WO2002048354A1 - Tumour suppressor gene identified on chromosome 18 - Google Patents

Abstract

The invention provides an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:1, or active fragment thereof, which encodes a polypeptide active in suppressing cellular proliferation. In particular, TSG18 functions as a tumour suppressor gene as well as having a role in immune/autoimmune/inflammatory disorders. It also provides variants of such DNA molecules which retain their function, polypeptides encoded by the DNA molecules and antibodies thereto, as well as the use of these molecules in diagnostic, prognostic and therapeutic procedures and other uses such as screening for candidate pharmaceuticals.

Description

Tumour Suppressor Gene Identified On Chromosome 18

Technical Field

The present invention relates to the identification of a novel gene, TSG18, with a role in suppressing cellular proliferation via a tumour suppressor mechanism as well as a role in immune/autoimmune/inflammatory functions and associated disorders. In view of this realisation, the invention is also concerned with the therapy of pathologies shown to be associated with TSG18, the screening of drugs to treat these pathologies, and the diagnosis and prognosis of pathologies shown to be associated with TSG18.

Background Art The development of human carcinomas has been shown to arise from the accumulation of genetic changes involving both positive regulators of cell function (oncogenes) and negative regulators (tumour suppressor genes) . For a normal somatic cell to evolve into a metastatic tumour it requires changes at the cellular level, such as immortalisation, loss of contact inhibition and invasive growth capacity, and changes at the tissue level, such as evasion of host immune responses and growth restraints imposed by surrounding cells, and the formation of a blood supply for the growing tumour.

Molecular genetic studies of colorectal carcinoma have provided substantial evidence that the generation of malignancy requires the sequential accumulation of a number of genetic changes within the same epithelial stem cell of the colon. For a normal colonic epithelial cell to become a benign adenoma, progress to intermediate and late adenomas, and finally become a malignant cell, inactivating mutations in tumour suppressor genes and activating mutations in proto-oncogenes are required (Fearon and Vogelstein, 1990) .

Tumour suppressor genes were first identified in the childhood cancer retinoblastoma. Both inherited and sporadic forms of this cancer exist, with the familial form inherited as a highly penetrant autosomal dominant trait, which was mapped to chromosome 13ql4 by genetic linkage analysis (Sparkes et al . , 1983). The observation that bilateral retinoblastoma was characteristic of the inherited disease and occurred at an early age, whereas unilateral retinoblastoma was characteristic of the sporadic form and occurred at a later age, led to the hypothesis that the tumour arises from two mutational steps (Knudson, 1971) . With this proposition, familial cancers would result from an inherited germline mutation of a gene suppressing the growth of cells (tumour suppressor gene) , such that all cells would carry this mutation. A second mutation or "hit" in any cell therefore resulted in the manifestation of the recessive mutation leading to cancer. The fact that only one more "hit" produces a cancerous cell meant that individuals with an inherited pre-disposition to the disease had an earlier age of onset and often bilateral tumours. In contrast, sporadic cases tended to be in one eye and later in onset because two "hits" were needed to the genes in the same cell.

This hypothesis was confirmed with the use of genetic markers mapping to 13ql4 to type DNA isolated from blood and tumour samples taken from the same affected individuals (Cavenee et al . , 1983). In several cases the constitutional DNA from lymphocytes was heterozygous for some markers but the tumour cells appeared homozygous for the same markers. The apparent reduction to homozygosity (or loss of heterozygosity, LOH) through the loss of one allele of these markers was suggested to be the second "hit" which was removing the remaining functional copy of the retinoblastoma gene in these individuals. The analysis of tumours in familial cases showed that the chromosome from the unaffected parent was in each instance the one eliminated from the tumour. A number of mechanisms were proposed including mitotic recombination, mitotic non- disjunction with loss of the wild-type allele or reduplication of the mutant allele, and gene conversion, deletion or mutation.

In addition to retinoblastoma, studies of other cancers have supported the model that LOH is a specific event in the pathogenesis of cancer. In Von Hippel-Lindau (VHL) syndrome both sporadic and inherited cases of the syndrome show LOH for the short arm of chromosome 3. Somatic translocations involving 3p in sporadic tumours, and genetic linkage to the same region in affected families has also been observed. Similarly, in colorectal carcinoma, inherited forms of the disease have been mapped to the long arm of chromosome 5 while LOH at 5q has been reported in both the familial and sporadic versions of the disease and the APC gene, mapping to this region, has been shown to be involved (Groden et al . , 1991). Other examples, which include the TP53 and NF2 genes, firmly establish the fact that a general mechanism in human cancer is the inactivation of tumour suppressor genes by LOH. Indeed LOH in tumour DNA is now taken as being strongly indicative of the presence and inactivation of a tumour suppressor gene.

Breast cancer is the most common malignancy seen in women, affecting approximately 10% of females in the Western world. The route to breast cancer is not as well mapped as that of colon cancer due in part to the histological stages of breast cancer development being less well defined. It is known however, that breast cancer is derived from the epithelial lining of terminal mammary ducts or lobuli. Hormonal influences, such as those exerted by oestrogen, are believed to be important because of the marked increase in breast cancer incidence in post- enopausal women, but the initial steps in breast cancer development probably occur before the onset of menopause. As with colon carcinoma, it is believed that a number of genes need to become involved in a stepwise progression during breast tumourigenesis . Certain women appear to be at an increased risk of developing breast cancer. Genetic linkage analysis has shown that 5 to 10% of all breast cancers are due to at least two autosomal dominant susceptibility genes. Generally, women carrying a mutation in a susceptibility gene develop breast cancer at a younger age compared to the general population, often have bilateral breast tumours, and are at an increased risk of developing cancers in other organs, particularly carcinoma of the ovary.

Genetic linkage analysis on families showing a high incidence of early-onset breast cancer (before the age of 46) was successful in mapping the first susceptibility gene, BRCA1, to chromosome 17q21 (Hall et al . , 1990). Subsequent to this, the BRCA2 gene was mapped to chromosome 13ql2-ql3 (Wooster et al., 1994) with this gene conferring a higher incidence of male breast cancer and a lower incidence of ovarian cancer when compared to BRCA1. Both BRCAl and BRCA2 have since been cloned (Miki et al., 1994; Wooster et al . , 1995) and numerous mutations have been identified in these genes in susceptible individuals with familial cases of breast cancer.

Additional inherited breast cancer syndromes exist, however they are rare. Inherited mutations in the TP53 gene have been identified in individuals with Li-Fraumeni syndrome, a familial cancer resulting in epithelial neoplasms occurring at multiple sites including the breast. Similarly, germline mutations in the MMAC1/PTEN gene involved in Cowden's disease and the ataxia telangiectasia (AT) gene have been shown to confer an increased risk of developing breast cancer, among other clinical manifestations, but together account for only a small percentage of families with an inherited predisposition to breast cancer. Somatic mutations in the TP53 gene have been shown to occur in a high percentage of individuals with sporadic breast cancer. However, although LOH has been observed at the BRCAl and BRCA2 loci at a frequency of 30 to 40% in sporadic cases (Cleton-Jansen et al . , 1995; Saito et al., 1993), there is virtually no sign of somatic mutations in the retained allele of these two genes in sporadic cancers (Futreal et al . , 1994; Miki et al . , 1996). Recent data suggests that DNA methylation of the promoter sequence of these genes may be an important mechanism of down- regulation. The use of both restriction fragment length polymorhisms and small tandem repeat polymorphic markers has identified numerous regions of allelic imbalance in breast cancer suggesting the presence of additional tumour suppressor genes, which may be implicated in breast cancer. Data compiled from more than 30 studies reveals the loss of DNA from at least 11 chromosome arms at a frequency of more than 25%, with regions such as 16q and 17p affected in more than 50% of tumours (Devilee and Cornelisse, 1994; Brenner and Aldaz, 1995) . However only some of these regions are known to harbour tumour suppressor genes shown to be mutated in individuals with both sporadic ( TP53 and RB genes) and familial (TP53, RB,

BRCAl, and BRCA2 genes) forms of breast cancer.

Cytogenetic studies have implicated loss of the long arm of chromosome 16 as an early event in breast carcinogenesis since it is found in tumours with few or no other cytogenetic abnormalities. Alterations in chromosome 1 and 16 have also been seen in several cases of duetal carcinoma i situ (DCIS), the preinvasive stage of ductal breast carcinoma. In addition, LOH studies on DCIS samples identified loss of 16q markers in 29 to 89% of the cases tested (Chen et al . , 1996; Radford efc al., 1995). Together, these findings suggest the presence of a tumour suppressor gene mapping to the long arm of chromosome 16 that is critically involved in the early development of a large proportion of breast cancers. Recently the TSG16 gene was identified from a region of restricted LOH at 16q24.3 (International Patent Application Number PCT/AU00/01329, the contents of which are incorporated herein by reference) . This gene encodes a polypeptide active in suppressing cellular proliferation. In particular, TSG16 functions as a tumour suppressor gene as well as having a role in immune/autoimmune/inflammatory disorders.

We have identified a gene that maps to chromosome 18pll.3 (TSG18), which together with TSG16 defines a new protein family. Chromosome 18pll alterations, as with 16q24.3 alterations, appear to be a common and early event in breast disease (Tran et al . , 1998; Kittiniyom et al . , 2001) . Allelic deletion of chromosome 18pll has also been observed in tumours of the lung and brain (Tran et al . , 1998), while other LOH studies have indicated the presence of tumour suppressor gene(s) mapping to the short arm of chromosome 18 in cervical carcinomas (Mitra et al . , 1994; Mullokandov et al., 1996). In addition, constitutional deletion of 18p has also been implicated in the early onset of gastric carcinomas (Dellavecchia et al . , 1999). Therefore, as with TSG16, the TSG18 gene may also be implicated in tumour suppression as well as other functions related to TSG16, such as those associated with immune/a toimmune/inflammatory responses .

Disclosure of the Invention The present invention provides an isolated mammalian nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, which encodes a polypeptide active in suppressing cellular functions associated with cancer. It will be understood that cellular functions associated with cancer include but are not restricted to, singly or in combination, cell proliferation, cell cycle, cell survival, invasion and growth receptor responses. The suppression of these cellular functions is frequently referred to as tumour suppression function and the genes which encode proteins having this function as tumour suppressor genes.

The invention also encompasses an isolated mammalian nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l and which encodes a polypeptide active in suppressing cellular functions associated with cancer. These include, but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses.

Such variants will have preferably at least about 85%, and most preferably at least about 95% sequence identity to the nucleotide sequence encoding TSG18. A particular aspect of the invention encompasses a variant of SEQ ID NO:l which has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% sequence identity to SEQ ID NO:l. Any one of the polynucleotide variants described above can encode an amino acid sequence, which contains at least one functional or structural characteristic of TSG18.

