AU783373B2 - Epithelial cell growth inhibitors - Google Patents

Description

WO 00/78955 PCT/US00/16900 EPITHELIAL CELL GROWTH INHIBITORS Field of the Invention This invention relates to a family of epithelial cell growth inhibitors useful in the diagnosis and treatment of epithelial cell cancers.

Background of the Invention Epithelial cell cancers, for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, cancer of the spleen, testicular cancer, cancer of the thymus, etc., are diseases characterized by abnormal, accelerated growth of epithelial cells.

This accelerated growth initially causes a tumor to form. Eventually, metastasis to different organ sites can also occur. Although progress has been made in the diagnosis and treatment of various cancers, these diseases still result in significant mortality.

The treatment of cancer is greatly enhanced by early detection. However, there are difficulties in detecting the disease in its early stages. For example, epithelial tissuecontaining organs such as the prostate, ovary, and others, are not easily palpated. The detection of abnormal tumor growth in such organs is difficult without frequent screening and appropriate markers. A substantial drawback of available cancer diagnostic assays is a high rate of false positive and negative results, making the available tests less reliable than desired.

For this reason, there is a great need to identify new diagnostic as well as new therapeutic agents to improve diagnosis and treatment of cancer, for example, prostate cancer, breast WO 00/78955 PCT/US00/1 6900 cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, cancer of the spleen, testicular cancer, cancer of the thymus, etc., A novel, specific, mammary cell growth inhibitor, Mammastatin, has recently been identified and characterized. Mammastatin has been expressed from variant clones, MammA (PCT/US97/18026, ATCC# 97451, deposited 22 February 1996); MammB (PCT/US97/27147, ATCC# deposited 15 June 2000); and MammC, described in copending PCT application No. PCT/US0/ filed on even date herewith (ATCC# deposited 15 June 2000).

Mammastatin is produced and secreted by normal mammary cells, and is detected in blood samples of normal individuals. Blood concentrations of the mammary cell growth inhibitor, and particularly of the active, phosphorylated form of Mammastatin, are reduced or absent in breast cancer patients. Administration of protein comprising active Mammastatin (secreted from normal human breast cancer cells) is effective to reduce tumor size and number, and to prevent tumor growth in late stage cancer patients.

Epithelial cell growth inhibitors having similarity to Mammastatin have now been discovered, isolated, and characterized. These inhibitors bear partial sequence identity to Mammastatin at the 5' end of the sequence, and have little or no identity at the 3' end of the molecule. Like Mammastatin, the newly discovered family of epithelial cell growth inhibitors (ECGI) are differentially expressed in normal epithelial cell tissues, but not in cancerous epithelial cell tissues. Also, like Mammastatin, the newly discovered family of epithelial cell growth inhibitors are detected in blood samples taken from normal individuals, but not in the blood of patients with epithelial cell cancers, as shown in the Examples below.

Summary of the Invention A family of epithelial cell growth inhibitors (ECGI) have now been identified in a number of different epithelial cells. These ECGI are differentially expressed in normal epithelial cells, but not in epithelial cancer cells. As shown in Examples below, Mammastatin-like ECGI proteins have been discovered in a variety of epithelial cell tissue, including prostate, colon, ovary, lung, spleen, testis, thymus, and others.

The ECGI of the invention are expressed in normal epithelial cells but not in cancerous epithelial cells. The Mammastatin-like ECGI proteins are encoded by nucleic acid sequences that hybridize to nucleic acid sequences encoding Mammastatin. The ECGI proteins also bind anti-Mammastatin antibody. A nucleic acid sequence encoding ECGI S. in prostate cells (PRT-6, SEQ ID NO: 4) has been isolated and characterized (PRT-6, deposited with ATCC on 15 June 2000), as described in the Examples below.

Because the ECGI of the invention are differently expressed by normal epithelial cells and not by cancerous epithelial cells, the presence or amount of the ECGI can be analyzed to diagnose cancer and/or to monitor treatment. The inventive ECGI proteins and nucleic acids encoding them also provide useful therapeutic agents to inhibit epithelial cell growth, prevent tumor formation, and treat cancer.

The present invention further provides an isolated and purified protein having the following characteristics: a molecular weight of approximately 50-60 kDa; (ii) produced by normal prostate, colon, or ovarian cells, but not by prostate, colon, or ovarian cancer cells; and (iii) binds antibody 7G6 (ATCC Accession No. PTA-4606).

The invention also provides a process for inhibiting growth of prostate, colon, or ovarian cells, comprising administering to said cell an isolated and purified protein having the following characteristics: a molecular weight of approximately 50-60 kDa; (ii) produced by normal prostate, colon, or ovarian cells, but not by prostate, colon, or ovarian cancer cells; and (iii) binds antibody 7G6 (ATCC Accession No. PTA-4606).

The invention further provides a method for detecting prostate, colon, or ovarian cell cancer, comprising analyzing body tissue or fluid for the protein of an isolated and purified protein having the following characteristics: a molecular weight of approximately 50-60 kDa; (ii) produced by normal prostate, colon, or ovarian cells, but not by prostate, colon, or ovarian cancer cells; and (iii) binds antibody 7G6 (ATCC Accession No. PTA-4606); (iv) is produced by normal prostate cells; and is encoded by SEQ ID NO:4 and correlating a reduction or absence of the protein, relative to a control sample, with the presence of prostate, colon, or ovarian cell cancer.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

•oooo* WO 00/78955 PCT/US00/16900 Brief Description of the Figures Figure 1A is a schematic diagram of an mRNA test panel showing locations of specific tissue mRNAs for analysis.

Figure 1B is a computer scanned image of a Northern blot showing hybridization of Mammastatin nucleic acid sequence to mRNA from a variety of tissues according to the plan shown in Figure 1A.

Figure 2 is a computer scanned image of a dot blot assay showing control, Mammastatin standard protein, serum samples from breast cancer patients, and conditioned medium from normal and cancerous human prostate cells probed with anti-Mammastatin antibody, 7G6.

Figure 3 is a computer scanned image of a Western blot assay, showing normal human mammary cell lysate human prostate cancer LnCap cell lysate MCF7 breast cancer cell lysate and normal human prostate cell lysate probed with anti-Mammastatin antibody, 7G6.

Figure 4 is a computer scanned image of a Western blot assay, showing cell lysates from normal prostate cells LnCap prostate cancer cells normal colon cells and colon cancer cells probed with anti-Mammastatin antibody, 7G6.

Figure 5 is a computer scanned image of a Western blot assay, showing cell lysates from human ovarian cancer cells normal human ovarian cells and normal human mammary cells probed with anti-Mammastatin antibody, 7G6. Lane A contained molecular weight standards.

WO 00/78955 PCT/US00/16900 Figure 6 is a computer scanned image of a dot blot assay showing serum samples from healthy male adults and from a prostate cancer patient probed with anti- Mammastatin antibody, 7G6.

Figure 7 is a computer scanned image of a DNA gel containing putative prostate ECGF DNA clones.

Figure 8 is a diagramatic representation of Prostate ECGI and its structural relationship to other sequences.

Detailed Description of the Invention Proteins of the invention: "Epithelial cell growth inhibitor (ECGI) proteins" of the invention are defined herein to mean Mammastatin-like proteins produced by and active to inhibit the growth of normal epithelial cells. Active, inhibitory ECGI proteins of the invention are reduced or absent in cancerous epithelial cells. The ECGI protein family disclosed herein appears to include inhibitors that are specific to each epithelial tissue, with little or no inhibitory activity across tissue types. As discussed more fully below, it is postulated that each ECGI protein contains a growth inhibitory domain and a tissue-specificity domain.

The ECGI proteins of the invention exhibit significant homology to Mammastatin, a mammary cell growth inhibitor produced by normal human mammary cells, and previously demonstrated be useful in the diagnosis and treatment of breast cancer (PCT/US97/18026).

ECGI proteins bind one or more anti-Mammastatin antibodies such as 7G6 (Neomarkers, Freemont, CA), and are encoded by nucleic acid sequences sharing significant homology with nucleic acid sequences encoding Mammastatin.

WO 00/78955 PCT/US00/16900 Studies reported in the Examples below demonstrate the differential expression of ECGI proteins in normal epithelial cell tissues, but not in cancerous epithelial cell tissues, including breast, prostate, ovary, and colon. Like Mammastatin, the ECGI proteins of the invention appear, for example, in Western blots, as doublets or triplet bands, with one major band and one or two smaller, less prominent bands. This pattern of expression was demonstrated for Mammastatin to be due to phosphorylation of the protein. Mammastatin has an approximate molecular weight of 53 kilodaltons when phosphorylated at two sites. Smaller sized Mammastatin, 49 and 44 kilodaltons, correspond to one or none of the sites being phosphorylated. Phosphorylation of the Mammastatin protein is correlated with its inhibitory activity.

