WO2000037677A1

WO2000037677A1 - Cancer related to human chromosome 16q tumor suppressor gene

Info

Publication number: WO2000037677A1
Application number: PCT/GB1999/004366
Authority: WO
Inventors: David John Porteous; Hani Gabra; Janet Elizabeth Vivienne Watson
Original assignee: Imperial Cancer Research Technology Limited
Priority date: 1998-12-22
Filing date: 1999-12-22
Publication date: 2000-06-29
Also published as: AU1876900A

Abstract

A polynucleotide comprising all, or a variant, or part thereof, of human chromosome 16q tumour suppressor gene or all, or a variant, or a part of an mRNA or cDNA derived from the tumour suppressor gene wherein the tumour suppressor gene contains at least part of the nucleotide sequences shown in any one of Figures 4, 5, 6, 7, 8, 9, 15, Z1, 23 or 31. A method of diagnosing cancer in a patient comprising the steps of (i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q tumour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor gene cDNA, or a mutant ellele thereof, or their complement. A method of treating a proliferative disease comprising the step of administering to the patient a nucleic acid which encodes the human chromosome 16q tumour suppressor polypeptide or a functional variant or portion or fusion thereof.

Description

CANCER RELATED TO HUMAN CHROMOSOME 16Q TUMOR SUPPRESSOR GENE

The present invention relates to cancer and in particular to ovarian, breast and prostate cancers.

Cancer is a serious disease and a major killer. Although there have been advances in the diagnosis and treatment of certain cancers in recent years, there is still a need for improvements in diagnosis and treatment.

Cancer is a genetic disease and in most cases involves mutations in one or more genes. There are believed to be around 200,000 genes in the human genome but only a handful of these genes have been shown to be involved in cancer. Although it is surmised that many more genes than have been presently identified will be found to be involved in cancer, progress in this area has remained slow despite the availability of molecular analytical techniques. This may be due to the varied structure and function of genes which have been identified to date which suggests that cancer genes can take many forms and have many different functions.

Ovarian cancer is the most frequent cause of death from gynaecological malignancies in the Western World, with an incidence of 5,000 new cases every year in England and Wales. It is the fourth most common cause of cancer mortality in American women. The majority of patients with epithelial ovarian cancer present at an advanced stage of the disease. Consequently, the 5 year survival rate is only 30% after adequate surgery and chemotherapy despite the introduction of new drugs such as platinum and taxol (Advanced Ovarian Cancer Trialists Group (1991) BMJ 303, 884-893; Ozols (1995) Semin Oncol. 22, 61-66). However, patients who have stage I disease (confined to the ovaries) do better with the 5 year survival rate being 70% . It is therefore desirable to have techniques to detect the cancer before metastasis to have a significant impact on survival.

Epithelial ovarian cancer constitutes 70-80% of ovarian cancer and encompasses a broad spectrum of lesions, ranging from localized benign tumours and neoplasms of borderline malignant potential to invasive adenocarcinomas. Histologically, the common epithelial ovarian cancers, are classified into several types, that is, serous, mucinous, endometrioid, clear cell, Brenner, mixed epithelial, and undifferentiated tumours. The heterogeneity of histological subtypes reflects the metaplastic potential of the ovarian surface Mullerian epithelium which shares a common embryological origin with the peritoneum and the rest of the uro-genital system. Germ cell, sex cord/stromal tumours and sarcomas represent the remainder of ovarian cancers. The histogenesis and biological characteristics of epithelial ovarian cancer are poorly understood as are the molecular genetic alterations that may contribute to the development of such tumours or their progression. Epidemiological factors related to ovulation seem to be important, whereby ovarian epithelial cells undergo several rounds of division and proliferative growth to heal the wound in the epithelial surface. These lead to the development of epithelial inclusion cysts and frank malignant tumours may arise from them (Fathalla (1971) Lancet 2, 163).

Genetic changes in the tumour are critical for the development of cancer. Many chromosomal regions (including chromosomes 3, 5, 6, 8, 11, 13, 17, 18, 22, and X) have been implicated to contain tumour suppressor genes involved in tumour progression of sporadic ovarian cancer, but only the p53 gene (chromosome arm 17p) has been found to be frequently mutated (Shelling et al (1995) Br. J. Cancer 72, 521-527). The BRCA1 gene (chromosome arm 17q) and the BRCA2 gene (chromosome arm 13q) isolated in 1994 and 1996 respectively, are mutated in a proportion of patients with familial breast/ovarian cancer (Ford & Easton (1995) Br. J. Cancer 72, 805-812). Familial ovarian cancer only accounts for 5-10% of all ovarian tumours. In tumours from patients with sporadic ovarian cancer, only five mutations in the BRCA1 gene and four in the BRCA2 gene have been reported (Takahashi et al (1995) Cancer Res. 55, 2998- 3002; Takahashi et al (1996) Cancer Res. 56, 2738-2741) suggesting that they are rare in sporadic ovarian cancer. Mutations in the mismatch repair genes have been reported at a frequency of 10% (Tangi et al (1996) Cancer Res. 56, 2501-2505; Fujita et al (1995) Int. J. Cancer 64, 361- 366; Orth et al (1994) Proc. Natl. Acad. Sci. USA 91, 9495-9499). Thus genes that may be more critical in tumour progression in sporadic ovarian cancer have not yet been fully characterised. Arnold et al (1996) Genes, Chromosomes & Cancer 16, 46-54 describes common genetic changes in human ovarian cancer.

Breast cancer is one of the most significant diseases that affects women. At the current rate, American women have a 1 in 8 risk of developing cancer by the age of 95 (American Cancer Society, Cancer Facts and Figures, 1992, American Cancer Society, Atlanta, Georgia, USA). Genetic factors contribute to an ill-defined proportion of breast cancer cases, estimated to be about 5% of all cases but approximately 25% of cases diagnosed before the age of 40 (Claus et al (1991) Am J. Hum. Genet. 48, 232-242). Breast cancer has been divided into two types, early-age onset and late stage onset, based on an inflection in the age- specific incidence curve at around the age of 50. Mutation of one gene, BRCAl, is thought to account for approximately 45% of familial breast cancer, but at least 80% of families with both breast and ovarian cancer (Easton et al (1993) Am. J. Hum. Genet. 52, 678-701).

WO 96/05306, WO 96/05307 and WO 96/05308 relate to methods and materials used to isolate and detect a human breast and ovarian cancer predisposing gene (BRCAl), some mutant alleles of which are alleged to cause susceptibility to cancer, in particular breast and ovarian cancer.

Carcinoma of the prostate has become a most significant disease in many countries. Over the last 20 years the mortality rates have doubled and it is now the second commonest cause of male cancer deaths in England and

Wales (Mortality Statistics: Cause England and Wales. OPCS DH2 19,

1993, Her Majesty's Stationery Office). The prevalence of prostate cancer has increased by 28% in the last decade and this disease now accounts for 12% of the total cancers of men in England and Wales (Cancer Statistics: Registrations England and Wales. OPCS MBI No 22,

1994, Her Majesty's Stationery Office). This increase and the recent deaths of many public figures from prostatic cancer have served to highlight the need to do something about this cancer. It has been suggested that the wider availability of screening may limit mortality from prostate cancer.

Prostate cancer screening currently consists of a rectal examination and measurement of prostate specific antigen (PSA) levels. These methods lack specificity as digital rectal examination has considerable inter- examiner variability (Smith & Catalona (1995) Urology 45, 70-74) and PSA levels may be elevated in benign prostatic hyperplasia (BPH), prostatic inflammation and other conditions. The comparative failure of PSA as a diagnostic test was shown in 366 men who developed prostate cancer while being included in the Physicians Health Study, a prospective study of over 22,000 men. PSA levels were measured in serum, which was stored at the start of the study, and elevated levels were found in only 47 % of men developing prostate cancer within the subsequent four years (Gann et al (1995) JAMA 273, 289-294).

Cytogenetic and allele loss studies have pointed to a number of chromosomal regions of potential involvement in prostate cancer. Cannon-Albright & Eeles (1995) Nature Genetics 9, 336-338 (Reference 1) discuss candidate regions for tumour suppressor prostate cancer susceptibility loci from loss-of-heterozygosity (LOH) studies which occur on human chromosome regions 3p, 7q, 8p, 9q, lOp, lOq, l ip, 13q, 16q, 17p, 18q and Y; whereas Brothman et al (1990) Cancer Res. 50 3795- 3803 surveyed cytogenetic information on human prostate adenocarcinoma which indicated loss of chromosomes 1, 2, 5 and Y and gain of 7, 14, 20 and 22, with rearrangements involving chromosome arms 2p, 7q and lOq being most common. Studies by Gao et al (1994) Oncogene 9, 2999-3003 indicate that a positive imitator phenotype in at least one of chromosomes 3p, 5q, 6p, 7p, 8p, lOq, l ip, 13q, 16q, 17p, 18q and Xq is found in prostate adenocarcinoma; and Massenkeil et al (1994) Anticancer Res. 14(6B), 2785-2790 indicates that LOH was observed at 8p, 17p, 18q in various prostate tumour samples but no deletions were observed on lOq in fourteen informative prostate tumours. Zenklusen et al (1994) Cancer Res. 54, 6370-6373 suggests that there is a possible tumour suppressor gene at 7q31.1. In addition, there have been other reports which describe other chromosome loss or abnormalities.

Loss of heterozygosity (LOH) studies have implicated chromosome 16 in a variety of cancers. For example, Elo et al (1997) Cancer Res. 57, 3356- 3359 alleges that LOH at 16q24.1-q24.2 is significantly associated with metastatic and aggressive behaviours of prostate cancer and that the most frequent area of LOH was located between the markers D16S504 and D16S422. By contrast, Chen et al (1996) Cancer Res. 56, 5605-5609 identifies a different region of 16q which is purportedly associated with breast cancer, and Latil et al (1997) Cancer Res. 57, 1058-1062 identifies three independent regions of chromosome 16q where LOH occurs in prostate adenocarcinoma. Latil and co-workers note that although several genes are located in the third region that they have identified, there are no obvious candidate tumour suppressor genes. A still further paper (Hansen et al (1998) Cancer Res. 58, 2166-2169) suggests that it is LOH at the marker D16S511 which is informative as an independent marker of good prognosis in primary breast cancer. Cleton-Jansen et al (1994) Genes, Chromosomes & Cancer 9, 101-107 alleges that at least two different regions are involved in allelic imbalance on Chromosome 16q in breast cancer; Sato et al (1991) Cancer Res. 51, 5118-5122 discusses allelotype of human ovarian cancer; Sato et al (1991) Cancer Res. 51, 5794-5799 describes the accumulation of genetic alterations and progression of primary breast cancer; and Callen et al (1992) Cytogen. Cell Genet. 60, 158-167 reports on a workshop on chromosome 16. Human chromosome 16 has also been implicated in various tumours, including Wilms' tumour and hepatocellular carcinoma, in the following papers: Chin et al (1998) Cancer Genet. Cytogenet. 103, 155-163; Cleton-Jansen et al (1994) Genes, Chromosomes & Cancer 9, 101-107; Sato et al (1990) Cancer Res. 50, 7184-7189; Tsuda et al (1990) Proc. Natl. Acad. Sci. USA 87, 6791- 6794; Maw et al (1992) Cancer Res. 52, 3094-3098; and Osman et al (1997) Int. J. Cancer 71, 580-584.

Figure 1 shows the relationship between various microsatellite markers on chromosome 16 as is presently understood. It is possible that as the map is refined the relative position of the markers may change.

Despite intensive study the involvement of LOH of chromosome 16q in various human cancers, and the role of any tumour suppressor gene, remain unclear.

There remains a need for new and better methods of diagnosing and treating cancer, especially cancers which affect large numbers of people such as breast and prostate cancer. Although there are fewer cases of ovarian cancer (20,000 per year in USA), mortality is higher.

Objects of the invention are to provide new and potentially better methods for the diagnosis of cancer and the treatment of proliferative diseases including cancer; to provide nucleic acids which are useful in such methods; and to provide a tumour suppressor gene associated with ovarian cancer.

A first aspect of the invention provides a poly nucleotide comprising all, or a variant, or part thereof, of human chromosome 16q tumour suppressor gene or all, or a variant, or a part of a mRNA or cDNA derived from the tumour suppressor gene wherein the tumour suppressor gene contains at least part of any of the nucleotide sequences shown in any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31. These nucleic acid sequences are derived from a minimal region of human chromosome 16q which we have shown is very likely to contain a tumour suppressor gene. The Figure 4 sequence is derived from a clone containing a marker we have called 4T7; the Figure 5 sequence is derived from a clone containing a marker we have called IM97; the Figure 6 sequence is derived from a clone containing a marker we have called 435E; the Figure 7 sequence is derived from a clone we have called ETAl; the Figure 8 sequence is derived from a clone containing a marker we have called 5.1A6; the Figure 9 sequence is derived from a clone containing a marker we have called 10102; the Figure 15 sequence is derived from a clone containing a marker we have called RD30; the Figure 21 sequence is derived from a clone containing a marker we have called IM29; the Figure 23 sequence is derived from a clone containing a marker we have called IM27; and the Figure 31 sequence is derived from a clone containing a marker we have called IM28. As is discussed in more detail below, at least some of these sequences contain sequences which are derived from, or are homologous to, expressed sequence tags (ESTs) which are cDNAs. Thus, a cDNA derived from the tumour suppressor gene is believed to contain sequence which is contiguous with at least a portion of the sequence given in the Figures. Since the tumour suppressor gene itself is believed to contain introns, the gene may contain sequence which is contiguous with all of the sequence given in the Figures because of the presence of introns in the gene but which is not present in sequence derived from cDNAs.

