential procedure that performs both RT and PCR in a simple reaction mixture. The procedure is carried out in about 200 μl of the reaction mixture containing (N, N-bis [2-hydroxyethyl] glycine) 50 mM, pH 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg / ml bovine serum albumin, 0.1 mM ethylenediaminetetraacetic acid, 0.02 mg / ml NaN3, glycerol 8% w / v, 1 50 μM each of dNTP, 0.25 μM of each primary, 5U of rTth polymerase, Mn (OAc) 2 3.25 mM, and 5 μl of target blood equivalents (see Example 3). Since RNA and the rTth polymerase enzyme are not stable in the presence of Mn (OAc) 2, Mn (OAc) 2 must be added just before the target addition. Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art. Conditions, which may be useful, include cDNA synthesis at 60 ° -70 ° for 1 5-45 min, and 30-45 amplification cycles at 94 ° C, 1 min; 55 ° C-70 ° C, 1 min; 72 ° C, 2 min. A one-step RT-PCR can also be performed by using a dual enzyme procedure with Taq polymerase and a reverse transcriptase enzyme, such as MMLV or AMV RT. B. Traditional RT-PCR. Alternatively, a traditional two-step RT-PCR reaction may be performed, as described by K.-Q. Hu et. al .. Virology 1 81: 721-726 (1 991), as follows: The ected mRNA is transcribed in 25 μl of reaction mixture containing 1.0 mM Tris-HCl, pH 8.3, 5 mM MgCl 2, 500 μM dNTP, 20 U of RNAsin, 1 μM antisense initiator and 25 U of AMV reverse transcriptase (avian myeloblastosis virus) or MMLV (Moloney murine leukemia virus). Reverse transcription is performed at 37-45 ° C for 30-60 min, followed by additional incubation at 95 ° C for 5 min to inactivate RT. PCR is performed using 1 μl of the cDNA reaction in a final PCR reaction volume of 50 μl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCI, 2 mM MgCl 2, 200 μM dNTP, 0.5 μM each initiator and 2.5 U polymerase Taq. Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler 480. Conditions, which may be useful, include 30-45 cycles of amplification (94 ° C, 1 min, 55 ° C-70 ° C, 1 min, 72 ° C, 2 min), final extension (72 ° C, 10 min) and soaking at 4 ° C. C. Analysis of PCR fragments. The correct products can then be verified by size determination using gel electrophoresis with fluorescent intercalators or by southern blotting techniques using labeled probes against the internal sequences of the PCR product. The probes can also be analogues of polynucleotides, such as morpholinos or peptide nucleic acids (PNA). The detection of a sequence selected from the group consisting of SECU ENCIA I D NO 1, or fragments or complements thereof is then indicative of the presence of the TREPA gene, suggesting the diagnosis of inflammatory disease.

D. Automated quantitative PCR. The amounts of specific PCR products in a given sample can be automatically verified by using a fluorescently labeled probe against the internal sequences of the PCR product coupled to an extinction portion. The decoupling of the fluorescent tag to the extinction portion of the internal probe hybridized to the PCR product by PCR polymerase during thermocycling can be detected by machines such as the Perkin-Elmer 7700 quantitative PCR machine, and is proportional to the product amount of PCR present in the sample. The detection of a sequence selected from the group consisting of SECU ENCIA ID NO 1, or fragments or complements thereof is then indicative of the presence of the TREPA gene, suggesting the diagnosis of inflammatory disease.

Example 9: OH-PCR A. Selection and labeling of probes. The target-specific primers and probes are designed to detect the above target sequence by PCR of oligonucleotide hybridization. Publications WP 92/1 0505, published June 25, 1992 and WO 92/1 1 388 published July 9, 1992, teach methods for labeling oligonucleotides at their 5 'and 3' ends, respectively. According to a known method for labeling an oligonucleotide, a phosphoramidite label reagent is prepared and used to add the label to the oligonucleotide during its synthesis. For example, see N.T. Thuong et. al .. Tet. Letters 29 (46): 5905-5908 (1988); or J.S. Cohen et. to the. , United States patent application published 07 / 246,688 (NTIS ORDER No. PAT-APPL-7-246, 688) (1989). Preferably, the probes are labeled at their 3 'end to avoid participation in PCR and the formation of unwanted extension products. For one-step OH-PCR, the probe must have a TM of at least 1 5 ° C below the TM of the primers. The primers and probes are labeled with either captureable or detectable portions using standard phosphoramidite chemistry, which is well known to one skilled in the art. B. PCR of oligo one-step hybridization. The OH-PCR is carried out in 200 μl of reaction containing (N, N-bis [2-hydroxyethyl] glycine) 50 mM, pH 8. 15, KOAc 81 .7 mM, KOH 33.33 mM, 0.01 mg / ml of albumin bovine serum, 0.1 mM ethylenediaminetetraacetic acid, 0.02 mg / ml NaN3, 8% w / v glycerol, 150 μM each of dNTP, 0.25 μM of each primer, 3.75 nM of probe, 5U of rTth polymerase, 3.25 mM of Mn (OAc) 2, and 5 μl of target blood equivalents (see Example 3). Since the RNA and the rTth polymerase enzyme are unstable in the presence of Mn (OAc) 2, the Mn (OAc) 2 must be added just before the addition of the target. The reaction is incubated in a Perkin-jElmer Thermal Cycler 480. Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art. Conditions, which may be useful, include cDNA synthesis (60 ° C, 30 min), 30-45 amplification cycles (94 ° C, 40 s, 55-70 ° C, 60 s), oligo-hybridization ( 97 ° C, 5 min, 1 5 ° C, 5 m in; 1 5 ° C soaking). The correct reaction product contains at least one of the filaments of the PCR product and an internally hybridized probe.

C. Analysis of OH-PCR product. The amplified reaction products are detected in an LCx® analyzer system (available from Abbott Laboratories, Abbott Park, I L). Briefly, the correct reaction product is captured by a microparticle labeled with antibody at a capture site in either the PCR product strand or the hybridization probe, and the complex is detected by ligation of a detectable antibody conjugate to either a detectable site in the PCR probe or filament. Only a complex containing a PCR filament hybridized to the internal probe is detectable. The detection of this complex is indicative, then, of the presence of the TREPA gene, suggesting the diagnosis of inflammatory disease, such as, rheumatoid arthritis. There are many other detection formats, which can be used to detect the presence of the nucleic acid sequence encoding TREPA. The sequence can also be detected by other methods including, but not limited to, ligase chain reaction (LCR, Abbott Laboratories, Abbott Park, IL), Q-beta replicase (Gene-Trak R, Naperville, Illinois), reaction in branched chain (Chiron, Emeryville, CA), and filament displacement tests (Becton Dickinson, Research Triangle Park, NC).

Example 1: Production of synthetic peptides Synthetic peptides (SEQ ID NOS 1 0-36 or fragments thereof) are prepared based on the predicted amino acid sequence of the TREPA polypeptide (see Example 1). All peptides are synthesized in an ABI Peptide Synthesizer (available from Applied Biosciences, LOCATION), Model 431 A, using FMOC chemistry, standard cycles and DCC-HOBt activation. The cut-off and deprotection conditions are as follows: The resin is added to 20 ml of trifluoroacetic acid (TFA), 0.3 ml of water, 0.2 ml of ethanedithiol, 0.2 ml of thioanisole and 100 mg of phenol, and stirred at room temperature for 1.5 hours. The resin is then filtered by suction and the peptide is obtained by precipitation of the TFA solution with ether followed by filtration. Each peptide is purified via preparative reverse phase HPLC, using a gradient of water / acetonitrile / 0.1% TFA and is lyophilized. The product is confirmed by mass spectrometry (see Example 12). Disulfide bond formation is achieved using auto-oxidation conditions, as follows: the peptide is dissolved in a minimum amount of DMSO (approximately 10 ml) before adding buffer (0.1 M Tris-HCl, pH 6.2) to a concentration of 0.3-0.8 mg / ml. The reaction is monitored by HPLC until complete formation of the disulfide bond, followed by preparative reverse phase HPLC using a gradient of water / acetonitrile / TFA 0. 1% and lyophilization. Then the product is confirmed by mass spectrometry (see Example 12). The purified peptides can be conjugated to keyhole limpet hemocyanin or other inm-reactive molecules with glutaraldehyde, mixed with auxiliary and injected into animals.