Western blots of ECGI probed with the anti-Mammastatin antibody 7G6, demonstrate the approximate size of ECGI produced by various epithelial cell tissues. As shown more fully in the Examples below (see, for example, Figures ECGI from prostate cells migrates in a Western blot to approximately 55 kilodaltons, with less prominent, smaller bands at 51 and 46 kilodaltons suggestive of phosphorylated forms similar to the pattern seen for Mammastatin. ECGI from colon cells migrates to approximately 50 KD, with less prominent bands at approximately 47 and 43 kilodaltons. ECGI from ovarian cells migrates to approximately 60 kilodaltons.

Nucleic Acid Sequences Encoding ECGI Nucleic acid sequences of the invention are defined herein as those nucleic acid sequences that encode ECGI proteins, as defined above. Nucleic acid sequences encoding ECGI proteins share significant sequence homology to nucleic acid sequences encoding WO 00/78955 PCT/US00/16900 Mammastatin, and hybridize to nucleic acid sequences encoding Mammastatin under conditions of high stringency.

Mammastatin-like epithelial cell growth inhibitors preferably have substantial identity (at least 90%, and preferably at least 95% identity) over approximately 1000 contiguous nucleotides of a nucleic acid sequence encoding Mammastatin. Nucleic acids encoding Mammastatin include those DNA inserts of MammA (PCT/US97/18026, ATCC# 97451, deposited 22 February 1996); MammB (PCT/US97/27147, ATCC# deposited June 2000); and MammC, described herein (ATCC# deposited 15 June 2000).

Consensus sequences determined for known Mammastatin clones are shown in the Comparative Sequence Table 5 below, and as SEQ ID NO: 1 (MammA); SEQ ID NO: 2 (MammB); SEQ ID NO: 3 (MammC). Prostate ECGI nucleic acid sequence (SEQ ID NO: 4) is shown in Tables 1, 2, and ECGI can be amplified from a specific epithelial cell nucleic acid library, for example, using internal Mammastatin primers and/or by hybridization to Mammastatin under conditions of strict stringency. As shown more fully in the Examples below, nucleic acid sequences hybridizing to Mammastatin have been demonstrated in numerous epithelial tissues, including central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphatic cells, bone marrow cells, lung and air passages, bladder, uterus, prostate, testis, ovary, liver, pancreas, adrenal gland, salivary gland, and mammary gland (See Figure 1).

The nucleic acid sequence of a ECGI isolated from prostate cells, for example, shares greater than 95% identity to Mammastatin at the 5' half of the molecule, with little or no identity of sequence, however, at the 3' half. It is postulated that the 5' end, sharing identity WO 00/78955 PCT/US00/16900 with Mammastatin, includes a growth inhibitory domain of the molecule, whereas the 3' end, having little identity to Mammastatin, includes a tissue-specificity domain.

Diagnostic Methods The invention further provides an in vitro assay for detecting active, inhibitory ECGI in patient samples, including tissues, cells, and fluids. Epithelial cell cancer and advancing metastatic disease is diagnosed by correlating the presence and type of ECGI protein in a patient's sample with that of normal or cancerous human epithelial cells. A patient's blood or tissue sample is analyzed for the ECGI protein, for the abundance of the ECGI protein and/or for its molecular weight forms. As discussed below, the absence or loss of ECGI protein, particularly of the higher molecular weight, phosphorylated forms, is correlated with a specific epithelial cell indicative of advancing metastatic disease.

Analysis of ECGI can be performed using a variety of known analytical tools and methods, including immunoassays, hybridization, PCR techniques, and the like. Preferred are immunoassay, including ELISA, Western Blot, and dot-blot analysis of a patient's sample methods, using anti-ECGI antibodies. Preferably, recombinant ECGI standards are used to provide a standard curve for reliable quantitation of inhibitor levels. Such immunoassays are exemplified by the dot-blot assays and Western blot assays shown in the examples below. In an alternative preferred embodiment of the invention, tissue samples, such as tumor biopsies, are analyzed by immunohistochemistry, or by culturing a patient's tumor cells and examining the cultures for expression of ECGI.

In a particularly preferred embodiment, an assay for the diagnosis of an epithelial cell cancer includes at least two specific antibodies: an antibody to identify the sampled tissue as WO 00/78955 PCT/US00/16900 epithelial tissue, such as an anti-cytokeratin antibody, and a specific anti-ECGI antibody. For example, using an immunoblot format, prostate tissue suspected of containing the prostate cancer cells is homogenized, separated on an SDS/PAGE gel, transferred to membrane, and probed with both anti-keratin and anti-prostate ECGI antibodies. Isotype specific second antibodies that are conjugated to a suitable marker system such as peroxidase or alkaline phosphates are used to detect bound antibodies. Membranes containing bound first and second antibodies are then developed using known colormetric or fluorometric techniques and quantitated by known methods.

In the most preferred embodiment, the sample is analyzed for the size and/or phosphorylated forms of the ECGI, such as by Western Blot, using anti-ECGI antibodies. A decline or absence of the high molecular weight ECGI protein form correlates with advancing cancer.

Diagnostic kits of the invention include ECGI protein or nucleic acid sequences encoding ECGI, for example, as controls. Optionally, the diagnostic kit contains one or more antibodies that bind the epithelial cell ECGI to be detected or quantified. The antibodies may bind a Mammastatin-like domain (for example, 7G6), or may be tissue-specific ECGI antibodies. Alternatively, the diagnostic kit includes one or more amplification primer or hybridization probe for the amplification and/or detection of nucleic acid sequences encoding an epithelial cell ECGI, for example, the primers used in the Examples below.

Therapeutic Use ECGI protein for therapeutic use is produced from epithelial cell cultures under serum free conditions or by recombinant means. Preferably, ECGI protein is produced in yeast or WO 00/78955 PCT/US00/16900 higher eucaryotic cells to achieve phosphorylation of the protein. Recombinant protein is produced in host cells or by synthetic means.

Functional ECGI is administered to patients by known method for the administration of phosphoprotein, preferably by injection, to increase inhibitor levels in the bloodstream and increase the inhibitor's interactions with the desired epithelial.

The protein may be delivered to the patient by methods known in the field for delivery of phosphorylated protein agents. In general, the inhibitor is mixed with the delivery vehicle and administered by injection.

The dosage of inhibitor to be administered may be determined by one skilled in the art, and will vary with the type of treatment modality and extent of disease. Since Mammastatin inhibits approximately 50% of mammary cancer cell growth at a concentration of 10 ng/ml and stops growth at about 20-25 ng/ml in vitro, a useful therapeutic dosage range of ECGI is about 2.5 jag to about 250 pg administered daily dose. Preferred is approximately 125 p.g daily administered dose. The aim of the administration is to result in a final body dose that is in the physiological 15-50 ng/ml) or slightly higher range (for example, 25-75 ng/ml).

For clinical use, the preferred dosage range is about 500 ng/ml for initial treatment of metastatic disease, followed by a maintenance dosage of about 50 ng/ml. In clinical studies using Mammastatin, an administered daily dose of about 50 ng/ml to about 750 ng/ml was sufficient to induce remission to Stage IV breast cancer patients.

Since active ECGI is a phosphorylated protein, it is anticipated that multiple doses of the inhibitor will be required to maintain growth inhibiting levels of ECGI in the patient's blood. Also, since ECGI generally acts as a cytostatic agent rather than a cytocidal agent, it is WO 00/78955 PCT/US00/16900 expected that a maximum effect of the inhibitor will require regular maintenance of inhibitor levels in epithelial cell cancer patients.

In its preferred use, the ECGI is administered in high dosages 50 ng/ml, preferably about 50-500 ng/ml) to induce tumor regression. Lower, maintenance doses (<50 ng/ml, preferably 20-50 ng/ml) are used to prevent cancer cell growth.

Clinical experience with administered Mammastatin in Stage IV breast cancer patients indicates a useful dose is that which maintains physiological levels of Mammastatin in the blood. Administration is preferably daily, but, may be, for example, by continuous infusion, by slow release depot, or by injection once every 2-3 days. Anecdotal evidence suggests continuous administration may induce feedback inhibition, thus, a preferred administration scheme is to administer daily dose of Mammastatin for approximately 25-28 days, followed by days without administration.

Diagnostic Assay Assays of the present invention for detecting the presence of the functional inhibitor in human tissue and serum are useful in screening patients for epithelial cell cancer, for screening the population for those at high risk of developing epithelial cell cancer, for detecting early onset of epithelial cancer, and for monitoring patient levels of inhibitor during treatment. For example, analysis of a patient's blood ECGI, for example, may indicate a reduced amount of high molecular weight, phosphorylated prostate ECGI, as compared with a normal control or with the patient's prior prostate ECGI profile. Such a change is correlated with increased risk of prostate cancer, with early onset of prostate cancer, and with advancing metastatic prostate cancer. Diagnostic assay for phosphorylated, active, 55 kD prostate ECGI preferably is by WO 00/78955 PCT/US00/16900 Western blot immunoassay, or ELISA using specific anti-ECGI antibodies. Screening, for example, in serum, is preferably by immunoassay, ELISA, Western blot, or dot blot assay.