The sequence of Figure 7 (ETAl) was obtained from an exon-trapping experiment and so is likely to contain coding exonic sequence. Thus, it is preferred that the poly nucleotide comprising all, or a variant, or part thereof, of the human chromosome 16q tumour suppressor gene of the invention or the poly nucleotide comprising all, or a variant, or part of a mRNA or cDNA derived from the tumour suppressor gene of the invention comprises at least part of the nucleotide sequence shown in Figure 7 (ETAl).

It is also particularly preferred that the polynucleotide comprising all, or a variant, or part thereof, of the human chromosome 16q tumour suppressor gene of the invention or the polynucleotide comprising all, or a variant, or part of a mRNA or cDNA derived from the tumour suppressor gene of the invention comprises at least part of the nucleotide sequence shown in any one of Figures 5, 15, 21, 23 or 31.

For the avoidance of doubt, by "nucleotide sequence shown in Figure 4" (or 5 or 6 or 7 or 8 or 9 or 15 or 21 or 23 or 31, as the case may be) we include the meaning that the polynucleotide contains the actual sequence given, or the reverse complement of the sequence given. It will be appreciated that a mRNA derived from the tumour suppressor gene will contain a RNA version of the sequence given (or of its reverse complement) in which T residues are replaced by U residues. We also include the meaning that the nucleotide sequence may vary slightly from that given. This may be a reflection of natural polymorphic variation; it may be a reflection of minor sequence determination errors in the sequences given in the said Figures.

The tumour suppressor gene of the invention is a gene which contains at least one set of 25 contiguous residues from the sequences shown in the relevant Figures. Preferably it contains at least one set of 50 contiguous residues from the sequences shown in the said Figures; more preferably it contains at least one set of 100 contiguous residues from the sequences shown in the relevant Figures. The gene and further cDNAs derivable from the gene are readily obtained using methods well known in the art. For example, further cDNAs can be isolated from a foetal brain or foetal heart or whole ovary cDNA library, using standard methods and using any of the ESTs as a probe. The sequence is readily determined using standard methods. Similarly, the gene can be isolated from a human genomic DNA library, such as an appropriate PI -artificial chromosome (PAC) clone (see below), using a suitable probe using standard methods.

Foetal brain and foetal heart cDNA libraries may be obtained using standard molecular biology methods or may be obtained from Clontech Laboratories, Ine, 1020 East Meadow Circle, Palo Alto, California 94303- 4230, USA. A whole ovary cDNA library may be obtained from Stratagene, Ine, 11011 North Torrey Pines Road, La Jolla, CA 92037.

Standard methods of screening DNA libraries, isolating and manipulating cloned DNA and sequencing DNA are described in Sambrook et al (1989) "Molecular cloning, a laboratory manual", 2nd Edition, Ed Sambrook et al, Cold Spring Harbor Press, Cold Spring Harbor, New York.

The predicted amino acid sequence encoded by the ETAl sequence (which contains exonic sequence) may be used to make peptides which can, in turn, be used to make antibodies. The antibodies can be used to screen a cDNA expression library or can be used to isolate the polypeptide encoded by the gene. Once the polypeptide is isolated its N-terminal sequence can be obtained using methods well known in the art. The amino acid sequence is then used to design an oligonucleotide probe which identifies the 5' coding region of a cDNA. Similarly, exonic sequence from Figures 4, 5, 6, 8, 9, 15, 21, 23 or 31 may also be used.

The tumour suppressor gene may also be readily identified by cDNA selection wherein, for example, cDNA from ovarian tumour cell lines is hybridised to genomic cDNA from the PAC clones (especially from PAC clones PAC4 and PAC22; see below). This approach has been used by Lovett et al (1991) Proc. Natl. Acad. Sci. USA 88, 9628-9632.

It will be appreciated that the 5' ends of cDNAs can be isolated by RACE (Rapid Amplification of cDNA Ends; Schaefer (1995) Anal. Biochem. 227, 255-273), a technique well known in the art. This approach, and related approaches, involve reverse transcription from mRNA using a primer based on the presently known 5' sequence which works back towards the 5' end of the mRNA transcript followed by PCR using random primers to prime from the "unknown" 5' end. Messenger RNA- based RACE can also be used for obtaining 5' ends by isolating mRNA, removing the 5' cap and then the 5' end is ligated to an adaptor sequence and PCR follows using one primer against the adaptor and one primer specific to the cDNA of interest.

Methods for isolating genes and parts of genes are described in Current Protocols in Human Genetics, 1996, Dracopoli et al (ed), John Wiley & Sons, incorporated herein by reference. One useful technique is "vectorette" PCR.

Vectorette PCR can be used for the identification of novel genes, or for the identification of additional sequence when part of the sequence of a gene is already known. The vectorette itself is a double stranded piece of synthetic DNA, with a mismatched central region and one end suitable for ligation to DNA cut by a restriction enzyme (described in Current Protocols in Human Genetics 1995 (see pages 5.9.15-5.9.21) and in Valdes et al (1994) Proc. Natl. Acad. Sci. USA 91, 5377-5381 and Allen et al PCR Methods and Applications 4, 71-75). Following ligation of the vectorette to restriction fragments derived from an appropriate DNA source (usually a large genomic DNA fragment such as a YAC clone), PCR amplification is performed using a primer derived from the target DNA in conjunction with a primer derived from the mismatched region of the vectorette. This vectorette primer has the same sequence as the bottom strand of this mismatched region and therefore has no complementary sequence to anneal to in the first cycle of PCR. The first round of amplification is unidirectional, as priming can only occur from the primer within the target DNA. This produces a complementary strand for the vectorette PCR primer to anneal to in the second PCR cycle. In the second and subsequent cycles of PCR, both primers can prime DNA synthesis with the end result being that the only fragment amplified contains the sequence of interest.

This technique can be used for the identification of intronic sequences within a gene based on a knowledge of the cDNA sequence for that gene. Following restriction digestion of a genomic DNA fragment bearing the gene of interest (such as a YAC clone) and subsequent ligation to the vectorette, a primer designed from the cDNA sequence is used in conjunction with the vectorette primer to PCR amplify a specific fragment of the gene. Exon/intron boundaries can be identified by comparison of the sequence of this fragment to that of the cDNA. Similarly, a vectorette approach can be used to identify the missing 5' end of a gene by using a primer derived from the 5' end of the known cDNA sequence to generate further 5' sequence data.

Vectorettes can also be used for the identification of completely novel gene sequences in a technique known as 'island rescue'. This approach exploits the fact that CpG-rich 'islands' exist within mammalian genomes and that such islands are associated with the 5' ends of genes. Certain restriction enzymes cut within CpG islands, for example, the enzyme Notl. Following Notl digestion of a genomic DNA fragment, a vectorette with a Nørl-compatible sticky end is ligated to the resulting sub-fragments. PCR amplification is then performed using the vectorette primer in conjunction with a primer derived from an Alu repeat element. Such elements occur at frequent intervals in the human genome, therefore it is likely that one or more will lie adjacent to the CpG island of interest and facilitate the generation of a PCR product. As a control, a second PCR reaction is executed, excluding the vectorette primer. Any fragments generated in the /w/vectorette primed reaction but absent from the Alu only control should represent part of the CpG island and can be gel- purified and analysed for coding sequences using standard methods.

The tumour suppressor gene is one which is involved in the origin or development of a cancer such as ovarian cancer, or breast cancer or prostate cancer, or gastric cancer or lung cancer or colon cancer.

A nucleic acid of the invention comprising a tumour suppressor gene or fragment or derivative thereof is readily identified; for example, the gene may be identified by screening a panel of RNAs from ovary or breast or prostate and other tumour cell lines in order to identify a reduced level of transcript. The transcript may be large, as it will probably have a complex function and several sites for disabling mutation 'hits' (as is the case with the tumour suppressor genes BRCAl, RB). Cross-species conservation indicates that the gene has a basic cell 'housekeeping' function, the loss of which may lead to loss of growth control and tumour formation; in particular, cross-species conservation of the human-derived nucleic acid in clone 435E has been demonstrated.

By "tumour suppressor gene" we include any gene for which loss or some reduction in any of its functions or activities can contribute to neoplasia.

Analysis of the entire coding region of the tumour suppressor gene in tumours indicates that the gene is a tumour suppressor gene when the gene has been altered compared to the gene in non-tumour tissue or to the gene in an individual who does not have, and who is not prone to, ovarian cancer or breast cancer or prostate cancer or gastric cancer or lung cancer or colon cancer, and that it is involved in the cancer. Suitable methods for mutation analysis include single-stranded conformation polymorphism (SSCP) analysis (or variations of this technique) and direct DNA sequencing. These are well known to the person skilled in the art, and SSCP, for example, is described in Current Protocols in Human Genetics, 1995, pp 7.4J-7.4.6. It will be appreciated that not all individuals with ovarian or breast or prostate or gastric or lung or colon cancer will have mutations in the human chromosome 16q tumour suppressor gene. However, the tumour cells PE04, WX330, HCT116, and AGS as described in more detail below, are believed to have deletions in the tumour suppressor gene. The tumour suppressor gene of the invention almost certainly contains introns and almost certainly is >0.5 kb, more likely > 1.0 kb and most likely between 1.0 kb and 500 kb. The tumour suppressor gene of the invention almost certainly is polymorphic in its DNA sequence. Thus, fragments (such as restriction fragments or fragments derived by enzymatic amplification) and variants (such as natural variants, eg allelic variants) or variants created by in vitro manipulation are part of the invention. Suitable such fragments include fragments which are useful as a hybridisation probe or fragments which are useful as an amplification primer. Suitable such variants include variants in which the coding sense of the gene is unaltered or variants in which the coding sequence is modified so as to alter the properties of the encoded polypeptide.

It is particularly preferred if the nucleic acid is a cDNA (copy DNA) derived from a mRNA transcribed from the tumour suppressor gene. Libraries of cDNA derived from selected tissues, such as breast, ovary or prostate, lung, stomach or colon are known in the art and can be prepared from suitable mRNA using methods known in the art for example as described in Molecular cloning, a laboratory manual (supra).

A second aspect of the invention provides a polynucleotide which selectively hybridises to the region of human chromosome 16q which is bounded by the markers Alu 11 and 10T7 in Figure 2 or which hybridises to the PAC clone 81N24 or the PAC clone 105K14 or the YAC clone 801B6, or a mutant allele thereof, or a polynucleotide which hybridises to a mRNA or cDNA derived from a gene from the region, or a mutant allele thereof. PAC clone 81N24 is referred to as "PAC4" in Figures 2 and 3; PAC clone 105K14 is referred to as "PAC22" in Figures 2 and 3. All of the PAC and YAC clones referred to in this patent application are publicly available. However, for the avoidance of doubt, PAC clones 81N24 and 105K14 have been deposited under the Budapest Treaty by Imperial Cancer Research Technology Limited at National Collection of Industrial and Marine Bacteria (NCIMB Ltd), 23 St Machar Drive, Aberdeen AB24 3RY, Scotland, UK under Accession No NCIMB 40993 (PAC clone 81N24) and NCIMB 40994 (PAC clone 105K14).

Preferably, the polynucleotide hybridises to the region of human chromosome 16q which is bounded by the markers Alu 29 and 7T7 in Figure 2.

More preferably the polynucleotide hybridises to the region of human chromosome 16q which is bounded by the markers 17SP6 and RD53 in Figure 2.

Preferred polynucleotides which selectively hybridise as said are described in more detail with respect to their use in various methods of the invention, particularly those described in respect of the sixth, seventh and eighth and ninth aspects (see below).

A third aspect of the invention provides a polynucleotide comprising all, or a variant, or part thereof, of human chromosome 16q tumour suppressor gene or all, or a variant, or a part of a mRNA or cDNA derived from the tumour suppressor gene wherein the tumour suppressor gene contains at least part of the nucleotide sequence shown in any of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31 further comprising a detectable label or a polynucleotide which selectively hybridises to the region of human chromosome 16q which is bounded by the markers Alu 11 and 10T7 in Figure 2 or which hybridises to the PAC clone 81N24 or PAC clone 105K14 or the YAC clone 801B6, or a mutant allele thereof, or a polynucleotide which hybridises to a mRNA or cDNA derived from a gene from the region, or a mutant allele thereof further comprising a detectable label.

Suitable detectable labels are disclosed in more detail below.

A fourth aspect of the invention provides a polypeptide capable of being encoded by the tumour suppressor gene of the invention or a fragment or variant or fusion thereof. The fragment or variant or fusion polypeptide preferably has tumour suppressor activity, especially in the ovary or breast or prostate or lung or stomach or colon, or cross-reacts with an antibody which is specific for the native polypeptide. A fragment may be made by deleting (typically using protein engineering methods) a non essential portion of the polypeptide. A variant may be one in which one or more amino acid residues are inserted, deleted or replaced with other amino acid residues.