Example 1 1: Expression of protein in a cell line A. Construction of EST expression plasmid. Plasmid 577, described in US Serial Patent Application no. 08 / 478,073, filed June 7, 1995 and incorporated herein by reference, has been constructed for the expression of secreted antigens in a permanent cell line. This plasmid contains the following DNA segments: (a) a 2.3 Kb fragment of pBR322 containing bacterial beta-lactamase and origin of DNA replication; (b) a 1.8 Kb cassette directing the expression of a neomycin resistance gene under the control of HSV-1 thymidine kinase promoter and poly-A addition signals; (c) a 1.9 Kb cassette directing the expression of a dihydrofolate reductase gene under the control of an SV-40 promoter and poly-A addition signals; (d) a 3.5 Kb cassette directing the expression of a rabbit immunoglobulin heavy chain signal sequence fused to a modified hepatitis C virus (HCV) E2 protein under the control of the SV40 T-AG promoter and enhancer of transcription, the hepatitis B virus supercritical antigen enhancer I (HbsAg) followed by a genome fragment of Herpes Simplex Virus-1 (HSV-1), providing signals of poly-A addition; and (e) a residual 0.7 kB fragment from the late SV40 genome region of no function in this plasmid. All segments of the vector will be coupled by standard methods known to those skilled in the art of molecular biology. Plasmids for the expression of secretable TREPA proteins are constructed by replacing the E2 protein coding sequence of hepatitis C virus on plasmid 577 with that of the EST sequence selected from the group consisting of SECU ENCIA ID NO 1 or fragments of the same, as follows: Digestion of plasmid 577 with Xbal releases the E2 gene fragment of hepatitis C virus. The resulting plasmid backbone allows insertion of the TRREP insert downstream of the immunoglobulin heavy chain signal sequence of rabbit, which directs the proteins expressed in the secretory pathway of the cell. The TREPA fragment is generated by PCR using standard procedures. Encoded in the sense PCR primer sequence is an Xba 1 site, followed immediately by a 12 nucleotide sequence encoding the amino acid sequence Ser-Asn-Glu-Leu ("SNEL") to promote signal protease processing, efficient secretion and final product stability in culture fluids. Following immediately to this 1 2 nucleotide sequence, the primer contains complementary nucleotides for template sequences encoding amino acids of the TREPA sequence. The antisense initiator incorporates a sequence encoding the eight amino acids Asp-Tyr-Lys-Asp-Asp-Asp-As p-Lys just before the stop codons. Within this sequence is the recognition site for a monoclonal antibody (Mab) designated anti-FLAG M2 (Eastman Kodak, Co., New Haven, CT). It is incorporated to aid in the analysis and purification of the EST protein product. PCR is performed using GeneAmp® reagents obtained from Perkin-Elmer-Cetus, essentially as directed by the supplier's instructions. PCR primers are used at a final concentration of 0.5 μM. The P R is carried out in the pSPORTI plasmid template in 1 00 μl of reaction for 35 cycles (94 ° C, 30 s; 55 ° C, 30 s; 72 ° C, 90 s) followed by an extension cycle of 72 ° C for 10 min. Another example of a plasmid for the expression of secretable TREPA proteins is created by fusing in frame the sequence encoding a modified cytomegalovirus UL4 gene leader sequence (SEQUENCE ID NO 4) to the synthetic octapeptide encoding the eight amino acids Asp-Tyr-Lys -Asp-Asp-Asp-Asp-Lys (recognized by the monoclonal antibody designated anti-FLAG M2; SEQUENCE ID NO 5), followed by an appropriate carboxyl-terminal portion of TREPA, which interacts with the affinal receptor, and consequently , can induce biological activity (SEQUENCE ID NO 3). One such form of soluble TRAI L is presented in SEQUENCE ID NO 3. This designed synthetic open reading frame (SEQUENCE ID NO 4 + SEQUENCE ID NO 5 + SEQUENCE ID NO 3) can be placed in an appropriate DNA backbone as appropriate. appropriate for the intended target cell, such as, pcDNA3 (Invitrogen Corp, San Diego, CA). B. Transfection of Chinese hamster ovary cells deficient in dihydrofolate reductase. The plasmid described above is transfected into CHO / dhfr- (DXB-1 1 1) cells (uriacio, et al., PNAS 77, 4451-4466; 1980); these cells are available from A.T.C.C. , 12301 Parkiawn Drive, Rockville, MD 20852, under ei no. CRL 9096), using the cationic liposome mediated procedure (PL Felgner et al., PNAS 84: 741 3-741 7 (1987), as follows: CHO / dhfr cells are grown in F-12 medium of Ham supplemented with 10% fetal calf serum, L-glutamine (1 mM) and seeded fresh in a 25 cm2 flask at a density of 5-8 x 105 cells per flask. and 80% confluency for transfection Twenty micrograms of plasmid DNA are added to 1.5 ml of Opti-MEM I medium and 100 μl of Lipofectin reagent (gibco-BRL, Grand Island, NY) is added to a second portion of 1.5 ml of Opti-MEM medium I. The two solutions are mixed and incubated at room temperature for 20 min.The culture medium is removed from the cells, and the cells are rinsed 3 times with 5 ml of Opti medium. -MEM I. The solution of Opti-MEM l-Lipofectin-DNA plasmid is then extended on the cells.The cells are incubated for 3 h 3 7 ° C, after which the Opti-M EM l-Lipofectin-DNA solution is replaced with culture medium for an additional 24 h before selection. C. Selection and amplification. One day after transfection, the cells are passaged 1: 3 and incubated with dhfr / G41 8 selection medium (hereinafter, "G medium minus F-12." The selection medium is F-12 of Ham with L-glutamine and without hypoxanthine, thymidine and glycine (JRH Biosciences, Lenexa, Kansas), and 300 μg / ml of G41 8 (Gibco-BRL, Grand Island, NY) The volume proportions of medium to area are maintained of surface area of 5 ml per 25 cm 2. After approximately two weeks, the DHFR / G41 8 cells expand to allow passage and continuous maintenance in the medium G minus F-12. Transfected ESTs are achieved by selection in the form of steps of DHFR +, G418 + cells with methotrexate (reviewed by R. Schimke, Cell 37: 705-71 3 [1984]) Cells were incubated with G medium minus F-12 containing methotrexate 150 nM (MTX) (Sigma, St. Louis, MO) for approximately two weeks until resistant colonies appear. No additional gene is achieved by selection of 150 nM adapted cells with 5 MTX uM. D. Production of protein / antigen. The G minus F-12 medium supplemented with 5 μM MTX extends just over confluent monolayers for 12 to 24 h at 37 ° C in 5% CO2. The growth medium is removed and the cells are rinsed 3 times with Dulbecco's phosphate buffered saline (PBS) (with calcium and magnesium) (Gibeco-BRL, Grand Island, NY), to remove remaining medium / serum that could be present. The cells are then incubated with VAS use medium (the formulation using VAS with L-glutamine with HEPES without phenol red, available from JRH Bioscience; Lenexa, KS, product number 52-08678P), for 1 h at 37 ° C in 5% CO2. The cells are then spread with VAS for production at 5 ml per 25 cm2 flask. The medium is removed after 7 days of incubation and then frozen to wait for purification with collections 2, 3 and 4. The monolayers are extended with VAS for 3 more collections of 7 days. E. Analysis of protein expression / TREPA antigen. The aliquots of VAS supernatants of the cells expressing the designed protein construct are analyzed either by polyacrylamide ida-S DS gel electrophoresis (S DS-PAG E), using standard methods and reagents known in the art (discontinuous Laemmli) or by mass spectrometry. F. Purification. The purification of the EST protein containing the FLAG sequence is performed by immunoaffinity chromatography using an affinity matrix comprising anti-FLAG M2 monoclonal antibody covalently linked to agarose by hydrazide linkage (Eastman Kodak Co., New Haven, CT). Prior to affinity purification, the protein in VAS media collections extracted from roller bottles is exchanged in 50 mM Tris-HCl buffer, pH 7.5, 50 mM NaCl using a Sephadex G-25 column (Pharmacia Biotech Inc., Uppsala, Sweden). The protein in this buffer is applied to the anti-FLAG M2 antibody affinity column, the unbound protein is levigated by washing the column with 50 mM Tris-HCl buffer, pH 7.5, 150 mM NaCl and the bound protein is levigated using an excess of FLAG peptide in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl 1. The excess FLAG peptide can be removed from the purified EST protein by gel electrophoresis. Then, the largest cloned insert containing the EST region is sub-cloned into either (i) a eukaryotic expression vector, which may contain a cytomegalovirus (CMV) promoter and / or fusionable protein sequences, which aid in protein expression and detection; or (ii) a bacterial expression vector containing superoxide dismutase (SOD) and CMP-KDO synthetase (CKS) or another protein fusion gene for the expression of the protein sequence. Methods and vectors, which are useful for the production of polypeptides, which contain SOD fusion sequences are described in EPO 0196056, published October 1, 1986, and those of CKS are described in EPO publication no. 0331 961, published September 1, 1989. The purified protein can be used in a variety of techniques including, but not limited to, animal immunization studies, solid phase immunoassays, etc.