For best results, the patient samples should be assayed within a short time of sampling (within one week), stored at 4°C (less than one year), or frozen for long term storage. Most preferably, samples are frozen until time of assay.

EXAMPLES

The invention may be better understood by reference to the following Examples, which are not intended to limit the invention in any way.

EXAMPLE 1 Multiple Tissue Expression of ECGI Northern blot analysis was performed on a multiple tissue expression array (Clonetech, Inc. #7775-1) to demonstrate the expression of ECGI in a variety of epithelial cell tissues. A digoxin-labeled EcoRl fragment of Mammastatin, containing approximately 1800 base pairs of the 3' region of pMammC, SEQ ID NO: 3 (approximately nucleotide 359 end) was used as a probe. The DIG-labeled Mammastatin cDNA was hybridized to the array in 10 ml easy HYB solution (Roche) for 16 hours at 650 C, with 65" C washes, anti-DIG antibody hybridization and CSPD development performed according to the manufacture's instructions.

The blot was then exposed to Kodak X-OMAT film for 30 minutes at room temperature.

The tissue plan of the multiple tissue expression array is shown in Figure 1A.

Hybridization of the Mammastatin cDNA to the mRNA of the array is shown in Figure 1B, WO 00/78955 PCT/US00/16900 and demonstrates the variety of epithelial cell tissues expressing a Mammastatin-like ECGI sequence. Specific tissues that hybridized to the Mammastatin cDNA included: central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphatic cells, bone marrow cells, lung and air passages, bladder, uterus, prostate, testis, ovary, liver, pancreas, adrenal gland, salivary gland, and mammary gland.

EXAMPLE 2 Normal Versus Cancerous Prostate Cells Normal prostate cells obtained from surgical samples and cancerous prostate cells, LnCap, obtained from the American Type Culture Collection (ATCC) were incubated and analyzed for the production of a prostate ECGI. The cells were cultured in DMEM/F12 media with 40 gM calcium, supplemented with 5% Chelex-treated horse serum, 10 ng/mL EGF, p.g/mL insulin, 100 ng/mL Cholera toxin and 1 jg/mL hydrocortisone for four days.

Conditioned media samples were then collected and analyzed.

Normal human mammary cells obtained from patient samples were incubated in the same medium and Mammastatin secreted into the culture medium was used as a control.

Serum obtained from breast cancer patients was also analyzed and used as a control.

Sample fluids were collected and loaded by suction onto a nitrocellulose membrane on a dot blot apparatus. The membranes were then probed with the anti-Mammastatin antibody 7G6, and antibody binding was detected with goat-anti mouse antibody labeled with alkaline phosphates. Color was developed with NBT/BCIP substrate system (Life Technologies). The results are shown in Figure 2.

WO 00/78955 PCT/US00/16900 The anti-Mammastatin antibody recognized a protein produced by normal prostate cells but not cancerous prostate cells. This is analgous to the antibody's recognition of the mammary cell growth inhibitor, Mammastatin, produced by normal mammary cells, but not breast cancer cells. This data, in combination with the data from Example 1, demonstrates the production of Mammastatin-like ECGI in other epithelial cell tissues, and particularly, in prostate cells.

EXAMPLE 3 Differential Expression of ECGI in Prostate, Colon, and Ovary Prostate Normal prostate cells (Clonetech, Inc.), LnCap prostate cancer cells MCF7 breast cancer cells and normal human mammary cells (obtained from hospital tissue) were incubated as described above for Example 2. After at least 48 hours incubation, cells were lysed in sample loading buffer and analyzed for the presence of ECGI by Western blot, using the anti-Mammastatin antibody, 7G6 as a probe. Normal human mammary cell protein (NHMC) lysate (1 mg/ml) was used as a Mammastatin control The data are shown in Figure 3.

Normal prostate cell lysate contained a protein that was recognized by anti- Mammastatin antibody, while prostate cancer cells (LnCap) and breast cancer cells (MCF7) did not. The protein recognized in the prostate cell lysate was of a similar size to that of Mammastatin WO 00/78955 PCT/US00/16900 Colon and Prostate Normal prostate cells (Clonetech, Inc.), LnCap prostate cancer cells Sw 948 colon cancer cells and normal colon epithelial cells (obtained from patient surgery tissue) were incubated as described above for Example 2. Cell lysates were prepared in sample loading buffer and analyzed for expression of ECGI by Western blot, using the anti- Mammastatin antibody, 7G6 as a probe.

As shown in Figure 4, normal prostate and normal colon epithelial cells expressed a protein that was recognized by the anti-Mammastatin antibody, while cancer cells from these tissues did not The differential expression of protein is similar to that demonstrated for Mammastatin in breast tissue. In addition, the pattern of bands shown in the Western blot for normal prostate and colon tissues is similar to the Phosphorylation pattern demonstrated for Mammastatin produced in normal human mammary cells. A larger prominent band is shown together with two smaller, fainter bands. This pattern has been correlated with Phosphorylation of Mammastatin.

Prostate ECGI is shown in the Western blot analysis (Figure 4) to have an approximate molecular weight of 51 kilodaltons; Colon ECGI is shown to have an approximate molecular weight of 50 kilodaltons.

Ovary OvCar-ovarian cancer cells normal human ovarian cells (patient surgery tissue) and normal human mammary cells (patient surgery tissue) were incubated as described above for Example 2. After an incubation period of at least 48 hours, direct lysates were prepared by removing growth media and rinsing cells with saline and SDS-PAGE sample loading buffer until viscous. Lysates were collected and separated on 10% SDS-PAGE, WO 00/78955 PCT/US00/16900 transferred electrophoretically onto nitrocellulose, and probed with the 7G6 anti-Mammastatin antibody. The data are shown in Figure 5, where lane A contains molecular weight standards; B, OvCar-ovarian cancer cell lysate; C, normal human ovarian cell lysate; and D, normal human mammary cell lysate.

Figure 5 demonstrates that a Mammastatin-like ECGI protein is produced in normal human ovarian tissues and is recognized by anti-Mammastatin antibody. The protein is not expressed in the ovarian cancer cells analyzed. The ovarian ECGI has an approximate molecular weight of 60 kilodaltons.

Example 4 Differential Detection of Prostate ECGI in Blood Serum samples from three healthy male volunteers were analyzed for the presence of the prostate ECGI, and compared with that of serum from a prostate cancer patient. Serum samples were loaded at 400 microliter and 200 microliter samples in duplicate. The samples were drawn onto nitrocellulose by vacuum in a 96 well dot blot apparatus. The filters were then probed with the anti-Mammastatin antibody, 7G6, and developed with NBT/BCIP substrate. The data are shown in Figure 6.

Normal human mammary cell (NHMC) cultures produced standard conditioned medium for comparison. Standards, in duplicate, contained 400, 200, 100, 50, 25, 12, and 6 microliters of NHCM medium. Serum samples from healthy adult males and from an adult prostate cancer patient were assayed using 400 and 200 microlites of serum sample.

A prominent signal from normal serum demonstrated the presence of prostate ECGI, while the prostate cancer patient's serum showed only a weak signal.

WO 00/78955 PCT/US00/16900 Example Inhibitory Activity of Prostate ECGI Normal prostate cells (Clonetech, Inc.), PC3 and LnCap prostate cancer cells were plated at a density of 5.0 x 104 cells per milliliter in 12 well plates in RPMI medium containing 10% fetal bovine serum. After 24 hours, the cultures were supplemented with 10% conditioned medium. Each sample was run in triplicate. Plates were allowed to incubate for six days at 37°C and 5% CO 2 and at the end of the incubation period, cells were lysed with Cetrimide and counted using a Colter Counter. Percent inhibition was calculated by comparing treated versus non-treated wells, and the data shown in the table below.

Androgen-insensitive PC3 cells were not inhibited by the normal prostate cell media or by the conditioned medium obtained from normal prostate cells. In contrast, LnCap cells were inhibited by the addition of growth medium, with the inhibition somewhat greater by media derived from normal prostate versus media derived from cancer cells.