The polypeptide capable of being encoded by the tumour suppressor gene, or a fragment or variant or fusions thereof is conveniently referred to as the "tumour suppressor polypeptide".

A fifth aspect of the invention provides an expression vector capable of expressing a polypeptide encoded by the human chromosome 16q tumour suppressor gene. Expression vectors and their uses are described in more detail below. Particularly preferred expression vectors are those that can be used to express the human chromosome 16q tumour suppressor polypeptide in mammalian cells, including human cells. It will be appreciated that such vectors may be useful in gene therapy applications.

The invention also includes recombinant molecules, especially molecules which can be propagated in or can, at least, be introduced into mammalian cells (including human cells) which molecules comprise at least part of the human chromosome 16q tumour suppressor gene. It will be appreciated that even if the whole gene or cDNA is not included, or the intact polypeptide is not encoded, these recombinant molecules may still be useful since they may be able to repair a cellular defect, for example by homologous recombination.

A particularly preferred embodiment of the fifth aspect of the invention is an expression vector, in particular an expression vector adapted for use as a gene therapy vector, which contains at least 20 nucleotides, preferably at least 30 nucleotides, more preferably at least 40 nucleotides and still more preferably at least 50 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31, or a complement thereof. It is particularly preferred if the contiguous nucleotides are from an exon of the tumour suppressor gene.

Typically the expression vector contains a promoter which can be used to transcribe RNA in a mammalian, preferably human, cell. Conveniently, the promoter may be a tissue or cell-type- selective promoter, examples of which are well known in the art.

A further aspect of the invention provides an expression vector which contains at least 20 nucleotides, preferably at least 30 nucleotides, more preferably at least 40 nucleotides and still more preferably at least 50 nucleotides which are contiguous from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31, or a complement thereof.

A still further aspect of the invention provides a vector adapted to replicate in a mammalian cell which contains at least 20 nucleotides, preferably at least 30 nucleotides, more preferably at least 40 nucleotides, and still more preferably at least 50 nucleotides which are contiguous from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31, or a complement thereof.

A further aspect of the invention comprises a molecule capable of specifically binding with a polypeptide of the fourth aspect of the invention. Suitably, the molecule is an antibody-like molecule comprising complementarity-determining regions specific for the said polypeptide.

Monoclonal antibodies which will bind to many of these antigens are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).

Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799). 0/37677

20

Suitably prepared non-human antibodies can be "humanized" in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.

It will be appreciated that antibody phage display libraries may also be used to select suitable antibody-like molecules as is well known in the art. As well as monoclonal antibodies, polyclonal antibodies which are made by standard immunological methods, are usefully made.

A sixth aspect of the invention provides a method for determining the susceptibility of a patient to cancer comprising the steps of (i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q tumour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor gene cDNA, or a mutant allele thereof, or their complement.

A seventh aspect of the invention provides a method of diagnosing cancer in a patient comprising the steps of (i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q tumour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor cDNA, or a mutant allele thereof, or their complement.

An eighth aspect of the invention provides a method of predicting the relative prospects of a particular outcome of a cancer in a patient 0/37677

21 comprising the steps of (i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q tumour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor gene cDNA, or a mutant allele thereof, or their complement.

By "human chromosome 16q tumour suppressor gene" we include the tumour suppressor gene at least part of which is found on human chromosome 16q between the markers Alu 11 and 10Sp6 in Figure 2.

The methods of determining susceptibility to cancer, and of diagnosis, and of predicting the relative prospects of a particular outcome of a cancer in a patient also include methods comprising the steps of (i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the region of human chromosome 16q which is bounded by the markers Alu 11 and 10T7 in Figure 2 or which hybridises to the PAC clone 81N2K or PAC clone 105K14 or the YAC clone 801B6, or a mutant allele thereof.

In a further embodiment, the above method makes use of nucleic acid which hybridises selectively to the nucleic acid whose sequence is shown in any one of Figures 4 to 31, preferably to that which is shown in any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23, or 31, or a complement thereof.

Figure 2 shows a physical map of contigs of PI -artificial chromosome (PAC) clones and DNA markers spanning the deleted regions from the following tumour samples and cell lines, all of which are believed to be 0/37677

22 deleted for at least part of the human chromosome 16q tumour suppressor gene.

PE04:

DNA was extracted from malignant cells isolated from an ascites specimen from a patient with stage III ovarian cancer. The same deletion is also found in a cell line (PE04) derived from the same sample. The primary sample and cell line were derived as shown in Wolf et al (1987) Int. J. Cancer 39, 695-702.

WX330:

WX330 cell line was derived from a pleural effusion from a patient with extensive small cell lung cancer (SCLC). The primary sample and cell line were derived as shown in Hay et al (1991) Br. J Cancer 63, Suppl XIV, 43-45.

HCT116:

HCT116 cell line was derived from malignant cells isolated from a male patient with colonic carcinoma (see Brattain et al (1981) Cancer Res. 41, 1751-1756). The culture was obtained from Susan Farrington (MRC HGU), who obtained it originally from the ATCC, USA under accession number CCL-247.

It is believed that at least part of the tumour suppressor gene is found between the markers Alu29 and 7T7; it is nirther believed that at least part of the tumour suppressor gene is found between the markers Alu20 and 0/37677

23

8T7; it is still further believed that at least part of the tumour suppressor gene is found between the markers 17SP6 and RD53; it is yet still further believed that at least part of the tumour suppressor gene is found between the markers 17Sp6 and RD30. The position of the markers are shown in Figure 2.

The nucleotide sequences of regions of human chromosome 16 which are defined by the markers are given in Figures 4 to 20. It is preferred if the tumour suppressor gene contains the human DNA sequence given in any one of Figures 4, 5, 6, 8, 9, 15, 21, 23 or 31. It is particularly preferred if the tumour suppressor gene contains the human DNA sequence given in RD30 (Figure 15(a) and (b) derived from a gene found in the region of chromosome 16q which is deleted from the tumour cells or cell lines PE04, WX330 and HCT116. It is further believed that at least part of the human chromosome 16q tumour suppressor gene is located on the yeast artificial chromosome (YAC) clone 801B6 (see Figure 2).

It is also further believed that at least part of the human chromosome 16q tumour suppressor gene is located on the PAC clones 81N24 and 105K14 (indicated as PAC clones 4 and 22 on Figure 2 and 3).

YAC clone 801B6 is described in Albertsen et al (1990) Proc. Natl. Acad. Sci. USA 87, 4256-4260 and is publicly available from Centre d'Etude du Polymorphisme Humain, 27 rue Juliette Dodu, 75010 Paris, France. PAC clones 4 to 31 are publicly available from the Human Genome Mapping Project Resource Centre, Hinxton Hall, Hinxton, Cambridgeshire CB10 IRQ, United Kingdom. PAC clones 33 to 38 are publicly available from the Resource Centre of the German Human Genome Project, Heubnerweg 6, Berlin, Germany. The Resource Centre names for the PAC clones are given below.

Thus, the invention includes methods for determining the susceptibility of a patient to cancer, of diagnosing cancer in a patient and predicting the relative prospects of a particular outcome of a cancer in a patient, the method comprising (i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with any nucleic acid which hybridises selectively to human DNA selected from human DNA which is bound by preferably (a) the markers Alu 11 and 10T7 in Figure 2; or more preferably (b) the markers Alu29 and 7T7 in Figure 2; or still more preferably (c) the markers Alu20 and 8T7; or yet still more preferably (d) the markers 17SP6 and RD53. Also preferably the nucleic acid which is to contact the sample nucleic acid in step (ii) may be nucleic acid which selectively hybridises to human chromosome 16 DNA which is present in YAC clone 801B6 or in PAC clones 81N24 or 105K14.

It will be appreciated that the tumour suppressor gene may exist as a "wild-type" gene or it may exist as mutant alleles which differ in sequence to the wild- type gene. By "mutant alleles" is included not only sequences which lead to changes in function or expression or stability of the tumour suppressor polypeptide, but allelic variants (or polymorphisms) which have no or only minor effect on the function or expression of the tumour suppressor polypeptide. Thus, the nucleic acids which selectively hybridise in the methods of the invention include those that selectively hybridise to the wild-type tumour suppressor gene sequence or to the wild- type tumour suppressor gene cDNA sequence (or mRNA sequence) as well as those which selectively hybridise to mutant alleles thereof. Also, it will readily be appreciated that, as is described in more detail herein, the skilled person can readily identify mutant alleles of the tumour suppressor gene and polymorphisms thereof. By "change in expression of the tumour suppressor polypeptide" is included any changes in the tumour suppressor gene which lead to changes in expression of the tumour suppressor polypeptide. For example, changes in the transcription of the tumour suppressor gene will lead to changes in the expression of the tumour suppressor polypeptide. Similarly, changes in the translation of tumour suppressor gene mRNA will lead to changes in the expression of the tumour suppressor polypeptide.

It will be appreciated that the nucleic acids which are useful in the method of the invention may readily be defined as those which selectively hybridise to the human DNA regions identified above.

By "selectively hybridising" is meant that the nucleic acid has sufficient nucleotide sequence similarity with the said human DNA or cDNA that it can hybridise under moderately or highly stringent conditions. As is well known in the art, the stringency of nucleic acid hybridization depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and CG or AT content of the sequence. Thus, any nucleic acid which is capable of selectively hybridising as said is useful in the practice of the invention.

Nucleic acids which can selectively hybridise to the said human DNA or cDNA include nucleic acids which have >95% sequence identity, preferably those with > 98%, more preferably those with > 99% sequence identity, over at least a portion of the nucleic acid with the said human DNA or cDNA. As is well known, human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene within the said human DNA would not match perfectly along its entire length with the said human DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said human DNA. Thus, the invention specifically includes the use of nucleic acids which selectively hybridise to tumour suppressor gene cDNA but may not hybridise to the tumour suppressor gene, or vice versa. For example, nucleic acids which span the intron-exon boundaries of the tumour suppressor gene may not be able to selectively hybridise to the tumour suppressor gene cDNA.

Typical moderately or highly stringent hybridisation conditions which lead to selective hybridisation are known in the art, for example those described in Molecular Cloning, a laboratory manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, incorporated herein by reference.

An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe nucleic acid is > 500 bases or base pairs is:

6 x SSC (saline sodium citrate)

0.5% sodium dodecyl sulphate (SDS)

100 μg/ml denatured, fragmented salmon sperm DNA

The hybridisation is performed at 68 °C. The nylon membrane, with the nucleic acid immobilised, may be washed at 68 °C in 1 x SSC or, for high stringency, O. l x SSC. 20 x SSC may be prepared in the following way. Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H₂0. Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 litre with H₂0. Dispense into aliquots. Sterilize by autoclaving.

An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe is an oligonucleotide of between 15 and 50 bases is:

3.0 M trimethylammonium chloride (TMAC1)

0.01 M sodium phosphate (pH 6.8)

1 mM EDTA (pH 7.6)

0.5% SDS

100 μg/ml denatured, fragmented salmon sperm DNA 0.1 % nonfat dried milk

The optimal temperature for hybridization is usually chosen to be 5°C below the T_t for the given chain length. Tj is the irreversible melting temperature of the hybrid formed between the probe and its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16, 4637 discusses the determination of TjS. The recommended hybridization temperature for 17- mers in 3 M TMAC1 is 48-50 °C; for 19-mers, it is 55-57 °C; and for 20- mers, it is 58-66 °C.

By "nucleic acid which selectively hybridises" is also included nucleic acids which will amplify DNA from the said region of human DNA by any of the well known amplification systems such as those described in more detail below, in particular the polymerase chain reaction (PCR). Suitable conditions for PCR amplification include amplification in a suitable 1 x amplification buffer:

10 x amplification buffer is 500 mM KC1; 100 mM Tris.Cl (pH 8.3 at room temperature); 15 mM MgCl₂; 0.1 % gelatin.

A suitable denaturing agent or procedure (such as heating to 95 °C) is used in order to separate the strands of double-stranded DNA.

Suitably, the annealing part of the amplification is between 37°C and 60°C, preferably 50°C.

Although the nucleic acid which is useful in the methods of the invention may be RNA or DNA, DNA is preferred. Although the nucleic acid which is useful in the methods of the invention may be double-stranded or single-stranded, single-stranded nucleic acid is preferred under some circumstances such as in nucleic acid amplification reactions.

The nucleic acid which is useful in the methods of the invention may be very large, such as 100 kb, if it is double stranded. For example, such large nucleic acids are useful as a template for making probes for use in FISH (fluorescence in situ hybridization) analysis. Typically, the labelled probes used in FISH are generally made by nick-translation or random priming from a genomic clone (such as an insert in a suitable PAC clone). Once made these probes are around 50-1000 nucleotides in length. The human DNA insert of PAC clone 81N24 is 188 kb; the human DNA insert of PAC clone 105K14 is 120 kb. These PAC clones may be useful probes in their own right, but they are more preferably used as a template for nick-translation or random primer extension as described above. However, for certain diagnostic, probing or amplifying purposes, it is preferred if the nucleic acid has fewer than 10 000, more preferably fewer than 1000, more preferably still from 10 to 100, and in further preference from 15 to 30 base pairs (if the nucleic acid is double-stranded) or bases (if the nucleic acid is single stranded). As is described more fully below, single-stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.