Typically sequence identity is calculated using the BLASTN algorithm with the BLOSSUM62 default matrix. The invention also encompasses an isolated mammalian nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer, including but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.

Under stringent conditions, hybridization with ³²P labelled probes will most preferably occur at 42°C in 750 mM NaCl, 75 mM trisodium citrate, 2% SDS, 50% formamide,

IX Denhart's, 10% (w/v) dextran sulphate and 100 μg/ml denatured salmon sperm DNA. Useful variations on these conditions will be readily apparent to those skilled in the art. The washing steps which follow hybridization most preferably occur at 65°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the ar .

The invention also provides an isolated mammalian nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. Still further, the invention encompasses an isolated mammalian nucleic acid molecule wherein the amino acid sequence has at least 70%, preferably 85%, and most preferably 95%, sequence identity to the sequence set forth in SEQ ID NO: 2. Preferably, sequence identity is determined using the BLASTP algorithm with the BLOSSUM62 default matrix.

The invention also encompasses an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.

Also envisaged is an isolated nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID

NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets. In a further aspect the invention is concerned with an isolated nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.

Still further, there is provided an isolated nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO: 2. In a still further aspect, there is provided an isolated nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l. Still further, there is provided an isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.

In a still further aspect, there is provided an isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l from base 215 to base 6,401.

In a further aspect the invention provides an isolated gene comprising the nucleotide sequence set forth in SEQ ID NO:l and TSG18 control elements, particularly the cis and trans elements which act in breast tissue.

The nucleotide sequences of the present invention can be engineered using methods accepted in the art so as to alter TSG18-encoding sequences for a variety of purposes. These include, but are not limited to, modification of the cloning, processing, and/or expression of the gene product. PCR reassembly of gene fragments and the use of synthetic oligonucleotides allow the engineering of TSG18 nucleotide sequences. For example, oligonucleotide- mediated site-directed mutagenesis can introduce mutations that create new restriction sites, alter glycosylation patterns and produce splice variants etc.

As a result of the degeneracy of the genetic code, a number of polynucleotide sequences encoding TSG18, some that may have minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention includes each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring TSG18, and all such variations are to be considered as being specifically disclosed.

The polynucleotides of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified, or may contain non-natural or derivatised nucleotide bases as will be appreciated by those skilled in the art . Such modifications include labels, methylation, intercalators, alkylators and modified linkages. In some instances it may be advantageous to produce nucleotide sequences encoding TSG18 or its derivatives possessing a substantially different codon usage than that of the naturally occurring TSG18. For example, codons may be selected to increase the rate of expression of the peptide in a particular prokaryotic or eukaryotic host corresponding with the frequency that particular codons are utilized by the host. Other reasons to alter the nucleotide sequence encoding TSG18 and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

The invention also encompasses production of DNA molecules, which encode TSG18 and its derivatives, or fragments thereof, entirely by synthetic chemistry. Synthetic sequences may be inserted into expression vectors and cell systems that contain the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host . These elements may include regulatory sequences, promoters, 5' and 3 ' untranslated regions and specific initiation signals (such as an ATG initiation codon and Kozak consensus sequence) which allow more efficient translation of sequences encoding TSG18. In cases where the complete TSG18 coding sequence including its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, additional control signals may not be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals as described above should be provided by the vector. Such signals may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf et al . , 1994).

The present invention allows for the preparation of purified TSG18 polypeptide or protein, from the polynucleotides of the present invention or variants thereof. In order to do this, host cells may be transfected with a DNA molecule as described above. Typically said host cells are transfected with an expression vector comprising a DNA molecule according to the invention. A variety of expression vector/host systems may be utilized to contain and express sequences encoding TSG18. These include, but are not limited to, microorganisms such as bacteria transformed with plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); or mouse or other animal or human tissue cell systems. Mammalian cells can also be used to express the TSG18 protein using various expression vectors including plasmid, cosmid and viral systems such as adenoviral, retroviral or vaccinia virus expression systems. The invention is not limited by the host cell employed.

The polynucleotide sequences, or variants thereof, of the present invention can be stably expressed in cell lines to allow long term production of recombinant proteins in mammalian systems. Sequences encoding TSG18 can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. The selectable marker confers resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode TSG18 may be designed to contain signal sequences which direct secretion of TSG18 through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, glycosylation, phosphorylation, and acylation. Post-translational cleavage of a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells having specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO or HeLa cells), are available from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the foreign protein.

When large quantities of TSG18 are needed such as for antibody production, vectors which direct high levels of expression of TSG18 may be used such as those containing the T5 or T7 inducible bacteriophage promoter. The present invention also includes the use of the expression systems described above in generating and isolating fusion proteins which contain important functional domains of the protein. These fusion proteins are used for binding, structural and functional studies as well as for the generation of appropriate antibodies.

In order to express and purify the protein as a fusion protein, the appropriate TSG18 cDNA sequence is inserted into a vector which contains a nucleotide sequence encoding another peptide (for example, glutathionine succinyl transferase) . The fusion protein is expressed and recovered from prokaryotic or eukaryotic cells. The fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the TSG18 protein obtained by enzymatic cleavage of the fusion protein.

Fragments of TSG18 may also be produced by direct peptide synthesis using solid-phase techniques. Automated synthesis may be achieved by using the ABI 431A Peptide Synthesizer (Perkin-Elmer) . Various fragments of TSG18 may be synthesized separately and then combined to produce the full length molecule. According to the present invention there is provided

According to a still further aspect of the invention there is provided an isolated mammalian polypeptide, comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, active in suppressing cellular functions associated with cancer, including but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses .

The invention also encompasses an isolated mammalian polypeptide active in suppressing cellular functions associated with cancer, including but not restricted to, one or more of cell proliferation, cell cycle, cell survival, invasion and growth receptor responses and having at least 70%, more preferably at least 85%, and most preferably at least 95%, sequence identity with the amino acid sequence set forth in SEQ ID NO: 2.

Preferably, sequence identity is determined using the BLASTP algorithm with the BLOSSUM62 default matrix.

In a further aspect the invention provides an isolated polypeptide, comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.

Still further, the invention provides an isolated polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% identity with the amino acid sequence set forth in SEQ ID NO: 2.

Also envisaged is an isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2. In a further aspect of the invention there is provided a method of preparing a polypeptide, comprising the steps of :

(1) culturing the host cells described above under conditions effective for production of the polypeptide; and

(2 ) harvesting the polypeptide.

Substantially purified TSG18 protein or fragments thereof can then be used in further biochemical analyses to establish secondary and tertiary structure for example by x-ray crystallography of TSG18 protein or by nuclear magnetic resonance (NMR) . Determination of structure allows for the rational design of pharmaceuticals to interact with the protein, alter protein charge configuration or charge interaction with other proteins, or to alter its function in the cell.

Chemical and structural similarity in the context of sequences and motifs, exists between regions of both TSG18 and TSG16 and the ankyrin repeat containing family of proteins including BARD1 and 1KB. TSG16 interacts via its ankyrin repeats with members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family, which are proteins that bind to STAT (signal transducer and activator of transcription) proteins to inhibit the immunological responses mediated by cytokine signalling. As TSG18 shows significant homology to TSG16, particularly across its ankyrin domains, TSG18 is likely to also interact with members of the PIAS family. Therefore abnormalities of TSG18 function may be associated not only with cancer but also with immune diseases including autoimmune/inflammatory disorders . The invention has provided the nucleotide and protein sequence of the TSG18 gene and therefore enables therapeutic methods for the treatment of all diseases shown to be associated with abnormalities of TSG18 function, including cancer and immune/autoimmune/inflammatory disorders and also enables methods for the diagnosis or prognosis of all diseases shown to be associated with abnormalities of TSG18 function.

Examples of such disorders include, but are not limited to, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, and stomach. Other cancers may include those of the adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland. Immune/autoimmune/inflammatory disorders include acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

In the treatment of diseases shown to be associated with decreased TSG18 expression and/or activity, it is desirable to increase the activity and/or expression of TSG18. In the treatment of disorders shown to be associated with increased TSG18 expression and/or activity, it is desirable to decrease the activity and/or expression of TSG18.

Enhancing TSG18 gene or protein function Enhancing, stimulating or re-activating TSG18 gene or protein function can be achieved in a variety of ways. In one aspect of the invention administration of an isolated DNA molecule, as described above, to a subject in need of such treatment may be initiated. Typically, TSG18 is administered to a subject to treat or prevent a disorder shown to be associated with decreased activity and/or expression of TSG18.

In a further aspect, there is provided the use of an isolated DNA molecule, as described above, in the manufacture of a medicament for the treatment of a disorder shown to be associated with decreased activity and/or expression of TSG18.

Typically, a vector capable of expressing TSG18 or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder shown to be associated with decreased activity and/or expression of

TSG18 including, but not limited to, those described above. Transducing retroviral vectors are often used for somatic cell gene therapy because of their high efficiency of infection and stable integration and expression. The full length TSG18 gene, or portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest. Other viral vectors can be used and include, as is known in the art, adenoviruses, adeno-associated virus, vaccinia virus, papovaviruses, lentiviruses and retroviruses of avian, murine and human origin.

Gene therapy would be carried out according to established methods (Friedman, 1991; Culver, 1996) . A vector containing a copy of the TSG18 gene linked to expression control elements and capable of replicating inside the cells is prepared. Alternatively the vector may be replication deficient and may require helper cells for replication and use in gene therapy.

Gene transfer using non-viral methods of infection can also be used. These methods include direct injection of DNA, uptake of naked DNA in the presence of calcium phosphate, electroporation, protoplast fusion or liposome delivery. Gene transfer can also be achieved by delivery as a part of a human artificial chromosome or receptor- mediated gene transfer. This involves linking the DNA to a targeting molecule that will bind to specific cell- surface receptors to induce endocytosis and transfer of the DNA into mammalian cells . One such technique uses poly-L-lysine to link asialoglycoprotein to DNA. An adenovirus is also added to the complex to disrupt the lysosomes and thus allow the DNA to avoid degradation and move to the nucleus. Infusion of these particles intravenously has resulted in gene transfer into hepatocytes . Although not identified to date, it is possible that certain individuals with TSG18-related disorders contain an abnormality in TSG18. In affected subjects that express a mutated form of TSG18 it may be possible to prevent the disorder by introducing into the affected cells a wild- type copy of the gene such that it recombines with the mutant gene. This requires a double recombination event for the correction of the gene mutation. Vectors for the introduction of genes in these ways are known in the art, and any suitable vector may be used. Alternatively, introducing another copy of the gene bearing a second mutation in that gene may be employed so as to negate the original gene mutation and block any negative effect.

In a still further aspect the invention provides a method for the treatment of a disorder shown to be associated with decreased activity and/or expression of TSG18, comprising administering a polypeptide as described above, or an agonist thereof, to a subject in need of such treatment .