Cell Type Inhibition by Inhibition by Normal Prostate Prostate Tumor medium medium LnCap #1 22.5 3.3 8.3 0.4 LnCap #2 22.7 0.6 16.7 15.8 PC3 0 0 Example 6 Isolation and Characterization of Prostate ECGI DNA Nucleic acid libraries were produced from the mRNA of normal prostate cells (patient surgery tissue) and from LnCap, prostate tumor cells WO 00/78955 PCT/US00/16900 The nucleic acid sequences in the normal and cancerous prostate cell libraries were incorporated into vectors and used to transform bacteria. Colonies of bacteria expressing the normal and cancer prostate cell nucleic acid sequences were screened by hybridization with a digoxin-labeled Mammastatin nucleic acid probe under stringent conditions, as described above.

The positive colonies were selected and grown in LB broth. Plasmids obtained from the positive colonies were purified and digested with ECO R1 and Xhol to release the CDNA inserts. The digested DNA was then separated on a 1 agarose gel (see Figure 7A) and the separated DNA was subjected to Southern blot analysis using the digoxin-labeled Mammastatin fragment as a probe. As shown in Figure 7 below, two prostate ECGI clones were isolated, each having an approximate size of 2 Kb: One clone was isolated from the normal prostate tissue library (PRN2.1) and one from the LnCap prostate tumor cell library (PRT-6).

PRT-6 was further characterized, and its nucleic acid sequence was determined. As shown below in Table 1, the nucleic acid sequence encoding Prostate ECGI has substantial identity to Mammastatin (greater than 90%) at the 5' end of the molecule (approximately nucleotides 15-1032 of MammC), with little or no identity at the 3' end of the molecule.

These regions of similarity and distinction are shown diagrammatically in Figure 8.

Example 7 Isolation and Characterization of Prostate ECGI DNA Nucleic acid libraries were constructed from the mRNA or normal prostate cells (obtained from patient surgery tissue) and from LnCap prostate tumor cells The library cDNA was used to transfer E.coli and plated out for colony hybridization. The WO 00/78955 PCT/US00/16900 colonies were screened with a digoxin-labeled Mammastatin C fragment generated by PCR using external PCR primers M200 and M2200.

[Sequence ID NO: 5] M200: GCGCCGGCCGGGCGCGACCCG [Sequence ID NO: 6] M2200: GCAATCTCAGCGCACTGCTGC Bacterial colonies expressing prostate ECGI clones were hybridized to the labeled Mammastatin probe under strict hybridization conditions, as described above.

Example 8 Homology of Prostate ECGI The prostate ECGI sequence was analyzed against nucleic acid sequences present in GenBank. Portions of two molecules showed some similarity to domains within the prostate ECGI sequence: 28SmRNA and 28SmRNA homology has been identified in many gene sequences with importance in growth regulation (Hu et al., 1999, PNAS 96:1339-1344; Mauro et al., 1997, PNAS 94:422- 427). Hip55 is a protein that binds to hematopoetic progenitor type 1 kinase, a protein involved in the src signal transduction pathway (Ensena et al, 1999, JBC 274:33945-50).

Using the open reading frame known for Hip55, a putative amino acid sequence was deducted for the prostate clone. As shown below in Table 3, the translation includes several internal stop codons.

Also using the Hip55 ORF, a putative amino acid sequence was deduced for MammB and MammC sequences, shown in Tables 4 and EDITORIAL NOTE APPLICATION NUMBER 60523/00 The following sequence listing pages 1-22 are part of the description. The claims pages follow on pages EPO -DG I 18. 10. 2000 SEQUENCE LISTING <110> BIOTHERAPIES, INC.

<120> EPITHELIAL CELL GROWTH INHIBITORS <130> 4273.3WOU1 <140> PCT/USOO/16900 <141> 2000-06-19 <150> 60/139,995 <151> 1999-06-18 <160> <170> Patentln Ver. 2.1 <210> 1 <211> 2418 <212> DNA <213> Homo sapiens <400> 1 tggggctcca tcggcacgag ccccccggtg gaaataccac aggggctctc cggggacagt ggtgtcctaa tcgcttgatc ctgacctttt gtggcgcca: tcattgtgta ctgggt ttag tggttatcct ctgaggagcc gaatcccc gccggtCCCC cqcqggagg gtcctgggaa cgttcgtgct atgtatcttt ctagatcacc gaggttgtac ggagaaatgc ggtacaggta ccccggtggC cacggtgaag tccccgcgag tactcttatc gcttctggqg gccaggtggg ggcgagctca ttgattttca gggttttaag agcgttcatt gcagaattca accgtcgtga gctcagtacg aatggggcga.

caggcggaac cgcctgtccc cgcgtgccc acggggcgcg cgtgccgaat atgatataat tggtagttct aaatttacat ccctcattag tttacaagtt ggccgctcta agacatgaga.

9ggCCC9ggg gttttttcac ccaagcgccc gagtttgact gggaggacag g9tacgaatac caggaggtgt aggacgtcgc ccaagcgttg gacaggttat agaggaaccg agctaccatc gatacggcag cgccggcggg gccqcgcgCC gccggaaagg tcggcacgag gaattCtttt atttctggtt ccctaccaaa tatccccctt tcattataca gaactagtgg ggtgtagaat cggggtCcgC tgacccggtc ggccgcgcgc ggggcggtac aaicctcccg agaccgtgta cagaaaagtt .tttitgatcc gattgttcac ttttacccta caqgttcaga tgtgggatta cgccgcqggag ccgccccccc gggaccqgg cggccgccC tgcacccatt cctttgggta tatttagaaa gtgatttttt ttacctctct gacaaattga atcccccggg ccgtgggagg cggccctgcg gagcgggqggg cggccgggcg acctgtcaaa tggagcagaa agcggggCct accacaggga ttcgatgtcg ccactaatag ctgatgattg catttggtgt tgactgacgc cctcggttgg ccctccacgc tccggtgcgq ctcgcccgtc cacaatatac gatatccagt tcttcatact taaatatgaa actgcagaat atattgaaat ctgcaggaat cccccggcgc ggccgccggt gcgagccccg 4;gacccgct~c cggtaacgca gqgcaaaagc cacgatcctt taactggctt gctcttccta.

ggaacgtga~g tttgttgcca aiqtqttgg tctaagtcat.

qctcggatta gccccgcgcg aqtgcccttc acgcaccgca atacaagtgc agtgggattg gatttccata agaatggtct gacttcaagg tttctgcata 120 180 240 300 360 420 480 540 600 660 720 780 8.40 900 960 J0 1080 1140 1200 1260 1320 1380 1440 ,Printed:2O':O9 2O1:- 1,6 bi J ,-o 2 agaggcacag caaagctgga tctactgaat gtttatatca agcattttaa acacigtgtt ggtgagaaa gagttcaact gagatctctc ttttatttcc tatttgttaa tataaagtac caggcagatc tctctacaaa ctactctgag gcgctgagat aaaaaaaaaa attttaggat attggaaatc tggcttt tgt aa tatacttt tttcaaaaca tgtcacattt tagtcttacc tqcgcaaagt cataccacac tttcccttcc ttacataact gggggagtga actttgagtc aaaatacaaa 4gctgaggtg tgcgccattg aaaaaaaa tcaaagttgt tcagatgaaa gatcicatt aaggtataaa taaaatagct caaaaatgtt gtagtaagac tagtaacagg tgctagcaca.