Example 12: Chemical analysis of TREPA proteins A. Analysis of fragments of normal peptides using MS. The serum of a patient with inflammatory disease is run on a polyacrylamide gel using standard procedures and stained with Coomassie Blue. Sections of the gel suspected of containing the unknown polypeptide are cut and subjected to in-gel reduction, acetamidation and tryptic digestion. P. Jeno et al, Anal. Bio. 224: 451-455 (1995), and J. Rosenfeld et al, Anal. Bio. 203: 1 73-179 (1992). The gel sections were washed with 100 mM N H4HCO3 and acetonitrile. The shrunk gel pieces are swollen in digestion buffer (50 mM NH 4 HCO 3, 5 mM CaCl 2, and 12.5 μg / ml trypsin) at 4 ° C for 45 min. The supernatant is aspirated and replaced with 5 to 10 μl digestion buffer without trypsin and allowed to incubate overnight at 37 ° C. The peptides are extracted with 3 changes of formic acid 5% and acetonitrile, and evaporated to dryness. Peptides are absorbed at approximately 0.1 μl of POROS R2 sorbent (Perseptive Biosystems, Framingham, Massachusetts) trapped on the tip of a capillary tube of entrained gas chromatography by dissolving in 10 μl of 5% formic acid and passing the solution through the capillary. The adsorbed peptides are washed with water and levigated with 5% formic acid in 60% methanol. The levigant is passed directly into the atomization capillary of an API I I I mass spectrometer (Perkin-Elmer Sciex, Thronhill, Ontario, Canada) for analysis by electroatomization mass spectrometry. M. Wilm et al. , Int. J. Mass Spectrom. Ion Process 136: 167-180 (1994), and M. Wilm et al. , Anal. Chem. 66: 1-8 (1994). The masses of the tryptic peptides are determined from the mass spectrum obtained from the first quadrupole. The masses corresponding to predicted peptides can be further analyzed in the MS / MS mode to give the amino acid sequence of the peptide. B. Analysis of peptide fragment using LC / MS. The presence of predicted polypeptides of mRNA sequences found in hyperplastic disease tissues can also be confirmed using liquid chromatography / double mass spectrometry (LC / MS / MS). D. Hess et al. , M ETHODS, A Companion to Methods in Enzvmology 6: 227-238 (1 994). The serum specimen or tumor extract of the patient is denatured with SDS and reduced with dithiothreitol (1.5 mg / ml) for 30 min at 90 ° C followed by alkylation with iodoacetamide (4 mg / ml) for 15 min at 25 min. ° C. Following the acrylamide electrophoresis, the polypeptides are "electroblotted" to a cationic membrane and are stained with Coomassie blue. Following the staining, the membranes are washed and sections that are thought to contain the unknown polypeptides are cut and dissected into small pieces. The membranes are placed in 500 μl of microcentrifuge tubes and immersed in 10 to 20 μi of proteolytic digestion buffer (100 mM Tris-HCl, pH 8.2, containing 0.1 M NaCl, 1 0% acetonitrile, 2 mM CaCl 2, and 5 ug / ml trypsin) (Sigma, St. Louis, MO). After 15 h at 37 ° C, 3 μl of saturated urea and 1 μl of 1 μg / ml of trypsin are added and incubated for an additional 5 h at 37 ° C. The digestion mixture is acidified with 3 μl of 10% trifluoroacetic acid and centrifuged to remove the supernatant from the membrane. The supernatant is injected directly onto a reversed-phase HPLC column, microbore, and levigated with a linear gradient of acetonitrile in 0.05% trifluoroacetic acid. The levigado is fed directly into an electrospray mass spectrometer, after passing through a current splitter if necessary to adjust the volume of the material. The data is analyzed following the procedures set forth in Example 12, section A.

Example 13: Gene immunization protocol A. Antigen expression in vivo. Gene immunization avoids protein purification steps by directly expressing an in antigen. live after inoculation of the appropriate expression vector. further, the production of antigen by this method can allow correct protein folding and glycosylation, since the protein is produced in mammalian tissue. The method uses the insertion of the gene sequence into a plasmid, which contains a CMV promoter, expansion and purification of the plasmid, and injection of the plasmid DNA into the muscular tissue of an animal. See, for example, H. Davis et al. , Human Molecular Genetics 2: 1847-1 851 (1 993). After one or two booster immunizations, the animal may be allowed to bleed, collect ascitic fluid or collect the spleen for the production of hybridomas. B. Preparation and purification of plasmids. The EST DNA sequences are generated from the pSPORTI EST vector using appropriate PCR primers containing suitable 5 'restriction sites. The PCR product is cut with the appropriate restriction enzymes and inserted into a vector, which contains the CMV promoter (e.g., pRc / CMV or pcDNA3 vectors from Invitrogen, San Diego, CA). The plasmid is then expanded into the appropriate bacterial strain and purified from the cell lysate using a CsCI gradient or a Qiagen plasmid DNA purification column. All these techniques are familiar to someone of ordinary skill in the molecular biology technique. C. Immunization protocol. The anesthetized animals are immunized intramuscularly with 0.1-1.00 μg of the purified plasmid diluted in PBS or other DNA-enhancing enhancers (Cardiotoxin, 25% sucrose). See, for example, H. Davis, et al, Human Gene Therapy 4: 733-740 (1993); and P.W. Wolff et al, Biotechniques 1 1: 474-485 (1991). One or two booster injections are given at monthly intervals. D. Test and use of antiserum. The animals are bled and the sera are tested by antibody using peptides synthesized from the known gene sequence (see Example 1 6), such as western blotting or EIA techniques. The antisera produced by this method can be used to detect the presence of the antigen in a patient's serum or tumor tissue extract by ELISA or Western blotting techniques.

Example 14: Production of antibodies against TREPA A. Production of polyclonal antisera. The antiserum against TREPA is prepared using peptides whose sequence is derived from that of the TREPA DNA sequence (SEQUENCE I D NO 1). The synthesis of the peptides is described in Example 1 0. 1. Conjugation of peptides. The peptide is conjugated to keyhole limpet hemocyanin activated maleimide (KLH, commercially available as Imject®, available from Pierce Chemical Company, Rockford, I L). Imject® contains approximately 250 moles of reactive maleimide groups per mole of hemocyanin. Activated KLH is dissolved in phosphate buffered saline (PBS, pH 8.4) at a concentration of approximately 7.7 mg / ml. The peptide is conjugated through the C-terminal cysteine previously added to the peptide synthesized in order to provide a monospecific binding site. The peptide is dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Company, St. Louis, MO) and reacted with the activated KLH at a molar ratio of about 1.5 mole of peptide per mole of reactive maleimide attached to the KLH. . A method for the conjugation of peptides is provided hereinbelow. It is known by the ordinary practitioner that the quantities, times and conditions of such a procedure can be varied to optimize the conjugation of peptides.