The PAC clones 81N24 and 105K14 overlap by about 60 kb and so the two clones define a minimal region of around 248 kb.

The nucleic acid for use in the methods of the invention may be a nucleic acid capable of hybridising to the tumour suppressor gene. Fragments and variants of this gene, and cDNAs derivable from the mRNA encoded by the gene are also preferred nucleic acids for use in the methods of the invention.

Clearly nucleic acids which selectively hybridise to the gene itself or variants thereof are particularly useful. Fragments of the gene are preferred for use in the method of the invention. Fragments may be made by enzymatic or chemical degradation of a larger fragment, or may be chemically synthesised. By "gene" is included not only the introns and exons but also regulatory regions associated with, and physically close to, the introns and exons, particularly those 5' to the 5 '-most exon. By "physically close" is meant within 50 kb, preferably within 10 kb, more preferably within 5 kb and still more preferably within 2 kb. However, tissue specific or inducible elements may be 50 kb in either direction of the coding regions (exons) or may be in the introns. Such elements of the tumour suppressor gene may be identified or located by DNAse hypersensitivity sites (detected on Southern blots) which indicate sites of regulatory protein binding. Alternatively, reporter constructs may be generated using the upstream genomic DNA (ie upstream of the 5 '-most exon) and, for example, β-galactosidase as a reporter enzyme. Serial deletions and footprinting techniques may also be used to identify the regulatory regions.

By "fragment" of a gene is included any portion of the gene of at least 15 nucleotides in length (whether single stranded or double stranded) but more preferably the fragment is at least 20 nucleotides in length, most preferably at least 50 nucleotides in length and may be at least 100 nucleotides in length or may be at least 500 nucleotides in length. Preferably the fragment is no more than 50 kb and, more preferably, no more than 100 kb.

By "variant" of a gene is included specifically a cDNA, whether partial or full length, or whether copied from any splice variants of mRNA. We also include specifically a nucleic acid wherein, compared to the natural gene, nucleotide substitutions (including inversions), insertions and deletions are present whether in the gene or a fragment thereof or in a cDNA. Both variants and fragments will be selected according to their intended purposes; for probing, amplifying or diagnostic purposes, shorter fragments but with a greater degree of sequence identity (eg at least 80%, 90% , 95% or 99%) will generally be required.

It is particularly preferred if the nucleic acid for use in the methods of the invention is an oligonucleotide primer which can be used to amplify a portion of the gene. The methods are suitable in respect of any cancer but it is preferred if the cancer is cancer of the ovary, breast, prostate, lung, stomach or colon. The methods are particularly suitable in respect of ovarian cancer. It will be appreciated that the methods of the invention include methods of prognosis and methods which aid diagnosis. It will also be appreciated that the methods of the invention are useful to the physician or surgeon in determining a course of management or treatment of the patient.

Although it is believed that any sample containing nucleic acid derived from the patient is useful in the methods of the invention, since mutations in the tumour suppressor gene may occur in familial cancers and not just sporadic cancers, it is, however, preferred if the nucleic acid is derived from a sample of the tissue in which cancer is suspected or in which cancer may be or has been found. For example, if the tissue in which cancer is suspected or in which cancer may be or has been found is ovary, it is preferred if the sample containing nucleic acid is derived from the ovary of the patient. Samples of ovary may be obtained by surgical excision, laproscopy and biopsy, endoscopy and biopsy, and image-guided biopsy. The image may be generated by ultrasound or technetium-99- labelled antibodies or antibody fragments which bind or locate selectively at the ovary. The well known monoclonal antibody HMFG1 is a suitable antibody for imaging ovarian cancer. Ascites/peritoneal cavity fluid, and peritoneal samples, may be obtained by surgery or laproscopy. It may also be possible to detect and isolate ovarian tumour cells in serum (Hibi et al (1998) Cancer Res. 58, 1405-1407). Similarly, if the tissue in which cancer is suspected or in which cancer may be or has been found is breast, it is preferred if the sample containing nucleic acid is derived from the breast of the patient; and so on. Breast samples may be obtained by excision, "true cut" biopsies, needle biopsy, nipple aspirate or image- guided biopsy.

In relation to prostate cancer, it is preferred that if blood, semen or urine is the source of the said sample containing nucleic acid derived from the patient that the sample is enriched for prostate-derived tissue or cells.

Enrichment for prostate cells may be achieved using, for example, cell sorting methods such as fluorescent activated cell sorting (FACS) using a prostate-selective antibody such as one directed to prostate-specific antigen (PSA). The source of the said sample also includes biopsy material and tumour samples, also including fixed paraffin mounted specimens as well as fresh or frozen tissue.

Other samples in which it may be beneficial to analyse the tumour suppressor gene or its product include lymph nodes, blood, serum and potential or actual sites of metastasis, for example bone.

The sample may be directly derived from the patient, for example, by biopsy of the tissue, or it may be derived from the patient from a site remote from the tissue, for example because cells from the tissue have migrated from the tissue to other parts of the body. Alternatively, the sample may be indirectly derived from the patient in the sense that, for example, the tissue or cells therefrom may be cultivated in vitro, or cultivated in a xenograft model; or the nucleic acid sample may be one which has been replicated (whether in vitro or in vivo) from nucleic acid from the original source from the patient. Thus, although the nucleic acid derived from the patient may have been physically within the patient, it may alternatively have been copied from nucleic acid which was physically within the patient. The tumour tissue may be taken from the primary tumour or from metastases.

It will be appreciated that a useful method of the invention includes the analysis of mutations in, or the detection of the presence or absence of, the tumour suppressor gene in any suitable sample. The sample may suitably be a freshly-obtained sample from the patient, or the sample may be an historic sample, for example a sample held in a library of samples.

Conveniently, the nucleic acid capable of selectively hybridising to the said human DNA and which is used in the methods of the invention further comprises a detectable label.

By "detectable label" is included any convenient radioactive label such as ³²P, ³³P or ³⁵S which can readily be incorporated into a nucleic acid molecule using well known methods; any convenient fluorescent or chemiluminescent label which can readily be incorporated into a nucleic acid is also included. In addition the term "detectable label" also includes a moiety which can be detected by virtue of binding to another moiety (such as biotin which can be detected by binding to streptavidin); and a moiety, such as an enzyme, which can be detected by virtue of its ability to convert a colourless compound into a coloured compound, or vice versa (for example, alkaline phosphatase can convert colourless o- nitrophenylphosphate into coloured ø-nitrophenol). Conveniently, the nucleic acid probe may occupy a certain position in a fixed assay and whether the nucleic acid hybridises to the said region of human DNA can be determined by reference to the position of hybridisation in the fixed assay. The detectable label may also be a fluorophore-quencher pair as described in Tyagi & Kramer (1996) Nature Biotechnology 14, 303-308. It will be appreciated that the aforementioned methods may be used for presymptomatic screening of a patient who is in a risk group for cancer. High risk patients for screening include patients over 50 years of age or patients who carry a gene resulting in increased susceptibility (eg predisposing versions of BRCAl, BRCA2 or p53); patients with a family history of breast/ovarian cancer; patients with affected siblings; nulliparous women; and women who have a long interval between menarche and menopause. For example, men older than about 60 years are at greater risk of prostate cancer than men below the age of 35. Similarly, the methods may be used for the pathological classification of tumours such as ovarian tumours. Similarly, the methods may be used for the pathological classification of tumours such as prostate tumours.

Conveniently, in the methods of the sixth, seventh and eighth aspects of the invention the nucleic acid which is capable of the said selective hybridisation (whether labelled with a detectable label or not) is contacted with a nucleic acid derived from the patient under hybridising conditions. Suitable hybridising conditions include those described above.

It is preferred that if the sample containing nucleic acid derived from the patient is not a substantially pure sample of the tissue or cell type in question that the sample is enriched for the said tissue or cells. For example, enrichment for ovarian cells in a sample such as a blood sample may be achieved using, for example, cell sorting methods such as fluorescent activated cell sorting (FACS) using an ovary cell-selective antibody, or at least an antibody which is selective for an epithelial cell. For example, Cam 5.2, anticytokeratin 7/8, from Becton Dickinson, 2350 Qume Drive, San Jose, California, USA, may be useful. The source of the said sample also includes biopsy material as discussed above and tumour samples, also including fixed paraffin mounted specimens as well as fresh or frozen tissue. The nucleic acid sample from the patient may be processed prior to contact with the nucleic acid which selectively hybridises to the tumour suppressor gene. For example, the nucleic acid sample from the patient may be treated by selective amplification, reverse transcription, immobilisation (such as sequence specific immobilisation), or incorporation of a detectable marker.

It is particularly preferred if the methods of the invention include the determination of mutations in, or the detection of the presence or absence of, the tumour suppressor gene.

The methods of the sixth, seventh and eighth aspects of the invention may involve sequencing of DNA at one or more of the relevant positions within the relevant region, including direct sequencing; direct sequencing of PCR-amplified exons; differential hybridisation of an oligonucleotide probe designed to hybridise at the relevant positions within the relevant region (conveniently this uses immobilised oligonucleotide probes in, so- called, "chip" systems which are well known in the art); denaturing gel electrophoresis following digestion with an appropriate restriction enzyme, preferably following amplification of the relevant DNA regions; SI nuclease sequence analysis; non-denaturing gel electrophoresis, preferably following amplification of the relevant DNA regions; conventional RFLP (restriction fragment length polymorphism) assays; heteroduplex analysis; selective DNA amplification using oligonucleotides; fluorescent in-situ hybridisation (FISH) of interphase chromosomes; ARMS-PCR (Amplification Refractory Mutation System-PCR) for specific mutations; cleavage at mismatch sites in hybridised nucleic acids (the cleavage being chemical or enzymic); SSCP single strand conformational polymorphism or DGGE (discontinuous or denaturing gradient gel electrophoresis); analysis to detect mismatch in annealed normal/mutant PCR-amplified DNA; and protein truncation assay (translation and transcription of exons - if a mutation introduces a stop codon a truncated protein product will result). Other methods may be employed such as detecting changes in the secondary structure of single-stranded DNA resulting from changes in the primary sequence, for example, using the cleavase I enzyme. This system is commercially available from GibcoBRL, Life Technologies, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, Scotland.

Another mutation detection method which may be used is Denaturing Gradient High Pressure Liquid Chromatography (DHPLC), also marketed under the name WAVE (Underhill et al (1997) Genome Res. 7, 996- 1005).

It will be appreciated that the methods of the invention may also be carried out on "DNA chips". Such "chips" are described in US 5,445,934 (Affymetrix; probe arrays), WO 96/31622 (Oxford; probe array plus ligase or polymerase extension), and WO 95/22058 (Affymax; fluorescently marked targets bind to oligomer substrate, and location in array detected); all of these are incorporated herein by reference.

Detailed methods of mutation detection are described in "Laboratory Protocols for Mutation Detection" 1996, ed. Landegren, Oxford University Press on behalf of HUGO (Human Genome Organisation). It is preferred if RFLP is used for the detection of fairly large (≥ 500bp) deletions or insertions. Southern blots may be used for this method of the invention.

PCR amplification of smaller regions (maximum 300bp) to detect small changes greater than 3-4 bp insertions or deletions may be preferred. Amplified sequence may be analysed on a sequencing gel, and small changes (minimum size 3-4 bp) can be visualised. Suitable primers are designed as herein described.

In addition, using either Southern blot analysis or PCR restriction enzyme variant sites may be detected. For example, for analysing variant sites in genomic DNA restriction enzyme digestion, gel electrophoresis, Southern blotting, and hybridisation specific probe (for example any suitable fragment derived from the tumour suppressor gene or its cDNA).

For example, for analysing variant sites using PCR DNA amplification, restriction enzyme digestion, gel detection by ethidium bromide, silver staining or incorporation of radionucleotide or fluorescent primer in the PCR.

Other suitable methods include the development of allele specific oligonucleotides (ASOs) for specific mutational events. Similar methods are used on RNA and cDNA for the suitable tissue, such as ovarian or breast or prostate tissue.

Whilst it is useful to detect mutations in any part of the tumour suppressor gene, it is preferred if the mutations are detected in the exons of the gene and it is further preferred if the mutations are ones which change the coding sense.

The methods of the invention also include checking for loss-of- heterozygosity (LOH; shows one copy lost). LOH may be a sufficient marker for diagnosis; looking for mutation/loss of the second allele may not be necessary. LOH of the gene may be detected using polymorphisms in the coding sequence, and introns, of the gene. LOH in a tumour cell, from whatever source, compared to blood is useful as a diagnostic tool, eg it may show that the tumour has progressed and requires more stringent treatment.

It is particularly preferred if LOH is assessed in relation to the portion of chromosome 16q which is bounded by the markers Alu 11 and 10T7 in Figure 2 since these markers define a region which we have found to be deleted in at least certain ovarian cancer cells. More preferably, LOH is assessed in relation to the portion of chromosome 16q which is bounded by the markers Alu 29 and 7T7; still more preferably LOH is assessed in relation to the portion of chromosome 16q which is bounded by the markers 17SP6 and RD53, or which is defined by reference to PAC clones 81N24 or 105K14 or YAC clone 801B6. A minimal region of deletion of around 250 kb is shown in Figure 2. It is particularly preferred if LOH is assessed in relation to the portion of chromosome 16q defined by this region or defined by the markers therein shown in Figure 2. Although none of the markers identified in the minimal region is itself polymorphic, primers which hybridise to these sequences may be used to detect polymorphic sequences. Particularly preferred nucleic acids for use in the aforementioned methods of the invention are those selected from the group consisting of primers suitable for amplifying nucleic acid.