In another aspect the invention provides the use of a polypeptide as described above, or an agonist thereof, in the manufacture of a medicament for the treatment of a disorder shown to be associated with decreased activity and/or expression of TSG18.

In a further aspect a pharmaceutical composition comprising a polypeptide as described above, typically a substantially purified TSG18, and a pharmaceutically acceptable carrier may be administered.

The pharmaceutical composition may be administered to a subject to treat or prevent a disorder associated with decreased activity and/or expression of TSG18 including, but not limited to, those provided above. Pharmaceutical compositions in accordance with the present invention are prepared by mixing TSG18 or active fragments or variants thereof having the desired degree of purity, with acceptable carriers, excipients, or stabilizers which are well known. Acceptable carriers, excipients or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including absorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitrol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG) .

In a further aspect, a suitable agonist may also include a small molecule that can mimic the function of TSG18.

Inhibiting TSG18 gene or protein function

Inhibiting the function of a mutated gene or protein can be achieved in a variety of ways . In one aspect of the invention there is provided a method of treating a disorder shown to be associated with increased activity and/or expression of TSG18, comprising administering an antagonist of TSG18 to a subject in need of such treatment .

In still another aspect of the invention there is provided the use of an antagonist of TSG18 in the manufacture of a medicament for the treatment of a disorder shovm to be associated with increased activity and/or expression of TSG18.

Such disorders may include, but are not limited to, those discussed above. In one aspect of the invention an isolated DNA molecule, which is the complement of any one of the DNA molecules described above and which encodes an RNA molecule that hybridises with the mRNA encoded by TSG18, may be administered to a subject in need of such treatment.

In a still further aspect of the invention there is provided the use of an isolated DNA molecule which is the complement of a DNA molecule of the invention and which encodes an RNA molecule that hybridises with the mRNA encoded by TSG18, in the manufacture of a medicament for the treatment of a disorder shown to be associated with increased activity and/or expression of TSG18.

Typically, a vector expressing the complement of the polynucleotide encoding TSG18 may be administered to a subject to treat or prevent a disorder shown to be associated with increased activity and/or expression of TSG18 including, but not limited to, those described above. Antisense strategies may use a variety of approaches including the use of antisense oligonucleotides, ribozymes, DNAzy es, injection of antisense RNA and transfection of antisense RNA expression vectors. Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (For example, see Goldman et al . , 1997).

According to still another aspect of the invention, there is provided a method of treating a disorder shown to be associated with increased activity and/or expression of

TSG18 comprising administering an antagonist of TSG18 to a subject in need of such treatment.

In still another aspect of the invention there is provided the use of an antagonist of TSG18 in the manufacture of a medicament for the treatment of a disorder shown to be associated with increased activity and/or expression of TSG18.

Such disorders may include, but are not limited to, those discussed above. In one aspect purified protein according to the invention may be used to produce antibodies which specifically bind TSG18. These antibodies may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues that express TSG18. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric and single chain antibodies as would be understood by the person skilled in the art .

For the production of antibodies, various hosts including rabbits, rats, goats, mice, humans, and others may be immunized by injection with a protein of the invention or with any fragment or oligopeptide thereof, which has immunogenic properties. Various adjuvants may be used to increase immunological response and include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin. Adjuvants used in humans include BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to TSG18 have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of amino acids from these proteins may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced. Monoclonal antibodies to TSG18 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (For example, see Kohler et al . , 1975; Kozbor et al . , 1985; Cote et al . , 1983; Cole et al . , 1984) . Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (For example, see Orlandi et al., 1989; Winter et al., 1991).

Antibody fragments which contain specific binding sites for TSG18 may also be generated. For example, such fragments include, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments . Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (For example, see Huse et al . , 1989). Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between a protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed.

In further embodiments, any of the genes, proteins, antagonists, antibodies, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents may be made by those skilled in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, therapeutic efficacy with lower dosages of each agent may be possible, thus reducing the potential for adverse side effects. Drug screening

According to still another aspect of the invention, peptides of the invention, particularly purified TSG18 polypeptides, and cells expressing these are useful for screening of candidate pharmaceutical agents in a variety of techniques for the treatment of TSG18-related disorders. Such techniques include, but are not limited to, utilising eukaryotic or prokaryotic host cells that are stably transformed with recombinant molecules expressing the TSG18 polypeptide or fragment thereof, preferably in competitive binding assays. Binding assays will measure for the formation of complexes between the TSG18 polypeptide, or fragments thereof, and the agent being tested, or will measure the degree to which an agent being tested will interfere with the formation of a complex between the TSG18 polypeptide, or fragment thereof, and a known ligand.

Another technique for drug screening provides high- throughput screening for compounds having suitable binding affinity to the TSG18 polypeptide (see PCT published application WO84/03564) . In this stated technique, large numbers of small peptide test compounds can be synthesised on a solid substrate and can be assayed through TSG18 polypeptide binding and washing. Bound TSG18 polypeptide is then detected by methods well known in the art . In a variation of this technique, purified polypeptides can be coated directly onto plates to identify interacting test compounds . An additional method for drug screening involves the use of host eukaryotic cell lines which carry mutations in the TSG18 gene. The host cell lines are also defective at the polypeptide level. Other cell lines may be used where the gene expression of TSG18 can be switched off. The host cell lines or cells are grown in the presence of various drug compounds and the rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of defective cells.

Mutant TSG18 polypeptide may also be used for screening compounds developed as a result of combinatorial library technology. This provides a way to test a large number of different substances for their ability to modulate activity of a polypeptide. The use of peptide libraries is preferred (see patent WO97/02048) with such libraries and their use known in the art.

A substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature. Non- peptide "small molecules" are often preferred for many in vivo pharmaceutical applications. In addition, a mimic or mimetic of the substance may be designed for pharmaceutical use. The design of mimetics based on a known pharmaceutically active compound ("lead" compound) is a common approach to the development of novel pharmaceuticals. This is often desirable where the original active compound is difficult or expensive to synthesise or where it provides an unsuitable method of administration. In the design of a mimetic, particular parts of the original active compound that are important in determining the target property are identified. These parts or residues constituting the active region of the compound are known as its pharmacophore . Once found, the pharmacophore structure is modelled according to its physical properties using data from a range of sources including x-ray diffraction data and NMR. A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be added. The selection can be made such that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, does not degrade in vivo and retains the biological activity of the lead compound. Further optimisation or modification can be carried out to select one or more final mimetics useful for in vivo or clinical testing.

It is also possible to isolate a target-specific antibody and then solve its crystal structure. In principle, this approach yields a pharmacophore upon which subsequent drug design can be based as described above. It may be possible to avoid protein crystallography altogether by generating anti-idiotypic antibodies (anti- ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analogue of the original binding site. The anti-id could then be used to isolate peptides from chemically or biologically produced peptide banks.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic and prognostic applications

Should abnormalities in TSG18 exist which give rise to TSG18-related disorders, the polynucleotide sequences of the invention may be used for the diagnosis or prognosis of these disorders, or a predisposition to such disorders. Examples of such disorders include, but are not limited to cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, and stomach. Other cancers may include those of the adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland.

Immune/autoimmune/inflammatory disorders include acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis. autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, poly yositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. Diagnosis or prognosis may be used to determine the severity, type or stage of the disease state in order to initiate an appropriate therapeutic intervention.

In another embodiment of the invention, the polynucleotides that may be used for diagnostic or prognostic purposes include oligonucleotide sequences, genomic DNA and complementary RNA and DNA molecules. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which mutations in TSG18 or abnormal expression of TSG18 may be correlated with disease. Genomic DNA used for the diagnosis or prognosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material. The DNA may be isolated and used directly for detection of a specific sequence or may be amplified by the polymerase chain reaction (PCR) prior to analysis. Similarly, RNA or cDNA may also be used, with or without PCR amplification. To detect a specific nucleic acid sequence, direct nucleotide sequencing, reverse transcriptase PCR (RT-PCR) , hybridization using specific oligonucleotides, restriction enzyme digest and mapping, PCR mapping, RNAse protection, and various other methods may be employed. Oligonucleotides specific to particular sequences can be chemically synthesized and labeled radioactively or non-radioactively and hybridised to individual samples immobilized on membranes or other solid-supports or in solution. The presence, absence or excess expression of TSG18 may then be visualized using methods such as autoradiography, fluorometry, or colorimetry.

In a particular aspect, the nucleotide sequences encoding TSG18 may be useful in assays that detect the presence of associated disorders, particularly those mentioned previously. The nucleotide sequences encoding TSG18 may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding TSG18 in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

In order to provide a basis for the diagnosis or prognosis of a disorder shown to be associated with a mutation in TSG18, the nucleotide sequence of the TSG18 gene can be compared between normal tissue and diseased tissue in order to establish whether the patient expresses a mutant gene. In order to provide a basis for the diagnosis or prognosis of a disorder shown to be associated with abnormal expression of TSG18, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding TSG18, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Another method to identify a normal or standard profile for expression of TSG18 is through quantitative RT-PCR studies. RNA isolated from body cells of a normal individual, particularly RNA isolated from tumour cells, is reverse transcribed and real-time PCR using oligonucleotides specific for the TSG18 gene is conducted to establish a normal level of expression of the gene.

Standard values obtained in both these examples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays or quantitative RT-PCR studies may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding TSG18 or closely related molecules may be used to identify nucleic acid sequences which encode TSG18. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding TSG18, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the TSG18 encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:1 or from genomic sequences including promoters, enhancers, and introns of the TSG18 gene (SEQ ID Numbers: 3-11) .

Means for producing specific hybridization probes for DNAs encoding TSG18 include the cloning of polynucleotide sequences encoding TSG18 or TSG18 derivatives into vectors for the production of mRNA probes . Such vectors are known in the art, and are commercially available. Hybridization probes may be labelled by radionuclides such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, or other methods known in the art .

According to a further aspect of the invention there is provided the use of a polypeptide as described above in the diagnosis or prognosis of a disorder shown to be associated with TSG18, or a predisposition to such disorders.

When a diagnostic or prognostic assay is to be based upon the TSG18 protein, a variety of approaches are possible. For example, diagnosis or prognosis can be achieved by monitoring differences in the electrophoretic mobility of normal and mutant proteins. Such an approach will be particularly useful in identifying mutants in which charge substitutions are present, or in which insertions, deletions or substitutions have resulted in a significant change in the electrophoretic migration of the resultant protein. Alternatively, diagnosis may be based upon differences in the proteolytic cleavage patterns of normal and mutant proteins, differences in molar ratios of the various amino acid residues, or by functional assays demonstrating altered function of the gene products.

In another aspect, antibodies that specifically bind TSG18 may be used for the diagnosis or prognosis of disorders characterized by abnormal expression of TSG18, or in assays to monitor patients being treated with TSG18 or agonists, antagonists, or inhibitors of TSG18. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic or prognostic assays for TSG18 include methods that utilize the antibody and a label to detect TSG18 in human body fluids or in extracts of cells or tissues . The antibodies may be used with or without modification, and may be labelled by covalent or non-covalent attachment of a reporter molecule.