The conjugation reaction described hereinafter is based on obtaining 3 mg of KLH peptide conjugate, which contains about 0.77 μmol of reactive maleimide groups. This amount of peptide conjugate is usually suitable for a primary injection and four booster injections for the production of polyclonal antisera in a rabbit. The amount of peptide conjugate produced can be increased or decreased, as appropriate. Briefly, the peptide is dissolved in DMSO at a concentration of 1.16 μmoles / 1000 μl (4.1 mg / 100 μl) of DMSO. One hundred microliters (100 μl) of the DMSO solution is added to 380 μl of the activated KLH solution prepared as described above, and 20 μl of PBS (pH 8.4) is added to bring the volume to 500 μl. The reaction is incubated overnight at room temperature with stirring. The degree of reaction is determined by measuring the amount of unreacted thiol in the reaction mixture. The difference between the initial concentration of thiol (1.1 1.6 mM) and the final concentration is considered to be the concentration of peptide, which has been coupled to the activated KLH. The amount of remaining thiol is measured using Ellman's reagent (5,5'-dithiobis (2-nitrobenzoic acid), Pierce Chemical Company, Rockford, IL). The cysteine standards are made at a concentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35 mg of cysteine HCl (Pierce Chemical Com pany, Rockford, IL) in 10 ml of PBS (pH 7.2) and diluting the stock solution to the desired concentration (s). The photometric determination of the thiol concentration is achieved by placing 200 μl of PBS (pH 8.4) in each cavity of a I mm ulon 2 ® microplate plate (Dynex Technologies, Chantilly, VA). Then, 10 μl of reaction mixture or standard is added to each cavity. Finally, 20 μl of Ellman's reagent at a concentration of 1 mg / ml in PBS (pH 8.4) is added to each well. The cavities are incubated for 10 minutes at room temperature, and the absorbance of all cavities is read at 415 nm with a microplate reader (such as the BioRad Model 3550, BioRad, Richmond, CA). The absorbance of the standards is used to construct a standard curve and the thiol concentration of the reaction mixture is determined from the standard curve. A decrease in free thiol concentration is indicative of a successful conjugation reaction. The unreacted peptide is removed by dialysis against PBS (pH 7.2) at room temperature for 6 hours. The conjugate is stored at 2-8 ° C if it is to be used immediately; otherwise, it is stored at -20 ° C or colder. 2. Animal immunization. New Zealand rabbits, whites, females, weighing 2 kg or more are used to produce polyclonal antiserum. Generally, an animal is immunized by peptide conjugate prepared as described hereinabove. One week before the first immunization, 5 to 10 ml of blood is obtained from the animal to serve as a non-immune pre-bled sample. The first immunogen used for the primary immunization consists of 1 mg of peptide conjugate prepared as described hereinbefore, emulsified in Freund's complex auxiliary (CFA) (Difco, Detroit, Ml). The primary immunogen is prepared by taking 0.5 ml of the diluted peptide conjugate at a concentration of 2 mg / ml and emulsifying it with 0.5 ml of CFA. The immunogen is injected into several sites of the animal, and injections may include subcutaneous, intraperitoneal and intramuscular injections. Four weeks following the primary immunization, a booster immunization is administered using a second immunogen consisting of 0.5 mg of emulsified peptide conjugate in incomplete Freund's adjuvant (IFA, available from Difco, Detroit, Ml). The immunogen used for the booster immunization dose is prepared by emulsifying 0.5 ml of peptide conjugate diluted to 1 mg / ml with 0.5 ml of I FA. Again, the booster dose is administered at several sites and can use subcutaneous, intraperitoneal and intramuscular injections. The animal is bled (5 ml) two weeks after the booster immunization, and the serum is tested by immuno-reactivity to the peptide, as described below. The reinforcement and bleeding program is repeated at 4-week intervals until an adequate degree is obtained. The titer or concentration of antiserum is determined by microtiter EIA as described in Example 1 7, below. An antibody titer of 1: 500 or greater is considered a suitable title for further study and use. B. Production of monoclonal antibody. 1 . Immunization protocol. Mice are immunized using the immunogen prepared as described herein above, except that the amount of the peptide conjugate for production of monoclonal antibody in mice is one tenth of the amount of the peptide conjugate used to produce polyclonal antisera in rabbits. Thus, the primary immunogen consists of 1 00 μg of peptide conjugate in 0.1 ml of CFA, while the immunogen used for booster immunizations consists of 50 μg of peptide conjugate in 0.1 ml of I FA. Hybridomas for the generation of monoclonal antibodies are prepared and classified using standard techniques. The methods used for monoclonal antibody development follow procedures known in the art and are detailed in Kohler and Milstein, Nature 256: 494 (1975), and reviewed in J.G. R. Hurrel, ed. , Monoclonal Hybridoma Antibodies: Techniques and Applications (Hybridoma Monoclonal Antibodies: Techniques and Applications), CRC Press, Inc., Boca Raton, FL (1982). Another method for the development of monoclonal antibodies, which is based on the Kohler and Milstein method, is that of L.T. Mimms et al. , Virology 176: 604-609 (1990), which is incorporated herein by reference. The immunization regime (5 mice) consists of a primary immunization with additional booster immunizations. The primary immunogen used for the primary immunization consists of 500 μg of peptide conjugate in 250 μl of PBS (pH 7.2) emulsified with 250 μl of CFA. Reinforcement immunizations performed at approximately two weeks and four weeks after immunization, consist of 250 μg of peptide conjugate in 250 μl of PBS (pH 7.2) emulsified with 250 μl of IFA. The immunogen used for booster immunizations is inoculated intraperitoneally and subcutaneously in each mouse, each mouse receiving a total of 100 μl of this immunogen thus prepared by immunization. The individual mice are classified for immunoresponse by a microtiter plate enzyme immunoassay (EIA), as described in Example 1, approximately four weeks after the third immunization. Mice are inoculated intravenously with 50 μg of peptide conjugate in PBS (pH 7.2) approximately fifteen weeks after the third immunization. Three days after this intravenous booster, the splenocytes are fused with, for example, Sp2 / 0-Ag14 myeloma cells from Milstein Laboratories, England, using the polyethylene glycol (PEG) method. The fusions are cultured in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal calf serum (FCS), plus 1% hypoxanthine, aminopterin and thymidine (HAT). Bulk cultures are classified by microtiter plate EIA following the protocol of Example 17. Clones reactive with the peptide used as the immunogen and not reactive with other peptides, are selected for final expansion. The clones thus selected are expanded, aliquoted and frozen in IMDM containing 10% FCS and 10% dimethyl sulfoxide. 2. Production of monoclonal antibodies containing ascitic fluid. The frozen hybridoma cells prepared as described hereinabove are thawed and placed in expansion culture. The viable hybridoma cells are inoculated intraperitoneally in mice treated with Pristane. The ascites fluid is removed from the mice, extracted, filtered through a 0.2 μ filter and subjected to an immunoglobulin class G (IgG) assay to determine the volume of the Protein A column required for purification. 3. Purification of monoclonal antibodies of ascitic fluid. Briefly, the filtered and thawed ascites fluid is mixed with an equal volume of Sepharose ligation buffer from Protein A (1.5 M glycine, 3.0 M NaCl, pH 8.9) and filtered again through a 0.2 μ filter. The volume of the Protein A column is determined by the amount of IgG present in the ascitic fluid. The levigado is then subjected to dialysis against PBS pH 7.2 overnight at 2-8 ° C. The dialyzed monoclonal antibody is filtered sterile and is dispensed in aliquots. The immuno-reactivity of the purified monoclonal antibody is confirmed by determining its ability to specifically bind to the peptide (SEQUENCE ID NO 6) used as the immunogen by using the EIA microtiter plate assay procedure of Example 17. The specificity of the antibody purified monoclonal is confirmed by determining its lack of ligation to irrelevant peptides. The purified monoclonal anti-TREPA thus prepared and characterized is placed either at 2-8 ° C for short-term storage and at -80 ° C for long-term storage. 4. Additional characterization of monoclonal antibody. The isotype and subtype of the monoclonal antibody produced as described hereinabove can be determined using commercially available pools (available from Amersham, Inc., Arlington Heights, IL). The stability test can also be performed on the monoclonal antibody by placing a copy of the monoclonal antibody in continuous storage at 2-8 ° C and testing the optical density (OD) readings over the course of a given period.

C. Use of recombinant proteins as immunogens. It is within the scope of the present invention that recombinant proteins made as described herein can be used as immunogens in the production of polyclonal and monoclonal antibodies, with corresponding changes in reagents and techniques known to those skilled in the art. D. Variation of immunogens. It is contemplated that the peptide unconjugated to a carrier, such as described hereinbefore in 1 (A) may be used at times as the immunogen for animal inoculation. This immunogen thus prepared can be ahed to auxiliaries, such as complete Freund's aids or complete infusion.

Example 1: Purification of TREPA peptide-specific antibodies from serum Immune sera are affinity purified using synthetic peptides immobilized by methods known in the art. The antiserum raised against a peptide as described in Example 10 is affinity purified in a variety of ways. An IgG fraction is obtained by passing the crude antiserum, diluted on a Protein A column (Affi-Gel protein A, Bio-Rad, Hercules, CA). The ligation with Binding Buffer supplied by the manufacturer removes all proteins that are not immunoglobulins. The levigation with pH 3, glycine, buffered 0. 1 M, gives an immunoglobulin preparation that is substantially free of albumin and other whey proteins.

The immunoaffinity chromatography is performed to obtain a preparation with a larger fraction of specific antigen binding antibody. The peptide used to produce antiserum is immobilized on a chromatography resin and the specific antibodies directed against its epitopes are adsorbed to the resin. After washing the non-ligation components, the specific antibodies are levigated with 0.1 M glycine buffer, pH 2.3, the antibody fractions are immediately neutralized with 1.0 M Tris buffer, pH 8.0, to preserve the immunoreactivity. The chosen resin depends on the reactive groups present in the peptide. If the peptide has an amino group, a resin is used, such as Affi-Gel 10 or Affi-Gel 15 (Bio-Rad, Hercules, CA). If coupling through a carboxy group in the peptide is desired, Affi-Gel 1 02 (Bio-Rad, Hercules, CA) can be used. If the peptide has a free sulfhydryl group, an organomercurial resin, such as Affi-Gel 501 (Bio-Rad, Hercules, CA) can be used. Alternatively, the spleens can be collected and used in the production of hybridomas to produce monoclonal antibodies.

Example 16: Western blotting of tissue samples Tissue samples are homogenized in SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 1 mM dithiotreitol, 2% SDS, 0.1% bromophenol blue, glycerol 1 0%), heated at 1 00 ° C for 1 0 min and run on an SDS-PAG E 14% with a run buffer of 25 mM Tris-HCl, pH 8.3, 250 mM glycine, 0.1% SDS. The proteins are transferred electrophoretically to nitrocellulose in a transfer buffer containing 39 mM glycine, 48 mM Tris-HCl, pH 8.3, 0.037% SDS or, 20% methanol. The nitrocellulose was dried at room temperature for 60 min and then blocked with a PBS solution containing either bovine serum albumin or 5% defatted milk powder for 2 h at 4 ° C. The filter is placed in a heat-sealable plastic bag containing a 5% defatted milk powder solution in PBS with a 1: 1000 to 1: 2000 dilution of affinity-purified anti-EST peptide antibodies (see Example 1 5) , incubated at 4 ° C for 2 h, followed by 3 washes of 10 min in PBS. A secondary antibody conjugated with alkaline phosphatase (ie, anti-mouse / rabbit IgG) is added at a 1: 200 to 1: 2000 dilution to the filter in a 150 mM NaCl buffer, 50 mM Tris-HCl, pH 7.5, and It is incubated for 1 h at room temperature. The bands are visualized on the addition and development of a chromogenic substrate, such as, 5-bromo-4-chloro-3-indolyl / nitro blue tetrazolium phosphate (BCI P / NBT). This chromogenic solution contains NBT 0.033% and BCI P 0.01 6% in a solution containing 100 mM NaCl, 5 mM MgCl 2 and 1 00 mM Tris-HCl, pH 9.5. The filter is incubated in the solution at room temperature until the bands develop to the desired intensity. The development is stopped in a PBS buffer containing 2 mM EDTA. Molecular mass determination is made based on the mobility of pre-stained molecular weight standards (Rainbow markers, Amersham, Arlington Heights, I L).