tcccacaacc aatacatgtt tagctcatcc cagagttcga aaatttagcc ggaagattga cactccagcc atgaacaagg tacatitcta aatacctact aatcaaatta accgtctat ctcatggtaa tattcagtaa actaggactt tgtgcctgtc cctttttccc tatgagaaca ctgtaatcct gaccagcctg gggcgtqctg ttgagcccag tgggtgagag acaagtgctc gtagtaccac t a ttaa aact taggtaaagc gggdatttat tgtt-acaataacgaaacct gaacctgaac atcttattct cccatttctt attgatatag agcactttgg ggcaacatgg gcacagacct gaggtggaag agagagaccc tagggacttg cagcatatat aa tgaaaag tgttttcttt 6a-tgtacgag aaitctgt -cg ctgaaccttg caicacacic tggctccctt tctttttttt cacaaaagga aaggccaagg tgaaaccctg gtagtctcag ctgcagcagt tgtctccaaa 1500 1560 -1620 1680 1740 1800 .1860 1920 1980 2040 2100 .2160 :2220 2280 2340 2400 2418 .e210> 2 <211> 2326 (212> DNA <213> Homo sapiens 400;>*2.cggcacgagc cccccggtgt cactactctg tcgcttctgg grtgccagtgg aggcgagctc tcagtacgaa gcaggaggtg agcgacgtcgf accaagcgtt a ga ca ggtt i agaggaaccg agctaccatc cgatacggca cgccggcggg ccgcgcgccg ccggaaaggc cggcacgagt aattcttttc tttctggttt cctaccaagt tccccctttt attatacaga acggtgaaga ccccgcgagg atcgtttttt cgccaagcgc ggagtttgac agggaggac-a tacagaccgjt tcagaaaagt ctttttgatc ggattgttca gtttacccta caggttcaga tgtgggatta gcgccgcgga cgcccccccc ggaccggggt ggccgCCCCC agcaccattc ctttggggag attgagaaat gattttttta acctctctac caaattgaat gacatgagag ggcccgcgcg -cactgacccg ccggccgcgc t ggggcggta aaacctcccg gaaagcgggg taccacaggg cttcgatgtc cccactaata ctgatgatgt catitggtgt ttactgaacg gcctcggttg cctccacgcg ccggtgcgga tcgcccgtca acaatagaca atatccagta cttcatactg aatatgaaag tgcagaatga attgaaattt gtgtagaata tccgCcggCC gtgaggcggg ,gccggccggg cacct gt caa tggagcagaa cctcagatct .ataactggct ggctcttcct gggaacgtga gttgttgcca atgtgcttgg cctctaagtc gcctcggatg ccccgcgcgc gtgcccttcg cgcaccgcac tacaagtgca gtgggattgc atttccatag aatggtctgg cttcaagggg ctgcattaga agtgggaggc cgcgggcgcc gggcgagcc cgcgacccgc acggtaacgc gggcaaaatg tctgaccttt tgtggcggcC atcAttggga ccccggcgcc ggtgaaatac cgaggggctc tceggggaca aggtgtccta atcttgattt tgggttttaa a agcgtt caa agcagaattc gctgggttta .gaccgtcgtg 600 tggtaatcct ctggggagcc .agaatcccgc gccggtcccc gcgggagggc tcctgggaaa gttcgtgctc tgtatcttta tagatcacct aggttgtaca agaaatgccc tacaggtatt ggcacagatt gctcagtacg aatggggcga ccaggcggaa cgcctgtccc gcgtgccccg cggggcgcgg gtgccgaatt ttatataatg ggtagttcta aatttacatc ctcattagta tacaagtttc ttaggattca 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 i'Pntd20L'9W2O1.

TT ?.Ou? iei In, s p MR

-Z

INN

aa9tt gt aag gaagaaatac atcatttata taaaaaaatc atagctaccg aatgttctca taagactatt, caggactagg.

cacatgtgc accccttttt tgtttatcag atccctgt aa agaccagcct gggcgtqctg t tgagcccag tgggtgagag aacaaggaca atttctagta cctacttatt aaattatagg tctattgggc.

tggtaatgtt cagaaacgaa acttgaacc t tgtcatctta ccccccattt aacaattgat tctagcactt.

gggcaacatg gcacacacct qaggtggaag agagagaccc agtgctctag gtaccaccag aaaactaatg aaaagctgt t atttatactg cacaataatt acctctgaac gaaccatcac.

ttcctggctc.

cttttctttc atagcacaaa tggaaggcca gtgaaaccct gtagtctcag ctgcaqcagt tgtcttcaaa ggacttgcaa catatattct aaaa gggttt ttcttttgca taccagacac :ctgtagggtg cttggagttc.

actccagatc cct kyttat t tttttatttg aggatataaa aggcaggpcag gtctctacaa ctactctgaq.

gcgctgagat aaaaaaaaaa agctggaatt ggaaatctca actgaattgg ctttgtgatc atatcaaata tactttaagg ttttaatttc: aaaacaaaaa tgtgtttgtc acatttcaaa gagaaatagt cttaccgtaq aacttgcgca. aagttagtaa tctccatacc tcctttccct ttaattacat gtacgggtga atcacttgat aaaaatacaa ggctgaggtg tgcgccatt~g aaaaaa acactgctag tcctcccaca aactaataca gtgatagctc ccagagttcg aaatttagcc ggaagattga cactccagcc 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 .2100 .2160 .2-220 -2280 2326 <210> 3 <211> 2355 <212> DNA <213> lomfo sapiens <400> 3 .gaattcggca. cgagcacqgt .gcgccccccc. ggtgtccccg cggtgaaata ccactActct cccgaggggc: tctcgcttct .gctccgggga cagtgccagg cgcaggtgtc ctaagqcgag aagctcgctt qatcttgatt ccttctgacc ttttgggttt gaagagacat qgaggggccc gatcgttttt ggcgccaagc.

tggggagtt ctcagggagg ttcagqtacga taagcaggag gPttgtaqg.

ccta tcattg4 tgagctggt.

gccatggtaa ttggct~g..

agtc -agaat~c gatagccggt cgcgcgcggg.

cttcgtcctg cgc acgttcg, gtgqatgtat attgctagat catagaggtt tctggagaaa aggggtacag taagaggcac gccaaggtt.catagcgac-g tgaagcagaa ttcaccaagc .ttagaccgtc. gtgagjacagg.

t cctgctag. Itacgagagga agccaatggg gcgaagctac ccgcccaggc ggaacgatac cccccgcctg tccccgqcgg.

agggcgcgtq 4cccgccgcg ggaaacgggg cgcqgccgga tgctcgtgcc gaattcggca ctttatgata taatgaattc cacctggtag ttctatttct 9taca.aattt acatccctac tgcccctcat tagtatcccc gtatttacaa gtttcattat agattttagg attcaaagtt ,gagaggtgta ggggqggggt tcactgaccc gcccggccgc.

gactggggqqg acagaaacct atacagacc.

.gtgtcagaaa tcgqttttt~g gttggattgt ttagttttac accgcaggtt catctg tggg ggcagcgc;cg.

cgggccg ccc cgccgggacc aaggcggccg cgagtagcac ttttcctttg, ggttiattga caagtgattt cttttacctc aca ga ca aat gtatgaacaa gaataagtgg .gaggcccccg ccgccggcpcc tg.cgggccgc -120 ggtgaggcgg .gggggcgagc 1-80 gcgccggc;cg. ggc~gcgaccc 2.40 g tac acctgt .caaacggqtaa *300 cccgtgagc agaagggcaa 360 tg aaagcggg gcctcacgat 420 agttaccaca gggataactg 480 atccttcgat. gtcggctctt 540 ,tcacccacta .atagggaacg. 600 cctactgatg .atgtgttgtt 6.60 cagacatttg %tgtatgtgc. 720 attatgActg aacgcctcta.780 cggagcctcg,gttggcctcg 840 cccccctcc acgcgccc 900 ggggtccggt gcggagtgcc 960 ccccctcqcc cgtcacgcac 1020 cattcacaat agacatacaa. 1080 ggtagatatc cagtagtgqg 1140 gaaatcttca tactgatttc 1200 ttttaaatat gaaagaatgg 1260 tctactgcag aatgacttca 1320 tgaatattga aatttctgca 1380.

ggacaagtgc tctagggact 1440

IMM.

.20,t '00-1 ~~71nr~d2O-O9QI T- I: TO! Ettgcaaagctg gaattggaaa tctcagatga aatacatttc tagtagtacc accaqcatat 1500 attctactga, qgtttatatc tagcatttta gacactgtgt gggtgagaaa agttcaactt agatctctcc atttcccttt ctttcttttt acaaaaggai aggccaaggc aaaccttgtc gtctcagcta cagcagtgcg *,cttaaaaaaa attggcttt~g aaatatactt atttcaaiaac ttgtcacatt tagtcttacc.

gcgcaaagt-t ataccacact tiatttctt aattgttaat ataaagtacg ag gcag8t ca tctacaaaaa ct ctgaggg&c ctgagattgc aaaaa tgatcatcat taatacctac taaggt ataa- aaatcaaatt ataaaatagc taccgtctat itcaaaaatgt tctcatggta :gtagtaagac tattcagtaa agtaacagga. ctaggacttg gct agcacat gtgcctgtcd tcccttcctc ccacaacccc tacataactai ata~catgctt gqtgagtgat agctcatdcdc cttg-agtcda gagttcgaga aatacaaaaa. tttagccggg tga~gfggga' aqatt~atiq qccattgcac tcdagcctg4 ttattaaaac ataggtaaag tgggcattta atgttcacaa adgaaacctc *aacctgaacc tcttattcct *tttttcccc atcagaacaa tgtaatccta ccagcctggg cgtgjctggca.