A variety of protocols for measuring TSG18, including ΞLISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of TSG18 expression. Normal or standard values for TSG18 expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to TSG18 under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of TSG18 expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Once an individual has been diagnosed with a disorder, effective treatments can be initiated. These may include administering a selective agonist to the mutant TSG18 so as to restore its function to a normal level or introduction of wild-type TSG18, particularly through gene therapy approaches as described above. Typically, a vector capable of expressing the appropriate full length TSG18 gene or a fragment or derivative thereof may be administered. In an alternative approach to therapy, substantially purified TSG18 polypeptide and a pharmaceutically acceptable carrier may be administered as described above or drugs which can replace the function of, or mimic the action of TSG18 may be administered.

In the treatment of disorders shown to be associated with increased TSG18 expression and/or activity, the affected individual may be treated with a selective antagonist such as an antibody to the relevant protein or an antisense (complement) probe to the corresponding gene as described above, or through the use of drugs which may block the action of TSG18.

In further embodiments, complete cDNAs, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose or prognose a disorder, and to develop and monitor the activities of therapeutic agents. Microarrays may be prepared, used, and analyzed using methods known in the art. (For example, see Schena et al., 1996; Heller et al., 1997).

Transformed hosts

The present invention also provides for the production of genetically modified (knock-out, knock-in and transgenic), non-human animal models transformed with the DNA molecules of the invention. These animals are useful for the study of the TSG18 gene function, to study the mechanisms of disease as related to the TSG18 gene, for the screening of candidate pharmaceutical compounds, for the creation of explanted mammalian cell cultures which express the protein or mutant protein and for the evaluation of potential therapeutic interventions. The TSG18 gene may have been inactivated by knock-out deletion, and knock-out genetically modified non-human animals are therefore provided.

Animal species which are suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates such as monkeys and chimpanzees. For initial studies, genetically modified mice and rats are highly desirable due to their relative ease of maintenance and shorter life spans. For certain studies, transgenic yeast or invertebrates may be suitable and preferred because they allow for rapid screening and provide for much easier handling. For longer term studies, non-human primates may be desired due to their similarity with humans.

To create an animal model for mutated TSG18 several methods can be employed. These include generation of a specific mutation in a homologous animal gene, insertion of a wild type human gene and/or a humanized animal gene by homologous recombination, insertion of a mutant (single or multiple) human gene as genomic or minigene cDNA constructs using wild type or mutant or artificial promoter elements or insertion of artificially modified fragments of the endogenous gene by homologous recombination. The modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase.

To create a transgenic mouse, which is preferred, a mutant version of TSG18 can be inserted into a mouse germ line using standard techniques of oocyte microinjection or transfection or microinjection into embryonic stem cells. Alternatively, if it is desired to inactivate or replace the endogenous TSG18 gene, homologous recombination using embryonic stem cells may be applied.

For oocyte injection, one or more copies of the mutant or wild type TSG18 gene can be inserted into the pronucleus of a just-fertilized mouse oocyte. This oocyte is then reimplanted into a pseudo-pregnant foster mother. The liveborn mice can then be screened for integrants using analysis of tail DNA for the presence of human TSG18 gene sequences . The transgene can be either a complete genomic sequence injected as a YAC, BAC, PAC or other chromosome DNA fragment, a cDNA with either the natural promoter or a heterologous promoter, or a minigene containing all of the coding region and other elements found to be necessary for optimum expression.

According to still another aspect of the invention there is provided the use of genetically modified non- human animals as described above for the screening of candidate pharmaceutical compounds. In a still further aspect of the invention there is provided a nucleic acid encoding a mutant TSG18 polypeptide which cannot form a complex with a wild- ype protein with which wild-type TSG18 does form a complex. Typically the protein is a member of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins, particularly PIAS1 or PIAS3.

According to a still further aspect of the invention there is provided a mutant TSG18 polypeptide which cannot form a complex with a wild-type protein with which wild- type TSG18 does form a complex. Typically the protein is a member of the PIAS family, particularly PIAS1 or PIAS3.

According to a still further aspect of the present invention there is provided a complex of wild-type TSG18 and a PIAS protein, particularly PIASl or PIAS3.

In a still further aspect of the invention, there is provided a complex of wild-type TSG18 and SUMO E3 ligase, an interaction mediated by the binding of TSG18 to PIASl.

In a still further aspect of the present invention there is provided the use of complexes as described above or the use of PIASl, PIAS3 or SUMO E3 ligase in screening for candidate pharmaceutical compounds . In the case of PIASl, PIAS3 and SUMO E3 ligase, one may screen for a drug which replaces the activity of TSG18 in a patient deficient in TSG18.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

Brief Description of the Drawings Figure 1A-1G. Genomic sequence of TSG18. Intron sequences are indicated in lowercase letters whereas exonic sequences are in uppercase. Intervals between larger introns are shortened by stretches of v's. The sequence of intron 1 is not known at this time. Figure 2. Northern blot analysis of the TSG18 gene.

Two bands were identified that indicated predominant expression of the TSG18 gene in skeletal muscle and placenta. The gene was observed to be expressed' to a lower level in the remaining tissues. RNA size markers are indicated on the left of the blot while the size of the mRNA species corresponding to TSG18 are indicated on the right of the blot. 1: Heart; 2: Brain; 3: Placenta; 4: Lung; 5: Liver; 6: Skeletal Muscle; 7: Kidney; 8: Pancreas . Figure 3. Amino acid sequence alignment between the

TSG18 and TSG16 proteins. Identical amino acids are boxed. The ankyrin domain of TSG18 is highlighted in bold italics. Modes for performing the invention Example 1: TSG18 identification

The amino acid sequence of the TSG16 gene was used to screen the htgs database at NCBI (www.ncbi.nlm.nih.gov/blast/blast .cgi?Jform=0) to identify potential homologous genes. The tblastn algorithm and the BLOSSUM62 default matrix were used. As expected, the TSG16 gene identified a chromosome 16 specific BAC (accession number ACO09098) in addition to two BAC clones from unknown chromosomal origin (accession numbers AC023256 and AC021009) . As these 2 BAC clones were 100% homologous to TSG16 sequences, they most likely arise from chromosome 16 also. Of note was the identification of two additional BAC clones (accession numbers AP001033 and AC015955) that are derived from chromosome 18 (18pll.3). The identity between TSG16 sequence and the sequence from these BACs ranged from 30-84% over the majority of the length of TSG16 indicating the presence of a closely related gene mapping to chromosome 18 (TSG18) . The sequence of the chromosome 18 BACs was assembled using the GAP4 software on a SUN workstation. To confirm the presence of transcribed sequences on chromosome 18 corresponding to TSG18, the assembled sequence was masked for repeats and analysed using the BLASTN algorithm and the BLOSSUM62 default matrix against the dbEST database at NCBI. A number of cDNA clones were identified that had identical 3 ' origins and were derived from a variety of tissues. Further homology searches of the non-redundant database identified additional cDNA clones. These included AB020681, which originated at the same 3' end as the previously identified EST cluster, as well as AK024808 which appeared to be located further 5' to these clones. Although AB020681 and AK024808 did not appear to overlap with each other, both showed homology to the TSG16 amino acid sequence. This suggests that they both are derived from a TSG16 homologous gene located on chromosome 18, namely TSG18. To confirm this, additional database screening was attempted to link the AB020681 and AK024808 cDNA clones. This involved BLASTN searches of the dbEST database using the chromosome 18 genomic sequence lying between the two clones. Using successive EST walking approaches, cDNA sequence was obtained that successfully linked both AB020681 and AK024808. Therefore the use of the sequence of TSG16 has enabled the identification and cloning of a homologous gene on chromosome 18 (TSG18) using an in silico approach.

Example 2: TSG18 sequence characterization

The TSG18 gene is 9,035 base pairs in length (SEQ ID NO:l) and is composed of 11 exons that span approximately 80 Kb of genomic DNA. Table 1 shows the genomic structure of the gene indicating the size of exons and introns. Analysis of exons 1 to 11 indicate an open reading frame of 6,186 nucleotides with a start codon in exon 1 at base 215 and a stop codon in exon 11 at base 6,401. This defines a protein of 2,062 amino acids (SEQ ID NO:2). Partial genomic DNA sequences indicating exon/intron junctions for TSG18 are set forth in Figure 1A-1G and SEQ ID Numbers: 3-11.

To confirm the size of the TSG18 gene. Northern blots obtained from Clontech were probed with a TSG18 specific probe. This probe was generated from RT-PCR of normal mammary gland RNA (Clontech) with TSG18 specific primers

(SEQ ID Numbers: 12 and 13). Reverse transcription and subsequent PCR reactions were conducted using the Omniscript and HotSTARTaq kits from Gibco BRL using manufacturers specifications. PCR products were excised from agarose gels and cleaned using the QIAquick PCR gel extraction kit according to supplied protocols. Cleaned PCR products were labeled with [α-³²P]dCTP using standard methodologies (Sambrook et al . , 1989) and hybridized to the Northern membrane overnight at 42°C in 10 ml of ExpressHyb solution (Clontech) - The membrane was washed the following day using manufacturers conditions. Figure 2 shows the results of the hybridization. As can be seen, two bands of approximately 9.5 Kb and 7 Kb in size are present predominantly in the skeletal muscle and placenta. Much weaker expression of the gene was seen in the remaining tissues. The larger band corresponds in size to the TSG18 gene sequence (SEQ ID NO:l) while the origin of the smaller band is unknown at this stage. It is possible that alternative splicing of TSG18 exons or the use of an alternative polyadenylation signal in the 3'UTR of TSG18 may account for the presence of this smaller TSG18 specific RNA species.

A large number of cDNA clones are present in dbEST which belong to the TSG18 gene. An observation of the tissues these cDNA clones were derived from indicates that the gene is expressed in the aorta, brain, breast, eye, germ cells, heart, kidney, lymphatic tissue, pancreas, placenta, prostate, smooth muscle, stomach, testis, tonsil, uterus, bone marrow, lung, ovary and thymus. The TSG18 nucleotide sequence also detects a number of mouse cDNA clones as well as clones derived from cow, rat, chicken, frog and pig cDNA libraries. The homology is as high as 91%, which suggests that this gene is highly conserved across species. A direct comparison between TSG16 and TSG18 indicates a high degree of conservation at both the physical and sequence level between the two proteins across their entire length. Both genes are of similar size (2,663 amino acids and 2,062 amino acids respectively), both genes span large genomic intervals, both genes share an extremely large exon (6,578 nucleotides and 4,721 nucleotides respectively) and both genes have extremely high exon/intron structure conservation. Amino acid sequence alignment between the two genes indicates that both have the same 5' and 3' termini, with the difference in length dictated by the fact that TSG18 has a shorter "large" exon. Sequence identity between TSG16 and TSG18 ranged from 22-67% across their entire lengths (Figure 3) with highest homology occurring between the ankyrin domains present in each gene (74% identity and 87% similarity) .