Example 17: EIA microtiter plate assay To demonstrate how relative immuno-reactivity is measured for synthetic EST peptides, cavities of 96-well microtiter plates (Dynatec Immunolon 4 polystyrene) are covered for 16 h at 4 ° C with 100 μl of the EST peptide in the following concentrations: 500 μM, 50 μM, 5 μM, 0.5 μM, 0.05 μM, and 0.005 μM. The buffer used for the application of these peptides is 100 mM ethano-morpholino sulfonic acid, pH 5.5. The cavities coated with EST peptides are then washed 3 times with wash buffer (8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM sodium chloride, 10 mM potassium chloride, 0.05% Tween 20, bovine serum albumin 0.1%, pH 7.4). The cavities are then blocked for 1 h at room temperature with 9% w / v of skimmed milk powder Carnation in phosphate buffered saline (8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM sodium chloride, potassium chloride 1.0mM, pH 7.4). The cavities are then washed 3 times with wash buffer. The test or control specimen (mouse or rabbit antiserum) is diluted 1 50 times with 4.5% Carnation nonfat dry milk in PBS. Then 1 00 μl of this sample is incubated in the cavities at 37 ° C for 1 h, followed by 3 washes with wash buffer. Anti-mouse / goat rabbit IgG conjugated with horseradish peroxidase is used as a second antibody label to bind with the anti-EST-antibody / EST antigen complex formed in the positive wells. 1000 μl of goat anti-mouse IgG / rabbit H RPO conjugate is added at a dilution of approximately 1: 5000 in wash buffer to each well, and incubated at room temperature for 1 hour. The cavities are washed 3 times with wash buffer. The positive cavities are identified by the absorbance readings at 405 nm after exposure of the cavities to 1 00 μl of ABTS solution (diammonium acid salt 2)., 2'-azinobis- [3-ethylbenzothizoline-6-sulfonic acid]) (Pierce Chemical Co., Rockford, I L, USA). Alternatively, the development of color can be achieved with the addition of each cavity of 1 00 μl of a solution of o-phenylenediamine (OPD) in hydrogen peroxide and a 1 0 min incubation at room temperature. The color development reaction is quenched with 1 00 μl of 1 N sulfuric acid. The colors in the cavities are read as absorbance with a Dynatech MR5000 plate reader at wavelengths of 490 nm and 630 nm. A positive signal is indicative of the presence of anti-EST peptide antibodies.

Example 1 8: Coating of solid phase particles A. Coating of microparticles with anti-EST peptide antibody. The affinity-purified anti-EST peptide antibodies (see Example 15) are coated on microparticles, which may include polystyrene, carboxylated polystyrene, polymethylacrylate or similar particles with a radius in the range of about 0.1 to 20 μ. The microparticles can be coated either passively or actively. One method is the coating of carboxylated latex microparticles activated with EDAC (1- (3-dimethylamidopropi) -3-ethylcarbodiimide hydrochloride) (Aldrich Chemical co., Milwaukee, Wl) with anti-EST antibody. Briefly, a final solid solution of 0.375% resin washed carboxylated latex microparticles is mixed in a solution containing 50 mM MES buffer, pH 4.0 and 150 mg / L affinity purified anti-EST antibody (see Example 1 5) for 15 min in an appropriate container. The EDAC coupling agent is added to a final concentration of 5.5 μg / ml for the mixture and is stirred for 2.5 h at room temperature. The microparticles are then washed with 8 volumes of a Tween 20® / sodium phosphate wash buffer, pH 7.2, by tangential flow filtration using a 0.2 μm Microgon filtration module. The washed microparticles are stored in an appropriate buffer, usually containing a dilute surfactant and irrelevant protein as a blocking agent, until needed. B. 0.635 cm pearl coating. Anti-EST antibodies can also be coated on the surface of 0.635 cm polystyrene beads by routine methods known in the art (Snitman et al, US Patent 5,273,882, incorporated herein by reference) and are used in sandwich assays. EIA or competitive ligation. The polystyrene beads are cleaned first by subjecting them to ultrasonication for approximately 1 5 seconds in 10 mM NaHCO3 buffer at pH 8.0. The beads are then washed in deionized water until all the fines are removed. The beads are then merged into an antibody solution in 1 0 mM carbonate buffer, pH 8 to 9.5. The antibody solution can be as dilute as 1 μg / ml in the case of high affinity monoclonal antibodies or as concentrated as approximately 500 μg / ml for polyclonal antibodies, which have not been purified by affinity. The beads are coated for at least 1 2 hours at room temperature and then washed with deionized water. The pearls can be dried or stored wet. They can be overcoated with protein stabilizers (sucrose) or nonspecific ligating blockers (irrelevant proteins, Carnation skim milk, or the like).

Example 1 9: Immunoassay of microparticle enzymes (MEIA) EST proteins and peptides are detected using a standardized antigen competition EIA assay or polyclonal antibody sandwich EIA assay in the I Mx® Analyzer (Abbott Laboratories, Abbott Park, IL). Briefly, samples suspected of containing the EST protein are incubated in the presence of anti-EST coated microparticles (see Example 1 6). The microparticles are washed and secondary polyclonal anti-EST antibodies conjugated with detectable entities (ie, alkaline phosphatase) are added and incubated with the microparticles. The microparticles are washed and the antibody / antigen / bound antibody complexes are detected by adding a substrate (i.e., 4-methyl umbelliferyl phosphate) (MUP) which will react with the secondary conjugate antibody to generate a detectable signal. A high signal, indicating the presence of EST protein, is a diagnosis of cancer.

A competitive ligation assay uses a detectably-labeled peptide that generates a specific support signal in the I Mx® analyzer when the peptide is bound to a microparticle coated with anti-peptide antibody. The labeled peptide is also added to the microparticles in the presence of samples from patients suspected of containing the EST protein. The EST protein in the patient sample will compete with the EST peptide labeled by ligation sites in the microparticle, resulting in decreased I Mx® signals. A diminished signal, indicating the presence of ESt protein in the patient sample, is indicative of the presence of the TREPA gene product, suggesting the diagnosis of inflammatory disease. Accordingly, it is thought that the TREPA gene polynucleotides and the proteins encoded therein, which are provided and discussed hereinabove, are useful as markers of TREPA associated disease. Samples based on the appearance of this marker in a test sample, such as blood, plasma or serum, can provide low-cost, non-invasive diagnostic information to assist the physician in making a cancer diagnosis to help select a therapy protocol, or to monitor the success of the chosen therapy. This marker can appear in readily accessible body fluids, such as blood, urine or stools, as antigens derived from diseased tissue, which are detectable by immunological methods. This marker can be elevated in a disease state, altered in a disease state, or be a normal protein, which appears in an inappropriate body compartment. In addition, the TREPA gene polynucleotides and the proteins encoded therefrom, provided by the present invention, are useful for treating a variety of disorders.

Example 20: HUVEC experiment Ligand preparation: UL4flagTREPA was transiently transfected into COS-7 cells using lipafectamine. Three days later the medium was changed, 2 ml of supernatant was collected from the cells. The supernatant was centrifuged at 1000 rpm to remove cell debris, and the protein was concentrated by centrifugation on a centricep 10 column (Pierce Chemical, Rockford, I L) at 500 x g for 1 hour. As a negative control, supernatants from cells treated in the same manner, except for the absence of DNA, were prepared in a similar manner (moc). Plaque preparation: The cavities of a 96-well tissue culture plate were coated with 10 ug / ml of anti-flag m2 antibody (Kodak) in 100 ul of PBS for 3 hours. The wells were washed three times with PBS and coated with the following overnight: Two wells were coated with 100 ul of concentrated supernatant of transfected moc cells. All the wells were washed with PBS three times before the addition of UVEC H cells (human umbilical endothelial cell). Cells: A 1 0 cm dish of HUVEC cells from confluent step 4 was washed 80% with PBS and then incubated with 5ml of PBS for 3 minutes at 37 ° C to remove the cells. 10 ml of serum-free medium were added, and the cells were counted and centrifuged at 1500 X g. The cells were resuspended in 10 ml of serum-free medium and counted. These cells were seeded at 1500 cells per well in 1000 ul of EGM medium (Clonetics, Waikersville, MD) with 2% fetal bovine serum. The plate was incubated at 3% CO 2, 37 ° C for six days. Results: The cavities were washed with PBS and stained with crystal violet stain (20% methanol, 0.1% crystal violet). The cavities were washed five times with water and photographed under a microscope with 10 times magnification. The number of cells per photograph was counted. The results are as follows: moc UL4flagTREPA 1 14 861 103 661 In another experiment, HUVEc cells were treated similarly, except that they were cultured in EGM medium without EGF and hydrocortisone, and 1% fetal bovine serum. Their numbers were measured by staining with the fluorescent dye calcien-AM (Molecular Probes, Eugene, OR). Fluorescence was measured at an excitation wavelength of 485 nm and read at 530 nm in a 2300 cytofluor system in sensitivity 2 (Millipore, Beford, MA). The fluorescence support of cavities with medium and no cells was deduced, and the results are as follows: moc UL4 flag TREPA -1 41 0 38 -1 41 -1 41 The results indicate that UL4flagTREPA is capable of inducing proliferation in HUVEC cells as measured by both direct cell counting and indirect fluorescent staining.