Suitably, the primers are selected from the group consisting of primers which hybridise to the nucleotide sequences shown in any of the Figures which show portions of human DNA which are present in chromosome 16q (see, for example, Figures 4 to 31).

Primers which are suitable for use in a polymerase chain reaction (PCR; Saiki et al (1988) Science 239, 487-491) are preferred. Suitable PCR primers may have the following properties:

It is well known that the sequence at the 5' end of the oligonucleotide need not match the target sequence to be amplified.

It is usual that the PCR primers do not contain any complementary structures with each other longer than 2 bases, especially at their 3' ends, as this feature may promote the formation of an artifactual product called "primer dimer". When the 3' ends of the two primers hybridize, they form a "primed template" complex, and primer extension results in a short duplex product called "primer dimer".

Internal secondary structure should be avoided in primers. For symmetric PCR, a 40-60% G+C content is often recommended for both primers, with no long stretches of any one base. The classical melting temperature calculations used in conjunction with DNA probe hybridization studies often predict that a given primer should anneal at a specific temperature or that the 72 °C extension temperature will dissociate the primer/ template hybrid prematurely. In practice, the hybrids are more effective in the PCR process than generally predicted by simple T_m calculations.

Optimum annealing temperatures may be determined empirically and may be higher than predicted. Taq DNA polymerase does have activity in the 37-55 °C region, so primer extension will occur during the annealing step and the hybrid will be stabilized. The concentrations of the primers are equal in conventional (symmetric) PCR and, typically, within 0.1- to 1- μM range.

Any of the nucleic acid amplification protocols can be used in the method of the invention including the polymerase chain reaction, QB replicase and ligase chain reaction. Also, NASBA (nucleic acid sequence based amplification), also called 3SR, can be used as described in Compton (1991) Nature 350, 91-92 and AIDS (1993), Vol 7 (Suppl 2), S108 or SDA (strand displacement amplification) can be used as described in Walker et al (1992) Nucl. Acids Res. 20, 1691-1696. The polymerase chain reaction is particularly preferred because of its simplicity.

When a pair of suitable nucleic acids of the invention are used in a PCR it is convenient to detect the product by gel electrophoresis and ethidium bromide staining. As an alternative to detecting the product of DNA amplification using agarose gel electrophoresis and ethidium bromide staining of the DNA, it is convenient to use a labelled oligonucleotide capable of hybridising to the amplified DNA as a probe. When the amplification is by a PCR the oligonucleotide probe hybridises to the interprimer sequence as defined by the two primers. The oligonucleotide probe is preferably between 10 and 50 nucleotides long, more preferably between 15 and 30 nucleotides long. The probe may be labelled with a radionuclide such as ³²P, ³³P and ³⁵S using standard techniques, or may be labelled with a fluorescent dye. When the oligonucleotide probe is fluorescently labelled, the amplified DNA product may be detected in solution (see for example Balaguer et al (1991) "Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescence adsorbent" Anal. Biochem. 195, 105-110 and Dilesare et al (1993) "A high-sensitivity electrochemiluminescence-based detection system for automated PCR product quantitation" BioTechniques 15, 152-157.

PCR products can also be detected using a probe which may have a fluorophore-quencher pair or may be attached to a solid support or may have a biotin tag or they may be detected using a combination of a capture probe and a detector probe.

Fluorophore-quencher pairs are particularly suited to quantitative measurements of PCR reactions (eg RT-PCR). Fluorescence polarisation using a suitable probe may also be used to detect PCR products.

Oligonucleotide primers can be synthesised using methods well known in the art, for example using solid-phase phosphoramidite chemistry.

The present invention provides the use of a nucleic acid which selectively hybridises to the human chromosome 16q mmour suppressor gene, or a mutant allele thereof, or a nucleic acid which selectively hybridises to human chromosome 16q tumour suppressor gene cDNA or a mutant allele thereof, or their complement in a method of diagnosing cancer or prognosing cancer or determining susceptibility to cancer; or in the manufacture of a reagent for carrying out these methods. The nucleic acids which selectively hybridise as said are preferably the same nucleic acids which selectively hybridise as said as described by reference to the first, second and third aspects of the invention.

Also, the present invention provides a method of determining the presence or absence, or mutation in, the said tumour suppressor gene. Preferably, the method uses a suitable sample from a patient.

The methods of the invention include the detection of mutations in the human chromosome 16q tumour suppressor gene.

The methods of the invention may make use of a difference in restriction enzyme cleavage sites caused by mutation. A non-denaturing gel may be used to detect differing lengths of fragments resulting from digestion with an appropriate restriction enzyme.

An "appropriate restriction enzyme" is one which will recognise and cut the wild-type sequence and not the mutated sequence or vice versa. The sequence which is recognised and cut by the restriction enzyme (or not, as the case may be) can be present as a consequence of the mutation or it can be introduced into the normal or mutant allele using mismatched oligonucleotides in the PCR reaction. It is convenient if the enzyme cuts DNA only infrequently, in other words if it recognises a sequence which occurs only rarely.

In another method, a pair of PCR primers are used which match (ie hybridise to) either the wild-type genotype or the mutant genotype but not both. Whether amplified DNA is produced will then indicate the wild- type or mutant genotype (and hence phenotype). However, this method relies partly on a negative result (ie the absence of amplified DNA) which could be due to a technical failure. It therefore may be less reliable and/or requires additional control experiments.

A preferable method employs similar PCR primers but, as well as hybridising to only one of the wild-type or mutant sequences, they introduce a restriction site which is not otherwise there in either the wild- type or mutant sequences.

The nucleic acids which selectively hybridise to the mmour suppressor gene or mmour suppressor gene cDNA, are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the tumour suppressor gene or mmour suppressor gene mRNA in a sample using other techniques. Mismatches can be detected using either enzymes (eg SI nuclease or resolvase), chemicals (eg hydroxylamine or osmium tetroxide and piperidine), or changes in electrophoretic mobility of mismatched hybrids as compared to totally matched hybrids. These techniques are known in the art. Generally, the probes are complementary to the mmour suppressor gene coding sequences, although probes to certain introns are also contemplated. A battery of nucleic acid probes may be used to compose a kit for detecting loss of or mutation in the wild- type tumour suppressor gene. The kit allows for hybridization to the entire tumour suppressor gene. The probes may overlap with each other or be contiguous. If a riboprobe is used to detect mismatches with mRNA, it is complementary to the mRNA of the human chromosome 16q mmour suppressor gene. The riboprobe thus is an anti-sense probe in that it does not code for the protein encoded by the tumour suppressor gene because it is of the opposite polarity to the sense strand. The riboprobe generally will be labelled, for example, radioactively labelled which can be accomplished by any means known in the art. If the riboprobe is used to detect mismatches with DNA it can be of either polarity, sense or anti- sense. Similarly, DNA probes also may be used to detect mismatches.

Nucleic acid probes may also be complementary to mutant alleles of the mmour suppressor gene. These are useful to detect similar mutations in other patients on the basis of hybridization rather than mismatches. As mentioned above, the tumour suppressor gene probes can also be used in Southern hybridizations to genomic DNA to detect gross chromosomal changes such as deletions and insertions.

According to the diagnostic and prognostic method of the present invention, loss of, or modification of, the wild-type gene function may be detected. The loss may be due to either insertional, deletional or point mutational events. If only a single allele is mutated, an early neoplastic state may be indicated. However, if both alleles are mutated then a malignant state is indicated or an increased probability of malignancy is indicated. The finding of such mutations thus provides both diagnostic and prognostic information. A mmour suppressor gene allele which is not deleted (eg that on the sister chromosome to a chromosome carrying a gene deletion) can be screened for other mutations, such as insertions, small deletions, and point mutations. We believe that detecting a mutation in a single copy (allele) of the gene is useful. Loss of the second allele may be necessary for carcinogenesis. If the second copy was lost routinely by a gross mechanism, this could be a useful event to detect. Some mutations of the gene may have a dominant negative effect on the remaining allele. Mutations leading to non-functional gene products may also lead to a malignant state or an increased probability of malignancy. Mutational events (such as point mutations, deletions, insertions and the like) may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of or alteration in the expression of the mmour suppressor gene product or to the mmour suppressor polypeptide being non-functional or having an altered expression. It is preferred if the amount of mmour suppressor gene mRNA in a test sample is quantified and compared to that present in a control sample. It is also preferred if the splicing patterns of mmour suppressor gene mRNA in a test sample is determined and compared to that present in a control sample.

The gene has two alleles, and it will be appreciated that alterations to both alleles may have a greater effect on cell behaviour than alteration to one. It is expected that at least one mutant allele has mutations which result in an altered coding sequence. Modifications to the second allele, other than to the coding sequence, may include total or partial gene deletion, and loss, mutation or modification of regulatory regions. Modification of regulatory regions may include changes in methylation status that can be assessed using methods well known in the art.

The amount of tumour suppressor gene mRNA is suitably determined per unit mass of sample tissue or per unit number of sample cells and compared this to the unit mass of known normal tissue or per unit number of normal cells. RNA may be quantitated using, for example, northern blotting or quantitative RT-PCR.

The invention also includes the following methods: in vitro transcription and translation of the mmour suppressor gene to identify truncated gene products, or altered properties such as substrate binding; immunohistochemistry of tissue sections to identify cells in which expression of the protein is reduced/lost, or its distribution is altered within cells or on their surface; and the use of RT-PCR using random primers, prior to detection of mutations in the region as described above.

A further aspect of the invention provides a system (or it could also be termed a kit of parts) for detecting the presence or absence of, or mutation in, the relevant region of human DNA, the system comprising a nucleic acid capable of selectively hybridising to the relevant region of human DNA and a nucleoside triphosphate or deoxynucleoside triphosphate or derivative thereof. Preferred nucleic acids capable of selectively hybridising to the relevant region of human DNA are the same as those preferred above.

The "relevant region of human DNA" includes the tumour suppressor gene, the mmour suppressor gene cDNA and the human-derived DNA present in YAC clone 801B6 or PAC clones 81N24 or 105K14.

By "mutation" is included insertions, substitutions and deletions.

By "nucleoside triphosphate or deoxynucleoside triphosphate or derivative thereof is included any naturally occurring nucleoside triphosphate or deoxynucleoside triophosphate such as ATP, GTP, CTP, and UTP, dATP dGTP, dCTP, TTP as well as non-naturally derivatives such as those that include a phosphorothioate linkage (for example αS derivatives).

Conveniently the nucleoside triphosphate or deoxynucleoside triphosphosphate is radioactively labelled or derivative thereof, for example with ³²P, ³³P or ³⁵S, or is fluorescently labelled or labelled with a chemiluminescence compound or with digoxygenin.

Conveniently deoxynucleotides are at a concentration suitable for dilution to use in a PCR.

Thus, the invention includes a kit of parts which includes a nucleic acid capable of selectively hybridising to the said relevant region of human DNA and means for detecting the presence or absence of, or a mutation in, the said region. Means for detecting the presence or absence of, or a mutation in, the said region include, for example, a diagnostic restriction enzyme or a mutant-specific nucleic acid probe or the like.

A further aspect of the invention provides a system for detecting the presence or absence of, or mutation in, the relevant region of DNA, the system comprising a nucleic acid which selectively hybridises to the relevant region of human DNA and a nucleic acid modifying enzyme. Preferred nucleic acids capable of selectively hybridising to the relevant region of human DNA are the same as those preferred above.

By "mutation" is included insertions, substitutions (including transversions) and deletions. By "nucleic acid modifying enzyme" is included any enzyme capable of modifying an RNA or DNA molecule.

Preferred enzymes are selected from the group consisting of DNA polymerases, DNA ligases, polynucleotide kinases or restriction endonucleases. A particularly preferred enzyme is a thermostable DNA polymerase such as Taq DNA polymerase. Nucleases such as Cleavase I which recognise secondary structure, for example mismatches, may also be useful.

Detecting mutations in the gene will be useful for determining the appropriate treatment for a patient, eg gene therapy using the mmour suppressor (see below). Detecting mutations in the gene may be useful to identify a subset of patients whose tumours have this shared characteristic, and can be analysed as a group for prognosis or response to various therapies.

Mutations in the gene may be related to response or resistance to certain treatments, this may be investigated using cell lines with known sensitivity to various therapies, or by clinical correlation studies.

It is possible that the tumour suppressor gene would be used as part of a panel of markers and tests, which the combined results of would direct therapy. Detecting mutations in the gene may be useful for monitoring disease spread and load.

Analysis of the mmour suppressor gene may be useful for differential diagnosis in the case where mutations in the gene are common in one mmour, but not another. For example, secondary tumours of gastrointestinal origin are frequently found in the ovaries and are difficult to distinguish from tumours of true ovarian origin.

A nirther aspect of the invention provides a method for determining the susceptibility of a patient to cancer comprising the steps of (i) obtaining a sample containing protein derived from the patient; and (ii) determining the relative amount, or the intracellular location, or physical form, of the human chromosome 16q mmour suppressor polypeptide, or the relative activity of, or change in activity of, or altered activity of, the human chromosome 16q tumour suppressor polypeptide.