Amino Acid Sequence

The amino acid sequence of TSG18 was used for in silico analysis to identify regions of homology to previously characterised proteins other than TSG16. Initially the BLASTP program was used to search for homologous sequences in the GenBank non-redundant protein database (http://www.ncbi.nlm.nih.gov/index.html). The search identified a number of proteins that exhibited homology to TSG18 in the region containing an ankyrin repeat motif (ANK), including Ankyrin 1, BARDl and IKB-R. Analyses of the TSG18 protein using the PfScan program (http://www.isrec.isb- sib.ch/software/ FSCAN_form.html) confirmed the presence of an ankyrin repeat domain with a PfScan score of 38.382. In comparison, Ankyrin 1 contains 23 ANK repeats and exhibits a PfScan score of 249.73, BARDl with 3 ANK repeats exhibits a score of 36.2 and Iκβ-α with 4 ANK repeats a score of 41.9. Ankyrin repeats have been identified in over 400 proteins ranging from transcription factors to toxins. The main function of ANK domains is to provide a site for protein-protein interactions. The ANK repeat unit contains 33 amino acids with a conserved consensus of XGXTPLHXAAXXGHXXXV/AXXLLXXGAXXN/DXXXX (where X can be any amino acid) . The number of repeats within a protein can vary widely from 3 in the rat Vlp to 23 in the human Ankyrin protein. TSG18, like TSG16 contains 3 ANK repeat units. X-ray structural analysis of the human p53 binding protein (53BP2), iKB- , and the yeast protein Swi6 ankyrin domains indicate that the ANK domain is an L- shaped structure, which consists of β-hairpins and α- helices. The α-helices create a pit which is surrounded by β-helical protrusions thus providing a docking site for interacting proteins. The highest BLASTP homology scores for TSG18 to characterized proteins were obtained with the ANK domains present in the proteins BARDl and KB-R. In addition to the conserved amino acids, which define the ANK repeat, homology extended to those residues, which are often non- conserved within the ANK motif. This suggests that TSG18, BARDl and/or IKB-R may have common protein interaction partners .

PfScan analysis also identified three putative bipartite nuclear localisation signals (BNLS) within TSG18. BNLS are characterised as stretches of sequence that consist of two leading basic amino acids - either arginine or lysine, separated from another three basic residues by ten amino acids. These sequences are recognised by nuclear importing machinery and allow the protein to be localised within the nucleus. The presence of multiple BNLS within proteins indicates that there does not necessarily need to be one dominating BNLS. It has been suggested that nuclear localisation can be due to the cumulative effect of all BNLS present rather than the effect of one signal.

Taken collectively the in silico analyses indicates that like TSG16, TSG18 is a nuclear protein that contains an ANK domain which may mediate interactions with proteins that also interact with the proteins BARDl and/or IKB-R.

BARDl has been shown to interact with the tumour suppressor protein BRCAl via its RING finger motif. The ankyrin domain of BARDl is responsible for the interaction with CstF-50, a member of the Cleavage stimulation factor complex. This complex, along with RNA polymerase II, has been shown to be involved in polyadenylation of mRNA precursors whereby the CstF complex specifies the site of processing (Takagaki et al., 1989). BARDl and BRCAl also interact with RNA polymerase II and the BARDl/CstF-50 interaction has been shown to inhibit polyadenylation in vitro (Kleiman and Manley, 1999) . It has been proposed that BARDl as part of the polyadenylation apparatus, senses sites of DNA damage and repair, and the inhibitory interaction with Cst ensures that nascent RNAs are not erroneously polyadenylated at such sites until the DNA has been repaired. IKB-R is involved in modulating the function of the transcription factor NF-KB. NF-KB transcription factors include a collection of proteins conserved from humans to Drosophila (reviewed in Gilmore, 1999) . These transcription factors are notably absent in yeast and C. elegans, probably as a result of the primary function of these factors, which is to control a variety of physiological aspects of immune responses, inflammation, and growth and development. The NF-KB proteins are related through a highly conserved DNA-binding/dimerisation domain called the Rel homology (RH) domain. NF-KB transcription factors bind to 10 base pair DNA sites (KB sites) as dimers. The activity of NF-KB is tightly regulated by interaction with inhibitory KB proteins. There are several 1KB proteins, each of which contains six to seven ANK repeats. However, each 1KB protein has different affinities for individual NF-KB complexes and each are expressed in a tissue specific manner. The binding of lκB-α to NF-KB blocks the ability of NF-KB to enter the nucleus and bind to DNA. From structural studies it is clear that IKBOC binding masks the nuclear localization sequence of NF-KB and also interferes with sequences important for DNA binding (Chen and Ghosh, 1999) . Therefore, in most cells, NF-KB is present as a latent and inactive iKB-bound complex in the cytoplasm. When a cell receives an extracellular signal, NF-KB rapidly enters the nucleus and activates gene expression. Virtually all signals that lead to activation of NF-KB converge on a complex that contains a serine- specific 1KB kinase (IKK) . Activation of IKK leads to the phosphorylation of two specific serine residues near the N terminus of lκB-α, which targets lκB-α for ubiquitination and degradation by the proteasome. The unmasked NF-KB can then enter the nucleus to activate target gene expression. There is a body of evidence linking deregulated NF-KB activity to oncogenesis in mammalian systems (reviewed in Gilmore et al . , 1999). In addition, alterations affecting the expression or function of the 1KB family members Bcl-3, iKB-α and iKB-ε have also been observed in several cancers. Together, these studies have identified tumour cells that display constitutively high levels of nuclear NF-KB activity due to hyperactivation of the NF-KB signaling pathway or to inactivating mutations in the regulatory 1KB subunits (reviewed in Rayet and Gelinas, 1999) .

The IKB-R protein was originally cloned by differential expression from a human lung epithelial cell line and has been shown to inhibit the DNA binding ability of an NF-KB complex present in nuclear extracts prepared from interleukin-1 activated HeLa cells (Ray et al., 1995) . It is therefore possible that this member of the 1KB family may play an important role in the regulation of NF- KB function in epithelial cells.

While not wishing to be bound by theory, the high homology of TSG18 ANK repeats to the ANK repeats of BARDl and IKB-R suggests that TSG18 may be a key protein in either or both of the pathways to which these important proteins belong, particularly in epithelial cells. Based on past studies it is possible that TSG18, like TSG16, may form a link connecting the BARD1/BRCA1 and NF-KB/IKB pathways .

Example 3 : Analysis of the TSG18 gene

The following methods are used to determine the structure and function of TSG18.

Biological studies

Mammalian expression vectors containing TSG18 cDNA can be transfected into breast or other carcinoma cell lines that have lesions in the gene. Phenotypic reversion in cultures (eg cell morphology, growth of transformants in soft-agar, growth rate) and in animals (eg tumourigenicity in nude mice) is examined. These studies can utilise wild-type or mutant forms of TSG18. Deletion and missense mutants of TSG18 can be constructed by in vitro mutagenesis.

Molecular biological studies

The ability of TSG18 protein to bind known and unknown protein can be examined. Due to the presence of an ANK domain region in TSG18 it is most likely that this gene participates in protein/protein interactions and procedures such as the yeast two-hybrid system are used to discover and identify any functional partners. The principle behind the yeast two-hybrid procedure is that many eukaryotic transcriptional activators, including those in yeast, consist of two discrete modular domains. The first is a DNA-binding domain that binds to a specific promoter sequence and the second is an activation domain that directs the RNA polymerase II complex to transcribe the gene downstream of the DNA binding site. Both domains are required for transcriptional activation as neither domain can activate transcription on its own. In the yeast two-hybrid procedure, the gene of interest or parts thereof (BAIT) , is cloned in such a way that it is expressed as a fusion to a peptide that has a DNA binding domain. A second gene, or number of genes, such as those from a cDNA library (TARGET) , is cloned so that it is expressed as a fusion to an activation domain. Interaction of the protein of interest with its binding partner brings the DNA-binding peptide together with the activation domain and initiates transcription of the reporter genes.

The first reporter gene will select for yeast cells that contain interacting proteins (this reporter is usually a nutritional gene required for growth on selective media) . The second reporter is used for confirmation and while being expressed in response to interacting proteins it is usually not required for growth. In previous studies of the TSG16 gene (International Patent Application Number PCT/AU00/01329) , yeast two- hybrid analysis using the displayGREEN-BASIC™ Two-Hybrid System kit (Display Systems Biotech) identified TSG16 ANK domain interacting proteins. From sequence analysis of these clones, members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins were identified to be interacting with the ANK domain of TSG16, in particular PIASl and PIAS3.

The PIASl protein has been found to bind to p53 (Kayho et al . , 2001), a tumour suppressor protein that plays a critical role in carcinogenesis. The amount of p53 and its transcriptional activity are increased in response to genotoxic stress through mechanisms such as phosphorylation, acetylation and sumoylation. Sumoylation involves the binding of an ubiquitin-like protein (SUMO- 1/sentrin/PICl) to target proteins. SUMO-1 conjugation to substrate protein appears to occur as in the ubiquitination reaction, with the E3 enzyme (SUMO ligase) being a key enzyme for recognition of the substrate to be sumoylated. A component of this enzyme has recently been shown to be the PIASl protein (Kahyo et al., 2001). As TSG16 also bind PIASl, it suggests that the either the TSG16 protein is a component of the SUMO E3 ligase complex and is involved in sumoylation of proteins such as p53, or PIAS proteins may affect the transcriptional activity of a range of tumour suppressor molecules, including p53 and TSG16. The PIAS family of proteins have also been shown to specifically inhibit STAT (signal transducer and activator of transcription) protein signaling (Liu et al . , 1998). STAT proteins are a family of cytoplasmic transcription factors that become activated, by tyrosine phosphorylation, following the binding of cytokines to their cell surface receptors. After phosphorylation, STATs dimerise, translocate to the nucleus and bind specific DNA elements in the promoters of responsive genes to activate transcription. STATl for example, plays an important role in mediating interferon-gamma (IFN-γ) , interleukin-6 (IL-6) type cytokine and epidermal growth factor (EGF) -dependant biological responses while STAT3 activation has been shown to be linked to oncogenic transformation.

IFN-γ is a cytokine that plays a fundamental role in several aspects of the immune response (Boehm et al., 1997) . Other properties include stimulation of bactericidal activity of phagocytes, stimulation of antigen presentation through class I and II major histocompatability complex molecules, as well as affects on cell proliferation and apoptosis. At the cellular level IFN-γ is able to mediate activation of an antiviral state and cause cell growth arrest at the Gi phase of the cell cycle.