-agcccaggag gtgagagaga' taatgaaaag 1560 ctgttttct-t 1620 tactgfacca*1680 taattctgta 1740 t gaaccittgg 1800 atcacadtcc 1860 -ggctcdtqtt 1920 catttftttt -1980 ttgatatigc 2040 gcactttga 2100 caacat -gotg'2160 ca'cacctgjta 2220 .gtggaagctg 2280 gagaccctgt 2340 2355 <210> 4 <211> 2333 ;<212> DNA *<213> Homo sapiens <400> 4 qcAcqagatt ttggcggaat tgtcccgcg actactctga, tcgctt ctgg gtgccaggtg aaggcgagct tcttgatttt ttgggtttta.

caagcgttca aagcagaatt agaccgtcgt ctgctcagta ccaatggggc gccaggcgg cccgcctgtc cgcgtgcccc acggggcgcg gaacctggcg tcctgcagaa.

tctcaaggcc tggtgcaggc agcagccccc agggccaggg cccactgtcccagc44ggaa aggccg tcgttttttc cgccaagcgc gggagtttga, cagggaggad cagtacgaat agcaggaggt tagcgacgtc caccaagcgt gagacaggtt cgagaggaac gaagctacca aacgatacgg cccgccggcg gccgcgcgcc gccggaaagg ctaaaccacc gcagctcac cagggcagat agaagaggag actggtgcag gctcagtggg ctatctacta tccagC1aa agaagaccct ggcggggt~c actgacccgg ccggccgcqc ctggggcggt agaa acctc acagaccgt~g gtcag aaaag gctttttgat tggattgttc agttttaccc cgcaggttca tctgtgggat cagcgccgcg ggecgcccc gggadcgggg cggccgcccc tccatctcca caaccagaga ctccctgctg gctgtgtatg cagcaaggtg caagggctct 4ttgagcttg gccggccctg tgaggcgggg 9CCggccggg a cacct gtca cgtggagdag aaagcgggg6 ttaccacagg ccttcgatgt acccactaat tactgatgat gacatt tggt tat qact gaa gagcctcggt ccctccacgc tccggtgcgg ctcgcccgtc gt cctcagcc cccactttgg aggagccggC aggaacctcc ctggctctga.

gtgcccgtgc ctacagoca:6 ggcccccggc cgggccgccg gggcgagccc cgCgacccgc aacggtaacg aagggcaaaa ctcacgatcc gataactggc cggctcttcc agggaacgtq gtgttgttgc gtatg tgctt cgcct ctaag tggcctcgga gccccgcgcg agtgcccttc acgcaccgca, tggcaAgctg cagagagcca gcccagcact agagcaggag qcacattgac cctgtacgac gcccccccgg gtgaaatac cgaggggctC t ccggg gaca caggtg tct gctcgcttgA ttctgacctt ttgtggcggc tatcattgtg' agctgggatt catggt aat c ggctgaggag tcagaatccc tagccggtcc cgcgggaggg gtcctgggaa cgttcgtggg aggagccicct gctgcigcca cctccatgtc accttctacg caccacattc taccaggcag 120 180 '240 300 360 480 540 600 660 7 IS0 840 900 960 1020 1080 1140 1200 1260 1320 13S0 1440 11Pr i ntebd 2019-2OO0.:1

I

-anze2: rY ccgacgacac acgaaggctg acgtggagct ttccitattg acccccagtg ccaaatgcag agacagcttg ctgagtggcc ggctgcCt tc caccactgtt ttgtgaccaa gccigctctg gatgcagatg ccac-gcggct ggaaaagtaa agagatctcc gtggcgtggc cattgagtga ctggaagagg aggatgaggc daatggcctg gctct tgcc actgccaagc tgcatttatt tagaatgacc Agtcaqagtg tgctcc cca C gccaagctcc -cactcqatgfd aaaaaaaaaa tttgaccccg t atgqgccgg ggctgagggc aggcctggga ctcagggctc gtgattccca ctgAcaggat tgcggggaag tgcctttttt cttgggaaca gatdatggtg tccatttctc caccctgggt ctgcaggccc aaaaaactcg agaacctcat atggccattt acatcttgcc gttgacattc cctccggctt cacatccttc actgagccaa ggtcctgagc ctttttctct gtgaacgtag gtttggcagc tgtccctct*9 attcaaaaac cagtgtgtgc agaagctttg cacgggcatc tggcatgttc cttcccctct agcactcttc ggcaqactca ctgcatcccc gccctgcctg aggggcatct tgcttctaag agaattgttt aggfgaatt tg cctgggctat ggcagacaca ctcaactgat gacttcttcg gaggtgatcg cctgccaact cagaca tggc caggaatagg gcctgtcacc cgaccctccc tggccaagcc gggaggctct gggtggtggc ttagcagagt tcttgttgga gggaagtg9-9 acatgttcct tctgacttca cca 1500 1560 1620 1680 17 1800 1860 1920 1980 2040 2100 2160 2220 2280 2333 <210> <211> 21 <212> DNA <213> Homo sapiens <400> gcgccggccq ggcgcgaccc g <210> 6 <211> 21* <212> DNA <213> Homo sapiens <400> 6 gcaatctcag cgcactgctg c <210> 7 <211> 2843 <212> DNA <213> Homo sapiens <400> 7 ctttgggag gttgcagtga aggccctgcc tcttctcagg ccttcggacg caccqcaacc gggattacag ccgaggccgt gtgagccata acaggcaggc gtaggaagc-a gagtttcact t ccacctccc ggaggagcca aggatccctc gt tgt g tca c aggcaggcag aaaataacag cttggtgccc gcgttcaagc ccacacccag gaggaatcgc tgtgctccag gcaggcagaa aatacagcac acgctggagt gattctcctg ctgattttgt ctaaccctgg tctggqcgaa agacaacagc ttaattaatt gcagtggcac cctcagcctc attgttagta ggaggttgag agacagaatg tgtattatg~t tttttttttt catctcggct ctgagtagct gaqacggcat Pri,6f*",d,!W 09 ~4'ON2bM 2-O'"Tensp Mrofl-

L

~1I ttctccatgt gggtcaqgct. ggtatcgaac, tggcgacccc agtggatctg -cccgccccgg 480 cctcccaaag aittattitt .tacatttatt .tctgctgccc gggttcacgcccgtgcccgg.

.Atggtctcgq acaggcgtga tgaaaccagt ccgtgt tgc .gatgAcaggq cccgacagac aaatttggga.

ctatcacagg tgtgatacag ttagcoctgg cagtcggtct ctgcgtcccc acccactgtg ttca ttccgg ctctgaaaac gcaactgtgt ctaggaaatc aaacagataa acccattaca.

atacaatagg atacaataca gatgccgagg agaaatcccg tcccagctgc agtgagccga aaaaataaat ataaattaaa cctgtcatc agggccagta gctgtgctgt gcttgaacct gggcgacaga aattaaaaag agaacaaccc tgctggggtg *ttaattattt tatttat tta aggctggagt cattc-tcdt4 ct a act tt t -tctcctgacc.

ta gttttgt .aaggct tgga .cgAg C cta8C agtttcacgg, .cgtcagcttc accqtacacg aaatacacgg tgtccgtgcc ctcgtctctg ccaggagccc ctgtgggagc gctgacacgc ggaggcctca cttctccacc gccactttga ataaataaaa atacaataag atacgataca atacaatacg tggacgcatc tctcaattga.

taggaaggct gattgcgcca acataaataa ataaataaat cctcactttg tggtgaaacc actgtctgta gggagcgga gcgagactc tgagtttctg caccgtgaca acaggqgtga i.ttatttact gcagcggcgt gtattttgagg ccgtgqtrcg ccggcctatt ccg agggatc cCggc.c!ga ca gaqcgltt tggctcacg taaggaggag attcacccaa agtgattctt gacggaagtt tggtcgatt a ctccatectt tcactggcag cagaggaagg gcccccgccc cgaccgggtc taacacaaaa atacgatacg atacaataca CcgggCgcgg acctgaagtc aaatacaaaa gaggcaggag tcgcactcca atacatacat aaaataaaat ggaggccaag ccgtctctac atcccagcta.

ggttgcagtg gtctccaaaa.

gggaaaaaga tac gccatcgtga tiagtticca tatttattta.

gatctcggct ccakgta gqt .tagagatggg tccacctcgg tatctattta t. it tttttt.

cacqggccct cccccccttt-.

ggctggcgtg gactctgagc..

aaaaatcgta.

aacacagaaa.

ttcggtttgg ccagatgatc gttgtgggga ccccccaccc gcgt cgggca gagcaccagg ccacctccaa tgattgacct gtaactaact ataggatgcg atacaataca tggctcatgc gggagttgga ctagccgggc aatcgcttga gtctgagcaa acatacatac aaataaatgg gccggtggat icacaataca ctcgggaggc agccgagatc aaigaaaatg agaaaagaaa ctggccggct acgtttattt ttttaatcta .tttatttatt tttt cgagac agactctcg(; cactgcaacg .tccgcctccc gqggatacag gtttcactgt .cctcccaaagttaactttga.

tttigaga cgg;Cctc6.qaL.

:cccttcccC.

.cttaaactca.

cgaggagtcc acgtt caaag ccagtCtttt accttgactg cgatgggtgg tcgtcttgga cctccccagg tcacctagcg ccgcctgcgc git C tcc ct ttgatcaggc aaataaaata ataggatacg atacaataca ctgtcatccc gacaagcccg gcggtggcac acctgggaag caagagcgaa atacatacat gccctgcgCg caagaggcgg caacattagc cgagctgagg gcgccactgc aaaatgaaac aaagaaaaaa gcgcccgcc~a ggtagccag tgctgggatg.