Example 21: Proliferation of three additional types of ligand preparation cells A soluble UL4flagTREPA construct (see SEQUENCE I D NO 3) was transfected into CHO cells. The clones were selected using geneticin selection at 500 ug / ml and assayed for the production of soluble flagTREPA by western blot of the conditioned media. A clone was propagated by expressing flagTREPA and used to produce conditioned medium. 500 ml of conditioned medium was collected and centrifuged at 1000 rpm to remove cell debris. Two μl of sepharose conjugated with M2 anti-flag were added to the supernatant and incubated with gentle stirring overnight at 4 ° C. The medium was centrifuged at 1,000 rpm to collect the beads. The medium was discarded. The beads were washed with 40 ml of PBS containing 2 mM MgCl2. 1 mM flag peptide was prepared in PBS with 2 mM MgCl2 and incubated with the perl for ten minutes. Seven fractions of 0.5 ml were levigated and analyzed by SDS-PAGE. The first four fractions were extracted and dialysed for 4 hours in 500 ml of PBS with 2 mM MgCl2 with a buffer change after the first two hours to remove the flag peptide. The resulting protein was analyzed by SDS PAGE and the protein was quantified by spectrophotometer using the total protein function of the GeneQuant DNA / RNA alculator (Pharmacia Biotech). Cells: Three types of additional endothelial cells were tested, HA (human aortic endothelial cells), H BE (human brain microvascular endothelial cells) and H UMVE (human microvascular endothelial cells). The three cell types are treated similarly. A 1 0 cm plate of 80% confluent passage under 9 was washed in PBS and incubated with 2 ml of PBS for 3 minutes at 37 ° C to remove the cells. The cells were washed in 10 ml of serum-free serum and counted. Cells were seeded at 1500 cells per well in 100 ul of EGM medium without EGF and hydrocortisone, and 1% fetal bovine serum. Five ng (final concentration of 50 ng / ml) of purified TREPA was added to each cavity. Five days later, the cell numbers were measured by staining with the fluorescent dye calcien-AM (Molecular Probes, Eugene, OR). Fluorescence was measured at an excitation wavelength of 485 nm and read at 530 nm in a 2300 cytofluor system at sensitivity 2 (Millipore, Bedford, MA). The cavity support fluorescence with medium and without cells was deduced, and the results are as follows: H U MVE cells untreated H UMVE treated with U L ^ IflagTREPA 6 26 6 25 5 21 5 21 Untreated HA cells HA cells treated with UL4flagTREPA 1 1 28 10 28 14 31 12 28 Untreated HBE cells HBE cells treated with UL4flagTREPA 8 21 14 25 6 21 12 22 The above results indicate that purified U L4flagTREPA is capable of inducing proliferation in a variety of endothelial cells as measured by indirect fluorescent staining. These cells include both large vessel (HA) endothelium and microvasculature endothelium (HBA and H UMVE).