The IFN-γ response has recently been postulated to be part of an endogenous tumour surveillance system (Kaplan et al . , 1998). To add support to this claim, additional experiments have shown that STATl interacts with the tumour suppressor BRCAl. This leads to differential activation of transcription of a subset of IFN-γ target genes leading to growth inhibition by this cytokine, with one of these genes being the cyclin-dependent kinase inhibitor, p2lWAFl (Ouchi et al . , 2000). It has been further shown that p2lWAFl activation is impaired in breast cancer cells lacking a functional BRCAl protein. Thus it is possible that the disturbance of the p21WAFl induction provides an early growth advantage to nascent tumour cells, which allows them to bypass the initial antitumour actions of IFN-γ.

EGF and IL-6 type cytokines also mediate their actions in part through the STAT pathway. While EGF is a mitogen for many cells, growth of some cultured cell lines, containing high numbers of EGF receptors, are inhibited by EGF. This growth inhibition has been shown for A431 cells to be mediated by the activation of STATl (via specific receptor kinase activity) and NF-KB (via 1KB degradation), which drive p21WAFl gene expression (Ohtsubo et al . , 2000). The IL-6 type cytokines signal through the common receptor subunit gpl30 and are involved in the regulation of many processes including gene expression, cell proliferation and differentiation. IL-6 has also been shown to stimulate inflammatory responses during wound healing. In fetal skin, this process is characterized by minimal inflammation and scarless repair and it has been suggested that diminished inflammation (due to diminished production of inflammatory cytokines such as IL-6) may provide a permissive environment for scarless wound healing (Liechty et al . , 2000).

PIAS proteins can modulate steroid receptor-dependent transcriptional activation and also have an established role in the negative regulation of STAT signaling. PIASl binds to the STATl dimer; this has been proposed to mask the DNA-binding activity of STATl (Liao et al . , 2000). Recent studies have suggested that the PIAS family of proteins may function to regulate other transcriptional responses (Moilanen et al . , 1999). Therefore the recruitment of PIASl to different transcription factors only after ligand stimulation may allow the targeting of PIASl to a specific transcriptional response induced by the corresponding signal. The binding of PIASl to the ANK domain of TSG16 may represent one of these novel transcriptional responses, and like STATl, may be linked to immunological responses, including those associated with tumour suppression. Given the interaction of more than one member of the PIAS family with the TSG16 ANK domain, it has been proposed that TSG16 may play a role in all PIAS associated functions.

The highest sequence homology between TSG16 and TSG18 occurs across their ankyrin domains suggesting that TSG18 also participates in PIAS protein interactions mediated by its ankyrin domain. Therefore, TSG16 and/or TSG18 may: (1) form part of the SUMO E3 ligase complex that sumoylates protein targets such as p53; or (2) be additional tumour suppressor genes (other than p53) that are sumoylated by PIASl containing SUMO E3 ligase complexes; or (3) represent a novel family of proteins involved in modulation of the STAT signaling pathway. Such signals are linked to immunological responses, including those associated with tumour suppression.

The nature of the TSG18 interacting genes and proteins can also be studied such that these partners can also be targets for drug discovery.

Structural studies

TSG18 recombinant proteins can be produced in bacterial, yeast, insect and/or mammalian cells and used in crystallographical and NMR studies. Together with molecular modeling of the protein, structure-driven drug design can be facilitated.

Example 4 : Generation of polyclonal antibodies against TSG18

The knowledge of the nucleotide and amino acid sequence of TSG18 allows for the production of antibodies, which selectively bind to TSG18 protein or fragments thereof. Following the identification of mutations in the gene, antibodies can also be made to selectively bind and distinguish mutant from normal protein. Antibodies specific for mutagenised epitopes are especially useful in cell culture assays to screen for malignant cells at different stages of malignant development. These antibodies may also be used to screen malignant cells, which have been treated with pharmaceutical agents to evaluate the therapeutic potential of the agent.

To prepare polyclonal antibodies, short peptides can be designed homologous to the TSG18 amino acid sequence. Such peptides are typically 10 to 15 amino acids in length. These peptides should be designed in regions of least homology to the mouse orthologue to avoid cross species interactions in further down-stream experiments such as monoclonal antibody production. Synthetic peptides can then be conjugated to biotin (Sulfo-NHS-LC Biotin) using standard protocols supplied with commercially available kits such as the PIERCE™ kit (PIERCE) . Biotinylated peptides are subsequently co plexed with avidin in solution and for each peptide complex, 2 rabbits are immunized with 4 doses of antigen (200 μg per dose) in intervals of three weeks between doses. The initial dose is mixed with Freund's Complete adjuvant while subsequent doses are combined with Freund's Immuno-adjuvant. After completion of the immunization, rabbits are test bled and reactivity of sera assayed by dot blot with serial dilutions of the original peptides. If rabbits show significant reactivity compared with pre-im une sera, they are then sacrificed and the blood collected such that immune sera can separated for further experiments.

Example 5 : Generation of monoclonal antibodies specific for TSG18

Monoclonal antibodies can be prepared for TSG18 in the following manner. Immunogen comprising intact TSG18 protein or TSG18 peptides (wild type or mutant) is injected in Freund's adjuvant into mice with each mouse receiving four injections of 10 to 100 ug of immunogen. After the fourth injection blood samples taken from the mice are examined for the presence of antibody to the immunogen. Immune mice are sacrificed, their spleens removed and single cell suspensions are prepared (Harlow and Lane, 1988). The spleen cells serve as a source of lymphocytes, which are then fused with a permanently growing myeloma partner cell (Kohler and Milstein, 1975) . Cells are plated at a density of 2X10⁵ cells/well in 96 well plates and individual wells are examined for growth. These wells are then tested for the presence of TSG18 specific antibodies by ELISA or RIA using wild type or mutant TSG18 target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality. Clones with the desired specificity are expanded and grown as ascites in mice followed by purification using affinity chromatography using Protein A Sepharose, ion-exchange chromatography or variations and combinations of these techniques.

Industrial Applicability

The tumour suppressor gene, TSG18, is implicated in cancers that arise from a number of tissues due to its ubiquitous expression pattern. In addition, this gene is implicated in other disease states due to the presence of specific functional domains within its encoded protein. The novel DNA molecules of the present invention are therefore useful in methods for the early detection of disease susceptible individuals as well as in diagnostic, prognostic and therapeutic procedures associated with these disease states.

References

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Claims

Claims :

1. An isolated nucleic acid molecule, comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses.

2. An isolated nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses .

3. An isolated nucleic acid molecule as claimed in claim 2 that is at least 85% identical.

4. An isolated nucleic acid molecule as claimed in claim 2 that is at least 95% identical.

5. An isolated nucleic acid molecule as claimed in claim 2 wherein sequence identity is determined using the BLASTN algorithm and the BLOSSUM62 default matrix.

6. An isolated nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.

7. An isolated nucleic acid molecule as claimed in claim 6 wherein the stringent conditions comprise hybridization at 42°C in 750 mM NaCl, 75 mM trisodium citrate, 2% SDS, 50% formamide, IX Denhart's, 10% w/v) dextran sulphate and 100 ug/ml denatured salmon sperm DNA.

8. An isolated nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.

9. An isolated nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO: 2.

10. An isolated nucleic acid molecule as claimed in claim 9 wherein the amino acid sequence has at least 85% sequence identity.

11. An isolated nucleic acid molecule as claimed in claim 9 wherein the amino acid sequence has at least 95% sequence identity.

12. An isolated nucleic acid molecule as claimed in claim

9 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix.

13. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.

14. An isolated nucleic acid molecule that is at least

70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID N0:1 in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.

15. An isolated nucleic acid molecule as claimed in claim 14 that is at least 85% identical.

16. An isolated nucleic acid molecule as claimed in claim 14 that is at least 95% identical.

17. An isolated nucleic acid molecule as claimed in claim 14 wherein sequence identity is determined using the BLASTN algorithm and the BLOSSUM62 default matrix.

18. An isolated nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.

19. An isolated nucleic acid molecule as claimed in claim 18 wherein the stringent conditions comprise hybridization at 42°C in 750 mM NaCl, 75 mM trisodium citrate, 2% SDS, 50% formamide, IX Denhart's, 10% w/v) dextran sulphate and

100 ug/ml denatured salmon sperm DNA.

20. An isolated nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO: 2.

21. An isolated nucleic acid molecule as claimed in claim

20 wherein the amino acid sequence has at least 85% sequence identity.

22. An isolated nucleic acid molecule as claimed in claim 20 wherein the amino acid sequence has at least 95% sequence identity.

23. An isolated nucleic acid molecule as claimed in claim 20 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix.

24. An isolated nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l.

25. An isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l.

26. An isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l from base 215 to base 6,401.

27. An isolated nucleic acid molecule as claimed in any one of claims 1 to 26 which is inactivated or whose expression is reduced through epigenetic mechanisms or as a result of mutation or polymorphism.

28. An isolated gene comprising the nucleotide sequence set forth in SEQ ID N0:1 from base 215 to base 6,401 and TSG18 control elements.

29. An isolated gene as claimed in claim 28 wherein the TSG18 control elements are those which mediate expression in breast tissue.

30. An expression vector which comprises a DNA molecule as defined in any one of claims 1 to 27 operably linked to suitable control elements .

31. A cell transformed with the expression vector of claim 30.

32. A cell as claimed in claim 31 in which TSG18 is expressed in a mutant form.

33. A cell as claimed in claim 31 in which TSG18 expression may be switched off.

34. A cell as claimed in any one of claims 31 to 33 which is an eukaryotic cell.

35. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions associated with cancer and/or modulating immunological responses .

36. An isolated polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and having at least 70% identity with the amino acid sequence set forth in SEQ ID NO: 2.

37. An isolated polypeptide as claimed in claim 36 with at least 85% sequence identity.

38. An isolated polypeptide as claimed in claim 36 with at least 95% sequence identity.

39. An isolated polypeptide as claimed in claim 36 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix.

40. An isolated polypeptide, comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets.

41. An isolated polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% identity with the amino acid sequence set forth in SEQ ID NO: 2.

42. An isolated polypeptide as claimed in claim 41 with at least 85% sequence identity.

43. An isolated polypeptide as claimed in claim 41 with at least 95% sequence identity.

44. An isolated polypeptide as claimed in claim 41 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix.

45. An isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

46. An isolated polypeptide as claimed in any one of claims 35 to 45 which is inactivated as a result of mutation or polymorphism.

47. A method of preparing a polypeptide, comprising the steps of:

(1) culturing the cells of claim 31 under conditions effective for production of the polypeptide; and

(2) harvesting the polypeptide.