A still further aspect of the invention provides a method of diagnosing cancer in a patient comprising the steps of (i) obtaining a sample containing protein derived from the patient; and (ii) determining the relative amount, or the intracellular location, or physical form, of the human chromosome 16q mmour suppressor polypeptide, or the relative activity of, or change in activity of, or altered activity of, the human chromosome 16q tumour suppressor polypeptide.

A yet still further aspect of the invention provides a method of predicting the relative prospects of a particular outcome of a cancer in a patient comprising the steps of (i) obtaining a sample containing protein derived from the patient; and (ii) determining the relative amount, or the intracellular location, or physical form of the human chromosome 16q mmour suppressor polypeptide, or the relative activity of, or change in activity of, or altered activity of, the human chromosome 16q mmour suppressor polypeptide. The methods of the invention also include the measurement and detection of the tumour suppressor polypeptide or mutants thereof in test samples and their comparison in a control sample. It may also be useful to detect altered activity of the polypeptide.

The sample containing protein derived from the patient is conveniently a sample of the tissue in which cancer is suspected or in which cancer may be or has been found. These methods may be used for any cancer, but they are particularly suitable in respect of cancer of the ovary, breast, or prostate. Methods of obtaining suitable samples are described in relation to earlier methods.

The methods of the invention involving detection of the tumour suppressor polypeptide are particularly useful in relation to historical samples such as those containing paraffin-embedded sections of mmour samples.

The relative amount of, or the intracellular location of, or the physical form of, the tumour suppressor polypeptide may be determined in any suitable way.

It is preferred if the relative amount of, or intracellular location of, or physical form of the tumour suppressor polypeptide is determined using a molecule which selectively binds to tumour suppressor polypeptide or which selectively binds to a mutant form of tumour suppressor polypeptide. Suitably, the molecule which selectively binds to mmour suppressor polypeptide or which selectively binds to a mutant of the tumour suppressor polypeptide is an antibody. The antibody may also bind to a namral variant or fragment of the tumour suppressor polypeptide. The antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press, 1982), both of which are incorporated herein by reference.

By "the relative amount of mmour suppressor polypeptide" is meant the amount of tumour suppressor polypeptide per unit mass of sample tissue or per unit number of sample cells compared to the amount of mmour suppressor polypeptide per unit mass of known normal tissue or per unit number of normal cells. The relative amount may be determined using any suitable protein quantitation method. In particular, it is preferred if antibodies are used and that the amount of tumour suppressor polypep tides is determined using methods which include quantitative western blotting, enzyme-linked immunosorbent assays (ELISA) or quantitative immunohistochemistry .

Other techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparations claimed in this invention. In a preferred embodiment of the invention, antibodies will immunoprecipitate the tumour suppressor polypeptide from solution as well as react with mmour suppressor polypeptide on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, antibodies will detect mmour suppressor polypeptides in paraffin or frozen tissue sections, using immunocytochemical techniques. Preferred embodiments relating to methods for detecting tumour suppressor polypeptide or its mutations include enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al in US Patent Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.

A further aspect of the invention provides a method of treating a proliferative disease comprising the step of administering to the patient a nucleic acid which selectively hybridises to the human chromosome 16q mmour suppressor gene, or a nucleic acid which selectively hybridises to human chromosome 16q mmour suppressor gene cDNA.

A further aspect of the invention provides a method of treating a proliferative disease comprising the step of administering to the patient a nucleic acid which encodes the human chromosome 16q mmour suppressor polypeptide or a functional variant or portion or fusion thereof.

Conveniently, the nucleic acid which encodes the human chromosome 16q mmour suppressor polypeptide or a functional variant or portion or fusion thereof includes at least 20 nucleotides, preferably at least 30 nucleotides, more preferably at least 40 nucleotides and still more preferably at least 50 nucleotides which are contiguous nucleotides from any of Figures 4, 5, 6, 7, 8, 9, 15, 21 , 23 or 31 or a complement thereof.

A still further aspect of the invention provides a method of treating a proliferative diseases comprising the step of administering to the patient a nucleic acid which contains at least 20 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31. Preferably the nucleic acid contains at least 30, or 40, or 50 of such contiguous nucleotides.

It is particularly preferred if the methods of the invention are used to treat cancer although, as discussed in more detail below, they may be used to treat other proliferative diseases. The methods may be used to treat cancer of the ovary, breast, prostate, lung, stomach, and colon.

The invention also includes the administration of all or part of the tumour suppressor gene or mmour suppressor gene cDNA to a patient with a cancer.

Suitably, the nucleic acid which is administered to the patient is a nucleic acid which encodes the tumour suppressor polypeptide or a functional variant or portion thereof. Preferably, the tumour suppressor polypeptide is a wild-type polypeptide or a variant polypeptide which has substantially wild-type activities. It is less preferred if the mmour suppressor polypeptide is a polypeptide with mutations which are found in cancer cells such as ovarian cancer cells; however, as discussed below, such polypeptides may be useful in provoking an anti-cancer cell immune response. Thus, according to the present invention, a method is also provided of supplying wild-type mmour suppressor gene function to a cell which carries mutant tumour suppressor gene alleles. Supplying such a function should suppress neoplastic growth of the recipient cells. The wild-type mmour suppressor gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene fragment is introduced and expressed in a cell carrying a mutant mmour suppressor allele, the gene fragment should encode a part of the mmour suppressor polypeptide which is required for non-neoplastic growth of the cell. The wild-type mmour suppressor gene or a part thereof may be introduced into the mutant cell in such a way that it recombines with the endogenous mutant tumour suppressor gene present in the cell. Such recombination requires a double recombination event which results in the correction of the mmour suppressor gene mutation. Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used. Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art, and the choice of method is within the competence of the routineer. Cells transformed with the wild-type mmour suppressor gene can be used as model systems to study cancer remission and drug treatments which promote such remission.

As generally discussed above, the mmour suppressor gene or fragment, where applicable, may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cancer cells. Such gene therapy is particularly appropriate for use in both cancerous and pre-cancerous cells, in which the level of mmour suppressor polypeptide is absent or diminished or otherwise changed compared to normal cells. It may also be useful to increase the level of expression of a mmour suppressor gene even in those mmour cells in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional or has an altered function. It may also be useful in the treatment of tumours with wild type mmour suppressor function. In this case it may also be used to treat proliferative diseases other than cancer. These include endometriosis, vascular proliferative disorders such as restenosis, kidney disorders such as glomerulosclerosis and interstitial fibrosis.

Gene therapy may be carried out according to generally accepted methods, for example, as described by Friedman, 1991. Cells from a patient's mmour would be first analyzed by the diagnostic methods described herein, to ascertain the production of tumour suppressor polypeptide and its physical form (ie what mutations it contains) in the mmour cells. A virus or plasmid vector (see further details below), containing a copy of the mmour suppressor gene linked to expression control elements and capable of replicating inside the tumour cells, is prepared. Suitable vectors are known, such as disclosed in US Patent 5,252,479 and PCT published application WO 93/07282. The vector is then injected into the patient, either locally at the site of the tumour or systemically (in order to reach any mmour cells that may have metastasized to other sites). If the transfected gene is not permanently incorporated into the genome of each of the targeted mmour cells, the treatment may have to be repeated periodically.

Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods. A number of viruses have been used as gene transfer vectors, including papovaviruses, eg SV40 (Madzak et al, 1992), adenovirus (Berkner, 1992; Berkner et al, 1988; Gorziglia and Kapikian, 1992; Quantin et al, 1992; Rosenfeld et al, 1992; Wilkinson et al, 1992; Stratford-Perricaudet et al, 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al, 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1987; Freese et al, 1990), and retroviruses of avian (Brandyopadhyay and Temin, 1984; Petropoulos et al, 1992), murine (Miller, 1992; Miller et al, 1985; Sorge et al, 1984; Mann and Baltimore, 1985; Miller et al, 1988), and human origin (Shimada et al, 1991; Helseth et al, 1990; Page et al, 1990; Buchschacher and Panganiban, 1992). To date most human gene therapy protocols have been based on disabled murine retroviruses.

Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al, 1980); mechamcal techniques, for example microinjection (Anderson et al, 1980; Gordon et al, 1980; Brinster et al, 1981; Constantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Feigner et al, 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et al, 1992; Nabel et al, 1990; Lim et al, 1992); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al, 1990; Wu et al, 1991; Zenke et al, 1990; Wu et al, 1989b; Wolff et al, 1991; Wagner et al, 1990; Wagner et al, 1991; Cotten et al, 1990; Curiel et al, 1991a; Curiel et al, 1991b). Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumour cells and not into the surrounding nondividing cells. Alternatively, the retroviral vector producer cell line can be injected into tumours (Culver et al, 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumours.

Other suitable systems include the retroviral-adenoviral hybrid system described by Feng et al (1997) Nature Biotechnology 15, 866-870, or viral systems with targeting ligands such as suitable single chain Fv fragments. In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine- conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.

Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in mmour deposits, for example, following direct in situ administration (Nabel, 1992).

Gene transfer techniques which target DNA directly to ovarian, breast, prostate, lung, colon or stomach tissues, eg epithelial cells of the breast or ovaries, is preferred. Receptor-mediated gene transfer, for example, is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/ tissue type. One appropriate receptor/ligand pair for introduction of the therapeutic gene into breast mmour cells may include the estrogen receptor and its ligand, estrogen (and estrogen analogues). These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function.

In the case where replacement gene therapy using a functionally wild-type tumour suppressor gene is used, it may be useful to monitor the treatment by detecting the presence of tumour suppressor gene mRNA or polypeptide, or functional tumour suppressor gene product, at various sites in the body, including the targeted tumour, sites of metastasis, blood serum, and bodily secretions/excretions, for example urine.

A further aspect of the invention provides a method of treating a proliferative disease, such as cancer, comprising the step of administering to the patient an effective amount of mmour suppressor polypeptide or a fragment or variant or fusion thereof to ameliorate the proliferative disease.

It will be appreciated that amelioration of the disease is included in the term treatment.

Peptides which have tumour suppressor activity can be supplied to cells which carry mutant or missing mmour suppressor gene alleles.

By "peptide" we include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237, incorporated herein by reference. This approach involves making pseudopep tides containing changes involving the backbone, and not the orientation of side chains. Meziere et al (1997) show that these pseudopeptides may be useful in some circumstances. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.

Similarly, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the Cα atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity of a peptide bond.

It will be appreciated that the peptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.

Thus, the term "peptide" includes peptidomimetics.

The mmour suppressor gene or mmour suppressor gene cDNA can be expressed by any suitable method. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co- transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus) , plant cells, animal cells and insect cells.

The vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p7rc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary mmour virus long terminal repeat to drive expression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps)

A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

A further aspect of the invention provides a method of treating cancer, the method comprising administering to the patient an effective amount of a mutant mmour suppressor polypeptide or fragment thereof, or an effective amount of a nucleic acid encoding a mutant tumour suppressor polypeptide or fragment thereof, wherein the said mutant tumour suppressor polypeptide is a mutant found in a cancer cell and the amount of said mutant polypeptide or amount of said nucleic acid is effective to provoke an anti-cancer cell immune response in said patient.

The mutant peptide or peptide-encoding nucleic acid constitutes a mmour or cancer vaccine. It may be administered directly into the patient, into the affected organ or systemically, or applied ex vivo to cells derived from the patient or a human cell line which are subsequently administered to the patient, or used in vitro to select a subpopulation from immune cells derived from the patient, which are then re-administered to the patient. If the nucleic acid is administered to cells in vitro, it may be useful for the cells to be transfected so as to co-express immune-stimulating cytokines, such as interleukin-2. The mutant mmour suppressor polypeptide or peptide fragment therefore comprising the mutation may be substantially pure, or combined with an immune-stimulating adjuvant, or used in combination with immune-stimulatory cytokines, or be administered with a suitable delivery system, for example liposomes. The peptide may also be tagged, or be a fusion protein. The nucleic acid may be substantially pure, or contained in a suitable vector or delivery system. The peptide or peptide encoded by the nucleic acid may be a fusion protein, for example with β2-microglobulin.

It is particularly useful if the cancer vaccine is administered in a manner which produces a cellular immune response, resulting in cy toxic mmour cell killing by NK cells or cytotoxic T cells (CTLs). Strategies of administration which activate T helper cells are particularly useful. It may also be useful to stimulate a humoral response. It may be useful to co- adminster certain cytokines to promote such a response, for example interleukin-2, interleukin-12, interleukin-6, or interleukin-10. It may also be useful to target the vaccine to specific cell populations, for example antigen presenting cells, either by the site of injection, use of targeting vectors and delivery systems, or selective purification of such a cell population from the patient and ex vivo administration of the peptide or nucleic acid (for example dendritic cells may be sorted as described in Zhou et al (1995) Blood 86, 3295-3301; Roth et al (1996) Scand. J. Immunology 43 , 646-651 ) . Patients to whom the therapy is to be given, may have their tumours typed for mutation so that the appropriate mutant peptide or nucleic acid can be used in the method or vaccine of the invention. Treatment may be monitored by determining the amount of mmour suppressor mRNA or polypeptide in the mmour pre- and post-treatment.