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Wiley, Steven R. (ii) TITLE OF THE INVENTION: MEMBER OF THE FAMILY OF TNF USEFUL FOR TREATMENT AND DIAGNOSIS OF DISEASE (ii) NUMBER OF SEQUENCES: 11 ( iv) ADDRESS FOR CORRESPONDENCE: (A) DESTINY: Abbott Laboratories, D377 / AP6D (B) STREET: 100 Abbott Park Road (C) CITY: Abbott Park (D) STATUS: IL (D) COUNTRY: USA (E) ZIP CODE : 60064 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Disquette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS (D) PACKAGE: FastSEQ Version 2.0 (vi) CURRENT REQUEST DATA: (A) ) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Becker, Cheryl L. (B) REGISTRATION NUMBER: 35,441 (C) REFERENCE NUMBER / CASE: 6048. US. P1 (¡x) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 847-935-1729 (B) TELEFAX: 847-938-2623 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1236 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESC RI PC OF THE S ECU ENCE: S EQ ID NO: 1: ATGGCCGCCC GTCGGAGCCA GAAGCGGAGG GGGCGCCGGG GGGAGCCGGG CACCGCCCTG 60 CTGGTCCCGC TCGCGCTGGG CCTGGGCCTG GCGCTGGCCT GCCTCGGCCT CCTGCTGGCC 120 GTGGTCAGTT TGGGGAGCCG GGCATCGCTG TCCGCCCAGG -AGCCTGCCCA GGAGGAGCTG 180 GTGGCAGAGG AGGACCAGGA CCCGTCGGAA CTGAATCCCC AGACAGAAGA AAGCCAGGAT 240 CCTGCGCCTT TCCTGAACCG ACTAGTTCGG CCTCGAAGAA GTGCACCTAA AGGCCGGAAA 300 ACACGGGCTC GAAGAGCGAT CGCAGCCCAT TATGAAGTTC ATCCACGACC TGGACAGGAC 360 GGAGCGCAGG CAGGTGTGGA CGGGACAGTG AGTGGCTGGG AGGAAGCCAG AATCAACAGC 420 TCCAGCCCTC TGCGCTACAA CCGCCAGATC GGGGAGTTTA TAGTCACCCG GGCTGGGCTC 480 TACTACCTGT ACTGTCAGGT GCACTTTGAT GAGGGGAAGG CTGTCTACCT GAAGCTGGAC 540 TTGCTGGTGG ATGGTGTGCT GGCCCTGCGC TGCCTGGAGG AATTCTCAGC CACTGCGGCG 600 AGTTCCCTCG GGCCCCAGCT CCGCCTCTGC CAGGTGTCTG GGCTGTTGGC CCTGCGGCCA 660 GGGTCCTCCC TGCGGATCCG CACCCTCCCC TGGGCCCATC TCAAGGCTGC CCCCTTCCTC 720 ACCTACTTCG GACTCTTCCA GGTTCACTGA GGGGCCCTGG TCTCCCCGCA GTCGTCCCAG 780 GCTGCCGGCT CCCCTCGACA GCTCTCTGGG CACCCGGTCC CCTCTGCCCC ACCCTCAGCC 840 GCTCTTTGCT CCAGACCTGC CCCTCCCTCT AGAGGCTGCC TGGGCCTGTT CACGTGTTTT 900 CCATCCCACA TAAATACAGT ATTCCCACTC TTATCTTACA ACAACCCCAC CGCCCACTCT 960 CCACCTCACT AGCTCCCCAA TCCCTGACCC TTTGAGGCCC CCAGTGATCT CGACTCCCCC 1020 CTGGCCACAG ACCCCCAGGG CATTGTGTTC ACTGTACTCT GTGGGCAAGG ATGGGTCCAG 1080 AAGACCCCAC TTCAGGCACT AAGAGGGGCT GGACCTGGCG GCAGCAAGCC AAAGAGACTG 1140 GGCCTAGGCC AGGAGTTCCC AAATGTGAGG GGCGAGAAAC AAGACAAGCT CCTCCCTTGA 1200 GAATTCCCTG TGGATTTTTA AAACAGATAT TATTTT 1236 • ~~ (2) I NFORMATION FOR S EQ ID NO: 2: (i) CHARACTERI STI CASE OF THE SECU ENCIA: (A) LENGTH: 249 amino acids (B) TI PO: amino acid (C) FI LAMENT: simple (D) TOPOLOGY: linear (ii) TI PO DE MOLÉCU LA: None (xi) DESCRI PCION OF SECU ENC IA: S EQ ID NO: 2: Met Ala Ala Arg Arg Ser Gln Lys Arg Arg Gly Arg Gly Glu Pro 1 5 10 15 Gly Thr Ala Leu Leu Val Pro Leu Ala Leu Gly Leu Gly Leu Ala Leu 20 25 30 Ala Cys Leu Gly Leu Leu Leu Ala Val Val Ser Leu Gly Ser Arg Ala 35 40 45 Ser Leu Ser Wing GIn Glu Pro Wing Gln Glu Glu Leu Val Wing Glu Glu 50 55 60 Asp Gln Asp Pro Ser Glu Leu Asn Pro Gln Thr Glu Glu Ser Gln Asp 65 70 75 80 Pro Wing Pro Phe Leu Asn Arg Leu Val Arg Pro Arg Arg Ser Wing Pro 85 90 95 Lys Gly Arg Lys Thr Arg Wing Arg Arg Wing Wing Wing Ala His Tyr Glu 100 105 110 Val His Pro Arg Pro Gly Gln Asp Gly Wing Gln Wing Gly Val Asp Gly 115 120 125 Thr Val Ser Gly Trp Glu Glu Wing Arg He Asn Ser Ser Pro Leu 130 135 140 Arg Tyr Asn Arg Gln He Gly Glu Phe He Val Thr Arg Ala Gly Leu 145 150 155 160 Tyr Tyr Leu Tyr Cys Gln Val His Phe Asp Glu Gly Lys Wing Val Tyr 165 170 175 Leu Lys Leu Asp Leu Leu Val Asp Gly Val Leu Ala Leu Arg Cys Leu 180 185 190 Glu Glu Phe Ser Ala Thr Ala Ala Ser Ser Leu Gly Pro Gln Leu Arg 195 200 205 Leu Cys Gln Val Ser Gly Leu Leu Ala Leu Arg Pro Gly Ser Ser Leu 210 215 220 Arg lie Arg Thr Leu Pro Trp Wing His Leu Lys Ala Wing Pro Phe Leu 225 230 235 240 Thr Tyr Phe Gly Leu Phe Gln Val His 245 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LH: 189 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Val Met Met Leu Arg Thr Trp Arg Leu Leu Pro Met Val Leu Leu 1 5 10 15 Ala Ala Tyr Cys Tyr Cys Ser Leu Ala Ala Pro Gly Ser Asp Tyr Lys 20 25 30 Asp Asp Asp Asp Lys Gly Arg Lys Thr Arg Wing Arg Arg Gly lie Wing 35 40 45 Wing His Tyr Glu Val His Pro Arg Pro Gly Gln Asp Gly Wing Gln Wing 50 55 60 Gly Val Asp Gly Thr Val Ser Gly Trp Glu Glu Wing Arg He Asn Ser 65 70 75 Ser Ser Pro Leu Arg Tyr Asn Arg Gln He Gly Glu Phe He Val Thr 85 90 95 Arg Ala Gly Leu Tyr Tyr Leu Tyr Cys Gln Val His Phe Asp Glu Gly 100 105 110 Lys Wing Val Tyr Leu Lys Leu Asp Leu Leu Val Asp Gly Val Leu Wing 115 120 125 Leu Arg Cys Leu Glu Glu Phe Ser Wing Thr Wing Wing Being Ser Leu Gly 130 135 140 Pro Gln Leu Arg Leu Cys Gln Val Be Gly Leu Leu Wing Leu Arg Pro 145 150 155 160 Gly Be Ser Leu Arg He Arg Thr Leu Pro Trp Wing His Leu Lys Wing 165 170 175 Wing Pro Phe Leu Thr Tyr Phe Gly Leu Phe Gln Val His 180 185 (2) I NFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LH: 24 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TI PO OF MOLECULE: protein (xi) DESCRITION OF SEQUENCE: SEQ ID NO: 4: Met Lys Trp Val Thr Phe lie Ser Leu Leu Phe Leu Phe Ser Ser I3 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg 20 (2) I NFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SECU ENCIA: (A) LH: 8 amino acids (B) TI PO: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear ( ii) TI PO OF MOLECULE: 5 (xi) DESCRI PTION OF SEQUENCE: SEQ ID NO: 5: Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LH: 146 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) ) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Arg Arg Ala lie Ala Ala His Tyr Glu Val His Pro Arg Pro Gly Gln 1 5 10 15 Asp Gly Ala Gln Ala Gly Val Asp Gly Thr Val Ser Gly Trp Glu Glu 20 25 30 Wing Arg He Asn Being Ser Pro Leu Arg Tyr Asn Arg Gln lie Gly 35 40 45 Glu Phe He Val Thr Arg Ala Gly Leu Tyr Tyr Leu Tyr Cys Gln Val 50 55 60 His Phe Asp Glu Gly Lys Ala Val Tyr Leu Lys Leu Asp Leu Leu Val 65 70 75 80 Asp Gly Val Leu Ala Leu Arg Cys Leu Glu Glu Phe Ser Ala Thr Wing 85 90 95 Ala Be Ser Leu Gly Pro Gln Leu Arg Leu Cys Gln Val Ser Gly Leu 100 105 110 Leu Ala Leu Arg Pro Gly Ser Ser Leu Arg lie Arg Thr Leu Pro Trp 115 120 125 Ala His Leu Lys Ala Ala Pro Phe Leu Thr Tyr Phe Gly Leu Phe Gln 130 135 140 Val His 145 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LH: 177 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Glu Lys Gln Gln Asn He Ser-Pro Leu Val Arg Glu Arg Gly Pro Gln 1 5 10 15 Arg Val Ala Ala His He Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu 20 25 30 Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys He Asn 35 40 45 Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His 50 55 60 Leu Arg Asn Gly Glu Leu Val He His Glu Lys Gly Phe Tyr Tyr lie 65 70 75 80 Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Lie Lys Glu Asn Thr 85 90 95 Lys Asn Asp Lys Gln Met Val Gln Tyr He Tyr Lys Tyr Thr Ser Tyr 100 105 110 Pro Asp Pro lie Leu Leu Met Lys Ser Wing Arg Asn Ser Cys Trp Ser 115 120 125 Lys Asp Wing Glu Tyr Gly Leu Tyr Ser NeTyrGIn Gly Gly He Phe 130 135 140 Glu Leu Lys Glu Asn Asp Arg He Phe Val Ser Val Thr Asn Glu His 145 150 155 160 Leu He Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe lie Val 165 170 175 Gly (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 183 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Glu Lys Gln Leu Ser Thr Pro Pro Leu Pro Arg Gly Gly Arg Pro Gln 1 5 10 15 Lys Val Wing Wing His He Thr Gly He Thr Arg Arg Ser Asn Wing 20 25 30 Leu lie Pro He Ser Lys Asp Gly Lys Thr Leu Gly GIn Lys lie Glu 35 40 45 Ser Trp Glu Ser Ser Arg Lys Gly His Ser Phe Leu Asn His Val Leu 50 55 60 Phe Arg Asn Gly Glu Leu Val He Glu Gln Glu Gly Leu Tyr Tyr He 65 70 75 80 Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Wing Glu Asp Wing Ser Lys 85 90 95 Met Val Ser Lys Asp Lys Val Arg Thr Lys Gln Leu Val GIn Tyr He 100 105 110 Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro He Val Leu Met Lys Ser Wing 115 120 125 Arg Asn Ser Cys Trp Ser Arg Asp Wing Glu Tyr Gly Leu Tyr Ser He 130 135 140 Tyr Gln Gly Glu Leu Phe Glu Leu Lys Lys Asn Asp Arg He Phe Val 145 150 155 160 Ser Val Thr Asn Glu His Leu Met Asp Leu Asp Gln Glu Wing Being Phe 165 170 175 Phe Gly Wing Phe lie He Asn 180 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 147 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Asp Lys Pro Val Wing His Val Val Wing Asn Pro Gln Wing Glu Gly Gln 1 5 10 15 Leu Gln Trp Leu Asn Arg Arg Wing Asn Wing Leu Leu Wing Asn Gly Val 20 25 30 Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu 35 40 45 lie Tyr Ser Gln Val Leu Phe Lys Gly Gln Giy Cys Pro Ser Thr His 50 55 60 Val Leu Leu Thr His Thr He Ser Arg He Wing Val Ser Tyr Gln Thr 65 70 75 80 Lys Val Asn Leu Leu Ser Ala lie Lys Ser Pro Cys Gln Arg Glu Thr 85 90 95 Pro Glu Gly Ala Glu Ala Lys Pro Trp Glu Pro He Tyr Leu Gly Gly 100 105 110 Val Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Wing Glu He Asn Arg 115 120 125 Pro Asp Tyr Leu Asp Phe Wing Glu Ser Gly Gln Val Tyr Phe Gly He 130 135 140 He Ala lie 145 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 145 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Leu Lys Pro Ala Ala His Leu lie Gly Asp Pro Ser Lys Gln Asn Ser 1 5 10 15 Leu Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe 20 25 30 Ser Leu Ser Asn Asn Ser Leu Leu Val Pro Thr Ser Gly He Tyr Phe 35 40 45 Val Tyr Ser Gln Val Val Phe Ser Gly Lys Wing Tyr Ser Pro Lys Wing 50 55 60 Thr Ser Ser Pro Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser 65 70 75 80 Gln Tyr Pro Phe His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr 85 90 95 Pro Gly Leu Gin Glu Pro Trp Leu His Ser Met Tyr His Gly Ser Ser 100 105 110 Gly Gln Leu Thr Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly He 115 120 125 Pro His Leu Val Leu Ser Pro Thr Val Phe Phe Gly Ala Phe Ala 130 135 140 Leu 145 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 165 amino acids (B) TYPE: amino acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 GIn Gly Met Phe Wing Gln Leu Val Wing Gln Asn Val Leu Leu lie Asp 1 5 10 15 Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Wing Gly Val Ser Leu 20 25 30 Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Wing 35 40 45 Lys Wing Gly Val Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val 50 55 60 Val Wing Gly Glu Gly Ser Gly Ser Val Ser Leu Wing Leu His Leu Pro 65 70 75 80 Gln Leu Arg Ser Wing Wing Gly Wing Wing Wing Wing Leu Thr Val Asp Leu 85 90 95 Pro Pro Wing Being Ser Glu Wing Arg Asn Being Wing Phe Gly Phe Gln Gly 100 105 110 Arg Leu Leu His Leu Be Wing Gly Gln Arg Leu Gly Val His Leu His 115 120 125 Thr Glu Wing Arg Wing Arg His Wing Trp Gln Leu Thr Gln Gly Wing Thr 130 135 140 Val Leu Gly Leu Phe Arg Val Thr Pro Glu Leu Pro Wing Gly Leu Pro 145 150 155 160 Ser Pro Arg Ser Glu 165

Claims (44)