48. An antibody which is immunologically reactive with a polypeptide as defined in any one of claims 35 to 46.

49. An antibody as claimed in claim 48 which is selected from the group consisting of a monoclonal antibody, a humanised antibody, a chimaeric antibody or an antibody fragment including a Fab fragment, (Fab')₂ fragment, Fv fragment, single chain antibodies and single domain antibodies.

50. A pharmaceutical composition comprising a polypeptide according to any one of claims 35 to 45, and a pharmaceutically acceptable carrier.

51. A method of treatment of a disorder associated with decreased expression or activity of TSG18, comprising administering a polypeptide as defined in any one of claims 35 to 45, or an agonist thereof, to a subject in need of such treatment .

52. A method according to claim 51 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired iimnunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, yocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring) , cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

53. The use of a polypeptide as defined in any one of claims 35 to 45, or an agonist thereof, in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG18.

54. The use as claimed in claim 53 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpastu e's syndrome, gout. Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring) , cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

55. A method of treating a disorder associated with increased expression or activity of TSG18, comprising administering an antagonist of TSG18 to a subject in need of such treatment.

56. A method according to claim 55 wherein the antagonist of TSG18 is an antibody as defined in claim 45 or 46.

57. The use of an antagonist of TSG18 in the manufacture of a medicament for the treatment of a disorder associated with increased expression or activity of TSG18.

58. The use as claimed in claim 57 wherein the antagonist of TSG18 is an antibody as defined in claim 48 or 49.

59. A method of treating a disorder associated with decreased expression or activity of TSG18, comprising gene therapy through administration of an isolated nucleic acid molecule as defined in any one of claims 1 to 26 to a subject in need of such treatment.

60. A method as claimed in claim 59 wherein an expression vector comprising the isolated nucleic acid molecule operably linked to suitable control elements is administered.

61. A method as claimed in claim 59 or claim 60 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis- ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

62. The use of an isolated nucleic acid molecule as defined in any one of claims 1 to 26 in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG18.

63. The use as claimed in claim 62 wherein the nucleic acid molecule is a part of an expression vector which also includes suitable control elements.

64. The use as claimed in claim 62 or claim 63 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis- ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring) , cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

65. A method of treating a disorder associated with increased activity or expression of TSG18, comprising administering an isolated nucleic acid molecule which is the complement of a nucleic acid molecule as defined in any one of claims 1 to 26 and which encodes a mRNA that hybridizes with the mRNA encoded by TSG18.

66. A method as claimed in claim 65 wherein an expression vector comprising the isolated nucleic acid molecule and suitable control elements is administered.

67. The use of an isolated DNA molecule which is the complement of a nucleic acid molecule as defined in any one of claims 1 to 26 and which encodes a mRNA that hybridizes with the mRNA encoded by TSG18, in the manufacture of a medicament for the treatment of a disorder associated with increased activity or expression of TSG18.

68. The use as claimed in claim 67 wherein the nucleic acid molecule is a part of an expression vector which also includes suitable control elements.

69. The use of an isolated nucleic acid molecule as claimed in any one of claims 1 to 26 for the diagnosis of disorders associated with TSG18, or a predisposition to such disorders.

70. The use as claimed in claim 69 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

71. The use of a polypeptide as defined in any one of claims 35 to 45 in the diagnosis of a disorder associated with TSG18, or a predisposition to such disorders.

72. The use of an antibody as defined in either one of claims 48 or 49 in the diagnosis of a disorder associated with TSG18, or a predisposition to such disorders.

73. A method for the diagnosis of a disorder associated with mutations in TSG18, or a predisposition to such disorders in a patient, comprising the steps of:

(1) obtaining a sample which includes TSG18 or a nucleic acid which codes for TSG18 from the patient; (2) comparing TSG18 or a nucleic acid which codes for TSG18 from the sample with wild-type TSG18 or a nucleic acid which codes for it in order to establish whether the person expresses a mutant TSG18.

74. A method as claimed in claim 73 wherein the nucleotide sequence of DNA from the patient is compared to the sequence of DNA encoding wild-type TSG18.

75. A method for the diagnosis of a disorder associated with abnormal expression or activity of TSG18, or a predisposition to such disorders, comprising the steps of:

(1) establishing a profile for normal expression of TSG18 in unaffected subjects;

(2) measuring the level of expression of TSG18 in a person suspected of abnormal expression or activity of TSG18; and

(3) comparing the measured level of expression with the profile for normal expression.

76. A method as claimed in claim 75 wherein reverse transcriptase PCR is employed to measure levels of expression.

77. A method as claimed in claim 75 wherein a hybridisation assay using a probe derived from TSG18, or a fragment thereof, is employed to measure levels of expression.

78. A method as claimed in claim 77 wherein the probe has at least 50% sequence identity to a nucleotide sequence encoding TSG18, or a fragment thereof.

79. A method for the diagnosis of a disorder associated with TSG18, or a predisposition to such disorders, comprising the steps of:

(1) establishing a physical property of wild- type TSG18; (2) obtaining TSG18 from a person suspected of an abnormality of TSG18; and

(3) measuring the property for the TSG18 expressed by the person and comparing it to the established property for wild-type TSG18 in order to establish whether the person expresses a mutant TSG18.

80. A method as claimed in claim 79 wherein the property is the electrophoretic mobility.

81. A method as claimed in claim 79 wherein the property is the proteolytic cleavage pattern.

82. A method as claimed in claim 79 wherein the property is the activity of TSG18 as measured by a functional assay.

83. A method as claimed in claim 79 wherein the property is binding of an antibody as defined in any either one of claims 48 or 49.

84. A method for determining whether a human tissue is pre-disposed to a neoplastic transformation, comprising, determining whether in a cell from the tissue a nucleic acid molecule that has the sequence shown in SEQ ID NO:l is absent or down-regulated.

85. A method according to claim 84 wherein the human tissue is breast tissue.

86. A method according to claim 84 comprising determining whether a peptide which has the sequence shown in SEQ ID NO: 2 is absent or expressed at reduced levels.

87. A method according to claim 86 comprising contacting the cell with an antibody for binding to the peptide under conditions which permit the antibody to bind to the peptide, if present.

88. A peptide for determining whether a human breast tissue is pre-disposed to neoplastic transformation, wherein the peptide has the sequence shown in SEQ ID NO: 2.

89. A fragment of the peptide according to claim 88,

90. An antibody for determining whether a human breast tissue is pre-disposed to neoplastic transformation, wherein the antibody is capable of binding to a peptide according to claim 89.

91. A nucleic acid molecule for determining whether a human breast tissue is pre-disposed to neoplastic transformation, wherein the nucleic acid molecule encodes a peptide according to claim 88 or claim 89.

92. A nucleic acid molecule according to claim 91, wherein the nucleic acid molecule has the sequence shown in SEQ ID NO:l.

93. A fragment of a nucleic acid molecule according to claim 92.

94. A fragment according to claim 93 wherein the fragment is about 50 nucleotides in length.

95. A hybridoma cell for secreting an antibody as defined in any one of claims 48, 49 or 90.

96. A genetically modified non-human animal transformed with an isolated nucleic acid molecule as defined in any one of claims 1 to 27.

97. A genetically modified non-human animal as claimed in claim 96 in which the animal is selected from the group consisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs and non-human primates such as monkeys and chimpanzees .

98. A genetically modified non-human animal as claimed in claim 96 wherein the animal is a mouse.

99. A genetically modified non-human animal in which TSG18 activity is reduced or absent.

100. A genetically modified non-human animal in which TSG18 gene function has been knocked out.

101. A genetically modified non-human animal in which TSG18 gene function is modified through transformation with a mutant TSG18 gene.

102. A genetically modified non-human animal as claimed in any one of claims 99 to 101 in which the animal is selected from the group consisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs and non-human primates such as monkeys and chimpanzees .

103. A genetically modified non-human animal as claimed in claim 99 wherein the animal is a mouse.

104. The use of a genetically modified non-human animal as claimed in any one of claims 97 to 103 in screening for candidate pharmaceutical compounds.

105. The use of a cell as defined in claim 31 to 33 in screening for candidate pharmaceutical compounds.

106. The use of an isolated polypeptide as defined in any one of claims 35 to 46 in screening for candidate pharmaceutical compounds.

107. A nucleic acid encoding a mutant TSG18 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG18 does form a complex.

108. A nucleic acid as claimed in claim 107 wherein the wild-type protein is a protein selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins.

109. A nucleic acid as claimed in claim 108 wherein the wild-type protein is PIASl or PIAS3.

110. A nucleic acid as claimed in any one of claims 107 to 109 wherein the mutant TSG18 polypeptide contains a mutation in an ankyrin repeat domain.

111. A mutant TSG18 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG18 does form a complex.

112. A mutant TSG18 as claimed in claim 111 wherein the wild-type protein is selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins .

113. A mutant TSG18 as claimed in claim 112 wherein the wild-type protein is PIASl or PIAS3.

114. A mutant TSG18 polypeptide as claimed in any one of claims 111 to 113 containing a mutation in an ankyrin repeat domain.

115. The use of mutant TSG18 polypeptide as defined in any one of claims 111 to 114 for the screening of candidate pharmaceutical compounds.

116. A complex of wild-type TSG18 and a protein selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins.

117. A complex as claimed in claim 111 wherein the protein is PIASl or PIAS3.

118. The use of a complex as claimed in either one of claims 116 or 117 or the use of PIASl or PIAS3 for the screening of candidate pharmaceutical compounds.

119. A nucleic acid encoding a mutant TSG18 polypeptide which cannot interact with a complex with which wild-type

TSG18 does interact.

120. A nucleic acid as claimed in claim 119 wherein the complex is the SUMO E3 ligase/TSG18 complex.

121. A mutant TSG18 polypeptide which cannot interact with a complex with which wild-type TSG18 does interact.

122. A mutant TSG18 as claimed in claim 121 wherein the complex is the SUMO E3 ligase/TSG18 complex.

123. The use of a mutant TSG18 polypeptide as defined in claim 121 or 122 for the screening of candidate pharmaceutical compounds.

124. A complex of wild-type TSG18 and SUMO E3 ligase.

125. The use of a complex as claimed in claim 124 or the use of SUMO E3 ligase for the screening of candidate pharmaceutical compounds.

126. The use of TSG18 to identify interacting proteins suitable as drug targets .

127. The use as claimed in claim 126 wherein TSG18 is fused to a DNA binding domain and used as the bait in a yeast two-hybrid system.

128. A fusion protein comprising TSG18 fused to a polypeptide whose function is DNA binding.

129. A fusion protein as claimed in claim 128 wherein TSG18 is fused to the DNA binding domain of the yeast GAL4 transcription factor.

130. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 3.

131. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:4.

132. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 5.

133. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 6.

134. An isolated DNA molecule consisting of the nucleotide sequence set forth in any one of SEQ ID NO:7.

135. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 8.

136. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 9.

137. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 10.

138. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 11.

[received by the International Bureau on 16 April 2002 (16.04.02); claims 1, 3, 23 and 48 find support in claims 35 - 45 prior to amendment and claims 11, 14, 17, 19, 41 and 47 find support in claims 1 - 26 prior to amendment.

(25 pages)]

1. A method for the treatment of a disorder associated with decreased expression or activity of TSG18, comprising administering a polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (c) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; and (d) a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, or an agonist thereof, to a subject in need of such treatment .

2. A method according to claim 1 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-eσtodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring) , cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

3. The use of a polypeptide comprising: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof which is active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (c) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; and (d) a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO:2, or an agonist thereof, in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG18.

4. The use as claimed in claim 3 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring) , cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

5. A method of treating a disorder associated with increased expression or activity of TSG18, comprising administering an antagonist of TSG18 to a subject in need of such treatment.

6. A method according to claim 5 wherein the antagonist of TSG18 is an antibody.

7. The use of an antagonist of TSG18 in the manufacture of a medicament for the treatment of a disorder associated with increased expression or activity of TSG18.

8. The use as claimed in claim 7 wherein the antagonist of TSG18 is an antibody.

9. An antibody which is immunologically reactive with a polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof which is active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (c) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; and (d) a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2.

10. An antibody as claimed in claim 9 which is selected from the group consisting of a monoclonal antibody, a humanised antibody, a chimaeric antibody or an antibody fragment including a Fab fragment, (Fab')₂ fragment, Fv fragment, single chain antibodies and single domain antibodies .

11. A method of treating a disorder associated with decreased expression or activity of TSG18, comprising gene therapy through administration of a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID N0:1 in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID N0:1; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID NO: 2; (f) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID NO: 2; and (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l, to a subject in need of such treatment .

12. A method as claimed in claim 11 wherein an expression vector comprising the nucleic acid molecule operably linked to suitable control elements is administered.

13. A method as claimed in claim 11 or claim 12 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis- ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout. Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma .

14. The use of an isolated nucleic acid molecule comprising: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID NO: 2; (f) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO:2; (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l, in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG18.

15. The use as claimed in claim 14 wherein the nucleic acid molecule is a part of an expression vector which also includes suitable control elements.

16. The use as claimed in claim 13 or claim 14 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis- ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

17. A method of treating a disorder associated with increased activity or expression of TSG18, comprising administering a nucleic acid molecule which is the complement of a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID N0:2; (f) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID N0:1 in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in

SEQ ID NO: 2; (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l, wherein the nucleic acid molecule encodes a mRNA that hybridizes with the mRNA encoded by TSG18.

18. A method as claimed in claim 17 wherein an expression vector comprising the nucleic acid molecule and suitable control elements is administered.

19. The use of an isolated DNA molecule which is the complement of a nucleic acid molecule comprising: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide seqiience set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID NO:2; (f) a nucleic acid molecule comprising the' nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO: 2; (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l, wherein the nucleic acid molecule encodes a mRNA that hybridizes with the mRNA encoded by TSG18, in the manufacture of a medicament for the treatment of a disorder associated with increased activity or expression of TSG18.

20. The use as claimed in claim 19 wherein the nucleic acid molecule is a part of an expression vector which also includes suitable control elements .

21. The use of an isolated nucleic acid molecule comprising: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID NO: 2; (f) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO:2; (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l, for the diagnosis of disorders associated with TSG18, or a predisposition to such disorders.

22. The use as claimed in claim 21 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast. prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED) , bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring) , cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

23. The use of a polypeptide comprising: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (c) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; or (d) a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% sequence identity with the amino acid seqiience set forth in SEQ ID NO: 2, in the diagnosis of a disorder associated with TSG18, or a predisposition to such disorders.

24. The use of an antibody as defined in either one of claims 9 or 10 in the diagnosis of a disorder associated with TSG18, or a predisposition to such disorders.

25. A method for the diagnosis of a disorder associated with mutations in TSG18, or a predisposition to such disorders in a patient, comprising the steps of:

(1) obtaining a sample which includes TSG18 or a nucleic acid which codes for TSG18 from the patient; (2) comparing TSG18 or a nucleic acid which codes for TSG18 from the sample with wild-type TSG18 or a nucleic acid which codes for it in order to establish whether the person expresses a mutant TSG18.

26. A method as claimed in claim 25 wherein the nucleotide sequence of DNA from the patient is compared to the sequence of DNA encoding wild-type TSG18.

27. A method for the diagnosis of a disorder associated with abnormal expression or activity of TSG18, or a predisposition to such disorders, comprising the steps of:

(1) establishing a profile for normal expression of TSG18 in unaffected subjects;

(2) measuring the level of expression of TSG18 in a person suspected of abnormal expression or activity of TSG18; and

(3) comparing the measured level of expression with the profile for normal expression.

28. A method as claimed in claim 27 wherein reverse transcriptase PCR is employed to measure levels of expression.

29. A method as claimed in claim 28 wherein a hybridisation assay using a probe derived from TSG18, or a fragment thereof, is employed to measure levels of expression.

30. A method as claimed in claim 29 wherein the probe has at least 50% sequence identity to a nucleotide sequence encoding TSG18, or a fragment thereof.

31. A method for the diagnosis of a disorder associated with TSG18, or a predisposition to such disorders, comprising the steps of:

(1) establishing a physical property of wild- type TSG18;

(2) obtaining TSG18 from a person suspected of an abnormality of TSG18; and (3) measuring the property for the TSG18 expressed by the person and comparing it to the established property for wild-type TSG18 in order to establish whether the person expresses a mutant TSG18.

32. A method as claimed in claim 31 wherein the property is the electrophoretic mobility.

33. A method as claimed in claim 31 wherein the property is the proteolytic cleavage pattern.

34. A method as claimed in claim 31 wherein the property is the activity of TSG18 as measured by a functional assay.

35. A method as claimed in claim 31 wherein the property is binding of an antibody as defined in either one of claims 9 or 10.

36. A method for determining whether a human tissue is pre-disposed to a neoplastic transformation, comprising, determining whether in a cell from the tissue a nucleic acid molecule that has the sequence shown in SEQ ID NO:l is absent or down-regulated.

37. A method according to claim 36 wherein the human tissue is breast tissue.

38. A method according to claims 36 or 37 comprising determining whether a peptide which has the sequence shown in SEQ ID NO: 2 is absent or expressed at reduced levels.

39. A method according to claim 38 comprising contacting the cell with an antibody for binding to the peptide under conditions which permit the antibody to bind to the peptide, if present.

40. A hybridoma cell for secreting an antibody as defined in either one of claims 9 or 10.

41. A genetically modified non-human animal transformed with a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID N0:1; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in SEQ ID NO: 2; (f) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets;

(h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO: 2; (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l.

42. A genetically modified non-human animal in which TSG18 activity is reduced or absent.

43. A genetically modified non-human animal in which TSG18 gene function has been knocked out.

44. A genetically modified non-human animal in which TSG18 gene function is modified through transformation with a mutant TSG18 gene.

45. A genetically modified non-human animal as claimed in any one of claims 41 to 44 in which the animal is selected from the group consisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs and non- human primates such as monkeys and chimpanzees.

46. The use of a genetically modified non-human animal as claimed in any one of claims 41 to 45 in screening for candidate pharmaceutical compounds.

47. The use of a cell transformed with a nucleic acid selected from the group consisting of: (a) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (c) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l; (d) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; (e) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, the polypeptide having an amino acid sequence with at least 70% sequence identity to that set forth in

SEQ ID NO: 2; (f) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:l, or a fragment thereof, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (g) a nucleic acid molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l in their respective coding regions, and which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; (h) a nucleic acid molecule that encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide seqiience set forth in SEQ ID NO:l; (i) a nucleic acid molecule which encodes a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, the polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO:2; (j) a nucleic acid molecule comprising exons 1 to 11 identified in the nucleotide sequence set forth in SEQ ID NO:l, in screening for candidate pharmaceutical compounds .

48. The use of a polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions associated with cancer and/or modulating immunological responses; (b) a polypeptide active in suppressing cellular functions associated with cancer and/or modulating immunological responses, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (c) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and which is active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets; or (d) a polypeptide active in suppressing cellular functions mediated through the STAT pathway and/or sumoylation of protein targets, and having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, in screening for candidate pharmaceutical compounds.

49. A nucleic acid encoding a mutant TSG18 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG18 does form a complex.

50. A nucleic acid as claimed in claim 49 wherein the wild-type protein is a protein selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins.

51. A nucleic acid as claimed in claim 50 wherein the wild-type protein is PIASl or PIAS3.

52. A nucleic acid as claimed in any one of claims 49 to 109 wherein the mutant TSG18 polypeptide contains a mutation in an ankyrin repeat domain.

53. A mutant TSG18 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG18 does form a complex.

54. A mutant TSG18 as claimed in claim 53 wherein the wild-type protein is selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins .

55. A mutant TSG18 as claimed in claim 54 wherein the wild-type protein is PIASl or PIAS3.

56. A mutant TSG18 polypeptide as claimed in any one of claims 53 to 55 containing a mutation in an ankyrin repeat domain.

57. The use of mutant TSG18 polypeptide as defined in any one of claims 53 to 55 for the screening of candidate pharmaceutical compounds.

58. A complex of wild-type TSG18 and a protein selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins.

59. A complex as claimed in claim 58 wherein the protein is PIASl or PIAS3.

60. The use of a complex as claimed in either one of claims 58 or 59 or the use of PIASl or PIAS3 for the screening of candidate pharmaceutical compounds.

61. A nucleic acid encoding a mutant TSG18 polypeptide which cannot interact with a complex with which wild-type

TSG18 does interact.

62. A nucleic, acid as claimed in claim 61 wherein the complex is the SUMO E3 ligase/TSG18 complex.

63. A mutant TSG18 polypeptide which cannot interact with a complex with which wild-type TSG18 does interact.

64. A mutant TSG18 as claimed in claim 63 wherein the complex is the SUMO E3 ligase/TSG18 complex.

65. The use of a mutant TSG18 polypeptide as defined in claim 63 or 64 for the screening of candidate pharmaceutical compounds.

66. A complex of wild-type TSG18 and SUMO E3 ligase.

67. The use of a complex as claimed in claim 66 or the use of SUMO E3 ligase for the screening of candidate pharmaceutical compounds.

68. The use of TSG18 to identify interacting proteins suitable as drug targets.

69. The use as claimed in claim 68 wherein TSG18 is fused to a DNA binding domain and used as the bait in a yeast two-hybrid system.

70. A fusion protein comprising TSG18 fused to a polypeptide whose function is DNA binding.

71. A fusion protein as claimed in claim 70 wherein TSG18 is fused to the DNA binding domain of the yeast GAL4 transcription factor.