A further aspect of the invention therefore provides a vaccine effective against cancer or cancer or tumour cells comprising an effective amount of a mutant tumour suppressor polypeptide or mutant-containing fragment thereof, or comprising a nucleic acid encoding such a polypeptide or mutant-containing fragment thereof.

A still further aspect of the invention provides a method of treating a proliferative disease, such as cancer, comprising the step of administering to the patient an effective amount of a compound which modulates mmour suppressor polypeptide function, or the function of a mutant mmour suppressor polypeptide found in a diseased cell, such as a cancer cell.

The compound which modulates mmour suppressor polypeptide function may suitably inhibit function, or it may suitably enhance function, of the tumour suppressor polypeptide.

Suitable compounds for use in this method of the invention include antibodies or fragments or variants thereof which modulate mmour suppressor polypeptide activity, or antisense molecules which modulate the expression of the mmour suppressor gene. It is also preferred if the modulators are ones which are selective for a mutant form of mmour suppressor polypeptide present in a cancer cell. Alternatively, suitable compounds may be obtained by screening.

Screening compounds by using the mmour suppressor polypeptide or binding fragment thereof in any of a variety of drug screening techniques may be used.

The mmour suppressor polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, for the formation of complexes between a tumour suppressor polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a mmour suppressor polypeptide, or fragment and a known ligand is interfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugs comprising contacting such an agent with a mmour suppressor polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the mmour suppressor polypeptide or fragment, or (ii) for the presence of a complex between the mmour suppressor polypeptide or fragment and a ligand, by methods well known in the art. In such competitive binding assays the mmour suppressor polypeptide or fragment is typically labeled. Free tumour suppressor polypeptide or fragment is separated from that present in a protein: protein complex and the amount of free (ie uncomplexed) label is a measure of the binding of the agent being tested to mmour suppressor polypeptide or its interference with mmour suppressor polypeptide: ligand binding, respectively.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the mmour suppressor polypeptide and is described in detail in Geysen, PCT published application WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the tumour suppressor polypeptide and washed. Bound mmour suppressor polypeptide is then detected by methods well known in the art.

Purified mmour suppressor polypeptide can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non- neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the tumour suppressor polypeptide on the solid phase.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the tumour suppressor polypeptide compete with a test compound for binding to the mmour suppressor polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the mmour suppressor polypeptide.

A further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a mutant mmour suppressor gene. These host cell lines or cells are defective at the mmour suppressor polypeptide level. The host cell lines or cells are grown in the presence of drug compound. The rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of mmour suppressor defective cells.

Additionally or alternatively, rational drug design may be used. The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (eg agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, eg enhance or interfere with the function of a polypeptide in vivo. See, eg Hodgson, 1991. In one approach, one first determines the three- dimensional structure of a protein of interest (eg tumour suppressor polypeptide) by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al, 1990). In addition, peptides (eg tumour suppressor polypeptide) are analyzed by an alanine scan (Wells, 1991). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide' s activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass 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 analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, for example, improved mmour suppressor polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc of mmour suppressor polypeptide activity.

Cells and animals which carry a mutant mmour suppressor allele can be used as model systems to study and test for substances which have potential as therapeutic agents. The cells are typically cultured epithelial cells. These may be isolated from individuals with tumour suppressor mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the mmour suppressor allele, using methods well known in the art. After a test substance is applied to the cells, the neoplastically transformed phenotype of the cell is determined. Any trait of neoplastically transformed cells can be assessed, including anchorage-independent growth, tumourigenicity in nude mice, invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in the art.

Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant tumour suppressor gene alleles, usually from a second animal species, as well as insertion of disrupted homologous genes. After test substances have been administered to the animals, the growth of tumours must be assessed. If the test substance prevents or suppresses the growth of tumours, then the test substance is a candidate therapeutic agent for the treatment of the cancers identified herein. These animal models provide an extremely important testing vehicle for potential therapeutic products.

Active mmour suppressor polypeptide molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Extracellular application of the mmour suppressor gene product may be sufficient to affect mmour growth. Supply of molecules with mmour suppressor polypeptide activity should lead to partial reversal of the neoplastic state. Other molecules with tumour suppressor polypeptide activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantially similar function are also used for peptide therapy.

Further aspects of the invention provide a pharmaceutical composition comprising a gene therapy vector including a nucleic acid which encodes the mmour suppressor polypeptide or a functional variant or portion or fusion thereof and pharmaceutically acceptable carrier; a pharmaceutical composition comprising a gene therapy vector including a nucleic acid which selectively hybridises to the mmour suppressor gene, or a mutant allele thereof, or a mmour suppressor gene cDNA, or a mutant allele thereof, and a pharmaceutically acceptable carrier; a pharmaceutical composition comprising mmour suppressor polypeptide or a fragment or variant or fusion thereof, and a pharmaceutically acceptable carrier.

Suitable gene therapy vectors are described above. Suitable mmour suppressor polypeptides are described above. By "pharmaceutically acceptable" is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy.

The invention will now be described in more detail.

Figure 1 is a map showing the relative positions of markers on human chromosome 16q around the region of interest. The homozygous deletion lies between D16S518 and D16S3029. The data are taken from part of the Whitehead Institute/MIT STS map which may be accessed at the following URL address http : //www . mpimg-berlin-dahlem. mpg . de/ ^"andy /GN/mithumanrh/ .

Figure 2 shows a map of the minimal PAC and YAC contig at chromosome 16q23 showing markers described in the application plus the closest flanking polymorphic markers D16S518, D16S3029 and D16S516.

Figure 3 is a detailed map of the PAC contig showing all the PAC clones and all the markers mapped on to them. Below are shown the regions of homozygous deletion found in the DNA from the three mmour types. The map is not to scale.

Figures 4 to 20 give the nucleotide sequence of part of human chromosome 16q at positions (as indicated in Figures 2 or 3 or 29 or 30): 4T7 (Figure 4); IM97 (Figure 5); 435E (Figure 6); ETAl (Figure 7); 5.1 A6 (Figure 8); 10102 (Figure 9); Alu 11 (Figure 10); Alu 29 (Figure 11); Alu 20 (Figure 12); 17SP6 (Figure 13); RD69 (Figure 14); RD30 (Figure 15); 5T7 (Figure 16); RD53 (Figure 17); 8T7 (Figure 18); 10T7 (Figure 19); 10SP6 (Figure 20). The sequences shown in Figures 5(b) and 15(b) are extended versions of the sequences shown in Figures 5(a) and 15(a), respectively.

The "mRNA" feature given on Figures 5 to 20 identifies the portion of the nucleotide sequence which is already in the database. For convenience, the following Table shows the relationship between the newly derived sequences in Figures 5 to 20 and the published sequences shown in the database.

Figures 21 to 28 and 31 give the nucleotide sequence of part of human chromosome 16q at positions (as indicated in Figures 29 or 30): IM29 (Figure 21); IM39 (Figure 22); IM27 (Figure 23); IM23 (Figure 24); IM25 (Figure 25); IM41 (Figure 26); IM43 (Figure 27); IM38 (Figure 28); and IM28 (Figure 31).

Figure 29 shows a more detailed map of the markers at the region of deletion on chromosome 16q. (a) The relative position of EST and STS markers are shown, including EST markers lying within the minimally deleted region, (b) PAC contig encompassing ~700kb. PAC clones were obtained from the RPCI-1 library or, where indicated, the RPCI-6 library. A scale bar with distances shown in kilobase pairs is given and the positions of Eagl, Bss HII and Sail sites are indicated as E, B and S respectively. A BssUll site was identified in PAC 253H19, shown as (B), but was not found to be present in any of the overlapping PAC clones, and was presumed to be created due to a polymorphism in that clone. The PAC clones around this site were aligned with respect to Eagl and Sail sites, and their STS content, (c) YAC contig encompassing ~3Mb. (Contig length based on the insert sizes provided by the Whitehead Institute for Biomedical Research). YAC clones were obtained from the CEPH Mega YAC library (Genethon). YACs are not shown to scale but are positioned with respect to the STS/EST map shown in (a), (d) Depiction of the 16q23 chromosome region from a small cell lung carcinoma (WC330), an ovarian adenocarcinoma ascitic specimen (PE04), a colonic adenocarcinoma (HCT116) and a gastric adenocarcinoma (AGS). The extent of the homozygous deletions in the three mmour s is shown by the unfilled, dashed bars, whilst the filled bars represent DNA which has been maintained.

Figure 30 shows the extent of the sequence analysis from various marker positions, and the order of the marker positions.

Some of the sequence figures contain the letters K, V or M. These are standard nucleotide ambiguity codes where K = G or T residue, V = A, C or G residue, and M = A or C residue.

TABLE

Where portions have been published, the database accession numbers are given. 00/37677

74

The STSs are derived from genomic DNA, whereas the ESTs are derived from cDNA.

In all instances in the specification where it is indicated that at least 20 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31 or a complement thereof, it is preferred if the at least 20 contiguous nucleotides are derived from the region of the sequence indicated under "Coordinates of transcribed sequence" referred to in the above Table. It is also preferred that the at least 20 nucleotides are homologous to or form part of the sequences given in the GenBank entries under the given Accession Nos. The above is particularly preferred in relation to the expression and gene therapy vectors of the invention.

Details of markers and clones shown on Figures 2 and 3

Markers Summary

00/37677

75

00/37677

76

00/37677

77

Yeast Artificial Chromosome (YAC) Clones

0/37677

78

For the avoidance of doubt the invention does not include ESTs or the clones from which they are derived which are in the public domain. The above table lists the clones and ESTs that we are aware of which are publicly known. 0/37677

79

REFERENCES

Anderson et al (1980) Proc. Natl. Acad. Sci. USA 77, 5399-5403.

Berkner (1992) Curr. Top. Microbiol. Immunol. 158, 39-61. Berkner et al (1988) BioTechniques 6, 616-629.

Brandyopadhyay and Temin (1984) Mol. Cell. Biol. 4, 749-754.

Breakfield and Geller (1987) Mol. Neurobiol. 1, 337-371.

Brinster et al (1981) Cell 27, 223-231.

Buchschacher and Panganiban (1992) J. Virol. 66, 2731-2739. Capecchi, M.R. (1989) Science 244, 1288.

Constantini and Lacy (1981) Nature 294, 92-9 .

Cotten et al (1990) Proc. Natl. Acad. Sci. USA 87, 4033-4037.

Culver et al (1992) Science 256, 1550-1552.

Curiel et al (1991a) Proc. Natl. Acad. Sci. USA 88, 8850-8854. Curiel et al (1991b) Hum. Gene Ther. 3, 147-154.

Donehower, L.A. et al (1992) Nature 356, 215.

Erickson, J. et al (1990) Science 249, 527-533.

Feigner et al (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417.

Fink et al (1992) Hum. Gene Ther. 3, 11-19. Freese et al (1990) Biochem. Pharmacol. 40, 2189-2199.

Friedman, T. (1991) In Therapy for Genetic Diseases, T.

Friedman, ed. , Oxford University Press, pp 105-121.

Gordon et al (1980) Proc. Natl. Acad. Sci. USA 11, 7380-7384.

Gorziglia and Kapikian (1992) J. Virol. 66, 4407-4412. Graham and van der Eb (1973) Virology 52, 456-467.

Hasty, P.K. et al (1991) Nature 350, 243.

Helseth et al (1990) J. Virol. 64, 2416-2420.

Hodgson, J. (1991) Bio/Technology 9, 19-21.

Johnson et al (1992) J. Virol. 66, 2952-2965. Kaneda et al (1989) J. Biol. Chem. 264, 12126-12129.

Lim et al (1992) Circulation 83, 2007-2011.

Madzak et al (1992) 7. Gen. Virol. 73, 1533-1536.

Mann and Baltimore (1985) J. Virol. 54, 401-407. Margolskee (1992) Curr. Top. Microbiol. Immunol. 158, 67-90.

Miller (1992) Curr. Top. Microbiol. Immunol. 158, 1-24.

Miller et al (1985) Mol. Cell. Biol. 5, 431-437.

Miller et al (1988) J. Virol. 62, 4337-4345.

Mombaerts, P. et al (1992) Cell 68, 869. Moss (1992) Curr. Top. Microbiol. Immunol. 158, 25-38.

Muzyczka (1992) Curr. Top. Microbiol. Immunol. 158, 97-123.

Nabel (1992) Hum. Gene Ther. 3, 399-410.

Ohi et al (1990) Gene 89, 279-282.

Page et al (1990) J. Virol. 64, 5370-5276. Pellicer et al (1980) Science 209, 1414-1422.

Petropoulos et al (1992) . Virol. 66, 3391-3397.

Philpott, K.L. et al (1992) Science 256, 1448.

Quantin et al (1992) Proc. Natl. Acad. Sci. USA 89, 2581-2584.

Rosenfeld et al (1992) Cell 68, 143-155. Shimada et al (1991) J. Clin. Invest. 88, 1043-1047.

Shinkai, Y. et al (1992) Cell 68, 855.

Snouwaert, J.N. et al (1992) Science 251, 1083.

Sorge et al (1984) Mol. Cell. Biol. 4, 1730-1737.

Stewart et al (1992) Hum. Gene Ther. 3, 267-275. Stratford-Perricaudet et al (1990) Hwm. Gene Ther. 1, 241-256.