1. REIVI NDICATIONS
2. 1 . A method for detecting the presence of TREPA target polynucleotide in a test sample, comprising: (a) contacting said test sample with at least one specific TREPA polynucleotide or complement thereof; and (b) detecting the presence of said TREPA target polynucleotide in the test sample, wherein said specific TREPA polynucleotide has at least 50% identity to the SEQUENCE ID NO 1 polynucleotide and fragments, analogs and complements thereof. The method of claim 1, wherein said target TREPA polynucleotide is attached to a solid phase before performing step (a).
3. 3. A method for detecting TREPA mRNA in a test sample, comprising: (a) performing reverse transcription with at least one primer in order to produce cDNA; (b) amplifying said cDNa obtained from step (a) by using another TREPA oligonucleotide primer (s) as a sense and antisense initiator (s) in a first step amplification to obtain a TREPA amplicon; and (c) detecting the presence of said TREPA amplicon in the test sample, wherein said TREPA oligonucleotide primers have at least 50% identity for SECU ENCIA ID NO 1 and fragments, analogues or complements thereof. the same.
4. 4. The method of separation 3, wherein said test sample is reacted with a solid phase before performing step (a) or step (b) or step (c).
5. The method of claim 3, wherein said detection step comprises using a detectable tag capable of generating a measurable signal.
6. 6. A method for detecting target TREPA polynucleotide in a test sample suspected of containing said target, comprising: (a) contacting said target TREPA polynucleotide with at least one TREPA oligonucleotide as a sense primer and with at least one TREPA oligonucleotide as an antisense initiator and amplifying them to obtain a first stage reaction product; (b) contacting said first step reaction product with at least one other TREPA oligonucleotide, with the proviso that the other TREPA oligonucleotide is located 3 'for the TREPA oligonucleotides used in step (a) and is complementary to said product of first stage reaction; and (c) detecting said TRE PA target nucleotide polynucleotide, wherein said TREPA oligonucleotides used in step (a) and step (b) have at least 50% identity for the S ECU E NC IA ID NO 1 and fragments, analogues or complements thereof.
7. 7. The method of claim 6, wherein said test sample is reacted with a solid phase before performing step (a) or step (b) or step (c).
8. 8. The method of claim 6, wherein said detection step comprises using a detectable label capable of generating a measurable signal.
9. The method of claim 8, wherein said detectable label is reacted with a solid phase. 1 0.
10. A test set useful for detecting TREPA polynucleotide in a test sample, comprising a container containing at least one TREPA polynucleotide having at least 50% for SEQUENCE I D NO 1 and fragments, analogues or complements thereof. eleven .
11. A purified polynucleotide or fragment thereof from the TREPA gene derivative, wherein said purified polynucleotide is capable of selectively hybridizing to the nucleic acid of said TREPA gene, and wherein said purified polynucleotide has at least 50% identity to SEQUENCE ID NO 1 and fragments, analogues or complements thereof.
12. 12. The purified polynucleotide of claim 1, wherein said purified polynucleotide is produced by recombinant techniques.
13. The purified polynucleotide of claim 12, wherein said polynucleotide produced by recombinant techniques comprises a sequence of at least one epitope encoded by TREPA. 1 4.
14. A system of recombinant expression comprising a nucleic acid sequence that encodes a reading frame derived from TREPA, which is operably linked to a control sequence compatible with a desired host and wherein said nucleic acid sequence has at least 50% identity for SECU ENCIA I D NO 1 and fragments, analogs or complements thereof. 5.
15. The recombinant expression system of claim 14 further comprising a cell transformed with said recombinant expression system.
16. 16. A polypeptide encoded by TREPA, wherein said polypeptide has at least 35% identity for amino acid sequence selected from the group consisting of SECUENCE I D NO 2, SEQUENCE I D NO 3 and fragments thereof. 7.
17. The polypeptide of claim 16, wherein said polypeptide is produced by recombinant technology.
18. The polypeptide of claim 16, wherein said polypeptide is produced by synthetic techniques. 1 9.
19. A compound which inhibits the activation of the TREPA polypeptide of claim 1 6.
20. The polypeptide of claim 16, wherein said polypeptide is a soluble fragment of the TREPA protein and is capable of binding to a receptor for TRIP twenty-one .
21. A method for treating a patient in need of inducing activation of the TREPA polypeptide of claim 16, comprising administering to a patient a therapeutically effective amount of a compound, which induces activation of the TREPA polypeptide of claim 16.
22. 22. A method for determining whether a compound is an agonist or antagonist for the TREPA protein, comprising: (a) contacting a cell having a TREPA protein expressed on its surface with said compound and a receptor ligand; (b) determining whether a biological effect is produced from the interaction of said cell and said compound; and (c) determining whether said compound is an agonist or antagonist.
23. 23. A method for determining whether a receptor binds to a TREPA ligand, comprising: (a) contacting a mammalian cell which expresses the TREPA ligand with a receptor; (b) detecting the presence of the receptor; and (c) determining whether the receptor binds to the TREPA ligand.
24. 24. An antibody, which binds specifically to at least one epitope encoded by TREPA, wherein said antibody is polyclonal or monoclonal, and wherein said epitope comprises an amino acid sequence having at least 35% identity for a sequence of amino acids selected from the group consisting of SEQUENCE ID NO 2, SEQUENCE ID NO 3 and fragments thereof.
25. 25. A test suite for determining the presence of TREPA antigen or antibody in a test sample, comprising a container containing a TREPA polypeptide having at least 35% identity for an amino acid sequence selected from the group consisting of SEQ ID NO. NO 2, SEQUENCE ID NO 3 and fragments of the same.
26. 26. The assay set of claim 25, wherein said polypeptide is bound to a solid phase.
27. 27. A test set for determining the presence of TREPA antigen or antibody in a test sample, comprising a container containing an antibody, which is specifically bound to TREPA antigen, wherein said antigen comprises at least one epitope of TREPA having at least 60% similarity for a sequence selected from the group consisting of SEQUENCE ID NO 2, SEQUENCE ID NO 3 and fragments thereof.
28. 28. The assembly of claim 27, wherein said antibody is bound to a solid phase.
29. 29. A method for producing a polypeptide comprising at least one TREPA epitope, said method comprises incubating transformed host cells with an expression vector, wherein said vector comprises a polynucleotide sequence encoding a polypeptide, said polypeptide comprises an amino acid sequence having at least 35% identity for a sequence of amino acids selected from the group consisting of SEQ ID NO. 2, SEQUENCE ID NO. 3 and fragments thereof.
30. 30. A method for detecting TREPA antigen in a test sample suspected of containing a TREPA antigen, comprising: (a) contacting said test sample with an antibody or fragment thereof, which is specifically binds to at least one TREPA antigen epitope selected from the group consisting of

    SECU ENC IA I D NO 2, S ECU ENC IA I D NO 3 and fragments thereof, for a time and under conditions sufficient for the formation of antibody / antigen complexes; and (b) detecting said complexes.
31. 31 The method of claim 30, wherein said antibody is bound to a solid phase.
32. 32. A method for detecting antibodies which bind to a TREPA antigen in a test sample suspected of containing said antibodies, comprising: (a) contacting said test sample with a TREPA polypeptide, wherein said polypeptide TREPA contains at least one TREPA epitope comprising an amino acid sequence or fragment thereof having at least 35% identity for an amino acid sequence selected from the group consisting of S ECU ENCIA ID NO 2, SEQUENCE ID NO 3 and fragments of them, for a time and under conditions sufficient to allow the antigen / antibody complexes to form; (b) detecting said complexes.
33. 33. The method of claim 32, wherein said TREPA polypeptide is attached to a solid phase.
34. 34. A cell grown in tissue culture comprising a nucleic acid sequence encoding at least one TREPA antigen epitope or a fragment thereof, wherein said nucleic acid sequence is transfected into said cell, and wherein said sequence of nucleic acids is the SEQUENCE ID NO 1 and fragments, analogues or complements of the same.
35. 35. A method for producing antibodies which specifically bind to TREPA antigen, comprising administering to an individual an isolated immunogenic polypeptide or fragment thereof, wherein said isolated immunogenic polypeptide comprises at least one TREPA epitope and has at least 35% identity for a sequence selected from the group consisting of SEQUENCE ID NO 2, SEQUENCE ID NO 3 and fragments thereof, in an amount sufficient to produce an immune response.
36. 36. A method for producing antibodies, which specifically bind to TREPA antigen, comprising administering to a mammal a plasmid comprising a sequence, which encodes at least one TREPA epitope, wherein said TREPA sequence is selected from the group consisting of of SEQUENCE ID NO 1 and fragments or complements thereof.
37. 37. A composition of matter comprising a TREPA polynucleotide or fragment thereof, wherein said nucleotide has at least 50% identity for SEQUENCE I D NO 1 and fragments, analogs or complements thereof.
38. 38. A composition of matter comprising a polypeptide containing at least one epitope encoded by TREPA, wherein said polypeptide has at least 35% > of identity for a selected sequence of the group consisting of SECTION I D NO 2, S ECU ENCE I D NO 3 and fragments thereof.
39. 39. The composition of matter of claim 38, wherein said polypeptide is a soluble fragment of the TREPA protein and is capable of going to a receptor for TREPA.
40. 40. The test set of claim 10, further comprising a container containing useful tools for collection of said sample selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups.
41. 41 The test set of claim 25, further comprising a container containing useful tools for collection of said sample selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups.
42. 42. The test set of claim 27, further comprising a container containing tools useful for collection of said sample selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups.
43. 43. A gene or fragment thereof, which encodes TREPA protein, which comprises an amino acid sequence that has at least 35% identity for SECU ENCIA ID NO 5.
44. 44. A gene or fragment thereof, comprising DNA that has at least 35% identity for SECU ENCIA ID NO 4.