.gtccAggtta Cgagg ttt q4 .aaagtgc4ggg .cgcttgtctt .ttctaaata~g ,C t ggtct g~t tcagtc attt agaaatggcc agaggattcc gggacttagg gggcgcggtg gggatcccaa -gtcactgtta acagcctggg cccttgttgC aaaaacgaac agica at aca ataggataca atacaataca gtcactttgg accaacatgg atgcctataa cggaggjttgc actccgtCtc acatacatac gtggctcaag tcagaccaac cgggcgctgt caggagaatc aacccagcct gcaacaaaat acaacaaaac 540 600 -66 720 .780 840 900 960 1020 1 080 '1140 1200 1 260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2843 <210> 8 <211> 1331 <212> DNA <213> Homo sapiens f:d rn~ed±2-09-;20'0j1 f, <400> 8 atggcggcga acc accgagaagt ccc cgcgtggctg gca aaggtgatgt acg ctcatcaact gga gtcagCaCCa tgcj gacgqatgtgjg agc agctttcaca agc gtgtaccaga ag~ gccaaagcag ag~ gcacagcggc ag gagcagcgct at caagaagtgg tt ttcaagcaga ag ctgaggagcc cc ccagCtgctg cc actcctccat 9t gagaccttct ac gaccaccaca tt gactaccagg

C

atcgaggtga tc ttccctgcca a agagaaaaaa c tUdJ'-'-Y ~~-nnne~a ac~CtgCaag aggcctaCgt gcgggtggtc tttacctatg :aatgaCatC aaggCaaCag

C

:aacagCggg ctgggccatC cgacCga cagggga rcctt ctg .caggcga jccagctt :ctgagtg ;agagtgg accaatgc aaggagga ctqgagca caggagca tcaaggaa gagagggc ttcctgca :atctcaac ,ctggtgcz :gagcagc ~cagggcci igccgacgi -gacgaag gggtggcctg cagagtgaag gggcgtgaaC ccigaagggg catcatggag ccgCttccag cqtgtctgag ggagaaccgt ggagcgccgg gqgtggcgag ccgaaatgac *catgtccacc gaagcagctC gcccagggcz ggcagaaga( -cccactggt4 999ggctcaq acacagagat g ctggtggcg gaggagatgg gaccccaact gatgtgcgga gcccatgtga aaggtgcca gacgtgggac attaaaaggg cggctggagg gagcgtgagc gccaqccccc caggagtctg acctccatrt acccaaccac Sgatctccctc.

;gaggctgtgt ;cagcagcaac t gggcaaggg c tcctttgav t ggctatggg, tggaggagct c ctggactgCcC agggagcctg1 ccatcaacgc aggcttcagg cccaggcccc ttggtaaaga aaaagcggcg tgcgtgaggC agaggacgtg ccgtgcacc ccagtcctca agacccactt ctgaggagcc atgaggaa~c I gtgCCggCtc :tctgtgcccg :ccgagaacct n cggatggcca a~aatttgtc tgccagccac acgggccgag tgccaactac agtgggCtCt cagcttctgg ggccgaggag tgcacgcc99 ggagcagcag gagggagatt gcctggcaag tggcagagag ggcgcccagc tccagagcai tgagcaca t tgccctgtac catcacggg( ttttggcat( agactagtC 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 ~1020 1080 1140 c:1200 g 12 t 1320 1331 ctacgtgg a gctcattgag tgaggctgag ggcggccgcIt <210> 9 <211> 760 <212>

PRT

<213> Homo sapiens <220> <221>

UNSURE

<222> (16) <220> <221>

UNSURE

<222> (41) <220> <221>

UNSURE

<222> (44) <220> <221>

UNSURE

<222> (48) <220> .Prifd2-Q9=200 1' 7i

I

<221> UNSURE <222> (64) <220> <221> UNSURE <C222> (96) <220> <221> UNSURE <222> (112) <22.0> <221> UNSURE <222> (118) <220> <221> UNSURE <222> (131) <220>.

<221> UNSURE <222> (144) <220> <221> UNSURE <222> (160) <220> <221> UNSURE <222> (168) <220> <221> UNSURE <222> (173) <220> <221> UNSURE <222> (192) <220> <221> UNSURE <222) (198) <220> <221) UNSURE <222> (208) <220> ~Printed:2O-O9~2OO1' rig

C

k~P~~

I

<221> UNSURE <222> (227) <220> <221> UNSURE <222> (240) <220> <221> UNSURE <222> (256) <220> <221> UNSURE <222> (264) <220> <221> UNSURE <222> (276) <220> <221> UNSURE <222> (288) <220> <221> UNSURE <222> (302) <220> <221> UNSURE <222> (304) <220> <221> UNSURE <222> (336) <220> <221> UNSURE <222> (352) <220> <221> UNSURE <222> (383) <220> <221> UNSURE <222> (399) <220> Prinited 2b-,2001; q jpetil pG M P <221> UNSURE <222> (431) <220> <221> UNSURE <222> (447) <220> <221> UNSURE <222> (479) <220> <221> UNSURE <222> (495) <220> <221> UNSURE <222> (520) <220> <221> UNSURE <222> (527) <220> <221> UNSURE <222> (543) <220> <221> UNSURE <222> (552) <220> <221> UNSURE <222> (575) <220> <221> UNSURE <222> (591) <220> <221> UNSURE <222> (622) <220> <221> UNSURE <222> (639) <220> P. inted:20-09-f2001- S i, i l- ion, h- e iF. l..r's ec o- t~L. Y <c221> <222> <220> <221> <222> <220> <C221> <222> <220> <221> <222> <400>

UNSURE

(671)

UNSURE

(687)

UNSURE

(719)

UNSURE

(735).

9 His Glu Ile Pro Thr Val Pro Thr Tyr Tyr Pro Ala Lys Pro Gin Xaa Leu. Leu Asn Lys Arq Gly Asp Arg Leu Ala Arg Ser 110 Gin Thr 125 Pro Trp Asn Thr Xaa Ala Ser Trp Gly Phe Ser Gi y Val Ser Asp Gly 175 Arg Lys Val Thr Thr Gly Ile Thr Gly Leu Trp Arg Pro Ser Val Xaa 180 185 190 Ser Asp Ala Glu 210 Trp Asp 225 Cys Cys His Leu Va'l Gly Thr Ile 290 Arq Leu 305 Gly Arg Val Pro Arg Pro Ser Ser 370 Leu Thr 385 Arg Pro Cys Leu Ala Phe Thr Lys Thr Val His Gly 245 Tyr Val 260 Xaa Leo Gin Arg Pro Pro Arg Ala 325 Val Leo 340 Arg Thr Gin Pro Pro Glu Ala Asp 405 Gin Ala 420 Phe Asp 200 Ile Val Gin Val Ala Gin Xaa Gly 265 Ser Lys 280 Ala Ser Arg Pro Arg Ala Gly Ala 345 Ser Trp 360 Leo Arg Phe Gly Ala Glu Glu Ala 425 Gly Pro Phe 235 Giu Asn Glu Gly Leu 315 Thr Pro Thr Pro Glu 395 Pro Tyr4 Tyr His Gly Asn Leu Met Arg Arg 255 Lys Leo 270 Pro Gly Xaa Pro Pro Arg Arg Cys 335 Arg Pro 350 Thr Ser Gin Xaa Ala Xaa Ser Pro 415 Pro Xaa 430 ~r~t6d;2:09'20.01 F4N51 W-Axw .1~ Gin Ser Leu 465 Ile Giy Asn Pro His 545 Ala Gly Ala Ser Ala 625 Se r As n Arg 09 d 0 1, 16L W-Ep hnspec 166.

Leu Cys 690 Ala Pro His Ser SeLeSe e LuGlGyLy Ser Leu Ser Leu Leu Gly Gly Lys Gly Cys Azg 'rrp Pro 710 Ser Ser His Pro Tyr Ser Lys Xaa Ala 720 Xaa Val 735 Asp Thr Thr Cys Ser Thr Arg Leu Arg Cys Leu Gin Cys Ala Ser Leu Glu Lys 755 Asp Ser Asp Phe Lys Ser Lys Lys Lys Lys Lys 750 Leu Trp Thr Ser <210> <211> <212> <213> 753

PRT

Homo sapiens <220> <221> UNSURE <222> <220> <221> UNSURE <222) (13) <220> <221> UNSURE <222> (32) <220> <221> UNSURE <222> (48) <220> <221> UNSURE <222> <220> <221> UNSURE <222> (87) <220> <221> UNSURE rnted:2'0-09-200-1 14 reppi ev ~8~9~11 i: <222> (96) <220> <221> UNSURE <222> (100) <220> <221> UNSURE <222> (127) <220> <221> UNSURE <222> (137) <220> <221> UNSURE <222> (142) <220> <221> UNSURE <222> (144) <220> <221> UNSURE <222> (166) <220> <221> UNSURE <222> (176) <220> <221> UNSURE <222> (192) <220> <221> UNSURE <222> (196) <220> <221> UNSURE <222> (224) <220> <221> UNSURE <222> (233) <220> <221> UNSURE ii..

rnsoeeOw.