Valancius, V. & Smithies, O. (1991) Mol. Cell Biol. 11, 1402.

Wagner et al (1990) Proc. Nat/. Acad. Sci. USA 87, 3410-3414.

Wagner et al (1991) Proc. Natl. Acad. Sci. USA 88, 4255-4259.

Wang and Huang (1989) Biochemistry 28, 9508-9514. Wells, J.A. (1991) Methods in Enzymol. 202, 390-411. Wilkinson et al (1992) Nucleic Acids Res. 20, 2233-2239. Wolff et al (1990) Science 247, 1465-1468. Wolff et al (1991) BioTechniques 11, 474-485. Wu et al (1989a) Genomics 4, 560-569.

Wu et al (1991) /. Biol. Chem. 266, 14338-14342.

Zenke et al (1990) Proc. Natl. Acad. Sci. USA 87, 3655-3659.

Claims

1. A polynucleotide comprising all, or a variant, or part thereof, of human chromosome 16q mmour suppressor gene or all, or a variant, or a part of a mRNA or cDNA derived from the mmour suppressor gene wherein the tumour suppressor gene contains at least part of the nucleotide sequences shown in any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31.

2. A polynucleotide which selectively hybridises to the region of human chromosome 16q which is bounded by the markers Alu 11 and

10T7 in Figure 2 or which hybridises to the PAC clone 81N24 or PAC clone 105K14 or the YAC clone 801B6, or a mutant allele thereof, or a polynucleotide which hybridises to a mRNA or cDNA derived from a gene from the region, or a mutant allele thereof.

3. A polynucleotide according to Claim 2 which hybridises to the region of human chromosome 16q which is bounded by the markers Alu 29 and 7T7 in Figure 2.

4. A polynucleotide according to Claim 3 which hybridises to the region of human chromosome 16q which is bounded by the markers 17SP6 and RD53 in Figure 2.

5. A polynucleotide according to any one of Claims 1 to 4 further comprising a detectable label.

6. A polypeptide encoded by the human chromosome 16q mmour suppressor gene as defined in Claim 1 or a variant or fragment or fusion of said polypeptide.

7. An expression vector capable of expressing a polypeptide according to Claim 6.

8. An expression vector according to Claim 7 which contains at least 20 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31 or a complement thereof.

9. An expression vector which contains at least 20 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15,

21, 23 or 31 , or a complement thereof.

10. An expression vector according to Claim 8 wherein the contiguous nucleotides are from an exon of the tumour suppressor gene.

11. An expression vector according to any one of Claims 7 to 10 for use in gene therapy.

12. A vector adapted to replicate in a mammalian cell which contains at least 20 nucleotides which are contiguous nucleotides from any of the

Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31, or a complement thereof.

13. A method for determining the susceptibility of a patient to cancer comprising the steps of

(i) obtaining a sample containing nucleic acid from the patient; and

(ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q tumour suppressor gene or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor gene cDNA, or a mutant allele thereof, or their complement.

14. A method of diagnosing cancer in a patient comprising the steps of

(i) obtaining a sample containing nucleic acid from the patient; and (ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q mmour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q mmour suppressor gene cDNA, or a mutant allele thereof, or their complement.

15. A method of predicting the relative prospects of a particular outcome of a cancer in a patient comprising the steps of

(i) obtaining a sample containing nucleic acid from the patient; and

(ii) contacting the said nucleic acid with a nucleic acid which hybridises selectively to the human chromosome 16q mmour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q mmour suppressor gene cDNA, or a mutant allele thereof, or their complement.

16. A method according to any one of Claims 13 to 15 wherein the cancer is ovarian cancer or breast cancer or prostate cancer.

17. A method according to any one of Claims 13 to 16 wherein the sample is a sample of the tissue in which cancer is suspected or in which cancer may be or has been found.

18. A method according to any one of Claims 13 to 17 wherein the sample is a sample of ovary and the cancer is ovarian cancer.

19. A method according to any one of Claims 13 to 18 wherein the nucleic acid which selectively hybridises to the human-derived DNA of said tumour suppressor gene or the said tumour suppressor gene cDNA sequence, or a mutant allele thereof, or their complement, further comprises a detectable label.

20. A method according to any one of Claims 13 to 19 wherein the nucleic acid which selectively hybridises as said is single-stranded.

21. A method according to any one of Claims 13 to 20 wherein the nucleic acid which selectively hybridises as said has fewer than 10000 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single-stranded.

22. A method according to any one of Claims 13 to 21 wherein the nucleic acid which selectively hybridises as said has fewer than 1000 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single-stranded.

23. A method according to any one of Claims 13 to 22 wherein the nucleic acid which hybridises as said has from 10 to 100 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single-stranded.

24. A method according to any one of Claims 13 to 23 wherein the nucleic acid which hybridises as said has from 15 to 30 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single-stranded.

25. A method according to any one of Claims 13 to 15 wherein the nucleic acid which hybridises as said hybridises to a portion human chromosome 16q DNA bounded by the markers Alu 11 and 10T7 in Figure 2 or which hybridises to the PAC clone 81N24 or PAC clone 105K14 or YAC clone 801B6 or which hybridises to a mRNA or cDNA derived from a gene from the region, or a mutant allele thereof.

26. A method according to Claim 25 wherein the portion is a single- stranded portion.

27. A method according to Claim 26 wherein said portion is capable of amplifying a portion of the said tumour suppressor said gene or the said tumour suppressor gene cDNA or mRNA in a nucleic acid amplification reaction.

28. A method for deterrnining the susceptibility of a patient to cancer comprising the steps of (i) obtaining a sample containing protein derived from the patient; and

(ii) determining the relative amount, or the intracellular location, or physical form, of the human chromosome 16q mmour suppressor polypeptide, or the relative activity of, or change in activity of, or altered activity of, the mmour suppressor polypeptide.

29. A method of diagnosing cancer in a patient comprising the steps of

(i) obtaining a sample containing protein derived from the patient; and

(ii) determining the relative amount, or the intracellular location, or physical form, of the human chromosome 16q tumour suppressor polypeptide, or the relative activity of, or change in activity of, or altered activity of, the mmour suppressor polypeptide.

30. A method of predicting the relative prospects of a particular outcome of a cancer in a patient comprising the steps of

(i) obtaining a sample containing protein derived from the patient; and

(ii) determining the relative amount, or the intracellular location, or physical form of the human chromosome 16q tumour suppressor polypeptide, or the relative activity of, or change in activity of, or altered activity of, the mmour suppressor polypeptide.

31. A method according to any one of Claims 28 to 30 wherein the cancer is ovarian cancer or breast cancer or prostate cancer.

32. A method according to any one of Claims 28 to 30 wherein the sample is a sample of the tissue in which cancer is suspected or in which cancer may be or has been found.

33. A method according to any one of Claims 28 to 32 wherein the sample is a sample of ovary and the cancer is ovarian cancer.

34. A method according to any one of Claims 28 to 33 wherein the relevant amount of the mmour suppressor polypeptide is determined using a molecule which selectively binds to tumour suppressor polypeptide or a natural variant or fragment thereof.

35. A method according to Claim 34 wherein the molecule which selectively binds tumour suppressor polypeptide or a natural variant or fragment thereof is an anti-tumour suppressor polypeptide antibody.

36. A method according to any one of Claims 28 to 33 wherein the relevant amount of the mmour suppressor polypeptide is determined by assaying or detecting the activity of the mmour suppressor polypeptide.

37. A method according to Claim 34 or Claim 35 wherein the molecule which selectively binds to the mmour suppressor polypeptide comprises a detectable label.

38. Use of a nucleic acid which selectively hybridises to the human chromosome 16q mmour suppressor gene, or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q mmour suppressor gene cDNA, or a mutant allele thereof, or their complement, in the manufacture of a reagent for diagnosing cancer.

39. Use of a molecule which selectively binds to human chromosome 16q mmour suppressor polypeptide or a namral fragment or variant thereof in the manufacture of a reagent for diagnosing cancer.

40. Use of a nucleic acid as defined in Claim 38 in a method of diagnosing cancer.

41. Use of a molecule which selectively binds to human chromosome 16q mmour suppressor polypeptide or a natural fragment or variant thereof in a method of diagnosing cancer.

42. A method of determining loss of heterozygosity in a tissue sample, the method comprising the steps of (i) obtaining a sample containing nucleic acid derived from the tissue and (ii) comparing a microsatellite marker product profile of the said nucleic acid with that of a reference (heterozygous) tissue, the microsatellite(s) being chosen by reference to the human chromosome 16q mmour suppressor gene.

43. A method of treating a proliferative disease comprising the step of administering to the patient a nucleic acid which selectively hybridises to the human chromosome 16q mmour suppressor gene as defined in Claim 1 or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor gene cDNA as defined in Claim 1.

44. A method of treating a proliferative disease comprising the step of administering to the patient a nucleic acid which encodes the human chromosome 16q tumour suppressor polypeptide or a functional variant or portion or fusion thereof as defined in Claim 6 to ameliorate the disease.

45. A method according to Claim 44 wherein the nucleic acid which encodes the human chromosome 16q tumour suppressor polypeptide or a functional variant or portion or fusion thereof includes at least 20 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31 or a complement thereof.

46. A method of treating a proliferative disease comprising the step of administering to the patient a nucleic acid which contains at least 20 nucleotides which are contiguous nucleotides from any one of Figures 4, 5, 6, 7, 8, 9, 15, 21, 23 or 31.

47. A method according to any one of Claims 43 to 46 wherein the proliferative disease is cancer.

48. Use of a nucleic acid as defined in any one of Claims 38 or 43 to 46 in the manufacture of a medicament for treating a proliferative disease.

49. A method of treating a proliferative disease comprising the step of administering to the patient an effective amount of human chromosome

16q mmour suppressor polypeptide or a fragment or variant or fusion thereof as defined in Claim 6 to ameliorate the disease.

50. A method according to Claim 49 wherein the proliferative disease is cancer.

51. Use of human chromosome 16q tumour suppressor polypeptide or a fragment or variant or fusion thereof as defined in Claim 6 in the manufacture of a medicament for treating a proliferative disease.

52. A method of treating a proliferative disease comprising the step of administering to the patient an effective amount of a compound which modulates human chromosome 16q mmour suppressor polypeptide function as defined in Claim 6, or the function of a mutant such polypeptide found in a diseased cell.

53. Use of a compound which modulates human chromosome 16q mmour suppressor polypeptide function as defined in Claim 6 in the manufacture of a medicament for treating a proliferative disease.

54. A method according to Claim 48 wherein the proliferative disease is cancer.

55. A method of treating cancer, the method comprising administering to the patient an effective amount of a mutant human chromosome 16q mmour suppressor polypeptide or a fragment or variant or fusion thereof as defined in Claim 6, or an effective amount of a nucleic acid encoding a said mutant polypeptide or fragment or variant or fusion thereof, wherein the said mutant said polypeptide is a mutant found in a cancer cell and the amount of said mutant polypeptide or amount of said nucleic acid is effective to provoke an anti-cancer cell immune response in said patient.

56. A cancer vaccine comprising a mutant human chromosome 16q tumour suppressor polypeptide or fragment thereof as defined in Claim 6, or a nucleic acid encoding a said mutant polypeptide or fragment thereof, wherein said mutant polypeptide is a mutant found in a cancer cell.

57. An antibody which reacts with human chromosome 16q mmour suppressor polypeptide as defined in Claim 6 or a mutant said polypeptide, or fragment thereof, wherein said mutant polypeptide is a mutant found in a cancer cell.

58. A nucleic acid which selectively hybridises to a nucleic acid encoding a mutant human chromosome 16q mmour suppressor polypeptide as defined in Claim 6, wherein said mutant polypeptide is a mutant found in a cancer cell.

59. A kit of parts comprising a nucleic acid which hybridises selectively to the human chromosome 16q tumour suppressor gene as defined in Claim 1 or a mutant allele thereof, or a nucleic acid which hybridises selectively to human chromosome 16q tumour suppressor cDNA as defined in Claim 1 or a mutant allele thereof, and means for detecting a mutation in the tumour suppressor gene wherein said mutation is a mutation in the mmour suppresor gene found in a cancer cell.

60. A pharmaceutical composition comprising an expression vector including a nucleic acid which encodes the human chromosome 16q tumour suppressor polypeptide or a functional variant or portion or fusion thereof as defined in Claim 6 and pharmaceutically acceptable carrier.

61. A pharmaceutical composition comprising a gene therapy vector including a nucleic acid which selectively hybridises to the human chromosome 16q tumour suppressor gene as defined in Claim 1, or a mutant allele thereof, or a human chromosome 16q mmour suppressor gene cDNA as defined in Claim 1, or a mutant allele thereof, and a pharmaceutically acceptable carrier.

62. A pharmaceutical composition comprising human chromosome 16q tumour suppressor polypeptide or a fragment or variant or fusion thereof as defined in Claim 6, and a pharmaceutically acceptable carrier.

63. A nucleic acid as defined in Claim 60 or 61 for use in medicine.

64. Human chromosome 16q tumour suppressor polypeptide or a fragment or variant or fusion thereof as defined in Claim 6, for use in medicine.

65. Any novel human tumour suppressor gene as herein disclosed.

66. Any novel method of treating cancer as herein disclosed.

67. Any novel method of diagnosing cancer as herein disclosed.