<222> (240) <220> <221> UNSURE <222> (245) <220> <221> UNSURE <222> (272) <220> <221> UNSURE <222> (288) <220> <221> UNSURE <222> (320) <220> <221> UNSURE <222> (336) <220> <221> UNSURE <222> (352)..(353) <220> <221> UNSURE <222> (363) <220> <221> UNSURE <222> (368) <220> <221> UNSURE <222> (371) <220> <221> UNSURE <222> (383)..(385) <220> <221> UNSURE <222> (410) <220> <221> UNSURE 16 firted 20-9a l00 in~P~ <222> (416) <220> <221> UNSURE <222> (432) <220> <221> UNSURE <222> (438) <220> <221> UNSURE <222> (464) <220> <221> UNSURE <222> (480) <220> <221> UNSURE <222> (487) <220> <221> UNSURE <222> (495)..(497) <220> <221> UNSURE <222> (505) <220> <221> UNSURE <222> (512)..

<220> <221> UNSURE <222> (522) <220> <221> UNSURE <222> (528) <220> <221> UNSURE <222> (550) <220> <221> UNSURE (513) P e:O w7 -to <222> (554) <220> <221> UNSURE <222> (560) <220> <221> UNSURE <222> (574) <220> <221> UNSURE <222> (576) <220> .<221> UNSURE <222> (589) <220> <221> UNSURE <222> (591) <220> <221> UNSURE <222> (608) <220> <221> UNSURE <222> (624) <220> <221> UNSURE <222> (652) <220> <221> UNSURE <222> (656)..(657) <220> <221> UNSURE <222> (662)..(663) <220> <221> UNSURE <222> (672) <220> <221> UNSURE LrV,..

T

JS

ON

<222> (702) <220> <221> UNSURE <222> (704) <220> <221> UNSURE <222> (718) <220> <221> UNSURE <222> (720) <220> <221> UNSURE <222> (723) <220> <221> UNSURE <222> (734) <220> <221> UNSURE <222> (743) <220> <221> UNSURE <222> (752) <400> ile Arg His 1 Ala Pro Gly Arg Arg Pro( Thr Asp Pro Ala Lys Arg 1 Ser Ala Arg Glu Glu Thr Xaa Glu Val Xaa Asn Val Ser Pro Arg Gly Ala Arg Gly Pro Val Lys Tyr His Tyr Ser Asp 40 jGly Gly Glu Pro Arg Gly Ala Leu 55 iArg Arg Pro Gly Ala Thr Arg Ser L Xaa Leu Gly Arg Tyr Thr Cys Gin epDI~bd h§1~ I Octfdon Pr;ied2-9-0 R S E-L,2Y;, Gin Arg Glu Val1 145 Ser Glu Ser Cys Arg 225 Ser Thr Pro Arg Gi u 305 Pro Ser Glu 105 Ile Xaa Thr Asp Ile 185 Gin Gin Xa a Se r Ser 265 Arg Arg Gi y ValI Thr Asp Phe Pro Gi y Val1 170 Val1 Val1 Tyr Gi y Lys 250 ValI Pro Al a Arg Arg 330 Ser Arg Ser Phe Leu 155 Gi y His Ser Glu Al a 235 Ser Gi y Pro Pro Gi y 315 Al a Ala Asn Thr Gly 140 Trp, Ser Pro Phe Arg 220 Asn Gi u Leu Pro Gly 300 Arg Arg Cys Leu Asn 125 Phe Arg Ser Leu Thr 205 Asn Gi y Ser Gly Ser 285 Pro Lys Al a Ile Pro Trp 110 Thr Asp Xaa Ala Pro Ser Tyr His 175 Ile Gly 190 Leu Leu Arg Mrg Ala Lys Arg Pro 255 Xaa Pro 270 Thr Arg Gly Ser Gly Gly Glu Phe 335 Phe Met Arg Xaa Xaa His 160 Xaa Xaa Met Xaa Xaa 240 Gly Xaa Xaa Ala Xaa 320 Xa a Xa a Pfi-nted:20-09-2001 :Pr~nted:2o!o9:2ooi File I~tir

D

Xaa Ile Leu Phe Leu Trp Val Asp Ile Gin Xaa Trp Asp Cys Xaa Xaa 355 365 Thr Xaa 385 Arg Leu Arg Gin Asn 465 Ser Xaa Xa a Pro Lys 545 Ser Phe Thr Trp 370 Arg Met Leu Gin Ser 450 Trp Thr Lys Vali Ser 530 Met Leu Asn Le u Phe Tyr Tyr Lys Trp Arg 405 Asn Asp 420 Giu Tyr Met Asn Ser Gin Leu Aia 485 Phe Ilie 500 Leu Phe Gly His Ser Trp Val Vai 565 Arc; Lys 580 Ile Ser Phe Trp 375 Phe Thr 390 Asn Aia Phe Lys Xaa Asn Lys Asp 455 Met Lys 470 Leu Xaa Ser Asn Ser Leu Leu Tyr 535 Xaa Cys 550 Arg Leu Vai Ser Pro Tyr Phe Ser Pro Gly Phe 440 Lys Tyr Ser Ile Ala 520 Cys Ser Phe Asn His Ile Leu His Tyr 425 Cys Cys Ile Ser Leu 505 Phe Thr Gin Se r Arg 585 Th r Giu Pro Xaa 410 Arg Ile Ser Ser Leu 490 Xaa Xaa Arg Xaa Lys 570 Thr Ala Lys Ser 395 Tyr Tyr Arc; Arg Ser 475 Ile Giy Phe His Phe 555 Arg Arg Ser Ser 380 Asp Pro Leu Giy Asp 460 Ser Pro Ile Gin Cys 540 Cys Asn Th r Thr Ser Phe Pro Gin Thr 445 Leu Thr Thr Lys Asn 525 Val.

Arc; Leu Xaa Cys Tyr Phe Phe Vai 430 Asp Gin Thr Tyr Ile 510 Ile Cys Val.

Xaa Thr 590 Ala Xaa Lys Thr 41i5 Ser Phe Ser Ser Xaa 495 Lys Lys His Arg Thr 575 Xaa Cys Xaa Giu 400 Xaa Xaa Ile Xaa Xaa 480 Xaa Xaa Xaa Ser Xaa 560 Xaa Ile Xaa ,Pfirited Fo MoS& -P td:6d-2001 Fepbl hb .;-rFjl.6A h s. -1 bt 0 A

Claims (11)

1. An isolated and purified protein having the following characteristics: a molecular weight of approximately 50-60 kDa; (ii) produced by normal prostate, colon, or ovarian cells, but not by prostate, colon, or ovarian cancer cells; and (iii) binds antibody 7G6 (ATCC Accession No. PTA-4606).

2. The protein of claim 1, wherein said protein is produced by normal prostate cells, and is encoded by SEQ ID NO:4.

3. A process for inhibiting growth of prostate, colon, or ovarian cells, comprising 10 administering to said cell the protein of claim 1.

4. A process for inhibiting growth of prostate cells comprising administering to said cells the protein of claim 2. A method for detecting prostate, colon, or ovarian cell cancer, comprising analyzing body tissue or fluid for the protein of claim 1 or claim 2; and 15 correlating a reduction or absence of the protein, relative to a control sample, with the presence of prostate, colon, or ovarian cell cancer.

6. The method of claim 5, wherein said analysing comprises immunoassay. *o

7. The method of claim 6, wherein said immunoassay is a dot blot assay or a Western blot assay.

8. The method of claim 5, wherein said body tissue or fluid is blood.

9. The method of claim 5, wherein said protein is produced by normal prostate cells. The method of claim 5, wherein said protein is encoded by SEQ ID NO:4.

11. A protein according to claim I substantially as herein described with reference to the examples.

12. A process according to claim 3 substantially as herein described with reference to example Dated this EIGHTEENTH day of MARCH

2005. Biotherapies. Inc. Applicant 4 6 .0 6 4 0* 4 60 6 E S 6 6 J 9 606 t6 C 6 0 @6 @0 6 S S 446 4 6.. 66 *6 *6 4 6 *606 06** 6* *6 0 44 4 6, 64* 666 Wray Associates Perth, Western Australia Patent Attorneys for the Applicant